Palm F, Onozato ML, Luo Z, Wilcox CS. Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems. Am J Physiol Heart Circ Physiol 293: H3227-H3245, 2007. First published October 12, 2007; doi:10.1152 doi:10. /ajpheart.00998.2007 )-dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS). ADMA is a risk factor for endothelial dysfunction, cardiovascular mortality, and progression of chronic kidney disease. Two isoforms of dimethylarginine dimethylaminohydrolase (DDAH) metabolize ADMA. DDAH-1 is the predominant isoform in the proximal tubules of the kidney and in the liver. These organs extract ADMA from the circulation. DDAH-2 is the predominant isoform in the vasculature, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibrils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH ϩ/Ϫ mice both have increased circulating ADMA, whereas gene silencing of DDAH-2 reduces vascular NO generation and endothelium-derived relaxation factor responses. DDAH-2 also is expressed in the kidney in the macula densa and distal nephron. Angiotensin type 1 receptor activation in kidneys reduces the expression of DDAH-1 but increases the expression of DDAH-2. This rapidly evolving evidence of isoform-specific distribution and regulation of DDAH expression in the kidney and blood vessels provides potential mechanisms for nephron site-specific regulation of NO production. In this review, the recent advances in the regulation and function of DDAH enzymes, their roles in the regulation of NO generation, and their possible contribution to endothelial dysfunction in patients with cardiovascular and kidney diseases are discussed. nitric oxide synthase; hypertension; diabetes mellitus; chronic kidney disease; asymmetric dimethylarginineis an endogenous methylated amino acid that inhibits the constitutive endothelial (e) or type III and neuronal (n) or type I isoforms of nitric oxide (NO) synthase (NOS) (49,91,103,199). It is a less potent inhibitor of the inducible (i) or type II NOS isoform (41,191,213). Proteins are subject to methylation of arginine residues by protein arginine methyltransferase (PRMT). S-adenosylmethionine, which is synthesized from methionine and ATP, serves as the methyl donor and, in the process, is converted to S-adenosylhomocysteine, which itself can be hydrolyzed to homocysteine. Remethylation of homocysteine in the "remethylation pathway" regenerates methionine (14,179). ADMA is released by protein hydrolysis and exported from the cell and taken up by other cells via system y ϩ carriers of the cationic amino acid (CAT) family (14,196,212). ADMA is eliminated both by renal excretion and metabolic degradation. Its metabolism is facilitated by dimethylarginine dimethylaminohydrolases (DDAHs), which are expressed as type 1 and 2 isoforms. Recent studies have shown differential sites of expression of DDAH-1 and -2 in blo...

Asymmetric (N G ,N G ) dimethylarginine (ADMA) is present in plasma and cells. It can inhibit nitric oxide synthase (NOS) that generates nitric oxide (NO) and cationic amino acid transporters (CAT) that supply intracellular NOS with its substrate, L-arginine from the plasma. Therefore, ADMA and its transport mechanisms are strategically placed to regulate endothelial function. This could have considerable clinical impact since endothelial dysfunction has been detected at the origin of hypertension and chronic kidney disease (CKD) in human subjects and may be a harbinger of large vessel disease and cardiovascular disease (CVD). Indeed, plasma levels of ADMA are increased in many studies of patients at risk for, or with overt CKD or CVD. However, the levels of ADMA measured in plasma of about 0.5 μmol · l −1 maybe below those required to inhibit NOS whose substrate, L-arginine, is present in concentrations manifold above the Km for NOS. However, NOS activity may be partially inhibited by cellular ADMA. Therefore, the cellular production of ADMA by protein arginine methyltransferase (PRMT) and protein hydrolysis, its degradation by N G , N G -dimethylarginine dimethylaminohydrolase (DDAH) and its transmembrane transport by CAT that determines intracellular levels of ADMA may also determine the state of activation of NOS. This is the focus of the review. It is concluded that cellular levels of ADMA can be 5-to 20-fold those in plasma and in a range that could tonically inhibit NOS. The relative importance of PRMT, DDAH and CAT for determining the intracellular NOS substrate: inhibitor ratio (L-arginine:ADMA) may vary according to the pathophysiologic circumstance. An understanding of this important balance requires knowledge of at least these three processes that regulate the intracellular levels of ADMA and arginine. KeywordsNitric oxide synthase (NOS); protein arginine methyl transferase (PRMT); cationic amino acid (CAA); cationic amino acid transporter (CAT); cardiovascular disease; chronic kidney disease (CKD); hypertension; reactive oxygen species and oxidative stress Address for correspondence: Christopher S. Wilcox, M.D., Ph.D., Division of Nephrology and Hypertension, Georgetown University Medical Center, 3800 Reservoir Road, NW, 6 PHC Bldg, F6003, Washington, DC 20007, wilcoxch@georgetown.edu, Phone: 1-202-444-9183, Fax: 1-202-444-7893. Conflict of InterestNone.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript Overview of ADMA Generation, Metabolism and TransportAsymme...

