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Angiotensin II (ANG II) increases proximal tubule superoxide (O2-) production more in rats fed a 20% fructose normal-salt diet compared with rats fed a 20% glucose normal-salt diet. A 20% fructose high-salt diet (FHS) increases systolic blood pressure (SBP), whereas a 20% glucose high-salt diet (GHS) does not. However, it is unclear whether FHS enhances ANG II-induced oxidative stress in proximal tubules and whether this contributes to increases in blood pressure in this model. We hypothesized that FHS augments the ability of ANG II to stimulate O2- production by proximal tubules, and this contributes to fructose-induced salt-sensitive hypertension. We measured SBP in male Sprague-Dawley rats fed FHS and GHS and determined the effects of 3 mM tempol and 50 mg/kg losartan for 7 days. We then measured basal and ANG II-stimulated (3.7 × 10-8 M) O2- production by proximal tubule suspensions and the role of protein kinase C. FHS increased SBP by 27 ± 5 mmHg (n = 6, P < 0.006) but GHS did not. Rats fed FHS + tempol and GHS + tempol showed no significant increases in SBP. ANG II increased O2- production by 11 ± 1 relative light units/µg protein/s in proximal tubules from FHS-fed rats (n = 6, P < 0.05) but not in tubules from rats fed GHS. ANG II did not significantly stimulate O2- production by proximal tubules from rats fed FHS + tempol or FHS + losartan. The protein kinase C inhibitor Gö6976 blunted ANG II-stimulated O2- production. In conclusion, FHS enhances the sensitivity of proximal tubule O2- production to ANG II, and this contributes to fructose-induced salt-sensitive hypertension. NEW & NOTEWORTHY A diet containing amounts of fructose consumed by 17 million Americans causes salt-sensitive hypertension. Oxidative stress is an initiating cause of this model of fructose-induced salt-sensitive hypertension increasing blood pressure. This salt-sensitive hypertension is prevented by losartan and thus is angiotensin II (ANG II) dependent. Fructose-induced salt-sensitive hypertension depends on ANG II stimulating oxidative stress in the proximal tubule. A fructose/high-salt diet augments the ability of ANG II to stimulate proximal tubule O2- via protein kinase C. [ABSTRACT FROM AUTHOR]

Spontaneously hypertensive rats (SHRs) have normal glomerular capillary pressure even though renal perfusion pressure is higher, suggesting that preglomerular vessels exhibit abnormally high resistance. This may be due to increased superoxide ([O.sup.-.sub.2]) production, which contributes to the vasoconstriction in hypertension. We tested the hypothesis that the myogenic response of the afferent arteriole (Af-Art) is exaggerated in SHRs because of increased levels of reactive oxygen species (ROS). Single Af-Arts were microdissected from kidneys of SHRs and Wistar-Kyoto (WKY) rats and microperfused in vitro. When perfusion pressure in the Af-Art was increased stepwise from 60 to 140 mmHg, the luminal diameter decreased by 8.4 [+ or -] 2.9% in WKY Af-Arts but fell by 29.3 [+ or -] 5.6% in SHR Af-Arts. To test whether ROS production is enhanced during myogenic response in SHRs, we measured chloromethyl-dichlorodihydrofluorescein diacetate acetyl ester (CM-[H.sub.2]DCFDA) florescence before and after increasing intraluminal pressure from 60 to 140 mmHg. Pressure-induced increases in ROS were fourfold greater in SHR Af-Arts compared with WKY Af-Arts (SHR, 48.0 [+ or -] 2.2%; and WKY, 12.2 [+ or -] 0.3%). To test whether [O.sup.-.sub.2] contributes to the myogenic response in SHRs, either the membrane-permeant [O.sup.-sub.2] scavenger Tempol or the nox2-based NADPH oxidase (NOX2) inhibitor gp91ds-tat were added to the Af-Art lumen and bath and the myogenic response was tested before and after treatment. Both Tempol ([10.sup.-4] M) and gp91ds-tat ([10.sup.-5] M) significantly attenuated the pressure-induced constriction in SHR Af-Arts but not in WKY Af-Arts. We conclude that 1) pressure-induced constriction is exaggerated in SHR Af-Arts, 2) NOX2-derived [O.sup.-.sub.2] may contribute to the enhanced myogenic response, and 3) [O.sup.-.sub.2] exerts little influence on the myogenic response under normotensive conditions. hypertension; superoxide doi: 10.1152/ajpheart.00537.2009.

We showed that luminal flow increases net superoxide ([O.sup.-.sub.2]) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net [O.sup.-.sub.2] production by thick ascending limbs. Initiation of flow (20 nl/min) increased net [O.sup.-.sub.2] production from 4 [+ or -] 1 to 61 [+ or -] 12 AU/s (P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net [O.sup.-.sub.2] production (2 [+ or -] 1 vs. 1 [+ or -] 1 AU/s; P > 0.05; n = 5). Flow-stimulated [O.sup.-.sub.2] was also blocked in [p47.sup.phox]-deficient mice. We measured flowstimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 [+ or -] 0.02 to 0.96 [+ or -] 0.04 AU (P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net [O.sup.-.sub.2] production from 5 [+ or -] 2 to 92 [+ or -] 6 AU/s (P < 0.001; n = 6). The PKC-[alpha]- and [beta]I-selective inhibitor G6 6976 (100 nM) decreased flow-stimulated net [O.sup.-.sub.2] production from 54 [+ or -] 15 to 2 [+ or -] 1 AU/s (P < 0.04; n = 5). Flow-induced net [O.sup.-.sub.2] production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-[alpha] but not dnPKC[beta]I or LacZ ([DELTA] = 11 [+ or -] 3 AU/s for dnPKC[alpha], 55 [+ or -] 7 AU/s for dnPKC[beta]I, and 63 [+ or -] 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net [O.sup.-.sub.2] production in thick ascending limbs via PKC-[alpha]-mediated activation of NADPH oxidase. reactive oxygen species; protein kinases; NADPH oxidase; luminal flow doi: 10.1152/ajprenal.00543.2009.

