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Effects on protein kinase C-� inhibition on glomerular vascular endothelialgrowth factor expression and endothelial cells in advanced experimentaldiabetic nephropathy

Darren J. Kelly,1 Danielle Buck,1 Alison J. Cox,1 Yuan Zhang,1 and Richard E. Gilbert1,2

1Department of Medicine, University of Melbourne, St. Vincent’s Hospital, Fitzroy, Australia; and 2Department of Medicine,University of Toronto, St. Michael’s Hospital, Toronto, Canada

Submitted 5 October 2006; accepted in final form 16 May 2007

Kelly DJ, Buck D, Cox AJ, Zhang Y, Gilbert RE. Effects on proteinkinase C-� inhibition on glomerular vascular endothelial growth factorexpression and endothelial cells in advanced experimental diabetic ne-phropathy. Am J Physiol Renal Physiol 293: F565–F574, 2007.First published May 23, 2007; doi:10.1152/ajprenal.00397.2006.—Ruboxistaurin is an inhibitor of the � isoform of protein kinase C(PKC-�) that reduces the actions of vascular endothelial growth factor(VEGF) and attenuates the progression of diabetic retinopathy. In theglomerulus VEGF is constitutively expressed where it likely has a rolein maintaining endothelial cell integrity, particularly in disease states.Given its potential use in diabetic nephropathy, we sought to deter-mine the effects of PKC-� inhibition on VEGF and glomerularendothelial cells in experimental diabetic nephropathy. Studies wereconducted in (mRen-2)27 rat, a transgenic rodent with hypertensionand an enhanced renin-angiotensin system that following induction ofdiabetes with streptozotocin develops many of the features of diabeticnephropathy. Moreover, to mimic the clinical context, the effects ofPKC-� inhibition were examined both with and without concomitantangiotensin-converting enzyme (ACE) inhibitor therapy. DiabeticRen-2 rats were randomized to receive either vehicle, the ACEinhibitor, perindopril (0.2 mg/l in drinking water), ruboxistaurin (10mg �kg�1 �day�1, admixed in chow), or their combination and studiedfor 12 wk. Diabetic Ren-2 rats displayed glomerular endothelial cellloss in association with overexpression of VEGF mRNA. Both cellloss and VEGF overexpression were attenuated by the administrationof either perindopril or ruboxistaurin, as single agent treatments withtheir combination providing additional, incremental improvements,reducing these manifestations of injury down to levels seen in non-diabetic, normotensive, nontransgenic animals. Combination therapywas also associated with additional improvements in albuminuria andglomerulosclerosis.

glomerular endothelial cells; glomerulosclerosis; albuminuria

RUBOXISTAURIN MESYLATE, A SPECIFIC and selective inhibitor ofPKC-� isoforms, has been shown to delay moderate visual lossin patients with diabetic retinopathy (12) and to reduce albu-minuria in patients with diabetic nephropathy (28). Its benefi-cial effects in diabetic eye disease are thought to relate, at leastin part, to its ability to reduce the actions of vascular endothe-lial growth factor (VEGF) (30), a pro-angiogenic and perme-ability-enhancing cytokine that is overexpressed in both human(1) and experimental diabetic retinopathy (10). However, todate, studies in the kidney have focused mostly on the role ofthe profibrotic growth factor, transforming growth factor-�,

and its effects on extracellular matrix expansion (19, 20), ratherthan on VEGF.

In addition to extracellular matrix expansion, the integrity ofthe glomerular capillary network is also recognized as a keydeterminant of renal pathology in both the experimental andhuman settings (23–25). The development of this network iscritically dependent on VEGF, a growth factor that not onlystimulates endothelial cell proliferation and permeability butmay also have a role in the maintenance of endothelial cellintegrity in the mature animal (6, 29). Accordingly, we soughtto examine the effects of PKC-� inhibition on glomerularendothelial cell and VEGF expression, as well as the moretraditional markers of glomerular injury of albuminuria andglomerulosclerosis. Moreover, since human subjects with dia-betic nephropathy mostly receive treatment with agents thatblock the renin-angiotensin system (RAS), we also sought toexamine the effects of PKC-� inhibition both with and withoutconcomitant angiotensin-converting enzyme (ACE) inhibitortherapy.

