research article classification of five uremic solutes
TRANSCRIPT
Research ArticleClassification of Five Uremic Solutes according toTheir Effects on Renal Tubular Cells
Takeo Edamatsu Ayako Fujieda Atsuko Ezawa and Yoshiharu Itoh
Pharmaceutical Division Kureha Corporation 3-26-2 Hyakunin-cho Shinjuku-ku Tokyo 169-8503 Japan
Correspondence should be addressed to Takeo Edamatsu edamatsukurehacojp
Received 25 June 2014 Revised 19 September 2014 Accepted 6 October 2014 Published 9 November 2014
Academic Editor Jochen Reiser
Copyright copy 2014 Takeo Edamatsu et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
BackgroundAims Uremic solutes which are known to be retained in patients with chronic kidney disease are considered to havedeleterious effects on disease progression Among these uremic solutes indoxyl sulfate (IS) has been extensively studied whileother solutes have been studied less to state We conducted a comparative study to examine the similarities and differences betweenIS p-cresyl sulfate (PCS) phenyl sulfate (PhS) hippuric acid (HA) and indoleacetic acid (IAA)Methods We used LLC-PK1 cellsto evaluate the effects of these solutes on viable cell number cell cycle progression and cell death Results All the solutes reducedviable cell number after 48-hour incubation N-Acetyl-L-cysteine inhibited this effect induced by all solutes except HA At theconcentration that reduced the cell number to almost 50of vehicle control IAA induced apoptosis but not cell cycle delay whereasother solutes induced delay in cell cycle progression with marginal impact on apoptosis Phosphorylation of p53 and Chk1 andexpression of ATF4 and CHOP genes were detected in IS- PCS- and PhS-treated cells but not in IAA-treated cells ConclusionsTaken together the adverse effects of PCS and PhS on renal tubular cells are similar to those of IS while those of HA and IAA differ
1 Introduction
Uremic solutes are a large number of compounds that areretained in chronic kidney disease (CKD) especially in end-stage renal disease resulting in elevated serum concentra-tions compared to normal condition [1] These solutes areexcreted in urine in healthy persons but are accumulatedas CKD progresses [2 3] Over a hundred uremic soluteshave been reported to date and these solutes are classifiedinto three groups according to the size and protein bindingproperties [4ndash6] Protein-bound solutes have attracted muchattention in the last decade because they are less efficientlyremoved by dialysis [7] and are possibly associated withCKD-related complications [8]
Indoxyl sulfate (IS) is a representative protein-boundsolute and its deleterious effects have been studied in variouscell types including renal tubular cells [9] mesangial cells[10] vascular endothelial cells [11] vascular smooth musclecells [12] osteoclasts [13] osteoblasts [14] erythrocytes [15]and monocytes [16] Moreover several studies demonstratethe adverse effects of IS on the kidney [17 18] and vascularsystems [19 20] of animal models In humans several
reports indicate the association of IS with impairment ofrenal function and development of cardiovascular disease inCKD patients [21ndash23] Although these studies imply a causalrelationship between IS and progression of CKD andorCKD-related complications it is necessary to clarify whetherother uremic solutes have similar effects
Our previous study demonstrated that the serum levelsof several solutes are elevated in CKD rats [24] Most of thesesolutes are also found to be increased in hemodialysis patients[25] Among these solutes we focused on five solutes whichwere IS p-cresyl sulfate phenyl sulfate hippuric acid andindoleacetic acidThe selection criteria for these solutes wereboth their high serum concentration in hemodialysis patientsand their effect on viable cell number of porcine renal tubularcells (unpublished observation)
Renal tubular cells are the cell component of renaltubules and tubular injury is thought to be one of the keyevents causing progression of CKD [26] Thus we evaluatedthe effects of the five uremic solutes on viable cell numberof renal tubular cells and investigated the underlying mecha-nisms
Hindawi Publishing CorporationInternational Journal of NephrologyVolume 2014 Article ID 512178 10 pageshttpdxdoiorg1011552014512178
2 International Journal of Nephrology
2 Materials and Methods
21 Cell Culture LLC-PK1 a porcine renal tubular epithelialcell line was obtained from American Type Culture Col-lection (ATCC Manassas VA USA) LLC-PK1 cells weremaintained in Medium 199 (Mediatech Manassas VA USA)supplemented with 10 FBS (BioWest Nuaille France)
22 Reagents Indoxyl sulfate was purchased from Biosynth(Staad Switzerland) p-Cresyl sulfate and phenyl sulfatewere synthesized at Eiweiss (Shizuoka Japan) Hippuric acidand indoleacetic acid were purchased from Tokyo chemicalindustry (Tokyo Japan) N-Acetyl-L-cysteine was purchasedfrom Sigma (St Louis MO USA)
23 Viable Cell Count Effects of uremic solutes on viablecell number were determined using the Cell Counting Kit-8 (Dojindo Wako Tokyo Japan) a water-soluble versionof the methyl thiazolyl tetrazolium assay according to themanufacturerrsquos instructions LLC-PK1 cells were suspendedin Medium 199 supplemented with 2 FBS and dispensedinto tubes Each uremic solute was added to the cell sus-pension and cell density was adjusted to 16 times 104 cellsmLOne milliliterwell of the cell suspension was seeded in a24-well plate and incubated for 48 hours After incubationthe medium was changed to a medium containing the CellCounting Kit-8 reagent The cells were incubated for another30min and the optical density at 450 nm (OD450) was mea-sured using a microplate reader (iMark Microplate ReaderBio-Rad Hercules CA USA) The OD450 of mediumcontaining Cell Counting Kit-8 reagent without cells wasmeasured and was subtracted from the OD450 of eachsample
24 Cell Cycle Analysis Effects of uremic solutes on growthrate were evaluated after adjusting the cell cycle at G1Sor G2M boundary Double thymidine block was used forsynchronization at G1S boundary Cells were incubatedovernight in serum-free medium containing 25mmolL ofthymidine changed to medium supplemented with 10 FBSand incubated for 7 hours and then in thymidine-containingmedium again overnight For G2M synchronization cellswere incubated overnight in serum-free medium containing10 120583molL of nocodazole After synchronization the cellswere treated with uremic solutes as described above for 4 or6 hours Then the cells were fixed in ethanol and stainedwith propidium iodide (Propidium IodideRNase StainingSolution Cell Signaling Technology Danvers MA USA)The stained cells were detected by flow cytometer (FACSCalibur Becton Dickinson Franklin Lakes NJ USA) and thedata were analyzed using the Flowjo software (Tomy DigitalBiology Tokyo Japan)
25 Apoptosis Cells were treated with uremic solutes asdescribed above for 48 hours After treatment culturemedium that might contain nonadherent cells and adherent
cells was harvested The cells were stained with FITC-conjugated annexin V and propidium (TACS Annexin V-FITC Apoptosis Detection Kit Trevigen Gaithersburg MDUSA) The stained cells were detected by flow cytometer(FACS Calibur) and the data were analyzed using the Flowjosoftware
26 Western Blotting Cells were treated with uremic solutesas described above for 5 hours After treatment the cellswere lysedwith RIPA Lysis Buffer (Santa Cruz BiotechnologySanta Cruz CA USA) Antibodies against phosphorylatedp53 (at Ser15) and Chk1 (at Ser345) were used (both aresupposed to be active forms and were purchased from CellSignaling Technology) Chemiluminescent signals generatedusing the ECL Select Western Blotting Detection System (GEHealthcare Buckinghamshire UK) were detected by Light-Capture II Cooled CCD Camera Systems (ATTO TokyoJapan) Antibody against 120573-actin was used as loading control(Biolegend San Diego CA USA)
27 Real-Time PCR Cells were treated with uremic solutesas described above for 5 or 24 hours After treatment thecells were lysed in ISOGEN (Nippon Gene Tokyo Japan)Total RNA was isolated according to the manufacturerrsquosinstructions and optical density at 260 nm was measuredusing a spectrophotometer (Gene Spec V Hitachi High-TechManufacturing amp Service Corporation Ibaraki Japan) TheRNA (300 ng) was reverse-transcribed using the PrimeScriptRT Reagent Kit (Takarabio Shiga Japan) in a 10 120583L reactionvolume Using 06 120583L of complementary DNA SYBR PremixEx Taq II Tli RNaseH Plus (Takarabio) and 10 pmol of eachof the primer sets for various target genes (Table 1) real-time PCR was performed in a 25 120583L reaction volume andanalyzed using the Thermal Cycler Dice Real Time System(TP800 Takarabio) at the following thermal cycling condi-tions denaturation at 95∘C for 30 sec followed by 40 cyclesof 95∘C denaturation for 5 sec and 60∘C annealingextensionfor 30 sec
28 Statistical Analysis Statistical analysis was performed byStudentrsquos 119905-test and 119875 lt 001 was considered as significant
3 Results
31 Effects on Cell Viability All five uremic solutes decreasedviable cell number dose-dependently upon incubating withLLC-PK1 cells for 48 hours (Figure 1) To compare the mech-anisms of action of the five solutes the concentration of eachsolute that reduces the cell number by almost 50 was usedin subsequent experiments For IAA two or three differentconcentrations were always used because the reduction ratesdiffered in different experiments In some experiments twoconcentrations of ISwere used for the same reasonViable cellnumbers were evaluated in each experiment for confirmationpurpose The results of viable cell numbers for variousexperimental conditions are shown in Supplementary Figuresavailable online at httpdxdoiorg1011552014512178
International Journal of Nephrology 3
Table 1 Primers for real-time PCR
Target Regulation Forward Reversep21 p53-regulated 51015840-catgtggacctgttgctgtc-31015840 51015840-cctcttggagaagatcagcc-31015840
ATF4 ER stress responsive 51015840-ggtcagtgcctcagacaaca-31015840 51015840-tctggcatggtttccaggtc-31015840
