University of Groningen

A translational approach to the genetics of renal diseaseDoorenbos, Carolina Roelfiena Cornelia

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Chapter 7

Urinary vitamin D binding protein: a potential novelmarker of interstitial inflammation and fibrosis

K Mirkovic*, CRC Doorenbos*, WA Dam, HJ Lambers Heerspink, MCJSlagman, FL Nauta, AB Kramer, RT Gansevoort, J van den Born, G Navis,MH de Borst* authors contributed equally

PLoS One. Provisionally accepted.

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Abstract

Non-invasive tubulointerstitial damage markers may allow better titration andmonitoring of renoprotective therapy. We investigated the value of urinary vitamin Dbinding protein excretion (uVDBP) as a tubulointerstitial inflammation and fibrosismarker in adriamycin rats, and tested whether uVDBP parallels renal damage andresponds to therapy intensification in humans. In adriamycin (ADR) rats, uVDBPwas strongly elevated vs. controls (CON) already at 6 wks after induction of nephrosis(ADR: 727 ± 674 [mean ± SD] vs. CON: 9 ± 12 µg/d, p < 0.01), i.e. before onsetof pre-fibrotic and inflammatory tubulointerstitial damage, and at all following 6-wktimepoints until end of followup at 30 wks (ADR: 1403 ± 1026 vs. CON: 206 ± 132µg/d, p < 0.01). In multivariate regression analysis, uVDBP was associated withtubulointerstitial macrophage accumulation (standardized beta=0.47, p=0.01) andcollagen III expression (standardized beta=0.44, p=0.02) independently of albuminuria.In humans, uVDBP was increased in 100 microalbuminuric subjects (44 ± 93 µg/d)and in 47 CKD patients with overt proteinuria (9.2 ± 13.0 mg/d) compared to 100normoalbuminuric subjects (12 ± 12 µg/d, p < 0.001). In CKD patients, uVDBPresponded to intensification of renoprotective therapy (ACEi+liberal sodium: 9.2 ±13.0 mg/d vs. dual RAAS blockade+low sodium: 2747 ± 4013 µg/d, p < 0.001), butremained still > 100-fold increased during maximal therapy vs. normoalbuminurics (p< 0.001), consistent with persisting tubulointerstitial damage. uVDBP was associatedwith tubular and inflammatory damage markers KIM-1 (standardized beta=0.52, p <0.001), beta-2-microglobuline (st.beta=0.45, p < 0.001), cystatin C (st.beta=0.40, p <0.001) and MCP-1 (st.beta=0.31, p < 0.001) independently of albuminuria. uVDBPmay be a novel urinary biomarker of tubulointerstitial damage. Prospectively designedstudies are required to validate our findings and confirm its relevance in the clinicalsetting.

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VDBP as a tubulointerstitial damage marker

Introduction

The severity of renal interstitial fibrosis has consistently been identified as the strongestpredictor of subsequent progressive renal function loss (1). Early profibrotic changes,on the other hand, might still be reversible (2). Reliable, non-invasive assessment oftubulointerstitial damage would therefore be of great value in development and moni-toring of renoprotective therapy (3). Vitamin D binding protein (VDBP), also knownas group-specific component (GC), is a glycosylated alpha-globulin of 58 kDa (4). Itsmain function is to transport vitamin D metabolites through the circulation. VDBP isfiltered by the glomerulus and subsequently reabsorbed by proximal tubular cells throughreceptor-mediated uptake. This process is crucial for retrieval of vitamin D for activationby 1-alpha hydroxylase, which is abundantly present in proximal tubular cells (5). Tubu-lar reabsorption of VDBP is mediated by megalin and cubulin receptors (6, 7). Sincereceptor-mediated uptake of proteins such as VDBP is energy-consuming, tubular injurywould be expected to result in urinary VDBP (uVDBP) loss (8). Indeed, a previous smallstudy suggested that uVDBP is grossly elevated in CKD patients and can be reversedby antiproteinuric treatment (9). In diabetic subjects, uVDBP is increased comparedto non-diabetics, especially when albuminuria is present (10). Whether uVDBP is re-lated to the extent of tubulointerstitial damage has not specifically been addressed. Wetherefore explored the potential role of uVDBP as a marker of tubulointerstitial damagein two independent settings. First we studied the time course of uVDBP in relation tothe development of interstitial fibrosis and inflammation in the rat model of adriamycin-induced nephropathy at several time points after induction of nephrosis. Subsequently,in human subjects we addressed the relation between uVDBP and established tubulardamage markers in patients with various stages of renal damage, i.e microalbuminuriaand overt proteinuria, respectively, as compared to normoalbuminuric controls. We alsostudied whether intensification of anti-proteinuric therapy would reduce urinary VDBPexcretion.

