Activation of Protein Kinase C-Mitogen Activated Protein Kinase Pathway and Diabetic Nephropathy

Daisuke Koya, Ryuichi Kikkawa, Atsunori Kashiwagi and Masakazu Haneda

Department of Medicine, Shiga University of Medical Science
Seta, Otsu, Shiga, Japan

koya@belle.shiga-med.ac.jp

Diabetic nephropathy is a leading cause of end-stage renal failure, accounting for 35-40% of all new cases requiring dialysis therapy worldwide. Recent clinical studies clearly indicate the not only hyperglycemia, but also systemic hypertension plays a crucial role in the development and progression of diabetic nephropathy. However, the incidence and prevalence of diabetic nephropathy has been rising despite ongoing advance in glycemic control and antihypertensive therapy with angiotensin converting enzyme inhibitors and/or angiotensin II receptor antagonists.

Therefore, a deeper understanding of diabetes-induced molecular events is needed to provide new insights into developing new therapeutic strategies for diabetic nephropathy. PKC activation, which is presumably caused by increased de novo synthesis of diacylglycerol (DG) from glycolytic intermediates, has emerged as a potential regulator of all aspects of the development and progression of diabetic nephropathy.

PKC activation immediately leads to extracellular-signal regulated-protein kinase (ERK), resulted in altering gene expression such as TGF- and extracellular matrix (ECM) proteins. To understand the pathophysiological significance of PKC-MAPK activation in diabetic nephropathy, the effects of PKC inhibitors, such as vitamin E (-tocopherol), thiazolidinediones, and a specific inhibitor for PKC isoform, ruboxistaurin (RBX; LY 333531), on renal dysfunction and pathologies were examined in diabetic rodent models. Vitamin E and thiazolidinediones prevented glomerular hyperfiltration as well as albuminuria in diabetic rats by inhibiting PKC-MAPK activation through enhancement of DG kinase activity. Oral administration of RBX to diabetic rats also normalized glomerular hyperfiltration as well as albuminuria through inhibiting PKC activity directly without affecting DG levels.

Inhibition of PKC-ERK activation also prevented the diabetes-induced alterations in mRNA expression of TGF- 1, type IV collagen, and fibronectin in diabetic glomeruli. Furthermore, mesangial expansion, one of the histological characteristics of diabetic nephropathy, was ameliorated in db/db mice by RBX treatment. Recently, we have also provided evidence that oxidative stress, another potential mechanism for diabetic nephropathy, was mediated by NADPH oxidase activation at least in part through PKC -dependent p47 phox and p67phox membranous translocation in the diabetic glomeruli. In conclusion, we highlight PKC-ERK activation as an important pathogenic factor how diabetes/hyperglycemia causes diabetic nephropathy, and the use of PKC inhibitors may be effective in treating diabetic nephropathy clinically.

Introduction

The Diabetes Control and Complications Trial (DCCT), the United Kingdom Prospective Diabetes Study (UKPDS), and the Kumamoto Study clearly demonstrated that the strict maintenance of euglycemia by intensive insulin treatment can prevent the onset and progression of diabetic nephropathy in patients with type 1 and type 2 diabetes mellitus ^{[1, 2, 3]}. Furthermore, recent clinical trials by using inhibitors for renin-angiotensin system (RAS) also provided the clinical impact in treating patients with diabetic nephropathy ^{[4, 5, 6]}.

However, diabetic nephropathy has been increasing in spite of intensive blood glucose control and antihypertensive therapy with angiotensin converting enzyme inhibitors and/or angiotensin II receptor antagonists. Therefore, an understanding of diabetes-induced molecular pathogenesis is needed to provide new insights into therapeutic strategies for the progression of diabetic nephropathy. Multiple biochemical mechanisms have been proposed to explain the adverse effects of hyperglycemia and/or diabetes.

Activation of diacylglycerol (DG)-protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) pathway ^{[7, 8]}, enhanced polyol pathway related with myo-inositol depletion ^[9], altered redox state ^[10], overproduction of advanced glycation end products ^[11], RAS activation^[12], and enhanced growth factor and cytokine production ^[13] have all been proposed as potential molecular mechanisms by which hyperglycemia and/or diabetes induces the chronic diabetic complications.

