Regulation of Microvascular Function by ROS

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Low rates of Angiotensin II (Ang II) infusion increase reactive oxygen species (ROS), upregulate key renal components of NADPH oxidase (p22phox, NOX-1) and downregulate EC-SOD. Antioxidant drugs implicate ROS in the development of hypertension, yet the specific roles of renal and systemic ROS in vasoconstriction, salt retention and the mediation by NADPH oxidase, or by reduced SOD-dependent defense in the kidney, are largely undefined. We will study BP homeostasis (telemetric BP), salt handling and sensitivity and microvascular mechanisms in mice (isolated, perfused renal afferent and mesenteric resistance vessels and in vivo intravital microscopy) to explore the roles of an endothelium-derived contracting factor (EDCF) and enhanced VSMC contractility in the microvessels. We will relate these functional studies to ROS, NO, and calcium activity in individual microdissected vessels and pre-glomerular vascular smooth muscle cells. Our primary strategy is the use of knockout models and small interference RNAs (siRNAs) directed at target genes delivered intrarenally to assess specifically renal mechanisms of hypertension. This will be extended with kidney cross-transplantation combined with a cre-lox strategy to knockout genes specifically in VSMC to establish, for the first time, the roles of the kidney and its afferent arterioles in mediating hypertension with ROS. Aim 1 will use the EC-SOD -/- mouse model of stable, sustained microvascular oxidative stress to test the hypothesis that oxidative stress releases endothelin, which acts on type A or B receptors to engage a cyclooxygenase-derived EDCF that activates adjacent VSMCs where contractility is enhanced by a Ca++ sensitizing pathway mediated by rho/rho kinase. Aim 2 will use EC-SOD -/- mice administered siRNA to IC-SOD delivered selectively to the kidney to test the hypothesis that IC-SOD is the major antioxidant defense in the kidney and that its renal deficiency promotes renal vasoconstriction, salt retention, and hypertension. Aim 3 will use normal mice with siRNA to p22phox delivered to the kidney or systemically to test the hypothesis that renal NADPH oxidase mediates increased afferent arteriolar contractility, RVR, salt retention and hypertension with Ang II. These projects are an integrated approach to dissect the roles of ROS in renal microvascular reactivity and salt handling that constitute renal mechanisms of hypertension.