CHOP ER stress responsive 51015840-cttcaccactcttgaccctg-31015840 51015840-gagctctgactggaatcagg-31015840
Cdc20 p53-regulated 51015840-aatgtcctggcaactggagg-31015840 51015840-tgagctccttgtagtgggga-31015840
Skp2 p53-regulated 51015840-acctccacccagatgtgact-31015840 51015840-gtgctgtacacgaaaagggc-31015840
Puma p53-regulated 51015840-gatctcaacgcgctgtacga-31015840 51015840-caggcacctaattaggctcc-31015840
GAPDH house-keeping gene 51015840-ccatcaccatcttccaggag-31015840 51015840-gagatgatgaccctcttggc-31015840
0
50
100
2 5 10 20 5 10 20 5 10 20
025 05 1 20 40 80
Cont IS PCS PhS IAA HA
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast lowastlowast
lowast
lowast
lowast
lowast
lowast
(mmolL)
Viab
le ce
ll nu
mbe
r (
of c
ontro
l)
Figure 1 Effects of five uremic solutes on viable cell number Porcinerenal tubular cells were treated with each uremic solute for 48 hoursViable cell number was evaluated using Cell Counting Kit-8 Opticaldensity at 450 nm was measured and the values were calculated aspercent of control Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant reduction of viable cell population compared tocontrol (119875 lt 001) Cont control IS indoxyl sulfate PCS p-cresylsulfate PhS phenyl sulfate HA hippuric acid
32 Effects on Cell Cycle Progression The decrease in viablecell number is supposed to be due to either reduction ingrowth rate or induction of cell death To investigate thesetwo possibilities the effects of uremic solutes on growth rateand apoptosis were evaluated
First to determine the effects on growth rate cells weresynchronized at G1S boundary and the synchronized cellswere released to grow with or without uremic solutes Inthis experimental setting the cell cytometry histogram shiftsfrom left to right in a time-dependent manner according tothe increase inDNA content (Figure 2(a))Therefore delayedgrowth will appear as a lag of the histogram on the lefthand side of the corresponding control Under these exper-imental conditions IS p-cresyl sulfate (PCS) and phenylsulfate (PhS) delayed growth rate (Figure 2(b)) whereashippuric acid (HA) and IAA (Figure 2(c)) had no impact ongrowth rate In this experiment 025 and 05mmolL of IAAdecreased viable cell number by 26 and 84 respectively(Supplementary Figure 1)
Next cells were synchronized at G2M boundary andthen the cells were released to grow with or without ure-mic solutes In this experimental setting the histogram ofuntreated control cells at 0 hours shows a predominantcell population on the right (cells in G2 phase Figure 3(a))
with negligible amount on the left (cells in G1 phase) Asmitosis progresses cells in G2 phase decrease and cells inG1 phase increase Data are shown as percent of cells inG1 and G2 phases IS PCS PhS and HA retarded boththe decrease of cells in G2 phase and the increase of cellsin G1 phase (Figure 3(b)) In sharp contrast to these foursolutes IAA had no effect on the rate of G2 to G1 transition(Figure 3(c)) In this experiment 025 05 and 1mmolLof IAA decreased viable cell number by 12 42 and 79respectively (Supplementary Figure 2)
33 Effects on Cell Death Because IAA had no effects ongrowth rate as described above the effect on cell death wasexamined Annexin V-FITC and propidium iodide were usedto detect dying and dead cells respectively As a result onlyIAA induced significant increase of dying cells (Figure 4)while marginal increases of dying cells were observed in PCSor HA-treated groups (119875 = 0044 and 0021 resp) IAA alsoinduced marginal increase of dead cells (119875 = 0013)
34 Inhibition of Cell Viability Reduction by Antioxidant Pre-vious studies have reported that IS induces reactive oxygenspecies (ROS) and that ROS could be involved in IS-inducedinhibition of cell proliferation in several cell types [27 28]Therefore we examined the effect of N-acetyl-L-cysteinean antioxidant on reduction of viable cell number inducedby each solute Incubation with N-acetyl-L-cysteine partiallyinhibited reduction of viable cell number in IS PCS or PhSgroups and completely inhibited the reduction in IAA group(Figure 5) Meanwhile N-acetyl-L-cysteine did not inhibitthe effect of HA
35 Phosphorylation of p53 and Chk1 IS has been reportedto inhibit cellular proliferation in a p53-dependent mannerin HK-2 a human proximal tubular cell line [29] Thereforewe examined phosphorylation of p53 We also examinedphosphorylation ofChk1 that is also involved inDNAdamageresponse like p53The results showed that IS andPCS inducedp53 phosphorylation (Figure 6) while IS PCS and PhSinduced Chk1 phosphorylation IAA and HA had no effecton phosphorylation of these proteins In this experiment025 and 05mmolL of IAA decreased viable cell numberby 30 and 55 respectively and 5 and 10mmolL of ISdecreased viable cell number by 42 and 76 respectively(Supplementary Figure 4)
4 International Journal of Nephrology
0 200 400 600 800
30
60
90
120
Cel
l cou
nt
01K
FL2-A
(a)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 800
0
20
40
60
0
20
40
60
0
20
40
60
0 200 400 600 8000
20
40
60
Cel
l cou
nt
IS (5) PCS (10) PhS (10) HA (40)
4h
6h
0 200 400 600 8000
20
40
60
Cel
l cou
nt
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
1K 1K 1K 1K
1K1K1K1KFL2-A FL2-A FL2-A FL2-A
(b)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 8000
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Cel
l cou
ntC
ell c
ount
IS (5) IAA (05)IAA (025)
4h
6h
1K 1K 1K
1K 1K 1KFL2-A FL2-A FL2-A
(c)
Figure 2 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G1S boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hours dottedline 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Histograms of uremicsolute-treated cells and corresponding control cells at indicated time points After synchronization cells were released to grow with (dashedline) or without (solid line) uremic solute for indicated periods Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Concentrations of solutes (mmolL) are shown in parentheses Duplicate data are shown Data arerepresentative of two independent experiments
International Journal of Nephrology 5
FL2-A0 200 400 600 800
50
100
150
200
0
Cel
l cou
nt
1K
(a)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)
PhS (10)HA (40)
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
0 4 6
(h) (h)0 4 6
lowast lowast
lowastlowast lowast
lowast
lowast
lowast lowastlowast
lowast lowastlowast
lowast
PCS (10)
ContIS (5)
PhS (10)HA (40)
PCS (10)
lowast
(b)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)IAA (025)
IAA (05)IAA (1)
ContIS (5)IAA (025)
IAA (05)IAA (1)
0
10
20
30
40
50
60
70
0 4 6
(h)0 4 6
(h)
80
0
10
20
30
40
50
60
70
lowast
lowastlowast
(c)
Figure 3 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G2M boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hoursdotted line 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Percent of cellsin G1 or G2 phase After synchronization cells were released to grow with or without uremic solutes for indicated periods Percent of cellsin each phase was analyzed and calculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acidIAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisks indicate significant difference compared to corresponding control(119875 lt 001) Concentrations of solutes (mmolL) are shown in parentheses
6 International Journal of Nephrology
5 10 10 025 05 40
Annexin V-FITC positive cells ()
0
5
10
15
20
25
0
5
10
15
20
25
Cont IS PCS PhS IAA HA
5 10 10 025 05 40
Cont IS PCS PhS IAA HA
Annexin V-FITCPI positive cells ()
lowast
Figure 4 Effects of uremic solutes on cell death After 48-hour incubation with each uremic solute porcine renal tubular cells were stainedwith annexin V-FITC and propidium iodide (PI) Percent of annexin V-positive cells and annexin VPI-positive cells was analyzed andcalculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAA indoleacetic acid Data areshown as mean plusmn SD (119899 = 4) Asterisk indicates significant difference compared to corresponding control (119875 lt 001) Sharp marks ()indicate 119875 lt 005
0
50
100
0 5 10 10 10 025 05 1 40
Cont IS PCS PhS IAA HA
NAC (0)NAC (01)NAC (1)
lowastlowast lowast
lowastlowast
lowast
lowast
lowast
lowast lowast
lowast
NS NSNS
Viab
le ce
ll nu
mbe
r(
of c
orre
spon
ding
cont
rol)
Figure 5 Effect of N-acetyl-L-cysteine (NAC) on uremic solute-induced decrease in viable cell number Porcine renal tubular cellswere treated with NAC (0 01 or 1mmolL) for 20 minutes andfurther treated with each uremic solute for 48 hours Viable cellnumber was evaluated using Cell Counting Kit-8 Optical densityat 450 nm was measured and the values were calculated as percentof the corresponding control Cont control IS indoxyl sulfatePCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAAindoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant difference each pairs described in this figure(119875 lt 001) NS means nonsignificant difference
36 Expression of Genes Involved in Growth Delay or CellDeath To further explore molecules which are involved inuremic solute-induced cell cycle delay andor cell death weexamined the expression of genes known to play importantroles in these processes (Figure 7) IS and PCS significantlyinduced p21 mRNA expression at 24 hours after treatment IS(10mmolL) PCS and HA significantly induced Puma (p53upregulated modulator of apoptosis) mRNA expression at 5hours after treatment PCS and PhS suppressed Cdc20 and
Phospho-p53 (Ser15)
Phospho-Chk1 (Ser345)C
ont
IS (5
)
IS (1
0)
PCS
(10
)
PhS
(10
)
IAA
(025
)
IAA
(05
)
HA
(40
)
120573-Actin
Figure 6 Effects of uremic solutes on p53 and Chk1 phosphoryla-tion Porcine renal tubular cells were treated with each uremic solutefor 5 hours Cell lysates were obtained and subjected to Westernblotting Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhSphenyl sulfate HA hippuric acid IAA indoleacetic acid Images arerepresentative of two independent experiments
Skp2 mRNA expression at 24 hours after treatment IS PCSPhS andHA induced ATF4 and CHOPmRNA On the otherhand IAA did not affect the expression of all these genes atall concentrations tested In this experiment 025 05 and1mmolL of IAA decreased viable cell number by 16 49 and72 respectively (Supplementary Figure 5)
