Induction of Glomerular Heparanase Expression in Rats with Adriamycin Nephropathy Is Regulated by Reactive Oxygen Species and the Renin-Angiotensin System
Andrea Kramer*,,
Mabel van den Hoven,,
Angelique Rops,,
Tessa Wijnhoven||,¶,
Lambert van den Heuvel¶,
Joost Lensen||,
Toin van Kuppevelt||,
Harry van Goor*,
Johan van der Vlag,,
Gerjan Navis* and
Jo H.M. Berden,
Departments of * Pathology and Nephrology, University Medical Center Groningen, Nephrology Research Laboratory and || Department of Matrix Biochemistry, Nijmegen Centre for Molecular Life Sciences, and Division of Nephrology and ¶ Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Address correspondence to: Dr. Jo H.M. Berden, Division of Nephrology (464), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: +31-24-3614761; Fax: +31-24-3540022; E-mail: j.berden{at}nier.umcn.nl
Received for publication February 28, 2006.
Accepted for publication June 29, 2006.
Heparan sulfate (HS) in the glomerular basement membrane (GBM)is important for regulation of the charge-dependent permeability.Heparanase has been implicated in HS degradation in severalproteinuric diseases. This study analyzed the role of heparanasein HS degradation in Adriamycin nephropathy (AN), a model ofchronic proteinuria-induced renal damage. Expression of heparanase,HS, and the core protein of agrin (to which HS is attached)was determined on kidney sections from rats with AN in differentexperiments. First, expression was examined in a model of unilateralAN in a time-course study at 6-wk intervals until week 30. Second,rats were treated with the hydroxyl radical scavenger dimethylthiourea(DMTU) during bilateral AN induction. Finally, 6 wk after ANinduction, rats were treated with angiotensin II receptor type1 antagonist (AT1A) or vehicle for 2 wk. Heparanase expressionwas increased in glomeruli of rats with AN, which correlatedwith HS reduction at all time points and in all experiments.Treatment with DMTU prevented the increased heparanase expression,the loss of GBM HS, and reduced albuminuria. Finally, treatmentof established proteinuria with AT1A significantly reduced heparanaseexpression and restored glomerular HS. In conclusion, an associationbetween heparanase expression and reduction of glomerular HSin AN was observed. The effects of DMTU suggest a role for reactiveoxygen species in upregulation of heparanase. Antiproteinurictreatment by AT1A decreased heparanase expression and restoredHS expression. These results suggest involvement of radicalsand angiotensin II in the modulation of GBM permeability throughHS and heparanase expression.
The glomerular basement membrane (GBM) consists of many extracellularmatrix proteins, including heparan sulfate (HS) proteoglycans,mainly agrin. Loss of negatively charged HS molecules resultsin an altered charge-dependent permeability of the GBM (1).The importance of HS in the charge-dependent permeability ofthe GBM has been demonstrated in several studies. First, digestionof HS by heparitinase resulted in increased permeability ofthe GBM for ferritin and albumin (2,3). Second, injection ofa specific mAb against HS caused massive albuminuria in rats(4). Third, loss of anionic HS has been reported in severalhuman and experimental glomerulopathies, which was inverselycorrelated with the degree of proteinuria (5,6). It is suggestedthat proteinuria-induced renal damage is associated with lossof glomerular HS in diabetic and nondiabetic renal disease (7–9).
HS loss in proteinuric renal disease can be attributed to severalmechanisms (reviewed in reference [1]), such as depolymerizationof HS by reactive oxygen species (ROS), masking of HS by nucleosome/Igcomplexes, reduction of both HS production and sulfation asa result of hyperglycemia, complement-mediated cleavage of HS,and proteolytic cleavage of the HS attachment site on the coreprotein by enzymes.
