Nitric oxide (NO) deficiency occurs in chronic kidney disease (CKD) irrespective of the primary cause and contributes to the cardiovascular complications and progression of CKD. There are many reasons for NO deficiency in CKD; 1 major factor is accumulation of the endogenous NOS inhibitor, asymmetric dimethylarginine, ADMA, due partly to a failure of the hydrolyzing enzymes dimethylarginine dimethylamino-hydrolases (DDAHs). The kidney is a major site of DDAH activity. Another cause is loss of NO synthase (NOS) protein and activity and our work suggests that in kidney the neuronal (n)NOS is a primary target and that several isoforms are present and altered by CKD. We propose in vivo and in vitro studies in 2 separate models of CKD, the 5/6 renal mass ablation/infarction (A/I) and chronic puromycin aminonucleoside nephrosis (PAN) models to determine the pattern of change of the different NOS and DDAH enzymes in the kidney and how this contributes to the progression of CKD and development of hypertension. Both DDAH and NOS protein abundance and activity are severely compromised by oxidative stress, which is high in CKD and we will use apocyanin to inhibit oxidant production and dietary manipulation to enhance oxidative stress as methods of changing DDAH and NOS activity. Nephron number is a major determinant of progression of CKD and development of hypertension and we will determine how reductions in nephron number impact on the renal NOS and DDAH enzymes. By using our newly developed method of selective kidney transfection with genes of interest we will manipulate the local intrarenal NOS and DDAH enzymes to determine the role of different nNOS (and other NOS) isoforms and the 2 DDAH isoforms in progression of CKD. We will conduct in vivo functional studies in the conscious rat as well as glomerular micropuncture using anesthetized preparations. We will also conduct a range of in vitro studies to determine enzyme location, abundance and activity. Studies will be in the Sprague Dawley rat a strain vulnerable to CKD and hypertension and in the Wistar Furth which is protected from multiple types of CKD and hypertension to gain insights into mechanisms of progression. The incidence of CKD is rapidly increasing in the US and these patients also have a very high rate of cardiovascular morbidity and mortality, which is clearly linked to NO deficiency. These proposed studies will determine the role of the renal NO system in the progression of CKD and its cardiovascular complications which will lead to new therapeutic approaches. ? ? ?
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