4 Discussion
To the best of our knowledge this is the first study thatsystematically compared the effects of five uremic soluteson porcine renal tubular cells Our results are schematizedin Figure 8 The five solutes evaluated in this study werecategorized into three groups by the difference in mechanismof reducing viable cell number upon 48-hour treatment
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 International Journal of Nephrology
2 Materials and Methods
21 Cell Culture LLC-PK1 a porcine renal tubular epithelialcell line was obtained from American Type Culture Col-lection (ATCC Manassas VA USA) LLC-PK1 cells weremaintained in Medium 199 (Mediatech Manassas VA USA)supplemented with 10 FBS (BioWest Nuaille France)
22 Reagents Indoxyl sulfate was purchased from Biosynth(Staad Switzerland) p-Cresyl sulfate and phenyl sulfatewere synthesized at Eiweiss (Shizuoka Japan) Hippuric acidand indoleacetic acid were purchased from Tokyo chemicalindustry (Tokyo Japan) N-Acetyl-L-cysteine was purchasedfrom Sigma (St Louis MO USA)
23 Viable Cell Count Effects of uremic solutes on viablecell number were determined using the Cell Counting Kit-8 (Dojindo Wako Tokyo Japan) a water-soluble versionof the methyl thiazolyl tetrazolium assay according to themanufacturerrsquos instructions LLC-PK1 cells were suspendedin Medium 199 supplemented with 2 FBS and dispensedinto tubes Each uremic solute was added to the cell sus-pension and cell density was adjusted to 16 times 104 cellsmLOne milliliterwell of the cell suspension was seeded in a24-well plate and incubated for 48 hours After incubationthe medium was changed to a medium containing the CellCounting Kit-8 reagent The cells were incubated for another30min and the optical density at 450 nm (OD450) was mea-sured using a microplate reader (iMark Microplate ReaderBio-Rad Hercules CA USA) The OD450 of mediumcontaining Cell Counting Kit-8 reagent without cells wasmeasured and was subtracted from the OD450 of eachsample
24 Cell Cycle Analysis Effects of uremic solutes on growthrate were evaluated after adjusting the cell cycle at G1Sor G2M boundary Double thymidine block was used forsynchronization at G1S boundary Cells were incubatedovernight in serum-free medium containing 25mmolL ofthymidine changed to medium supplemented with 10 FBSand incubated for 7 hours and then in thymidine-containingmedium again overnight For G2M synchronization cellswere incubated overnight in serum-free medium containing10 120583molL of nocodazole After synchronization the cellswere treated with uremic solutes as described above for 4 or6 hours Then the cells were fixed in ethanol and stainedwith propidium iodide (Propidium IodideRNase StainingSolution Cell Signaling Technology Danvers MA USA)The stained cells were detected by flow cytometer (FACSCalibur Becton Dickinson Franklin Lakes NJ USA) and thedata were analyzed using the Flowjo software (Tomy DigitalBiology Tokyo Japan)
25 Apoptosis Cells were treated with uremic solutes asdescribed above for 48 hours After treatment culturemedium that might contain nonadherent cells and adherent
cells was harvested The cells were stained with FITC-conjugated annexin V and propidium (TACS Annexin V-FITC Apoptosis Detection Kit Trevigen Gaithersburg MDUSA) The stained cells were detected by flow cytometer(FACS Calibur) and the data were analyzed using the Flowjosoftware
26 Western Blotting Cells were treated with uremic solutesas described above for 5 hours After treatment the cellswere lysedwith RIPA Lysis Buffer (Santa Cruz BiotechnologySanta Cruz CA USA) Antibodies against phosphorylatedp53 (at Ser15) and Chk1 (at Ser345) were used (both aresupposed to be active forms and were purchased from CellSignaling Technology) Chemiluminescent signals generatedusing the ECL Select Western Blotting Detection System (GEHealthcare Buckinghamshire UK) were detected by Light-Capture II Cooled CCD Camera Systems (ATTO TokyoJapan) Antibody against 120573-actin was used as loading control(Biolegend San Diego CA USA)
27 Real-Time PCR Cells were treated with uremic solutesas described above for 5 or 24 hours After treatment thecells were lysed in ISOGEN (Nippon Gene Tokyo Japan)Total RNA was isolated according to the manufacturerrsquosinstructions and optical density at 260 nm was measuredusing a spectrophotometer (Gene Spec V Hitachi High-TechManufacturing amp Service Corporation Ibaraki Japan) TheRNA (300 ng) was reverse-transcribed using the PrimeScriptRT Reagent Kit (Takarabio Shiga Japan) in a 10 120583L reactionvolume Using 06 120583L of complementary DNA SYBR PremixEx Taq II Tli RNaseH Plus (Takarabio) and 10 pmol of eachof the primer sets for various target genes (Table 1) real-time PCR was performed in a 25 120583L reaction volume andanalyzed using the Thermal Cycler Dice Real Time System(TP800 Takarabio) at the following thermal cycling condi-tions denaturation at 95∘C for 30 sec followed by 40 cyclesof 95∘C denaturation for 5 sec and 60∘C annealingextensionfor 30 sec
28 Statistical Analysis Statistical analysis was performed byStudentrsquos 119905-test and 119875 lt 001 was considered as significant
3 Results
31 Effects on Cell Viability All five uremic solutes decreasedviable cell number dose-dependently upon incubating withLLC-PK1 cells for 48 hours (Figure 1) To compare the mech-anisms of action of the five solutes the concentration of eachsolute that reduces the cell number by almost 50 was usedin subsequent experiments For IAA two or three differentconcentrations were always used because the reduction ratesdiffered in different experiments In some experiments twoconcentrations of ISwere used for the same reasonViable cellnumbers were evaluated in each experiment for confirmationpurpose The results of viable cell numbers for variousexperimental conditions are shown in Supplementary Figuresavailable online at httpdxdoiorg1011552014512178
International Journal of Nephrology 3
Table 1 Primers for real-time PCR
Target Regulation Forward Reversep21 p53-regulated 51015840-catgtggacctgttgctgtc-31015840 51015840-cctcttggagaagatcagcc-31015840
ATF4 ER stress responsive 51015840-ggtcagtgcctcagacaaca-31015840 51015840-tctggcatggtttccaggtc-31015840
CHOP ER stress responsive 51015840-cttcaccactcttgaccctg-31015840 51015840-gagctctgactggaatcagg-31015840
Cdc20 p53-regulated 51015840-aatgtcctggcaactggagg-31015840 51015840-tgagctccttgtagtgggga-31015840
Skp2 p53-regulated 51015840-acctccacccagatgtgact-31015840 51015840-gtgctgtacacgaaaagggc-31015840
Puma p53-regulated 51015840-gatctcaacgcgctgtacga-31015840 51015840-caggcacctaattaggctcc-31015840
GAPDH house-keeping gene 51015840-ccatcaccatcttccaggag-31015840 51015840-gagatgatgaccctcttggc-31015840
0
50
100
2 5 10 20 5 10 20 5 10 20
025 05 1 20 40 80
Cont IS PCS PhS IAA HA
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast lowastlowast
lowast
lowast
lowast
lowast
lowast
(mmolL)
Viab
le ce
ll nu
mbe
r (
of c
ontro
l)
Figure 1 Effects of five uremic solutes on viable cell number Porcinerenal tubular cells were treated with each uremic solute for 48 hoursViable cell number was evaluated using Cell Counting Kit-8 Opticaldensity at 450 nm was measured and the values were calculated aspercent of control Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant reduction of viable cell population compared tocontrol (119875 lt 001) Cont control IS indoxyl sulfate PCS p-cresylsulfate PhS phenyl sulfate HA hippuric acid
32 Effects on Cell Cycle Progression The decrease in viablecell number is supposed to be due to either reduction ingrowth rate or induction of cell death To investigate thesetwo possibilities the effects of uremic solutes on growth rateand apoptosis were evaluated
First to determine the effects on growth rate cells weresynchronized at G1S boundary and the synchronized cellswere released to grow with or without uremic solutes Inthis experimental setting the cell cytometry histogram shiftsfrom left to right in a time-dependent manner according tothe increase inDNA content (Figure 2(a))Therefore delayedgrowth will appear as a lag of the histogram on the lefthand side of the corresponding control Under these exper-imental conditions IS p-cresyl sulfate (PCS) and phenylsulfate (PhS) delayed growth rate (Figure 2(b)) whereashippuric acid (HA) and IAA (Figure 2(c)) had no impact ongrowth rate In this experiment 025 and 05mmolL of IAAdecreased viable cell number by 26 and 84 respectively(Supplementary Figure 1)
Next cells were synchronized at G2M boundary andthen the cells were released to grow with or without ure-mic solutes In this experimental setting the histogram ofuntreated control cells at 0 hours shows a predominantcell population on the right (cells in G2 phase Figure 3(a))
with negligible amount on the left (cells in G1 phase) Asmitosis progresses cells in G2 phase decrease and cells inG1 phase increase Data are shown as percent of cells inG1 and G2 phases IS PCS PhS and HA retarded boththe decrease of cells in G2 phase and the increase of cellsin G1 phase (Figure 3(b)) In sharp contrast to these foursolutes IAA had no effect on the rate of G2 to G1 transition(Figure 3(c)) In this experiment 025 05 and 1mmolLof IAA decreased viable cell number by 12 42 and 79respectively (Supplementary Figure 2)
33 Effects on Cell Death Because IAA had no effects ongrowth rate as described above the effect on cell death wasexamined Annexin V-FITC and propidium iodide were usedto detect dying and dead cells respectively As a result onlyIAA induced significant increase of dying cells (Figure 4)while marginal increases of dying cells were observed in PCSor HA-treated groups (119875 = 0044 and 0021 resp) IAA alsoinduced marginal increase of dead cells (119875 = 0013)
34 Inhibition of Cell Viability Reduction by Antioxidant Pre-vious studies have reported that IS induces reactive oxygenspecies (ROS) and that ROS could be involved in IS-inducedinhibition of cell proliferation in several cell types [27 28]Therefore we examined the effect of N-acetyl-L-cysteinean antioxidant on reduction of viable cell number inducedby each solute Incubation with N-acetyl-L-cysteine partiallyinhibited reduction of viable cell number in IS PCS or PhSgroups and completely inhibited the reduction in IAA group(Figure 5) Meanwhile N-acetyl-L-cysteine did not inhibitthe effect of HA