Materials and Methods

Animal model

Male Wistar rats (n = 90 ) were housed in a temperature and light controlled room, withfree access to food and water. Twentyfour hour urine was collected every two weeks byhousing in metabolic cages. Surgical procedures took place under isoflurane anesthesia.At the end of the study, the abdominal aorta was cannulated and kidneys were perfusedin situ with saline during removal. Proteinuria was measured on a BNII third-generationnephelometer (Dade Behring, Mannheim, Germany). The protocols were approved by theAnimal Experiments Committee of the University of Groningen, the Netherlands.

Rats were assigned to two groups: unilateral adriamycin proteinuric (UAP) nephropa-thy (n = 60 ), or controls (n = 30 ). Unilateral adriamycin nephrosis was induced bytemporarily clipping the left renal artery through a midline abdominal incision, followedby adriamycin (1.5 mg/kg) injection via the tail vein, as described previously (11). Af-ter 12 min, when adriamycin has been cleared from the circulation (12), the clamp wasremoved. Control rats underwent the same procedure but were injected with saline. To

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study renal damage over time, groups of 12 UAP and six control rats were sacrificed atweeks 6, 12, 18, 24, and 30 after surgery. Kidneys were fixed in formalin and embeddedinto paraffin.

Patients

For clinical studies, 100 normoalbuminuric and 100 microalbuminuric subjects were ran-domly selected from Prevention of REnal and Vascular ENd stage Disease (PREVEND),a prospective cohort study investigating the association of albuminuria with renal andcardiovascular disease progression in the general population (13). In short, between 1997and 1998, participants of the PREVEND cohort were recruited from 40,856 inhabitantsof the city of Groningen, the Netherlands. To obtain a cohort enriched for the presence ofelevated albuminuria levels, selection was based on the albumin concentration in a spotmorning urine sample. The baseline screening round was completed in by 8,592 partic-ipants. Thereafter, participants were invited to visit an outpatient clinic for a medicalexamination at approximately 3-year intervals. For the present study, data obtained atbaseline were used. The PREVEND study was approved by the medical ethics committeeof our institution and conducted in accordance with the guidelines of the Declaration ofHelsinki. All participants gave written informed consent. Anthropometrical, clinical andbiochemical data were collected as reported previously (13). Glomerular filtration ratewas estimated (eGFR) with the modified Modification of Diet in Renal Disease (MDRD)formula, taking into account gender, age, race, and serum creatinine concentration. Ateach screening round two 24-h urine samples were collected after thorough oral and writteninstructions on how to collect these samples. Urinary albumin concentration was deter-mined in fresh urine samples by nephelometry with a threshold of 2.3 mg/L and intra-and inter-assay coefficients of variation (CV) of 2.2 and 2.6%, respectively (BNII; DadeBehring Diagnostic, Marburg, Germany). Albuminuria is given as the mean of the two24-hour urine albumin excretion measurements. Urine samples for biomarker measure-ments were stored at -20°C. VDBP was measured in urine samples collected at baselinefrom 100 randomly selected normo- and microalbuminuric subjects.

Furthermore, urine samples and clinical data were used from a previously performedrandomized controlled trial in patients with established non-diabetic chronic kidney dis-ease (14). Briefly, in this study, 52 patients were subjected to a protocol including a 6-weekstudy period with standard anti-proteinuric therapy (i.e. the ACE inhibitor lisinopril 40mg/day and a regular sodium diet of 200 mmol Na+ per day), and another 6-week studyperiod with intensified anti-proteinuric therapy (lisinopril 40 mg/day combined with theAT1 receptor blocker valsartan 320 mg/day and a low sodium diet, target 50 mmol Na+

per day). At the end of each study period, clinical data were recorded and blood and urinesamples were collected for analysis. Data and urine samples from both study periods wereavailable for 47 patients for the current post-hoc analyses (14).

