caspases, bcl-2 proteins and apoptosis in autosomal-dominant polycystic kidney disease

Kidney International, Vol. 61 (2002), pp. 1220–1230

GENETIC DISORDERS – DEVELOPMENT

Caspases, Bcl-2 proteins and apoptosis in autosomal-dominantpolycystic kidney disease

TEVFIK ECDER, VYACHESLAV Y. MELNIKOV, MELINDA STANLEY, DIDEM KORULAR, M. SCOTT

LUCIA, ROBERT W. SCHRIER, and CHARLES L. EDELSTEIN

Departments of Medicine and Pathology, University of Colorado School of Medicine, Denver, Colorado, USA

Caspases, Bcl-2 proteins and apoptosis in autosomal-dominant stage renal failure in the USA requiring dialysis and renalpolycystic kidney disease. transplantation [1]. There have been major advances in

Background. Apoptosis is a characteristic feature of human the study of the pathophysiology of cyst formation andautosomal-dominant polycystic kidney disease (ADPKD). Thethe progression of renal failure in ADPKD [2]. TubularHan:Sprague-Dawley (SPRD) rat model closely resembles hu-epithelial hyperplasia, tubular fluid secretion and alter-man ADPKD and presents an opportunity to investigate the

apoptotic pathway in the pathogenesis of this disease. ations in extracellular matrix are established characteris-Methods. Han:SPRD rats were studied during the early tics of ADPKD. Another potential factor in cyst forma-

stages of ADPKD (newborn, 2 and 6 weeks old). Apoptotic tion and loss of non-cystic tubules leading to renal failurecells were detected by the TUNEL (Tdt-mediated dUTP nickin ADPKD is apoptosis.end-labeling) assay. Caspase-3 activity was measured using the

Increased levels of apoptosis are observed in humanfluorescent substrate DEVD-AMC and cleavage of poly(ADP-ribose) polymerase [PARP]. Expression of pro- and ADPKD [3] and experimental models of polycystic kid-anti-apoptotic B-cell lymphoma (Bcl-2) proteins was detected ney disease (PKD) [3–7]. In human ADPKD, apoptosison Western blot analysis. was detected in non-cystic tubules in pre-uremic humanResults. TUNEL (�) cells, caspase-3 activity and caspase-

polycystic kidneys, suggesting that it may lead to themediated PARP breakdown were significantly increased inprogressive loss of normal nephrons in PKD [3]. Apopto-2-week-old heterozygous (Cy/�) and homozygous (Cy/Cy) rat

kidneys compared to normal littermate controls. In Cy/� rat sis may be causally linked to the development of renalkidneys, decreased expression of anti-apoptotic Bcl-XL coin- cystic disease, as it is essential for Madin-Darby caninecided with increased caspase-3 activity at 2 weeks of age while kidney (MDCK) cell cyst cavitation in collagen type 1expression of Bcl-2, another anti-apoptotic protein, increased

matrix. Cystogenesis in this system is inhibited by overex-at 6 weeks of age. In Cy/Cy rat kidneys, decreased expressionof Bcl-XL and increased expression of Bcl-2 was present at pression of the anti-apoptotic gene, Bcl-2 [8]. The central2 weeks of age. Pro-apoptotic Bax and Bad expression was component of this apoptotic machinery is a proteolyticunchanged at 2 weeks of age in both Cy/� and Cy/Cy rat system involving the family of cysteine proteases calledkidneys.

caspases of which caspase-3 is the “executioner” caspaseConclusions. Activation of caspase-3 and dysregulation of[9]. The role of caspases in ADPKD is not known.the balance between pro- and anti-apoptotic Bcl-2 family mem-

bers, specifically a down-regulation of anti-apoptotic Bcl-XL, The balance between the pro- and anti-apoptotic Bcl-2correlates with increased apoptosis in polycystic Han:SPRD protein family members has been found to control cas-rat kidneys. pase-3 activity [10–12]. For example, anti-apoptotic Bcl-2

can inhibit procaspase-3 activation by preventing cyto-chrome c release from the mitochondria [13, 14]. The

Autosomal-dominant polycystic kidney disease relationship between caspase-3 and dysregulation of(ADPKD) is the most common life-threatening heredi- Bcl-2 proteins in ADPKD has not yet been studied.tary disease in the USA, affecting from 1:400 to 1:1000 The Han:SPRD rat closely resembles ADPKD andpeople. ADPKD accounts for about 5 to 10% of end- therefore is a worthy model to study mechanisms of

apoptosis. Also, apoptosis and its molecular pathwayshave not been characterized in the Han:SPRD rat. InKey words: caspase-3, Bcl-2, apoptosis, polycystic kidneys, Han:SPRD

rats, ADPKD, progressive renal disease. the present study, the role of the “executioner” caspase-3and the balance between pro- and anti-apoptotic pro-Received for publication November 21, 2000teins in apoptosis in cystic and non-cystic tubules wereand in revised form October 30, 2001