35 Phosphorylation of p53 and Chk1 IS has been reportedto inhibit cellular proliferation in a p53-dependent mannerin HK-2 a human proximal tubular cell line [29] Thereforewe examined phosphorylation of p53 We also examinedphosphorylation ofChk1 that is also involved inDNAdamageresponse like p53The results showed that IS andPCS inducedp53 phosphorylation (Figure 6) while IS PCS and PhSinduced Chk1 phosphorylation IAA and HA had no effecton phosphorylation of these proteins In this experiment025 and 05mmolL of IAA decreased viable cell numberby 30 and 55 respectively and 5 and 10mmolL of ISdecreased viable cell number by 42 and 76 respectively(Supplementary Figure 4)
4 International Journal of Nephrology
0 200 400 600 800
30
60
90
120
Cel
l cou
nt
01K
FL2-A
(a)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 800
0
20
40
60
0
20
40
60
0
20
40
60
0 200 400 600 8000
20
40
60
Cel
l cou
nt
IS (5) PCS (10) PhS (10) HA (40)
4h
6h
0 200 400 600 8000
20
40
60
Cel
l cou
nt
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
1K 1K 1K 1K
1K1K1K1KFL2-A FL2-A FL2-A FL2-A
(b)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 8000
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Cel
l cou
ntC
ell c
ount
IS (5) IAA (05)IAA (025)
4h
6h
1K 1K 1K
1K 1K 1KFL2-A FL2-A FL2-A
(c)
Figure 2 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G1S boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hours dottedline 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Histograms of uremicsolute-treated cells and corresponding control cells at indicated time points After synchronization cells were released to grow with (dashedline) or without (solid line) uremic solute for indicated periods Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Concentrations of solutes (mmolL) are shown in parentheses Duplicate data are shown Data arerepresentative of two independent experiments
International Journal of Nephrology 5
FL2-A0 200 400 600 800
50
100
150
200
0
Cel
l cou
nt
1K
(a)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)
PhS (10)HA (40)
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
0 4 6
(h) (h)0 4 6
lowast lowast
lowastlowast lowast
lowast
lowast
lowast lowastlowast
lowast lowastlowast
lowast
PCS (10)
ContIS (5)
PhS (10)HA (40)
PCS (10)
lowast
(b)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)IAA (025)
IAA (05)IAA (1)
ContIS (5)IAA (025)
IAA (05)IAA (1)
0
10
20
30
40
50
60
70
0 4 6
(h)0 4 6
(h)
80
0
10
20
30
40
50
60
70
lowast
lowastlowast
(c)
Figure 3 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G2M boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hoursdotted line 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Percent of cellsin G1 or G2 phase After synchronization cells were released to grow with or without uremic solutes for indicated periods Percent of cellsin each phase was analyzed and calculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acidIAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisks indicate significant difference compared to corresponding control(119875 lt 001) Concentrations of solutes (mmolL) are shown in parentheses
6 International Journal of Nephrology
5 10 10 025 05 40
Annexin V-FITC positive cells ()
0
5
10
15
20
25
0
5
10
15
20
25
Cont IS PCS PhS IAA HA
5 10 10 025 05 40
Cont IS PCS PhS IAA HA
Annexin V-FITCPI positive cells ()
lowast
Figure 4 Effects of uremic solutes on cell death After 48-hour incubation with each uremic solute porcine renal tubular cells were stainedwith annexin V-FITC and propidium iodide (PI) Percent of annexin V-positive cells and annexin VPI-positive cells was analyzed andcalculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAA indoleacetic acid Data areshown as mean plusmn SD (119899 = 4) Asterisk indicates significant difference compared to corresponding control (119875 lt 001) Sharp marks ()indicate 119875 lt 005
0
50
100
0 5 10 10 10 025 05 1 40
Cont IS PCS PhS IAA HA
NAC (0)NAC (01)NAC (1)
lowastlowast lowast
lowastlowast
lowast
lowast
lowast
lowast lowast
lowast
NS NSNS
Viab
le ce
ll nu
mbe
r(
of c
orre
spon
ding
cont
rol)
Figure 5 Effect of N-acetyl-L-cysteine (NAC) on uremic solute-induced decrease in viable cell number Porcine renal tubular cellswere treated with NAC (0 01 or 1mmolL) for 20 minutes andfurther treated with each uremic solute for 48 hours Viable cellnumber was evaluated using Cell Counting Kit-8 Optical densityat 450 nm was measured and the values were calculated as percentof the corresponding control Cont control IS indoxyl sulfatePCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAAindoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant difference each pairs described in this figure(119875 lt 001) NS means nonsignificant difference
36 Expression of Genes Involved in Growth Delay or CellDeath To further explore molecules which are involved inuremic solute-induced cell cycle delay andor cell death weexamined the expression of genes known to play importantroles in these processes (Figure 7) IS and PCS significantlyinduced p21 mRNA expression at 24 hours after treatment IS(10mmolL) PCS and HA significantly induced Puma (p53upregulated modulator of apoptosis) mRNA expression at 5hours after treatment PCS and PhS suppressed Cdc20 and
Phospho-p53 (Ser15)
Phospho-Chk1 (Ser345)C
ont
IS (5
)
IS (1
0)
PCS
(10
)
PhS
(10
)
IAA
(025
)
IAA
(05
)
HA
(40
)
120573-Actin
Figure 6 Effects of uremic solutes on p53 and Chk1 phosphoryla-tion Porcine renal tubular cells were treated with each uremic solutefor 5 hours Cell lysates were obtained and subjected to Westernblotting Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhSphenyl sulfate HA hippuric acid IAA indoleacetic acid Images arerepresentative of two independent experiments
Skp2 mRNA expression at 24 hours after treatment IS PCSPhS andHA induced ATF4 and CHOPmRNA On the otherhand IAA did not affect the expression of all these genes atall concentrations tested In this experiment 025 05 and1mmolL of IAA decreased viable cell number by 16 49 and72 respectively (Supplementary Figure 5)
4 Discussion
To the best of our knowledge this is the first study thatsystematically compared the effects of five uremic soluteson porcine renal tubular cells Our results are schematizedin Figure 8 The five solutes evaluated in this study werecategorized into three groups by the difference in mechanismof reducing viable cell number upon 48-hour treatment
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Nephrology 3
Table 1 Primers for real-time PCR
Target Regulation Forward Reversep21 p53-regulated 51015840-catgtggacctgttgctgtc-31015840 51015840-cctcttggagaagatcagcc-31015840
ATF4 ER stress responsive 51015840-ggtcagtgcctcagacaaca-31015840 51015840-tctggcatggtttccaggtc-31015840
CHOP ER stress responsive 51015840-cttcaccactcttgaccctg-31015840 51015840-gagctctgactggaatcagg-31015840
Cdc20 p53-regulated 51015840-aatgtcctggcaactggagg-31015840 51015840-tgagctccttgtagtgggga-31015840
Skp2 p53-regulated 51015840-acctccacccagatgtgact-31015840 51015840-gtgctgtacacgaaaagggc-31015840
Puma p53-regulated 51015840-gatctcaacgcgctgtacga-31015840 51015840-caggcacctaattaggctcc-31015840
GAPDH house-keeping gene 51015840-ccatcaccatcttccaggag-31015840 51015840-gagatgatgaccctcttggc-31015840
0
50
100
2 5 10 20 5 10 20 5 10 20
025 05 1 20 40 80
Cont IS PCS PhS IAA HA
lowast
lowast
lowast
lowast
lowast
lowast
lowast
lowast lowastlowast
lowast
lowast
lowast
lowast
lowast
(mmolL)
Viab
le ce
ll nu
mbe
r (
of c
ontro
l)
Figure 1 Effects of five uremic solutes on viable cell number Porcinerenal tubular cells were treated with each uremic solute for 48 hoursViable cell number was evaluated using Cell Counting Kit-8 Opticaldensity at 450 nm was measured and the values were calculated aspercent of control Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant reduction of viable cell population compared tocontrol (119875 lt 001) Cont control IS indoxyl sulfate PCS p-cresylsulfate PhS phenyl sulfate HA hippuric acid
32 Effects on Cell Cycle Progression The decrease in viablecell number is supposed to be due to either reduction ingrowth rate or induction of cell death To investigate thesetwo possibilities the effects of uremic solutes on growth rateand apoptosis were evaluated
First to determine the effects on growth rate cells weresynchronized at G1S boundary and the synchronized cellswere released to grow with or without uremic solutes Inthis experimental setting the cell cytometry histogram shiftsfrom left to right in a time-dependent manner according tothe increase inDNA content (Figure 2(a))Therefore delayedgrowth will appear as a lag of the histogram on the lefthand side of the corresponding control Under these exper-imental conditions IS p-cresyl sulfate (PCS) and phenylsulfate (PhS) delayed growth rate (Figure 2(b)) whereashippuric acid (HA) and IAA (Figure 2(c)) had no impact ongrowth rate In this experiment 025 and 05mmolL of IAAdecreased viable cell number by 26 and 84 respectively(Supplementary Figure 1)
Next cells were synchronized at G2M boundary andthen the cells were released to grow with or without ure-mic solutes In this experimental setting the histogram ofuntreated control cells at 0 hours shows a predominantcell population on the right (cells in G2 phase Figure 3(a))
with negligible amount on the left (cells in G1 phase) Asmitosis progresses cells in G2 phase decrease and cells inG1 phase increase Data are shown as percent of cells inG1 and G2 phases IS PCS PhS and HA retarded boththe decrease of cells in G2 phase and the increase of cellsin G1 phase (Figure 3(b)) In sharp contrast to these foursolutes IAA had no effect on the rate of G2 to G1 transition(Figure 3(c)) In this experiment 025 05 and 1mmolLof IAA decreased viable cell number by 12 42 and 79respectively (Supplementary Figure 2)
