Protein Kinase C ß Inhibition Attenuates the Progression of Experimental Diabetic Nephropathy in the Presence of Continued Hypertension
1 Department of Medicine, University of Melbourne, St. Vincent’s Hospital, Fitzroy, Victoria, Australia&6'(0d;, 百拇医药
2 Department of Physiology, University of Melbourne, Parkville, Victoria, Australia&6'(0d;, 百拇医药
3 St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia&6'(0d;, 百拇医药
ABSTRACT&6'(0d;, 百拇医药
In addition to hyperglycemia, hypertension and the renin-angiotensin system have been consistently implicated in the pathogenesis of diabetic nephropathy. Each of these pathogenetic factors may induce changes in cellular function by a common intracellular signaling pathway, the activation of protein kinase C (PKC) ß. The present study thus sought to determine the in vivo effect of PKC ß inhibition in experimental diabetic nephropathy in the setting of continued hyperglycemia, hypertension, and activation of the RAS. Studies were conducted in the (mRen-2)27 rat, a rodent that is transgenic for the entire mouse renin gene (Ren-2) and develops many of the structural, functional, and molecular characteristics of human diabetic nephropathy when experimental diabetes is induced with streptozotocin (STZ). Six-week-old female Ren-2 rats received an injection of STZ or vehicle and were maintained for 6 months. Within 24 h, diabetic rats were further randomized to receive treatment with the specific PKC ß inhibitor, LY333531, admixed in diet (10 mg · kg-1 · d-1) or no treatment (n = 8/group). Diabetic rats developed albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis with a concomitant increase in transforming growth factor-ß (TGF-ß). Western blot analysis demonstrated increased PKC ß in diabetic animals, localized by immunofluorescence to the glomerular mesangium. In vivo inhibition of PKC ß with LY333531 led to a reduction in albuminuria, structural injury, and TGF-ß expression, despite continued hypertension and hyperglycemia.
Diabetic nephropathy is a leading cause of end-stage renal disease throughout much of the world (1). In addition to elevated blood glucose, hypertension and inappropriate activation of the renin-angiotensin system (RAS) have been identified as contributing to the development and progression of diabetic renal disease. Indeed, clinical studies not only have demonstrated a key role for good glycemic control in reducing the development and progression of diabetic nephropathy (2,3) but also have shown the importance of blood pressure reduction (4,5) and blockade of the RAS (6–8) in slowing the progression of renal dysfunction in both type 1 and type 2 diabetes. However, despite these advances, the incidence of end-stage renal disease as a result of diabetes continues to rise in the United States and other countries where diabetes is an important problem.?jx, 百拇医药
In addition to their frequent coexistence in patients with diabetic renal disease, hyperglycemia and hypertension may induce changes in cellular function by common intracellular signaling pathways (9). Indeed, several components of the diabetic milieu, implicated in the pathogenesis of diabetic nephropathy, induce activation of protein kinase C (PKC) ß. These include both glucose-dependent pathways such as the formation of advanced glycation end products and hyperglycemia per se in addition to glucose-independent mechanisms such as hypertension and activation of the RAS (10–13).
Previous studies have shown that not only is PKC ß the major isoform of PKC activated by hyperglycemia in the diabetic kidney (14), but also its inhibition of the ß isoform reduces albuminuria (15) and mesangial expansion (16). However, whether this therapeutic strategy is also effective in the setting of coexistent hypertension and inappropriate activation of the RAS (17), as in human diabetic nephropathy, remains uncertain. Furthermore, the effects of PKC ß inhibition on tubulointerstitial injury in diabetes, a major histological feature of the disease (18), also has not been reported.w@6?\*, http://www.100md.com
The present study thus sought to examine the effects of PKC ß inhibition in the diabetic (mRen-2)27 rat model of experimental diabetic nephropathy. This transgenic animal has the entire mouse renin gene (Ren-2) inserted into the genome of a Sprague-Dawley rat (19) and develops hypertension and many of the structural and functional characteristics of human diabetic nephropathy when diabetes is induced with streptozotocin (STZ) (20).
RESEARCH DESIGN AND METHODS;lrn, 百拇医药
Animals.;lrn, 百拇医药
Six-week-old female, heterozygous (mRen-2)27 rats that weighed 125 ± 5 g were randomized to receive either 55 mg/kg STZ (Sigma, St. Louis, MO) diluted in 0.1 mol/l citrate buffer (pH 4.5) or citrate buffer alone (nondiabetic) by tail-vein injection after an overnight fast. Diabetic Ren-2 (n = 8 per group) were treated with the PKC ß inhibitor LY333531 (Eli Lilly and Co., Indianapolis, IN) 10 mg · kg-1 · d-1, milled into rat food for 6 months after STZ or vehicle. Treatment commenced within 24 h of STZ injection. All rats were housed in a stable environment (maintained at 22 ± 1°C with a 12-h light/dark cycle) and allowed free access to tap water. Each week, rats were weighed and blood glucose was determined using an AMES glucometer (Bayer Diagnostics, Melbourne, Australia). Every 4 weeks, systolic blood pressure (SBP) was recorded in preheated conscious rats by tail-cuff plethysmography (21). Diabetic rats received a daily injection of insulin (2–4 units intraperitoneally; Ultratard, Novo Nordisk, Bagsraerd, Denmark) to promote weight gain and to reduce mortality. Experimental procedures adhered to the guidelines of the National Health and Medical Research Council of Australia’s Code for the Care and Use of Animals for Scientific Purposes and were approved by the Bioethics Committee of the University of Melbourne.
Albuminuria.moy-%ew, 百拇医药
Rats were individually housed in metabolic cages every 2 months and habituated for 2–3 h, and measurements of urinary albumin excretion were obtained over 24 h. Animals continued to have free access to tap water and standard laboratory diet during this period. After 24 h in metabolic cages, an aliquot of urine (5 ml) was collected from the 24-h urine sample and stored at -70°C for subsequent analysis of albumin. Albuminuria was determined by a double-antibody radioimmunoassay, as previously performed in our laboratories (22).moy-%ew, 百拇医药
Tissue preparation.moy-%ew, 百拇医药
Rats were anesthetized (Nembutal 60 mg/kg body wt intraperitoneally; Boehringer-Ingelheim, North Ryde, Australia), and the abdominal aorta was cannulated with an 18-G needle. Perfusion-exsanguination commenced at SBP (180–220 mmHg) via the abdominal aorta with 0.1 mol/l PBS (pH 7.4; 20–50 ml) to remove circulating blood, and the inferior vena cava adjacent to the renal vein was simultaneously severed allowing free flow of the perfusate. After clearance of circulating blood, 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4) was perfused for another 5 min (100–200 ml of fixative) to fix the tissues. Kidneys were removed from the animal, decapsulated, sliced transversely, and either frozen-embedded in OCT (Tissue-Tek; Miles, Elkhart, IN) for immunofluorescence or paraffin-embedded for light microscopic evaluation.
