There is considerable indirect evidence that in established chronic renal disease (CRD) of various causes, there is net renal and possibly systemic nitric oxide (NO) deficiency which probably contributes to both the progression of the renal disease and to the secondary hypertension. One factor which may make an important contribution to reduced NO synthesis (NOS) is accumulation of the endogenous NOS inhibitor ADMA. We propose a comprehensive series of studies, in rats, which will establish the relative contributions of different aspects of the NO system during the evolution of renal failure. These studies will include an investigation into the metabolism of ADMA. Two distinct models of CRD will be used, one of which (the 5/6th reduction of renal mass) will be employed with, and without, severe systemic hypertension. We will use in vivo functional studies to investigate renal function and blood pressure regulation in the conscious rat as well as regulation of the glomerular and peripheral microcirculation, using anesthetized preparations. Most studies will be in the Sprague Dawley rat, but in addition, rats will be studied who are susceptible to CRD (Dahl salt sensitive rats) and resistant to CRD (Wistar-Furth rats), to determine whether baseline NOS activity is related to the CRD susceptibility. Further, metabolic cage and in vitro studies will be conducted in various mice with targeted deletions of one or more of the NOS enzymes. In rats, interventions will be used to either ameliorate (L-arginine supplementation; angiotensin inhibition) or exacerbate (low dose NOS inhibition, urea supplementation) progression of CRD, and their impact on the NOS system will be evaluated. Harvested rat plasma will be bioassayed in vitro with cultured endothelial cells to determine its impact on NOS activity, based on our clinical observations which suggest that CRD plasma may contain NOS inhibitory elements. Cell culture studies will also explore the mechanism(s) by which uremic levels of urea inhibit L-arginine transport. These studies will, in aggregate, allow us to test the hypotheses 1) That NO production decreases progressively during CRD due to a series of alterations in the NOS system including increased levels of endogenous inhibitors. 2) That once established, the etiology of the disease is irrelevant to this progressive loss of NO function 3) That the severity and rate of development of CRD is determined in part by the initial level of NOS activity, 4) That uremic levels of urea impact on NO production via reducing substrate availability.
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