Accepted for publication October 31, 2001 investigated in the early stages of ADPKD in this ratmodel. 2002 by the International Society of Nephrology

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Ecder et al: Apoptosis and PKD 1221

METHODS Positive and negative controls for TUNEL stain wereperformed. A histological section of a normal Han:AnimalsSPRD rat kidney was treated with TACS-Nuclease be-

The study was conducted in normal (�/�), heterozy-fore the TUNEL stain was performed. The vast majority

gous (Cy/�) and homozygous (Cy/Cy) Han:SPRD rats.of the cells exhibited pale blue nuclear staining which

All the normal rats and Cy/� rats studied were males.is characteristic of apoptosis in this assay. This TACS-

The Cy/Cy rats studied were both male and female. A Nuclease positive control confirmed that the permeabili-colony of Han:SPRD rats was established in our animal zation and the labeling reaction had worked. In contrast,care facility from a litter that was obtained from the a histological section of normal Han:SPRD rat kidneyPolycystic Kidney Program at the University of Kansas stained with the TUNEL stain in the absence of TdTMedical Center, Kansas City, Kansas. The study protocol enzyme demonstrated the absence of apoptosis and thewas approved by the University of Colorado Health Sci- presence of the counterstain in all the cells.ences Center Animal Care and Use Committee. Ratshad free access to tap water and standard rat chow. Quantification of TUNEL positive cells

TUNEL positive cells in the non-cystic and cystic tu-Tissue preparationbules and interstitium were counted in 10 random areas

Newborn, 2- and 6-week-old Han:SPRD rats were in the cortical region. Non-cystic tubules were definedused in our experiments. Rats were anesthetized by intra- as tubules with a diameter of less than 50 �m. TUNELperitoneal injection of pentobarbital sodium (50 mg/kg positive cells were expressed as a percentage of totalbody wt) and kidneys were removed. The kidneys were tubular cells. TUNEL positive cells were also quantifiedweighed after removal. The left kidney was fixed in 4% in cystic versus non-cystic tubules. TUNEL positive cellsparaformaldehyde in phosphate-buffered saline (PBS) in the interstitium were expressed per square millimeterfor 120 minutes and then put into 70% ethanol for histo- of tissue.logical examinations. The right kidney was frozen in All cells staining positive with the TUNEL assay wereliquid nitrogen and stored at �80�C for caspase assays counted. This includes darkly stained nuclei, lightlyand Western blot analyses. stained nuclei and pyknotic nuclei with apoptotic bodies

that stained positive.Cyst volume density

Hematoxylin-eosin stained sections were used to de- Quantification of interstitial infiltrationtermine the cyst volume density. This was performed by Tissue sections were graded by a reviewer, blinded toa reviewer, blinded to the identity of the rat kidney, the identity of the rat kidney, as to the extent and severityusing point counting stereology [15]. Areas of the cortex of interstitial infiltration by inflammatory cells [16]: 0 �at 90 degrees, 180 degrees and 270 degrees from the absence of infiltrate; 1 � focal, mild; 2 � focal, moderate;hilum of each section were selected to guard against field 3 � diffuse, mild; 4 � diffuse, moderate or severe.selection variation.

Caspase-3 assayIn situ detection of DNA fragmentation The activity of caspase 3 was determined by use of

The terminal deoxynucleotidyl transferase (TdT) me- fluorescent substrates as we have previously describeddiated nick-end labeling (TUNEL) method was used to [17] with modifications [18]. Renal cortex was mixeddetect in situ DNA strand breaks. TACS 2 TdT-blue with a lysis buffer containing 25 mmol/L Na� Hepes, 2label in situ apoptosis detection kit (Trevigen, Gaithers- mmol/L dithiothreitol (DTT), 1 mmol/L ethylenediam-burg, MD, USA) was used. Paraffin blocks were sec- inetetraacetic acid (EDTA), 0.1% 3-[(3-cholamidopropyl)tioned at 4-�m thickness on slides. Paraffin sections were dimethylammonio]-1-pro-panesulfonate (CHAPS), 10%deparaffinized in xylene and rehydrated in graded etha- sucrose, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF),nols and PBS. The tissue sections were incubated with and 1 �mol/L pepstatin A, pH 7.2; it was homogenizedproteinase K for one hour at 37�C for permeabilization. with 10 strokes in a glass-Teflon homogenizer. The lysateEndogenous peroxidase activity was quenched by in- was then centrifuged at 4�C at 100,000 � g in a Beckmancubating the tissue sections with 3% hydrogen peroxide Ti70 rotor for one hour. The resultant supernatants werein methanol for five minutes. The sections were then immediately frozen in liquid N2 and stored at �70�Ctreated with biotinylated nucleotide, manganese cation until use. Lysate protein was measured by the Bradfordand TdT enzyme for one hour at 37�C. After treating method as described in the Bio-Rad protein assay kitwith streptavidin-horseradish peroxidase (HRP) for ten with bovine serum albumin as standards.minutes at room temperature, the sections were stained The caspase assay was then performed on this superna-with blue label for seven minutes. The tissues were coun- tant as follows: 90 �L of supernatant (200 to 400 �g

protein) and 10 �L of the substrate (final concentration,terstained with nuclear fast red.

