Diminished Loss of Proteoglycans and Lack of in Protein Kinase C-␣–Deficient Diabetic Mice Jan Menne,1,2 Joon-Keun Park,2 Martin Boehne,2 Marlies Elger,2 Carsten Lindschau,2 Torsten Kirsch,2 Matthias Meier,2 Faikah Gueler,2 Annette Fiebeler,3 Ferdinand H. Bahlmann,2 Michael Leitges,4 and Hermann Haller2

Activation of protein kinase C (PKC) isoforms has been implicated in the pathogenesis of diabetic nephropathy. We showed earlier that PKC-␣ is activated in the kid- iabetes affects Ͼ300 million people worldwide; -neys of hyperglycemic animals. We now used PKC-␣؊/؊ 20–40% will develop overt nephropathy. Diabe mice to test the hypothesis that this PKC isoform tes is the most common cause of end-stage mediates streptozotocin-induced diabetic nephropathy. Drenal disease. The earliest clinical sign of ne- We observed that renal and glomerular hypertrophy was phropathy is . Microalbuminuria also ؊ ؊ similar in diabetic wild-type and PKC-␣ / mice. How- heralds impending cardiovascular morbidity and mortality ever, the development of albuminuria was almost absent (1–4). Microalbuminuria predicts overt , which ؊/؊␣ in the diabetic PKC- mice. The hyperglycemia-in- is now believed to actively promote renal insufficiency (5). duced downregulation of the negatively charged base- Therefore, successful treatment of diabetic patients ment membrane heparan sulfate proteoglycan perlecan -؊ ؊ should aim for the prevention or regression of albumin was completely prevented in the PKC-␣ / mice, com- pared with controls. We then asked whether transform- uria. Hyperglycemia seems to cause microalbuminuria in ␤ ␤ diabetic patients (6,7). However, how the metabolic dis- ing growth factor- 1 (TGF- 1) and/or vascular endothelial growth factor (VEGF) is implicated in the turbance causes cellular effects is incompletely under- PKC-␣–mediated changes in the basement membrane. stood. The serine-threonine kinase, protein kinase C The hyperglycemia-induced expression of VEGF165 and (PKC), has been implicated (8,9). PKC consists of at least its receptor VEGF receptor II (flk-1) was ameliorated in 12 different isoforms with distinct cofactor activation, ␤ ؊/؊␣ PKC- mice, whereas expression of TGF- 1 was not expression patterns, and cellular functions. From various affected by the lack of PKC-␣. Our findings indicate that PKC isotypes, PKC-␣,-␤I, -␤II, -␦,-␴, and -␨ were reported two important features of diabetic nephropathy—glo- to be activated by high- concentrations in various merular hypertrophy and albuminuria—are differen- cell culture models and in the diabetic kidney (8,9). In a tially regulated. The glucose-induced albuminuria recent study, we investigated diabetic rats and found that ␣ seems to be mediated by PKC- via downregulation of PKC-␣ is markedly increased in renal glomeruli and inter- proteoglycans in the basement membrane and regula- stitial capillaries as well as in the endothelial cells of larger tion of VEGF expression. Therefore, PKC-␣ is a possible therapeutic target for the prevention of diabetic albu- arteries. Other isoforms were less distinctly affected (10). minuria. 