Original Contribution
Aldose reductase inhibition counteracts nitrosative stress and poly(ADP-ribose) polymerase activation in diabetic rat kidney and high-glucose-exposed human mesangial cells

https://doi.org/10.1016/j.freeradbiomed.2005.12.034Get rights and content

Abstract

Both increased aldose reductase (AR) activity and oxidative/nitrosative stress have been implicated in the pathogenesis of diabetic nephropathy, but the relation between the two factors remains a subject of debate. This study evaluated the effects of AR inhibition on nitrosative stress and poly(ADP-ribose) polymerase (PARP) activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. In animal experiments, control (C) and streptozotocin-diabetic (D) rats were treated with/without the AR inhibitor fidarestat (F, 16 mg kg−1 day−1) for 6 weeks starting from induction of diabetes. Glucose, sorbitol, and fructose concentrations were significantly increased in the renal cortex of D vs C (p < 0.01 for all three comparisons), and sorbitol pathway intermediate, but not glucose, accumulation, was completely prevented in D + F. F at least partially prevented diabetes-induced increase in kidney weight as well as nitrotyrosine (NT, a marker of peroxynitrite-induced injury and nitrosative stress), and poly(ADP-ribose) (a marker of PARP activation) accumulation, assessed by both immunohistochemistry and Western blot analysis, in glomerular and tubular compartments of the renal cortex. In vitro studies revealed the presence of both AR and PARP-1 in human mesangial cells, and none of these two variables were affected by high glucose or F treatment. Nitrosylated and poly(ADP-ribosyl)ated proteins (Western blot analysis) accumulated in cells cultured in 30 mM D-glucose (vs 5.55 mM glucose, p < 0.01), but not in cells cultured in 30 mM L-glucose or 30 mM D-glucose plus 10 μM F. AR inhibition counteracts nitrosative stress and PARP activation in the diabetic renal cortex and high-glucose-exposed human mesangial cells. These findings reveal new beneficial properties of the AR inhibitor F and provide the rationale for detailed studies of F on diabetic nephropathy.

Introduction

Diabetes accounts for at least ∼35% of all new cases of end-stage renal disease in the United States [1], and diabetic patients make up the fastest growing group of renal dialysis and transplant recipients. The Diabetes Control and Complications Trial [2] and the U.K. Prospective Diabetes Study [3] strongly suggest the importance of hyperglycemia in the pathogenesis of chronic complications of diabetes mellitus including diabetic renal disease. Hyperglycemia leads to diabetic nephropathy (DN) via multiple mechanisms, and among them increased aldose reductase (AR) activity [4], [5], nonenzymatic glycation and glycooxidation [6], [7], activation of protein kinase C (PKC) and hexosamine pathway [8], [9], [10], arachidonic acid metabolism via 12/15-lipoxygenase pathway [11], [12], and triose phosphate accumulation [13] are the best studied. Growing evidence obtained in diabetic animals (primarily, STZ (streptozotocin)-diabetic rats and mice) [14], [15], [16], [17] as well as cell culture models [18], [19], [20] implicates free radicals and the potent oxidant peroxynitrite (a product of superoxide anion radical reaction with nitric oxide) in both hemodynamic and metabolic abnormalities leading to DN.

Oxidative stress affects all three compartments of the renal cortex, i.e., glomeruli [21], tubulo-interstitium [22], and vasculature [23]. Renal hydrogen peroxide overproduction and lipid peroxide accumulation occur at a very early stage of STZ-diabetes [24] and are associated with clearly manifest impairment of antioxidative defense and, in particular, GSH and ascorbate (AA) depletion, changes in glutathione and ascorbate redox states reflecting in increased oxidized glutathione(GSSG)/GSH and dehydroAA/AA ratios, and upregulated superoxide dismutase, GSH peroxidase, GSH transferase, and GSSG reductase activities [14], [25]. Enhanced lipid peroxidation and GSH depletion have also been documented in the model of advanced DN [15]. Evidence for the presence of peroxynitrite-induced injury in the diabetic kidney is emerging [17], [26].

