Diabetic nephropathy (DN) is the major cause of chronic kidney disease and end-stage renal failure (ESRD) in USA as well as for our veterans. Development of new effective therapies is hindered by the lack of a clear disease pathogenesis at the early stage of DN. Loss of podocytes occurs at the early stage of DN and is thought to contribute to the development of DN. The mechanism of podocyte apoptosis remains unclear. Advanced Glycation Endproducts (AGE), generated by high glucose in diabetes, induces ROS production and inflammatory response through the receptor for AGE (RAGE). In diabetic kidneys, podocytes line on AGE-modified GBM and are exposure to both high glucose and circulating AGE. Our preliminary findings show that podocytes cultured either on matrix-bound AGE or with soluble AGE underwent significant apoptosis. The apoptotic effects of AGE were inhibited when RAGE was knocked down by siRNA. AGE stimulated ROS production and scavenge of ROS reduced AGE-induced podocyte apoptosis. AGE reduced AKT phosphorylation leading to the activation of FOXO4. FOXO4 is a key transcription factor mediating AGE/ROS-induced podocyte apoptosis through activation of a pro-apoptotic gene (BIM). AGE suppressed SIRT1, a deacetylase, and therefore increased FOXO4 acetylation leading to activation of pro-apoptotic genes and inhibition of anti-ROS genes. Finally, SIRT1 expression was also suppressed in kidneys of patients with DN. Therefore, we hypothesize that AGE causes podocyte apoptosis, an early event of DN, through inhibition of SRIT1 expression and increase of ROS-mediated FOXO4 acetylation which leads to the activation of pro-apoptotic genes in podocytes. The following specific aims will be determined in our proposal:
in specific aim 1, we will determine the cellular and molecular mechanism of Sirt1/FOXO4 in mediating AGE-induced ROS production and podocyte apoptosis in vitro. We will assess the role of Sirt1 and histone acetyltransferase (p300/CBP) in mediating AGE/ROS-induced FOXO4 acetylation. We will further identify podocyte-specific FOXO4-target genes using ChiP-ChiP assay. We will determine the role of Sirt1 and FOXO4 target genes in podocyte apoptosis.
In specific aim 2, we will assess the role of the Sirt1/FOXO4 in animal models of DN. We will determine whether prevention of AGE formation by pyridoxamine will reduce ROS production and podocyte apoptosis in diabetic db/db mice. We will assess the role of FOXO4 and Sirt1 in the development of DN using FOXO4 knockout mice and podocyte-specific Sirt1 transgenic mice after induction of diabetes by Streptozotocin. We believe that the proposed studies will help us not only to illustrate new mechanisms of podocyte injury but also to identify new targets of intervention in patients with DN.
7. Project Narrative Potential Impact on Veterans health Care: Many Veterans suffer from diabetes and diabetic complications including diabetic kidney disease or nephropathy (DN). DN is the leading cause of End-stage renal failure (ESRD) in our Veterans patient population. In any dialysis center at VA hospital, renal failure in about 50% patients is caused by diabetic kidney disease. The Veterans on chronic dialysis have a high rate of mobility and mortality and poor quality of life. Dialysis also adds additional emotional burden for our Veterans who have been already suffering from PTSD and other pschychological disorders such as major depression. Therefore, the research to identify the better treatment regimes for patients with DN will help to reduce the incidence of ESRD in our Veterans and will have enormous impact on Veterans health care. We believe that our study will provide a new approach for the treatment of patients with DN.