Perez-Rojas JM, Kassem KM, Beierwaltes WH, Garvin JL, Herrera M. Nitric oxide produced by endothelial nitric oxide synthase promotes diuresis. Am J Physiol Regul lntegr Comp Physiol 298: R1050-R1055, 2010. First published February 10, 2010; doi: 10.1152/ajpregu.00181.2009.--Exwacellular fluid volume is highly regulated, at least in part, by peripheral resistance and renal function. Nitric oxide (NO) produced by NO synthase type 3 (NOS 3) in the nonrenal vasculature may promote fluid retention by reducing systemic vascular resistance and arterial pressure. In contrast, NO produced by renal NOS 3 promotes water excretion by reducing renal vascular resistance, increasing glomerular filtration, and inhibiting reabsorption along the nephron. Thus, the net effect of NO from NOS 3 on urinary volume (UV) is unclear. We hypothesized that NO produced by NOS 3 promotes water excretion primarily due to renal tubular effects. We gave conscious wild-type and NOS 3 -/- mice an acute volume load and measured UV, blood pressure, plasma renin concentration (PRC), [Na.sup.+], vasopressin, and urinary [Na.sup.+] and creatinine concentrations. To give the acute volume load, we trained mice to drink a large volume of water while in metabolic cages. On the day of the experiment, water was replaced with 1% sucrose, and mice had access to it for 1 h. Volume intake was similar in both groups. Over 3 h, wild-type mice excreted 62 [+ or -] 10% of the volume load, but NOS 3 -/- excreted only 42 [+ or -] 5% (P < 0.05). Blood pressure in NOS 3 -/- was 118 [+ or -] 3 compared with 110 [+ or -] 2 mmHg in wild-type mice (P < 0.05), but it did not change following volume load in either strain. PRC, vasopressin, and glomerular filtration rate were similar between groups. Urinary [Na.sup.+] excretion was 49.3 [+ or -] 7.0 in wild-type vs. 37.8 [+ or -] 6.4 [micro]mol/3 h in NOS 3 -/- mice (P < 0.05). Bumetanide administration eliminated the difference in volume excretion between wild-type and NOS 3 -/- mice. We conclude that 1) NO produced by NOS 3 promotes water and [Na.sup.+] excretion and 2) the renal epithelial actions of NO produced by NOS 3 supersede the systemic and renal vascular actions. nitric oxide; nitric oxide synthase 3; diuresis; transport doi: 10.1152/ajpregu.00181.2009.

Superoxide (0-2) produced by NADPH oxidase regulates Na absorption and renal hemodynamics. Increased NaC1 in the thick ascending limb (TAL) stimulates [O.sub.2] generation. However, we do not know whether physiological changes in NaCI concentration augment [O.sub.2] generation, nor do we know the mediator(s) involved. In other cells, Racl, a regulatory subunit of NADPH oxidase, is activated by elevated NaC1. We hypothesized that increasing luminal NaC1 within the physiological range activates Rac1 and NADPH oxidase and, thereby, increases 0-2 production. We increased NaC1 from 10 to 57 mM in medullary TAL suspensions and used lucigenin to measure 0-2 generation and Western blot to measure Racl activity. Increasing NaC1 stimulated 0-2 generation from 1.41 _+ 0.16 to 2.71 [+ or -] 0.30 nmol [[sigma].sub.2] * [min.sup.-1] * mg [protein.sup.-1] (n = 6, P < 0.05). This increase was blocked by the Na-K-2C1 cotransporter inhibitor furosemide and the NADPH oxidase inhibitor apocynin. To examine the role of Racl in NaCl-induced [[sigma].sub.2] production, we measured Rac1 translocation by Western blot. When we added NaC1, Racl in the particulate fraction increased from 6.8 + 0.8 to 11.7 [+ or -] 2.4% of total Racl (n = 7, P < 0.05). Then we measured 0-2 generation in the presence and absence of the Racl inhibitor. In the absence of the Racl inhibitor, NaC1 increased [O.sub.2] generation from 1.07 [+ or -] 0.24 to 2.02 + 0.49 nmol [[sigma].sub.2] * [min.sup.-1] * mg [protein.sup.-1], and this increase was completely blocked by the inhibitor. Similarly, in vivo treatment of TALs with adenovirus expressing dominant-negative Racl decreased NaCl-induced 0-2 generation by 60% compared with control (0.33 [+ or -] 0.04 vs. 0.81 [+ or -] 0.17 nmol [O.sub.2] * [min.sup.-l] * mg [protein.sup.-1], n = 6, P < 0.05). We concluded that physiological increases in NaCI stimulate TAL [[sigma].sub.2] generation by activating Racl. reactive oxygen species; sodium-potassium-chloride cotransport(er); NaC1 transport doi: 10.1152/ajprenal.00472.2009

Absorption of NaCl by the thick ascending limb (TAL) involves active transport and therefore depends on oxidative phosphorylation. Extracellular ATP has pleiotropic effects, including both stimulation and inhibition of transport and inhibition of oxidative phosphorylation. However, it is unclear whether ATP alters TAL transport and how this occurs. We hypothesized that ATP inhibits TAL Na absorption by reducing Na entry. We measured oxygen consumption in TAL suspensions. ATP reduced oxygen consumption in a concentration-dependent manner. The purinergic (P2) receptor antagonist suramin (300 [micro]M) blocked the effect of ATP on TAL oxygen consumption (147 [+ or -] 15 vs. 146 [+ or -] 16 nmol [O.sub.2] x [min.sup.-1] x mg [protein.sup.-1]). In contrast, the adenosine receptor antagonist theophylline did not block the effect of ATP on oxygen consumption. When Na-K-2Cl cotransport and Na/H exchange were blocked with furosemide (100 [micro]M) plus dimethyl amiloride (100 [micro]M), ATP did not inhibit TAL oxygen consumption (from 78 [+ or -] 13 to 98 [+ or -] 5 nmol [O.sub.2] x [min.sup.-1] x mg [protein.sup.-1]). The Na ionophore nystatin (200 U/ml) increased TAL oxygen consumption to a similar extent in both ATP- and vehicle-treated samples (368 [+ or -] 41 vs. 397 [+ or -] 47 nmol [O.sub.2] x [min.sup.-1] x mg [protein.sup.-1]). The nitric oxide synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (3 mM) blocked the ATP effects on TAL oxygen consumption (157 [+ or -] 10 vs. 165 [+ or -] 15 nmol [O.sub.2] x [min.sup.-1] x mg [protein.sup.-1]). The P2X-selective receptor antagonist NF023 blocked the effect of ATP on oxygen consumption, whereas the P2X-selective agonist [beta]-[gamma]-Me-ATP reduced oxygen consumption in a concentration-dependent manner. We conclude that ATP inhibits Na transport-related oxygen consumption in TALs by reducing Na entry and P2X receptors and nitric oxide mediate this effect. NKCC2; purinergic signaling; NHE doi: 10.1152/ajprenal.00325.2009.