METHODS

Animals. Eight-week-old female, homozygous (mRen-2)27 rats (St.Vincent’s Hospital Animal House, Melbourne, Australia) weighing170 � 20 g were assigned to receive either 55 mg/kg of streptozotocin(STZ; Sigma, St. Louis, MO) diluted in 0.1 M citrate buffer, pH 4.5,or citrate buffer alone (nondiabetic) by tail vein injection following anovernight fast. Diabetic rats were then randomized into four groups,receiving either treatment with the selective PKC-� inhibitorruboxistaurin mesylate 10 mg �kg�1 �day�1 (LY333531, Eli Lilly,Indianapolis, IN) in rat chow, the ACE inhibitor perindopril 0.2mg �kg�1 �day�1 in drinking water (Technologie Servier Labora-tories, Paris, France), combination therapy (ruboxistaurin 10mg �kg�1 �day�1 and perindopril 0.2 mg �kg�1 �day�1), or no treat-ment for 12 wk. Treatment commenced within 24 h of STZ or citratebuffer injection. Untreated rats received nondrug control chow (Cer-tified Rodent Diet no. 5002, LabDiet) and untreated drinking water adlibitum. A group of 10 nontransgenic Sprague-Dawley (SD) ratsserved as nondiabetic, normotensive controls receiving nondrug con-trol chow. All animals were housed in a stable environment main-tained at 22 � 1°C with a 12:12-h light-dark cycle commencingat 6 AM.

Each week, rats were weighed and their blood glucose levels weremeasured (Accu-check Advantage II Blood Glucose Monitor, RocheDiagnostics) and only STZ-treated animals with blood glucose �15mmol/l were considered diabetic. Every 4 wk, systolic blood pressure(SBP) was determined in preheated conscious rats via tail-cuff ple-

Address for reprint requests and other correspondence: D. J. Kelly, Dept. ofMedicine, St. Vincent’s Hospital, Fitzroy, Victoria 3065, Australia (e-mail:[email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Renal Physiol 293: F565–F574, 2007.First published May 23, 2007; doi:10.1152/ajprenal.00397.2006.

0363-6127/07 $8.00 Copyright © 2007 the American Physiological Societyhttp://www.ajprenal.org F565

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Fig. 1. In situ hybridization autoradiographs for vascular endothe-lial growth factor (VEGF) mRNA in nondiabetic Ren-2 (A), dia-betic Ren-2 (B), diabetic Ren-2 treated with ruboxistaurin (C),perindopril (D), their combination (E), and nondiabetic, nontrans-genic rats (F). Autoradiographs are presented as pseudocolorizedimages (green, low expression; yellow, moderate expression; red,high expression). Magnification �8.

Table 1. Animal characteristics

N Body Weight, g SBP, mmHg Plasma Glucose, mmol/l HbA1c, % Plasma Creatinine, �mol/l AER, mg/day

Nondiabetic Ren-2 � vehicle 8 289�15 204�14 5.78�0.51 3.16�0.24 55�4 14 �/�1.2Diabetic Ren-2 � vehicle 8 271�5.0* 208�10 25.7�1.94† 10.7�0.35† 70�4* 81 �/�1.3†Diabetic Ren-2 � ruboxistaurin 8 258�4.0* 218�7 28.5�0.84† 11.2�0.34† 60�2* 37 �/�1.2†‡Diabetic Ren-2 � perindopril 8 265�4.0* 147�8† 28.8�0.88† 10.5�0.44† 66�4* 7 �/�1.4‡Diabetic Ren-2 � perindopril �

ruboxistaurin 10 255�4.0* 145�5† 27.6�1.15† 11.0�0.34† 65�2* 2 �/�1.2†§Nondiabetic SD � vehicle 8 560�16†‡§ 137�3† 5.50�0.43 3.62�0.21 49�1 3 �/�1†

Data are expressed as means � SE except for albumin excretion rate (AER) which is expressed as geometric mean �/� tolerance factor. *P 0.05 and†P 0.01 vs. nondiabetic Ren-2. ‡P 0.05 vs. untreated diabetic Ren-2. §P 0.05 vs. monotherapy-treated diabetic Ren-2. SBP, systolic blood pressure;HbA1c, hemoglobin A1c; SD, Sprague-Dawley rats.

F566 PKC-� INHIBITION IN THE DIABETIC NEPHROPATHY

AJP-Renal Physiol • VOL 293 • AUGUST 2007 • www.ajprenal.org

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thysmography (4) using a noninvasive blood pressure (NIBP) con-troller and Powerlab (AD Instruments, New South Wales, Australia).Hemoglobin A1c (HbA1c) was measured by HPLC at the end of thestudy. Diabetic rats received a daily injection of insulin (2–4 U ip;Humulin NPH, Eli Lilly) to reduce mortality and to promote weightgain. Experimental procedures adhered to the guidelines of the Na-tional Health and Medical Research Council of Australia’s Code forthe Care and Use of Animals for Scientific Purposes and wereapproved by the Animal Research Ethics Committee of St. Vincent’sHospital.

Tissue preparation. Rats were anesthetized (60 mg/kg body wt ipNembutal, Boehringer-Ingelheim) and the kidneys were excised,decapsulated, sliced transversely, and fixed in neutral bufferedformalin and paraffin-embedded for subsequent light microscopicevaluation.

