Investigation of Genetic and Molecular Basis of Diabetic Nephropathy Susceptibility in Mice

Diabetic nephropathy (DN) is one of the major causes of morbidity and mortality in diabetic patients and also the leading single cause of end stage renal disease (ESRD) in the United States. However, approximately 40% of diabetic patients develop DN, demonstrating a significant genetic component of disease susceptibility and progression. Differential susceptibilities to DN have also been observed between well-defined strains of inbred rodents. For example, DBA/2J mice represent a susceptible, while C57BL/6J mice demonstrate a resistant genetic background to the development of DN. However, in both humans and animal models, the molecular and genetic mechanisms underlying DN susceptibility have not been fully elucidated.;In kidney diseases, progressive loss of filtration and glomerulosclerosis are typically associated with permanent podocyte injury/depletion and expansion of mesangial matrix with mesangial hypertrophy. In this study, we determined that DBA/2J inbred strain manifest podocyte loss in response to diabetes induced by either streptozotocin injection or Insulin2 Akita genetic mutations, while C57BL/6J strain was protected from podocyte loss. When compared with non-diabetic controls, podocyte number per glomerular section area decreased by 27% in diabetic DBA/2J mice, but no reduction of podocytes was observed in diabetic C57BL/6J mice. Thus we demonstrated that DBA/2J strain is susceptible and C57BL/6J is resistant to diabetes-induced podocyte depletion and that actually goes along with the susceptibility to diabetic nephropathy.;In order to characterize glomerular transcriptome profiles associated with differential susceptibility to glomerular manifestations of DN in diabetic DBA/2J mice in comparison with diabetic C57BL/6J mice, glomerular microarray analysis was performed and glomerular transcriptomic profiles were compared between diabetic and non-diabetic mice. Based on pathway analysis of expression profiles, mitochondrial dysfunction and impaired oxidative phosphorylation were characteristic glomerular trancriptome profiles in DN-susceptible diabetic DBA/2J mice, compared with DN-resistant diabetic C57BL/6J mice. These computational predictions were confirmed by functional tests (mitochondrial oxygen consumption rate tests and anti-8 oxoG staining), demonstrating that glomerular mitochondrial dysfunction and oxidative damage were detectable already during initial stages of diabetes in DBA/2J mice, but not in C57BL/6J mice.;To investigate the genetic basis of DN susceptibility, we used the BXD recombinant inbred panel to map genetic loci (QTL) associated with number of podocytes after long-term diabetes (6 months of diabetes). We identified two suggestive QTLs on chromosome 13 (LDPD1) and 17 (LDPD2) influencing the number of podocytes in diabetic mice, in which 32 genes reside. In an effort to determine candidate gene(s) underlying these two QTLs, we utilized extensive available information regarding sequence variants (SNPs) and differential expression of these genes in the C57BL/6J and DBA/2J parental strains, as well as the functional pathway activity and the information of cis-acting expression regulatory QTL mapping of these genes. Based on above criteria, xanthine dehydrogenase (Xdh) was prioritized as the candidate gene, contributing to podocyte reduction and DN susceptibility of DBA/2J mice.;Xdh gene was localized under the peak of the LDPD2 QTL on Chromosome 17. Xdh expression was strongly increased by diabetes in glomeruli of DBA/2J, but not C57BL/6J mice. Xdh and xanthine oxidase (XO) are interconvertible forms of the same enzyme encoded by the Xdh gene. XO is known to produce ROS. To validate a functional role of XO contribution to podocyte depletion and DN, we subjected diabetic DBA/2J mice to XO inhibitor, and found significant amelioration of DN, including reduced albuminuria and podocyte loss in diabetic mice. In addition, protein oxidation damage in the glomeruli of diabetic mice was also reduced by the XO inhibitor treatment. as shown by anti-3 nitrotyrosine staining. Thus, we have identified XDH/XO as a critical player in the DN of DBA/2J mice, probably through altering ROS generation in the glomeruli.