Heparanase is an endo-(1,4)-d-glucuronidase that is involvedin the cleavage of HS and hence is associated with extracellularmatrix degradation and tissue remodeling (10–13). A recentstudy in patients with diabetic nephropathy suggested that lossof HS in the GBM is attributable to accelerated HS degradationby increased heparanase expression (14). Studies in experimentalrenal diseases (passive Heymann nephritis, puromycin aminonucleosidenephrosis, and anti-GBM nephritis) suggest that heparanase alsomay be involved in nondiabetic proteinuric disease (15–18).
In chronic proteinuric renal diseases, blockade of the renin-angiotensinsystem (RAS) reduces proteinuria and thereby reduces progressiverenal function loss. In Adriamycin nephropathy (AN), a modelof chronic proteinuric renal damage (19), we previously reporteda decrease of HS expression, which was caused partially by hydroxylradicals (7). Furthermore, we found that RAS blockade protectsagainst loss of HS in established AN (9). However, whether changesin heparanase may be involved in the decrease of HS expressionin AN and/or the effects of RAS blockade is unknown. Therefore,in this study we first determined the time course of heparanaseand HS expression in relation to the development of proteinuriaand renal structural damage in AN. For this purpose, we usedthe unilateral variant of the AN model to allow good resolutionover time. Next, the effect of scavenging of ROS during theperiod of induction of AN on heparanase and HS expression andon proteinuria was studied. Finally, we evaluated the potentialof antiproteinuric treatment with an angiotensin I receptorantagonist (AT1A) to restore the disbalance between heparanaseand HS in established AN with persistent proteinuria.
Animals and Experimental Design
Male Wistar rats were housed in a temperature-controlled roomwith a 12-h light-dark cycle and with free access to food andwater. Twenty-four-hour urine samples were collected every 2wk in metabolic cages, with measurement of water and food intake.Surgical procedures took place under isoflurane anesthesia inN2O/O2 (1:2). Systolic BP was measured weekly by the tail-cuffmethod in conscious rats (20). At the end of the study, theabdominal aorta was cannulated, a 2-ml blood sample was taken,and kidneys were perfused in situ with saline and removed. Proteinuriawas measured on a BNII third-generation nephelometer (Dade Behring,Mannheim, Germany) by using a 20% trichloroacetic acid solution.The Committees for Animal Experiments of the University of Groningenand the Radboud University Nijmegen, The Netherlands, approvedall studies.
Experiment 1.
Unilateral AN was induced by temporarily clipping the left renalartery and vein through a midline abdominal incision (20), followedby Adriamycin (1.5 mg/kg body wt) injection via the tail vein.After 12 min, when Adriamycin had been cleared from the circulation(19), the clamp was removed. For studying expression of heparanase,HS and renal damage over time, eight rats (at each time point)were killed at weeks 6, 12, 18, 24, and 30. In this experiment,the right kidney—exposed to Adriamycin—was comparedwith the control (nonexposed, left) kidney. Previously, we showedthat the clipped left kidney did not differ from healthy controlkidneys, not even after 30 wk (21).
Experiment 2.
Bilateral AN was induced by intravenous injection of 5 mg/kgbody wt Adriamycin via the tail vein. One group of rats (n =8) received an initial intraperitoneal injection of the hydroxylscavenger dimethylthiourea (DMTU; 500 mg/kg body wt; Sigma,St. Louis, MO) 6 h before Adriamycin injection, followed bytreatment with DMTU (1.25 mg/kg body wt) twice a day for 7 d.The control group (n = 7) received saline instead of DMTU. Ratswere killed 4 wk after the injection of Adriamycin. Urine wascollected at weeks 0 and 4 for determination of urinary albuminexcretion.
Experiment 3.
Bilateral AN was induced by intravenous injection of Adriamycin(2 mg/kg body wt) via the tail vein. At week 6, a renal biopsywas performed via a dorsolateral incision. After removal ofa part of the lower pole from the left kidney, gel foam (Spongostan;Ferrosan, Copenhagen, Denmark) was applied for hemostasis. Afterrecovery, rats were treated with the angiotensin II type 1 receptorantagonist (AT1A) L158,809 (150 mg/L drinking water; n = 20)or vehicle (n = 10). In previous experiments, the biopsy didnot affect the course of renal damage (9,22). Treatment wascontinued until the rats were killed at week 8. Eight healthyrats that were killed at week 6 were used as time controls.