Measurement of urinary VDBP and other biomarkers

After thawing urine and plasma samples were vortexed and subsequently centrifuged(14.000 rpm). The supernatant was used for measurements. Human VDBP was measuredwith a commercially available sandwich ELISA (Immundiagnostik, catalog # K2314, Ben-

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VDBP as a tubulointerstitial damage marker

sheim, Germany), according to the manufacturer’s instructions. Briefly, plasma (diluted1:40000) or urine (diluted 1:2 - 1:5 for controls and 1:10 - 1:3000 for the other groups de-pending on the concentration of VDBP) was incubated in a microtiter plate coated withpolyclonal anti-VDBP antibodies for one hour. Subsequently, a polyclonal peroxidase-labeled rabbit-anti-VDBP detection antibody was added and incubated for one hour.After washing, tetramethylbenzidine was added as substrate for 15 minutes. After addinga stop solution, absorbance at 450 nm was measured by a spectrophotometer (BenchMarkPlus, Bio-Rad Laboratories, Veenendaal, The Netherlands). Using a standard curve gen-erated with VDBP protein as provided by the manufacturer, final VDBP concentrationswere calculated. The detection limit of this ELISA is 1.23 ng/ml; intra-assay CV < 5.0%for 16 replicate determinations at concentrations of 24.2 and 42.9 mg/dl and inter-assayCV < 12.7% for a concentration of 19.3 mg/dl in 14 different assays on two differentlots; recovery ranges from 85-116% and linearity was acceptable (r2 =0.998). Rat VDBPwas measured using another commercially available ELISA kit (Alpco, Salem, NH), ac-cording to the manufacturer’s instructions. Detection limit as provided by manufacturer:3.125 ng/ml, range 3.125-100 ng/ml. VDBP excretion was calculated from the VDBPconcentration in urine collected over a 24-hour period. The established proximal tubulardamage markers KIM-1, beta-2-microglobulin and cystatin C as well as the inflammationmarker MCP-1 were determined as described previously (15, 16). Briefly, we developeddirect sandwich-enzyme-linked immunosorbent assays using monoclonal coating antibod-ies and labeled polyclonal detection antibodies on a Maxisorp plate (Nunc, Denmark).The concentration of the analyte was determined spectrophotometrically by conversion ofo-phenylenediamine by a horseradish peroxidase label. KIM-1, beta-2-microglobulin, andMCP-1 antibodies were obtained from R&D systems (Minneapolis, USA). The intra-assaycoefficients of variation (CV) of these ELISAs were 7.4%, 9.7%, and 6.8% respectively.Cystatin C was measured by nephelometry (reagents obtained from Siemens [Marburg,Germany]). Samples were diluted to obtain optimal concentration for measurement. Allsamples were measured in duplicate.

Immunohistochemistry

Paraffin sections were stained with periodic acid-Schiff to evaluate focal glomerulosclero-sis (FGS). Immunostaining for collagen type III (Biogenesis, Poole, UK), α-SMA (clone1A4; Sigma), or macrophages (ED1; Serotec, Oxford, UK) was performed as previouslydescribed (17). An automated staining system (DAKO Autostainer, Carpinteria, CA,USA) was used to obtain comparable staining results for all slides. Immunostaining wasquantified by computerized morphometry as described previously (17).

Statistical analyses

Analyses were performed with PASW Statistics 18.0.3 (SPSS, Armonk, NY). Parametricvariables are expressed as mean ± standard deviation (SD), whereas non-parametric vari-ables are given as median (interquartile range). Differences between groups were testedusing student’s t-test or Mann-Whitney test where appropriate. Linear regression analysiswas performed to address the association between uVDBP and parameters of tubuloin-terstitial damage, both at the time point of sacrifice. These associations were studied

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in univariate models as well as in multivariate models adjusting for albuminuria to ad-dress whether the relation between uVDBP and parameters of structural renal damagedepended on albuminuria. In these analyses, all adriamycin animals were considered to-gether in a regression model adjusted for the timepoint after induction of nephrosis. Thisallowed us to study the relation between uVDBP and renal damage parameters acrossthe spectrum of renal damage. Distribution of the variables was verified by Q-Q plotsand histograms. Non-normally distributed variables were transformed to the natural logbefore entering the regression model. For all analyses a two-sided p < 0.05 was consideredstatistically significant.