Oxidative-nitrosative stress triggers several important downstream mechanisms, i.e., activation of mitogen-activated protein kinases [20], [27], the nuclear transcription factor NF-κB [28], [29], and upregulation of growth factors such as transforming growth factor-β (TGF-β) [14], [15], cytokines [30], [31], and vascular endothelial growth factor [32] implicated in diabetic renal disease [12], [27], [28], [29], [33], [34]. In vitro and in vivo studies in nondiabetic models of oxidative injury as well as animal and cell culture models of diabetic complications revealed that free radical and peroxynitrite-induced DNA single-strand breakage is also responsible for activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and resultant energy failure, profound metabolic imbalances, and changes in transcriptional regulation and gene expression [35], [36], [37]. Recent studies including those from our group generated evidence of an important role of PARP activation in diabetic endothelial [38] and myocardial [39] dysfunction, peripheral neuropathy [40], [41], and retinopathy [42], [43]. We have also demonstrated (1) tubular PARP activation manifested by increased poly(ADP-ribose) immunoreactivity, and (2) the key role of PARP in upregulation of endothelin-1 and endothelin (A) and (B) receptors, known to play an important role in DN, in the renal cortex of STZ-diabetic rats with 4-week duration of diabetes [44].

The interactions among various hyperglycemia-initiated mechanisms are not completely understood, and the relation between increased AR activity and oxidative-nitrosative stress/PARP activation in the renal cortex has never been explored. The present study was designed to evaluate the effect of pharmacological AR inhibition with the potent and highly specific AR inhibitor (ARI) fidarestat [45], [46], [47] on nitrosative stress and PARP activation in diabetic rat kidney and high-glucose-exposed human mesangial cells. Our animal studies performed in the STZ-diabetic rat model as well as in vitro studies in high-glucose-exposed human mesangial cells provide evidence of the major contribution of increased AR activity to diabetes- and hyperglycemia-associated nitrosative stress and PARP activation in the renal cortex, and, specifically, the key cell target in DN, i.e., glomerular mesangial cells. Furthermore, they identify abundant AR protein expression and early high-glucose-induced peroxynitrite formation and PARP activation in human mesangial cells, thus suggesting the importance of these mechanisms in human DN.

Section snippets

Reagents

Unless otherwise stated, all chemicals were of reagent-grade quality, and were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol (HPLC grade), perchloric acid, hydrochloric acid, and sodium hydroxide were obtained from Fisher Scientific (Pittsburgh, PA). Reagents for immunohistochemistry were purchased from Vector Laboratories, Inc., Burlingdale, CA and Dako Laboratories, Inc. (Santa Barbara, CA) as specified in the procedures. Monoclonal anti-nitrotyrosine (NT) antibody, clone 1A6

Results

The final body weights were comparably lower in untreated and fidarestat-treated diabetic rats than in the control group (Table 1). The final blood glucose concentrations were similarly elevated in untreated and fidarestat-treated diabetic rats compared with the control rats.

Kidney weights were increased in diabetic rats compared with controls (2.41 ± 0.059 vs 1.79 ± 0.061 g, p < 0.01), and this increase was slightly, but significantly, reduced by fidarestat (2.2 ± 0.088 g, p < 0.01 vs

Discussion

Our results provide evidence of clearly manifest nitrosative stress in early experimental DN. Increased immunoreactivity of NT (a marker of peroxynitrite-induced injury) in both glomerular and tubular compartments of the renal cortex of STZ-diabetic rats in the present study is consistent with elevated renal nitrosylated protein content (assessed by Western blot analysis) in another report [49]. Furthermore, increased NT immunoreactivity has also been documented in vasculature of diabetic rats

Acknowledgments

The study was supported by the National Institutes of Health Grant 1R21DK070720-01, grants from American Diabetes Association and Juvenile Diabetes Research Foundation (all to Irina G. Obrosova), and the Juvenile Diabetes Research Foundation Center for the Study of Complications of Diabetes Grant 4-200-421 (to I. G.Obrosova and M.J. Stevens). The authors are grateful to Drs. D. Carper, R.L. Sorenson, and J.M. Petrash for their gifts of AR antibodies, and human AR enzyme.

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