Immunofluorescence Staining
To determine heparanase, HS, and agrin core protein expression,we performed indirect immunofluorescence staining on 2-µmcryostat kidney sections. Tissue sections were fixed in 100%acetone for 10 min at 4°C, followed by 1 h of incubationof primary antibodies diluted in PBS that contained 1% BSA and0.01% sodium azide at room temperature. After washing in PBS,the appropriate secondary antibodies were diluted in PBS andincubated for 1 h at room temperature, with 5% normal rat serumand 10% normal goat serum. Specific primary and secondary antibodiesthat were used are summarized in Table 1. Subsequently, thesections were washed in PBS and embedded in Vectashield mountingmedium H-1000 (Vector Laboratories, Burlingame, CA).
For investigation of which glomerular cell type was responsiblefor the expression of heparanase in AN, sections were double-stainedwith anti-heparanase and (1) anti-agrin, to distinguish betweenthe outside of the GBM covered by podocytes and the inside withendothelial cells lining the GBM; (2) anti-synaptopodin, whichstains the cytoskeleton of podocytes; and (3) anti-Thy1.1, whichis a mesangial cell marker. For investigation of in which tubularcell type heparanase was expressed, double stainings were performedwith anti-heparanase and (1) anti–aquaporin 2, a markerfor collecting ducts; (2) anti–calbindin D-28k, a markerfor distal convoluted tubuli; and (3) anti–P-glycoprotein,a marker for proximal tubuli (summarized in Table 1). Confocallaser scanning microscopy (Leica, Heidelberg, Germany) was usedto evaluate the stainings.
Quantification of Immunofluorescence Staining and Histochemistry
The kidney sections were randomly coded and evaluated by twoindependent observers on a Zeiss Axioskop microscope (equippedwith an epi-illuminator). For agrin and HS expression, linearityof the GBM was scored at an arbitrary scale of 0 to 10 (0 =no staining, 1 = 10% linear GBM staining, etc., with a maximumscore of 10 for 100% staining). Heparanase expression was scoredfrom 0 to 5 arbitrary units for staining intensity. Expressionwas analyzed in 25 glomeruli per animal.
Focal glomerulosclerosis (FGS) was scored semiquantitativelyon periodic acid-Schiff–stained paraffin sections (4 µm),as described previously (23). FGS lesions were defined as glomerularareas with mesangial expansion and adhesion formation simultaneouslypresent in one segment. Scoring was performed on a scale of0 to 4 in 50 glomeruli per kidney moving from outer to innercortex. The FGS scores presented in Tables 2 and 3 are the medianFGS scores per 50 glomeruli per experimental animal group multipliedby 100 (score in arbitrary units from 0 to 400).
Table 3. Characteristics of the intervention study in bilateral AN (experiment 3)a
Statistical Analyses
Data are expressed as median and 95% confidence intervals. Differencesbetween groups were determined by Kruskal-Wallis and Mann-WhitneyU tests. Linear regression was performed to detect whether proteinuriaand heparanase and HS expression were associated. Analyses wereperformed using SPSS version 12.0 (SPSS, Inc., Chicago, IL)and GraphPad Prism, version 4.0 software (GraphPad Software,Inc., San Diego, CA). Statistical significance was regardedat P < 0.05.