Extracellular ATP regulates many physiological processes via release of nitric oxide (NO). ATP stimulates NO in thick ascending limbs (TALs), but the signaling cascade involved in the cells of this nephron segment, as well as many other types of cells, is poorly understood. We hypothesized that ATP enhances NO synthase (NOS) activity by stimulating PI3 kinase and Akt. We measured 1) NO in TALs using the NO-sensitive dye DAF-2 DA and 2) Akt activity by fluorescence resonance energy transfer and phosphorylation of Akt isoforms. ATP (100 [micro]M) stimulated NO in wild-type mice [26 [+ or -] 4 arbitrary units (AU)], but not in NOS3 -/- mice (2 [+ or -] 2 AU; P < 0.04). In the presence of the NOS1- and NOS2-selective inbibitors 7-NI and 1400W, ATP stimulated NO by 30 [+ or -] 2 and 33 [+ or -] 3 AU, respectively (not significant vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5 [+ or -] 2 vs. 28 [+ or -] 4 AU; P < 0.02). ATP alone increased Akt activity and this effect was significantly blocked by suramin, a P2 receptor antagonist. In the presence of an Akt-selective inhibitor, ATP-induced NO was blocked by 90 [+ or -] 4%. ATP significantly stimulated Akt1 phosphorylation at [Ser.sup.473] by 91 [+ or -] 13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominant-negative Aktl significantly decreased ATP-induced NO by 88 [+ or -] 6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL. purinergic signaling; adenosine triphosphate; Na-K-2Cl cotransporter

We have shown that increased luminal flow induces [O.sup.-.sub.2] and nitric oxide (NO) production in thick ascending limbs (TALs). However, the interaction of flow-stimulated NO and [O.sup.-.sub.2] in TALs is unclear. We hypothesized that NO inhibits flow-induced [O.sup.-.sub.2] production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated [O.sup.-.sub.2] production in rat TALs using dihydroethidium in the absence and presence of L-arginine (0.3 mM), the substrate for NO synthase. The addition of L-arginine reduced flow-induced net [O.sup.-.sub.2] production from 68 [+ or -] 9 to 17 [+ or -] 4 AU/s (P < 0.002). The addition of the NO synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME; 5 mM) in the presence of L-arginine stimulated production (L-arginine: 15 [+ or -] 4 AU/s vs. L-arginine + L-NAME: 63 [+ or -] 7 AU/s; P < 0.002). The guanylate cyclase inhibitor LY-83583 (10 [micro]M) also enhanced flow-induced net [O.sup.-.sub.2] production in the presence of L-arginine (L-arginine: 7 [+ or -] 4 AU/s vs. L-arginine + LY-83583:53 [+ or -] 7 AU/s; P < 0.01). In the presence of LY-83583, L-arginine only reduced flow-induced net [O.sup.-.sub.2] by 36% (LY-83583:80 [+ or -] 7 AU/s vs. LY-83583 + L-arginine: 51 [+ or -] 3 AU/s; P < 0.006). The cGMP analog dibutyryl (db)-cGMP reduced flow-induced net [O.sup.-.sub.2] from 39 [+ or -] 9 to 7 [+ or -] 3 AU/s (P < 0.03). The PKG inhibitor KT-5823 (5 [micro]M) partially restored flow-induced net [O.sup.-.sub.2] in the presence of L-arginine (L-arginine: 4 [+ or -] 4 AU/s vs. L-arginine + KT-5823:32 [+ or -] 9 AU/s; P < 0.03) and db-cGMP (db-cGMP: 9 [+ or -] 7 AU/s vs. db-cGMP + KT-5823:54 [+ or -] 5 AU/s; P < 0.01). Phosphodiesterase H inhibition had no effect on arginine-inhibited [O.sup.-.sub.2] production. We conclude that 1) NO reduces flow-stimulated [O.sup.-.sub.2] production, 2) this occurs primarily via the cGMP/PKG pathway, and 3) [O.sup.-.sub.2] scavenging by NO plays a minor role. reactive oxygen species; soluble guanylate cyclase; phosphodiesterase; scavenging

Tubuloglomerular feedback (TGF) is the mechanism by which the macula densa (MD) senses increases in luminal NaCl concentration and sends a signal to constrict the afferent arteriole (Af-Art). The kidney expresses constitutively heine oxygenase-2 (HO-2) and low levels of HO-1. HOs release carbon monoxide (CO), biliverdin, and free iron. We hypothesized that renal HOs inhibit TGF via release of CO and biliverdin. Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the MD perfusate. When HO activity was inhibited by adding stannous mesoporphyrin (SnMP) to the MD perfusate, the TGF response increased from 2.1 [+ or -] 0.2 to 4.1 [+ or -] 0.4 [micro]m (P = 0.003, control vs. SnMP, n = 7). When a CO-releasing molecule, (CORM-3; 50 [micro]M), was added to the MD perfusate, the TGF response decreased by 41%, from 3.6 [+ or -] 0.3 to 2.1 [+ or -] 0.2 [micro]m (P < 0.001, control vs. CORM-3, n = 12). When CORM-3 at 100 [micro]M was added to the perfusate, it completely blocked the TGF response, from 4.2 [+ or -] 0.4 to -0.2 [+ or -] 0.3 [micro]m (P < 0.001, control vs. CORM-3, n = 6). When biliverdin was added to the perfusate, the TGF response decreased by 79%, from 3.4 [+ or -] 0.3 to 0.7 [+ or -] 0.4 [micro]m (P = 0.001, control vs. biliverdin, n = 6). The effects of SnMP and CORM-3 were not blocked by inhibition of nitric oxide synthase. We concluded that renal HO inhibits TGF probably via release of CO and biliverdin. HO regulation of TGF is a novel mechanism that could lead to a better understanding of the control of renal microcirculation and function. carbon monoxide; biliverdin; afferent arteriole; macula densa

Extracellular ATP is an autocrine/paracrine factor that regulates renal function. Transient receptor potential vanilloid (TRPV) 4 is a cation channel that mediates release of autocrine/paracrine factors by acting as an osmosensor. The renal medulla, and therefore the thick ascending limb, is exposed to osmotic stress. We hypothesize that reduced osmolality stimulates ATP release from the thick ascending limb via transient receptor potential vanilloid (TRPV) 4 activation. We measured ATP release by medullary thick ascending limb suspensions after reducing bath osmolality from 350 to 323 mosmol/kg[H.sub.2]O, using the luciferin-luciferase assay. Decreasing osmolality stimulated ATP release compared with control (38.9 [+ or -] 7.2 vs. 2.4 [+ or -] 1.0 pmol/mg protein; n = 6, P < 0.01). To examine the role of TRPV4, we used 1) Ca-free solutions, 2) a TRPV4 inhibitor, 3) small interfering (si) RNA against TRPV4, and 4) a TRPV4 activator. Removal of Ca completely blocked osmolality-induced ATP release (42.2 [+ or -] 5.9 vs. 2.6 [+ or -] 1.5 pmol/mg protein; n = 6, P < 0.01). In the presence of the TRPV4-selective inhibitor ruthenium red, osmolality-induced ATP release was blocked by 73% (56.4 [+ or -] 19.9 vs. 8.8 [+ or -] 2.3 pmol/mg protein; n = 6; P < 0.03). In vivo treatment of thick ascending limbs with siRNA against TRPV4 decreased osmolality-induced ATP release by 62% (31.5 [+ or -] 3.4 vs. 12.4 [+ or -] 1.1 pmol/mg protein; n = 6; P < 0.01), while reducing TRPV4 expression by 74% compared with the nontreated kidney. Treatment with scrambled siRNA did not affect TRPV4 expression and/or osmolality-induced ATP release. Finally, in the absence of changes in osmolality, the specific TRPV4 agonist 4[alpha]-PDD increased ATP release (3.6 [+ or -] 0.9 vs. 25.4 [+ or -] 7.4 pmol/mg protein; n = 6; P < 0.04). We concluded that decreases in osmolality stimulate ATP release by thick ascending limbs and this effect is mediated by TRPV4 activation. osmosensor; cell swelling, calcium channels, hyposmolality