In situ hybridization. In situ hybridization was performed using acDNA encoding VEGF-A, as previously reported (10). In situ hybrid-ization was then performed on 4-�m paraffin sections using33P-labeled antisense riboprobe, as previously reported (10). In brief,tissue sections were dewaxed in histosol, hydrated through gradedethanol, and immersed in distilled water. Sections were then washedin 0.1 M PBS, pH 7.4, and hybridized with [33P]-labeled antisense-and sense-specific probes (5 � 105 cpm/25 �l hybridization buffer)which were added to hybridization buffer (300 mM NaCl, 10 mMTris �HCl, pH 7.5, 10 mM Na2HPO4, pH 6.8, 5 mM EDTA, pH 8.0,1 � Denhardt’s solution, 0.8 mg yeast RNA/ml, 50% deionizedformamide, and 10% dextran sulphate), heated to 85°C, and 25 �lwere added to the sections. Coverslips were placed on the sections andthe slides were incubated in a humidified chamber (50% formamide)at 60°C for 14 to 16 h. Slides were then washed in 2� SSC (0.3 MNaCl, 0.33 M Na3C6H5O7 �2H2O) containing 50% formamide at 50°Cto remove the coverslips. The slides were again washed with 2� SSC,50% formamide for a further 1 h at 55°C. Sections were then rinsedthree times in RNase buffer (10 mM Tris �HCl, pH 7.5, 1 mM EDTA,pH 8.0, 0.5 M NaCl) at 37°C and treated with 150 �g RNase A/ml inRNnase buffer for a further 1 h at 37°C and then washed with 2� SSCat 55°C for 45 min. Finally, sections were dehydrated through gradedethanol, air dried, and exposed to Kodak Biomax MR Autoradiogra-phy film for 4 days at room temperature. Slides were coated withIlford K5 emulsion (Ilford, 1:1 with distilled water), stored withdesiccant at room temperature for 21 days, developed in Ilfordphenisol, fixed in Ilford Hypam, and stained with hematoxylin andeosin (H & E).

Quantitative autoradiography. Quantitative in situ hybridization byautoradiographic film densitometry which permits the assessment ofgene expression equivalent to Northern blot analysis was used todetermine the magnitude of gene expression, as previously reportedby our group (11). In brief, film densitometry of autoradiographicimages was performed by computer-assisted image analysis as previ-ously described (2) using a micro computer imaging device (MCID;Imaging Research, St. Catherine’s, Ontario, Canada). With thismethod quantitation of transcript is based on the changes in X-rayfilm density that follows exposure to the radioactive emissions ofradiolabeled VEGF mRNA. In situ autoradiographic images wereplaced on a uniformly illuminating fluorescent light box (NorthernLight Precision Luminator model C60, Image Research, Ontario,Canada) and captured using a video camera (Sony Video CameraModule CCD) connected to an IBM AT computer with a 512 �512 pixel array imaging board with 256 gray levels. In view of thefocal nature of rat glomerular VEGF mRNA, the outline of 20glomeruli/section was defined by interactive tracing for each kid-ney section, as previously described (5). Following appropriatecalibration by constructing a curve of optical density vs. radioac-tivity, quantitation of digitalized autoradiographic images wasperformed using MCID software. Data were expressed as opticaldensity per centimeter squared relative to control kidneys [relativeoptical density (ROD)].

All sections were cut in a uniform manner in the midsaggital plane,hybridized in the same experiment, and analyzed in duplicate underidentical conditions. All analyses were performed with the observermasked to the animal study group.

Immunohistochemistry. Glomerular capillary endothelial cell den-sity was evaluated by immunostaining with JG-12 (Bender MedSystems, Burlingame, CA), a rat endothelial cell-specific monoclonalantibody (14), monoclonal anti-rat endothelial cell antigen-1(RECA-1) antibody (Serotec, Oxford, UK), polyclonal anti-TGF-�(R&D Systems, Minneapolis, MN), and anti-rat VEGF antibodies(R&D Systems). Three-micrometer sections were placed into histosolto remove the paraffin wax, hydrated in graded ethanol, and immersedin tap water before being incubated for 20 min with normal goatserum (NGS) diluted 1:10 with 0.1 M PBS at pH 7.4. Sections werethen incubated for 18 h at 4°C with JG-12 or RECA-1. Sectionsincubated with 1:10 NGS instead of the primary antiserum served asthe negative control. After thorough washing with PBS (3 � 5-minchanges), the sections were flooded with a solution of 5% hydrogenperoxide, rinsed with PBS (2 � 5 min), and incubated with biotinyl-ated goat anti-mouse IgG (Dakopatts, Glostrup, Denmark) diluted1:200 with PBS. Sections were rinsed with PBS (2 � 5 min) andincubated with an avidin-biotin peroxidase complex (Vector, Burlin-game, CA) diluted 1:200 with PBS. Following rinsing with PBS (2 �5 min), sections were incubated with 0.05% diaminobenzidine and0.05% hydrogen peroxide (Pierce, Rockford, IL) in PBS at pH 7.6 for1 to 3 min, rinsed in tap water for 5 min, counterstained in Mayer’shematoxylin, differentiated in Scott’s tap water, dehydrated, cleared,and mounted in Depex.