Clinical and Morphologic Data
Data on proteinuria and BP for the time-course study (experiment1) and the intervention study (experiment 3) are shown in Tables 2and 3, respectively. In unilateral AN, proteinuria was increasedat all time points and BP was normal. The score for FGS wasincreased in the Adriamycin-exposed kidney compared with thenonexposed control kidney and progressed over time. When ratswere treated with DMTU before induction of bilateral AN, albuminuriaat week 4 was lower compared with that in saline-treated ratswith AN (255 [185 to 276] versus 353 [274 to 436] mg/24 h; P< 0.05). In the intervention study at week 6 (before treatmentwith AT1A started), proteinuria was markedly increased comparedwith healthy controls. Treatment during 2 wk with AT1A reducedproteinuria and BP. The score for FGS did not change duringthe 2 wk of treatment. In the vehicle-treated group, proteinuriaand BP and FGS score remained stable.
Heparanase Overexpression Is Associated with Reduced HS Expression in AN: Time Course
Glomerular HS and heparanase expression in the exposed and nonexposedkidney, assessed by indirect immunofluorescence staining oncryostat sections, is shown in Figure 1. HS showed a nice linearstaining along the GBM in control kidneys (Figure 1A). However,in Adriamycin-exposed kidneys, the intensity of HS expressionin the GBM was reduced, whereas agrin expression remained unaltered(Figure 1B). Semiquantitative analysis revealed that HS wassignificantly reduced in Adriamycin-exposed kidneys at all timepoints without changes over time, whereas agrin core proteinexpression did not differ between control and Adriamycin-exposedkidneys (Figure 2, A and B). Heparanase expression was markedlyincreased in all Adriamycin-exposed kidneys (Figure 1D) as comparedwith the control kidneys (Figure 1C), which was confirmed bysemiquantitative analysis. Heparanase expression was increasedsignificantly at all time points (Figure 2C) without changesover time. The reduction in glomerular HS expression significantlycorrelated with an increase in heparanase expression (R2 = 0.34,P < 0.001; Figure 2D). Taken together, these results indicatethat increased heparanase expression and loss of glomerularHS are early events in the time course of the Adriamycin-inducednephrotic syndrome.
Figure 1. Heparan sulfate (HS; proteoglycans) and heparanase expression in Adriamycin nephropathy (AN) and control kidneys (indirect immunofluorescence staining). (A) JM403 staining for HS and MI91 staining for agrin core protein in a control kidney; a nice linear staining of the glomerular basement membrane (GBM) is observed for both HS and agrin. (B) HS and agrin staining in an Adriamycin-exposed kidney: Glomerular HS staining is decreased, whereas agrin staining remains linear. (C) HS and heparanase staining in a control kidney: Heparanase is present in the tubuli but absent in the glomerulus. (D) HS and heparanase expression in an Adriamycin-exposed kidney: A glomerulus with reduced HS expression and increased heparanase expression. Magnification, x40.
Figure 2. Semiquantitative analysis of glomerular HS, agrin, and heparanase expression in arbitrary units (A.U.) at different time points in unilateral AN (experiment 1). (A) HS expression in the GBM was significantly decreased in AN at all time points. (B) Agrin core protein expression did not differ between Adriamycin-exposed and control kidneys. (C) Glomerular heparanase expression was upregulated in AN at all time points. (D) Correlation between HS staining and heparanase expression in unilateral AN. Open symbols indicate score of nonexposed kidneys; filled symbols indicate score of Adriamycin-exposed kidneys. *P < 0.05.
DMTU Reduces the Early Effect of Adriamycin on HS and Heparanase Expression
Because HS reduction in AN has been attributed to a depolymerizationof HS by ROS (7), we studied whether the increased heparanaseexpression could be attributed to ROS by treating rats withthe hydroxyl radical scavenger DMTU during disease inductionby Adriamycin and the first week afterward. When the rats werekilled, 3 wk later, a loss of HS was observed in the saline-treatedrats that was partly prevented by the treatment with DMTU (Figure 3A),whereas expression of agrin was comparable in both groups (datanot shown), which is in line with previous experiments (7).It is interesting that the glomerular heparanase expressionin AN rats that were treated with the ROS scavenger DMTU wassignificantly lower compared with that in saline-treated ANrat (Figure 3B).