Ecder et al: Apoptosis and PKD1222

25 �mol/L) were added to 100 �L of caspase assay buffer. quent detection was carried out by enhanced chemilumi-nescence (Amersham), according to the manufacturer’sThe assay buffer contained 250 mmol/L K� Hepes, 50

mmol/L KCl, 1 mmol/L DTT, 1 mmol/L EDTA, and instructions. Prestained protein markers (Bio-Rad) wereused for molecular mass determination.0.1% CHAPS, pH 7.4. Ac-Asp-Glu-Val-Asp-7-amido-

4-methyl coumarin (Ac-DEVD-AMC) in 10% dimethylImmunohistochemistrysulfoxide (DMSO) was used as a susceptible substrate

for caspase-3 [19]. The solution was pre-incubated for Immunohistochemical localization was performed onparaformaldehyde-fixed and paraffin-embedded tissuesten minutes at 30�C before the substrate was added. The

reaction was then initiated by addition of the substrate. using the avidin-biotin immunoperoxidase method. Par-affin blocks were sectioned at 4-�m thickness on slides.Peptide cleavage was measured over one hour at 30�C

using a Cytofluor 4000 series fluorescent plate reader Paraffin sections were deparaffinized in xylene and rehy-drated in graded ethanols. Endogenous peroxidase activ-(Perseptive Biosystems) at an excitation wavelength of

380 nm and an emission wavelength of 460 nm. An AMC ity was quenched by incubating the tissue sections with3% hydrogen peroxide in water for five minutes. Thestandard curve was determined for each experiment.

Caspase activity was expressed in nmol AMC released sections were then treated with 1.5% goat serum in phos-phate buffered saline (PBS) for one hour to block non-per minute of incubation time per mg of lysate protein.specific binding of the biotinylated secondary antibody.

Immunoblot analysis The sections were incubated with anti-Bcl-XL antibody(1:50 dilution; rabbit polyclonal, cat# sc1041; Santa CruzImmunoblot analysis was performed as we have pre-

viously described [17]. Renal cortex was homogenized Biotechnology) in 1.5% goat serum in PBS for two hoursin humidity chamber at room temperature. After wash-in lysis buffer (in mmol/L: 5 Na2HPO4, 5 NaH2PO4, 150

NaCl, 1 EDTA, 0.1% Triton X-100, 50 NaF, and 0.2 ing in PBS, tissues were incubated with biotinylated sec-ondary antibody (Santa Cruz Biotechnology) at RT forNa3VO4, and 0.1% �-mercaptoethanol, pH 7.2) plus

proteinase inhibitors (100,000 �U/mL aprotinin, 20 30 minutes. Following rinses in PBS, tissues were incu-bated in avidin-biotin-peroxidase complex (Santa Cruz�mol/L leupeptin, 20 �mol/L pepstatin A, and 200

�mol/L PMSF). The homogenates were centrifuged Biotechnology) at room temperature for 30 minutes andrinsed in PBS. The antibody-bound peroxidase was visu-(14,000 RPM at 4�C for 10 min) to remove unbroken

cells and debris. Supernatants were mixed with sample alized by 3,3�-diaminobenzidine (DAB) chromogen. Tis-sue sections were counterstained with methyl green.buffer containing 50 mmol/L Tris-base (pH 6.8), 0.5%

glycerol, 0.01% bromphenol blue, and 0.75% sodium Control sections were prepared by using rabbit IgG (cat# I-1000; Vector Laboratories, Burlingame, CA, USA)dodecyl sulfate (SDS) and heated at 95�C for five min-

utes. Equal amounts of protein (50 to 100 �g/lane) were instead of the primary antibody. Control sections showedno staining with DAB.fractionated by Tris-glycine-SDS-7.5 or 12.5% PAGE.

The electrophoretically separated proteins were thenChemistrytransferred to a nitrocellulose membrane (Millipore,

Bedford, MA, USA) by wet electroblotting for 90 min- Serum urea nitrogen and creatinine levels were mea-sured using a Beckman autoanalyzer (Beckman Instru-utes. The membranes were blocked with 5% nonfat dry

milk in TBST [50 mmol/L Tris (pH 7.5), 150 mmol/L ments, Fullerton, CA, USA).NaCl, and 0.1% Tween 20] buffer at pH 7.5 for one

Statistical analysishour at room temperature. Immunoblot analyses wereperformed with the following primary antibodies: anti- Unpaired two group comparisons were analyzed by

the unpaired t test. Multiple group comparisons wereparent-PARP (1:1000 dilution; polyclonal; Biosource,Camarillo, CA, USA), anti-cleaved-PARP (1:1000 dilu- performed using analysis of variance (ANOVA) with

post-test according to Newman-Keuls. A P value oftion; monoclonal; Serotec, Raleigh, NC, USA), anti-Bcl-2 (1:500 dilution; monoclonal; Transduction Labora- 0.05 was considered statistically significant. Values are

expressed as means SE.tories, Lexington, KY, USA), anti-Bax (1:5000 dilution;polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA,USA), anti-Bad (1:500 dilution; monoclonal; Transduc-

