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NDT Advance Access originally published online on April 6, 2009
Nephrology Dialysis Transplantation 2009 24(7):2021-2023; doi:10.1093/ndt/gfp150
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© The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Inhibition of protein kinase C in diabetic nephropathy—where do we stand?

Jan Menne, Matthias Meier, Joon-Keun Park and Hermann Haller

Department of Nephrology, Hanover Medical School, Hanover, Germany

Correspondence and offprint requests to: Jan Menne; E-mail: menne.jan{at}mh-hannover.de

Keywords: diabetic nephropathy; protein kinase C

Hyperglycaemia plays a key role in the pathogenesis of microvascular diabetic complications. More than 20 years ago, it was described that the activation of the protein kinase C (PKC) system by hyperglycaemia may represent an important mediator of glucotoxicity in diabetic nephropathy [1,2]. The putative intracellular mechanism is the glucose-induced de novo synthesis of diacylglycerol that is one of the intracellular activators of PKC. Although hyperglycaemia is a major PKC activator, several other PKC stimuli related to the diabetic state such as increased production of reactive oxygen species, free fatty acids or various growth factors and angiotensin II have been identified over the last two decades [3,4] (Figure 1). Thus, inhibition of PKC to prevent complications of diabetes is an attractive hypothesis. However, there are several obstacles to such a strategy.


Figure 1
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  		Fig. 1 In the diabetic milieu, metabolic and haemodynamic factors lead to PKC activation and vice versa. Although initiated by the same external stimuli, mainly hyperglycaemia, intracellular signalling cascades activate distinct PKC isoforms each with its own specific role. Thus, PKC isoform specificity and cellular diversity seem to be responsible for the diverge outcome leading to albuminuria and/or renal fibrosis. Importantly, some isoforms, such as PKC{varepsilon}, seem to be protective and should therefore not be medically inhibited. AGE’s: advanced glycation endproducts; RAS: renin–angiotensin system; HSPG: heparin sulphate proteoglycan; VEGF: vascular endothelial growth factor; TGFβ1: transforming growth factor; CTGF: connective tissue growth factor.

 
PKC constitutes a family of homologous serine/threonine kinases that are involved in many signalling events [5]. In mammals, a gene family of nine independent gene loci are distributed over the whole genome [6]. Due to biochemical properties and sequence homologies, the PKC family is divided into classical ({alpha}, β I, β II, {gamma}), novel ({delta}, {varepsilon}, {eta}, {theta}) and atypical ({zeta}, {iota}/{lambda}) isoforms. Despite a similar structure and overlapping substrate specificities in vitro, isoform specificity under in vivo conditions is remarkable and defined via unique expression patterns, intracellular localization and specific binding partners of the isoforms [5,7]. Initially, based on experimental in vitro data, PKCβ was favoured as the main culprit of hyperglycaemia-induced cellular damage [8]. However, soon other PKC isoforms were implicated in the cellular response to hyperglycaemia. The ensuing debate has not been completely resolved; however, recently the analysis of PKC isoform-specific knock-out mice has helped to clarify the role of the different isoforms in diabetic nephropathy [9].

PKC{alpha} seems to be important in the regulation of the glomerular barrier and albuminuria. The analysis of PKC{alpha} knock-out mice showed that these mice when made hyperglycaemic do not develop albuminuria [10]. Indeed, deletion of PKC{alpha} in vivo abolished nephrin loss in STZ-induced murine diabetic nephropathy [11]. Furthermore, PKC{alpha} deficiency prevented VEGF upregulation and the loss of negatively charged heparin sulfate proteoglycans (HSPG) under diabetic conditions [10]. The effect on VEGF is interesting because it is known that VEGF blockade by antibodies reduces albuminuria in type 1 and 2 diabetic animal models [12,13]. Surprisingly, glomerular and renal hypertrophy was not affected by deletion of PKC{alpha} [10].

This function seems to be occupied by PKCβ. PKCβ knock-out mice are protected against the development of renal and glomerular hypertrophy as well as mesangial expansion, and transforming growth factor-beta 1 (TGFβ1), connective tissue growth factor (CTGF) and matrix molecule expression are reduced under diabetic conditions [14,15]. However, albuminuria is not prevented in PKCβ-deficient mice [14,15]. Consistent with this finding is the observation that neither the loss of nephrin nor the upregulation of VEGF is abolished in diabetic PKCβ knock-out mice [15].

In addition to the described molecular effects of these two isoforms, it is noteworthy that PKC{alpha} and β are involved in many intracellular molecular pathways important for the development of diabetic complications. Several authors have demonstrated that PKC{alpha} as well as PKCβ activation is associated with increased NADPH activity and NADPH-dependent superoxide production [14,16]. As excessive production of reactive oxygen species has been implicated in the pathogenesis of diabetic nephropathy, this observation might be clinically relevant. It has also been demonstrated that advanced glycation endproducts (AGE’s) mediate some of their damaging effects via PKC{alpha} signalling [17].