Histopathology.wk{77, http://www.100md.com
Changes in kidney structure were assessed in a masked protocol in at least 25 randomly selected tissue sections from each group studied. Sections were stained with either Mayer’s hematoxylin and eosin to examine cell structure, periodic acid Schiff to identify changes in basement membrane architecture and glycogen deposition, or Masson’s modified trichrome to demonstrate collagen matrix (23).wk{77, http://www.100md.com
Glomerulosclerotic index.wk{77, http://www.100md.com
In 3-µm kidney sections stained with periodic acid Schiff, 150–200 glomeruli from rats were examined. The degree of sclerosis in each glomerulus was graded on a scale of 0 to 4, as previously described (24), as follows: grade 0, normal; grade 1, sclerotic area up to 25% (minimal); grade 2, sclerotic area 25–50% (moderate); grade 3, sclerotic area 50–75% (moderate to severe); and grade 4, sclerotic area 75–100% (severe). A glomerulosclerotic index was then calculated using the formula: 4 GSI = {Sigma} Fi (i) i = 0
where Fi is the percentage of glomeruli in the rat with a given score (i).-.u(*26, http://www.100md.com
Immunofluorescence.-.u(*26, http://www.100md.com
Six-micron frozen sections were postfixed in 4% paraformaldehyde for 20 min and incubated for 20 min with normal goat serum (NGS) diluted 1:10 with 0.1 mol/l PBS at pH 7.4. Sections were then incubated for 18 h at 4°C with specific mouse anti-rat monoclonal PKC ß antibody (1:250; Zymed, San Francisco, CA). Sections incubated with 1:10 NGS instead of the primary antiserum served as the negative control. After thorough washing with PBS (3 x 5 min changes), the sections were incubated with FITC-labeled goat anti-mouse IgG (Dakopatts, Glostrup, Denmark) diluted 1:200 with PBS for 1 h at room temperature. Sections were rinsed with PBS (2 x 5 min), rinsed in tap water for 5 min, and mounted for microscope viewing.-.u(*26, http://www.100md.com
Immunohistochemistry.-.u(*26, http://www.100md.com
Three-micron sections were placed into histosol, hydrated in graded ethanol, and immersed in tap water before being incubated for 20 min with NGS diluted 1:10 with 0.1 mol/l PBS at pH 7.4. Sections were then incubated for 18 h at 4°C with specific primary to transforming growth factor-ß (TGF-ß) (1:250, Zymed). Sections incubated with 1:10 NGS instead of the primary antiserum served as the negative control. After thorough washing with PBS (3 x 5 min changes), the sections were flooded with a solution of 5% hydrogen peroxide, rinsed with PBS (2 x 5 min), and incubated with biotinylated goat anti-rabbit IgG (Dakopatts) diluted 1:200 with PBS. Sections were rinsed with PBS (2 x 5 min) and incubated with an avidin-biotin peroxidase complex (Vector, Burlingame, CA) diluted 1:200 with PBS. After rinsing with PBS (2 x 5 min), sections were incubated with 0.05% diaminobenzidine and 0.05% hydrogen peroxide (Pierce, Rockford, IL) in PBS at pH 7.6 for 1–3 min, rinsed in tap water for 5 min, counterstained in Mayer’s hematoxylin, differentiated in Scott’s tap water, dehydrated, cleared, and mounted in Depex (20).
Quantification of matrix deposition and immunohistochemistry.-#k[, http://www.100md.com
The accumulation of matrix within the tubulointerstitium was assessed on Masson’s trichrome-stained sections using computer-assisted image analysis, as previously reported (25,26). Briefly, five random nonoverlapping fields from six rats per group were captured and digitized using a BX50 microscope attached to a Fujix HC5000 digital camera, then loaded onto a Pentium III IBM computer. An area of blue on a trichrome-stained section or brown on TGF-ß–stained sections was selected for its color range, and the proportional area of tissue with this range of color was then quantified. Calculation of the proportional area was then determined using image analysis (AIS, Analytical Imaging Station Version 6.0, ON, Canada) for quantification of histological sections.-#k[, http://www.100md.com
Western blot analysis.-#k[, http://www.100md.com
Protein concentration of whole kidney samples was determined by the Bradford assay, using BSA as a standard. Samples containing 10 µg of protein were diluted to 30 µl in loading buffer, denatured for 5 min at 95°C, and separated by electrophoresis in 12.5% SDS-PAGE gels. After electroblotting onto Hybond transfer membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.), gel loading and transfer efficiency were assessed by staining the blot with 0.1% Ponceau’s Solution (Sigma Chemical Co.). Blots were blocked overnight at 4°C in 5% wt/vol nonfat dry milk before incubation with the primary antibody for 60 min at room temperature. Anti–PKC ß antibody (dilution 1:750; Zymed) was used to demonstrate the 81-kDa protein band. After the blot was incubated with a horseradish peroxidase–conjugated anti-mouse secondary antibody (dilution 1:1,000; Amersham) for 60 min at room temperature, antibody binding was visualized by enhanced chemiluminescence detection reagents. The bands of the resulting autoradiographs were compared for optical density using ImageQuaNT software (Version 4.2a, Build 13, Amersham). Relative quantities were compared normalized to control values, arbitrarily assigned as 100%.
Statistics.3kv'a{%, http://www.100md.com
Data are expressed as mean ± SE unless otherwise stated. Statistical significance was determined by a two-way ANOVA with a Fisher’s post hoc comparison. Albuminuria was analyzed using log-transformed data and represented as geometric means x/÷ tolerance factors. Analyses were performed using Statview II + Graphics package (Abacus Concepts, Berkeley, CA) on an Apple Macintosh G4 computer (Apple Computer, Cupertino, CA). A P < 0.05 was regarded as statistically significant.3kv'a{%, http://www.100md.com
RESULTS3kv'a{%, http://www.100md.com
Renal functional and biochemical studies.3kv'a{%, http://www.100md.com
In comparison with control animals, diabetic rats had reduced body weight, which was unaffected by treatment (P < 0.01). All rats, both diabetic and nondiabetic, were hypertensive with elevated SBP that was not altered by LY333531 treatment (P < 0.01; ). Plasma glucose was elevated to a similar extent in treated and untreated diabetic rat groups (P < 0.01; ). Diabetes was associated with an increase in urinary albumin excretion when compared with controls. Treatment with LY333531 reduced albumin excretion in diabetic rats.
fig.ommtted8+7:', 百拇医药
Body weight, kidney weight, SBP, and plasma glucose of control and diabetic transgenic (mRen-2)27 rats treated for 6 months with LY3335318+7:', 百拇医药
Renal structure.8+7:', 百拇医药
Glomerular injury was a prominent feature of diabetic rats, with evidence of both diffuse and nodular glomerulosclerosis . These changes were significantly attenuated by treatment with LY333531 . In addition to these glomerular changes, tubulointerstitial pathology was present in diabetic rats . These changes were also substantially reduced in diabetic rats treated with LY333531 .8+7:', 百拇医药
fig.ommtted8+7:', 百拇医药
Representative PAS-stained sections from control diabetic and diabetic LY333531-treated Ren-2 rats. In control rats (A), there is only minimal glomerulosclerosis, whereas diabetes is associated with a dramatic increase in glomerulosclerosis (B). Treatment of diabetic rats with the PKC ß inhibitor LY333531 (C) was associated with a reduction in the number and extent of glomerusclerosis. Magnification x350.
fig.ommtted2n, http://www.100md.com
Glomerulosclerosis expressed as glomerulosclerotic index (top) and tubulointerstitial fibrosis expressed as percentage area occupied by extracellular matrix (blue) on trichrome-stained sections (bottom) in control, diabetic, and diabetic + LY333531–treated Ren-2 rats. *P < 0.01 diabetic vs. control; {dagger} P < 0.01 diabetic + LY333531 vs. untreated diabetic.2n, http://www.100md.com
fig.ommtted2n, http://www.100md.com
Representative Masson’s trichrome-stained sections from control, diabetic, and diabetic + LY333531–treated Ren-2 rats. In control rats (A), there is sparse collagen (blue staining) within the interstitium, whereas diabetes is associated with substantial fibrosis (B). Treatment of diabetic rats with the PKC ß inhibitor LY333531 (C) was associated with a reduction in the extent of fibrosis. Magnification x350.2n, http://www.100md.com
PKC ß Western blotting and immunofluorescence.2n, http://www.100md.com
Western blot analysis demonstrated increased PKC ß in the kidneys of diabetic compared with control animals. This increased expression of PKC ß in diabetic animals was significantly reduced by treatment with LY333531. The cell-specific localization of PKC ß was examined using immunofluorescence microscopy. These studies demonstrated that PKC ß was expressed in the glomeruli of control rats, in a pattern consistent with its presence in mesangial cells . Immunostaining for PKC ß was increased in glomeruli of diabetic rats, although its pattern of distribution was unchanged. Treatment of diabetic rats with LY333531 was associated with a reduction in the overexpression of PKC ß when compared with untreated diabetic animals. No immunostaining of PKC ß was detected in the tubulointerstitium in either control or diabetic rats.