RESULTStion Laboratories), anti-Bcl-XL (1:1000 dilution; poly-

Renal mass and function of Han:SPRD ratsclonal; Santa Cruz Biotechnology). The membranes wereincubated overnight at 4�C with primary antibodies, Two-kidney/total body weight (%) of newborn and

2-week-old Cy/Cy Han:SPRD rats were significantlywashed in TBST buffer, and further incubated with sheepanti-mouse IgG or donkey anti-rabbit IgG coupled to higher compared to �/� and Cy/� littermates (Table

1). Cy/Cy rats also had a significantly increased serumhorseradish peroxidase (Amersham) at 1:1000 dilutionin TBST buffer for one hour at room temperature. Subse- urea nitrogen and serum creatinine at 2 weeks of age


Table 1. Two-kidney weight/total body weight (2KW/TBW %) and renal functions of Han:SPRD rats

�/� Cy/� Cy/Cy P value

2KW/TBW %Newborn 1.060.06 1.220.07 2.310.10 0.001a

2 week old 1.060.03 1.300.03 9.980.37 0.001a

6 week old 0.960.02 1.310.05 — 0.0001b

Serum urea nitrogen mg/dLNewborn 28.31.7 24.01.4 35.57.5 NS2 week old 19.61.8 17.41.5 41.93.8 0.001a

6 week old 16.61.0 160.6 — NSSerum creatinine mg/dl

Newborn 0.400.00 0.350.03 0.350.05 NS2 week old 0.280.02 0.240.02 0.360.02 0.01a

6 week old 0.440.04 0.430.06 — NSa Cy/Cy vs. �/� and Cy/�b Cy/� vs. �/�

Fig. 1. Detection of apoptosis on TUNEL and hematoxylin-eosin staining of 2-week-old kidney sections. The TUNEL stain was performed inkidney sections from �/�, Cy/� and Cy/Cy Han:SPRD rat kidneys (A, B and C). A representative area from each section is shown. Kidneysfrom �/� rats did not demonstrate apoptosis (A). Polycystic kidneys from Cy/� rats demonstrated apoptotic nuclei in cystic tubules (arrows) aswell as non-cystic tubules (arrowhead; B). In panel B, an apoptotic cell is “sloughing off” into the cyst lumen. Polycystic kidneys from Cy/Cy ratsdemonstrated massive cyst formation and apoptotic nuclei (condensed pyknotic nuclei with apoptotic bodies) in the cells lining the cysts (arrows;C). Hematoxylin-eosin staining also was performed (D, E and F ). Kidneys from �/� rats did not demonstrate apoptotic nuclei (D). Polycystickidneys from Cy/� rats demonstrated condensed pyknotic apoptotic nuclei in cystic tubules (arrows) as well as non-cystic tubules (arrowhead;E). Polycystic kidneys from Cy/Cy rats demonstrated massive cyst formation and condensed pyknotic apoptotic nuclei in the cells lining the cysts(arrows; F). Magnification �600.

(Table 1) and died at 4 weeks of age with massively TUNEL assayenlarged kidneys and uremia (data not shown). Two- TUNEL staining is demonstrated in Figure 1. TUNELweek-old Cy/� rats had slightly enlarged kidneys that positive cells were not seen in normal littermate controlscontinued to enlarge and became significantly larger (Fig. 1A). TUNEL positive cells were seen in both cystic

and non-cystic tubules of cystic kidneys of Han:SPRDcompared to normals by 6 weeks of age (Table 1).


Fig. 2. Quantification of TUNEL positive cells in normal littermatecontrols (�/�), heterozygous (Cy/�) and homozygous (Cy/Cy) Han:SPRD rat kidneys. TUNEL positive cells were quantified in tubular

Fig. 3. Caspase-3 activity is increased in 2- and 6-week-old Han:SPRDcells in newborns and at 2 and 6 weeks of age. Apoptotic cells wererat kidneys. Cytosolic extracts of renal cortex were prepared. The ac-expressed as a percentage of total tubular cells as determined from antivity of caspase 3 was determined using the fluorescent substrateaverage of all tubules within ten random high power fields (�400) fromAc-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin (Ac-DEVD-AMC).the same area of the kidney. A minimum of 100 tubules per kidneyThere was an increase in caspase-3 activity in 2-week-old heterozygouswas studied. *P � 0.002 vs. Cy/� and �/�; **P 0.02 vs. �/�; ***P (Cy/�) and homozygous (Cy/Cy) rat kidneys compared to normal lit-0.001 vs. Cy/� and �/�; ****P 0.05 vs. �/�. N � 4 each for newborntermate controls. There was an increase in caspase-3 activity in 6-week-�/�, Cy/� and Cy/Cy; N � 3 each for 2-week-old �/� and Cy/Cy;old Cy/� rat kidneys compared to normal littermate controls (�/�).N � 5 for 2-week-old Cy/�; N � 3 for 6-week-old �/� and N � 6 for*P 0.05 vs. �/�; **P 0.01 vs. Cy/�; P 0.001 vs. �/�; ***P 6-week-old Cy/�.0.05 vs. �/� (N � 6).

