Lifestyle and overeating with consequent obesity are major triggering factors for type-2 diabetes epidemic but important underlying pathogenic elements appear to be genetic factors. Positive family history confers a 2.4 fold increased risk and 15–25% of first degree relatives of patients with type-2 diabetes develop impaired glucose tolerance. The lifetime risk for type-2 diabetes is 38% if one parent has diabetes and 60% if both the parents are affected 1. Multiple susceptibility genes have been reported in pathogenesis of type-2 diabetes as well as diabetic complications such as nephropathy. Clinical diabetic nephropathy results from glomerular, tubular, interstitial and vascular lesions. Genesis of diabetic kidney disease involves both hemodynamic and metabolic pathways and a subset of diabetic patients who develop nephropathy may have a genetic susceptibility to develop renal injury in response to abnormal physiological milieu that is associated with diabetes. There is interplay of metabolic, biochemical and hemodynamic abnormalities that contribute to development of diabetic renal disease and it has been proposed that renin-angiotensin-aldosterone system (RAAS), nitric oxide, and transforming growth factor (TGF)-β1 pathways are important 2.

As a part of an extensive analysis of genetic susceptibility to diabetic renal disease, we had earlier reported the role of RAAS gene polymorphisms in diabetic CRI. A highly significant association of Met235Thr, and a weaker association of T>C (-344) and G>A (-1903) SNPs with CRI, independent of hypertension, was observed there in 3. It suggested that the effect of RAAS may not be mediated through modulation of hypertension as a crucial mechanism for development of CRI but it may operate by stimulation of chemokines like transforming growth factor β1 (TGFβ1), tumor necrosis factor α (TNFα) and interleukin 1 (IL1). Hyperglycemia and systemic hypertension leads to glomerular hypertension as a result of ineffective preglomerular resistances, which are possibly genetically determined 45. Glomerular hypertension in early stages of nephropathy leads to changes in endothelial and mesangial cells. The resultant increased expression of glucose transporter-1 (GLUT-1) leads to increased production of TGFβ1 and formation of advanced glycation end-products (AGE) and its receptor 67. This promotes deposition of collagens I, III and fibronectin. Activation of mitochondrial protein kinase-C (PKC) also leads to production of TGFβ1, superoxide and peroxynitrite and decreases activity of nitric oxide. This decreased activity increases angiotensin II and angiotensinogen II type-1 receptor which loop back upstream in the cascade increasing glomerular hypertension and expression of TGFβ1 and GLUT-1 thereby continuing the vicious cycle of glomerular injury 2. In addition, angiotensin II also activates PKC and MAPK pathways, which are abundantly implicated in activation of TGFβ1, TNFα and IL1, in various cells 891011. Over expression of TNFα and IL-1 stimulates the expression of chemokines like monocyte chemoattractant protein1 (MCP1) and "Regulated upon activation and normal T cell expressed and secreted" (RANTES) in human mesangial cells 121314. Up regulation of MCP1 and RANTES triggers recruitment of monocytes and macrophage infiltration in glomerulus of diabetic rats 15 and DN subjects 16. Chemokine receptors, CCR2 and CCR5, are the major receptors for MCP1 and RANTES respectively and are expressed on the surface of monocytes. Wada et al 17 have reported that CCR5 positive cells were detected in both the glomeruli and the interstitium of human crescentic glomerulonephritis (GN) and therefore, it is speculated that CCR2 and CCR5 might be involved in the recruitment of macrophages in human DN. CCR2 and CCR5 mediated monocyte recruitment and differentiation to macrophage in the glomerulus and interstitium has been speculated to play a role in development of glomerulosclerosis and fibrosis in progressive diabetic kidney disease 18. Considering such importance of inflammatory genes in development and progression of diabetic renal disease, we tested association of nine SNPs from TGFβ1, TNFα, CCR2, and CCR5 genes with diabetic chronic renal insufficiency (CRI) using a case-control design.