fig.ommtted$u, 百拇医药
Representative Western blot (top) and analysis (bottom) for PKC ß isoform in control (lanes 1 and 2), diabetic (lanes 3 and 4), and Diabetic + LY333531–treated Ren-2 rats (lanes 5 and 6). Densitometry measurements (mean ± SE) are expressed as a percentage of control (100%). *P < 0.01 diabetic vs. control; {dagger} P < 0.05 diabetic + LY333531 vs. untreated diabetic.$u, 百拇医药
fig.ommtted$u, 百拇医药
Representative photomicrograph of PKC ß immunofluorescence in control, diabetic, and diabetic + LY333531–treated Ren-2 rats. In control rats (A), PKC ß immunofluorescence was detected with more intense PKC ß labeling noted in glomeruli of diabetic rats (B). Treatment of diabetic rats with LY333531 was associated with a reduction in PKC ß immunofluorescence to levels similar to that of control animals (C). Magnification x350.$u, 百拇医药
TGF-ß immunohistochemistry.$u, 百拇医药
Minimal immunostainable TGF-ß was present in the kidneys of control rats (1.68 ± 0.58% proportional area). In contrast, abundant TGF-ß was expressed in the kidney (15.63 ± 2.5%; P < 0.001 versus control) of diabetic rats. This overexpression was attenuated in diabetic rats treated with LY333531 (4.38 ± 0.70%, P < 0.001 versus diabetic; ).
fig.ommttedg^r@4[x, 百拇医药
Representative photomicrograph of TGF-ß immunohistochemistry in control, diabetic, and diabetic + LY333531–treated Ren-2 rats. In control rats (A), TGF-ß immunostaining was detected with more intense TGF-ß labeling noted in glomeruli of diabetic rats (B). Treatment of diabetic rats with LY333531 was associated with a reduction in TGF-ß immunostaining to levels similar to that of control animals (C). Magnification x420.g^r@4[x, 百拇医药
DISCUSSIONg^r@4[x, 百拇医药
The present study demonstrates several novel findings in relation to the pathogenesis of diabetic nephropathy. First, despite the presence of continuing hyperglycemia and hypertension, PKC ß inhibition with LY333531 reduced the development of structural and functional manifestations of renal injury in this model. Second, although immunofluorescence microscopy localized PKC ß to the glomerulus, inhibition of this enzyme also attenuated injury in the tubulointerstitium. Third, diabetes was accompanied by increased immunoreactive PKC ß, and this was also reduced with LY333531.
The pathogenesis of diabetic nephropathy is complex and involves both glucose-dependent and glucose-independent pathways. In both type 1 and type 2 diabetes, the degree of hyperglycemia influences both the likelihood of developing nephropathy and the rate of its progression (2,27,28). High intracellular glucose concentrations, per se, may lead to activation of PKC (29) and in particular the ß isoform, which has been shown to be activated in the glomeruli in experimental diabetes (15,30). However, in addition to these glucose-dependent mechanisms, other glucose-independent components of the diabetic state contribute to the development and progression of diabetic nephropathy. In particular, both experimental and clinical studies indicate that hypertension and inappropriate activation of the RAS are likely key contributors (4,6,17). Both angiotensin II (31), the effector molecule of the RAS, and cell stretch, the in vitro counterpart of hypertension, activate PKC (10). The m(Ren-2)27 rat, used in the present studies (19), is not only hypertensive but also displays overactivity of the intrarenal RAS (20,32), both key features in the pathophysiology of progressive kidney disease in humans with diabetes. In the present study, LY333531 significantly attenuated the structural and functional manifestations of diabetic renal injury along with a reduction in the overexpression of the profibrotic growth factor TGF-ß. The finding that these beneficial changes occurred despite the continued presence of hyperglycemia, hypertension, and constitutive activation of the RAS is consistent with PKC ß activation as a final common pathway for these pathogenetic attributes of the diabetic milieu.
Although the glomerulus, in particular the mesangium, has largely been the focus of studies in diabetes, tubulointerstitial injury is also a major feature of diabetic nephropathy and an important predictor of both renal dysfunction (33,34) and its response to therapeutic interventions (35–37). In the present study, PKC ß was localized to the mesangial region of the glomerulus but was not detected in tubular epithelium of either control or diabetic animals. However, despite the pattern of distribution, inhibition of PKC ß with LY333531 attenuated tubulointerstitial as well as glomerular injury. These findings suggest that tubulointerstitial injury in diabetic nephropathy may develop as a consequence of glomerular damage. Indeed, experimental studies have indicated that multiple pathogenetic mechanisms may account for the tubulointerstitial injury that follows glomerular injury (8). These include excessive protein load to the proximal tubule leading to peritubular inflammation and fibrosis, postglomerular vasoconstriction with peritubular capillary rarefaction, tubular ischemia and atrophy, and misdirection of filtrate into the periglomerular and peritubular space (38).
PKC is a ubiquitously expressed large family of serine-threonine kinases that transduce a wide range of cell-signaling processes by substrate-specific phosphorylation (30,39). Of the 11 identified PKC isoenzymes, a preferential increase in the ß isoform has been described in experimental diabetes and in nondiabetic renal disease in humans (9,15,40), although this has not been a universal finding (41). Although enhanced PKC activity in diabetes occurs as a consequence of glucose-induced generation of diacyl glycerol and the resultant membrane translocation (9,42), recent studies suggest that other mechanisms may also contribute. Indeed, in the present study, diabetes was associated with an increase in immunoreactive PKC ß as determined by both Western blot analysis and immunofluorescence. These findings suggest that high glucose, and possibly other aspects of the diabetic milieu, not only induce activation of PKC enzymatic activity but also lead to increased PKC ß protein expression. Similar changes have also been demonstrated in the in vitro setting, in which exposure of cultured mesangial cells to 48 h of high glucose resulted in a doubling of total PKC ßII protein, detected by immunoblotting (43). In the present study, the diabetes-associated increase in PKC ß expression was attenuated by LY333531, suggesting that PKC ß activation may induce its own expression. The mechanisms underlying the possible autoinduction of PKC ß in the diabetic kidney is uncertain. Recent studies suggest that the epidermal growth factor receptor may be involved in both the induction and response to PKC activation (43,44). Alternatively, the increase in PKC ß may reflect a mesangial cell expansion in the setting of diabetes.
