Early in diabetic nephropathy glomerular mesangium and renal tubules hypertrophy. Mesangial hypertrophy is an important, perhaps reversible, step preceding glomeruloscierosis and overt nephropathy. Using unique transgenic mice overexpressing retinoblastoma protein (Rb) and mesangial cells expressing Rb from a tetracycline-confrollable promoter system, we have discovered that renal and glomerular mesangial cell hypertrophy occur with both diabetes mellitus and with over expression of hypophosphorylated Rb. Preliminary data has shown that when Rh is over expressed, neither kidneys nor mesangial cells hypertrophy further in diabetic conditions. Our hypothesis to explain these observations is that excess glucose results in specific, cyclin-dependent phosphorylation of Rb protein during early Gi phase, and that Rb is involved distally in a pathway of glomerular mesangial cell hypertrophy. To test this hypothesis, three Specific Aims are proposed: (1) Determine whether renal and glomerular hypertrophy caused by diabetes mellitus in vivo and mesangial cell hypertrophy caused by high glucose concentrations ex vivo are dependent on activation of specific C1 phase cyclin-cdk complexes and specific phosphorylations of Rb protein. (2) Determine how high glucose regulates cdk4-cyclin Dl activity and Rb phosphorylation in mesangial cells by examining patterns of gene regulation including transcription of the cyclin D1 gene itself and of events controlled by relevant transcription factors including AP-1. (3) Test the requirement for Cl cyclin dependent kinase activation and specific cdk4-dependent phosphorylations of Rb protein for diabetic renal hypertrophy in vivo and mesangial cell hypertrophy in culture. Transgenic mice with strategic phosphorylation sites in Rb inactivated by site-directed mutagenesis will be generated and the effects of type 1 diabetes mellitus will be examined in them. Primary mesangial cells will also be cultured from the mice and more detailed studies on hypertrophy and GI cell cycle regulation by cdks and Rb conducted in vitro.