Results

Rat unilateral adriamycin model

The relation between urinary VDBP excretion and structural renal abnormalities inchronic kidney disease was addressed in the rat unilateral adriamycin nephropathy model.This model is characterized by relatively mild albuminuria as only one kidney is affected,followed by the development of renal inflammation, focal glomerulosclerosis (FGS), andtubulointerstitial fibrosis in the affected kidney, as described in detail previously (18).Massive urinary VDBP excretion was already present at 6 weeks after adriamycin injec-tion (Figure 7.1 A, (adriamycin: 727 ± 674 µg/d vs. controls: 9 ± 12 µg/d, p < 0.01),when interstitial alpha-smooth muscle actin (α-SMA) and collagen III expression as wellas interstitial macrophage accumulation were still similar to control kidneys (Figure 7.1B). Also at all other time points until end of followup at 30 wks (ADR: 1403 ± 1026 vs.CON: 206 ± 132 µg/d, p < 0.01), VDBP remained elevated in adriamycin rats comparedto controls at the same time point after start of the experiment.

The relation between uVDBP and renal damage markers were subsequently addressedin multivariate regression analysis where all adriamycin rats were analyzed together anddata were adjusted for the timepoint of sacrifice. This approach allowed us to address therelation between uVDBP and renal damage markers across the spectrum of renal dam-age, analyzing each animal once (at the time of sacrifice). uVDBP was strongly associatedwith interstitial expression of the pre-fibrotic marker α-SMA and with collagen III expres-sion, as well as with interstitial macrophage accumulation (Table 7.1). uVDBP was alsoassociated with albuminuria and FGS. Subsequently the associations between uVBDPand the parameters of renal damage were adjusted for albuminuria in multivariate regres-sion analysis. uVDBP remained significantly associated with α-SMA and collagen III aswell as with interstitial macrophage accumulation, independently of albuminuria. Theassociation between uVDBP and FGS, however, did depend on albuminuria (Table 7.1).Together, these data suggest that uVDBP is already increased before the onset of (early)tubulointerstitial fibrosis and inflammation, and uVDBP increases further in associationwith increasing tubulointerstitial damage severity.

Clinical studies

We further characterized urinary VDBP excretion in the clinical setting. Table 7.2 showsbaseline parameters at inclusion for the study groups: subjects with microalbuminuria

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VDBP as a tubulointerstitial damage marker

Figure 7.1: A. Graphs illustrating the development of albuminuria (orange) anduVDBP (purple) over time in the unilateral adriamycin nephropathy model and insham-operated control animals. B. Development of tubulointerstitial damage as re-flected by interstitial alpha-smooth muscle actin (blue bars) and collagen III staining(green bars), and interstitial macrophage accumulation (red bars) in adriamycin rats(dark bars) and controls (light bars).

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Table 7.1: Associations of urinary VDBP with renal histological parameters in theaffected kidney in the rat unilateral adriamycin nephropathy model.

Univariate analysis Adjusted for albuminuria

Damage marker Standardized beta P Standardized beta P

FGS score 0.56 < 0.001 0.28 0.52Interstitial collagen III 0.32 0.005 0.44 0.02Interstitial macrophagecount

0.46 < 0.001 0.47 0.01

Interstitial SMA proteinexpression

0.46 < 0.001 0.43 0.01

(n=100 ) and normoalbuminuric controls (n=100 ), both derived from a general popu-lation cohort study, and subjects with non-diabetic chronic kidney disease (CKD) withovert proteinuria (n=47 ). The CKD group was studied during two different treatmentperiods of 6 weeks, one period with an ACE inhibitor under a liberal sodium diet, andone period with intensified renoprotective therapy: dual RAAS blockade (ACE inhibitorand AT1 receptor blocker) under dietary sodium restriction. As shown in Figure 7.2,uVDBP was low in normoalbuminuric subjects, higher in microalbuminurics, and high-est in patients with established CKD (p < 0.001). In CKD patients, intensification ofanti-proteinuric therapy (dual RAAS blockade and dietary sodium restriction) reduceduVDBP from median 4 mg/day to 1 mg/24h (p < 0.001); however urinary VDBP wasstill > 100-fold increased as compared to normoalbuminuric subjects (median 7 µg/24h; p< 0.001 vs. CKD+ACEi+ARB+low sodium), consistent with persistent tubulointerstitialdamage despite maximal therapy.

