A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients RODRIGO GONZÁLEZ1, ARES TIRADO1, MONSERRAT BALANDA1, MIRIAM ALVO2, INÉS BARQUIN3, PILAR DURRUTY4, SERGIO LOBOS1 and DANIELA

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls. Key terms: GAGA box, gene promoter, nephrin, polymorphism. Corresponding author: Daniela Seelenfreund, Laboratorio de Bioquímica, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile. Vicuña Mackenna 20, Santiago de Chile, Chile. e-mail: [email protected], Fax: 56-2-222-7900, Phone: 56-2-978-1677 Received: April 13, 2007. Accepted: October 16, 2007 INTRODUCTION Diabetic nephropathy (DN) is one of the major complications of chronic diabetes, greatly affecting the quality of life and survival of diabetic patients. As global prevalence of type 2 diabetes is steadily increasing, the social and economic impact of patients with renal complications is an ever growing burden on health systems. DN is now the leading cause of end stage renal disease (ESRD), a disease that is reaching epidemic proportions and whose growing incidence is associated to diabetes (ADA, 2004, Wolf et al., 2005 and Jones et al., 2005). DN is explained in part by a poor metabolic control and high arterial blood pressure. Yet, there is also evidence for a genetic involvement in the pathogenesis of diabetic nephropathy. Risk factors include constitutional factors such as low birthweight; haemodynamic factors, including activation of the renin-angiotensin system (RAS) and hypertension, metabolic factors such as hyperglycaemia, genetic and additional factors such as urinary albumin excretion rate (AER) and smoking (Rossing, 2006). Among the genetic factors, the involvement of several candidate genes in the development of DN has been studied, namely diabetes susceptibility genes, nephrin gene, growth factor genes, glucose metabolism genes and genes of the RAS, such as angiotensin converting enzyme (ACE); nevertheless no clear picture has yet emerged from these studies (Rossing, 2006). GONZÁLEZ ET AL. Biol Res 40, 2007, 357-364 358 NPHS1 codes for nephrin, a protein that is expressed mainly in the kidney (Routsalainen et al., 1996; Holthofer et al. 1999; Holzman et al., 1999), but also in pancreas (Zanone et al, 2005) and in the central nervous system (Beltcheva et al., 2003). In the kidney, nephrin is expressed in glomeruli and is located at the slit diaphragm, where it plays a crucial role in the renal filtration barrier at the foot processes of podocytes (Routsalainen et al., 1996, Kestilä et al., 1998). The human nephrin gene NPHS1 was mapped on chromosome 19q13.1 (Kestilä et al., 1994) and found to cause the Finnish type congenital nephrotic syndrome (CNF) (Lenkkeri et al., 1999). At least 60 diseasecausing mutations in the nephrin gene have been identified and result in a lack of functional protein, some of them leading to massive proteinuria already in utero (Lenkkeri et al., 1999; Beltcheva et al., 2001; Liu et al., 2001). Exonic polymorphisms in the nephrin gene have been associated with diverse acquired proteinuric kidney diseases and failure at the glomerular slit diaphragm (Kim et al., 2002, Koop et al., 2003), as well as ectopic glomerular nephrin expression in different pathogenic conditions (Doublier et al., 2001). The FinnDiane study of the three nonsynonymous polymorphisms (E117K, R408Q and N1077S searched for an association of polymorphisms of the nephrin gene with DN; it however found no involvement of these particular coding regions in this disease in a large Finnish population of type 1 diabetic patients (Petterson-Fernholm et al., 2003). Another study showed that two silent and one intronic polymorphism on the nephrin gene are associated with type 2 diabetes in a Japanese population, suggesting that these variations may have a functional influence on nephrin expression in the pancreas (Daimon et al., 2006). DN is clinically characterized by proteinuria and progressive renal insufficiency and diagnosed through the measurement of albuminuria (Durruty, 2003). The presence of albuminuria >30 mg/24 hrs indicates renal damage and is also associated with functional changes in the filtration barrier (Wolf et al., 2005). In general, nephrin protein production is decreased in most proteinuric kidney diseases (Koop et al., 2003), and also an impairment of nephrin assembly with other podocyte proteins, such as podocin and C2AP, has been reported (Benigni et al., 2004). In diabetic patients, nephrin mRNA is significantly reduced in the kidney compared to control subjects (Toyoda et al., 2004). The decreased expression of the nephrin gene in DN suggests that its regulation at a transcriptional level could be impaired. A purine-rich element has been described as an important transcriptional control region in the insulin gene promoter (Kennedy and Rutter, 1992). Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the NPHS1 promoter region. In this work we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study on 100 individuals in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. MATERIALS AND METHODS