Novel mechanisms of diabetic nephropathy

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DESCRIPTION (provided by applicant): Energy status of cells is altered by diabetes; its role in diabetes-induced kidney injury is not well studied. Diabetic nephropathy is characterized by renal hypertrophy and extracellular matrix accumulation. mRNA translation, an energy consuming process, is the rate-limiting step in protein synthesis and is a site of regulation even when gene expression is controlled by transcription. However, the role of energy sensors in regulation of mRNA translation underlying diabetes-induced renal hypertrophy and matrix synthesis has not been addressed. We wish to examine the role of an important energy sensor, AMP-activated protein kinase (AMPK), in renal hypertrophy and matrix accumulation. The rationale for these studies is based on the following preliminary data. Phosphorylation of AMPK and its activity are reduced in association with high glucose-induced increase in protein synthesis and hypertrophy, and increase in matrix protein synthesis in renal cells, as well as, in renal cortex and glomeruli of rodents with type 1 or type 2 diabetes at the stage of renal hypertrophy. Administration of metformin and AICAR restores reduced AMPK phosphorylation and inhibits renal hypertrophy seen in the type 1 diabetic rat. However, the role of AMPK in progressive renal injury in type 1 or type 2 diabetes is not known. We wish to test the hypothesis that AMPK activity regulates renal hypertrophy and matrix accumulation in type 1 and type 2 diabetes. Our Specific Aims are: Specific aim 1. In vitro studies. (A) To explore mediators and signaling pathways involved in high glucose regulation of AMPK phosphorylation. Glomerular epithelial, mesangial and proximal tubular epithelial cells in culture will be studied under conditions of hyperglycemia that induce cell hypertrophy, and stimulate mRNA translation and synthesis of matrix proteins. We will investigate if altered AMP-ATP content, activity of LKB-1, an upstream kinase for AMPK, PI 3-kinase-Akt axis, and PKC-TGF-3 axis serve as upstream cues to regulate changes in AMPK phosphorylation and activity. (B) To investigate AMPK regulation of initiation and elongation phases of mRNA translation. The downstream effects of AMPK in regulation of critical events initiation and elongation phases of mRNA translation will be studied; signaling pathways mediating AMPK effect will be elucidated. (C) To explore the role of AMPK in high glucose induced matrix synthesis by renal cells in the context of augmented mRNA translation of matrix proteins. Polyribosomal assays and in vitro translation assays will be employed to define the role of AMPK. Specific aim 2. In vivo studies. To explore role of AMPK in renal pathology in type 1 or type 2 diabetes. Regulation of AMPK phosphorylation by aforementioned upstream factors and its activity and regulation of downstream effectors will be studied in the early stage (4 -14 days) of renal hypertrophy and established stage (3 months) of matrix accumulation and correlated with functional changes in albuminuria and changes in GFR. The ability of AICAR and metformin to inhibit both the anatomical and functional changes induced by diabetes in rodent models of type 1 and type 2 diabetes will be explored. AMPK alphal knock out mice will be studied for evolution of renal abnormalities in type 1 and type 2 diabetes. These studies may identify AMPK as a novel mediator of injury and a treatment target in diabetic nephropathy.