Figure 3. Effect of treatment with the hydroxyl radical scavenger dimethylthiourea (DMTU) before disease induction by Adriamycin and the first week afterward (experiment 2). (A) Decrease of HS expression in AN could be partly prevented by treatment with DMTU. (B) Heparanase expression was significantly lower after treatment with DMTU. *P < 0.05.
Reduction of Heparanase and Increase in HS Expression after Treatment with AT1A
Next, we investigated whether, in established proteinuria, antiproteinurictreatment by AT1A can reduce glomerular heparanase expressionalong with restoration of HS expression in the GBM. HS (proteoglycans)and heparanase expression was determined in bilateral AN, before(week 6) and after 2 wk of treatment with AT1A or vehicle (week8). Glomerular HS expression was decreased in all Adriamycin-exposedanimals at week 6, and heparanase expression was markedly increased,which was comparable with the results observed in the unilateralmodel of AN (Figure 4). Treatment with AT1A significantly increasedglomerular HS expression compared with the vehicle-treated animals(Figure 4A), which was, interestingly, accompanied by a significantreduction in heparanase (Figure 4B). However, AT1 receptor blockadedid not completely restore HS expression to normal levels asobserved in the healthy controls. For the individual Adriamycinanimals (vehicle and AT1A at weeks 6 and 8), the reduction inglomerular HS expression was significantly correlated with increasedheparanase expression (R2 = 0.63, P < 0.001; Figure 4C).
Figure 4. Effects of treatment with angiotensin type I receptor antagonist (AT1A) or vehicle (VEH) on heparanase and HS expression in bilateral AN (Experiment 3) compared with healthy control rats at week 6. Indirect immunofluorescence double staining was performed on kidney biopsies before (week 6) and after treatment (week 8). (A) Glomerular HS expression was reduced before treatment but restored after 2 wk of treatment with AT1A. Treatment with VEH did not restore HS expression. (B) After 2 wk of treatment with AT1A, heparanase expression decreased, and it did not change with VEH treatment. (C) Correlation between HS staining and heparanase expression in bilateral AN. *P < 0.05.
Heparanase Expression in Glomeruli and Tubuli
Heparanase expression in AN is located at the outer side ofthe GBM and is minimally expressed within the capillary loops(Figure 5, A through C). Co-localization with synaptopodin confirmsthat heparanase is expressed by the podocytes and not by mesangiumcells, because no co-localization with anti-Thy1.1 could beobserved.
Figure 5. Immunofluorescence double staining with anti-heparanase (green) and anti-agrin (red) (A), and with anti-heparanase (red) and anti-synaptopodin (B), or anti-Thy1.1 (C) (both in green). Glomerular heparanase is expressed mainly at the outside of the GBM, confirmed by co-localization with the podocyte marker synaptopodin. Minimal staining was observed in the capillary loops (according to the agrin/heparanase staining). No co-localization with mesangium cells was shown. For tubular heparanase expression, we used immunofluorescence double staining with anti-heparanase (red) and anti–calbindin D-28k (D), anti–aquaporin 2 (E), and anti–P-glycoprotein (F; all green). Tubular heparanase is expressed mainly in the proximal tubuli, because it co-localized with P-glycoprotein. No co-localization with the distal convoluting tubuli and collecting ducts was shown. Magnification, x63.
Heparanase is expressed in tubuli of both normal and diseasedanimals. Tubular HS and heparanase expression was not alteredby either Adriamycin injection or AT1A treatment compared withcontrols. Using specific tubular markers, we observed that heparanaseis expressed mainly in proximal tubuli. No expression was foundin the distal convoluted tubuli or collecting ducts (Figure 5,D through F).