rats (Fig. 1B). TUNEL positive cells with condensed tection of condensed, pyknotic nuclei on hematoxylin-pyknotic nuclei and apoptotic bodies in 2-week-old eosin staining.Cy/Cy kidneys are demonstrated in Figure 1C. In 2-week- TUNEL positive cells also were quantified in the inter-old Cy/� Han-SPRD rats, 75% of the tubular TUNEL stitium of Han:SPRD rat kidneys. There were no apo-positive cells were in cystic and 25% were in non-cystic ptotic cells in the interstitium of newborn, 2- and 6-week-tubules. In 6-week-old Cy/� Han-SPRD rats, 59% of old normal littermate controls. There were 0.26 apoptoticthe tubular TUNEL positive cells were in cystic and 41% cells in the interstitium/mm2 kidney in newborn Cy/Cywere in non-cystic tubules. (P 0.05 vs. normal littermate controls), 12 cells in

Tubular cells with condensed pyknotic nuclei charac- the interstitium/mm2 in 2-week-old Cy/Cy (P 0.01 vs.teristic of apoptosis also were identified on hematoxylin- controls) and 0.4 cells in the interstitium/mm2 in 6-week-eosin stained sections of Cy/� and Cy/Cy kidneys com- old Cy/� kidneys (P 0.05 vs. controls; N � 3).pared to normal littermate controls (Fig. 1 D, E and F). Quantification of cyst volume density, apoptosis and

To eliminate the possibility that the tip of a large cyst interstitial inflammation was performed at 32 weeks ofwas being called a non-cystic tubule, serial sections were age, a time point when renal dysfunction is evident inperformed followed by TUNEL analysis. Apoptotic tu- males but not females. At this time point, the male ratsbular cells on hematoxylin-eosin staining were identified have renal failure compared to normal renal function inin non-cystic tubules (less than 50 �m diameter). Eight the females [20]. The cyst volume density was 39.3% serial back-to-back sections, 10 �m apart, were followed 2.7 in males versus 14.4% 1.5 in females (P 0.01, N �for the development of cysts in “normal caliber tubules.” 4). There was no difference in the number of TUNELIn the region of these normal caliber tubules that had positive cells in non-cystic tubules (% of tubular cells)apoptosis, no cysts were identified, suggesting that apo- in males and females at this time: 7% 1.5 in malesptotic nuclei were not lying on the edge of a cystic tubule. versus 9.6% 1.1 in females (P � NS, N � 4). There

Quantification of TUNEL positive cells is demon- was a significant difference in the number of TUNELstrated in Figure 2. There were significant increases in positive cells in cystic tubules (% of tubular cells) inTUNEL positive cells in newborn Cy/Cy kidneys, 2-week- males and females: 7.9% 1 in males versus 3.4% old Cy/� and Cy/Cy kidneys and 6-week-old Cy/� 0.3 in females (P 0.01, N � 4). The males had morekidneys compared to the appropriate normal littermate interstitial inflammation than the females. Scoring of thecontrols (Fig. 2). The presence of apoptotic cells corre- interstitial infiltrate was 3.5 in males and 1.0 in females

(P 0.01, N � 4). This experiment suggests that thesponding with TUNEL positivity was confirmed by de-


Fig. 4. PARP breakdown is increased in2-week-old Han:SPRD rat kidneys. Renalcortex was immunoblotted with separate anti-bodies that recognize parent PARP (116 kD)(upper panel) and caspase-mediated PARPcleavage products (89 kD and 64 kD) (lowerpanel). In 2-week-old heterozygous (Cy/�)and homozygous (Cy/Cy) rat kidneys, thereis a decrease in parent PARP protein com-pared to normal littermate controls (�/�)(upper panel). In 2-week-old heterozygous(Cy/�) and homozygous (Cy/Cy) rat kidneys,there is an increase in a 64 kD cleavage prod-uct of PARP compared to normal littermatecontrols (�/�) (lower panel). Positive control(Pos) is apoptotic Jurkat cells. (N � 3 miceeach for 2-week-old �/�, Cy/� and Cy/Cy).