Clinical details of the subjects included in the study are reported and discussed somewhere else (3; See additional file 1). In brief, No significant difference was observed in body-mass index, total serum cholesterol and triglyceride levels between the DM and CRI groups (p > 0.05). However, serum creatinine and blood pressure and proportion of hypertensive individuals and those with diabetic retinopathy were significantly greater (p < 0.05) among diabetic CRI subjects compared to DM controls.

Chronic renal insufficiency due to diabetes is the most frequent cause of death due to end stage renal disease and thus a major health concern world over 23. Contemporary evidences both from in vitro and in vivo studies in experimental animals and renal biopsy specimens from patients with progressive diabetic renal disease suggest that cytokines play crucial role in development and progression of diabetic kidney disease 242526. Moreover, results from our previous report 3 suggested that the effect of RAAS may not be mediated through modulation of hypertension as a crucial mechanism for development of CRI but it may operate by stimulation of inflammatory genes. Therefore, in this case-control study genetic contribution of nine SNPs from TGFβ1, TNFα, CCR2 and CCR5 genes for CRI among individuals with type2 diabetes have been analysed. We observed a significant allelic association of allele "A" of SNP 59029 G>A in chemokine receptor-5 gene with diabetic CRI.

Neither of the two promoter SNPs (G>A -800 and C>T -509) of TGFβ1 gene showed association with CRI in this study. These SNPs have not been tested for association with diabetic nephropathy in any other population and thus no comparison is possible. However, a study reported a significant association of C>T-509 (p = 0.02) SNPs in TGFβ1 gene polymorphisms with chronic kidney failure in European population 27. Another study, which investigated association of two polymorphisms inTGFβ1 gene, reported a significant association of Arg25Pro inTGFβ1 gene with end-stage renal disease 28. However, in our population since SNP Arg25Pro was found to be a minor variant (with frequency of variant allele = 0.01, unpublished data), it was non-informative. Lack of association of CCR2 Val64Ile SNP in our study is similar to other reports from Japanese population 14. As for association of CCR5 gene polymorphisms with CRI, we observed a strong trend of association of 59029 G>A SNP. This is in conformity with two other available reports of association of this marker with DN among Japanese population 1429. Though the allele 'A' is predominant in both control (DM) and case (CRI) groups, in our sample set, the allele frequency is significantly higher in CRI group (P = 0.02) and seems to be predisposing based on OR estimates (OR: 1.39; CI: 1.04–1.84). An enhanced expression of CCR5 by peripheral blood mononuclear cells has been seen in individuals with the CCR5 59029A-genotype 3031, thereby suggesting that the genotype could regulate CCR5 gene expression. This further corroborates our observation of association of allele 'A' with the disease. Considering the important role of CCR5 gene in macrophage infiltration into glomerulus and interstitium, our observation of association of promoter SNP in seems exciting but in view of the sub-optimal power of the sample analysed (G = 29%), a replication study seems warranted.

Following analysis on the two sub groups of CRI patients, one with and the other without proliferative retinopathy (PR), a compound group of genotypes 'GA and AA' of SNP G>A -800 in TGFβ1 gene was found to confer predisposition (P = 0.035; OR: 3.028; CI: 1.079–8.50) only in those CRI patients with proliferative retinopathy (Table 3). TGFβ1 gene has been known to influence almost every pathway implicated in development of diabetic CRI 32. Therefore, considering that TGFβ1 plays a very crucial role in diabetic CRI progression and development, our finding seems promising. However, due to relatively small sample size after sub-grouping in this analysis, we would be cautious in interpreting our results.

In conclusion, our results suggest that 59029 G>A SNP in CCR5 gene may play a role in CRI susceptibility in the Asian Indian population.

The author(s) declare that they have no competing interests.

All authors have read and approved the final Ms. PP was involved in the study design, carried out molecular genetics and statistical analyses, compiled the data, wrote the Ms.; AKT was involved in molecular genetic analysis; KMPK, ACA, AG, and RG were the principal clinical investigators involved in study design, defining exclusion and inclusion criteria of study subjects and were mainly responsible for identification of study subjects from their respective clinical centres; BKT was the principal geneticist and coordinator of the project, involved in conceptualization of the project, study design, oversee complete genetic analyses in the laboratory, critical inputs and finalization of the manuscript.