Recently, much attention has been paid on PKC-MAPK hypothesis since many vascular abnormalities of diabetes can be mimicked by PKC-MAPK activation and inhibited by PKC-MAPK inhibitors in both diabetic animal models and in vitro experiments using cultured glomerular mesangial cells. Here, we focus on the role of PKC-MAPKs activation in the development and progression of diabetic nephropathy and emerging evidence regarding the activation of PKC-MAPK pathway will be briefly reviewed (Fig. 1).

Pathophysiological significance of PKC-MAPK activation in diabetes

PKC, a family of serine-threonine kinases, has at least eleven PKC isoforms and can be categorized into classical PKC (, ₁, ₂, ), novel PKC (, , , , ), and atypical PKC (, ) on the basis of their common structural features ^[14]. The classical PKC enzymes contain two cysteine-rich zinc finger-like motifs (C₁ region), which are essential for interaction with phorbol ester and diacylglycerol (DG), and a Ca^[2+]-binding domain (C₂ region) in their regulatory domain. The novel PKC enzymes do not require Ca^[2+] since C₂ region is absent. The novel PKC enzymes are activated by phosphatidylserine and DG or phorbol esters. The atypical PKC enzymes, which lack the C₂ region and one of the cysteine-rich zinc finger-like motifs in the C₁ region, are not activated by Ca^[2+], DG, or phorbol esters, but their activation depends on phosphatidylserine and cis-unsaturated fatty acids.

PKC activation regulates a number of vascular functions such as vascular permeability, contractility, cell proliferation, extracellular matrix protein synthesis, and signal transduction cascade for hormones and growth factors ^[14]. Our laboratory and Craven et al. reported that high glucose increases DG levels and subsequently activates PKC in vascular cells and tissues ^[15-17]. The activation of PKC by high glucose appears to be related to an elevation of de novo DG level from glycolytic intermediates, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, but not to be dependent on hydrolysis of phosphatidylinositol by phospholipase C and phosphatidylcholine by phospholipase D since inositol phosphates production, which is derived from hydrolysis of phosphatidylinositol, has been found to be unchanged in cultured aortic smooth muscle cells ^[18] and mesangial cells exposed to elevated glucose levels ^[19]. Among eleven PKC isoforms, PKC , ₁, , and were shown to be activated in the glomeruli of diabetic rats as well as in mesangial cells exposed to high glucose by using a western blot analysis and/or a confocal microscopy ^{[ 20, 21]}. In addition, the activation of PKC isoforms has been shown in cultured mesangial cells exposed to high glucose ^[22], although the mechanism for activation of PKC , which is independent on DG, is unclear.

Mesangial expansion and capillary basement membrane thickening, mainly composed of type IV collagen, fibronectin, and laminin, are pathological manifestations of diabetic nephropathy. High glucose-induced type IV collagen expression can be prevented by PKC inhibitors such as staurosporine or calphostin C and the treatment with PKC agonists stimulates type IV collagen expression and fibronectin accumulation ^[23], suggesting that the effects of high glucose on increasing production of extracellular matrix proteins are mediated through PKC activation. PKC activation can induce the expression of transforming growth factor 1 (TGF- 1), a prototypical multifunctional cytokine, which is one of the crucial growth factors in regulating extracellular matrix protein accumulation in diabetic nephropathy. One possible mechanism by which high glucose induces the expression and synthesis of TGF- 1 and extracellular matrix proteins is by PKC’s actions via the transcription factors c-fos and c-jun, which forms complexs for activated protein-1 (AP-1) binding site, since mRNA expression of c-fos and c-jun protooncogenes has been demonstrated to be increased in cultured mesangial cells exposed to high glucose and in rat glomeruli after induction of diabetes ^{[24, 25]}.

Mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated protein kinase-1/2 (ERK 1/2), stress-activated c-Jun N-amino terminal kinase (JNK), and p38 MAPK, play a key role in the intracellular signal transduction cascade to integrate the transcription of genes for a variety of cellular responses relevant to diabetic nephropathy such as cell growth, differentiation, and extracellular matrix synthesis ^[26]. We and others have shown that ERK as well as p38 MAPK is activated in mesangial cells exposed to high glucose and in the glomeruli of diabetic animals ^{[8, 27]}. The co-activation of PKC and MAPKs by high glucose reinforces the notion that PKC plays an important role in the process of ERK activation. Indeed, we found that a general PKC inhibitor, calphostin C prevented the activation of ERK under high glucose. Furthermore, PKC inhibitor (RBX) can also inhibit hyperglycemia-induced glomerular ERK activation in diabetic rats (personal communication by Kitada M, Koya D, Haneda M). Recently, Isono et al. have also reported that the activation of ERK possibly through PKC is responsible for the overproduction of TGF- and extracellular matrix proteins, such as type 1 collagen and fibronectin in mesangial cells under high glucose conditions, possibly through activated protein-1 (AP-1) activation ^[28].