In summary, the present study demonstrates that, in a model of advanced diabetic nephropathy, inhibition of PKC ß significantly attenuated the structural and functional manifestations of injury despite continued hyperglycemia and hypertension. These findings suggest the potential role for this therapeutic strategy in the treatment and prevention of diabetic kidney disease.#il(hm#, 百拇医药
ACKNOWLEDGMENTS#il(hm#, 百拇医药
This project was supported by a program grant from the Juvenile Diabetes Foundation International and the NHMRC Australia. D.J.K. is a recipient of a Career Development Award from the Juvenile Diabetes Foundation International.#il(hm#, 百拇医药
The authors thank Mariana Pacheco and Giao Tran for expert technical assistance.#il(hm#, 百拇医药
REFERENCES#il(hm#, 百拇医药
Ritz E, Rychlik I, Locatelli F, Halimi S: End-stage renal failure in type 2 diabetes: a medical catastrophe of worldwide dimensions. Am J Kidney Dis34 :795 –808,1999#il(hm#, 百拇医药
Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med329 :977 –986,1993]]v^, 百拇医药
Fioretto P, Steffes MW, Sutherland DER, Goetz FC, Mauer M: Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med339 :69 –75,1998]]v^, 百拇医药
Parving H-H, Andersen ER, Smidt U, Hommel E, Mathiesen E: Antihypertensive treatment postpones endstage renal failure in diabetic nephropathy. Br Med J294 :1443 –1447,1987]]v^, 百拇医药
Bakris GL, Williams M, Dworkin L, Elliott WJ, Epstein M, Toto R, Tuttle K, Douglas J, Hsueh W, Sowers J: Preserving renal function in adults with hypertension and diabetes: a consensus approach. National Kidney Foundation Hypertension and Diabetes Executive Committees Working Group. Am J Kidney Dis36 :646 –61,2000]]v^, 百拇医药
Lewis EJ, Hunsicker LG, Bain RP, Rohde RD, for the Collaborative Study Group: The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. N Engl J Med329 :1456 –1462,1993
Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, Ritz E, Atkins RC, Rohde R, Raz I: Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med345 :851 –60,2001q.f(fs, 百拇医药
Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi G, Snapinn SM, Zhang Z, Shahinfar S: Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med345 :861 –869,2001q.f(fs, 百拇医药
Koya D, King GL: Protein kinase C activation and the development of diabetic complications. Diabetes47 :859 –866,1998q.f(fs, 百拇医药
Gruden G, Thomas S, Burt D, Lane S, Chusney G, Sacks S, Viberti G: Mechanical stretch induces vascular permeability factor in human mesangial cells: mechanisms of signal transduction. Proc Natl Acad Sci U S A94 :12112 –12116,1997q.f(fs, 百拇医药
Osicka TM, Yu Y, Panagiotopoulos S, Clavant SP, Kiriazis Z, Pike RN, Pratt LM, Russo LM, Kemp BE, Comper WD, Jerums G: Prevention of albuminuria by aminoguanidine or ramipril in streptozotocin-induced diabetic rats is associated with the normalization of glomerular protein kinase C. Diabetes49 :87 –93,2000
Weiss RH, Ramirez A: TGF-beta- and angiotensin-II-induced mesangial matrix protein secretion is mediated by protein kinase C. Nephrol Dial Transplant13 :2804 –2813,1998/u&, 百拇医药
Kreisberg JI, Kreisberg SH: High glucose activates protein kinase C and stimulates fibronectin gene expression by enhancing a cAMP response element. Kidney Int Suppl51 :S3 –S11,1995/u&, 百拇医药
Craven PA, DeRubertis FR: Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose. J Clin Invest83 :1667 –1675,1989/u&, 百拇医药
Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell S-E, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunction in diabetic rats by an oral PKC ß inhibitor. Science272 :728 –731,1996/u&, 百拇医药
Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, Kikkawa R: Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J14 :439 –447,2000
Anderson S: Role of local and systemic angiotensin in diabetic renal disease. Kidney Int52 (Suppl. 63) :S107 –S110,1997iz(){oi, 百拇医药
Gilbert RE, Cooper ME: The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int56 :1627 –1637,1999iz(){oi, 百拇医药
Mullins JJ, Peters J, Ganten D: Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature344 :541 –544,1990iz(){oi, 百拇医药
Kelly DJ, Wilkinson-Berka JL, Allen TJ, Cooper ME, Skinner SL: A new model of diabetic nephropathy with progressive renal impairment in the transgenic (mRen-2)27 rat. Kidney Int54 :343 –352,1998iz(){oi, 百拇医药
Bunag RD: Validation in awake rats of a tail-cuff method for measuring systolic pressure. J Appl Physiol34 :279 –282,1973iz(){oi, 百拇医药
Jerums G, Allen TJ, Cooper ME: Triphasic changes in selectivity with increasing proteinuria in type I and type II diabetes. Diabet Med6 :772 –779,1989iz(){oi, 百拇医药
Masson P: Trichrome stainings and their preliminary technique. J Tech Methods2 :75 –90,1929
Scholey JW, Miller PL, Rennke HG, Meyer TW: Effect of converting enzyme inhibition on the course of adriamycin-induced nephropathy. Kidney Int36 :816 –822,1989$0a[, 百拇医药
Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM: Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem45 :1559 –1565,1997$0a[, 百拇医药
Lehr HA, van der Loos CM, Teeling P, Gown AM: Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem47 :119 –126,1999$0a[, 百拇医药
Stratton IM, Adler AI, Neil HA, Matthews DR, Manley SE, Cull CA, Hadden D, Turner RC, Holman RR: Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ321 :405 –412,2000$0a[, 百拇医药
Gilbert RE, Tsalamandris C, Bach L, Panagiotopoulos S, O’Brien RC, Allen TJ, Goodall I, Seeman E, Cooper ME, Murray R, Jerums G: Glycemic control and the rate of progression of early diabetic kidney disease. Kidney Int44 :855 –859,1993
Newton AC: Protein kinase C: structure, function, and regulation. J Biol Chem270 :28495 –28498,1995syho, 百拇医药
Koya D, Jirousek MR, You-Wei L, Ishii H, Kuboki K, King GL: Characterization of protein kinase C ß isoform activation on gene expression of transforming growth factor-ß, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. J Clin Invest100 :115 –126,1997syho, 百拇医药
Feng X, Zhang J, Barak LS, Meyer T, Caron MG, Hannun YA: Visualization of dynamic trafficking of a protein kinase C ßII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization. J Biol Chem273 :10755 –10762,1998syho, 百拇医药
Kelly DJ, Skinner SL, Gilbert RE, Cox AJ, Cooper ME, Wilkinson-Berka JL: Effects of endothelin or angiotensin II receptor blockade on diabetes in the transgenic (mRen-2)27 rat. Kidney Int57 :1882 –1894,2000syho, 百拇医药
Bader R, Bader E, Grung KE, Markensen-Haen S, Christ H, Bohle A: Structure and function of the kidney in diabetic glomerulosclerosis: correlations between morphological and functional parameters. Pathol Res Pract167 :204 –216,1980
Lane P, Steffes MW, Fioretto P, Mauer SM: Renal interstitial expansion in insulin-dependent diabetes mellitus. Kidney Int43 :661 –667,1993m%t, 百拇医药
Gilbert RE, Cox A, Wu LL, Allen TJ, Hulthen L, Jerums G, Cooper ME: Expression of transforming growth factor-ß1 and type IV collagen in the renal tubulointerstitium in experimental diabetes: effects of angiotensin converting enzyme inhibition. Diabetes47 :414 –422,1998m%t, 百拇医药
Kelly DJ, Gilbert RE, Cox AJ, Soulis T, Jerums GT, Cooper ME: Aminoguanidine ameliorates overexpression of prosclerotic growth factors and collagen deposition in experimental diabetic nephropathy. J Am Soc Nephrol12 :2098 –2107,2001m%t, 百拇医药
Cordonnier DJ, Pinel N, Barro C, Maynard C, Zaoui P, Halimi S, De Ligny BH, Reznic Y, Simon D, Bilous RW: Expansion of cortical interstitium is limited by converting enzyme inhibition in type 2 diabetic patients with glomerulosclerosis. J Am Soc Nephrol10 :1253 –1263,1999m%t, 百拇医药
Kriz W, Hosser H, Hahnel B, Gretz N, Provoost AP: From segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies. Nephrol Dial Transplant13 :2781 –2798,1998
Murphy M, McGinty A, Godson C: Protein kinases C: potential targets for intervention in diabetic nephropathy. Curr Opin Nephrol Hypertens7 :563 –570,1998p]}@o+, 百拇医药
Ganz MB, Abunader R, Saxena R, Grond J: Protein kinase C ß(Ii) isoform is up-regulated in human proliferative glomerulonephritis. Exp Nephrol5 :225 –232,1997p]}@o+, 百拇医药
Kang N, Alexander G, Park JK, Maasch C, Buchwalow I, Luft FC, Haller H: Differential expression of protein kinase C isoforms in streptozotocin-induced diabetic rats. Kidney Int56 :1737 –1750,1999p]}@o+, 百拇医药
Craven PA, Studer RK, Negrete H, DeRubertis FR: Protein kinase C in diabetic nephropathy. J Diabetes Complications9 :241 –245,1995p]}@o+, 百拇医药
Kapor-Drezgic J, Zhou X, Babazono T, Dlugosz JA, Hohman T, Whiteside C: Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent. J Am Soc Nephrol10 :1193 –1203,1999p]}@o+, 百拇医药
Banan A, Fields JZ, Farhadi A, Talmage DA, Zhang L, Keshavarzian A: The ß1 isoform of protein kinase c mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells. J Pharmacol Exp Ther301 :852 –866,2002(Darren J. Kelly Yuan Zhang Claire Hepper Renae M. Gow Kassie JaworskiBruce E. Kemp Jennifer L. Wilki)
2 Department of Physiology, University of Melbourne, Parkville, Victoria, Australia&6'(0d;, 百拇医药
3 St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia&6'(0d;, 百拇医药
ABSTRACT&6'(0d;, 百拇医药
In addition to hyperglycemia, hypertension and the renin-angiotensin system have been consistently implicated in the pathogenesis of diabetic nephropathy. Each of these pathogenetic factors may induce changes in cellular function by a common intracellular signaling pathway, the activation of protein kinase C (PKC) ß. The present study thus sought to determine the in vivo effect of PKC ß inhibition in experimental diabetic nephropathy in the setting of continued hyperglycemia, hypertension, and activation of the RAS. Studies were conducted in the (mRen-2)27 rat, a rodent that is transgenic for the entire mouse renin gene (Ren-2) and develops many of the structural, functional, and molecular characteristics of human diabetic nephropathy when experimental diabetes is induced with streptozotocin (STZ). Six-week-old female Ren-2 rats received an injection of STZ or vehicle and were maintained for 6 months. Within 24 h, diabetic rats were further randomized to receive treatment with the specific PKC ß inhibitor, LY333531, admixed in diet (10 mg · kg-1 · d-1) or no treatment (n = 8/group). Diabetic rats developed albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis with a concomitant increase in transforming growth factor-ß (TGF-ß). Western blot analysis demonstrated increased PKC ß in diabetic animals, localized by immunofluorescence to the glomerular mesangium. In vivo inhibition of PKC ß with LY333531 led to a reduction in albuminuria, structural injury, and TGF-ß expression, despite continued hypertension and hyperglycemia.