33 Effects on Cell Death Because IAA had no effects ongrowth rate as described above the effect on cell death wasexamined Annexin V-FITC and propidium iodide were usedto detect dying and dead cells respectively As a result onlyIAA induced significant increase of dying cells (Figure 4)while marginal increases of dying cells were observed in PCSor HA-treated groups (119875 = 0044 and 0021 resp) IAA alsoinduced marginal increase of dead cells (119875 = 0013)
34 Inhibition of Cell Viability Reduction by Antioxidant Pre-vious studies have reported that IS induces reactive oxygenspecies (ROS) and that ROS could be involved in IS-inducedinhibition of cell proliferation in several cell types [27 28]Therefore we examined the effect of N-acetyl-L-cysteinean antioxidant on reduction of viable cell number inducedby each solute Incubation with N-acetyl-L-cysteine partiallyinhibited reduction of viable cell number in IS PCS or PhSgroups and completely inhibited the reduction in IAA group(Figure 5) Meanwhile N-acetyl-L-cysteine did not inhibitthe effect of HA
35 Phosphorylation of p53 and Chk1 IS has been reportedto inhibit cellular proliferation in a p53-dependent mannerin HK-2 a human proximal tubular cell line [29] Thereforewe examined phosphorylation of p53 We also examinedphosphorylation ofChk1 that is also involved inDNAdamageresponse like p53The results showed that IS andPCS inducedp53 phosphorylation (Figure 6) while IS PCS and PhSinduced Chk1 phosphorylation IAA and HA had no effecton phosphorylation of these proteins In this experiment025 and 05mmolL of IAA decreased viable cell numberby 30 and 55 respectively and 5 and 10mmolL of ISdecreased viable cell number by 42 and 76 respectively(Supplementary Figure 4)
4 International Journal of Nephrology
0 200 400 600 800
30
60
90
120
Cel
l cou
nt
01K
FL2-A
(a)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 800
0
20
40
60
0
20
40
60
0
20
40
60
0 200 400 600 8000
20
40
60
Cel
l cou
nt
IS (5) PCS (10) PhS (10) HA (40)
4h
6h
0 200 400 600 8000
20
40
60
Cel
l cou
nt
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
1K 1K 1K 1K
1K1K1K1KFL2-A FL2-A FL2-A FL2-A
(b)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 8000
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Cel
l cou
ntC
ell c
ount
IS (5) IAA (05)IAA (025)
4h
6h
1K 1K 1K
1K 1K 1KFL2-A FL2-A FL2-A
(c)
Figure 2 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G1S boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hours dottedline 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Histograms of uremicsolute-treated cells and corresponding control cells at indicated time points After synchronization cells were released to grow with (dashedline) or without (solid line) uremic solute for indicated periods Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Concentrations of solutes (mmolL) are shown in parentheses Duplicate data are shown Data arerepresentative of two independent experiments
International Journal of Nephrology 5
FL2-A0 200 400 600 800
50
100
150
200
0
Cel
l cou
nt
1K
(a)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)
PhS (10)HA (40)
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
0 4 6
(h) (h)0 4 6
lowast lowast
lowastlowast lowast
lowast
lowast
lowast lowastlowast
lowast lowastlowast
lowast
PCS (10)
ContIS (5)
PhS (10)HA (40)
PCS (10)
lowast
(b)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)IAA (025)
IAA (05)IAA (1)
ContIS (5)IAA (025)
IAA (05)IAA (1)
0
10
20
30
40
50
60
70
0 4 6
(h)0 4 6
(h)
80
0
10
20
30
40
50
60
70
lowast
lowastlowast
(c)
Figure 3 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G2M boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hoursdotted line 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Percent of cellsin G1 or G2 phase After synchronization cells were released to grow with or without uremic solutes for indicated periods Percent of cellsin each phase was analyzed and calculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acidIAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisks indicate significant difference compared to corresponding control(119875 lt 001) Concentrations of solutes (mmolL) are shown in parentheses
6 International Journal of Nephrology
5 10 10 025 05 40
Annexin V-FITC positive cells ()
0
5
10
15
20
25
0
5
10
15
20
25
Cont IS PCS PhS IAA HA
5 10 10 025 05 40
Cont IS PCS PhS IAA HA
Annexin V-FITCPI positive cells ()
lowast
Figure 4 Effects of uremic solutes on cell death After 48-hour incubation with each uremic solute porcine renal tubular cells were stainedwith annexin V-FITC and propidium iodide (PI) Percent of annexin V-positive cells and annexin VPI-positive cells was analyzed andcalculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAA indoleacetic acid Data areshown as mean plusmn SD (119899 = 4) Asterisk indicates significant difference compared to corresponding control (119875 lt 001) Sharp marks ()indicate 119875 lt 005
0
50
100
0 5 10 10 10 025 05 1 40
Cont IS PCS PhS IAA HA
NAC (0)NAC (01)NAC (1)
lowastlowast lowast
lowastlowast
lowast
lowast
lowast
lowast lowast
lowast
NS NSNS
Viab
le ce
ll nu
mbe
r(
of c
orre
spon
ding
cont
rol)
Figure 5 Effect of N-acetyl-L-cysteine (NAC) on uremic solute-induced decrease in viable cell number Porcine renal tubular cellswere treated with NAC (0 01 or 1mmolL) for 20 minutes andfurther treated with each uremic solute for 48 hours Viable cellnumber was evaluated using Cell Counting Kit-8 Optical densityat 450 nm was measured and the values were calculated as percentof the corresponding control Cont control IS indoxyl sulfatePCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAAindoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant difference each pairs described in this figure(119875 lt 001) NS means nonsignificant difference
36 Expression of Genes Involved in Growth Delay or CellDeath To further explore molecules which are involved inuremic solute-induced cell cycle delay andor cell death weexamined the expression of genes known to play importantroles in these processes (Figure 7) IS and PCS significantlyinduced p21 mRNA expression at 24 hours after treatment IS(10mmolL) PCS and HA significantly induced Puma (p53upregulated modulator of apoptosis) mRNA expression at 5hours after treatment PCS and PhS suppressed Cdc20 and
Phospho-p53 (Ser15)
Phospho-Chk1 (Ser345)C
ont
IS (5
)
IS (1
0)
PCS
(10
)
PhS
(10
)
IAA
(025
)
IAA
(05
)
HA
(40
)
120573-Actin
Figure 6 Effects of uremic solutes on p53 and Chk1 phosphoryla-tion Porcine renal tubular cells were treated with each uremic solutefor 5 hours Cell lysates were obtained and subjected to Westernblotting Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhSphenyl sulfate HA hippuric acid IAA indoleacetic acid Images arerepresentative of two independent experiments
Skp2 mRNA expression at 24 hours after treatment IS PCSPhS andHA induced ATF4 and CHOPmRNA On the otherhand IAA did not affect the expression of all these genes atall concentrations tested In this experiment 025 05 and1mmolL of IAA decreased viable cell number by 16 49 and72 respectively (Supplementary Figure 5)
4 Discussion
To the best of our knowledge this is the first study thatsystematically compared the effects of five uremic soluteson porcine renal tubular cells Our results are schematizedin Figure 8 The five solutes evaluated in this study werecategorized into three groups by the difference in mechanismof reducing viable cell number upon 48-hour treatment
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 International Journal of Nephrology
0 200 400 600 800
30
60
90
120
Cel
l cou
nt
01K
FL2-A
(a)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 800
0
20
40
60
0
20
40
60
0
20
40
60
0 200 400 600 8000
20
40
60
Cel
l cou
nt
IS (5) PCS (10) PhS (10) HA (40)
4h
6h
0 200 400 600 8000
20
40
60
Cel
l cou
nt
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
1K 1K 1K 1K
1K1K1K1KFL2-A FL2-A FL2-A FL2-A
(b)
0 200 400 600 800 0 200 400 600 800 0 200 400 600 800
0 200 400 600 8000 200 400 600 8000 200 400 600 8000
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Cel
l cou
ntC
ell c
ount
IS (5) IAA (05)IAA (025)
4h
6h
1K 1K 1K
1K 1K 1KFL2-A FL2-A FL2-A
(c)
Figure 2 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G1S boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hours dottedline 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Histograms of uremicsolute-treated cells and corresponding control cells at indicated time points After synchronization cells were released to grow with (dashedline) or without (solid line) uremic solute for indicated periods Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Concentrations of solutes (mmolL) are shown in parentheses Duplicate data are shown Data arerepresentative of two independent experiments
International Journal of Nephrology 5
FL2-A0 200 400 600 800
50
100
150
200
0
Cel
l cou
nt
1K
(a)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)
PhS (10)HA (40)
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
0 4 6
(h) (h)0 4 6
lowast lowast
lowastlowast lowast
lowast
lowast
lowast lowastlowast
lowast lowastlowast
lowast
PCS (10)
ContIS (5)
PhS (10)HA (40)
PCS (10)
lowast
(b)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)IAA (025)
IAA (05)IAA (1)
ContIS (5)IAA (025)
IAA (05)IAA (1)
0
10
20
30
40
50
60
70
0 4 6
(h)0 4 6
(h)
80
0
10
20
30
40
50
60
70
lowast
lowastlowast
(c)
Figure 3 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G2M boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hoursdotted line 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Percent of cellsin G1 or G2 phase After synchronization cells were released to grow with or without uremic solutes for indicated periods Percent of cellsin each phase was analyzed and calculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acidIAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisks indicate significant difference compared to corresponding control(119875 lt 001) Concentrations of solutes (mmolL) are shown in parentheses
6 International Journal of Nephrology
5 10 10 025 05 40
Annexin V-FITC positive cells ()
0
5
10
15
20
25
0
5
10
15
20
25
Cont IS PCS PhS IAA HA
5 10 10 025 05 40
Cont IS PCS PhS IAA HA
Annexin V-FITCPI positive cells ()
