We have found that endothelial dysfunction develops early in human hypertension and chronic kidney disease (CKD). It is accompanied by increased reactive oxygen species (ROS) which can inactivate nitric oxide (NO). However, there is also an increase in asymmetric dimethylarginine (ADMA) which can inhibit NOS. Plasma levels of ADMA and markers of ROS predict cardiovascular disease and CKD progression. ADMA is generated intracellularly by protein arginine methyltransferases (PRMTs), metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and exported by cationic amino acid transporters (CATs). The hypothesis is that ROS, generated within preglomerular vascular smooth muscle cells (PGVSMCs), renal glomerular endothelial cells (RGECs) and renal proximal tubule cells (RPTCs) enhance intracellular ADMA which reduces vascular NO generation and angiogenesis and reduces absolute proximal reabsorption (APR). Therefore, this hypothesis links these two major risk factors. The first aim will contrast cell stably transfected with p22phox (S-p22phox) with empty vector wild type cells (Wt). We will study the effects of superoxide (O2.-) vs. H2O2 on the activities and expression of the enzymes and transporters that regulate intracellular ADMA in these three cell types. We will test our hypothesis that PGVSMCs generate a myocyte derived endothelial regulator factor driven by NADPH oxidase that signals to adjacent ECs to reduce NO activity and prevent proliferation and angiogenesis via ADMA and/or H2O2. This is a novel pathway in the vessel wall to compliment endothelium to VSMC communication. Similar studies in S-p22phox PTCs will probe the role of ADMA in the impaired 22Na+ uptake and O2 usage. We have found profound reductions in APR in the SHR model of oxidative stress that are dependent on p22phox, ROS and DDAH-1. A reduced Na+ reabsorption by the energy-efficient proximal tubule dictates enhanced delivery and reabsorption by downstream nephron segments. This could account for the decreased efficiency of O2 usage and the hypoxia of kidneys from animals with oxidative stress. Therefore, Aim 2 will investigate these interactions in vivo. We will study these effects on the NHE inhibitory protein-2 (NHERF-2) in vivo in the microperfused proximal tubules of WKY and SHR, by the addition of drugs to the perfusates, or use of small interference RNAs targeting NHERF-2 or specific proteins involved in production, metabolism and transport of ADMA and ROS and in vascular function in mice transgenic for p22phox in VSMCs. These studies are an escalating series of mechanistic studies in cells and functional studies in the kidney and vessels to test the hypothesis that ADMA mediates the effects of ROS on blood vessels and proximal tubular transport which could lead to new targets for intervention to prevent progressive cardiovascular and kidney diseases.