Diabetic nephropathy is a leading cause of end-stage renal disease throughout much of the world (1). In addition to elevated blood glucose, hypertension and inappropriate activation of the renin-angiotensin system (RAS) have been identified as contributing to the development and progression of diabetic renal disease. Indeed, clinical studies not only have demonstrated a key role for good glycemic control in reducing the development and progression of diabetic nephropathy (2,3) but also have shown the importance of blood pressure reduction (4,5) and blockade of the RAS (6–8) in slowing the progression of renal dysfunction in both type 1 and type 2 diabetes. However, despite these advances, the incidence of end-stage renal disease as a result of diabetes continues to rise in the United States and other countries where diabetes is an important problem.?jx, 百拇医药
In addition to their frequent coexistence in patients with diabetic renal disease, hyperglycemia and hypertension may induce changes in cellular function by common intracellular signaling pathways (9). Indeed, several components of the diabetic milieu, implicated in the pathogenesis of diabetic nephropathy, induce activation of protein kinase C (PKC) ß. These include both glucose-dependent pathways such as the formation of advanced glycation end products and hyperglycemia per se in addition to glucose-independent mechanisms such as hypertension and activation of the RAS (10–13).
Previous studies have shown that not only is PKC ß the major isoform of PKC activated by hyperglycemia in the diabetic kidney (14), but also its inhibition of the ß isoform reduces albuminuria (15) and mesangial expansion (16). However, whether this therapeutic strategy is also effective in the setting of coexistent hypertension and inappropriate activation of the RAS (17), as in human diabetic nephropathy, remains uncertain. Furthermore, the effects of PKC ß inhibition on tubulointerstitial injury in diabetes, a major histological feature of the disease (18), also has not been reported.w@6?\*, http://www.100md.com
The present study thus sought to examine the effects of PKC ß inhibition in the diabetic (mRen-2)27 rat model of experimental diabetic nephropathy. This transgenic animal has the entire mouse renin gene (Ren-2) inserted into the genome of a Sprague-Dawley rat (19) and develops hypertension and many of the structural and functional characteristics of human diabetic nephropathy when diabetes is induced with streptozotocin (STZ) (20).
RESEARCH DESIGN AND METHODS;lrn, 百拇医药
Animals.;lrn, 百拇医药
Six-week-old female, heterozygous (mRen-2)27 rats that weighed 125 ± 5 g were randomized to receive either 55 mg/kg STZ (Sigma, St. Louis, MO) diluted in 0.1 mol/l citrate buffer (pH 4.5) or citrate buffer alone (nondiabetic) by tail-vein injection after an overnight fast. Diabetic Ren-2 (n = 8 per group) were treated with the PKC ß inhibitor LY333531 (Eli Lilly and Co., Indianapolis, IN) 10 mg · kg-1 · d-1, milled into rat food for 6 months after STZ or vehicle. Treatment commenced within 24 h of STZ injection. All rats were housed in a stable environment (maintained at 22 ± 1°C with a 12-h light/dark cycle) and allowed free access to tap water. Each week, rats were weighed and blood glucose was determined using an AMES glucometer (Bayer Diagnostics, Melbourne, Australia). Every 4 weeks, systolic blood pressure (SBP) was recorded in preheated conscious rats by tail-cuff plethysmography (21). Diabetic rats received a daily injection of insulin (2–4 units intraperitoneally; Ultratard, Novo Nordisk, Bagsraerd, Denmark) to promote weight gain and to reduce mortality. Experimental procedures adhered to the guidelines of the National Health and Medical Research Council of Australia’s Code for the Care and Use of Animals for Scientific Purposes and were approved by the Bioethics Committee of the University of Melbourne.
Albuminuria.moy-%ew, 百拇医药
Rats were individually housed in metabolic cages every 2 months and habituated for 2–3 h, and measurements of urinary albumin excretion were obtained over 24 h. Animals continued to have free access to tap water and standard laboratory diet during this period. After 24 h in metabolic cages, an aliquot of urine (5 ml) was collected from the 24-h urine sample and stored at -70°C for subsequent analysis of albumin. Albuminuria was determined by a double-antibody radioimmunoassay, as previously performed in our laboratories (22).moy-%ew, 百拇医药
Tissue preparation.moy-%ew, 百拇医药
Rats were anesthetized (Nembutal 60 mg/kg body wt intraperitoneally; Boehringer-Ingelheim, North Ryde, Australia), and the abdominal aorta was cannulated with an 18-G needle. Perfusion-exsanguination commenced at SBP (180–220 mmHg) via the abdominal aorta with 0.1 mol/l PBS (pH 7.4; 20–50 ml) to remove circulating blood, and the inferior vena cava adjacent to the renal vein was simultaneously severed allowing free flow of the perfusate. After clearance of circulating blood, 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4) was perfused for another 5 min (100–200 ml of fixative) to fix the tissues. Kidneys were removed from the animal, decapsulated, sliced transversely, and either frozen-embedded in OCT (Tissue-Tek; Miles, Elkhart, IN) for immunofluorescence or paraffin-embedded for light microscopic evaluation.