The magnitude of glomerular JG-12, RECA-1, and TGF-�immunostaining was quantified using image analysis as previouslydescribed (22). Briefly, the glomerulus, considered as the areainternal to and including Bowman’s capsule, was outlined byinteractive tracing, as previously described (16). Images were thencaptured using a BX50 Olympus microscope attached to a FujixHC-2000 digital camera and a Pentium III IBM computer. Thecolor range for immunolabeled cells (brown on immunoperoxi-dase-labeled sections) was selected and image analysis was per-formed using a chromogen-separating technique (22). The propor-tional glomerular area showing positive immunostaining was mea-sured from three sections per rat (n 6/group), providing in excessof 50 glomeruli/treatment group.

To quantify JG-12 immunolabeling in the tubulointerstitium, fiverandom nonoverlapping fields of renal cortex without glomeruli fromsix rats per group were captured, digitized, and quantified as describedabove. All tissues were processed in an identical manner with theobserver masked to study group identity.

Histopathology. Changes in glomerular structure were also as-sessed in a masked protocol in at least 25 randomly selected tissuesections from each group studied. Sections were stained with eitherH & E and periodic acid Schiff’s stain (PAS). In 3-�m kidney

Table 2. Kidney gene expression of VEGF as assessed bydensitometric quantification of in situ hybridizationautoradiographic films

VEGF mRNA, density/cm2

Nondiabetic Ren-2 1.0�0.2Diabetic Ren-2 1.4�0.1*Diabetic Ren-2 � perindopril 0.8�0.2†Diabetic Ren-2 � ruboxistaurin 0.8�0.1†Diabetic Ren-2 � perindopril � ruboxistaurin 0.6�0.1†‡§Nondiabetic SD 0.6�0.1†‡§

Data are shown as means � SE for density/cm2 relative to nondiabeticRen-2 kidneys, arbitrarily assigned a value of 1. *P 0.05 vs. nondiabeticRen-2. †P 0.05 vs. diabetic Ren-2. ‡P 0.05 vs. diabetic Ren-2 rats treatedwith either perindopril or ruboxistaurin. §P 0.05 vs. nondiabetic Ren-2.

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sections stained with PAS, 150 to 200 glomeruli from rats wereexamined in a masked protocol. The extent of sclerosis in eachglomerulus was subjectively graded on a scale of 0 to 4, aspreviously described (17), with grade 0, normal; grade 1, scleroticarea up to 25% (minimal); grade 2, sclerotic area 25–50% (mod-erate); grade 3, sclerotic area 50 –75% (moderate to severe); andgrade 4, sclerotic area 75–100% (severe). A glomeruloscleroticindex (GSI) was then calculated using the formula

GSI � �i0

4

Fi �i

where Fi is the percent of glomeruli in the rat with a given score (i).

Albuminuria. Rats were individually housed in metabolic cages at12 wk, habituated for 2 to 3 h, and urine was collected over 24 h.Animals continued to have free access to tap water and standardlaboratory chow during this period. After 24 h in metabolic cages, analiquot of urine (5 ml) was collected from the 24-h urine sample andstored at �70°C for subsequent analysis of albumin by radioimmu-noassay, as previously performed (17).

Statistics. Data are expressed as means � SE unless otherwisestated. Statistical significance was determined by a two-wayANOVA with a Fisher’s post hoc comparison. Albuminuria wasskew distributed and was analyzed following log transformationand presented as geometric means �/� tolerance factors. Analyses

Fig. 2. Representative photomicrographs of glomeruli labeledwith anti-VEGF antibody showing immunolabeling in podo-cytes from nondiabetic Ren-2 (A), diabetic Ren-2 (B), diabeticRen-2 treated with perindopril (C), ruboxistaurin (D), theircombination (E), and nondiabetic, nontransgenic rats (F). Orig-inal magnification �350.

Fig. 3. Representative photomicrographs of glomeruli labeled with JG-12 (A–F) and RECA-1 antibodies (G–L) showing endothelial cell immunostaining fromnondiabetic Ren-2 (A, G), diabetic Ren-2 (B, H), diabetic Ren-2 treated with ruboxistaurin (C, I), perindopril (D, J), their combination (E, K), and nondiabetic,nontransgenic rats (F, L). Original magnification �350. Quantitation of glomerular endothelial cell density as the proportional area immunostained with JG-12(M) and RECA-1 (N) antibodies. Data are shown as means � SE. *P 0.01 vs. nondiabetic Ren-2 rats. †P 0.05 vs. untreated diabetic Ren-2 rats. ‡P 0.05vs. perindopril or ruboxistaurin monotherapy-treated diabetic Ren-2 rats. §P 0.05 vs. nondiabetic Ren-2 rats.