53:2101–2109, 2004 Recently, we showed that glucose-induced activation of PKC-␣ in vitro leads to an increased expression of trans- ␤ ␤ forming growth factor- 1 (TGF- 1) (11). We tested the hypothesis that high-glucose–induced PKC-␣ activation in From 1Phenos, Hannover, Germany; the 2Department of Nephrology, Han- vivo is a mediator of functional and structural alterations nover Medical School, Hannover, Germany; the 3Franz Vollhard Clinic, Charite´, Berlin, Germany; and the 4Max-Planck-Institute for Experimental in experimental diabetic nephropathy. We used PKC-␣– , Hannover, Germany. deficient hyperglycemic mice. Address correspondence and reprint requests to Prof. Dr. Hermann Haller, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. E-mail: [email protected]. RESEARCH DESIGN AND METHODS Received for publication 24 November 2003 and accepted in revised form 30 Experiments were performed with male 129/SV PKC-␣Ϫ/Ϫ mice (12) and April 2004. 129/SV wild-type (WT) animals from the strain that was used to generate the J.M. and J.-K.P. contributed equally to this work. ␣Ϫ/Ϫ H.H. is a member of the advisory panel of Aventis, Bayer, MSD, Sankyo, 129/SV PKC- mice. The animals received a standard diet with free access Novartis, and Lilly, manufacturers of pharmaceuticals related to the treatment to tap water. All procedures were carried out according to guidelines from the of diabetes. He has received honoraria for speaking engagements from American Physiological Society and were approved by local authorities. AstraZeneca, Aventis, Bayer, Baxter, Berlin-Chemie, Boehringer Ingelheim, Seven-week-old weight-matched mice received either 125 mg/kg body wt MSD, Novartis, Roche, Sanofi, and Sankyo. He is also a paid consultant for streptozotocin (Sigma-Aldrich) in 50 mmol/l sodium citrate (pH 4.5; n ϭ 20 per Amgen and Sankyo. Bayer, Berlin-Chemie, MSD, Sanofi, and Sankyo provide group) or sodium citrate buffer (n ϭ 8 per group) intraperitoneally on days 1 funds to H.H.’s laboratory to conduct studies on a new drug to treat diabetic and 4. Glucose levels from tail blood were measured with the Glucometer complications. Elite (Bayer, Leverkusen, Germany) every other day. Animals with glucose A , tuft area; A៮ , average tuft area; GBM, glomerular basement membrane; T T Ͼ PFA, paraformaldehyde; PKC, protein kinase C; TGF, transforming growth levels 16 mmol/l on two consecutive measurements were regarded as hyperglycemic, and glucose measurements were extended to once weekly. factor; VEGF, vacular endothelial growth factor; VEGFR, VEGF receptor; VT, tuft volume; WT, wild type. Animals that were not hyperglycemic within 14 days after the first injection © 2004 by the American Diabetes Association. were excluded. The mice received no within the complete study

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FIG. 1. Nonfasting serum glucose in WT and PKC-␣؊/؊ mice after injection of streptozotocin or Na-citrate (control) intraperitoneally. WT control; Œ, WT diabetic; Ⅺ, PKC-␣؊/؊ control; f, PKC-␣؊/؊ ,‚ diabetic. FIG. 2. Albumin excretion after 2 and 8 weeks. The median is shown as a solid bar. *P 0.05 vs. control; **P 0.01 vs. control; ***P 0.01 vs. period. Ketonuria did not occur (data not shown). After 2 or 8 weeks of < < < WT diabetic. hyperglycemia, the animals were killed according to the following protocol. After anesthesia with Avertin (2.5%), a laparotomy was performed and was collected by puncturing the bladder with a 23-gauge needle. Then, the PaeselϩLorei, Frankfurt, Germany), anti–type IV collagen (catalog no. 1340- abdominal aorta was cannulated with a 23-gauge needle, and the organs were 01; Southern Biotechnology, Birmingham, AL), and anti–type III collagen perfused with lactated Ringer solution. After ligation of the left renal artery, (catalog no. 234189; Calbiochem). For indirect immunofluorescence, nonspe- the left kidney was removed, weighed, and