Myogenic and angiotensin contractions of afferent arterioles generate reactive oxygen species. Resistance vessels express NOX-2 and -4. Angiotensin II activates p47phox/NOX-2 whereas it downregulates NOX-4. Therefore, we tested the hypothesis that p47phox enhances afferent arteriolar angiotensin contractions. Angiotensin II infusion in p47phox +/+, but not -/- mice, increased renal cortical nicotinamide adenine dinucleotide phosphate oxidase activity (7±1 to 12±1; P<0.01 vs 5±1 to 7±1; NS, 103 · RLU · min-1 · μg protein-1), mean arterial pressure (77±2 to 91±2; P<0.005 vs 74±2 to 77±1; NS, mmHg) and renal vascular resistance (7.5±0.4 to 10.1±0.7; P<0.01 vs 7.9±0.4 to 8.3±0.4 NS, mmHg/ml·min-1·gkwt-1). Afferent arterioles from p47phox -/- mice had a lesser myogenic response (3.1±0.4 vs 1.4±0.2 dynes·cm-1·mmHg-1; P<0.02) and a lesser (P<0.05) contraction to 10-6M angiotensin II (diameter change +/+: 9.3±0.2 to 3.4±0.6 μm vs -/-: 9.9±0.6 to 7.5±0.4 μm). Angiotensin and increased perfusion pressure generated significantly (P<0.05) more reactive oxygen species in p47phox +/+ than -/- arterioles. Angiotensin II infusion increased the maximum responsiveness of afferent arterioles from p47phox +/+ mice to 10-6 M angiotensin II yet decreased the response in p47phox -/- mice. The angiotensin infusion increased the sensitivity to angiotensin II only in p47phox +/+ mice. We conclude that p47phox is required to enhance renal nicotinamide adenine dinucleotide phosphate oxidase activity and basal afferent arteriolar myogenic and angiotensin II contractions and to switch afferent arteriolar tachyphylaxis to sensitization to angiotensin during a prolonged angiotensin infusion. These effects likely contribute to hypertension and renal vasoconstriction during infusion of angiotensin II.

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Abstract-Proximal tubule reabsorption is regulated by systemic and intrinsic mechanisms, including locally produced autocoids. Superoxide, produced by NADPH oxidase enhances NaCl transport in the loop of Henle and the collecting duct, but its role in the proximal tubule is unclear. We measured proximal tubule fluid reabsorption (Jv) in WKY rats and compared that with Jv in the spontaneously hypertensive rat (SHR), a model of enhanced renal superoxide generation. Rats were treated with the NADPH oxidase inhibitor apocynin (Apo) or with small interfering RNA for p22 phox , which is the critical subunit of NADPH oxidase. Jv was lower in SHR compared with Wistar-Kyoto rats (WKY; WKY: 2.3Ϯ0.3 vs SHR: 1.1Ϯ0.2 nL/min per millimeter; nϭ9 to 11; PϽ0.001). Apo and small interfering RNA to p22 phox normalized Jv in SHRs but had no effect in WKY rats. Jv was reduced in proximal tubules perfused with S-1611, a highly selective inhibitor of the Na ϩ /H ϩ exchanger 3, the major Na ϩ uptake pathway in the proximal tubule, in WKY rats but not in SHRs. Pretreatment with Apo restored an effect of S-1611 to reduce Jv in the SHRs (SHRϩApo: 2.9Ϯ0.4 vs SHRϩApoϩS-1611: 1.0Ϯ0.3 nL/min per millimeter; PϽ0.001). However, because expression of the Na ϩ /H ϩ exchanger 3 was similar between SHR and WKY rats, this suggests that superoxide affects Na ϩ /H ϩ exchanger 3 activity. Direct microperfusion of Tempol or Apo into the proximal tubule also restored Jv in SHRs. In conclusion, superoxide generated by NADPH oxidase inhibits proximal tubule fluid reabsorption in SHRs. This finding implies that proximal tubule fluid reabsorption is regulated by redox balance, which may have profound effects on ion and fluid homeostasis in the hypertensive kidney. Key Words: proximal reabsorption Ⅲ superoxide Ⅲ Tempol Ⅲ apocynin Ⅲ hypertension I n the kidney, the proximal tubule (PT) reabsorbs 60% to 70% of filtered NaCl and fluid. Therefore, changes in PT reabsorption can have profound effects on renal and body fluid balance and may contribute to the development of hypertension. The normal kidney protects against acute increases in blood pressure by excreting NaCl rapidly. The PT is thought to mediate much of this pressure-natriuresis response. In young spontaneously hypertensive rats (SHRs), before the onset of hypertension, expression of the major Na ϩ transport systems in the PTs was higher 1 and Na ϩ excretion was lower compared with normotensive rats (Wistar-Kyoto [WKY]). 2 This was accompanied by an increase in fluid reabsorption in the PT in young (5-week-old) prehypertensive SHRs compared with WKY. These observations suggest that an exaggerated NaCl and fluid reabsorption in the PT may contribute to the development of hypertension in young SHRs, which persists in the adult animal. However, the increased reabsorption seen in young animals is not consistently observed in adult SHRs. For example, in 7-and 12-week-old SHRs, at a time when hypertension was established, baseline proximal tubule fluid reabsorption (Jv) in the PT was lower compared with that...