Fig. 5. Anti-apoptotic Bcl-XL protein (32 kD)expression is decreased in 2-week-old Han:SPRD rat kidneys. Renal cortex was immu-noblotted for Bcl-XL protein. Bcl-XL proteinexpression in newborns is unchanged in het-erozygous (Cy/�) and homozygous (Cy/Cy)rat kidneys compared to normal littermatecontrols (�/�). Bcl-XL protein expression at2 weeks of age is decreased in heterozygous(Cy/�) and homozygous (Cy/Cy) rat kid-neys compared to normal littermate controls(�/�). This decrease in Bcl-XL expression cor-responds with the increase in apoptosis (Fig.2), caspase-3 activity (Fig. 3) and PARPbreakdown (Fig. 4) in Cy/� and Cy/Cy kid-neys at 2 weeks. Positive control (Pos) is apo-ptotic Jurkat cells. (N � 6).

better renal function in 32-week-old females compared of age, caspase-3 activity was 5.4 0.9 nmol/min/mg in�/� and 7.5 1.6 nmol/min/mg in Cy/� (P 0.05 vs.to males is associated with less cyst formation, less apo-�/�, N � 6; Fig. 3).ptosis in cysts and less interstitial inflammation rather

than to less apoptosis in normal tubules of female cysticPARP expressionkidneys.

The DNA repair enzyme poly(ADP-ribose) polymer-ase (PARP) is a major player in apoptosis. Cleavage ofCaspase-3 assayPARP into characteristic fragments by caspase-3 is aThe caspase pathways that are centrally important alldefining characteristic of apoptosis [23]. PARP activa-involve the “executioner” caspase-3 [21]. The centraltion is an extremely sensitive indicator of DNA damagerole of caspase-3 in apoptosis is supported by caspase-3[24].knockout mice that have strong phenotypes based on

At 2 weeks of age, there was a decrease in parentcell death defects, developmental defects and usually PARP expression and an increase in a 64 kD PARPfetal/perinatal mortality [22]. cleavage product in both Cy/� and Cy/Cy kidneys com-

There was a significant increase in caspase-3 activity pared to normal littermate controls (Fig. 4).in Han:SPRD rat kidneys at 2 and 6 weeks of age com- The cleavage of 116 kD parent PARP to 89 kD andpared to normal littermate controls. At 2 weeks of age, 24 kD fragments has classically been described as duecaspase-3 activity was 7.3 0.5 nmol/min/mg in �/�, to caspase-3 [25]. We detected a 64 kD fragmentation9.9 0.6 nmol/min/mg in Cy/� (P 0.05 vs. �/�, N � product in Han:SPRD rat kidneys. Recently, formation6) and 16.5 1.0 nmol/min/mg in Cy/Cy rats (P 0.01 of a specific 64 kD fragment of PARP has been described

during apoptosis [25].vs. Cy/�; P 0.001 vs. �/�, N � 6; Fig. 3). At 6 weeks


Fig. 6. Anti-apoptotic Bcl-2 protein expres-sion is increased in 2-week-old homozygous(Cy/Cy) and in 6-week-old heterozygous(Cy/�) rat kidneys. Renal cortex was immu-noblotted for Bcl-2 protein. Bcl-2 protein ex-pression in newborns is unchanged in Cy/�and Cy/Cy rat kidneys compared to normallittermate controls (�/�) (upper panel). Bcl-2protein expression is increased in Cy/Cy ratkidneys at 2 weeks (middle panel) and inCy/� rat kidneys at 6 weeks of age (lowerpanel). Positive control (Pos) is apoptotic Jur-kat cells (N � 3).

Fig. 7. Pro-apoptotic Bax protein expressionis unchanged in newborn and 2-week-oldHan:SPRD rat kidneys. Renal cortex was im-munoblotted for Bax protein. Bax proteinexpression in newborns (upper panel) and2-week-olds (lower panel) is unchanged inheterozygous (Cy/�) and homozygous (Cy/Cy) rat kidneys compared to normal litter-mate controls (�/�) (N � 3).

Bcl-2 family proteins old rats. Bcl-2 protein expression was increased in2-week-old Cy/Cy kidneys. Bcl-2 protein expression wasThe Bcl-2 family of proteins consists of pro- and anti-not increased at 2 weeks but increased at 6 weeks inapoptotic proteins. Pro-apoptotic proteins include BaxCy/� kidneys (Fig. 6).and Bad, while anti-apoptotic proteins include Bcl-2 and

Expression of the pro-apoptotic protein Bax was un-Bcl-XL.changed between Cy/Cy, Cy/� and �/� littermate con-In newborns, Bcl-XL expression was unchanged be-trols in newborns (Fig. 7), 2-week-olds (Fig. 7) andtween Cy/Cy, Cy/� and �/� littermate controls (Fig. 5).6-week-olds (data not shown). Expression of anotherAt 2 weeks of age, anti-apoptotic Bcl-XL expression waspro-apoptotic protein Bad also was unchanged betweendecreased in Cy/� and Cy/Cy rat kidneys compared toCy/Cy, Cy/� and �/� littermate controls in newborns,normal littermate controls (Fig. 5). The decrease in2-week-olds and 6-week-olds (data not shown).Bcl-XL expression at 2 weeks corresponds with the in-

crease in apoptosis (Fig. 2), caspase-3 activity (Fig. 3) andImmunohistochemistryPARP breakdown (Fig. 4) in Cy/� and Cy/Cy kidneys.

