Angiotensin II–NAD(P)H Oxidase–Stimulated Superoxide Modifies Tubulovascular Nitric Oxide Cross-Talk in Renal Outer Medulla

Abstract

Abstract-The source of superoxide (O 2 ⅐Ϫ) production and cell-to-cell interactions of O 2 ⅐Ϫ and nitric oxide (NO) in response to angiotensin II (AngII) were studied by fluorescence microscopic techniques to image rat renal outer medullary microtissue strips. Changes in intracellular O 2 ⅐Ϫ were determined by dihydroethidium-ethidium ratios, and NO was determined with 4,5-diaminofluorescein diacetate. AngII (1 mol/L) significantly increased O 2 ⅐Ϫ in the isolated, medullary thick ascending limb (mTAL). These responses were inhibited by the superoxide dismutase mimetic 4-hydroxytetramethylpiperidine-1-oxyl (TEMPOL) and by the NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin. AngII did not increase O 2 ⅐Ϫ in either pericytes of isolated, intact vasa recta (VR) or pericytes of VR with a disrupted endothelium, even when surrounded by mTAL. However, AngII did increase O 2 ⅐Ϫ when the tissue strips were preincubated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), indicating that cross-talk of O 2 ⅐Ϫ from mTAL to the VR occurred but was normally inhibited by NO. Also, tissue O 2 ⅐Ϫ reduction by TEMPOL increased the diffusion of NO from mTAL to the pericytes, indicating that cross-talk of NO from the mTAL to the VR is also inhibited by O 2 ⅐Ϫ. We conclude that AngII stimulates O 2 ⅐Ϫ production in mTAL via the NAD(P)H oxidase pathway and that interactions of O 2 ⅐Ϫ and NO ultimately determine the effectiveness of in situ free-radical cross-talk between the mTAL and the VR. Key Words: free radicals Ⅲ rats Ⅲ oxidative stress Ⅲ renal blood flow Ⅲ angiotensin II O xidative stress in the kidney has been found to play an important role in the regulation of renal medullary blood flow (MBF), tubular sodium reabsorption, and the long-term control of arterial pressure. 1,2 It has been shown in short-term, anesthetized-rat studies that medullary interstitial infusion of the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid (DETC) reduces MBF and sodium excretion without a change in blood pressure. 1 Opposite changes were observed with medullary interstitial infusion of the cellpermeable SOD mimetic 4-hydroxytetramethylpiperidine-1-oxyl (TEMPOL). 1 Consistent with these observations, longterm, medullary interstitial infusion of DETC led to a reduction of MBF and produced hypertension in the absence of changes in cortical blood flow. 2 Because the same dose of DETC when given intravenously neither reduced MBF nor changed arterial pressure, that study demonstrated that an increase in oxidative stress, specifically within the renal medulla, would result in hypertension.Techniques for fluorescence imaging of both nitric oxide (NO) and Ca 2ϩ in the vasa recta (VR) and tubules of microtissue strips obtained from the medulla were developed recently in our laboratory. 3,4 We have demonstrated that NO serves as a paracrine substance that mediates cross-talk between the tubular epithelium of the medullary thick ascending limb (mTAL) and contractile VR pericyte...

Expression of Heme Oxygenase-1 in Thick Ascending Loop of Henle Attenuates Angiotensin II-Dependent Hypertension

“…NO produced in the TALH can diffuse into surrounding vasa recta capillaries to serve as a buffer against vasoconstrictors such as Ang II. 18 Ang IImediated increases in TALH superoxide production results in an attenuation of NO release, which decreases medullary blood flow and increases sodium reabsorption. It has previously been reported that superoxide-mediated decrease in NO crosstalk contributes to the decreased medullary blood flow observed in the hypertensive Dahl salt-sensitive rat.…”

Strain vessel hypothesis: a viewpoint for linkage of albuminuria and cerebro-cardiovascular risk

“…62 When the juxtamedullary glomeruli are injured by hypertension, the impairment of oxygen delivery downstream from the vasa recta vessels would be expected to result in tubulointerstitial injuries in the outer medulla. Superimposed on these factors, a number of other elements, including neurohormonal factors, 56 inflammatory pathways, 53 oxidative stress 63,64 and tubular transport, 65,66 are involved in the pathogenesis of the injuries, among which tubulovascular crosstalk is of particular interest. 63,64,67,68 We have recently shown that an increase in tubular transport at the mTAL enhances superoxide production and reduces nitric oxide synthesis.…”

“…Superimposed on these factors, a number of other elements, including neurohormonal factors, 56 inflammatory pathways, 53 oxidative stress 63,64 and tubular transport, 65,66 are involved in the pathogenesis of the injuries, among which tubulovascular crosstalk is of particular interest. 63,64,67,68 We have recently shown that an increase in tubular transport at the mTAL enhances superoxide production and reduces nitric oxide synthesis. [63][64][65]67 These changes would be transmitted to the neighboring vasa recta and would therefore facilitate vasoconstriction and reduction of medullary blood flow.…”

“…63,64,67,68 We have recently shown that an increase in tubular transport at the mTAL enhances superoxide production and reduces nitric oxide synthesis. [63][64][65]67 These changes would be transmitted to the neighboring vasa recta and would therefore facilitate vasoconstriction and reduction of medullary blood flow.…”

β1 Receptors Protect the Renal Afferent Arteriole of Angiotensin-Infused Rabbits from Norepinephrine-Induced Oxidative Stress

“…Single-agent signal capture was achieved by cycling at 3-s intervals between a 460-and 605-nm filter. Changes in O 2 ⅐ Ϫ were expressed as the ratio of ethidium:DHE fluorescence (26). The system used an Olympus IX70 fluorescence microscope equipped with dual photomultipliers (PMT, Photon Technology Int., Lawrenceville, NJ).…”