These experimental data suggest that it is necessary to inhibit PKC{alpha} and β simultaneously in the treatment of diabetic nephropathy; otherwise important components of long-term kidney damage (albuminuria, interstitial structural changes and/or reactive oxygen stress) will not be sufficiently inhibited (Figure 1). Interestingly, inhibition of PKC{alpha} seems not only to protect the kidney but also to preserve heart function and to prevent heart failure [18]. Recent work has also shown that PKC{alpha} activation leads to arterial thrombus formation [19] making PKC{alpha} inhibition even more appealing as a new treatment strategy in diabetic patients.

What do these experimental data tell us with regard to the studies in diabetic patients with PKC inhibition? Based on the original findings by King and co-workers, a specific PKCβ inhibitor was developed and used in clinical trials [20]. However, the initial results of these studies are not completely convincing. Treatment with the selective PKCβ inhibitor ruboxistaurin (LY333531, Arxxant®) did not clearly prevent diabetic nephropathy in human trials. In a double-blind, placebo-controlled study in type 2 diabetic patients with proteinuria, the effect of 32 mg ruboxistaurin (n = 59) versus placebo (n = 62) as add-on medication to standard treatment with an ACE inhibitor or angiotensin II receptor blocker was analysed [21]. After 1 year, the UACR was reduced by 24% from baseline in the active treatment arm (P < 0.02) versus 9% in the placebo group (n.s.). The eGFR decline was less rapid in the active treatment group (–2.5 ± 1.9 ml/min versus –4.8 ± 1.8 ml/min) [21]. However, no significant difference between placebo and active treatment was demonstrated for either of the two mentioned parameters and it was discussed that the statistical power of this study was not sufficient. To overcome this problem, a post hoc analysis of the renal outcome from three diabetic retinopathy trials (PKC-DRS, PKC-DME, PKC-DRS2) was published more recently [22]. In these studies, patients with diabetic eye disease were treated with either 32 mg ruboxistaurin (n = 580) or placebo (n = 577) for an average of 3 years with moderate improvement of vision. Disappointingly, renal function was not improved. Doubling of the serum creatinine was similar to 5.9% versus 6.1%, and the decline in eGFR/year was comparable. Furthermore, the number of patients who had micro- or macroalbuminuria at the end of the study was similar [22]. However, this analysis has several limitations (post hoc analysis, nearly normal renal function at baseline, a high drop-out rate, missing baseline proteinuria levels and UACR measurements only at the end of the DRS-2 study). These data and the data from the prospective intervention study still suggest that ruboxistaurin has only little effect on albuminuria [22,23].

This is not completely surprising. Taking into account the results from the above-mentioned studies in gene-deficient mice, inhibition of PKCβ should only have a small effect on albuminuria. A specific inhibition of PKC{alpha} would be needed. Interestingly, in the clinical setting, ruboxistaurin treatment prevented an increase of the urinary TGFβ1 excretion [24] supporting the concept that PKCβ inhibition is important to prevent interstitial fibrosis development. Therefore, a larger and longer prospective ruboxistaurin trial might have resulted in a significant clinical finding in patients with existing diabetic nephropathy.

There is still one problem with regard to specificity of ruboxistaurin. In contrast to the finding in the human trials, ruboxistaurin was able to prevent albuminuria in the streptozotozin rat model of type 1 diabetes [20,25], in the db/db mice model of type 2 diabetes [26] and in hypertensive mREN-2 transgenic diabetic rats [27]. So, why does ruboxistaurin prevent albuminuria in rodent models but not in humans? One explanation is a decreased selectivity for the two isoforms in rodents. The PKCβ selectivity of ruboxistaurin was determined using a PKC activity assay performed with cloned human PKC isoforms. Even if these PKC isoforms are strongly conserved across several species they are not completely homologous [28]. Therefore, it might well be that ruboxistaurin inhibits not only PKCβ but also other closely related PKC isoforms, e.g. PKC{alpha}, in rodents. Such an effect could explain why ruboxistaurin is able to prevent albuminuria in rodents more efficiently than in humans.

What do we need for a bright ‘PKC future’? As suggested above, new highly selective PKC{alpha}/β should be developed and we believe that such compounds would overcome the limitations observed with the selective PKCβ inhibitor ruboxistaurin. Unpublished data from the analysis of PKC{alpha}/β double knock-out mice and with a PKC inhibitor are supporting such an approach. However, inhibition of PKC{varepsilon} should be strictly avoided, as hyperglycaemia-induced PKC{varepsilon} activation protects against the development of albuminuria and glomerulosclerosis [29]. Lately, the three-dimensional structures of distinct PKC isoforms have been identified that will certainly provide valuable information for more selective drug design [6,30].

In summary, the existing data obtained by analysing PKC isoform-specific knock-out mice and use of the PKCβ inhibitor ruboxistaurin suggest that the diabetes-induced activation of PKC{alpha} is crucial for the development of albuminuria, whereas PKCβ activation is important for mesangial expansion, basement membrane thickening and renal hypertrophy. Therefore, simultaneous inhibition of PKC{alpha} and β seems to be a very promising new treatment strategy for the prevention and treatment of diabetic nephropathy. The story of PKC inhibition in diabetic nephropathy shows both the beauty and the problems of translational research in medicine.

Conflict of interest statement. J. M. and H. H. are shareholders of Phenos GmbH.



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Received for publication: 9. 3.09
Accepted in revised form: 13. 3.09


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