Original Article Altered Gene Expression Related to Glomerulogenesis and Podocyte Structure in Early Diabetic Nephropathy of db/db Mice and Its Restoration by Pioglitazone Hisashi Makino, 1 Yoshihiro Miyamoto, 1 Kazutomo Sawai, 2 Kiyoshi Mori, 2 Masashi Mukoyama, 2 Kazuwa Nakao, 2 Yasunao Yoshimasa, 1 and Shin-ichi Suga 3 Glomerular injury plays a pivotal role in the development of diabetic nephropathy. To elucidate molecular mecha- nisms underlying diabetic glomerulopathy, we compared glomerular gene expression proﬁles of db/db mice with those of db/m control mice at a normoalbuminuric stage characterized by hyperglycemia and at an early stage of diabetic nephropathy with elevated albuminuria, using cDNA microarray. In db/db mice at the normoalbuminuric stage, hypoxia-inducible factor-1 (HIF-1), ephrin B2, glomerular epithelial protein 1, and Pod-1, which play key roles in glomerulogenesis, were already upregulated in parallel with an alteration of genes related to glucose metabolism, lipid metabolism, and oxidative stress. Podo- cyte structure-related genes, actinin 4 and dystroglycan 1 (DG1), were also signiﬁcantly upregulated at an early stage. The alteration in the expression of these genes was conﬁrmed by quantitative RT-PCR. Through pioglitazone treatment, gene expression of ephrin B2, Pod-1, actinin 4, and DG1, as well as that of oxidative stress and lipid metabolism, was restored concomitant with attenuation of albuminuria. In addition, HIF-1 protein expression was partially attenuated by pioglitazone. These results suggest that not only metabolic alteration and oxidative stress, but also the alteration of gene expression related to glomeru- logenesis and podocyte structure, may be involved in the pathogenesis of early diabetic glomerulopathy in type 2 diabetes. Diabetes 55:2747–2756, 2006 D iabetic nephropathy is the leading cause of end-stage renal disease in the U.S., Japan, and most of Europe (1). Clinical features of diabetic nephropathy are development of albuminuria followed by persistent proteinuria and, later, reduction of glomerular ﬁltration rate (2). Increased thickness of glo- merular basement membrane and augmentation of glomer- ular extracellular matrix are recognized as pathological hallmarks of diabetic nephropathy (2). Thus, glomerular injury is apparently critical for the initiation and progres- sion of the disease. Several pathways are postulated as potential mechanisms of diabetic nephropathy, including renal hemodynamic changes, accretion of advanced glyca- tion end products, intracellular accumulation of sorbitol, oxidation of glycoproteins by reactive oxygen species, and activation of protein kinase C (2,3). Recently, much atten- tion has been paid to the role of podocyte injury in glomerular diseases, including diabetic nephropathy (3– 6). However, the precise molecular mechanisms underly- ing diabetic glomerulopathy still remain unclear. Microarray is a novel tool by which whole-genome analysis can identify new genes and pathways that are important for the pathophysiology of diabetic nephropathy (7). Although several laboratories recently performed cDNA microarray analyses of diabetic kidney (8 –12), most of them examined gene expression of whole kidney, despite the importance of glomerular injury in diabetic nephropathy. In addition, analysis of whole kidney often makes it difﬁcult to select genes associated with diabetic glomerulopathy because glomeruli occupy only a small part of the kidney. Only one of these reports showed the gene expression proﬁle of glomeruli (12). However, be- cause the report analyzed glomeruli from advanced dia- betic nephropathy patients with apparent histological changes, it did not provide much information about the mechanism of early diabetic glomerulopathy. In this study, we performed microarray analysis using isolated glomeruli from diabetic mice at a normoalbumin- uric stage and an early stage of diabetic nephropathy with no apparent histological change in order to ﬁnd the genes that are strongly associated with diabetic glomerular in- jury. This approach also enabled us to avoid the modiﬁca- tion of gene expression proﬁles by cell component alteration. We analyzed db/db mice, a genetic model of type 2 diabetes with obesity and insulin resistance (13), because they exhibited histological changes resembling those in human diabetic nephropathy (13,14). Because accumulating evidence indicates that insulin resistance participates in the pathogenesis of diabetic nephropathy in type 2 diabetes (15), we also examined the effects of pioglit- azone, one of the insulin sensitizers that improves insulin sensitivity, on the gene expression proﬁle of db/db mice. RESEARCH DESIGN AND METHODS Male diabetic db/db mice and their nondiabetic db/m littermates were used for this study. All mice were purchased from CLEA Japan (Tokyo). These db/db From the 1 Department of Atherosclerosis and Diabetes, National Cardiovas- cular Center, Suita City, Osaka, Japan; the 2 Department of Endocrinology and Metabolism, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the 3 National Cardiovascular Center Research Institute, Suita City, Osaka, Japan. Address correspondence and reprint requests to Shin-ichi Suga, MD, PhD, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita City, Osaka 565-8565, Japan. E-mail: s-suga@umin.ac.jp. Received for publication 27 December 2005 and accepted in revised form 27 June 2006. DG1, dystroglycan 1; GLEPP1, glomerular epithelial protein 1; HIF-1, hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor. DOI: 10.2337/db05-1683 © 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. DIABETES, VOL. 55, OCTOBER 2006 2747