Immunohistochemical detection of Bcl-XL is demon-Expression of one of the other anti-apoptotic proteins,Bcl-2, also was determined in newborns, 2- and 6-week- strated in Figure 8. In �/�, Bcl-XL is localized to mainly


Fig. 8. Immunohistochemistry of Bcl-XL in 2-week-old Han:SPRD ratkidneys (magnification �400). Immunohistochemical localization ofBcl-XL was performed on paraformaldehyde-fixed and paraffin-embed-ded kidney tissues using the avidin-biotin immunoperoxidase method.A polyclonal antibody from Santa Cruz Laboratories (1:50 dilution)was used. The antibody-bound peroxidase was visualized by 3,3�-diami-nobenzidine (DAB) chromogen. Tissue sections were counterstainedwith methyl green. Negative controls were prepared by using rabbitIgG substituted for Bcl-XL antibody. Negative control sections of normallittermate controls (�/�) (A), heterozygous (Cy/�) (C) and homozy-gous (Cy/Cy) (E) rat kidneys showed no staining. Bcl-XL staining in�/� (B), Cy/� (D) and Cy/Cy (F ) kidneys is localized to predominantlyproximal tubules. There is decreased Bcl-XL staining in Cy/� and Cy/Cykidneys compared to �/�, confirming the changes seen on immunoblotanalysis of Bcl-XL (Fig. 5).

proximal tubules (Fig. 8B). In Cy/� and Cy/Cy rat kid- cystic tubules of pre-uremic human polycystic kidneys,which suggests that apoptosis contributes to the progres-neys Bcl-XL expression is decreased (Fig. 8D and 8F).

These immunohistochemical findings confirm the de- sive loss of renal function in ADPKD. The Han:SPRDrat model closely resembles human ADPKD. The PKDcrease in Bcl-XL protein expression on immunoblot anal-

ysis (Fig. 5). In addition, the immunohistochemistry of in this rat model has been extensively characterized[20, 28]. Heterozygote Han:SPRD rats develop slowlyBcl-XL confirms that the changes in this anti-apoptotic

protein are occurring in tubular cells as opposed to other progressive renal cystic disease, whereas homozygoteanimals develop massive cystic enlargement leading toparts of the kidney, for example, interstitium.early death from uremia. Apoptosis, however, has notbeen characterized in the Han:SPRD rat. Thus, we first

DISCUSSION characterized the time course of apoptosis during earlyResearch into apoptosis has grown into an exciting ADPKD in Han:SPRD rats. Our data demonstrate that

and rapidly expanding field that will have an important increased apoptosis is present at an early stage of theeffect on the understanding of renal disease [26]. In- disease in both heterozygous and homozygous rats (Figs.creased levels of apoptosis are observed in human 1 and 2).ADPKD [3, 27], cpk model of autosomal-recessive poly- The role of the “executioner” caspase-3 in this in-cystic kidney disease [3] and dysplastic renal disease [7]. creased apoptosis in Han:SPRD rat kidneys was studiedIncreased apoptosis is also a feature of experimental next. The caspases are a family of intracellular cysteinemodels of PKD, such as transgenic mice overexpressing proteases. The term “caspase” embodies two propertiesthe proto-oncogene c-myc (SBM mice) [6], mice lacking of these proteases in which “c” refers to “cysteine” andthe transcription factor AP-2 beta [5] and Bcl-2 deficient “aspase” refers to their specific ability to cleave sub-mice [4]. Apoptosis was detected in kidneys of humans strates after an aspartate residue. Caspase-3 has beenwith ADPKD regardless of renal function, but not in shown to play a crucial role in the promotion of apoptoticnormal kidneys [3]. In this latter study, apoptosis was cell death [29, 30]. Increased caspase activity has been

detected in cystic kidneys in cpk mice, a model of autoso-detected not only in cyst epithelium, but also in non-


mal-recessive polycystic kidney disease [18]. In this and die from severe PKD [4, 41, 42]. If the anti-apoptoticBcl-2 regulates apoptosis in ADPKD, then it may bestudy, apoptosis was localized primarily to the intersti-

tium with little evidence of cell death in cyst epithelium decreased in the presence of excess apoptosis in thiscondition. However, Bcl-2 protein expression was foundor non-cystic tubules. The present results, however, are

the first demonstration of increased caspase-3 activity in to be increased in the presence of apoptosis in cystichuman kidneys and cystic kidneys of cpk mice [18, 27]. InADPKD (Fig. 3). The increase in caspase-3 occurred

early in the time course of the disease, corresponding to contrast, AP-2� knockout mice die of polycystic kidneydisease just after birth and expression of anti-apoptoticthe major peaks of tubular apoptosis in both Cy/� and