snap frozen in isopentane (Ϫ40°C). cific binding sites were blocked with 10% normal donkey serum (Jackson The right kidney was perfused with 3% paraformaldehyde (PFA) in 0.1 mol/l ImmunoResearch Laboratory, West Grove, PA) for 30 min. Then sections were Soerensen’s phosphate buffer. The right kidney was fixed for an additional incubated with the primary antibody for 1 h. For fluorescent visualization of 20 h in 3% PFA in Soerensen’s phosphate buffer and embedded in paraffin. bound primary antibodies, sections were further incubated with Cy3-conju- Albuminuria. Albumin concentration in spot urine samples was measured gated secondary antibodies (Jackson ImmunoResearch Laboratory) for 1 h. with a commercially available competitive enzyme-linked immunosorbent Specimens were analyzed using a Zeiss Axioplan-2 imaging microscope with assay following the instructions of the manufacturer (Exocell, Philadelphia, the computer program AxioVision 3.0 (Zeiss). Semiquantitative analysis of ␤ PA) and was normalized to urine . VEGF, VEGF-RII, and TGF- 1 expression was done by counting the numbers Histology. Histological and morphometric analysis was carried out on of glomeruli with high, moderate, and weak expression. A total of 40 paraffin sections (3-␮m thickness) cut on a rotation microtome (Microm) and glomeruli/animal were counted. The scoring was done without knowledge of stained with trichrome stain after Masson-Goldner. Glomerular tuft volume the identity of the animal group by two independent observers. was estimated as described before (13,14). In each animal, 50 random Protein chemistry. For Western blotting, the frozen kidneys were pulverized cross-sectional profiles of superficial to midcortical glomeruli (first two rows in liquid nitrogen and resuspended in 2 ml of lysis buffer (20 mmol/l Tris buffer of glomeruli beneath the kidney capsule) were recorded with a digital video [pH 7.5] containing 10 mmol/l glycerophosphate, 2 mmol/l pyrophosphate, 1 camera (Axiocam; Zeiss, Jena, Germany) connected to a light microscope mmol/l sodium fluoride, 1 mmol/l phenylmethylsulfonyl fluoride, 1 g/ml (Axioplan-2; Zeiss), and the glomerular tuft area (A ) was measured using an leupeptin, 1 mmol/l dithiothreitol, and 1 mmol/l EDTA). Homogenates were T ៮ image analysis system (Axiovision; Zeiss). Average tuft area (AT) was used to sonicated for three 20-s bursts on ice and centrifuged at 500g for 1 min to Ϫ calculate an average glomerular tuft volume (VT) for each animal by the remove cell debris. Aliquots of the supernatants were stored at 80°C. The ϭ␤ ϫ ៮ ␤ϭ ␮ formula VT /k (AT)3/2, where 1.38 (shape coefficient for spherical par- protein amount was measured using the Lowry assay. A total of 70 gof ϭ ticles) and k 1.1 (size distribution coefficient) (15). Average VT was cor- protein of each sample was resuspended in loading buffer and run on a 10% rected for the effects of shrinkage (roughly 48%) during paraffin embedding (16). polyacrylamide gel and electrophoretically transferred to nitrocellulose mem- Immunochemistry. Immunohistochemistry was performed on cryostat sec- brane. Membranes were blocked in 5% skim milk and 1% BSA for1hatroom ␤ tions of the frozen kidneys or on paraffin sections using the following primary temperature. Primary antibody against factor TGF- 1 (sc-146; Santa Cruz) was antibodies: anti–PKC-␣ (catalog no. sc-208; Santa Cruz Biotechnologies, Santa applied with gentle rocking overnight at 4°C. After three 10-min washing steps ␤ Cruz, CA) anti–TGF- 1 (catalog no. sc-146; Santa Cruz), anti–vascular endo- with TBST buffer (50 mmol/l Tris HCl [pH 7.5], 150 mmol/l NaCl, 0.01% Tween thelial growth factor (VEGF; catalog no. sc-152; Santa Cruz), VEGF RII 20), incubation with horseradish peroxidase–conjugated goat anti-rabbit (catalog no. sc-504; Santa Cruz), anti-fibronectin (catalog no. 14-109-0568; secondary antibody (Dianova, Hamburg, Germany) was performed for 1 h at