VDBP is associated with established renal damage markers

The relation between uVDBP and established markers of proximal tubular damage and re-nal inflammation was further addressed in normo- and microalbuminuric subjects (n=200 ).uVDBP was strongly associated with urinary kidney injury molecule-1 (KIM-1), beta-2-microglobulin and cystatin C (Table 7.3 and Figure 7.3). Of interest, urinary VDBP wasalso associated with urinary excretion of the inflammation marker monocyte chemoattrac-tant protein-1 (MCP-1). These associations were independent of albuminuria (Table 7.3,Figure 7.3).

Discussion

To our knowledge this study is the first to demonstrate that urinary VDBP is a biomarkerof tubulointerstitial damage, independent of albuminuria, in an animal model of renal dam-age. In humans, urinary VDBP increased along with the severity of renal damage, wasassociated with tubular and inflammatory markers independently of albuminuria, andresponded to intensification of renoprotective therapy. Yet, the current cornerstone ofrenoprotective therapy (maximal RAAS blockade combined with dietary sodium restric-tion) did not achieve normalization of uVDBP, suggesting persistent tubulointerstitial

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VDBP as a tubulointerstitial damage marker

Figure 7.2: Box-whisker plots indicating the median, interquartile range, and rangeof 24-h uVDBP per patient category. general population with normoalbuminuria, generalpopulation with microalbuminuria, chronic kidney disease with overt proteinuria treated withACE inhibitor and liberal sodium diet, and chronic kidney disease with overt proteinuria treatedwith ACE inhibitor + ARB + low sodium diet. uVDBP increased with albuminuria (first threegroups, p < 0.001) and responded to intensification of anti-proteinuric treatment (two last groups,p < 0.001).

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Tab

le7.2

:B

ase

line

chara

cte

ristics

of

gen

era

lp

op

ula

tion

coh

ort.

Gen

era

lp

op

ula

tion

coh

ort

CK

Dw

ithovert

pro

tein

uria

Norm

oalb

um

inu

rian

=100

Mic

roalb

um

inu

rian

=100

AC

Ei+

libera

lso

diu

mn

=47

AC

Ei+

AR

B+

low

sod

ium

n=

47

P*

Age

(yr)

60±

11

60±

11

51±

13

51±

13

-M

ale

(%)

76

76

83

83

-B

aselin

ehisto

ryC

VD

(%)

22

39

66

-B

MI

(kg/m

2)27±

429±

528±

428±

4-

SB

P(m

mH

g)

140±

21

144±

21

135±

20

122±

18

0.0

01

Blo

od

pressu

relow

ering

dru

gs

(%)

25

35

100

100

-A

CE

i/A

RB

(%)

78

100

100

-C

holestero

l(m

mol/

L)

5.2±

1.2

5.1±

1.2

5.1±

1.1

4.9±

1.1

0.2

7G

luco

se(m

mol/

L)

5.1±

1.1

5.4±

1.2

5.5±

0.6

5.3±

0.6

0.3

9eG

FR

(ml/

min

)78±

15

77±

15

57±

25

48±

5<

0.0

01

Alb

um

inuria

(mg/24h)

14±

959±

51

2191±

1734

949±

1113

<0.0

01

*P

valu

eca

lcula

tedby

paired

ttest

com

parin

ggro

up

on

AC

Ein

hibito

r(A

CE

i)+libera

lsod

ium

diet

versus

AC

Ei+

an

gioten

sinrecep

tor

blocker(A

RB

)+lo

wsod

ium

diet.

CV

D=

card

iova

scula

rd

isease,

BM

I=

body

ma

ssin

dex,

SB

P=

systolic

bloodp

ressure,

eGF

R=

estima

tedglo

meru

lar

filtra

tion

rate.

116

VDBP as a tubulointerstitial damage marker

Figure 7.3: Box-whisker plots illustrating the relation between uVDBP and estab-lished renal tubular damage markers kidney injury molecule-1 (KIM-1) (A), beta-2-microglobulin (B2M) (B), Cystatin C (CysC) (C) and monocyte chemoattractantprotein-1 (MCP-1) (D) in normo- and microalbuminuric subjects (both n=100). TheuVDBP is presented per quartile of the established renal damage marker studied. In all pan-els (A-D), the left four box whisker plots represent the unadjusted VDBP excretion, and theright four box whisker plots represent the uVDBP adjusted for albuminuria. All biomarkers arepositively associated with uVDBP, also when uVDBP was adjusted for albuminuria (p < 0.001,Kruskal Wallis).

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Table 7.3: Associations of VDBP with renal damage markers across the clinicalcohorts.