We hypothesized that elevated macula densa intracellular pH ([pH.sub.i]) during tubuloglomerular feedback enhances [O.sup.-.sub.2] production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, [O.sup.-.sub.2] production increased from 0.53 [+ or -] 0.09 to 2.62 [+ or -] 0.54 U/min (P < 0.01). To determine whether inhibiting the Na/H exchanger blocks [O.sup.-.sub.2] production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, [O.sup.-.sub.2] production induced by NaCl was blunted by 40%. To study the effect of [pH.sub.i] on [O.sup.-.sub.2] in intact macula densa cells, we measured [O.sup.-.sub.2] while pHi was changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, [O.sup.-.sub.2] production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of [O.sup.-.sub.2], we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin ([10.sup.-5] M), [O.sup.-.sub.2] production induced by elevating pHi was blocked. Finally, we measured the optimum pH for [O.sup.-.sub.2] production by the macula densa and found optimum extracellular pH is at 7.7 and optimum [pH.sub.] is ~8 for [O.sup.-.sub.2] production. We found that elevated [pH.sub.i] enhances [O.sup.-.sub.2] production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and [O.sup.-.sub.2] production is pH sensitive, with an optimum [pH.sub.i] of 8. tubuloglomerular feedback; pH optimum; Na/H exchange; reactive oxygen species

Superoxide ([O.sup.-.sub.2]) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of [O.sup.-.sub.2] production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces [O.sup.-.sub.2] production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect [O.sup.-.sub.2] production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, [O.sup.-.sub.2] production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in [O.sup.-.sub.2], we used tempol ([10.sup.-4] M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of [O.sup.-.sub.2], we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, [O.sup.-.sub.2] production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, [O.sup.-.sub.2] production was blocked. To test whether depolarization of the macula densa induces [O.sup.-.sub.2] production, we artificially induced depolarization by adding valinomycin ([10.sup.-6] M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases [O.sup.-.sub.2] production. We conclude that increasing luminal NaC1 induces [O.sup.-.sub.2] production during tubuloglomerular feedback. [O.sup.-.sub.2] generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization. tubuloglomerular feedback doi:10.1152/ajprenal.00515.2006

Nitric oxide (NO) produced by endothelial cells diffuses to vascular smooth muscle cells to cause dilatation of the renal vasculature and other vessels. Although it is generally assumed that NO moves from cell to cell by free diffusion, we recently showed that aquaporin-1 (AQP-1) transports NO across cell membranes. AQP-1 is expressed in endothelial and vascular smooth muscle cells. We hypothesized that diffusion of NO into vascular smooth muscle cells and out of endothelial cells is facilitated by AQP-1, and transport of NO by AQP-1 is involved in endothelium-dependent relaxation. In intact aortic rings from AQP-1 -/- mice, vasorelaxation induced by acetylcholine (which increases endogenous NO) was reduced (P < 0.0001 vs. control). No differences were found in the relaxation caused by intracellular delivery of NO or intracellular cGMP between strains. In endothelium-denuded aortic rings from AQP-1 -/- mice, the vasorelaxant capability of NO released in the extracellular space was reduced (P < 0.0001 vs. control). Influx of NO (5 [micro]M) into vascular smooth muscle cells was 0.17 [+ or -] 0.02 f.u./s for control and 0.07 [+ or -] 0.01 f.u./s for AQP-1 -/- mice, 62% lower (P < 0.002). NO released by endothelial cells in response to 1 [micro]M acetylcholine was 96.2 [+ or -] 17.7 pmol NO/rag for control and 41.9 [+ or -] 13.4 pmol NO/mg for AQP-1 -/- mice, 56% reduction (P < 0.04). NOS3 expression was 1.33 [+ or -] 0.29 O.D. units for control and 3.84 [+ or -] 0.76 O.D. units for AQP-1 -/- mice, 188% increase (P < 0.01). We conclude that 1) AQP-1 facilitates NO influx into vascular smooth muscle cells, 2) AQP-1 facilitates NO diffusion out of endothelial cells, and 3) transport of NO by AQP-1 is required for full expression of endothelium-dependent relaxation. aquaporin-1; nitric oxide; transport; relaxation; aquaporin-1 knockout doi:10.1152/ajprenal.00353.2006

Superoxide ([O.sup.-.sub.2]) regulates renal function and is implicated in hypertension. [O.sup.-.sub.2] production increases in response to increased ion delivery in thick ascending limbs (TALs) and macula densa and mechanical strain in other cell types. Tubular flow in the kidney acutely varies causing changes in ion delivery and mechanical stress. We hypothesized that increasing luminal flow stimulates [O.sup.-.sub.2] production by NADPH oxidase in TALs via activation of Na-K-2Cl cotransport. We measured intracellular [O.sup.-.sub.2] in isolated rat TALs using dihydroethidium in the presence and absence of luminal flow and inhibitors of NADPH oxidase, Na-K-2Cl cotransport, and Na/H exchange. In the absence of flow, the rate of 02 production was 5.8 [+ or -] 1.4 AU/s. After flow was initiated, it increased to 29.7 [+ or -] 4.3 AU/s (P < 0.001). [O.sup.-.sub.2] production was linearly related to flow. Tempol alone and apocynin alone blocked the flow-induced increase in [O.sup.-.sub.2] production (3.5 [+ or -] 1.7 vs. 4.5 [+ or -] 2. 8 AU/s and 8.2 [+ or -] 2.1 vs. 10.6 [+ or -] 2.8 AU/s, respectively). Furosemide decreased flow-induced [O.sup.-.sub.2] production by 55% (37.3 [+ or -] 5.2 to 16.8 [+ or -] 2.8 AU/s; P < 0.002); however, dimethylamiloride had no effect. Finally, we examined whether changes in mechanical forces are involved in flow-induced [O.sup.-.sub.2] production by using a Na-free solution to perfuse TALs. In the absence of NaCl, luminal flow enhanced [O.sup.-.sub.2] production (1.5 [+ or -] 0.5 to 13.5 [+ or -] 1.1 AU/s; P < 0.001), ~50% less stimulation than when flow was increased in the presence of luminal NaCl. We conclude that flow stimulates [O.sup.-.sub.2] production in TALs via activation of NADPH oxidase and that NaCl absorption due to Na-K-2Cl cotransport and flow-associated mechanical factors contribute equally to this process. reactive oxygen species; ion delivery; Na transport; mechanical strain

Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) and, if so, elucidated the cellular mechanisms involved. Changes in [[Ca.sup.2+]]i responses were studied by fluorescence imaging using the [Ca.sup.2+] indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (A[T.sub.1]) receptors were blocked by losartan (10 [micro]M). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [[[Ca.sup.2+]].sub.i] that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [[[Ca.sup.2+]].sub.i] that peaked at 30 s (basal: 2.2 [+ or -] 0.3 vs. ANG II: 14.9 [+ or -] 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular [Ca.sup.2+] with EGTA (2 mM) did not alter microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses ([Ca.sup.2+] free + ANG II: 12.3 [+ or -] 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular [Ca.sup.2+] stores or with U-73122 to inhibit phospholipase C (1 [micro]M each) markedly attenuated microinjected ANG II-induced [[[Ca.sup.2+].sub.i] responses. Combined microinjection of ANG II and losartan abolished [[[Ca.sup.2+]].sub.i] responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [[[Ca.sup.2+]].sub.i] by stimulating intracellular A[T.sub.1] receptors and releases [Ca.sup.2+] from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function. intracellular calcium; kidney: microinjection; proximal tubules; receptor-mediated endocytosis

Nitric oxide (NO) plays a role in many physiological and pathophysiological processes. In the kidney, NO reduces renal vascular resistance, increases glomerular filtration rate, alters renin release, and inhibits transport along the nephron. The thick ascending limb is responsible for absorbing 20-30% of the filtered load of NaCl, much of the bicarbonate that escapes the proximal nephron, and a significant fraction of the divalent cations reclaimed from the forming urine. Additionally, this nephron segment plays a role in [K.sup.+] homeostasis. This article will review recent advances in our understanding of the role NO plays in regulating the transport processes of the thick ascending limb. NO has been shown to inhibit NaCl absorption primarily by reducing [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransport activity. NO also inhibits bicarbonate absorption by reducing [Na.sup.+]/[H.sup.+] exchange activity. It has also been reported to enhance luminal [K.sup.+] channel activity and thus is likely to alter [K.sup.+] secretion. The source of NO may be vascular structures such as the afferent arteriole or vasa recta, or the thick ascending limb itself. NO is produced by NO synthase 3 in this segment, and several factors that regulate its activity both acutely and chronically have recently been identified. Although the effects of NO on thick ascending limb transport have received a great deal of attention recently, its effects on divalent ion absorption and many other issues remain unexplored. sodium-potassium-2 chloride cotransport; sodium/hydrogen exchanger; cGMP; bicarbonate; calcium; magnesium; [N.sup.a+]-[K.sup.+]-ATPase

Superoxide ([O.sup.-.sub.2]) increases [Na.sup.+] reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of [O.sup.-.sub.2] on other THAL transporters, such as [Na.sup.+]/[H.sup.+] exchangers, are unknown. We hypothesized that [O.sup.-.sub.2] stimulates [Na.sup.+]/[H.sup.+] exchange in the THAL. We assessed total [Na.sup.+]/[H.sup.+] exchange activity by measuring recovery of intracellular pH ([pH.sub.i]) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pHi recovery rate from 0.26 [+ or -] 0.05 to 0.21 [+ or -] 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state [pH.sub.i] from 7.52 to 7.37. Because THALs have different [Na.sup.+]/[H.sup.+] exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral [Na.sup.+]/[H.sup.+] exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal [Na.sup.+]/[H.sup.+] exchange from 3.5 [+ or -] 0.8 to 6.7 [+ or -] 1.4 pmol x [min.sup.-1] x [mm.sup.-1] (P < 0.01) but decreased basolateral [Na.sup.+]/[H.sup.+] exchange from 10.8 [+ or -] 1.8 to 6.8 [+ or -] 1.1 pmol x [min.sup.-1] x [mm.sup.-1] (P < 0.007). To ascertain whether these effects were caused by [O.sup.-.sub.2] or [H.sub.2][O.sub.2], we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral [Na.sup.+]/[H.sup.+] exchange. We conclude that [O.sup.2] inhibits basolateral and stimulates luminal [Na.sup.+]/[H.sup.+] exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral [Na.sup.+]/[H.sup.+] exchange may enhance stimulation of luminal [Na.sup.+]/[H.sup.+] exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of [O.sup.-.sub.2] on [Na.sup.+]/[H.sup.+] exchange may increase net HC[O.sup.-.sub.3] reabsorption by the THAL. reactive oxygen species; intracellular pH; superoxide dismutase

Superoxide ([O.sup.-.sub.2]) enhances Na reabsorption by the thick ascending limb (THAL). Na absorption in this segment involves the Na-K-2Cl cotransporter, K channel, and Na-K-ATPase. We hypothesized that [O.sup.-.sub.2] stimulates NaCl absorption primarily by enhancing Na-K-2Cl cotransport. First, we measured steady-state intracellular Na ([Na.sub.i]) and chloride ([Cl.sub.i]). Xanthine oxidase (XO; 0.75 mU/ml) and hypoxanthine (HX; 0.125 mM) were added to the bath to increase [O.sup.-.sub.2]. During the control period, [Na.sub.i] was 12.2 [+ or -] 1.9 mM. After treatment with [O.sup.-.sub.2], it rose to 20.9 [+ or -] 3.3 mM, a 71% increase (P < 0.01). [Cl.sub.i], also increased (P < 0.01). Neither XO nor HX alone had a significant effect on [Na.sub.i] or [Cl.sub.i]. Next, we tested cotransport activity by measuring the initial rate of increase in [Na.sub.i] caused by changing luminal Na-Cl-K from 50/0/0 to 140/134/4 mM. During the control period, the initial rate of increase was 0.13 [+ or -] 0.02 arbitrary units (AU)/min. After treatment with [O.sup.-.sub.2], it was 0.22 [+ or -] 0.04 AU/min /P < 0.025), a 69% increase. Neither XO nor HX alone had a significant effect. Furosemide completely blocked the increase in intracellular Na in the control and [O.sup.-.sub.2] treatment periods. Next, we studied K channel activity by measuring the depolarization caused by increasing luminal K from 1 to 25 mM using a voltage-sensitive dye. During the control period, maximum depolarization was 7.31 [+ or -] 0.77 AU. After [O.sup.-.sub.2] treatment, it was 6.18 [+ or -] 0.90 AU (P < 0.05), a 15% decrease. Finally, we assessed the effects of [O.sup.-.sub.2] on Na-K-ATPase activity in THAL suspensions by measuring ATP hydrolysis. [V.sub.max] and [K.sub.1/2] for Na were not affected by [O.sup.-.sub.2]. We concluded that [O.sup.-.sub.2] stimulates THAL NaCl absorption primarily by enhancing Na entry via Na-K-2Cl cotransport. reactive oxygen species; transport; Na-K-ATPase; K channels; NKCC2