Histopathology.wk{77, http://www.100md.com
Changes in kidney structure were assessed in a masked protocol in at least 25 randomly selected tissue sections from each group studied. Sections were stained with either Mayer’s hematoxylin and eosin to examine cell structure, periodic acid Schiff to identify changes in basement membrane architecture and glycogen deposition, or Masson’s modified trichrome to demonstrate collagen matrix (23).wk{77, http://www.100md.com
Glomerulosclerotic index.wk{77, http://www.100md.com
In 3-µm kidney sections stained with periodic acid Schiff, 150–200 glomeruli from rats were examined. The degree of sclerosis in each glomerulus was graded on a scale of 0 to 4, as previously described (24), as follows: grade 0, normal; grade 1, sclerotic area up to 25% (minimal); grade 2, sclerotic area 25–50% (moderate); grade 3, sclerotic area 50–75% (moderate to severe); and grade 4, sclerotic area 75–100% (severe). A glomerulosclerotic index was then calculated using the formula: 4 GSI = {Sigma} Fi (i) i = 0
where Fi is the percentage of glomeruli in the rat with a given score (i).-.u(*26, http://www.100md.com
Immunofluorescence.-.u(*26, http://www.100md.com
Six-micron frozen sections were postfixed in 4% paraformaldehyde for 20 min and incubated for 20 min with normal goat serum (NGS) diluted 1:10 with 0.1 mol/l PBS at pH 7.4. Sections were then incubated for 18 h at 4°C with specific mouse anti-rat monoclonal PKC ß antibody (1:250; Zymed, San Francisco, CA). Sections incubated with 1:10 NGS instead of the primary antiserum served as the negative control. After thorough washing with PBS (3 x 5 min changes), the sections were incubated with FITC-labeled goat anti-mouse IgG (Dakopatts, Glostrup, Denmark) diluted 1:200 with PBS for 1 h at room temperature. Sections were rinsed with PBS (2 x 5 min), rinsed in tap water for 5 min, and mounted for microscope viewing.-.u(*26, http://www.100md.com
Immunohistochemistry.-.u(*26, http://www.100md.com
Three-micron sections were placed into histosol, hydrated in graded ethanol, and immersed in tap water before being incubated for 20 min with NGS diluted 1:10 with 0.1 mol/l PBS at pH 7.4. Sections were then incubated for 18 h at 4°C with specific primary to transforming growth factor-ß (TGF-ß) (1:250, Zymed). Sections incubated with 1:10 NGS instead of the primary antiserum served as the negative control. After thorough washing with PBS (3 x 5 min changes), the sections were flooded with a solution of 5% hydrogen peroxide, rinsed with PBS (2 x 5 min), and incubated with biotinylated goat anti-rabbit IgG (Dakopatts) diluted 1:200 with PBS. Sections were rinsed with PBS (2 x 5 min) and incubated with an avidin-biotin peroxidase complex (Vector, Burlingame, CA) diluted 1:200 with PBS. After rinsing with PBS (2 x 5 min), sections were incubated with 0.05% diaminobenzidine and 0.05% hydrogen peroxide (Pierce, Rockford, IL) in PBS at pH 7.6 for 1–3 min, rinsed in tap water for 5 min, counterstained in Mayer’s hematoxylin, differentiated in Scott’s tap water, dehydrated, cleared, and mounted in Depex (20).
Quantification of matrix deposition and immunohistochemistry.-#k[, http://www.100md.com
The accumulation of matrix within the tubulointerstitium was assessed on Masson’s trichrome-stained sections using computer-assisted image analysis, as previously reported (25,26). Briefly, five random nonoverlapping fields from six rats per group were captured and digitized using a BX50 microscope attached to a Fujix HC5000 digital camera, then loaded onto a Pentium III IBM computer. An area of blue on a trichrome-stained section or brown on TGF-ß–stained sections was selected for its color range, and the proportional area of tissue with this range of color was then quantified. Calculation of the proportional area was then determined using image analysis (AIS, Analytical Imaging Station Version 6.0, ON, Canada) for quantification of histological sections.-#k[, http://www.100md.com
Western blot analysis.-#k[, http://www.100md.com
Protein concentration of whole kidney samples was determined by the Bradford assay, using BSA as a standard. Samples containing 10 µg of protein were diluted to 30 µl in loading buffer, denatured for 5 min at 95°C, and separated by electrophoresis in 12.5% SDS-PAGE gels. After electroblotting onto Hybond transfer membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.), gel loading and transfer efficiency were assessed by staining the blot with 0.1% Ponceau’s Solution (Sigma Chemical Co.). Blots were blocked overnight at 4°C in 5% wt/vol nonfat dry milk before incubation with the primary antibody for 60 min at room temperature. Anti–PKC ß antibody (dilution 1:750; Zymed) was used to demonstrate the 81-kDa protein band. After the blot was incubated with a horseradish peroxidase–conjugated anti-mouse secondary antibody (dilution 1:1,000; Amersham) for 60 min at room temperature, antibody binding was visualized by enhanced chemiluminescence detection reagents. The bands of the resulting autoradiographs were compared for optical density using ImageQuaNT software (Version 4.2a, Build 13, Amersham). Relative quantities were compared normalized to control values, arbitrarily assigned as 100%.
Statistics.3kv'a{%, http://www.100md.com
Data are expressed as mean ± SE unless otherwise stated. Statistical significance was determined by a two-way ANOVA with a Fisher’s post hoc comparison. Albuminuria was analyzed using log-transformed data and represented as geometric means x/÷ tolerance factors. Analyses were performed using Statview II + Graphics package (Abacus Concepts, Berkeley, CA) on an Apple Macintosh G4 computer (Apple Computer, Cupertino, CA). A P < 0.05 was regarded as statistically significant.3kv'a{%, http://www.100md.com
RESULTS3kv'a{%, http://www.100md.com
Renal functional and biochemical studies.3kv'a{%, http://www.100md.com
In comparison with control animals, diabetic rats had reduced body weight, which was unaffected by treatment (P < 0.01). All rats, both diabetic and nondiabetic, were hypertensive with elevated SBP that was not altered by LY333531 treatment (P < 0.01; ). Plasma glucose was elevated to a similar extent in treated and untreated diabetic rat groups (P < 0.01; ). Diabetes was associated with an increase in urinary albumin excretion when compared with controls. Treatment with LY333531 reduced albumin excretion in diabetic rats.
fig.ommtted8+7:', 百拇医药
Body weight, kidney weight, SBP, and plasma glucose of control and diabetic transgenic (mRen-2)27 rats treated for 6 months with LY3335318+7:', 百拇医药
Renal structure.8+7:', 百拇医药
Glomerular injury was a prominent feature of diabetic rats, with evidence of both diffuse and nodular glomerulosclerosis . These changes were significantly attenuated by treatment with LY333531 . In addition to these glomerular changes, tubulointerstitial pathology was present in diabetic rats . These changes were also substantially reduced in diabetic rats treated with LY333531 .8+7:', 百拇医药
fig.ommtted8+7:', 百拇医药
Representative PAS-stained sections from control diabetic and diabetic LY333531-treated Ren-2 rats. In control rats (A), there is only minimal glomerulosclerosis, whereas diabetes is associated with a dramatic increase in glomerulosclerosis (B). Treatment of diabetic rats with the PKC ß inhibitor LY333531 (C) was associated with a reduction in the number and extent of glomerusclerosis. Magnification x350.
fig.ommtted2n, http://www.100md.com
Glomerulosclerosis expressed as glomerulosclerotic index (top) and tubulointerstitial fibrosis expressed as percentage area occupied by extracellular matrix (blue) on trichrome-stained sections (bottom) in control, diabetic, and diabetic + LY333531–treated Ren-2 rats. *P < 0.01 diabetic vs. control; {dagger} P < 0.01 diabetic + LY333531 vs. untreated diabetic.2n, http://www.100md.com
fig.ommtted2n, http://www.100md.com
Representative Masson’s trichrome-stained sections from control, diabetic, and diabetic + LY333531–treated Ren-2 rats. In control rats (A), there is sparse collagen (blue staining) within the interstitium, whereas diabetes is associated with substantial fibrosis (B). Treatment of diabetic rats with the PKC ß inhibitor LY333531 (C) was associated with a reduction in the extent of fibrosis. Magnification x350.2n, http://www.100md.com
PKC ß Western blotting and immunofluorescence.2n, http://www.100md.com
Western blot analysis demonstrated increased PKC ß in the kidneys of diabetic compared with control animals. This increased expression of PKC ß in diabetic animals was significantly reduced by treatment with LY333531. The cell-specific localization of PKC ß was examined using immunofluorescence microscopy. These studies demonstrated that PKC ß was expressed in the glomeruli of control rats, in a pattern consistent with its presence in mesangial cells . Immunostaining for PKC ß was increased in glomeruli of diabetic rats, although its pattern of distribution was unchanged. Treatment of diabetic rats with LY333531 was associated with a reduction in the overexpression of PKC ß when compared with untreated diabetic animals. No immunostaining of PKC ß was detected in the tubulointerstitium in either control or diabetic rats.