lowast
Figure 4 Effects of uremic solutes on cell death After 48-hour incubation with each uremic solute porcine renal tubular cells were stainedwith annexin V-FITC and propidium iodide (PI) Percent of annexin V-positive cells and annexin VPI-positive cells was analyzed andcalculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAA indoleacetic acid Data areshown as mean plusmn SD (119899 = 4) Asterisk indicates significant difference compared to corresponding control (119875 lt 001) Sharp marks ()indicate 119875 lt 005
0
50
100
0 5 10 10 10 025 05 1 40
Cont IS PCS PhS IAA HA
NAC (0)NAC (01)NAC (1)
lowastlowast lowast
lowastlowast
lowast
lowast
lowast
lowast lowast
lowast
NS NSNS
Viab
le ce
ll nu
mbe
r(
of c
orre
spon
ding
cont
rol)
Figure 5 Effect of N-acetyl-L-cysteine (NAC) on uremic solute-induced decrease in viable cell number Porcine renal tubular cellswere treated with NAC (0 01 or 1mmolL) for 20 minutes andfurther treated with each uremic solute for 48 hours Viable cellnumber was evaluated using Cell Counting Kit-8 Optical densityat 450 nm was measured and the values were calculated as percentof the corresponding control Cont control IS indoxyl sulfatePCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAAindoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant difference each pairs described in this figure(119875 lt 001) NS means nonsignificant difference
36 Expression of Genes Involved in Growth Delay or CellDeath To further explore molecules which are involved inuremic solute-induced cell cycle delay andor cell death weexamined the expression of genes known to play importantroles in these processes (Figure 7) IS and PCS significantlyinduced p21 mRNA expression at 24 hours after treatment IS(10mmolL) PCS and HA significantly induced Puma (p53upregulated modulator of apoptosis) mRNA expression at 5hours after treatment PCS and PhS suppressed Cdc20 and
Phospho-p53 (Ser15)
Phospho-Chk1 (Ser345)C
ont
IS (5
)
IS (1
0)
PCS
(10
)
PhS
(10
)
IAA
(025
)
IAA
(05
)
HA
(40
)
120573-Actin
Figure 6 Effects of uremic solutes on p53 and Chk1 phosphoryla-tion Porcine renal tubular cells were treated with each uremic solutefor 5 hours Cell lysates were obtained and subjected to Westernblotting Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhSphenyl sulfate HA hippuric acid IAA indoleacetic acid Images arerepresentative of two independent experiments
Skp2 mRNA expression at 24 hours after treatment IS PCSPhS andHA induced ATF4 and CHOPmRNA On the otherhand IAA did not affect the expression of all these genes atall concentrations tested In this experiment 025 05 and1mmolL of IAA decreased viable cell number by 16 49 and72 respectively (Supplementary Figure 5)
4 Discussion
To the best of our knowledge this is the first study thatsystematically compared the effects of five uremic soluteson porcine renal tubular cells Our results are schematizedin Figure 8 The five solutes evaluated in this study werecategorized into three groups by the difference in mechanismof reducing viable cell number upon 48-hour treatment
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Nephrology 5
FL2-A0 200 400 600 800
50
100
150
200
0
Cel
l cou
nt
1K
(a)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)
PhS (10)HA (40)
0
10
20
30
40
50
60
70
0
10
20
30
40
50
60
70
0 4 6
(h) (h)0 4 6
lowast lowast
lowastlowast lowast
lowast
lowast
lowast lowastlowast
lowast lowastlowast
lowast
PCS (10)
ContIS (5)
PhS (10)HA (40)
PCS (10)
lowast
(b)
Cells in G1 phase () Cells in G2 phase ()
ContIS (5)IAA (025)
IAA (05)IAA (1)
ContIS (5)IAA (025)
IAA (05)IAA (1)
0
10
20
30
40
50
60
70
0 4 6
(h)0 4 6
(h)
80
0
10
20
30
40
50
60
70
lowast
lowastlowast
(c)
Figure 3 Effects of uremic solutes on cell cycle progression after porcine renal tubular cells were synchronized at G2M boundary (a) Time-dependent shift of histograms of untreated control cells Each line indicates time after release from synchronization Solid line 0 hoursdotted line 4 hours and dashed line 6 hours Duplicate data for each time point are shown except for 0 hours (b) and (c) Percent of cellsin G1 or G2 phase After synchronization cells were released to grow with or without uremic solutes for indicated periods Percent of cellsin each phase was analyzed and calculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acidIAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisks indicate significant difference compared to corresponding control(119875 lt 001) Concentrations of solutes (mmolL) are shown in parentheses
6 International Journal of Nephrology
5 10 10 025 05 40
Annexin V-FITC positive cells ()
0
5
10
15
20
25
0
5
10
15
20
25
Cont IS PCS PhS IAA HA
5 10 10 025 05 40
Cont IS PCS PhS IAA HA
Annexin V-FITCPI positive cells ()
lowast
Figure 4 Effects of uremic solutes on cell death After 48-hour incubation with each uremic solute porcine renal tubular cells were stainedwith annexin V-FITC and propidium iodide (PI) Percent of annexin V-positive cells and annexin VPI-positive cells was analyzed andcalculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAA indoleacetic acid Data areshown as mean plusmn SD (119899 = 4) Asterisk indicates significant difference compared to corresponding control (119875 lt 001) Sharp marks ()indicate 119875 lt 005
0
50
100
0 5 10 10 10 025 05 1 40
Cont IS PCS PhS IAA HA
NAC (0)NAC (01)NAC (1)
lowastlowast lowast
lowastlowast
lowast
lowast
lowast
lowast lowast
lowast
NS NSNS
Viab
le ce
ll nu
mbe
r(
of c
orre
spon
ding
cont
rol)
Figure 5 Effect of N-acetyl-L-cysteine (NAC) on uremic solute-induced decrease in viable cell number Porcine renal tubular cellswere treated with NAC (0 01 or 1mmolL) for 20 minutes andfurther treated with each uremic solute for 48 hours Viable cellnumber was evaluated using Cell Counting Kit-8 Optical densityat 450 nm was measured and the values were calculated as percentof the corresponding control Cont control IS indoxyl sulfatePCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAAindoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant difference each pairs described in this figure(119875 lt 001) NS means nonsignificant difference
36 Expression of Genes Involved in Growth Delay or CellDeath To further explore molecules which are involved inuremic solute-induced cell cycle delay andor cell death weexamined the expression of genes known to play importantroles in these processes (Figure 7) IS and PCS significantlyinduced p21 mRNA expression at 24 hours after treatment IS(10mmolL) PCS and HA significantly induced Puma (p53upregulated modulator of apoptosis) mRNA expression at 5hours after treatment PCS and PhS suppressed Cdc20 and
Phospho-p53 (Ser15)
Phospho-Chk1 (Ser345)C
ont
IS (5
)
IS (1
0)
PCS
(10
)
PhS
(10
)
IAA
(025
)
IAA
(05
)
HA
(40
)
120573-Actin
Figure 6 Effects of uremic solutes on p53 and Chk1 phosphoryla-tion Porcine renal tubular cells were treated with each uremic solutefor 5 hours Cell lysates were obtained and subjected to Westernblotting Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhSphenyl sulfate HA hippuric acid IAA indoleacetic acid Images arerepresentative of two independent experiments
Skp2 mRNA expression at 24 hours after treatment IS PCSPhS andHA induced ATF4 and CHOPmRNA On the otherhand IAA did not affect the expression of all these genes atall concentrations tested In this experiment 025 05 and1mmolL of IAA decreased viable cell number by 16 49 and72 respectively (Supplementary Figure 5)
4 Discussion
To the best of our knowledge this is the first study thatsystematically compared the effects of five uremic soluteson porcine renal tubular cells Our results are schematizedin Figure 8 The five solutes evaluated in this study werecategorized into three groups by the difference in mechanismof reducing viable cell number upon 48-hour treatment
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 International Journal of Nephrology
5 10 10 025 05 40
Annexin V-FITC positive cells ()
0
5
10
15
20
25
0
5
10
15
20
25
Cont IS PCS PhS IAA HA
5 10 10 025 05 40
Cont IS PCS PhS IAA HA
Annexin V-FITCPI positive cells ()
lowast
Figure 4 Effects of uremic solutes on cell death After 48-hour incubation with each uremic solute porcine renal tubular cells were stainedwith annexin V-FITC and propidium iodide (PI) Percent of annexin V-positive cells and annexin VPI-positive cells was analyzed andcalculated Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAA indoleacetic acid Data areshown as mean plusmn SD (119899 = 4) Asterisk indicates significant difference compared to corresponding control (119875 lt 001) Sharp marks ()indicate 119875 lt 005
0
50
100
0 5 10 10 10 025 05 1 40
Cont IS PCS PhS IAA HA
NAC (0)NAC (01)NAC (1)
lowastlowast lowast
lowastlowast
lowast
lowast
lowast
lowast lowast
lowast
NS NSNS
Viab
le ce
ll nu
mbe
r(
of c
orre
spon
ding
cont
rol)
Figure 5 Effect of N-acetyl-L-cysteine (NAC) on uremic solute-induced decrease in viable cell number Porcine renal tubular cellswere treated with NAC (0 01 or 1mmolL) for 20 minutes andfurther treated with each uremic solute for 48 hours Viable cellnumber was evaluated using Cell Counting Kit-8 Optical densityat 450 nm was measured and the values were calculated as percentof the corresponding control Cont control IS indoxyl sulfatePCS p-cresyl sulfate PhS phenyl sulfate HA hippuric acid IAAindoleacetic acid Data are shown as mean plusmn SD (119899 = 4) Asterisksindicate significant difference each pairs described in this figure(119875 lt 001) NS means nonsignificant difference
36 Expression of Genes Involved in Growth Delay or CellDeath To further explore molecules which are involved inuremic solute-induced cell cycle delay andor cell death weexamined the expression of genes known to play importantroles in these processes (Figure 7) IS and PCS significantlyinduced p21 mRNA expression at 24 hours after treatment IS(10mmolL) PCS and HA significantly induced Puma (p53upregulated modulator of apoptosis) mRNA expression at 5hours after treatment PCS and PhS suppressed Cdc20 and
Phospho-p53 (Ser15)
Phospho-Chk1 (Ser345)C
ont
IS (5
)
IS (1
0)
PCS
(10
)
PhS
(10
)
IAA
(025