The dietary sodium requirements for rats have been a matter of debate. Our hypothesis was that normal commercial rodent chow contains sodium in excess of dietary needs and that this could have a significant impact on cardiovascular and renal physiology. To investigate dietary sodium requirements, 3-wk-old weanling Sprague-Dawley rats were fed a custom pelleted diet containing no sodium that was isocaloric to normal commercial rodent chow. These rats were provided with two drinking bottles; one contained water, and the other contained 0.5% NaCl. Thus they could choose and consume sodium as needed. Age-matched controls received normal pelleted Harlan Teklad 22/5 rodent diet (0.5% sodium content) and water ad libitum. Body weight and liquid intake were monitored over 7 wk until the rats were 10 wk old. At the end of the study, blood pressure was recorded. Weekly sodium intake in the experimental group was only 15% of that reported for rats fed normal rodent chow beginning in the first week postweaning. Growth was identical in the two groups (7.8 [+ or -] 0.1 vs. 7.6 [+ or -] 0.1 g/day), as was the total fluid volume intake. Blood pressure was significantly lower in the experimental rats compared with controls (96 [+ or -] 4 vs. 122 [+ or -] 4 mmHg, P < 0.05). These data suggest that, when given the choice, rats will consume significantly less sodium than provided in commercial chow, without any alteration in their growth rate. Rats fed standard commercial rodent chow may consume at least seven times more sodium than is necessary. This suggests commercial rodent diets may force excess sodium to accommodate caloric intake. calories; growth; metabolic requirements; blood pressure

A high-salt diet increases renal endothelin (ET) production and thick ascending limb (THAL) endothelial nitric oxide synthase (eNOS) expression. ET stimulates THAL eNOS expression via E[T.sub.B] receptors. The tonicity of the renal medulla is highly variable, and hyperosmolality stimulates ET-1 synthesis by endothelial cells. We hypothesized that a high-salt diet raises medullary osmolality, increases ET release by the THAL, and thus enhances eNOS expression. Seven days of high salt (1% NaCl in drinking water) increased eNOS expression in THALs by 125 [+ or -] 31%. High salt increased outer medullary osmolality from 362 [+ or -] 13 to 423 [+ or -] 6 mosmol/kg[H.sub.2]O (P < 0.05). Bosentan, a dual-ET receptor antagonist, blocked the increase in THAL eNOS expression caused by high salt (2.66 [+ or -] 0.44 absorbance units with bosentan vs. 5.15 [+ or -] 0.67 for vehicle; P < 0.05). Conscious systolic blood pressure did not differ between the two groups. In primary cultures of medullary THALs, raising osmolality from 300 to 350 and 400 mosmol/kg[H.sub.2]O using NaCl increased eNOS expression by 39 [+ or -] 11% (P < 0.05) and 71 [+ or -] 16%, respectively (P < 0.05). In primary cultures of THALs, raising osmolality from 300 to 400 mosmol/kg[H.sub.2]O for 1 h increased ET-1 release from 62 [+ or -] 7 to 113 [+ or -] 2 pg/mg protein (P < 0.05). BQ-788, an E[T.sub.B] receptor antagonist (1 [micro]M), blocked the stimulatory effect of 400 mosmol/kg[H.sub.2]O on eNOS expression (70 [+ or -] 13% vs. -5 [+ or -] 10%; paired difference, 74 [+ or -] 15%; P < 0.05). BQ-788 alone had no significant effect. We concluded that high salt stimulates THAL eNOS expression by increasing outer medullary osmolality, ET-1 release by the THAL and E[T.sub.B] receptor activation. This may be an important regulatory mechanism of THAL NaCl absorption when dietary salt intake is increased. nitric oxide; kidney; outer medulla; Henle's loop

Endothelial nitric oxide synthase (eNOS) regulates NaCl absorption by the thick ascending limb of the loop of Henle (THAL). We found that augmenting luminal flow induces eNOS activation and translocation to the apical membrane of THALs (Ortiz PA, Hong NJ, and Garvin JL. Am J Physiol Renal Physiol 287: F274-F280, 2004). In other cells, eNOS activation by shear stress is mediated by phosphatidylinositol 3-OH kinase (PI3)-kinase. We hypothesized that luminal flow induces eNOS activation via PI3-kinase. Pretreatment of THALs with wortmannin, a PI3kinase inhibitor, significantly reduced flow-induced nitric oxide (NO) release by 75% (from 53.6 [+ o -] 6 to 13.2 [+ or -] 5.7 pA/mm). Increasing luminal flow from 0 to 20 nl/min induced eNOS translocation to the apical membrane, whereas in the presence of wortmannin eNOS translocation was prevented (basolateral = 32 [+ or -] 2%, middle = 38 [+ or -] 1%, apical = 30 [+ or -] 1%, n = 5, not significant vs. no flow). We next studied which PI3-kinase product mediates eNOS translocation. Addition of PI(3,4,5)[P.sub.3] (5 [micro]M) in the absence of flow increased NO levels (P < 0.05) and induced eNOS translocation to the apical membrane (from 40 [+ or-] 4 to 60 [+ or -] 2% of total eNOS, n = 6, P < 0.05). Incubation with PI(3,4)[P.sub.2] or PI(4,5)[P.sub.2] did not change eNOS localization. We next tested whether heat shock protein (Hsp)90 is involved in eNOS translocation. The Hsp90 inhibitor geldanamycin blocked flow-induced eNOS translocation to the apical membrane (n = 6). Flow also induced translocation of Hsp90 to the apical membrane (from 35 [+ or -] 2 to 57 [+ or -] 2%; P < 0.05) in a PI3-kinase-dependent manner. We conclude that luminal flow induces eNOS translocation and activation in the THAL via PI3-kinase and that Hsp90 is involved in eNOS translocation to the apical membrane. nitric oxide; renal tubules; trafficking; phosphatidylinositol; heat shock protein; endothelial nitric oxide synthase

