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.

Public Health Relevance

All the common risk factors for cardiovascular or progressive chronic kidney diseases (CKD) are associated with increased oxidative stress and endothelial dysfunction of small blood vessels. Oxidative stress implies an increase in reactive oxygen species (ROS). ROS formed in blood vessel inactive nitric oxide, and thereby create peroxynitrite that inactivates the enzyme that forms prostacyclin. Nitric oxide and prostacyclin released from endothelial cells in blood vessels maintain normal endothelial function. More recent evidence implicates asymmetric dimethylarginine (ADMA) as well as ROS in endothelial dysfunction. Similar to ROS, circulating levels of ADMA also correlate closely with cardiovascular risk factors and also predict future cardiovascular events and progression of CKD but presently, less is known about the regulation of vascular and kidney levels of ADMA. ADMA is synthesized by enzymes called protein arginine methyltransferases which are widely distributed in cells. It is metabolized intracellularly by dimethylarginine dimethylaminohydrolases (DDAHs) and is transported across cell membranes by cationic amino acid transporters (CATs). Our preliminary studies in cells and in the kidney indicate that intracellular ROS increase PRMT and decrease DDAH and CAT activity, thereby enhancing cellular levels of ADMA. The first aim is to study these events in the blood vessel wall in a simplified model system where vascular smooth muscle cells are grown in culture alone or with endothelial cells. Transfection of these vascular smooth muscle cells with the gene for p22phox which activates nicotinamide adenine dinucleotide phosphate oxidase to produce ROS reduced to nitric oxide and reduced the proliferation of the endothelial cells. The candidate mediators for this novel myocyte derived endothelial regulatory factor that signals between oxidatively modified vascular smooth muscle cells and neighboring endothelial cells are ADMA and hydrogen peroxide (H2O2) which is a stable form of ROS. We will study the interaction between ROS and the systems that generate, metabolize and transport ADMA in these model systems and in proximal tubule cells in culture to understand the interaction between these two important cardiovascular and kidney regulatory systems. The second aim is to translate these findings from cells to the intact kidney of the living rat. We propose to microperfuse proximal tubules of normotensive WKY and spontaneously hypertensive (SHR) rats which we have shown to have severe oxidative stress and a reduced ability to reabsorb fluid and salt. We propose to add drugs to the perfusate of the tubular lumen or to administer small interference RNAs to the rats to knock down targeted genes for key mediators in the ADMA and ROS pathways to study the mechanism of the profoundly altered proximal reabsorption in rat with oxidative stress and the roles of H2O2, ADMA and their interaction with the sodium hydrogen exchanger regulatory factor 2. The proposal is for an initial series of mechanistic studies under closely controlled conditions in isolated cells from resistance vessel to the kidney (preglomerular vascular smooth muscle cells and renal glomerular endothelial cells) and of the proximal tubule. They will be followed by translational studies in rat models of oxidative stress to determine how these findings impact kidney function. It is anticipated that conclusions from these studies could improve the understanding of microvascular function and tubular reabsorption and thereby the pathogenesis of hypertension, cardiovascular disease, and chronic kidney disease. They could identify new molecular mechanisms that underlie endothelial and kidney dysfunction, and could provide new targets for therapy for patients with these conditions.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Hypertension and Microcirculation Study Section (HM)
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Ketchum, Christian J
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Georgetown University
Internal Medicine/Medicine
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