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were performed using Statview II � Graphics package (AbacusConcepts, Berkeley, CA) on an Apple Macintosh G4 computer(Apple Computer, Cupertino, CA). A P value 0.05 was regardedas statistically significant.

RESULTS

Animal characteristics. In comparison with Ren-2 controlanimals, diabetic rats had reduced body weight that was unaf-fected by any of the assigned treatments (P 0.01). Untreated

diabetic and nondiabetic rats were hypertensive with elevatedSBP. Perindopril treatment reduced SBP while ruboxistaurintreatment did not affect SBP when administered either as singleagent treatment or in combination with perindopril (Table 1).Plasma glucose and HbA1c were elevated to a similar extent in alldiabetic rat groups, irrespective of assigned treatment (Table 1).SD control rats were normotensive and normoglycemic (Table 1).

VEGF expression. In situ hybridization autoradiographyrevealed punctate cortical expression of VEGF mRNA consis-

Fig. 4. Representative photomicrographs of cortical tubuloint-erstitium labeled with JG-12 showing endothelial cell immuno-staining from nondiabetic Ren-2 (A), diabetic Ren-2 (B), dia-betic Ren-2 treated with perindopril (C), ruboxistaurin (D), theircombination (E), and nondiabetic, nontransgenic Sprague-Daw-ley (SD) rats (F). Original magnification �350. G: quantitationof tubulointerstitial endothelial cell density as the proportionalarea immunostained with JG-12 antibody. Data are shown asmeans � SE. *P 0.05 vs. nondiabetic Ren-2 rats. †P 0.05vs. untreated diabetic Ren-2 rats.



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tent with localization of transcript to glomeruli (Fig. 1).Densitometric analysis of autoradiographic images con-firmed a significant increase in VEGF expression in nondi-abetic Ren-2 rats, compared with control SD animals (Table2). Diabetes was associated with a further incremental

increase in VEGF, which was attenuated by both perindopriland ruboxistuarin and to a greater extent with their combi-nation (Fig. 1, Table 2). Immunohistochemistry showedVEGF protein localized to podocytes within the glomerulus(Fig. 2).

Fig. 5. Representative photomicrographs of glomerular trans-forming growth factor (TGF)-� immunostaining from nondia-betic Ren-2 (A), diabetic Ren-2 (B), diabetic Ren-2 treated withperindopril (C), ruboxistaurin (D), their combination (E), andnondiabetic, nontransgenic SD rats (F). Original magnification�350. G: quantitation of glomerular TGF-� expressed as theproportional area immunostained. Data are shown as means �SE. *P 0.05 vs. nondiabetic Ren-2 rats. †P 0.05 vs.untreated diabetic Ren-2 rats.



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Microvascular changes. Intense localization of JG-12 to theendothelial cells of glomerular capillary loops was noted in allgroups. When compared with control SD rats, JG-12 immuno-labeling was reduced in the glomeruli of nondiabetic Ren-2 ratswith further reduction evident in diabetic Ren-2 animals (Fig.3). This decrease was attenuated by both perindopril andruboxistuarin to levels found in nondiabetic Ren-2 rats. Fur-thermore, the combination of perindopril with ruboxistuarin

resulted in a further incremental increase in glomerular JG-12labeling, beyond that of single agent treatment, reaching levelssimilar to those of SD control rats (Fig. 3). GlomerularRECA-1 labeling was similar to that of JG-12.

In addition to the changes within the glomerulus, diabeteswas also associated with a reduction in JG-12 immunolabelingwithin the tubulointerstitium that was incrementally improvedwith perindopril, ruboxistaurin, and their combination (Fig. 4).

Fig. 6. Representative periodic acid Schiff’s-stained sectionsfrom control and diabetic Ren-2 rats treated with ruboxistaurin,perindopril, or their combination. In control (A) rats onlyminimal glomerular thickening was noted, while diabetes (B)was associated with a dramatic glomerulosclerosis. Treatmentof diabetic rats with ruboxistaurin (C) or perindopril (D) wasassociated with a reduction in the glomerulosclerosis. Glomer-uli from combination therapy-treated rats showed only minimalsclerosis (E), similar to nondiabetic SD animals (F). Originalmagnification �350. G: glomerulosclerotic index (GSI) in SDand Ren-2 rats treated with ruboxistaurin, perindopril, or theircombination. Data are shown as means � SE. *P 0.05 vs.nondiabetic Ren-2 rats. †P 0.05 vs. untreated diabeticRen-2 rats. ‡P 0.05 vs. ruboxistaurin-treated and perin-dopril monotherapy-treated diabetic Ren-2 rats. §P 0.05vs. ruboxistaurin � perindopril-treated Ren-2 diabetic rats.