fig.ommtted$u, 百拇医药
Representative Western blot (top) and analysis (bottom) for PKC ß isoform in control (lanes 1 and 2), diabetic (lanes 3 and 4), and Diabetic + LY333531–treated Ren-2 rats (lanes 5 and 6). Densitometry measurements (mean ± SE) are expressed as a percentage of control (100%). *P < 0.01 diabetic vs. control; {dagger} P < 0.05 diabetic + LY333531 vs. untreated diabetic.$u, 百拇医药
fig.ommtted$u, 百拇医药
Representative photomicrograph of PKC ß immunofluorescence in control, diabetic, and diabetic + LY333531–treated Ren-2 rats. In control rats (A), PKC ß immunofluorescence was detected with more intense PKC ß labeling noted in glomeruli of diabetic rats (B). Treatment of diabetic rats with LY333531 was associated with a reduction in PKC ß immunofluorescence to levels similar to that of control animals (C). Magnification x350.$u, 百拇医药
TGF-ß immunohistochemistry.$u, 百拇医药
Minimal immunostainable TGF-ß was present in the kidneys of control rats (1.68 ± 0.58% proportional area). In contrast, abundant TGF-ß was expressed in the kidney (15.63 ± 2.5%; P < 0.001 versus control) of diabetic rats. This overexpression was attenuated in diabetic rats treated with LY333531 (4.38 ± 0.70%, P < 0.001 versus diabetic; ).
fig.ommttedg^r@4[x, 百拇医药
Representative photomicrograph of TGF-ß immunohistochemistry in control, diabetic, and diabetic + LY333531–treated Ren-2 rats. In control rats (A), TGF-ß immunostaining was detected with more intense TGF-ß labeling noted in glomeruli of diabetic rats (B). Treatment of diabetic rats with LY333531 was associated with a reduction in TGF-ß immunostaining to levels similar to that of control animals (C). Magnification x420.g^r@4[x, 百拇医药
DISCUSSIONg^r@4[x, 百拇医药
The present study demonstrates several novel findings in relation to the pathogenesis of diabetic nephropathy. First, despite the presence of continuing hyperglycemia and hypertension, PKC ß inhibition with LY333531 reduced the development of structural and functional manifestations of renal injury in this model. Second, although immunofluorescence microscopy localized PKC ß to the glomerulus, inhibition of this enzyme also attenuated injury in the tubulointerstitium. Third, diabetes was accompanied by increased immunoreactive PKC ß, and this was also reduced with LY333531.
The pathogenesis of diabetic nephropathy is complex and involves both glucose-dependent and glucose-independent pathways. In both type 1 and type 2 diabetes, the degree of hyperglycemia influences both the likelihood of developing nephropathy and the rate of its progression (2,27,28). High intracellular glucose concentrations, per se, may lead to activation of PKC (29) and in particular the ß isoform, which has been shown to be activated in the glomeruli in experimental diabetes (15,30). However, in addition to these glucose-dependent mechanisms, other glucose-independent components of the diabetic state contribute to the development and progression of diabetic nephropathy. In particular, both experimental and clinical studies indicate that hypertension and inappropriate activation of the RAS are likely key contributors (4,6,17). Both angiotensin II (31), the effector molecule of the RAS, and cell stretch, the in vitro counterpart of hypertension, activate PKC (10). The m(Ren-2)27 rat, used in the present studies (19), is not only hypertensive but also displays overactivity of the intrarenal RAS (20,32), both key features in the pathophysiology of progressive kidney disease in humans with diabetes. In the present study, LY333531 significantly attenuated the structural and functional manifestations of diabetic renal injury along with a reduction in the overexpression of the profibrotic growth factor TGF-ß. The finding that these beneficial changes occurred despite the continued presence of hyperglycemia, hypertension, and constitutive activation of the RAS is consistent with PKC ß activation as a final common pathway for these pathogenetic attributes of the diabetic milieu.
Although the glomerulus, in particular the mesangium, has largely been the focus of studies in diabetes, tubulointerstitial injury is also a major feature of diabetic nephropathy and an important predictor of both renal dysfunction (33,34) and its response to therapeutic interventions (35–37). In the present study, PKC ß was localized to the mesangial region of the glomerulus but was not detected in tubular epithelium of either control or diabetic animals. However, despite the pattern of distribution, inhibition of PKC ß with LY333531 attenuated tubulointerstitial as well as glomerular injury. These findings suggest that tubulointerstitial injury in diabetic nephropathy may develop as a consequence of glomerular damage. Indeed, experimental studies have indicated that multiple pathogenetic mechanisms may account for the tubulointerstitial injury that follows glomerular injury (8). These include excessive protein load to the proximal tubule leading to peritubular inflammation and fibrosis, postglomerular vasoconstriction with peritubular capillary rarefaction, tubular ischemia and atrophy, and misdirection of filtrate into the periglomerular and peritubular space (38).
PKC is a ubiquitously expressed large family of serine-threonine kinases that transduce a wide range of cell-signaling processes by substrate-specific phosphorylation (30,39). Of the 11 identified PKC isoenzymes, a preferential increase in the ß isoform has been described in experimental diabetes and in nondiabetic renal disease in humans (9,15,40), although this has not been a universal finding (41). Although enhanced PKC activity in diabetes occurs as a consequence of glucose-induced generation of diacyl glycerol and the resultant membrane translocation (9,42), recent studies suggest that other mechanisms may also contribute. Indeed, in the present study, diabetes was associated with an increase in immunoreactive PKC ß as determined by both Western blot analysis and immunofluorescence. These findings suggest that high glucose, and possibly other aspects of the diabetic milieu, not only induce activation of PKC enzymatic activity but also lead to increased PKC ß protein expression. Similar changes have also been demonstrated in the in vitro setting, in which exposure of cultured mesangial cells to 48 h of high glucose resulted in a doubling of total PKC ßII protein, detected by immunoblotting (43). In the present study, the diabetes-associated increase in PKC ß expression was attenuated by LY333531, suggesting that PKC ß activation may induce its own expression. The mechanisms underlying the possible autoinduction of PKC ß in the diabetic kidney is uncertain. Recent studies suggest that the epidermal growth factor receptor may be involved in both the induction and response to PKC activation (43,44). Alternatively, the increase in PKC ß may reflect a mesangial cell expansion in the setting of diabetes.
