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Deficient Diabetic Mice Diminished Loss of Proteoglycans and Lack of Albuminuria 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. 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 proteinuria, 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-glucose 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. Diabetes 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-␣– Endocrinology, 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 insulin within the complete study DIABETES, VOL. 53, AUGUST 2004 2101 PKC-␣ AND ALBUMINURIA 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 urine 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 creatinine. 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
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