)
IAA
(05
)
HA
(40
)
120573-Actin
Figure 6 Effects of uremic solutes on p53 and Chk1 phosphoryla-tion Porcine renal tubular cells were treated with each uremic solutefor 5 hours Cell lysates were obtained and subjected to Westernblotting Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhSphenyl sulfate HA hippuric acid IAA indoleacetic acid Images arerepresentative of two independent experiments
Skp2 mRNA expression at 24 hours after treatment IS PCSPhS andHA induced ATF4 and CHOPmRNA On the otherhand IAA did not affect the expression of all these genes atall concentrations tested In this experiment 025 05 and1mmolL of IAA decreased viable cell number by 16 49 and72 respectively (Supplementary Figure 5)
4 Discussion
To the best of our knowledge this is the first study thatsystematically compared the effects of five uremic soluteson porcine renal tubular cells Our results are schematizedin Figure 8 The five solutes evaluated in this study werecategorized into three groups by the difference in mechanismof reducing viable cell number upon 48-hour treatment
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Nephrology 7
5h24h
5h24h
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
0
05
1
15
2
25Puma
5
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0 5 10 10 10 025 05 1 40
0
02
04
06
08
1
12
14Skp2
0
1
2
3
4
CHOP
lowastlowast lowast
lowast lowast
lowastlowast lowast
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
lowast
lowastlowast
lowast
lowast
lowast lowast lowast
Relat
ive g
ene e
xpre
ssio
n
0
05
1
15
2
25
3p21
0 5 10 10 10 025 05 1 40
Cdc20
Cont IS
IS IS
PCS PhS IAA HA
Cont PCS PhS IAA HA
Relat
ive g
ene e
xpre
ssio
n
0 5 10 10 10 025 05 1 400
1
2
3
4ATF4
ISCont PCS PhS IAA HA ISCont PCS PhS IAA HA
Cont PCS PhS IAA HA
Cont IS PCS PhS IAA HA
Figure 7 Effects of uremic solutes on gene expression Porcine renal tubular cells were treatedwith each uremic solute for 5 and 24 hours TotalRNA was extracted and subjected to real-time PCR Gene expression level relative to GAPDH expression was calculated by ΔΔCt methodData are expressed as relative values to the corresponding control Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenyl sulfateHA hippuric acid IAA indoleacetic acid Data are shown as mean plusmn SD (119899 = 6) Asterisks indicate more than 15-fold increase or decreasewith statistical significance compared to corresponding control (119875 lt 001)
The first group is composed of IS PCS and PhS Thisgroup of solutes induces delay in cell cycle progressionrather than cell death to reduce the viable cell numberOxidative stress is presumably involved in these processesbecause viable cell number was partly recovered by incu-bation with N-acetyl-L-cysteine Moreover IS is reported toinduce oxidative stress in several cell types including renaltubular cells [27ndash32]This oxidative stress would induceDNAdamage which is followed by activation of Chk1 and p53 [33]Subsequent modulation or regulation of their downstreamtargets would eventually delay cell cycle progression [3132 34ndash38] Meanwhile upregulation of ATF4 and CHOPknown as a hallmark of one of the three pathways ofendoplasmic reticulum stress (ER stress) response was alsoobserved ER stress response is an adaptive response in
nature but prolonged ER stress overwhelms the adaptiveresponse and eventually results in apoptosis through CHOPupregulation [39] Thus upregulation of these genes couldinduce apoptosis but only marginal increase of dying cellswas observed with this group of solutes A possible reason isthat the incubation time was too short to observe apoptosisOn the other hand it is reported that CHOP induction byIS inhibits cellular proliferation in human proximal tubularcells [40] Therefore upregulation of ATF4 and CHOP mightalso contribute to delay in cell cycle progression in additionto activation of the p53 pathway
IS has been reported to inhibit proliferation of humanproximal tubular cells through inducing oxidative stressp53 activation and ER stress [29 32 40] The presentstudy confirms these phenomena in porcine tubular cells
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 International Journal of Nephrology
DNA damage
p53Chk1
p21 Puma
ER stress
ATF4
CHOP
Oxidative stress
NAC
NAC
Cdc20 Skp2
ApoptosisCell cycle progression
Oxidative stress
Group1 IS PCS PhS
Group3 HA
Group2 IAA
[33]
[34]
[40][39]
[39]
[39 40]
[49][31 32 40]
[27ndash32]
[29ndash32]
[35ndash38]
Figure 8 Schematic diagram of the effects of uremic solutes onporcine renal tubular cells Group 1 exclusively induces cell cycledelay which would be mediated by oxidative stress and ER stressIS is reported to induce oxidative stress [27ndash32] and ER stress[40] Oxidative stress can cause DNA damage which is followedby Chk1 and p53 activation [33] Activated Chk1 can arrest cellcycle progression [34] Activation of p53 by IS is also reported[29ndash32] Activated p53 can repress transcription of Cdc20 [38]and Skp2 [36 37] Because both Cdc20 and Skp2 have importantroles in cell cycle progression [35] their repression might delaycell cycle Another downstream target of p53 p21 which is alsoan important regulator of cell cycle is also reported to be inducedby IS [31 32 40] Meanwhile ER stress can induce ATF4 andCHOP expression which is followed by apoptosis [39] Howeverit is also reported that IS induced CHOP expression and CHOPmediates inhibition of proliferation instead of induction of apoptosisin human renal tubular cells [40] Thus observed ATF4 and CHOPmRNA induction might be involved in cell cycle delay rather thanapoptosisGroup 2 exclusively induces apoptosis but its mechanismof action is largely unknown Group 3 marginally induces bothcell cycle delay and apoptosis which might be partly mediatedby p53 and ER stress More detailed explanation is in Section 4Cont control IS indoxyl sulfate PCS p-cresyl sulfate PhS phenylsulfate HA hippuric acid IAA indoleacetic acid NAC N-acetyl-L-cysteine Number in brackets corresponds to the number ofreference literature
and additionally reveals that PCS and PhS induce growthretardation in a similar manner as IS Administration of PCSto CKD rat model has been shown to cause further renaltubular damage by mechanisms similar to that of IS [41]Moreover another report reveals that IS and PCS inducesimilar inflammatory gene expressions in renal tubular cells[42] Thus IS PCS and PhS might aggravate renal functionvia p53 activation and ER stress in an additive mannerActivation of p53 and resultant cellular senescence have beenproposed to increase sensitivity to insult and decrease repairability in the renal system further impairing renal function[43] ER stress is also known to be involved in the progressionof kidney disease [44] Future studies are necessary to clarifythe involvement of these solutes in the induction of cellularsenescence andor ER stress in clinical settings
IS also induces cellular senescence via p53 activation inother cell types such as endothelial cells [30] and vascularsmooth muscle cells [31] which implies involvement of ISin the progression of cardiovascular disease (CVD) in CKDpatients as has been suggested in several epidemiologicalstudies [22 23] The association of PCS with CVD hasalso been reported [45ndash47] Thus it would be interesting toevaluate the effects of PCS and PhS in vascular cells
The second group comprises only IAA but other solutesnot tested in this studymay also belong to this group In sharpcontrast to IS IAA induces cell death rather than delay ofcell cycle progression to reduce viable cell number Althoughthe effects of both IAA and IS seemed to be mediated byROS the results were different This might be due to thedifference of subcellular organelle where ROS productiontakes place However future studies are required to clarifythemechanisms in detail Interestingly senescent endothelialcells are more susceptible to apoptotic stimuli [48] Thus onemight consider the possibility that IS (a cellular senescence-inducing solute) and IAA (a cell death-inducing solute) mayaffect viability of certain cell types in a cooperative manner
The third group comprises HA but again other solutesmay also belong to this group HA marginally induces delayboth in cell cycle progression and in cell death whichdiffers from the other two groups N-Acetyl-L-cysteine didnot inhibit the effect of HA further supporting the notionthat HA forms another group Although phosphorylation ofp53 was not observed in HA-treated cells HA did modu-late the expression of p53-regulated genes (Puma) [49] tosome extent Thus HA may induce p53 activation slightlyFurthermore HA would induce ER stress as indicated byupregulation of the marker gene ATF4 and CHOP Theseresults suggest that HA may affect the cellular system in asomewhat overlapping manner with the first group
This study has some limitations First the concentrationsof the uremic solute tested in this experiment were muchhigher than the serum concentrations in CKD patients [25 25] In clinical settings multiple uremic solutes exist andmay affect biological systems in an additive or synergisticmanner However only a single solute was tested in eachexperiment Thus high concentrations were necessary toobtainmeasurable effects on cellular function and to evaluatethe mechanisms of each solute
In conclusion this study suggests that uremic solutes canbe categorized into groups depending on their mechanismsof action on cells We speculate that solutes within a groupexert their effects in an additive manner while solutes indifferent groups act in a cooperative manner Future studiesare necessary to verify these possibilities
Conflict of Interests
All four authors are employees of Kureha Corporation
References
[1] R Vanholder R de Smet G Glorieux et al ldquoReview onuremic toxins classification concentration and interindividual
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
International Journal of Nephrology 9
variabilityrdquo Kidney International vol 63 no 5 pp 1934ndash19432003
[2] J Boelaert F Lynen G Glorieux et al ldquoA novel UPLC-MS-MS method for simultaneous determination of seven uremicretention toxins with cardiovascular relevance in chronic kid-ney disease patientsrdquo Analytical and Bioanalytical Chemistryvol 405 no 6 pp 1937ndash1947 2013