To define the pathophysiological significance of PKC-MAPK activation, the effects of vitamin E (d- tocopherol), thioazolidinediones, and a specific inhibitor for PKC isoform LY 333531 ^[17], on renal dysfunction and pathologies were examined in diabetic animal models and in mesangial cells exposed to elevated levels of glucose. Vitamin E and thiazolidinediones can prevent glomerular hyperfiltration as well as albuminuria in diabetic rats by inhibiting PKC-MAPK activation through decreasing DG levels ^{[29, 30]}. Oral administration of PKC inhibitor to diabetic rats can also normalize glomerular filtration rate as well as albuminuria in parallel with inhibition of PKC activity whereas PKC inhibitor did not affect DG levels. PKC inhibitor can also prevent the diabetes-induced abnormalities in mRNA expression of TGF 1, type IV collagen, and fibronectin in glomeruli of diabetic rats ^[20]. In addition, mesangial expansion, one of the important histological characters of diabetic nephropathy, was significantly ameliorated in db/db mice by the treatment with PKC inhibitor ^[31]. The enhanced expression of TGF- and extracellular matrix proteins in glomeruli of db/db mice was also shown to be prevented by PKC inhibitor ^[31].

RESULTS FROM CLINICAL TRIALS

The safety and vascular effects of PKC inhibitor were evaluated in a one month clinical study on 29 patients with type 1 or 2 diabetes of less than 10 years, with no or minimal retinopathy. The double-blind, placebo-controlled randomized trial showed significant improvement in retinal blood flow and mean circulation time with no change in glycemic indices ^[32].

Results of the clinical trial of PKC inhibitor in diabetic neuropathy have also been reported recently. A 1-year double-blind, randomized, placebo-controlled trial with PKC inhibitor at 32 mg or 64 mg was carried out in 205 type 1 or 2 diabetic patients with peripheral neuropathy. The results showed that PKC inhibitor improved both neuropathic symptons and vibration detection threshold ^[33]. The Protein Kinase C Diabetic Retinopathy Study (PKC-DRS), a multi-national, multi-center, placebo-controlled, randomized, double-masked, 4-arm clinical trial designed to evaluate the effects of PKC inhibitor on the progression of diabetic retinopathy is also nearing completion and its results should be available soon ^[32]. Beckman et al. have reported that PKC β isoform inhibitor, LY333531, was able to normalize endothelial-induced vasodilation in hyperglycemia-induced endothelial dysfunction ^[34].

SUMMARY

Emerging evidence has accumulated to indicate that PKC-MAPK activation can cause many of the pathophysiological abnormalities associated with the development and progression of diabetic nephropathy ^[35]. The ability of PKC inhibitors such as vitamin E, thiazolidinediones, and PKC β specific inhibitor to prevent diabetes induced glomerular hyperfiltration, albuminuria, and glomerular overexpression of TGF-β and extracellular matrix components suggests that PKC-MAPK activation induced by diabetes and hyperglycemia lies in intracellular signaling pathway leading to these abnormalities (Fig. 2).

REFERENCES

1 The 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 Med 329: 977-86, 1993

2 UK Prospective Diabetes Study (UKPDS) Group: Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet 352: 837-53, 1998

3 Ohkubo Y, Kishikawa H, Araki E, Miyata T, Isami S, Motoyoshi S, Kojima Y, Furuyoshi N, Shichiri M.: Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract. 28:103-17, 1995

4 Lewis EJ, Hunsicker LG, Bain RP, Rohde RD. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N Engl J Med 329:1456-62, 1993

5. Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi G, Snapinn SM, Zhang Z, Shahinfar S; RENAAL Study Investigators. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med 345:861-69, 2001

6. Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, Ritz E, Atkins RC, Rohde R, Raz I; Collaborative Study Group. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med 345:851-60, 2001

7 Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes 47: 859-66, 1998

8 Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, Kikkawa R. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes 46: 847-53, 1997

9 Haneda M, Kikkawa R, Arimura T, Ebata K, Togawa M, Maeda S, Sawada T, Horide N, Shigeta Y. Glucose inhibits myo-inositol uptake and reduces myo-inositol content in cultured rat glomerular mesangial cells. Metabolism 39: 40-45, 1990