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TGF-�. Glomeruli from untreated diabetic rats displayedprominent immunolabeling for TGF-� when compared withnondiabetic rats, which was substantially diminished by treat-ment with perindopril, ruboxistuarin, and their combination(Fig. 5).

Glomerulosclerosis. Glomerular injury was a prominent fea-ture of diabetic rats, with evidence of both diffuse and nodularglomerulosclerosis (Fig. 6A). These changes were attenuatedby treatment with perindopril and ruboxistaurin (Fig. 6, A andB). When compared with single agent treatment, combinationtherapy with perindopril and ruboxistaurin resulted in a furtherreduction in the extent of glomerulosclerosis (Fig. 6, A and B).However, despite combination treatment, the glomerular struc-ture was not normalized and the extent of sclerosis remainedgreater than in SD control rats.

Albuminuria. Diabetes was associated with an increase inurinary albumin excretion. Treatment with perindopril, andruboxistaurin to a lesser extent, both reduced albuminuria indiabetic Ren-2 rats (Table 1). Combination therapy withboth perindopril and ruboxistaurin resulted in a furtherincremental reduction in albuminuria, beyond that of singleagent treatment, reaching levels similar to those of SDcontrol rats (Table 1).

DISCUSSION

With blockade of the RAS an established therapy for dia-betic nephropathy, new therapies need to show incrementalefficacy. In the present report, the addition of the PKC-�inhibitor ruboxistaurin to background ACE inhibition hadadditive effects in attenuating glomerular injury. These effectsincluded incremental improvements not only in traditionalmeasurements of glomerular structure and function such asglomerulosclerosis and albuminuria but also on glomerularcapillary endothelial cells, recently recognized as a key deter-minant in recovery from injury (24, 25).

Consistent with findings in human diabetic nephropathy(23), we also noted a reduction in glomerular endothelial cellsin the advanced disease that is characteristic of the diabeticRen-2 rat (18). Paradoxically, this endothelial cell loss oc-curred in the setting of increased glomerular VEGF, a growthfactor named for its potent induction of endothelial cell pro-liferation (7). However, while traditionally viewed as pro-proliferative, recent studies showed that these actions arecontext dependent. In particular, Ferrari and colleagues (8)reported that in the setting of high levels of TGF-�, VEGF actstogether with TGF-� to induce endothelial cell apoptosis.Consistent with that report, the present study found that VEGFand TGF-� were both elevated in the glomeruli of diabetic ratsin the setting of endothelial cell loss. Moreover, the aforemen-tioned interaction between VEGF and TGF-� may also con-tribute to the beneficial effects of VEGF blockade (9, 27) aswell as TGF-� inhibition (31) in experimental diabetic ne-phropathy.

Angiotensin II (15) and PKC (21) are potent inducers ofVEGF expression in cultured podocytes. Consistent with thesein vitro findings, we noted that intervention with either aPKC-� or ACE inhibitor in the in vivo context attenuated thediabetes-associated increase in VEGF mRNA, restoring levelsto those of nondiabetic Ren-2 rats. Moreover, the combinationof perindopril and ruboxistaurin led to a further reduction in

VEGF down to levels found in control SD rats. Similar to thefindings in untreated diabetic animals, the relationship betweenVEGF expression and endothelial cell number in the interven-tion groups of this study may at first glance also seem contra-dictory. That is, when compared with untreated animals, thosereceiving drug therapy had lower levels of glomerular VEGFbut a greater number of endothelial cells. We speculate that anumber of mechanisms may account for this paradox. First, inaddition to inducing VEGF expression in podocytes, angioten-sin II and PKC also cause endothelial cell injury. Thus reduc-ing the levels of these injurious agents with perindopril andruboxistaurin would likely lead to endothelial cell preservation,independent of VEGF. Furthermore, ACE and PKC inhibitionboth led to reduced TGF-� expression thereby attenuating theproapoptotic effect of this growth factor combination, as dis-cussed above.

As in previous studies (13, 17, 19), the present report alsoshows that ruboxistaurin, when used as single agent therapy,reduces albuminuria, albeit to a lesser extent than perindoprilmonotherapy. However, the effects of ACE inhibition reflectcontributions from both blood pressure lowering as wellas diminution in angiotensin II production. In contrast,ruboxistaurin’s effects were independent of any change inblood pressure. Furthermore, when used in combination withthe ACE inhibitor perindopril, ruboxistaurin provided a furtherincremental reduction in albuminuria, a key marker and patho-genetic factor in renal disease progression (26). Indeed, ani-mals receiving combination therapy had urinary albumin ex-cretion levels that were similar to those of nondiabetic, nor-motensive, nontransgenic animals.

