Original Article Poly(ADP-Ribose) Polymerase Inhibitors Ameliorate Nephropathy of Type 2 Diabetic Lepr db/db Mice Csaba Szabo ´, 1 Alisha Biser, 2 Rita Benko ˝ , 3 Erwin Bo ¨ ttinger, 4 and Katalin Suszta ´k 2 The activation of the poly(ADP-ribose) polymerase (PARP) plays an important role in the pathophysiology of various diseases associated with oxidative stress. We found increased amounts of poly(ADP) ribosylated pro- teins in diabetic kidneys of Lepr db/db (BKsJ) mice, suggest- ing increased PARP activity. Therefore, we examined the effects of two structurally unrelated PARP inhibitors (INO- 1001 and PJ-34) on the development of diabetic nephrop- athy of Lepr db/db (BKsJ) mice, an experimental model of type 2 diabetes. INO-1001 and PJ-34 were administered in the drinking water to Lepr db/db mice. Both INO-1001 and PJ-34 treatment ameliorated diabetes-induced albumin ex- cretion and mesangial expansion, which are hallmarks of diabetic nephropathy. PARP inhibitors decreased diabetes- induced podocyte depletion in vivo and blocked hypergly- cemia-induced podocyte apoptosis in vitro. High glucose treatment of podocytes in vitro led to an early increase of poly(ADP) ribosylated modified protein levels. Reactive oxygen species (ROS) generation appears to be a down- stream target of hyperglycemia-induced PARP activation, as PARP inhibitors blocked the hyperglycemia-induced ROS generation in podocytes. INO-1001 and PJ-34 also normalized the hyperglycemia-induced mitochondrial de- polarization. PARP blockade by INO-1001 and PJ-34 pre- vented hyperglycemia-induced nuclear factor-B (NFB) activation of podocytes, and it was made evident by the inhibitor of B phosphorylation and NFB p50 nuclear translocation. Our results indicate that hyperglycemia- induced PARP activation plays an important role in the pathogenesis of glomerulopathy associated with type 2 diabetes and could serve as a novel therapeutic target. Diabetes 55:3004 –3012, 2006 D iabetic nephropathy is the leading cause of end-stage renal disease in the U.S. (1). Charac- teristic morphological lesions of diabetic ne- phropathy initially present in the renal glomerulus; these include glomerular hypertrophy, thick- ening of the basement membrane, and mesangial expan- sion (2). Several interventions have been shown to slow the progression of diabetic nephropathy, including tight glucose and blood pressure control and the blockade of the renin-angiotensin system (3–5). However, none of these can cure or prevent the development of diabetic nephropathy. Recent observations indicate important roles for glo- merular epithelial cells (podocytes) in the pathogenesis of diabetic nephropathy (6 –9). The density of glomerular visceral epithelial cells is reduced in kidneys of individuals with diabetic nephropathy. Among various glomerular morphological characteristics, the decreased podocyte density is one of the strongest predictors of disease progression (10). Apoptosis and detachment of podocytes have been implicated as a potential mechanism of podo- cyte loss in animal models of diabetic nephropathy (7,11). We recently reported increased apoptosis of podocytes in type 1 diabetic Akita and type 2 diabetic Lepr db/db mice at the time of development of hyperglycemia. In vitro treat- ment of podocytes with high glucose also leads to in- creased apoptosis rate (7,12). Podocyte apoptosis seems to contribute significantly to the development of diabetic nephropathy, as prevention of podocyte apoptosis in vivo was associated with a decrease in albuminuria and mes- angial expansion in the Lepr db/db model of type 2 diabetes. Brownlee (13) has pioneered the concept that hypergly- cemia-induced overproduction of superoxide is the single unifying link to diabetes complications, including cellular activation of protein kinase C, hexosamine pathway, and advanced glycation formation, which are the major path- ways of hyperglycemic damage in endothelial cells. This process occurs via inhibition of glyceraldehyde-3-phos- phate dehydrogenase activity, which is likely to be the consequence of poly(ADP) ribosylation of the enzyme by active poly(ADP-ribose) polymerase (PARP)-1 (14). Since uncoupling protein 1 or manganese superoxide dismutase overexpressions blocked the activation of PARP-1, it has been hypothesized that the high-glucose–induced PARP-1 activation is the consequence of the increased intracellular reactive oxygen species (ROS) and subsequent DNA breakage in endothelial cells (14). PARP-1 is one of the most abundant nuclear proteins. The catalytic function of PARP-1 relates to its role as a DNA damage sensor and signaling molecule. The zinc From the 1 Department of Surgery, University of Medicine and Dentistry of New Jersey, Newark, New Jersey; the 2 Division of Nephrology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York; the 3 Depart- ment of Human Physiology and Clinical Experimental Research, Semmelweis University Medical School, Budapest, Hungary; and the 4 Department of Medicine, Mount Sinai School of Medicine, New York, New York. Address correspondence and reprint requests to Katalin Susztak, Division of Nephrology, Albert Einstein College of Medicine, Bronx, NY 10461. E-mail: ksusztak@aecom.yu.edu. Received for publication 1 February 2006 and accepted in revised form 1 August 2006. C.S. is a stockholder of Inotek Pharmaceuticals, a firm involved in the development of PARP inhibitors. Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. ELISA, enzyme-linked immunosorbent assay; IB, inhibitor of B; NFB; nuclear factor-B; PARP, poly(ADP-ribose) polymerase; PAS, periodic acid Schiff; ROS, reactive oxygen species. DOI: 10.2337/db06-0147 © 2006 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3004 DIABETES, VOL. 55, NOVEMBER 2006