10 Williamson JR, Chang K, Frangos M, Hasan KS, Ido Y, Kawamura T, Nyengaard JR, van den Enden M, Kilo C and Tilton RG: Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 42: 801-13, 1993

11 Brownlee M, Cerami A and Vlassara H: Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 318: 1315-21, 1988

12 Abbott KC, Bakris GL: Treatment of the diabetic patient: focus on cardiovascular and renal risk reduction. Prog Brain Res. 139:289-98, 2002

13 Sharma K and Ziyadeh FN: Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-beta as a key mediator. Diabetes 44: 1139-46, 1995

14 Nishizuka Y: Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9: 484-96, 1995

15 Craven PA, Davidson CM and DeRubertis FR: Increase in diacylglycerol mass in isolated glomeruli by glucose from de novo synthesis of glycerolipids. Diabetes 39: 667-74, 1990

16 Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W and King GL: Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci U S A 89: 11059-63, 1992

17 Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell SE, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP and King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 272: 728-31, 1996

18 Inoguchi T, Xia P, Kunisaki M, Higashi S, Feener EP and King GL: Insulin's effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues. Am J Physiol 267: E369-79, 1994

19 Haneda M, Kikkawa R, Koya D, Uzu T, Maeda S, Togawa M, Shigeta Y: Alteration of mesangial response to ANP and angiotensin II by glucose. Kidney Int. 44:518-26, 1993

20 Koya D, Jirousek MR, Lin YW, Ishii H, Kuboki K and King GL: Characterization of protein kinase C beta isoform activation on the gene expression of transforming growth factor-beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. J Clin Invest 100: 115-26, 1997

21 Babazono T, Kapor-Drezgic J, Dlugosz JA, Whiteside C: Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat glomerular cells. Diabetes. 47:668-76, 1998

22 Kikkawa R, Haneda M, Uzu T, Koya D, Sugimoto T and Shigeta Y: Translocation of protein kinase C alpha and zeta in rat glomerular mesangial cells cultured under high glucose conditions. Diabetologia 37: 838-41, 1994

23 Fumo P, Kuncio GS, Ziyadeh FN: PKC and high glucose stimulate collagen alpha 1 (IV) transcriptional activity in a reporter mesangial cell line. Am J Physiol. 267(4 Pt 2):F632-8, 1994

24 Shankland SJ, Scholey JW: Expression of growth-related protooncogenes during diabetic renal hypertrophy. Kidney Int. 47:782-8, 1995

25 Kreisberg JI, Radnik RA, Ayo SH, Garoni J, Saikumar P: High glucose elevates c-fos and c-jun transcripts and proteins in mesangial cell cultures. Kidney Int. 46:105-12, 1994

26 Tomlinson DR: Mitogen-activated protein kinases as glucose transducers for diabetic complications. Diabetologia. 42:1271-81, 1999

27 Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, Kuboki K, Meier M, Rhodes CJ and King GL: Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest 103: 185-95, 1999

28 Isono M, Cruz MC, Chen S, Hong SW, Ziyadeh FN: Extracellular signal-regulated kinase mediates stimulation of TGF-beta1 and matrix by high glucose in mesangial cells. J Am Soc Nephrol. 11:2222-30, 2000

29 Koya D, Lee IK, Ishii H, Kanoh H and King GL: Prevention of glomerular dysfunction in diabetic rats by treatment with d-alpha-tocopherol. J Am Soc Nephrol 8: 426-35, 1997

30 Isshiki K, Haneda M, Koya D, Maeda S, Sugimoto T and Kikkawa R: Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats. Diabetes 49: 1022-32, 2000

31 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 J. 14:439-47, 2000

32 Aiello LP: The Potential Role of PKC beta in Diabetic Retinopathy and Macular Edema. Surv Ophthalmol 47 Suppl 2: S263-9, 2002

33 Vinik AI: Neuropathy: new concepts in evaluation and treatment. South Med J 95: 21-3, 2002

34 Beckman JA, Goldfine AB, Gordon MB, Garrett LA and Creager MA: Inhibition of protein kinase Cbeta prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circ Res 90: 107-11, 2002

35 Haneda M, Koya D, Isono M, Kikkawa R: Overview of glucose signaling in mesangial cells in diabetic nephropathy. J Am Soc Nephrol. 14:1374-82, 2003