Histopathologically, diabetic nephropathy is characterizedby glomerulosclerosis, an important predictor of decliningrenal function (23). In the present study, when compared withSD controls, mild glomerulosclerosis was noted in nondiabeticRen-2 rats, consistent with the effects of hypertension and anactive tissue-based RAS on glomerular structure (3). However,as previously reported, diabetes led to a further substantialincrease in the extent of glomerulosclerosis that was attenuatedby both single agent treatment with either perindopril orruboxistaurin (17, 18), and as shown in the present study, afurther, albeit small reduction with combination therapy wasalso noted. However, even this combination of agents failed tonormalize the extent of glomerulosclerosis to levels seen innondiabetic, normotensive, nontransgenic SD rats.

In summary, combination therapy with inhibitors of PKC-�and ACE has additive effects in attenuating endothelial cellloss, reducing glomerular VEGF expression, lowering albu-minuria, and lessening glomerulosclerosis.

ACKNOWLEDGMENTS

The authors thank M. Pacheco, J. Court, S. Glowacka, and L. Di Rago forexpert technical assistance. We also thank Sirirat Reungjui and Richard J.Johnson for assistance with immunohisochemical techniques.

GRANTS

This project was supported by a team grant from the Canadian Institutes ofHealth Research, Juvenile Diabetes Research Foundation, and the NationalHealth and Medical Research Council of Australia. D. Kelly is a recipient ofa Career Development Award from the Juvenile Diabetes Research Founda-tion.



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REFERENCES

1. Aiello LP, Bursell SE, Clermont A, Duh E, Ishii H, Takagi C, Mori F,Ciulla TA, Ways K, Jirousek M, Smith LE, King GL. Vascularendothelial growth factor-induced retinal permeability is mediated byprotein kinase C in vivo and suppressed by an orally effective �-isoform-selective inhibitor. Diabetes 46: 1473–1480, 1997.

2. Baskin DG, Stahl W. Fundamentals of quantitative autoradiography bycomputer densitometry for in situ hybridization with emphasis on 33P.J Histochem Cytochem 41: 1767–1773, 1993.

3. Brenner BM. Hemodynamically mediated glomerular injury and theprogressive nature of kidney disease. Kidney Int 23: 647–655, 1983.

4. Bunag RD. Validation in awake rats of a tail-cuff method for measuringsystolic pressure. J Appl Physiol 34: 279–282, 1973.

5. Cooper ME, Vranes D, Youssef S, Stacker SA, Cox AJ, Rizkalla B,Casley DJ, Bach LA, Kelly DJ, Gilbert RE. Increased renal expressionof vascular endothelial growth factor (VEGF) and its receptor VEGFR-2in experimental diabetes. Diabetes 48: 2229–2239, 1999.

6. Ferrara N. Vascular endothelial growth factor: basic science and clinicalprogress. Endocr Rev 25: 581–611, 2004.

7. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growthfactor. Endocr Rev 18: 4–25, 1997.

8. Ferrari G, Pintucci G, Seghezzi G, Hyman K, Galloway AC, MignattiP. VEGF, a prosurvival factor, acts in concert with TGF-�1 to induceendothelial cell apoptosis. Proc Natl Acad Sci USA 103: 17260–17265,2006.

9. Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, TiltonRG, Rasch R. Amelioration of long-term renal changes in obese type 2diabetic mice by a neutralizing vascular endothelial growth factor anti-body. Diabetes 51: 3090–3094, 2002.

10. Gilbert RE, Vranes D, Berka JL, Kelly DJ, Cox A, Wu LL, StackerSA, Cooper ME. Vascular endothelial growth factor and its receptors incontrol and diabetic rat eyes. Lab Invest 78: 1017–1027, 1998.

11. Gilbert RE, Wu LL, Kelly DJ, Cox A, Wilkinson-Berka JL, JohnstonCI, Cooper ME. Pathological expression of renin and angiotensin II in therenal tubule after subtotal nephrectomy–implications for the pathogenesisof tubulointerstitial fibrosis. Am J Pathol 155: 429–440, 1999.

12. Group TPDS. The effect of ruboxistaurin on visual loss in patients withmoderately severe to very severe nonproliferative diabetic retinopathy:initial results of the protein kinase C � inhibitor diabetic retinopathy study(PKC-DRS) multicenter randomized clinical trial. Diabetes 54: 2188–2197, 2005.

13. Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, BursellSE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, KingGL. Amelioration of vascular dysfunction in diabetic rats by an oral PKC� inhibitor. Science 272: 728–731, 1996.