TABLE 1 Body weight, kidney weight, and glomerular volume in WT and PKC-␣Ϫ/Ϫ mice

Body weight Body weight Kidney weight Kidney–to–body Glomerular VT Study group n day 0 (g) week 8 (g) week 8 (mg) weight ratio (%) (␮m3 ϫ 103)1 week 8* WT control 8 21.8 Ϯ 1.1 25.9 Ϯ 1.5 229 Ϯ 18.9 8.8 Ϯ 0.4 226 Ϯ 27 WT diabetic 16 20.7 Ϯ 1.0 22.7 Ϯ 1.6† 257 Ϯ 38.4 11.3 Ϯ 1.4† 452 Ϯ 79† PKC-␣Ϫ/Ϫ control 8 19.5 Ϯ 2.0‡ 24.3 Ϯ 2.5 191 Ϯ 24.0 7.8 Ϯ 0.4 205 Ϯ 27 PKC-␣Ϫ/Ϫ diabetic 14 20.4 Ϯ 1.3 21.9 Ϯ 1.6§ 236 Ϯ 33.4§ 10.8 Ϯ 1.5† 387 Ϯ 115† Data are means Ϯ SD. *Glomerular tuft volume of superficial to midcortical glomeruli (n ϭ 5 animals per group); †P Ͻ 0.01 versus control; ‡P Ͻ 0.05 versus WT control; §P Ͻ 0.05 versus PCK-␣Ϫ/Ϫ control.

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FIG. 3. Light (A and C) and electron (B and D) microscopy of the kidney from diabetic WT (A and B) and PKC-␣؊/؊ (C and D) animals. No difference in mesan- gial expansion was apparent between the two diabetic groups. The electron micrographs showed no signs of endo- thelial cell swelling or podocyte dam- age. A and C: Masson-Goldner trichrome .stain ؋200. B and D: TEM ؋17,000 room temperature. After three additional TBST washes, the membrane was We then analyzed PKC-␣ expression in the kidney by incubated with Renaissance reagent (NEN Life Science, Zaventem, Belgium) immunohistochemistry. In WT animals, PKC-␣ was pre- according to the manufacturer’s instructions and exposed to X-ray film (Kodak). Quantification was done by measuring relative density (Scion dominantly expressed in the glomeruli. Under diabetic Image). conditions, an increased expression of PKC-␣ in glomeruli Electron microscopy. For electron microscopic investigation, additional was observed (data not shown). No glomerular PKC-␣ animals were anesthetized with 2.5% avertin (150 ␮l/10 g body wt) and the expression was found in control or diabetic PKC-␣Ϫ/Ϫ kidneys were fixed by perfusion via the abdominal aorta. After flushing of the vasculature with Ringer’s saline, the animals were perfused with fixative mice (data not shown). containing 1.5% PFA and 1.5% glutaraldehyde in 0.1 cacodylate buffer (pH 7.4) During the study period, a significant increase in body for 5 min. Tissue slices were postfixed in the same solution for 2 h and with weight was observed (Table 1). After 8 weeks of diabetes, 1% OsO in cacodylate buffer for 1 h and embedded in Epon. Thin sections (70 4 the animals were killed and the kidneys removed and nm) were stained with uranyl acetate and lead citrate and examined in a Zeiss EM 10 electron microscope. analyzed further. A significant increase in the kidney weight TaqMan PCR. For real-time qPCR, 2 ␮g of DNase-treated total RNA was and the kidney–to–body weight ratio during hyperglyce- reverse transcribed using a mix of random hexamers and oligo(dT)12–15 mia was observed in WT and PKC-␣Ϫ/Ϫ mice. The kidney– oligonucleotides (Stratagene, Amsterdam, Netherlands) and Superscript II to–body weight ratio increased significantly from 8.8 Ϯ Reverse Transcriptase (Invitrogen, Carlsbad, CA). qPCR