Abstract-Asymmetrical dimethylarginine inhibits nitric oxide synthase, cationic amino acid transport, and endothelial function. Patients with cardiovascular risk factors often have endothelial dysfunction associated with increased plasma asymmetrical dimethylarginine and markers of reactive oxygen species. We tested the hypothesis that reactive oxygen species, generated by nicotinamide adenine dinucleotide phosphate oxidase, enhance cellular asymmetrical dimethylarginine. Incubation of rat preglomerular vascular smooth muscle cells with angiotensin II doubled the activity of nicotinamide adenine dinucleotide phosphate oxidase but decreased the activities of dimethylarginine dimethylaminohydrolase by 35% and of cationic amino acid transport by 20% and doubled cellular (but not medium) asymmetrical dimethylarginine concentrations (PϽ0.01). This was blocked by tempol or candesartan. Cells stably transfected with p22 phox had a 50% decreased protein expression and activity of dimethylarginine dimethylaminohydrolase despite increased promoter activity and mRNA. The decreased DDAH protein expression and the increased asymmetrical dimethylarginine concentration in p22phox -transfected cells were prevented by proteosomal inhibition. These cells had enhanced protein arginine methylation, a 2-fold increased expression of protein arginine methyltransferase-3 (PϽ0.05) and a 30% reduction in cationic amino acid transport activity (PϽ0.05). Asymmetrical dimethylarginine was increased from 6Ϯ1 to 16Ϯ3 mol/L (PϽ0.005) in p22 phox -transfected cells. Thus, angiotensin II increased cellular asymmetrical dimethylarginine via type 1 receptors and reactive oxygen species. Nicotinamide adenine dinucleotide phosphate oxidase increased cellular asymmetrical dimethylarginine by increasing enzymes that generate it, enhancing the degradation of enzymes that metabolize it, and reducing its cellular transport. This could underlie increases in cellular asymmetrical dimethylarginine during oxidative stress. Ⅲ tempol Ⅲ cationic amino acid transferase (CAT) Ⅲ hypertension A symmetrical dimethylarginine (ADMA) inhibits nitric oxide synthase (NOS) and cationic amino acid transport (CAT). 1 ADMA is generated by protein arginine methyltransferases (PRMTs) and, after proteolysis, cellular ADMA is metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) or exported by CATs. 2,3 Angiotensin II (Ang II) can generate reactive oxygen species (ROS) in blood vessels by activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. 4 Patients with early hypertension or kidney disease have elevated plasma levels of ADMA and markers of ROS, 5 which may contribute to endothelial dysfunction and subsequent cardiovascular or renal events. Although increased ADMA occurs in several conditions associated with ROS, 6 it is unclear how ROS increase ADMA. Moreover, infusions of Ang II sufficient to increase ROS have variable effects on plasma ADMA. [7][8][9] The present studies were designed to test the hypothesis that NADPH oxidase enhances PRMT an...

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with ⅚ surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O(2) usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na(+) transport per O(2) consumed (T(Na/)Q(O2)), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F(2α) and H(2)O(2), renal expression of UCP-2 and renal O(2) extraction, and reduced T(Na/)Q(O2) (sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po(2) (sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22(phox), NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione-S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O(2) usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

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