During the early stage of type 1 diabetes mellitus (DM) in the rat, accelerated superoxide anion (O2 -) production fuels degradation of nitric oxide (NO) via peroxynitrite (ONOO.) formation, resulting in protein tyrosine nitration and alterations in vascular reactivity. The mechanisms engendering renal oxidative stress in DM have not been established. We have found that rats with DM exhibit significant increases in both circulating angiotensin II (Angll) levels and renal cortical levels of the Angll type 1 (AT 1) receptor protein. AT 1 receptor activation can provoke O2.- production via NAD(P)H oxidase activation. We have also found evidence of reduced Hsp90 levels in the renal cortex of rats with DM. Emerging evidence indicates that Hsp90 represents an essential step in NO production, functioning at least in part to limit O2.- production by NO synthase (NOS). On the basis of these preliminary observations, we hypothesize that NAD(P)H oxidase activation and NOS-uncoupling contribute to renal cortical oxidative and nitrosative stress in DM, with consequent effects on NO bioavailability and action that ultimately diminish its impact on renal function. The STZ-induced model of DM in the rat will be utilized to evaluate: 1) the role of Angll-dependent NAD(P)H oxidase activation in the renal cortical O2.- production during DM; 2) the contribution of Hsp90- or BH4-dependent NOS-uncouplin9 to renal cortical O2.- production during DM; and 3) the potential role of tyrosine nitration of guanylyl cyclase in altering in renal microvascular function during DM. Tin accord with RFA-DK-O2-O23, this application requests support for a collaborative partnership between two independent, established investigators - Pamela K. Carmines (a renal physiologist with expertise in the renal microvascular complications of DM)) and Jennifer S. Pollock (a protein chemist with particular expertise in NOS). Drs. Carmines and Pollock recently documented their success as collaborators in a joint publication providing much of the preliminary data for this project. Completion of the proposed work should position Dr. Pollock as an active diabetes researcher. Funding of this research partnership would also strengthen the ongoing efforts of Dr. Carmines' laboratory by facilitating access to expertise and technologies that are complementary to those currently available to her laboratory for studies of renal microvascular function and oxidative stress in DM. The proposed collaboration thus allows a biochemical, cellular, and functional approach that could unveil novel targets for therapeutic interventions that limit the development or consequences of oxidative and nitrosative stress during the early stage of DM, thereby delaying or preventing development of diabetic nephropathy.