Endothelin-1 (ET-1) acutely inhibits NaC1 reabsorption by the thick ascending limb (THAL) by activating the E[T.sub.B] receptor, stimulating endothelial nitric oxide synthase (eNOS), and releasing nitric oxide (NO). In nonrenal tissue, chronic exposure to ET-I stimulates eNOS expression via the E[T.sub.B] receptor and activation of phosphatidylinositol 3-kinase (PI3K). We hypothesized that ET-1 increases eNOS expression in the THAL by binding to E[T.sub.B] receptors and stimulating PI3K. In primary cultures of medullary THALs treated for 24 h, eNOS expression increased by 36 [+ or -] 18% with 0.01 nM ET-1, 123 [+ or -] 30% with 0.1 nM (P < 0.05; n = 5), and 71 [+ or -] 30% with 1 nM, whereas 10 nM had no effect. BQ-788, a selective E[T.sub.B] receptor antagonist, completely blocked stimulation of eNOS expression caused by 0.1 nM ET-1 (12 [+ or -] 25 vs. 120 [+ or -] 40% for ET-1 alone; P < 0.05; n = 5). BQ-123, a selective E[T.sub.A] receptor antagonist, did not affect the increase in eNOS caused by 0.1 nM ET-1. Sarafotoxin c ($6c; 0.1 [micro]M), a selective E[T.sub.B] receptor agonist, increased eNOS expression by 77 [+ or -] 30% (P < 0.05; n = 6). Wortmannin (0.01 [micro]M), a PI3K inhibitor, completely blocked the stimulatory effect of 0.1 [micro]M $6c (77 [+ or -] 30 vs. -28 [+ or -] 9%; P < 0.05; n = 6). To test whether the increase in eNOS expression heightens activity, we measured NO release in response to simultaneous treatment with L-arginine, ionomycin, and clonidine using a NO-sensitive electrode. NO release by control cells was 337 [+ or -] 61 and 690 [+ or -] 126 pA in ET-1-treated cells (P < 0.05; n = 5). Taken together, these data suggest that ET-1 stimulates THAL eNOS, activating E[T.sub.B] receptors and PI3K and thereby increasing NO production. endothelial nitric oxide synthase; phosphatidylinositol 3-kinase

A high-salt diet enhances nitric oxide (NO)-induced inhibition of transport in the thick ascending limb (THAL). Long exposures to NO inhibit Na-K-ATPase in cultured cells. We hypothesized that NO inhibits THAL Na-K-ATPase after long exposures and a high-salt diet would augment this effect. Rats drank either tap water or 1% NaC1 for 7-l0 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 [micro]M) reduced Na-K-ATPase activity from 0.44 [+ or -] 0.03 to 0.30 [+ or -] 0.04 nmol P[sub.i] x [micro]g [protein.sup.-1] x [min.sup.-1] in THALs from rats on a normal diet (P < 0.03). Nitroglycerin also reduced Na-K-ATPase activity (P < 0.04). After 20 min, SPM had no effect (change -0.07 [+ or -] 0.05 nmol P[sub.i] x [micro]g [protein.sup.-1] x [min.SUP.-1]). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONO[O.sup.-] formed by NO reacting with [O.sup.-.sub.2] was involved, we measured [O.sup.-.sub.2] production. THALs from rats on normal and high salt produced 35.8 [+ or -] 0.3 and 23.7 [+ or -] 0.8 nmol [O.sup.-.sub.2] x [min.sup.-1] x mg [protein.sup.-1], respectively (P < 0.01). Because [O.sup.-.sub.2] production differed, we studied the effects of the [O.sup.-.sub.2] scavenger tempol. In the presence of 50 [micro]M tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 [+ or -] 0.05 vs. 0.52 [+ or -] 0.07 nmol P[sub.i] x [micro]g [protein.sup.-1] x [min.sup.-1]). Propyl gallate, another [O.sup.-.sub.2] scavenger, also prevented SPM-induced inhibition of Na-K-ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when [O.sup.-.sub.2] levels were increased with xanthine oxidase and hypoxanthine. We concluded that NO inhibits Na-K-ATPase after long exposures when rats are on a normal diet and this inhibition depends on [O.sup.-.sub.2]. NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased [O.sup.-.sub.2] production. superoxide; nitric oxide; Na transport; hypertension

Nitric oxide (NO) plays an essential role in the maintenance of cardiovascular and renal homeostasis. Endogenous NO is produced by three different NO synthase (NOS) isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). To investigate which NOS is responsible for NO production in different tissues, NOS knockout (-/-) mice have been generated for the three isoforms. This review focuses on the regulation of cardiovascular and renal function in relation to blood pressure homeostasis in the different NO[S.sup.-/-] mice. Although regulation of vascular tone and cardiac function in eNO[S.sup.-/-] has been extensively studied, far less is known about renal function in these mice. eNO[S.sup.-/-] mice are hypertensive, but the mechanism responsible for their high blood pressure is still not clear. Less is known about cardiovascular and renal control in nNO[S.sup.-/-] mice, probably because their blood pressure is normal. Recent data suggest that nNOS plays important roles in cardiac function, renal homeostasis, and regulation of vascular tone under certain conditions, but these are only now beginning to be studied. Inasmuch as iNOS is absent from the cardiovascular system under physiological conditions, it may become important to blood pressure regulation only during pathological conditions related to inflammatory processes. However, iNOS is constitutively expressed in the kidney, where its function is largely unknown. Overall, the study of NOS knockout mice has been very useful and produced many answers, but it has also raised new questions. The appearance of compensatory mechanisms suggests the importance of the different isoforms to specific processes, but it also complicates interpretation of the data. In addition, deletion of a single gene may have physiologically significant effects in addition to those being studied. Thus the presence or absence of a specific phenotype may not reflect the most important physiological function of the absent gene. endothelial nitric oxide synthase; neuronal nitric oxide synthase; inducible nitric oxide synthase; knockout mice; blood pressure