14. Kang DH, Hughes J, Mazzali M, Schreiner GF, Johnson RJ. Impairedangiogenesis in the remnant kidney model. II. Vascular endothelial growthfactor administration reduces renal fibrosis and stabilizes renal function.J Am Soc Nephrol 12: 1448–1457, 2001.

15. Kang YS, Park YG, Kim BK, Han SY, Jee YH, Han KH, Lee MH,Song HK, Cha DR, Kang SW, Han DS. Angiotensin II stimulates thesynthesis of vascular endothelial growth factor through the p38 mitogenactivated protein kinase pathway in cultured mouse podocytes. J MolEndocrinol 36: 377–388, 2006.

16. Kelly DJ, Hepper C, Wu LL, Cox AJ, Gilbert RE. Vascular endothelialgrowth factor expression and glomerular endothelial cell loss in theremnant kidney model. Nephrol Dial Transplant 18: 1286–1292, 2003.

17. Kelly DJ, Hepper C, Zhang Y, Jaworski K, Wilkinson-Berka JL,Gilbert RE. Protein kinase C � inhibition attenuates the progression ofexperimental diabetic nephropathy in the presence of continued hyperten-sion. Diabetes 52: 512–518, 2003.

18. Kelly DJ, Wilkinson-Berka JL, Allen TJ, Cooper ME, Skinner SL. Anew model of diabetic nephropathy with progressive renal impairment inthe transgenic (mRen-2)27 rat. Kidney Int 54: 343–352, 1998.

19. Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S,Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, KikkawaR. Amelioration of accelerated diabetic mesangial expansion by treatmentwith a PKC � inhibitor in diabetic db/db mice, a rodent model for type 2diabetes. FASEB J 14: 439–447, 2000.

20. Koya D, Jirousek MR, You-Wei L, Ishii H, Kuboki K, King GL.Characterization of protein kinase C � isoform activation on gene expres-sion of transforming growth factor-�, extracellular matrix components,and prostanoids in the glomeruli of diabetic rats. J Clin Invest 100:115–126, 1997.

21. Lee EY, Chung CH, Kim JH, Joung HJ, Hong SY. Antioxidantsameliorate the expression of vascular endothelial growth factor mediatedby protein kinase C in diabetic podocytes. Nephrol Dial Transplant 21:1496–1503, 2006.

22. Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM. Appli-cation of photoshop-based image analysis to quantification of hormonereceptor expression in breast cancer. J Histochem Cytochem 45: 1559–1565, 1997.

23. Mauer S, Steffes M, Ellis E, Sutherland D, Brown D, Goetz F.Structural-functional relationships in diabetic nephropathy. J Clin Invest74: 1143–1155, 1984.

24. Remuzzi A, Gagliardini E, Sangalli F, Bonomelli M, Piccinelli M,Benigni A, Remuzzi G. ACE inhibition reduces glomerulosclerosis andregenerates glomerular tissue in a model of progressive renal disease.Kidney Int 69: 1124–1130, 2006.

25. Remuzzi G, Benigni A, Remuzzi A. Mechanisms of progression andregression of renal lesions of chronic nephropathies and diabetes. J ClinInvest 116: 288–296, 2006.

26. Remuzzi G, Bertani T. Pathophysiology of progressive nephropathies.N Engl J Med 339: 1448–1456, 1998.

27. Sung SH, Ziyadeh FN, Wang A, Pyagay PE, Kanwar YS, Chen S.Blockade of vascular endothelial growth factor signaling amelioratesdiabetic albuminuria in mice. J Am Soc Nephrol 17: 3093–3104, 2006.

28. Tuttle KR, Bakris GL, Toto RD, McGill JB, Hu K, Anderson PW. Theeffect of ruboxistaurin on nephropathy in type 2 diabetes. Diabetes Care28: 2686–2690, 2005.

29. Wakelin SJ, Marson L, Howie SE, Garden J, Lamb JR, Forsythe JL.The role of vascular endothelial growth factor in the kidney in health anddisease. Nephron Physiol 98: p73–p79, 2004.

30. Xia P, Aiello LP, Ishii H, Jiang ZY, Park DJ, Robinson GS, Takagi H,Newsome WP, Jirousek MR, King GL. Characterization of vascularendothelial growth factor’s effect on the activation of protein kinase C, itsisoforms, and endothelial cell growth. J Clin Invest 98: 2018–2026, 1996.

31. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, HongSW, Isono M, Chen S, McGowan TA, Sharma K. Long-term preventionof renal insufficiency, excess matrix gene expression, and glomerularmesangial matrix expansion by treatment with monoclonal antitransform-ing growth factor-� antibody in db/db diabetic mice. Proc Natl Acad SciUSA 97: 8015–8020, 2000.



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