In summary, the present study demonstrates that, in a model of advanced diabetic nephropathy, inhibition of PKC ß significantly attenuated the structural and functional manifestations of injury despite continued hyperglycemia and hypertension. These findings suggest the potential role for this therapeutic strategy in the treatment and prevention of diabetic kidney disease.#il(hm#, 百拇医药
ACKNOWLEDGMENTS#il(hm#, 百拇医药
This project was supported by a program grant from the Juvenile Diabetes Foundation International and the NHMRC Australia. D.J.K. is a recipient of a Career Development Award from the Juvenile Diabetes Foundation International.#il(hm#, 百拇医药
The authors thank Mariana Pacheco and Giao Tran for expert technical assistance.#il(hm#, 百拇医药
REFERENCES#il(hm#, 百拇医药
Ritz E, Rychlik I, Locatelli F, Halimi S: End-stage renal failure in type 2 diabetes: a medical catastrophe of worldwide dimensions. Am J Kidney Dis34 :795 –808,1999#il(hm#, 百拇医药
Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med329 :977 –986,1993]]v^, 百拇医药
Fioretto P, Steffes MW, Sutherland DER, Goetz FC, Mauer M: Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med339 :69 –75,1998]]v^, 百拇医药
Parving H-H, Andersen ER, Smidt U, Hommel E, Mathiesen E: Antihypertensive treatment postpones endstage renal failure in diabetic nephropathy. Br Med J294 :1443 –1447,1987]]v^, 百拇医药
Bakris GL, Williams M, Dworkin L, Elliott WJ, Epstein M, Toto R, Tuttle K, Douglas J, Hsueh W, Sowers J: Preserving renal function in adults with hypertension and diabetes: a consensus approach. National Kidney Foundation Hypertension and Diabetes Executive Committees Working Group. Am J Kidney Dis36 :646 –61,2000]]v^, 百拇医药
Lewis EJ, Hunsicker LG, Bain RP, Rohde RD, for the Collaborative Study Group: The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. N Engl J Med329 :1456 –1462,1993
Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, Ritz E, Atkins RC, Rohde R, Raz I: Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med345 :851 –60,2001q.f(fs, 百拇医药
Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi G, Snapinn SM, Zhang Z, Shahinfar S: Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med345 :861 –869,2001q.f(fs, 百拇医药
Koya D, King GL: Protein kinase C activation and the development of diabetic complications. Diabetes47 :859 –866,1998q.f(fs, 百拇医药
Gruden G, Thomas S, Burt D, Lane S, Chusney G, Sacks S, Viberti G: Mechanical stretch induces vascular permeability factor in human mesangial cells: mechanisms of signal transduction. Proc Natl Acad Sci U S A94 :12112 –12116,1997q.f(fs, 百拇医药
Osicka TM, Yu Y, Panagiotopoulos S, Clavant SP, Kiriazis Z, Pike RN, Pratt LM, Russo LM, Kemp BE, Comper WD, Jerums G: Prevention of albuminuria by aminoguanidine or ramipril in streptozotocin-induced diabetic rats is associated with the normalization of glomerular protein kinase C. Diabetes49 :87 –93,2000
Weiss RH, Ramirez A: TGF-beta- and angiotensin-II-induced mesangial matrix protein secretion is mediated by protein kinase C. Nephrol Dial Transplant13 :2804 –2813,1998/u&, 百拇医药
Kreisberg JI, Kreisberg SH: High glucose activates protein kinase C and stimulates fibronectin gene expression by enhancing a cAMP response element. Kidney Int Suppl51 :S3 –S11,1995/u&, 百拇医药
Craven PA, DeRubertis FR: Protein kinase C is activated in glomeruli from streptozotocin diabetic rats. Possible mediation by glucose. J Clin Invest83 :1667 –1675,1989/u&, 百拇医药
Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell S-E, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunction in diabetic rats by an oral PKC ß inhibitor. Science272 :728 –731,1996/u&, 百拇医药
Koya D, Haneda M, Nakagawa H, Isshiki K, Sato H, Maeda S, Sugimoto T, Yasuda H, Kashiwagi A, Ways DK, King GL, Kikkawa R: Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J14 :439 –447,2000
Anderson S: Role of local and systemic angiotensin in diabetic renal disease. Kidney Int52 (Suppl. 63) :S107 –S110,1997iz(){oi, 百拇医药
Gilbert RE, Cooper ME: The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int56 :1627 –1637,1999iz(){oi, 百拇医药
Mullins JJ, Peters J, Ganten D: Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene. Nature344 :541 –544,1990iz(){oi, 百拇医药
Kelly DJ, Wilkinson-Berka JL, Allen TJ, Cooper ME, Skinner SL: A new model of diabetic nephropathy with progressive renal impairment in the transgenic (mRen-2)27 rat. Kidney Int54 :343 –352,1998iz(){oi, 百拇医药
Bunag RD: Validation in awake rats of a tail-cuff method for measuring systolic pressure. J Appl Physiol34 :279 –282,1973iz(){oi, 百拇医药
Jerums G, Allen TJ, Cooper ME: Triphasic changes in selectivity with increasing proteinuria in type I and type II diabetes. Diabet Med6 :772 –779,1989iz(){oi, 百拇医药
Masson P: Trichrome stainings and their preliminary technique. J Tech Methods2 :75 –90,1929
Scholey JW, Miller PL, Rennke HG, Meyer TW: Effect of converting enzyme inhibition on the course of adriamycin-induced nephropathy. Kidney Int36 :816 –822,1989$0a[, 百拇医药
Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM: Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem45 :1559 –1565,1997$0a[, 百拇医药
Lehr HA, van der Loos CM, Teeling P, Gown AM: Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem47 :119 –126,1999$0a[, 百拇医药
Stratton IM, Adler AI, Neil HA, Matthews DR, Manley SE, Cull CA, Hadden D, Turner RC, Holman RR: Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ321 :405 –412,2000$0a[, 百拇医药
Gilbert RE, Tsalamandris C, Bach L, Panagiotopoulos S, O’Brien RC, Allen TJ, Goodall I, Seeman E, Cooper ME, Murray R, Jerums G: Glycemic control and the rate of progression of early diabetic kidney disease. Kidney Int44 :855 –859,1993
Newton AC: Protein kinase C: structure, function, and regulation. J Biol Chem270 :28495 –28498,1995syho, 百拇医药
Koya D, Jirousek MR, You-Wei L, Ishii H, Kuboki K, King GL: Characterization of protein kinase C ß isoform activation on gene expression of transforming growth factor-ß, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. J Clin Invest100 :115 –126,1997syho, 百拇医药
Feng X, Zhang J, Barak LS, Meyer T, Caron MG, Hannun YA: Visualization of dynamic trafficking of a protein kinase C ßII/green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization. J Biol Chem273 :10755 –10762,1998syho, 百拇医药
Kelly DJ, Skinner SL, Gilbert RE, Cox AJ, Cooper ME, Wilkinson-Berka JL: Effects of endothelin or angiotensin II receptor blockade on diabetes in the transgenic (mRen-2)27 rat. Kidney Int57 :1882 –1894,2000syho, 百拇医药
Bader R, Bader E, Grung KE, Markensen-Haen S, Christ H, Bohle A: Structure and function of the kidney in diabetic glomerulosclerosis: correlations between morphological and functional parameters. Pathol Res Pract167 :204 –216,1980
Lane P, Steffes MW, Fioretto P, Mauer SM: Renal interstitial expansion in insulin-dependent diabetes mellitus. Kidney Int43 :661 –667,1993m%t, 百拇医药
Gilbert RE, Cox A, Wu LL, Allen TJ, Hulthen L, Jerums G, Cooper ME: Expression of transforming growth factor-ß1 and type IV collagen in the renal tubulointerstitium in experimental diabetes: effects of angiotensin converting enzyme inhibition. Diabetes47 :414 –422,1998m%t, 百拇医药
Kelly DJ, Gilbert RE, Cox AJ, Soulis T, Jerums GT, Cooper ME: Aminoguanidine ameliorates overexpression of prosclerotic growth factors and collagen deposition in experimental diabetic nephropathy. J Am Soc Nephrol12 :2098 –2107,2001m%t, 百拇医药
Cordonnier DJ, Pinel N, Barro C, Maynard C, Zaoui P, Halimi S, De Ligny BH, Reznic Y, Simon D, Bilous RW: Expansion of cortical interstitium is limited by converting enzyme inhibition in type 2 diabetic patients with glomerulosclerosis. J Am Soc Nephrol10 :1253 –1263,1999m%t, 百拇医药
Kriz W, Hosser H, Hahnel B, Gretz N, Provoost AP: From segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies. Nephrol Dial Transplant13 :2781 –2798,1998
Murphy M, McGinty A, Godson C: Protein kinases C: potential targets for intervention in diabetic nephropathy. Curr Opin Nephrol Hypertens7 :563 –570,1998p]}@o+, 百拇医药
Ganz MB, Abunader R, Saxena R, Grond J: Protein kinase C ß(Ii) isoform is up-regulated in human proliferative glomerulonephritis. Exp Nephrol5 :225 –232,1997p]}@o+, 百拇医药
Kang N, Alexander G, Park JK, Maasch C, Buchwalow I, Luft FC, Haller H: Differential expression of protein kinase C isoforms in streptozotocin-induced diabetic rats. Kidney Int56 :1737 –1750,1999p]}@o+, 百拇医药
Craven PA, Studer RK, Negrete H, DeRubertis FR: Protein kinase C in diabetic nephropathy. J Diabetes Complications9 :241 –245,1995p]}@o+, 百拇医药
Kapor-Drezgic J, Zhou X, Babazono T, Dlugosz JA, Hohman T, Whiteside C: Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent. J Am Soc Nephrol10 :1193 –1203,1999p]}@o+, 百拇医药
Banan A, Fields JZ, Farhadi A, Talmage DA, Zhang L, Keshavarzian A: The ß1 isoform of protein kinase c mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells. J Pharmacol Exp Ther301 :852 –866,2002(Darren J. Kelly Yuan Zhang Claire Hepper Renae M. Gow Kassie JaworskiBruce E. Kemp Jennifer L. Wilki)