Our data demonstrate that glomerular heparanase expression isincreased in Adriamycin-induced nephropathy and associated witha decreased HS expression in the GBM. These changes occur earlyafter disease induction and remain stable during follow-up,whereas renal structural damage progresses. We previously foundROS to induce loss of glomerular HS (7); our data show thattreatment with the ROS scavenger DMTU reduces heparanase expressioncompared with saline-treated Adriamycin rats, with subsequentlya better preservation of glomerular HS expression and ameliorationof albuminuria. Finally, glomerular heparanase expression inestablished AN was reversible by antiproteinuric treatment withRAS blockade, along with an increased glomerular HS expression.
In a previous study, we reported that by treatment with a ROSscavenger, HS expression was not completely restored and albuminuriawas not completely prevented in AN (7), suggesting that additionalmechanisms are involved in the reduction of HS in this model.Our study demonstrates that heparanase expression is increasedin AN and correlated with the loss of HS in the GBM, suggestingthat heparanase may play an important role in HS reduction.The increased expression of heparanase in AN is in line withthe findings in puromycin aminonucleoside nephrosis, passiveHeymann nephritis, and anti-GBM nephritis (15–18). Theloss of glomerular HS also is in accordance with previous studiesin proteinuric renal disease (7–9). Our data on a consistentassociation between HS and heparanase suggest that heparanaseis an important factor involved in the breakdown of HS and therebyin the development of proteinuria. The pathogenic potentialfor heparanase in proteinuria is supported by a recent studyshowing that inhibition of heparanase prevented both proteinuriaand loss of HS in passive Heymann nephritis (16). Moreover,heparin and heparin derivatives that inhibit heparanase havebeen shown to exert antiproteinuric effects in diabetic nephropathy,further supporting the impact of heparanase (24).
As mentioned before, ROS have already been demonstrated to beinvolved in the loss of HS in AN, which we explained by ROS-mediateddepolymerization of HS (7). In this study, treatment of AN withDMTU prevented the increase in heparanase expression and theloss of HS in the GBM. Whether ROS production in this modelis persistent seems unlikely, because treatment with DMTU (6h before and for only 1 wk after Adriamycin administration)partially prevents proteinuria, loss of HS, and heparanase overexpressionat later stages.
Treatment with an angiotensin-converting enzyme (ACE) inhibitoror AT1A ameliorates proteinuria in both human and experimentalrenal diseases, including AN, and provides renoprotection (21,25–30).Antiproteinuric treatment with AT1A in this study led to a partiallyrestored glomerular HS expression. This effect of AT1A on HSexpression in AN is comparable with the effect of ACE inhibitionthat preserved glomerular HS expression in rats with AN (9).Along with the restored HS expression after treatment with AT1A,we observed a markedly reduced glomerular heparanase expression.In vitro experiments show that angiotensin II reduces HS expressionin the extracellular matrix of human podocytes, raising thepossibility of a direct effect (31). No data on intrarenal angiotensinII are available in AN; however, we have reported elevated intrarenalactivity of ACE in AN, consistent with a local activation ofRAS (32).
We used three different approaches to study the expression ofglomerular heparanase and HS in AN. The first was a unilateralmodel with a relatively low (1.5 mg/kg body wt) Adriamycin dosageto allow good resolution over time. In the second study, theacute bilateral AN model (5 mg/kg body wt) with DMTU treatmentwas used with short-term follow-up. Finally, in the chronicbilateral AN model with AT1 intervention, we used 2 mg/kg bodywt. Despite variations in the induction and severity of themodel, the observed correlation between HS and heparanase waspresent consistently.
