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Background/Aims: Tempol is a protective antioxidant against ischemic injury in many animal models. The molecular mechanisms are not well understood. Nuclear factor erythroid 2-related factor (Nrf2) is a master transcription factor during oxidative stress, which is enhanced by activation of protein kinase C (PKC) pathway. Another factor, tubular epithelial apoptosis, is mediated by activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) signaling pathway during renal ischemic injury. We tested the hypothesis that tempol activates PKC or PI3K/Akt/Nrf2 pathways to transcribe many genes that coordinate endogenous antioxidant defense. Methods: The right renal pedicle was clamped for 45 minutes and the left kidney was removed to study renal ischemia/reperfusion (I/R) injury in C57BL/6 mice. The response was assessed from serum parameters, renal morphology and renal expression of PKC, phosphorylated-PKC (p-PKC), Nrf2, heme oxygenase-1 (HO-1), Akt, phosphorylated-Akt (p-Akt), pro-caspase-3 and cleaved caspase-3 in groups of sham and I/R mice given vehicle, or tempol (50 or 100 mg/kg, intraperitoneal injection). Results: The serum malondialdehyde (MDA, marker of reactive oxygen species) doubled and the BUN and creatinine increased 5- to 10-fold after I/R injury. Tempol (50 or 100 mg/kg) prevented the increases in MDA but only tempol (50 mg/kg) lessened the increases in BUN and creatinine and moderated the acute tubular necrosis. I/R did not change expression of PKC or p-PKC but reduced renal expression of Nrf2, p-Akt, HO-1 and pro-caspase-3 and increased cleaved caspase-3. Tempol (50 mg/kg) prevented these changes produced by I/R whereas tempol (100 mg/kg) had lesser or inconsistent effects. Conclusion: Tempol (50 mg/kg) prevents lipid peroxidation and attenuates renal damage after I/R injury. The beneficial pathway apparently is not dependent on upregulation or phosphorylation of PKC, at lower tempol doses, does implicate upregulation of Akt with expression of Nrf2 that could account for the increase in the antioxidant gene HO-1 and a reduction in the cleavage of the cellular damage marker pro-caspase-3. [ABSTRACT FROM AUTHOR]

Adenosine can mediate the tubuloglomerular (TGF) response via activation of [A.sub.1] receptors on the afferent arteriole, but both adenosine [A.sub.1] and [A.sub.2] receptors can regulate preglomerular resistance. We tested the hypothesis that adenosine [A.sub.2] receptors offset the effect of [A.sub.1] receptors and modulate the TGF. Maximal TGF responses were measured in male Sprague-Dawley rats as changes in proximal stop-flow pressure ([DELTA][P.sub.SF]) in response to increased perfusion of the loop of Henle (0 to 40 nl/min) with artificial tubular fluid (ATF). The maximal TGF response was studied after 5 rain of intratubular perfusion (10 nl/min) with ATF alone, or with ATF plus the [A.sub.2A] receptor antagonist (ZM-241385; [10.sup.-7] or [10.sup.-5] tool/l), [A.sub.1] receptor antagonist (PSB-36; [10.sup.-8] mol/l), or with a combination of [A.sub.1] (PSB-36; [10.sup.-8] mol/l) and [A.sub.2A] (ZM-241385; [10.sup.-7] mol/1) antagonists. The maximal TGF response ([DELTA][P.sub.SF]) with ATF alone was 11.7 [+ or -] 1.0 mmHg. Specific [A.sub.2] inhibition (low dose) enhanced the maximal TGF response (15.7 [+ or -] 0.8 mmHg; P < 0.01), whereas a high dose (unspecific inhibition) attenuated the response (5.0 [+ or -] 0.4 mmHg; P < 0.001). [A.sub.1] inhibition alone led to a paradoxical TGF response, with an increase in [P.sub.SF] of 3.1 [+ or -] 0.5 mmHg (P < 0.05). Simultaneous application of [A.sub.1] and [A.sub.2] antagonists abolished the TGF response ([DELTA][P..sub.SF]: 0.4 [+ or -] 0.3 mmHg). In conclusion, adenosine [A.sub.2] receptors modulate the TGF response by counteracting the effects of adenosine [A.sub.1] receptors. adenosine receptors; kidney; micropuncture; renal microcirculation doi: 10.1152/ajprenal.00211.2010.

Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.38-kb fragment of the rat smooth muscle [alpha]-actin promoter and first intron (12). Two founder lines with highest expression in the kidney [353 [+ or -] 42 and 575 [+ or -] 43% compared with the wild type (WT)] were used in the experiments. Enhanced expression of A1AR at the expected site in these lines was confirmed by augmented constrictor responses of isolated afferent arterioles to administration of the A1AR agonist [N.sup.6]-cyclohexyladenosine. Maximum TGF responses (0-30 nl/min flow step) were increased from 8.4 [+ or -] 0.9 mmHg in WT (n = 21) to 14.2 [+ or -] 0.7 mmHg in A1AR-transgene (tg) 4 (n = 22; P < 0.0001), and to 12.6 [+ or -] 1.2 mmHg in A1AR-tg7 (n = 12; P < 0.02). Stepwise changes in perfusion flow caused greater numerical TGF responses in A1AR-tg than WT in all flow ranges with differences reaching levels of significance in the intermediate flow ranges of 7.5-10 and 10-15 nl/min. Proximal-distal single-nephron glomerular filtration rate (SNGFR) differences (free-flow micropuncture) were also increased in A1AR-tg, averaging 6.25 [+ or -] 1.5 nl/min compared with 2.6 [+ or -] 0.51 nl/min in WT (P = 0.034). Basal plasma renin concentrations as well as the suppression of renin secretion after volume expansion were similar in A1AR-tg and WT mice, suggesting lack of transgene expression in juxtaglomerular cells. These data indicate that A1AR expression in vascular smooth muscle cells is a critical component for TGF signaling and that changes in renal vascular A1AR expression may determine the magnitude of TGF responses. single nephron glomerular filtration rate; smooth muscle; juxtaglomerular apparatus doi: 10.1152/ajprenal.00264.2009.

We reported impaired endotheliumderived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means [+ or -] SD) plasma levels of ADMA (PADMA, 766 [+ or -] 217 VS. 393 [+ or -] 57 nmol/1) and symmetric dimethylarginine (PsDMA: 644 [+ or -] 140 VS. 399 [+ or -] 70 nmol/1) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 [+ or -] 9 vs. 14 [+ or -] 5 nmol/ [micro] mo1 creatinine) and decreased rates of renal ADMA clearance (18 [+ or -] 3 vs. 28 [+ or -] 5 ml/min). They had significantly increased plasma levels of HODE (PHODE: 309 [+ or -] 30 vs. 226 [+ or -] 24 nmol/1) and renal HODE excretion (433 [+ or -] 93 vs. 299 [+ or -] 67 nmol/[micro]mol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure. blood pressure; 13-hydroxyoctadecadienoic acid; endothelium-derived relaxation factor/nitric oxide; albuminuria

Acute intravenous Tempol reduces mean arterial pressure (MAP) and heart rate (HR) in spontaneously hypertensive rats. We investigated the hypothesis that the antihypertensive action depends on generation of hydrogen peroxide, activation of heme oxygenase, glutathione peroxidase or potassium conductances, nitric oxide synthase, and/or the peripheral or central sympathetic nervous systems (SNSs). Tempol caused dose-dependent reductions in MAP and HR (at 174 [micro]mol/kg; [DELTA]MAP, -57 [+ or -] 3 mmHg; and [DELTA]HR, -50 [+ or -] 4 beats/min). The antihypertensive response was unaffected by the infusion of a pegylated catalase or by the inhibition of catalase with 3-aminotriazole, inhibition of glutathione peroxidase with buthionine sulfoximine, inhibition of heme oxygenase with tin potassium channels with iberiotoxin. However, the antihypertensive response was significantly (P < 0.01) blunted by 48% by the activation of adenosine 5'-triphosphate-sensitive potassium ([K.sub.ATP]) channels with cromakalim during maintenance of blood pressure with norepinephrine and by 31% by the blockade of these channels with glibenclamide, by 40% by the blockade of nitric oxide synthase with [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME), and by 40% by the blockade of ganglionic autonomic neurotransmission with hexamethonium. L-NAME and hexamethonium were additive, but glibenclamide and hexamethonium were less than additive. The central administration of Tempol was ineffective. The acute antihypertensive action of Tempol depends on the independent effects of potentiation of nitric oxide and inhibition of the peripheral SNS that involves the activation of [K.sub.ATP] channels. superoxide dismutase; nitric oxide synthase; sympathetic nervous system; catalase; adenosine 5'-triphosphate-activated potassium channels

Asymmetric ([N.sup.G], [N.sup.G])-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 vasculatare, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibtils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH +/- 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-specifiic 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 dimethylarginine

Angiotensin (ANG) II activating type 1 receptors (ATIRs) enhances superoxide anion ([O.sub.2.sup.*-]) and arachidonate (AA) formation. AA is metabolized by cyclooxygenases (COXs) to [PGH.sub.2], which is metabolized by thromboxane (Tx)[A.sub.2] synthase to Tx[A.sub.2] or oxidized to 8-isoprostane [PGF.sub.2[alpha]] (8-Iso) by [O.sub.*-]. [PGH.sub.2], Tx[A.sub.2], and 8-Iso activate thromboxane-prostanoid receptors (TPRs). We investigated whether blood pressure in a rat model of early (3 wk) two-kidney, one-clip (2K,1C) Goldblatt hypertension is maintained by [AT.sub.1]Rs or [AT.sub.2]Rs, driving COX-1 or -2-dependent products that activate TPRs. Compared with sham-operated rats, 2K,1C Goldblatt rats had increased mean arterial pressure (MAP; 120 [+ or -] 4 vs. 155 [+ or -] 3 mmHg; P < 0.001), plasma renin activity (PRA; 22 [+ or -] 7 vs. 48 [+ or -] 5 ng x [m1.sup.-1] x [h.sup.-l]; P < 0.01), plasma malondialdehyde (1.07 [+ or -] 0.05 vs. 1.58 [+ or -] 0.16 nmol/1; P < 0.01), and Tx[B.sub.2] excretion (26 [+ or -] 4 vs. 51 [+ or -] 7 ng/24 h; P < 0.01). Acute graded intravenous doses of benazeprilat (angiotensin-converting enzyme inhibitor) reduced MAP at 20 min (-36 [+ or -] 5 mmHg; P < 0.001) and excretion of Tx[A.sub.2] metabolites. Indomethacin (nonselective COX antagonist) or SC-560 (COX-1 antagonist) reduced MAP at 20 min (-25 [+ or -] 5 and -28 [+ or -] 7 mmHg; P < 0.001), whereas valdecoxib (COX-2 antagonist) was ineffective (-9 [+ or -] 5 mmHg; not significant). Losartan ([ATI.sub.1]R antagonist) or SQ-29548 (TPR antagonist) reduced MAP at 150 min (-24 [+ or -] 6 and -22 [+ or -] 3 mmHg; P < 0.001), whereas PD-123319 ([AT.sub.2]R antagonist) was ineffective. Acute blockade of TPRs, COX-1, or COX-2 did not change PRA, but Tx[B.sub.2] generation by the clipped kidney was reduced by blockade of COX-1 and increased by blockade of COX-2.2K, 1C hypertension in rats activates renin, [O.sub.2.sup*-], and vasoconstrictor PGs. Hypertension is maintained by [AT.sub.1]Rs and by COX-1, but not COX-2, products that activate TPRs. renovascular hypertension; angiotensin receptor blocker; thromboxane [A.sub.2]; isoprostane; angiotensin-converting enzyme inhibition