Association with uVDBPUnivariate Adjusted for albuminuria

Damage marker Standardized beta P Standardized beta P

KIM-1 0.77 < 0.001 0.52 < 0.001Beta-2-microglobulin 0.54 < 0.001 0.45 < 0.001Cystatin C 0.49 < 0.001 0.4 < 0.001MCP-1 0.28 < 0.001 0.31 0.001

KIM-1 = kidney injury molecule-1, MCP-1 = monocyte chemoattractant protein-1

damage in CKD patients. These findings suggest that uVDBP could be developed into anon-invasive urinary marker to monitor tubulointerstitial inflammation and fibrosis.

The urinary loss of VDBP in the setting of renal damage has been reported previouslyin a rat nephrotoxicity model using urinary proteomics analysis (19), as well as in thesetting of chronic kidney disease (9, 10), but its mechanism has not been documented.Interestingly, we found that uVDBP was strongly and consistently elevated in a veryearly stage of adriamycin-induced nephropathy, even before a detectable increase in theearly pre-fibrotic marker α-SMA. uVDBP was also strongly associated with markers ofboth early and late tubulointerstitial fibrosis (α-SMA and collagen III, respectively) inlinear regression analysis adjusted for the time after induction of nephrosis. Of interest,VDBP excretion was also associated with interstitial inflammation, even independent ofalbuminuria. Its potential as a marker of interstitial inflammation renders VDBP an evenmore interesting candidate biomarker. The fact that these associations persisted afteradjustment for albuminuria suggests that not only protein overload of the megalin/cubulincomplex plays a role in urinary VDBP loss. Rather, damaged tubular epithelial cells inareas of tubulointerstitial fibrosis may no longer be able to handle VDBP, resulting in grossVDBP loss into the urine. Indeed, major factors involved in tubulointerstitial fibrosis andinflammation (e.g. TGF-beta and angiotensin II) negatively regulate receptor-mediatedendocytosis (20, 21).

We and others reported previously that uVDBP is increased in patients with chronickidney disease (9, 10), and that it can be modulated by anti-proteinuric treatment inpatients (9), although our previous study was of limited sample size. Our study is in linewith previous work showing that uVDBP increases with increasing severity of diabeticnephropathy (10). Although the combination of optimal RAAS blockade and dietarysodium restriction, an intervention considered optimal for renoprotection, considerablyreduced VDBP excretion, it remained > 100-fold increased as compared to healthy nor-moalbuminuric subjects. This suggests that tubulointerstitial damage, considered the finalcommon pathway towards end-stage renal disease (ESRD), persists to a considerable ex-tent despite current best available therapy. This is in line with previous preclinical studiesshowing progression of pre-fibrotic tubulointerstitial lesions - in spite of optimal reductionof proteinuria - by ACE inhibition and (very strict) dietary sodium restriction (22). Thismay explain at least partly why many patients progress to ESRD despite optimal RAAS

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VDBP as a tubulointerstitial damage marker

blockade and dietary sodium restriction - although progression to ESRD is more commonin those with high sodium intake (23).

Limitations of our study include the lack of histopathological data in humans to whichuVDBP could be related, and the largely cross-sectional design. Although in the animalstudies uVDBP was tracked over time, this study does not contain prospective data al-lowing validation of uVDBP as a biomarker. Strengths of our study, on the other hand,include the use of 24h-urine samples in both human and the animal studies, and theequivocal findings in patients and animals, using two different ELISA systems to detecturinary VDBP. Therefore we believe that our observations can be considered hypothesis-generating and warrant further studies on the predictive value of urinary VDBP in awell-characterized cohort with longitudinal follow-up, as well as studies addressing theassociations between uVDBP and histopathological findings in the clinical setting.

In conclusion, we demonstrated that urinary VDBP excretion is increased early afterrenal injury, and is associated with tubulointerstitial inflammation and fibrosis indepen-dently of albuminuria in a rat model of proteinuric nephropathy. In humans, uVDBPincreased with increasing severity of renal damage, and responded to renoprotective ther-apy. Yet, persisting uVDBP at > 100 times above normal suggested persistent tubuloin-terstitial damage despite optimal renoprotective therapy. Future studies should addresswhether urinary VDBP has predictive value for progression of renal function loss and isa more suitable early marker of tubulointerstitial damage than those currently available,to better guide therapy in chronic kidney disease patients.

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