The changes in glomerular heparanase expression in the Adriamycinmodel and its reversibility by ROS scavenging or RAS blockadeobserved here theoretically could be due to different factors,namely (1) ROS, (2) angiotensin II, and/or (3) proteinuria.A single intrarenal administration of Adriamycin leads to achain of reactions. Generation of ROS leads to injury of thepodocytes, resulting in persistent proteinuria and increasedproduction of (local) angiotensin II. All three elements (ROS,angiotensin II, and proteinuria), in our opinion, could be sequentiallyresponsible for the persistence of heparanase overexpression.Although the generation of ROS can be induced by angiotensinII (33–35), ROS also are thought to be induced by Adriamycinper se. The beneficial effects of DMTU treatment during theinduction phase of AN support a role for ROS in the inductionof changed charge-selective properties of the GBM, as a resultof both a direct effect of ROS on HS and ROS-induced heparanaseexpression. The effects of RAS blockade, showing reversibilityof heparanase upregulation in established nephropathy, supporta role for angiotensin II in heparanase expression. As to proteinuria,the time course study with early changes in heparanase and HSexpression, which were stable during long-term follow-up despiteprolonged exposure to proteinuria, suggests that increased heparanaseand decreased HS expression are causal to proteinuria ratherthan a consequence. Furthermore, in a model of protein-overloadnephropathy (36), we could not demonstrate a reduction in HSor an increase in heparanase expression (data not shown).
Our results suggest that both ROS and RAS are involved in upregulationof heparanase expression. Several studies have demonstratedthat there is a link between ROS and RAS signaling. AngiotensinII has been shown to induce ROS production, whereas ROS mediatesseveral effects of angiotensin II, such as on protein synthesis,cell hypertrophy, and vascular endothelial growth factor production.Scavengers of ROS ameliorate angiotensin II–induced orangiotensin II–mediated effects, whereas ACE inhibitionor angiotensin II receptor blockade were able to reverse oxidativestress (37–40). Further studies would be needed to addressthe possible interactions between ROS and RAS in regulationof heparanase expression in AN.
Heparanase plays an important role in the loss of HS in AN.Heparanase expression is increased early in the time courseof AN and shows a clear association with the loss of HS in theGBM and proteinuria, suggesting that heparanase is an importantmediator of loss of glomerular HS and development of proteinuriain AN. Scavenging of ROS prevented upregulation of heparanaseand loss of HS. In addition to direct ROS-mediated depolymerizationof HS, which we have shown previously in vitro, ROS indirectlymay contribute to the HS loss in AN by upregulation of heparanaseexpression. It is feasible that in AN, both direct and indirectROS-mediated mechanisms are operative. Reduction of heparanaseand the subsequent restoration of glomerular HS contribute tothe beneficial effects of RAS blockade. Our results suggestthat both ROS and RAS play a role in heparanase induction andin the breakdown of HS in AN. However, the interplay betweenROS and RAS in the induction of heparanase requires furtherinvestigation.
Acknowledgments
This project was supported by grants from the Groningen UniversityInstitute of Drug Exploration, from the Dutch Diabetes ResearchFoundation (grant 2001.00.048), and from The Netherlands Organizationfor Scientific Research (grant 902-27-292).
We thank Goos Laverman, Lotte Vis, Bianca Meijeringh, AllardWagenaar, Marian Bulthuis, Inge Hamming, Jaenine Beukema, Mirjanvan Timmeren, Marinka Bakker, and Mieke Baselmans for assistance.We also thank Dr. P. Deen and Dr. J. Hoenderop (Department ofPhysiology) and Dr. R. Masereeuw (Department of Pharmacologyand Toxicology) from the Radboud University Nijmegen MedicalCentre for the generous gifts of the aquaporin-2, calbindinD-28k, and P-glycoprotein antibodies, respectively. L158,809was a kind gift from Merck Sharp & Dome Research Laboratories(Rahway, NJ).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
A.K. and M.v.d.H. contributed equally to this work.
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Abstract
Introduction
(1,4)-d-glucuronidase that is involved in the cleavage of HS and hence is associated with extracellular matrix degradation and tissue remodeling (10–13). A recent study in patients with diabetic nephropathy suggested that loss of HS in the GBM is attributable to accelerated HS degradation by increased heparanase expression (14). Studies in experimental renal diseases (passive Heymann nephritis, puromycin aminonucleoside nephrosis, and anti-GBM nephritis) suggest that heparanase also may be involved in nondiabetic proteinuric disease (15–18). 