[3] C-J Lin H-H Chen C-F Pan et al ldquop-Cresylsulfate andindoxyl sulfate level at different stages of chronic kidneydiseaserdquo Journal of Clinical Laboratory Analysis vol 25 no 3pp 191ndash197 2011
[4] N Neirynck R Vanholder E Schepers S Eloot A Pletinckand G Glorieux ldquoAn update on uremic toxinsrdquo InternationalUrology and Nephrology vol 45 no 1 pp 139ndash150 2013
[5] F Duranton G Cohen R De Smet et al ldquoNormal andpathologic concentrations of uremic toxinsrdquo Journal of theAmerican Society of Nephrology vol 23 no 7 pp 1258ndash12702012
[6] R Vanholder S van Laecke and G Glorieux ldquoThe middle-molecule hypothesis 30 years after lost and rediscovered in theuniverse of uremic toxicityrdquo Journal of Nephrology vol 21 no2 pp 146ndash160 2008
[7] R Vanholder R De Smet and N Lameire ldquoProtein-bounduremic solutes the forgotten toxinsrdquo Kidney International vol59 pp S266ndashS270 2001
[8] S Liabeuf T B Drueke and Z A Massy ldquoProtein-bounduremic toxins new insight from clinical studiesrdquo Toxins vol 3no 7 pp 911ndash919 2011
[9] M Motojima A Hosokawa H Yamato T Muraki and TYoshioka ldquoUremic toxins of organic anions up-regulate PAI-1expression by induction of NF-120581B and free radical in proximaltubular cellsrdquo Kidney International vol 63 no 5 pp 1671ndash16802003
[10] A K Gelasco and J R Raymond ldquoIndoxyl sulfate inducescomplex redox alterations in mesangial cellsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 290 no 6 ppF1551ndashF1558 2006
[11] L Dou E Bertrand C Cerini et al ldquoThe uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation andwound repairrdquo Kidney International vol 65 no 2 pp 442ndash4512004
[12] G Muteliefu A Enomoto P Jiang M Takahashi and T NiwaldquoIndoxyl sulphate induces oxidative stress and the expressionof osteoblast-specific proteins in vascular smooth muscle cellsrdquoNephrology Dialysis Transplantation vol 24 no 7 pp 2051ndash2058 2009
[13] A Mozar L Louvet C Godin et al ldquoIndoxyl sulphate inhibitsosteoclast differentiation and functionrdquo Nephrology DialysisTransplantation vol 27 no 6 pp 2176ndash2181 2012
[14] T Nii-Kono Y Iwasaki M Uchida et al ldquoIndoxyl sulfateinduces skeletal resistance to parathyroid hormone in culturedosteoblastic cellsrdquo Kidney International vol 71 no 8 pp 738ndash743 2007
[15] M S E Ahmed M Abed J Voelkl and F Lang ldquoTriggeringof suicidal erythrocyte death by uremic toxin indoxyl sulfaterdquoBMC Nephrology vol 14 no 1 article 244 2013
[16] S Ito Y Higuchi Y Yagi et al ldquoReduction of indoxyl sulfate byAST-120 attenuates monocyte inflammation related to chronickidney diseaserdquo Journal of Leukocyte Biology vol 93 no 6 pp837ndash845 2013
[17] T Miyazaki M Ise M Hirata et al ldquoIndoxyl sulfate stimulatesrenal synthesis of transforming growth factor-1205731 and progres-sion of renal failurerdquo Kidney International vol 51 no 63 ppS211ndashS214 1997
[18] T Miyazaki M Ise H Seo and T Niwa ldquoIndoxyl sulfateincreases the gene expressions of TGF-1205731 TIMP-1 and pro-1205721(I) collagen in uremic rat kidneysrdquo Kidney InternationalSupplement vol 51 no 62 pp S15ndashS22 1997
[19] A Adijiang S Goto S Uramoto F Nishijima and T NiwaldquoIndoxyl sulphate promotes aortic calcification with expressionof osteoblast-specific proteins in hypertensive ratsrdquo NephrologyDialysis Transplantation vol 23 no 6 pp 1892ndash1901 2008
[20] S Ito M Osaka Y Higuchi F Nishijima H Ishii and MYoshida ldquoIndoxyl sulfate induces leukocyte-endothelial inter-actions through up-regulation of E-selectinrdquo The Journal ofBiological Chemistry vol 285 no 50 pp 38869ndash38875 2010
[21] I-W Wu K-H Hsu C-C Lee et al ldquoP-cresyl sulphate andindoxyl sulphate predict progression of chronic kidney diseaserdquoNephrology Dialysis Transplantation vol 26 no 3 pp 938ndash9472011
[22] C-J Lin H-L Liu C-F Pan et al ldquoIndoxyl sulfate predictscardiovascular disease and renal function deterioration inadvanced chronic kidney diseaserdquoArchives of Medical Researchvol 43 no 6 pp 451ndash456 2012
[23] F C Barreto D V Barreto S Liabeuf et al ldquoSerum indoxyl sul-fate is associated with vascular disease and mortality in chronickidney disease patientsrdquoClinical Journal of the American Societyof Nephrology vol 4 no 10 pp 1551ndash1558 2009
[24] K Kikuchi Y Itoh R Tateoka A Ezawa K Murakami andT Niwa ldquoMetabolomic analysis of uremic toxins by liquidchromatographyelectrospray ionization-tandem mass spec-trometryrdquo Journal of Chromatography B Analytical Technologiesin the Biomedical and Life Sciences vol 878 no 20 pp 1662ndash1668 2010
[25] Y Itoh A Ezawa K Kikuchi Y Tsuruta and T Niwa ldquoProtein-bound uremic toxins in hemodialysis patients measured byliquid chromatographytandem mass spectrometry and theireffects on endothelial ROS productionrdquo Analytical and Bioan-alytical Chemistry vol 403 no 7 pp 1841ndash1850 2012
[26] K S Hodgkins and H W Schnaper ldquoTubulointerstitial injuryand the progression of chronic kidney diseaserdquo PediatricNephrology vol 27 no 6 pp 901ndash909 2012
[27] Z Tumur and T Niwa ldquoIndoxyl sulfate inhibits nitric oxideproduction and cell viability by inducing oxidative stress invascular endothelial cellsrdquoThe American Journal of Nephrologyvol 29 no 6 pp 551ndash557 2009
[28] V-C Wu G-H Young P-H Huang et al ldquoIn acute kidneyinjury indoxyl sulfate impairs human endothelial progenitorcells modulation by statinrdquoAngiogenesis vol 16 no 3 pp 609ndash624 2013
[29] H Shimizu D Bolati A Adijiang et al ldquoSenescence anddysfunction of proximal tubular cells are associated with acti-vated p53 expression by indoxyl sulfaterdquo American Journal ofPhysiology Cell Physiology vol 299 no 5 pp C1110ndashC1117 2010
[30] Y Adelibieke H Shimizu G Muteliefu D Bolati and TNiwa ldquoIndoxyl sulfate induces endothelial cell senescence byincreasing reactive oxygen species production and p53 activityrdquoJournal of Renal Nutrition vol 22 no 1 pp 86ndash89 2012
[31] G Muteliefu H Shimizu A Enomoto F Nishijima MTakahashi and T Niwa ldquoIndoxyl sulfate promotes vascularsmooth muscle cell senescence with upregulation of p53 p21
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 International Journal of Nephrology
and prelamin A through oxidative stressrdquo American Journalof PhysiologymdashCell Physiology vol 303 no 2 pp C126ndashC1342012
[32] H Shimizu D Bolati A Adijiang et al ldquoNF-120581b plays animportant role in indoxyl sulfate-induced cellular senescencefibrotic gene expression and inhibition of proliferation inproximal tubular cellsrdquo American Journal of Physiology CellPhysiology vol 301 no 5 pp C1201ndashC1212 2011
[33] H Niida and M Nakanishi ldquoDNA damage checkpoints inmammalsrdquoMutagenesis vol 21 no 1 pp 3ndash9 2006
[34] C S Soslashrensen and R G Syljuasen ldquoSafeguarding genomeintegrity the checkpoint kinases ATR CHK1 and WEE1restrain CDK activity during normal DNA replicationrdquo NucleicAcids Research vol 40 no 2 pp 477ndash486 2012
[35] P Fasanaro M C Capogrossi and F Martelli ldquoRegulation ofthe endothelial cell cycle by the ubiquitin-proteasome systemrdquoCardiovascular Research vol 85 no 2 pp 272ndash280 2010
[36] T Zuo R Liu H Zhang et al ldquoFOXP3 is a novel transcriptionalrepressor for the breast cancer oncogene SKP2rdquo Journal ofClinical Investigation vol 117 no 12 pp 3765ndash3773 2007
[37] D-J JungD-H Jin S-WHong et al ldquoFoxp3 expression in p53-dependent DNA damage responsesrdquo The Journal of BiologicalChemistry vol 285 no 11 pp 7995ndash8002 2010
[38] T Banerjee S Nath and S Roychoudhury ldquoDNA damageinduced p53 downregulates Cdc20 by direct binding to its pro-moter causing chromatin remodelingrdquo Nucleic Acids Researchvol 37 no 8 pp 2688ndash2698 2009
[39] R Sano and J C Reed ldquoER stress-induced cell death mecha-nismsrdquo Biochimica et Biophysica ActamdashMolecular Cell Researchvol 1833 no 12 pp 3460ndash3470 2013
[40] T Kawakami R Inagi T Wada T Tanaka T Fujita and MNangaku ldquoIndoxyl sulfate inhibits proliferation of human prox-imal tubular cells via endoplasmic reticulum stressrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 3 ppF568ndashF576 2010
[41] H Watanabe Y Miyamoto D Honda et al ldquop-Cresyl sulfatecauses renal tubular cell damage by inducing oxidative stress byactivation ofNADPHoxidaserdquoKidney International vol 83 no4 pp 582ndash592 2013
[42] C-Y Sun H-H Hsu and M-S Wu ldquoP-Cresol sulfate andindoxyl sulfate induce similar cellular inflammatory geneexpressions in cultured proximal renal tubular cellsrdquo Nephrol-ogy Dialysis Transplantation vol 28 no 1 pp 70ndash78 2013
[43] H Yang and A B Fogo ldquoCell senescence in the aging kidneyrdquoJournal of the American Society of Nephrology vol 21 no 9 pp1436ndash1439 2010
[44] R Inagi ldquoEndoplasmic reticulum stress as a progression factorfor kidney injuryrdquoCurrent Opinion in Pharmacology vol 10 no2 pp 156ndash165 2010
[45] B K I Meijers K Claes B Bammens et al ldquo119901-Cresoland cardiovascular risk in mild-to-moderate kidney diseaserdquoClinical Journal of the American Society of Nephrology vol 5no 7 pp 1182ndash1189 2010
[46] I-WWuK-HHsuH-JHsu et al ldquoSerum free p-cresyl sulfatelevels predict cardiovascular and all-cause mortality in elderlyhemodialysis patientsmdasha prospective cohort studyrdquoNephrologyDialysis Transplantation vol 27 no 3 pp 1169ndash1175 2012
[47] C-J Lin C-K Chuang T Jayakumar et al ldquoSerum p-cresylsulfate predicts cardiovascular disease and mortality in elderlyhemodialysis patientsrdquo Archives of Medical Science vol 9 no 4pp 662ndash668 2013
[48] J Hoffmann J Haendeler A Aicher et al ldquoAging enhancesthe sensitivity of endothelial cells toward apoptotic stimuliimportant role of nitric oxiderdquo Circulation Research vol 89 no8 pp 709ndash715 2001
[49] K Nakano and K H Vousden ldquoPUMA a novel proapoptoticgene is induced by p53rdquo Molecular Cell vol 7 no 3 pp 683ndash694 2001
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom