Maristela Lika Onozato

Isoform-Specific Regulation by N G ,N G -Dimethylarginine Dimethylaminohydrolase of Rat Serum Asymmetric Dimethylarginine and Vascular Endothelium-Derived Relaxing Factor/NO

Asymmetric dimethylarginine (ADMA), which inhibits NO synthase, is inactivated by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH). We tested whether DDAH-1 or -2 regulates serum ADMA (SADMA) and/or endothelium-derived relaxing factor (EDRF)/NO. Small inhibitory (si)RNAs targeting DDAH-1 or -2, or an siRNA control were given intravenously to rats. After 72 hours, EDRF/NO was assessed from acetylcholine-induced, NO synthase–dependent relaxation and 4-amino-5-methylamino-2′,7′-diflouroflourescein diacetate for NO activity in isolated mesenteric resistance vessels (MRVs). Expression of mRNA for DDAH-1 versus -2 was 2- and 7-fold higher in the kidney cortex and liver, respectively, whereas expression of DDAH-2 versus -1 was 5-fold higher in MRVs. The proteins and mRNAs for DDAH-1 or -2 were reduced selectively by 35% to 85% in the kidney cortex, liver, and MRVs 72 hours following the corresponding siRNA. SADMA was increased only after siDDAH-1 (266±25 versus 342±39 [mean±SD] nmol · L−1; P<0.005), whereas EDRF/NO responses and NO activity were not changed consistently by siDDAH-1 but were greatly reduced after siDDAH-2. Mean arterial pressure was not changed significantly by any siRNA. In conclusion, SADMA is regulated by DDAH-1, which is expressed at sites of ADMA metabolism in the kidney cortex and liver, whereas EDRF/NO is regulated primarily by DDAH-2, which is expressed strongly in blood vessels. This implies specific functions of DDAH isoforms.

Angiotensin II and Oxidative Stress in Dahl Salt-Sensitive Rat With Heart Failure

Abstract—Reactive oxygen species have an important pathogenic role in organ damage. We investigated the role of oxidative stress via nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the kidney of the Dahl salt-sensitive (DS) rats with heart failure (DSHF). Eleven-week-old DS rats fed an 8%-NaCl diet received either vehicle or imidapril (1 mg/kg per day) for 7 weeks. The renal expression of the NAD(P)H oxidase p47phox and endothelial NO synthase were evaluated. In DSHF rats, associated with increased renal angiotensin II, mRNA and protein expression of NAD(P)H oxidase p47phox were enhanced with an increase in renal lipid peroxidation production (0.33±0.03 versus 0.22±0.01 nmol/mg protein, P <0.05) and urinary excretion of hydrogen peroxide (26.9±6.6 versus 9.5±2.1 U/mg creatinine, P <0.01) compared with levels in Dahl salt-resistant rats. The endothelial NO synthase expression was decreased in the kidney. Treatment with imidapril reduced renal angiotensin II and NAD(P)H oxidase expression and the oxidative products (kidney lipid peroxidation product: 0.16±0.02, P <0.001; urinary hydrogen peroxide: 3.1±0.2, P <0.01 versus DSHF rats). Imidapril significantly decreased albuminuria and reduced glomerulosclerosis without changes in the blood pressure. In conclusion, DSHF rats showed increased oxidative stress in the kidney via NAD(P)H oxidase. Blockade of local angiotensin II with subpressor dose of imidapril inhibited NAD(P)H oxidase and prevented renal damage.

Eplerenone Shows Renoprotective Effect by Reducing LOX-1–Mediated Adhesion Molecule, PKCε-MAPK-p90RSK, and Rho-Kinase Pathway

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) may play an important role in atherosclerosis by inducing leukocyte adhesion molecules, such as intercellular and vascular cell adhesion molecule-1 (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]). We hypothesized that eplerenone, a novel selective aldosterone blocker, produces inhibition of LOX-1–mediated adhesion molecules, suppresses mitogen-activated protein (MAP) kinase and its downstream effector p90 ribosomal S6 kinase (p90RSK) through the protein kinase C&egr; (PKC&egr;) pathway, and improves endothelial function by inhibition of Rho-kinase in the renal cortex of Dahl salt-sensitive hypertensive (DS) and salt-resistant (DR) rats. Eplerenone (10, 30, and 100 mg/kg per day) was given from the age of 6 weeks to the left ventricular hypertrophy stage (11 weeks) for 5 weeks. At 11 weeks, expression levels of LOX-1, ICAM-1, VCAM-1, and Rho-kinase were higher in DS rats than in DR rats and were decreased by eplerenone. Similarly, upregulated phosphorylation of PKC&egr;, MAP kinase, and p90RSK in DS rats was also inhibited by eplerenone. In contrast, downregulated endothelial nitric oxide synthase mRNA was increased by eplerenone to a similar degree as after treatment with Y-27632, a selective Rho-kinase inhibitor. Eplerenone administration resulted in significant improvement in glomerulosclerosis (eplerenone 10 mg, −61%; 30 mg, −78%; and 100 mg, −84% versus DS; P<0.01, respectively) and urinary protein (10 mg, −78%; 30 mg, −87%; and 100 mg, −88% versus DS; P<0.01, respectively). These results suggest that the renoprotective effects of eplerenone may be partly caused by inhibition of LOX-1–mediated adhesion molecules and PKC&egr;–MAP kinase–p90RSK pathway, and improvement in endothelial function.

Suppressing renal NADPH oxidase to treat diabetic nephropathy

Renal nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase is an important source of oxidative stress and its expression is enhanced in the glomerulus and distal tubules of diabetic nephropathy. High glucose-induced protein kinase C signalling or renal angiotensin II signalling increases the membrane translocation of cytosolic component p47phox. NADPH oxidase-derived reactive oxygen species (ROS) in the podocytes damage the glomerular basement membrane and the slit diaphragm causing proteinuria, and mesangial and glomerular endothelial NADPH oxidase increase TGF-β and cause collagen and fibronectin accumulation. Tubular NADPH oxidase stimulated by angiotensin II or aldosterone contributes to sodium retention and to tubulointerstitial damage. Thus, inhibition of the renal renin–angiotensin II–aldosterone system with angiotensin-converting enzyme inhibitor, angiotensin II type 1 receptor blocker or selective aldosterone inhibitor indirectly suppresses NADPH oxidase reducing renal ROS, proteinuria and glomerulosclerosis. Statins are also effective in blocking the membrane translocation of Rac, especially in diabetes with hypercholesterolemia where ROS is produced by the intrinsic NADPH oxidase and by the activated macrophages. A medical herb, picrorhiza, inhibits the membrane translocation of p47phox, is a specific inhibitor of NADPH oxidase and, more so than superoxide dismutase mimetics, may be a promising strategy for the treatment of diabetic nephropathy.

Endogenous Adrenomedullin Protects Against Vascular Response to Injury in Mice

Background—In our previous study, adrenomedullin (AM) overexpression could limit the arterial intimal hyperplasia induced by cuff injury in rats. However, it remains to be elucidated whether endogenous AM plays a role against vascular injury. Methods and Results—We used the AM knockout mice to investigate the effect of endogenous AM. Compared with wild-type (AM+/+) mice, heterozygous AM knockout (AM+/−) mice had the increased intimal thickening of the cuff-injured femoral artery, concomitantly with lesser AM staining. In AM+/− mice, cuff placement increased both the production of superoxide anions (O2−) measured by coelentarazine chemiluminescence and the immunostaining of p67phox and gp91phox, subunits of NAD(P)H oxidase in the adventitia, associated with the increment of CD45-positive leukocytes, suggesting that the stimulated formation of radical oxygen species accompanied chronic adventitial inflammation. Not only the AM gene transfection but also the treatment of NAD(P)H oxidase inhibitor apocynin and membrane-permeable superoxide dismutase mimetic tempol could limit cuff-induced intimal hyperplasia in AM+/− mice, associated with the inhibition of O2− formation in cuff-injured artery. Conclusions—The overproduction of oxidative stress induced by the increased NAD(P)H oxidase activity might be involved in cuff-injured arterial intimal hyperplasia in AM+/− mice. Thus, it is suggested that endogenous AM possesses a protective action against the vascular response to injury, possibly through the inhibition of oxidative stress production.

Expression of NG,NG-dimethylarginine dimethylaminohydrolase and protein arginine N-methyltransferase isoforms in diabetic rat kidney: effects of angiotensin II receptor blockers.

OBJECTIVE—The nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) is generated by protein arginine N-methyltransferase (PRMT)-1 and is metabolized by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH). We tested the hypothesis that increased serum ADMA (SADMA) in the streptozotocin (STZ)-induced diabetic rat model of diabetes is mediated by an angiotensin receptor blocker–sensitive change in DDAH or PRMT expression. RESEARCH DESIGN AND METHODS—Data were compared from four groups of rats: sham-injected controls, untreated STZ-induced diabetic rats at 4 weeks, STZ-induced diabetic rats administered the angiotensin II (Ang II) receptor blocker telmisartan for 2 weeks, and control rats administered telmisartan for 2 weeks. RESULTS—Immunostaining and Western blotting of microdissected nephron segments localized DDAH I in the proximal tubules and DDAH II in the glomeruli, afferent arterioles, macula densa, and distal nephron. Renal Ang II and SADMA increased with diabetes but were normalized by 2 weeks of telmisartan. DDAH I expression was decreased in diabetic kidneys, while DDAH II expression was increased. These changes were reversed by telmisartan, which also reduced expression of PRMT-1 and -5. Telmisartan increased expressions of DDAH I but decreased DDAH II in Ang II-stimulated kidney slices ex vivo. CONCLUSIONS—Renal Ang II and SADMA are increased in insulinopenic diabetes. They are normalized by an Ang II receptor blocker, which increases the renal expression of DDAH I, decreases PRMT-1, and increases renal NO metabolites.

Interaction of human and rat organic anion transporter 2 with various cephalosporin antibiotics

Cephalosporin antibiotics are thought to be excreted into the urine via organic anion transporters (OATs) and OAT can mediate nephrotoxicity by cephalosporins, particularly by cephaloridine. The purpose of this study was to elucidate the interaction of human-OAT2 and rat-OAT2 with cephalosporin antibiotics using proximal tubule cells stably expressing human-OAT2 and rat-OAT2. Human-OAT2 is localized to the basolateral side of the proximal tubule, whereas rat-OAT2 is localized to the apical side of the proximal tubule. Cephalosporins tested were cephalothin, cefoperazone, cefazolin, ceftriaxone, cephaloridine, cefotaxime, cefadroxil and cefamandole. These cephalosporins dose-dependently inhibited organic anion uptake mediated by human-OAT2 and rat-OAT2. There was no species difference observed for the effects of OAT2 with cephalosporins between human and rat transporters. Kinetic analysis revealed that the inhibitory effects for human-OAT2 were competitive. Cephaloridine significantly decreased the viability of cells stably expressing human-OAT2, human-OAT1, human-OAT3 and human-OAT4. The decreased viability of cells stably expressing human-OAT1, human-OAT3 and human-OAT4 but not human-OAT2 was reversed by probenecid. In conclusion, human-OAT2 interacts with cephalosporins, and thus, human-OAT2 may mediate the uptake of cephalosporins on the basolateral side of the proximal tubule. The interaction of human-OAT2 with cephalosporins was the weakest among the basolateral human-OATs tested. In addition, it is suggested that human-OATs mediate cephaloridine-induced nephrotoxicity.

Expression of DDAH and PRMT isoforms in the diabetic rat kidney; effects of angiotensin II receptor blocker

OBJECTIVE—The nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) is generated by protein arginine N-methyltransferase (PRMT)-1 and is metabolized by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH). We tested the hypothesis that increased serum ADMA (SADMA) in the streptozotocin (STZ)-induced diabetic rat model of diabetes is mediated by an angiotensin receptor blocker–sensitive change in DDAH or PRMT expression. RESEARCH DESIGN AND METHODS—Data were compared from four groups of rats: sham-injected controls, untreated STZ-induced diabetic rats at 4 weeks, STZ-induced diabetic rats administered the angiotensin II (Ang II) receptor blocker telmisartan for 2 weeks, and control rats administered telmisartan for 2 weeks. RESULTS—Immunostaining and Western blotting of microdissected nephron segments localized DDAH I in the proximal tubules and DDAH II in the glomeruli, afferent arterioles, macula densa, and distal nephron. Renal Ang II and SADMA increased with diabetes but were normalized by 2 weeks of telmisartan. DDAH I expression was decreased in diabetic kidneys, while DDAH II expression was increased. These changes were reversed by telmisartan, which also reduced expression of PRMT-1 and -5. Telmisartan increased expressions of DDAH I but decreased DDAH II in Ang II-stimulated kidney slices ex vivo. CONCLUSIONS—Renal Ang II and SADMA are increased in insulinopenic diabetes. They are normalized by an Ang II receptor blocker, which increases the renal expression of DDAH I, decreases PRMT-1, and increases renal NO metabolites.

Protective Effect of Dietary Potassium Against Vascular Injury in Salt-Sensitive Hypertension

Hypertensive cardiovascular damage is accelerated by salt loading but counteracted by dietary potassium supplementation. We suggested recently that antioxidant actions of potassium contribute to protection against salt-induced cardiac dysfunction. Therefore, we examined whether potassium supplementation ameliorated cuff-induced vascular injury in salt-sensitive hypertension via suppression of oxidative stress. Four-week–old Dahl salt-sensitive rats were fed a normal-salt (0.3% NaCl), high-salt (8% NaCl), or high-salt plus high-potassium (8% KCl) diet for 5 weeks, and some of the rats fed a high-salt diet were also given antioxidants. One week after the start of the treatments, a silicone cuff was implanted around the femoral artery. Examination revealed increased cuff-induced neointimal proliferation with adventitial macrophage infiltration in arteries from salt-loaded Dahl salt-sensitive rats compared with that in arteries from non–salt-loaded animals (intima/media ratio: 0.471±0.070 versus 0.302±0.037; P<0.05), associated with regional superoxide overproduction and reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and mRNA overexpression. On the other hand, simultaneous potassium supplementation attenuated salt-induced neointimal hyperplasia (intima/media ratio: 0.205±0.012; P<0.001), adventitial macrophage infiltration, superoxide overproduction, and reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and overexpression. Antioxidants, which decrease vascular oxidative stress, also reduced neointima formation induced by salt excess. In conclusion, high-potassium diets seems to have a protective effect against the development of vascular damage induced by salt loading mediated, at least in part, through suppression of the production of reactive oxygen species probably generated by reduced nicotinamide-adenine dinucleotide phosphate oxidase.

Antioxidative effect of p38 mitogen-activated protein kinase inhibitor in the kidney of hypertensive rat.

Objective Nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase is regulated by angiotensin II, interleukin (IL)-1β and tumor necrosis factor (TNF)-α via p38 mitogen-activated protein kinase (MAPK). We hypothesized that p38 MAPK inhibitor, FR167653, may suppress NAD(P)H oxidase and its oxygen radical production and ameliorate renal damage in Dahl salt-sensitive rats with heart failure (DSHF). Methods DSHF rats were fed with 8% NaCl diet from 6 to 18 weeks old. Eleven-week-old DSHF rats received either vehicle or FR167653 (2 mg/kg per day) for 7 weeks and the renal NAD(P)H oxidase p47phox and nitric oxide synthase (NOS), superoxide production and renal damage were evaluated in comparison with the control Dahl salt-resistant rat fed with 8% NaCl diet. Results In the kidney of DSHF rat, phosphorylated p38 MAPK was enhanced with an increased IL-1β and TNF-α production compared with control rats. Treatment with FR167653 significantly suppressed p38 MAPK, IL-1β and TNF-α. Renal NAD(P)H oxidase p47phox expression and superoxide production were significantly increased in the DSHF rats and treatment with FR167653 suppressed NAD(P)H oxidase expression and reduced superoxide formation. Renal endothelial and inducible NOS were reduced in DSHF rats compared with control rats, but FR167653 increased NOS and NO production in the kidney. Proteinuria, glomerulosclerosis and interstitial macrophage migration via intercellular adhesion molecule-1 (ICAM-1) were enhanced in DSHF rat and they were ameliorated by FR167653. Conclusion The inhibition of p38 MAPK by FR167653 reduced renal IL-1β and TNF-α production and ameliorated renal damage in hypertensive rat via suppression of NAD(P)H oxidase and enhanced NO bioavailability.