The thick ascending limb of the loop of Henle (THAL) plays an important role in the regulation of NaCl and water reabsorption. In vivo studies have shown that the free radical superoxide ([O.sup.-.sub.2]) stimulates Na and water reabsorption by the kidney. However, it is not known whether [O.sup.-.sub.2] regulates transport along the nephron in general or in the THAL specifically. We hypothesized that [O.sup.-.sub.2] stimulates THAL NaCl reabsorption. Cl absorption was measured in isolated, perfused THALs from Sprague-Dawley rats. First, we tested whether extracellular [O.sup.-.sub.2] stimulates Cl absorption. Addition of the [O.sup.-.sub.2]-generating system xanthine oxidase/hypoxanthine increased Cl absorption from 112.7 [+ or -] 12.0 to 146.2 [+ or -] 13.9 pmol*[mm.sup.-1]*[min.sup.-1], a 33% increase (P < 0.03). When superoxide dismutase (300 U/ml) was present in the bath, addition of xanthine oxidase/hypoxanthine did not significantly increase Cl absorption (116.9 [+ or -] 13.8 vs. 102.5 [+ or -] 8.5 pmol*[mm.sup.-1]*[min.sup.-1]). Furthermore, adding 200 nM [H.sub.2][O.sub.2] to the bath did not significantly affect Cl absorption (from 130.3 [+ or -] 13.7 to 125.3 [+ or -] 19.6 pmol*[mm.sup.-1]*[min.sup.-1]). Because extracellular [O.sup.-.sub.2] stimulated Cl absorption, we next tested whether endogenously produced [O.sup.-.sub.2] could stimulate transport. Under basal conditions, THALs produced detectable amounts of [O.sup-.sub.2], as measured by lucigenin-enhanced chemiluminescence. Adding the [O.sup.-.sub.2] scavenger tempol to the bath decreased Cl absorption from 198.1 [+ or -] 35.4 to 132.4 [+ or -] 23.5 pmol*[mm.sup.-1]*[min.sup.-1], a 31% decrease (P < 0.02). To make sure tempol was not exerting cytotoxic effects, we tested whether its effect was reversible. With tempol in the bath, Cl absorption was 117.2 [+ or -] 9.3 pmol*[mm.sup.-1]*[min.sup.-1]. Sixty minutes after tempol was removed from the bath, Cl absorption had increased to 149.2 [+ or -] 9.1 pmol*[mm.sup.1]*[min.sup.-1] (P < 0.05). We concluded that both exogenous and endogenous [O.sup-.sub.2] stimulate THAL NaCl absorption. To our knowledge, these are the first data showing a direct effect of [O.sup.-.sub.2] on nephron transport. superoxide dismutase; sodium-potassium-2 chloride cotransport; loop of Henle; salt-sensitive hypertension; urinary sodium excretion

Nitric oxide (NO) plays an important role in various physiological processes in the kidney. In vivo experiments first suggested that the natriuretic and diuretic effects caused by NO may be due to decreased NaCl and fluid absorption by the nephron. In the last 10 years, several reports have directly demonstrated a role for NO in modulating transport in different tubule segments. The effects of NO on proximal tubule transport are still controversial. Both stimulation and inhibition of net fluid and bicarbonate have been reported in this segment, whereas only inhibitory effects of NO have been found in Na/H exchanger and Na/K-ATPase activity. The effects of NO in the thick ascending limb are more homogeneous than in the proximal tubule. In this segment, NO decreases net Cl and bicarbonate absorption. A direct inhibitory effect of NO on the Na-K-2Cl cotransporter and the Na/H exchanger has been reported, while NO was found to stimulate apical K channels in this segment. In the collecting duct, NO inhibits Na absorption and vasopressin-stimulated osmotic water permeability. An inhibitory effect of NO on H-ATPase has also been reported in intercalated cells of the collecting duct. Overall, the reported effects of NO in the different nephron segments mostly agree with the natriuretic and diuretic effects observed in vivo. However, the net effect of NO on transport is still controversial in some segments, and in cases like the distal tubule, it has not been studied. sodium/hydrogen exchange; sodium-potassium-2 chloride costransport; epithelial sodium channel; sodium/potassium-adenosine triphosphatase; proximal tubule; thick ascending limb; collecting duct

Ren, Yilin, Hong Yu, Hong Wang, Oscar A. Carretero, and Jeffrey L. Garvin. Nystatin and valinomycin induce tubuloglomerular feedback. Am J Physiol Renal Physiol 281: F1102--F1108, 2001. First published September 21, 2001; 10.1152/ajprenal.00357.2001.--The macula densa expresses a luminal [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter and a basolateral [Cl.sup.-] conductance. Although it is known that cotransport of [Na.sup.+], [K.sup.+], and [Cl.sup.-] is the first step in tubuloglomerular feedback (TGF), subsequent steps are unclear. We hypothesized that [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] entry via the luminal [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter elevates intracellular [Cl.sup.-], increases electrogenic Clefflux across the basolateral membrane, and depolarizes the macula densa, initiating TGF. We perfused afferent arterioles with macula densa attached. The macula densa was perfused with solutions containing either 5 mM [Na.sup.+] and 3 mM [Cl.sup.-] (low NaCl) or 80 mM [Na.sup.+] and 77 mM [Cl.sup.-] (high NaCl). When the macula densa perfusate was changed from low to high NaCl, afferent arteriole diameter decreased from 15.8 [+ or -] 0.8 to 13.1 [+ or -] 0.7 mm (P < 0.05). Adding 10 [micro] M furosemide to the macula densa lumen blocked TGF. When nystatin, a group I cation ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl, it induced TGF (from 18.0 [+ or -] 1.5 to 15.6 [+ or -] 1.6 mm; P = 0.003). When valinomycin, a [K.sup.+]-selective ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl containing 5 mM [K.sup.+], it did not induce TGF. Subsequent addition of 50 mM KCl to the macula densa perfusate induced TGF (from 21.7 [+ or -] 0.8 to 17.5 [+ or -] 1.3 mm; P = 0.0047; n = 6). Adding 50 mM KCl without valinomycin did not induce TGF. When 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1 [micro] M), a [Cl.sup.-] channel blocker, was added to the bath, it blocked TGF induced by high NaCl, but did not block TGF induced by valinomycin plus 50 mM KCl. NPPB did not alter afferent arteriole constriction induced by norepinephrine. We concluded that increased NaCl in the lumen of the macula densa leads to influx of [Cl.sup.-] via the [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter. The accelerated transport increases intracellular [Cl.sup.-]. The subsequent exit of [Cl.sup.-] across the basolateral membrane via [Cl.sup.-] channels in turn leads to depolarization of the macula densa and thereby induces TGF. afferent arteriole; transport; chloride channel Received 1 December 2000; accepted in final form 16 July 2001

A study was conducted to test the hypothesis that endogenously produced nitric oxide (NO) directly reduces NaCl transport by the thick ascending limb of the loop of Henle (TALH). Results indicated a significant role for locally produced NO, which may act through an autocrine mechanism to directly affect TALH sodium chloride transport. Hence, TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO seen in vivo.