was performed on an Ϯ Ͻ SDS 7700 system (Applied Biosystems, Darmstadt, Germany) using 10 ng of 0.4 to 11.3 1.4% (P 0.01) in the WT animals and from Ϫ/Ϫ transcribed RNA, Rox dye as internal control (Invitrogen), FastStart Taq 7.8 Ϯ 0.4 to 10.8 Ϯ 1.5% (P Ͻ 0.01) in the PKC-␣ mice Polymerase (Roche Diagnostics, Mannheim, Germany), and gene-specific (Table 1). primers in combination with SYBR-Green chemistry (Molecular Probes, In contrast to our observations on renal hypertrophy, Eugene, OR). PCR amplification was carried out for 10 min at 95°C, 40 cycles ␣ for10sat95°C, and 1 min at 60°C. Specificity of the amplification product was we found a significant effect of PKC- deficiency on albu- verified by melting curve analysis. For each group, three RNA samples were min excretion. After 2 weeks of hyperglycemia, a slight used. For normalization of the samples, distribution of 18S ribosomal RNA increase in albumin excretion was present in the WT ani- was measured. Quantification was carried out using qgene software. Primers mals, whereas no albuminuria was observed in PKC-␣Ϫ/Ϫ were designed using Primer Express software (Applied Biosystems) based on mice (Fig. 2). After 8 weeks of diabetes, a further increase Unigene clusters and GenBank accession numbers, respectively (given in parentheses). The primer sequences are the following: 5Ј-3Ј direction, r18S was observed in the WT mice, whereas only a few of the Ϫ/Ϫ (NC_001665) ACATCCAAGGAAGGCAGCAG (primer 1) and TTTTCGTCACT PKC-␣ animals showed slight albuminuria (Fig. 2). The ACCTCCCCG (primer 2); and for Perlecan (Mm.273662) GGGAGGCCCGTCT albumin–to–creatinine ratio increased from a median of TGTCT (primer 1) and GTGTTGACCGCCACATTAGGA (primer 2). 7.38 to 21.43 g/mol in the diabetic WT animals (P Ͻ 0.01). Statistics. Data are shown as mean Ϯ SD. The data were compared by ANOVA, and the Bonferroni multiple comparison test was used as posttest. In contrast, the albumin–to–creatinine ratio remained sta- The Mann-Whitney U test was used as posttest when analyzing the albumin- ble, with a median of 8.03 g/mol in control and 10.79 g/mol Ϫ/Ϫ uria. Significant differences were accepted when P Ͻ 0.05. Data analysis was in hyperglycemic PKC-␣ mice. performed using InStat. The lack of albuminuria in hyperglycemic PKC-␣Ϫ/Ϫ mice indicates that PKC-␣ is involved in the diabetes- RESULTS induced changes of the glomerular basement membrane Hyperglycemia was induced in 7-week-old mice by intra- (GBM) or the slit membrane. We therefore analyzed the peritoneal injection of streptozotocin on days 1 and 4. glomeruli using histology and electron microscopy (Fig. Approximately 80% of the animals were diabetic 14 days 3). No apparent difference in glomerular volume and after the first injection. The hyperglycemia persisted in the mesangial expansion was observed between the two diabetic animals during the 8-week study period (Fig. 1). diabetic groups (Fig. 3A and C). Hyperglycemia induced

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FIG. 4. Immunohistochemistry of glomerular perlecan (expression in WT (A and B) and PKC-␣؊/؊ (C and D mice. Under hyperglycemic conditions, no or only a little perlecan expression was detectable in WT mice (B) compared with both nondiabetic control animal groups (A and C). In contrast, in hyperglycemic PKC- .(؊/؊ animals, perlecan expression was not changed (D␣ ␮m. E: TaqMan PCR results 50 ؍ Cryostat sections. Bar for perlecan expression in whole kidney extracts. *P < 0.05 versus WT control.