Cy/Cy rats. Understanding of the molecular control of Bcl-2 is down-regulated in parallel to massive apoptotictubular death [5]. Thus, in the present study changes inapoptosis in ADPKD may be not only pathogenetically

relevant, but may also lead to novel treatments for protein expression of the major pro- and anti-apoptoticBcl-2 family proteins next were investigated. Pro-apo-ADPKD. For example, in animal studies, caspase inhibi-

tion has remarkable effects on organ injury due to apoptotic proteins Bax and Bad were unchanged in newborn,2- and 6-week-old Cy/� and Cy/Cy rats. However, inptosis in cerebral ischemia [31], myocardial ischemia-

perfusion injury [32], liver disease [33] and renal ischemia 2-week-old Cy/� and Cy/Cy rat kidneys, there was adramatic decrease in the anti-apoptotic Bcl-XL protein.[34].

Caspase-3 participates in apoptosis by cutting off con- In the heterozygotes, expression of the other anti-apo-ptotic protein, Bcl-2, increased later at 6 weeks of age.tacts between surrounding cells, reorganizing the cy-

toskeleton, marking the cell for phagocytosis and disinte- This later increase in Bcl-2 may represent a compensa-tory mechanism to the already increased apoptosis. Ingrating the cell into apoptotic bodies. While caspase-3

disrupts the nuclear structure by shutting down DNA Cy/Cy kidneys at 2 weeks there was increased expressionof anti-apoptotic Bcl-2 at the same time as decreasedreplication, interrupting splicing and destroying DNA,

it also activates the DNA repair enzyme poly (ADP- expression of Bcl-XL. This may represent an acceleratedanti-apoptotic response to the peak of apoptosis ob-ribose) polymerase (PARP). Caspase-3 cleavage of

PARP is a commonly assessed molecular event to dem- served in 2-week-old homozygotes. Alternatively, theremay be different “initiator” pathways in Cy/� versusonstrate caspase-3 activation and represents an early mo-

lecular hallmark of apoptosis [23, 35]. PARP activation Cy/Cy rats leading to the increased caspase-3 activityand apoptosis. These results in Han:SPRD rats are com-is an extremely sensitive indicator of DNA damage, ap-

pearing much earlier and exceeding in magnitude the patible with what is known about the regulation of apo-ptosis.DNA nicks monitored by TUNEL staining [24]. Cleav-

age of PARP during apoptosis that is blocked by cas- The balance between pro- and anti-apoptotic Bcl-2family proteins has been found to control caspase-3 activ-pase-3 inhibition has been detected in renal epithelial

cells [36, 37]. On this background, parent and cleaved ity [10–12]. For example, anti-apoptotic Bcl-2 and Bcl-XL

can indirectly inhibit procaspase-3 activation by pre-PARP in Han:SPRD rats with ADPKD were studiednext. A novel 64 kD PARP cleavage product associated venting cytochrome c release from the mitochondria

[13, 14]. The decrease in anti-apoptotic Bcl-XL proteinwith the early apoptosis in these animals was detected.These data confirm recent results describing the forma- (Fig. 5) corresponded with the peaks of apoptosis and

caspase-3 activity during early ADPKD. Caspase-3 hastion of a similar specific 64 kD fragment of the parentPARP during apoptosis [25]. Inhibition of PARP is been shown to cleave Bcl-XL and convert it from a cyto-

protective protein to a cytodestructive protein that pro-therefore a potential protective strategy that needs tobe examined in experimental ADPKD. In this regard, motes apoptosis and cytochrome c release [43].

The role of apoptosis in the pathogenesis of cyst for-the PARP inhibitor, 3-aminobenzamide, and PARPknockout mice afford protection against apoptosis and mation is multifaceted. In our study, increased apoptosis

was demonstrated at an early stage of cyst formation,cerebral ischemia [38].The role of pro- and anti-apoptotic proteins were next that is, at birth in Cy/Cy and at 2 weeks of age in Cy/�,

suggesting that it may play a causative role in cyst forma-studied in Han:SPRD rats with PKD. Bcl-2 family pro-teins are critical regulators of the main apoptosis path- tion rather than being a late marker of epithelial cell

dysfunction. The kidneys of Bcl-2 knockout mice [4],ways [39]. The ratio of pro- and anti-apoptotic Bcl-2proteins can determine which cells live or die under the AP-2� knockout mice [5] and c-myc transgenic mice

[6] have increased apoptosis. The presence of polycysticpresence of adverse stimuli. When pro-apoptotic Bax orBad is in excess, cells execute a death command [39]; kidneys in these mouse models suggests that apoptosis

may play a causative role in cyst formation. Also, apopto-but, when anti-apoptotic Bcl-2 or Bcl-XL dominate, apo-ptosis is inhibited and cells survive [40]. In support of a sis is essential for Madin-Darby canine kidney (MDCK)

cell cyst cavitation in collagen type 1 matrix and cysto-role of the Bcl-2 protein family in PKD is the demonstra-tion that Bcl-2 deficient mice have increased apoptosis genesis in this system is inhibited by overexpression of


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