Abstract-The present study characterized the biochemical pathways responsible for superoxide (O 2 Ϫ· ) production in different regions of the rat kidney and determined the role of O 2 Ϫ· in the control of renal medullary blood flow (MBF) and renal function. By use of dihydroethidium/DNA fluorescence spectrometry with microtiter plates, the production of O 2 Ϫ· was monitored when tissue homogenate from different kidney regions was incubated with substrates for the major O 2 Ϫ· -producing enzymes, such as NADH/NADPH oxidase, xanthine oxidase, and mitochondrial respiratory chain enzymes. The production of O 2 Ϫ· via NADH oxidase was greater (PϽ0.05) in the renal cortex and outer medulla (OM) than in the papilla. The mitochondrial enzyme activity for O 2 Ϫ· production was higher (PϽ0.05) in the OM than in the cortex and papilla. Compared with NADH oxidase and mitochondrial enzymes, xanthine oxidase and NADPH oxidase produced much less O 2 Ϫ· in the kidney under this condition. Overall, the renal OM exhibited the greatest enzyme activities for O 2 Ϫ· production. In anesthetized rats, renal medullary interstitial infusion of a superoxide dismutase inhibitor, diethyldithiocarbamate, markedly decreased renal MBF and sodium excretion. Diethyldithiocarbamate (5 mg/kg per minute by renal medullary interstitial infusion [RI]) reduced the renal medullary laser-Doppler flow signal from 0.6Ϯ0.04 to 0.4Ϯ0.03 V, a reduction of 33%, and both urine flow and sodium excretion decreased by 49%. In contrast, a membrane-permeable superoxide dismutase mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (TEMPOL, 30 mol/kg per minute RI) increased MBF and sodium excretion by 34% and 69%, respectively. These effects of TEMPOL on renal MBF and sodium excretion were not altered by pretreatment with N G -nitro-L-arginine methyl ester (10 g/kg per minute RI). We conclude that (1) Key Words: free radicals Ⅲ oxygen Ⅲ hemodynamics, renal Ⅲ kidney I n contrast to the conventional idea that reactive oxygen species (ROS) are of only pathological consequence, recent studies have indicated that under physiological conditions, low concentrations of ROS play an important role in the normal regulation of cell and organ function. 1-4 Redoxmediated signaling is emerging as a fundamental regulatory mechanism in cell biology and physiology. 2,4 In this regard, ROS have been reported to participate in the control of vascular tone, and the interaction of superoxide (O 2 Ϫ· ) and NO has been considered as one of the important mechanisms regulating cardiovascular function. 1,2,4,5 It has been demonstrated that O 2 Ϫ· inactivates the endothelium-dependent relaxing factor, thereby reducing the arteriolar dilation to acetylcholine or other endothelium-dependent vasodilators 6 and that endothelial superoxide dismutase (SOD) activity significantly increased the half-life of the endothelium-dependent relaxing factor produced by acetylcholine. 5 Recent studies have provided direct evidence that inactivation of SOD activity with diethyldithiocarbamate (DETC) selectively in...

The present study examined whether preglomerular arterioles of the rat produce 20-hydroxyeicosatetraenoic acid (20-HETE) and whether 20-HETE is vasoactive on these vessels. Raf preglomerular arterioles produced 20-HETE (4.8 +/- 1.0 pmol.min-1.mg-1, n = 7) and, to a lesser extent, 14-, 15-, 11-, and 12-dihydroxyeicosatetraenoic acid, 6-ketoprostaglandin F/alpha and prostaglandin E2 when incubated with [14C]larachidonic acid. The results of immunoblotting and reverse-transcription polymerase chain reaction experiments indicate that these vessels express mRNA and protein for a P-450 4A2 enzyme. With the use of a rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free media, addition of 20-HETE (1 nM-1 microM) to the bath reduced the diameter of proximal and distal portions of the efferent arterioles. At a concentration of 1 microM, the diameter of the proximal and distal portions of the afferent arteriole fell by 14 +/- 1 and 16 +/- 3% after 20-HETE. The response to 20-HETE (1 microM) was not altered by blockade of cyclooxygenase, lipoxygenase, and p-450 pathways. Blockade of the large-conductance Ca(2+)-activated K+ channel with tetraethylammonium (1 mM) reduced the diameter of afferent arterioles by 10% and blocked the vasoconstrictor response to 20-HETE (1 microM). These results indicate that 20-HETE is an endogenous constrictor of preglomerular arterioles and suggest a role for the P-450 4A2 enzyme in the regulation of renal vascular tone.

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

. Role of renal NO production in the regulation of medullary blood flow. Am J Physiol Regul Integr Comp Physiol 284: R1355-R1369, 2003 10.1152/ajpregu.00701.2002The unique role of nitric oxide (NO) in the regulation of renal medullary function is supported by the evidence summarized in this review. The impact of reduced production of NO within the renal medulla on the delivery of blood to the medulla and on the long-term regulation of sodium excretion and blood pressure is described. It is evident that medullary NO production serves as an important counterregulatory factor to buffer vasoconstrictor hormoneinduced reduction of medullary blood flow and tissue oxygen levels. When NO synthase (NOS) activity is reduced within the renal medulla, either pharmacologically or genetically [Dahl salt-sensitive (S) rats], a super sensitivity to vasoconstrictors develops with ensuing hypertension. Reduced NO production may also result from reduced cellular uptake of L-arginine in the medullary tissue, resulting in hypertension. It is concluded that NO production in the renal medulla plays a very important role in sodium and water homeostasis and the long-term control of arterial pressure. renal medulla; Dahl salt-sensitive rats; hypertension; L-arginine THE RENAL MEDULLARY CIRCULATION is now recognized to be of importance for reasons that extend far beyond providing the economy of countercurrent exchange to concentrate large volumes of filtrate to produce small volumes of concentrated urine. It is now recognized that medullary blood flow (MBF) can play an important role in determining long-term levels of arterial pressure and that 15-30% reductions of blood flow to the renal medulla in the face of imperceptibly small changes of total renal blood flow can lead to the development of hypertension (16,21). Evidence is reviewed showing that nitric oxide (NO) plays a unique role in the acute and chronic regulation of renal MBF, sodium homeostasis, and arterial pressure. More specifically, the role of NO in the regulation of MBF, in linking tubular metabolic needs with delivery of nutrients and as a counterregulatory system to protect the medulla from the consequences of underperfusion, are the focus of this review. The influence of NO on the renal cortex and such issues that relate to pressure-natriuresis, autoregulation of total renal blood flow, tubuloglomerular feedback, and glomerular filtration rate (GFR) regulation are important subjects of renal function that either have been reviewed by others (2,29,43,55,96) or are beyond the scope of this relatively brief review.

Abstract-This study exammed the effect of intravenous mfuslon of subpressor doses of anglotensm (Ang II) on renal medullary blood flow (MBF), medullary partial oxygen pressure (PoJ, and mtnc oxide (NO) concentration under normal condltlons and during reduction of the medullary nitric oxide synthase (NOS) activity m anesthetized rats With laser Doppler flowmetry and polarographlc measurement of PO, with mlcroelectrodes, Ang II (5 rig/kg per mmute) did not alter renal cortical and medullary blood flows or medullary PO, iV,-mtro-L-argmme methyl ester (L-NAME) was infused mto the renal medullary mterstltlal space at a dose of 1 4 pg/kg per minute, a dose that did not slgmficantly alter basal levels of MBF or PO, Intravenous mfuslon of Ang II at the same dose m the presence of L-NAME decreased MBF by 23% and medullary PO, by 28%, but it had no effect on cortical blood flow or arterial blood pressure An m vlvo rmcrodlalysls-oxyhemoglobm NO trappmg technique was used m other rats to determine tissue NO concentrations using the $ame protocol Ang II infusion mcreased tissue NO concentrations by 85% m the renal cortex and 150% m the renal medulla Renal medullary mterstltlal mfuclon of L-NAME(1 4 Fg/kg per mmute) reduced medullary NO concentrations and substantially blocked Ang II-induced increases m NO concentrations m the renal medulla, but not m the renal cortex Tissue slices of the renal cortex and medulla were studled to determine the effects of Ang II and L-NAME on the mtnte/mtrate production Ang II stimulated the rntrlte/mtrate production predominately m the renal medulla, which was slgmficantly attenuated by L-NAME We conclude that small elevations of clrculatmg Ang II levels increase medullary NO production and concentrations, which plays an important role m buffering the vasoconstrlctor effects of this peptlde and m mamtammg a constancy of MBF (Hypertension.1997;31[part 2]:271-276.)Key Words: nitric oxide n anglotensm II n renal hemodynamlcs n renal medulla n laser Doppler flowmetry T here have been many stumes defining the role of Ang II m the regulation of total renal blood flow, glomemlar filtration, and tubular transport The role of Ang II m the regulation of blood flow to the renal medulla, however, remains poorly understood It has been demonstrated that the vasa recta bundles exhibit a high density of Ang II receptors' and that Ang II can produce a remarkable vasoconstncaon of the Isolated perfused vasa recta * These observations have suggested that Ang II could pamclpate m the control of MBF as m the renal cortex However, m vlvo stu&es using laser Doppler flowmetry and vldeormcroscopy techmques have demonstrated that intravenous mfuslon of Ang II has mmlmal effects on MBF and can even increase papillary blood 5ow at high doses 3-6 This suggested that the vasoconstnctlve effects of Ang II on the medullary vessels could be modulated by stlmulatlon of a local counterregulatory mechamstn Gwen the importance of Ml3F m the long-term control of blood pressure,'" this counterregulatory mechanism may play an import...