␣Ϫ/Ϫ a significant increase of the mean glomerular VT from PKC- mice as compared with diabetic WT animals 226 Ϯ 27 to 452 Ϯ 79 ␮m3 ϫ 103 (P Ͻ 0.01) in WT mice (Fig. 3B and D). and from 205 Ϯ 27 to 387 Ϯ 115 ␮m3 ϫ 103 (P Ͻ 0.01) in We next investigated the molecular composition of the PKC-␣Ϫ/Ϫ mice (Table 1). The electron micrographs GBM using immunohistochemistry. We first assessed the showed no signs of endothelial cell swelling or podocyte expression of perlecan, a major heparan sulfate proteogly- damage. Podocytes displayed well-developed foot pro- can of the GBM. As shown in Fig. 4A and C, there was a cesses without an obvious difference between both strong expression of perlecan in nondiabetic animals. The diabetic groups. Inspection of the GBM revealed a wide expression of perlecan was greatly reduced in the glomer- variation of GBM thickening in the diabetic animals. uli of diabetic WT animals (Fig. 4B). This diabetes-induced Thickened GBM segments with formation of subepithe- loss of glomerular perlecan expression was completely lial “humps” seemed to be less frequent in diabetic prevented in diabetic PKC-␣Ϫ/Ϫ mice (Fig. 4D). In addi-

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␤ ؊/؊␣ ␤ FIG. 5. Immunohistochemistry of TGF- 1 expression in WT (A and B) and PKC- (C and D) mice. Semiquantitative analysis of TGF- 1 ؊/؊␣ ␤ expression was performed (E). TGF- 1 expression was defined as strong, moderate, or weak. In WT (B) and PKC- (D) mice, hyperglycemia ␤ resulted in a significantly higher number of glomeruli with a strong expression of TGF- 1 expression than in control animals (P < 0.05). Paraffin ␤ ␮ ؍ sections. Bar 50 m. Also by Western blot analysis, an increase in TGF- 1 expression was detectable in both hyperglycemic groups (F). However, these differences were not significant. *P < 0.01. tion, we analyzed the perlecan mRNA expression. We collagen, and fibronectin showed an increase under dia- found a significant reduction of perlecan mRNA levels in betic conditions in both groups (data not shown). whole-kidney extracts from diabetic versus control WT To elucidate the possible mediators between PKC-␣ and mice (0.08 Ϯ 0.14 vs. 1.0 Ϯ 0.24 relative units; P Ͻ 0.05) perlecan expression in the GBM, we analyzed the role of ␤ (Fig. 4E). No reduction of perlecan mRNA levels was TGF- 1 and VEGF and VEGF receptor II (VEGFR-II). The ␣Ϫ/Ϫ Ϯ ␤ observed in diabetic versus control PKC- mice (1.43 results for TGF- 1 are shown in Fig. 5. Immunohistochem- 0.59 vs. 0.76 Ϯ 0.26 relative units). Further analysis of the istry showed a comparable increase of the glomerular ␤ extracellular matrix molecules type III collagen, type IV TGF- 1 expression in hyperglycemia with no significant

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FIG. 6. Immunohistochemistry (A–D) and semiquanti- tative analysis (E) of VEGF expression in WT (A and B) and PKC-␣؊/؊ (C and D) mice. The number of glomeruli with a strong VEGF expression was en- hanced in both diabetic WT mice (B) and diabetic PKC-␣؊/؊ mice (D) compared with control PKC-␣؊/؊ mice (A and C). However, this increase was amelio- ;rated in diabetic PKC-␣؊/؊ mice (compare B and D .␮m. *P < 0.01 50 ؍ P < 0.01). Paraffin sections. Bar difference between WT and PKC-␣Ϫ/Ϫ mice (Fig. 5A–E). Under hyperglycemic conditions, a significant increase of ␤ Furthermore, no significant difference in the TGF- 1 ex- the VEGFR-II expression was observed in WT animals pression between WT and PKC-␣Ϫ/Ϫ diabetic mice was compared with nondiabetic mice (Fig. 7). This increase detected by Western blot (Fig. 5F). was significantly reduced but not abolished in PKC-␣Ϫ/Ϫ To elucidate the possible role of VEGF, we analyzed the mice (P Ͻ 0.01). These results indicate that the expression glomerular expression of VEGF165 by immunohistochem- of VEGF and VEGFR-II is regulated by PKC-␣. istry. These results are shown in Fig. 6. Only a weak expression of VEGF165, mainly located in podocytes, was observed in nondiabetic control animals of both groups. DISCUSSION Under hyperglycemic conditions, a significant increase of The most important finding of our study is that diabetic VEGF was observed in the WT animals. In contrast, this PKC-␣Ϫ/Ϫ animals were protected from albuminuria, increase was significantly reduced but not completely whereas WT mice were not. By electron microscopy, we abolished in PKC-␣Ϫ/Ϫ mice (Fig. 6). These results with found no convincing evidence that a different basement VEGF165 prompted us to investigate the influence of membrane thickness or deterioration of podocyte struc- PKC-␣ expression on the diabetes-induced expression of ture explained the finding. However, the hyperglycemia- VEGFR-II. These results are shown in Fig. 7. The VEGFR-II induced downregulation of the negatively charged heparan expression was weak in the nondiabetic animals, as shown sulfate proteoglycan perlecan was completely prevented by immunohistochemistry. The fluorescence signal was in the PKC-␣Ϫ/Ϫ mice. The glucose-induced albuminuria mainly located in the endothelial and mesangial cells. seems to be mediated by the PKC isoform ␣ via downregu-

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FIG. 7. Immunohistochemistry (A–D) and semiquantita- tive analysis (E) of VEGFR-II expression in WT (A and -B) and PKC-␣؊/؊ (C and D) mice. The number of glo meruli with a strong expression of VEGFR-II was signif- icantly higher in diabetic WT mice (B) than in diabetic > ␮m. *P 50 ؍ PKC-␣؊/؊ mice (D). Paraffin sections. Bar 0.01. lation of heparan sulfate proteoglycans in the GBM. It is (19). This hypothesis is based on immunohistochemical interesting that the lack of albuminuria in the diabetic findings from diabetic patients with early nephropathy. PKC-␣Ϫ/Ϫ animals is dissociated from the diabetes-in- These patients have a reduced amount of heparan sulfate duced renal and glomerular hypertrophy. We then asked in the GBM (20,21). Perlecan is the most common heparan ␤ whether expression of TGF- 1 and/or VEGF is implicated sulfate proteoglycan in the body (22,23). Perlecan was not in the PKC-␣–mediated changes of the basement mem- detectable by immunohistochemistry in WT diabetic ani- brane. The hyperglycemia-induced glomerular expression mals; however, downregulation was prevented in the of VEGF165 and its receptor flk-1 was ameliorated in PKC-␣Ϫ/Ϫ diabetic mice. Therefore, we assume that the ␣Ϫ/Ϫ ␤ ␣Ϫ/Ϫ PKC- mice, whereas expression of TGF- 1 was not missing loss of negative charges in the PKC- diabetic affected by the lack of PKC-␣. Our findings indicate that mice might be responsible for the lack of albuminuria in two important features of diabetic nephropathy—glomer- these mice. It has been demonstrated that perlecan core ular hypertrophy and albuminuria—are differentially reg- protein is downregulated in the of diabetic patients ulated. In addition, glucose-induced glomerular expression (24). In the retinal basement membrane of diabetic pa- of VEGF in diabetes may be mediated by PKC-␣. tients, a diminished or unaltered perlecan expression has It is assumed that the loss of negative charges in the been reported (25,26). Thus far, no report exists about the basement membrane might be causative for the develop- expression of perlecan in the kidney of humans or animals ment of albuminuria (17,18). According to the Steno with diabetes. However, incubation of glomerular epithe- hypothesis, diabetic complications result from the loss of lial cells with 30 mmol/l glucose resulted in a reduction of heparan sulfate proteoglycans in the basement membrane the heparan sulfate proteoglycan synthesis (27). In diabe-

DIABETES, VOL. 53, AUGUST 2004 2107 PKC-␣ AND ALBUMINURIA tes, heparan sulfate proteoglycan side chains are dimin- tend the findings by the fact that PKC-␣ signaling cascade ␤ ished or altered (28,29). It has been suggested that PKC is not involved in the regulation of TGF- 1 in the diabetic isoforms may be involved in the regulation of heparan milieu. Furthermore, our findings suggest that hyperglyce- sulfate production (30). Our data suggest that PKC-␣ plays mia induces the pathological changes in the kidney by a key role in the synthesis of heparan sulfate proteoglycan activation of different mediator systems whereby VEGF is perlecan mRNA expression. Further studies will be need- associated with endothelial and podocyte function, where- ␤ ed to elucidate the molecular pathway of this interaction. as TGF- 1 plays a role in mesangial expansion. It will also be necessary to analyze the expression of Inoguchi et al. (45) observed an increased expression of heparan sulfate synthesis and other heparan sulfate pro- the PKC isoform ␤II in tissues from diabetic animals and teoglycans such as agrin (31). It has been suggested that hypothesized that this PKC isoform is primarily responsi- perlecan plays a role in the development of arterioscle- ble for the glucose-induced effects in diabetes. Further rotic diseases. Therefore, PKC-␣ might be involved in the evidence for this hypothesis stems from reports that development of other diabetes-related complications. Fur- a specific PKC-␤ inhibitor ameliorates hyperglycemia- ther studies are necessary to clarify this topic. induced changes in the kidney. In rats with streptozotocin- Our study suggests a role for PKC-␣ in the regulation of induced and db/db mice with type 2 VEGF165 and its receptor in diabetes. VEGF is a cytokine diabetes, the development of albuminuria and mesangial that potentially induces angiogenesis, endothelial perme- expansion could be prevented by the administration of the aforementioned inhibitor (46–48). However, it is interest- ability, and endothelium-dependent vasodilation and rep- ␤ ␤ resents a key factor for the development of (proliferative) ing that only PKC- I but not PKC- II is expressed in diabetic retinopathy (32). However, the role of VEGF in glomerular cells (49,50). From the existing data, it can be concluded that PKC-␣ and -␤ are both important in the the development of diabetic nephropathy has been less development of diabetic complications. We assume that thoroughly investigated. VEGF and its receptor are ex- PKC-␤ is more important in the upregulation of TGF-␤ pressed in the healthy human kidney (33). VEGF expres- 1 and for the development of glomerular hypertrophy under sion is increased in patients with diabetic nephropathy diabetic conditions, whereas PKC-␣ seems to play a criti- (34,35), and patients with type 1 diabetes and diabetic cal role in the development of albuminuria by perpetuating nephropathy have increased plasma levels of VEGF (34). the loss of the negatively charged heparan sulfate and Furthermore, blockade of VEGF with systemic antibody upregulation of glomerular VEGF expression. As the struc- administration decreased albuminuria in rats with strep- ture of PKC-␣ and -␤ are very similar, it is conceivable tozotocin-induced hyperglycemia or db/db mice with type that inhibition of one of the two isoforms will lead to a 2 diabetes (36,37). Various cell culture studies have proved compensatory rise in the activity of the other isoform. that high-glucose–induced stimulation of VEGF expres- Therefore, blockade of both PKC isoforms may be neces- sion is regulated via a PKC-dependent pathway (35,38,39). sary to achieve a therapeutic effect. However, the specific PKC isoform involved in this cas- cade has not been identified yet (38,40,41). Our data suggest that the PKC-␣ isotype is involved in the regulation ACKNOWLEDGMENTS of VEGF and its receptor (42). Therefore, our finding of a This work was supported by a grant-in-aid from the reduced glomerular expression of VEGF and VEGFR-II in Deutsche Forschungsgemeinschaft to H.H. (Ha 1388-7/1) diabetic PKC-␣Ϫ/Ϫ compared with WT mice could be one and from the EFSD (European Foundation for the Study of possible explanation for the observed difference in the Diabetes)-Servier grant for vascular complications of type development of albuminuria. 2 diabetes to M.M. 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