Nancy J. Hong

Superoxide Stimulates NaCl Absorption in the Thick Ascending Limb Via Activation of Protein Kinase C

Abnormal production of superoxide (O2−) contributes to hypertension, in part because of its effects on the kidney. The thick ascending limb absorbs 20% to 30% of the filtered load of NaCl. O2− stimulates NaCl absorption by the thick ascending limb by enhancing Na+/K+/2Cl− cotransporter activity; however, the signaling mechanism is unknown. We hypothesized that O2− stimulates NaCl absorption by activating protein kinase C (PKC). To test this, we measured the effect of O2− on: (1) Cl− absorption in the presence and absence of PKC inhibitors, (2) total PKC activity, and (3) activation of specific PKC isoforms. Isolated perfused medullary thick ascending limbs were exposed to O2− generated by xanthine oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L). O2− increased Cl− absorption by 42% (from 76.2±3.6 to 108.2±11.9 pmol/min per millimeter; n=5; P<0.05). After treatment with the general PKC inhibitor staurosporine (10 nmol/L), O2− did not stimulate Cl− absorption (Δ−5.7±8.6%; n=6). In thick ascending limb suspensions, O2− increased total PKC activity by 33% (from 66±11 to 88±12 mU/mg protein; n=5; P<0.05) and increased PKC-α and PKC-δ activity by 1.75- and 0.37-fold, respectively. The PKC-α/β–selective inhibitor Gö976 (100 nmol/L) blocked the ability of O2− to stimulate Cl− absorption by isolated perfused medullary thick ascending limbs (Δ4.5±15.0%; n=5). The role of PKC-δ could not be studied because of cell necrosis caused by the selective inhibitor rottlerin. We conclude that PKC-α is required for O2−-stimulated NaCl absorption in the thick ascending limb.

Gene Transfer of eNOS to the Thick Ascending Limb of eNOS-KO Mice Restores the Effects of l-Arginine on NaCl Absorption

Abstract—The thick ascending limb of the loop of Henle (THAL) plays an essential role in the regulation of sodium and water homeostasis by the kidney. l-Arginine, the substrate for nitric oxide synthase (NOS), decreases NaCl absorption by THALs. We hypothesized that eNOS produces the NO that regulates THAL NaCl transport and that selective expression of eNOS in the THAL of eNOS knockout(−/−) mice would restore the effects of l-arginine on NaCl absorption. eNOS−/− mice were anesthetized, the left kidney was exposed, and the renal interstitium was injected with recombinant adenoviral vectors that expressed green fluorescent protein (GFP) or eNOS driven by the promoter of the Na/K/2Cl cotransporter Ad-NKCC2GFP and Ad-NKCC2eNOS, respectively. In Ad-NKCC2eNOS–transduced kidneys, eNOS expression was detected 7 days after injection but was absent in Ad-NKCC2GFP–transduced kidneys. In THALs from eNOS−/− mice transduced with Ad-NKCC2eNOS, adding l-arginine increased DAF-2DA fluorescence, a measure of NO production, by 9.1±1.1% (P <0.05; n=5), but not in THALs transduced with Ad-NKCC2GFP. In THALs from eNOS−/− mice transduced with Ad-NKCC2eNOS, Cl absorption averaged 85.9±11.8 pmol/min per millimeter. Adding l-arginine (1 mmol/L) to the bath decreased Cl absorption to 59.7±11.0 pmol/min per millimeter (P <0.05; n=6). In THALs transduced with Ad-NKCC2GFP, Cl absorption averaged 96.0±21.0 pmol/min per millimeter. Adding l-arginine to the bath did not significantly affect Cl absorption (100.6±20.6 pmol/min per millimeter; n=4). We concluded that gene transfer of eNOS to the THAL of eNOS−/− mice restores l-arginine–induced inhibition of NaCl transport and NO production. These data indicate that eNOS is essential for the regulation of THAL NaCl transport by NO.

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 ± 0.24 to 2.81 ± 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3–/– mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 ± 0.13 versus 3.31 ± 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 ± 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 ± 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 ± 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 ± 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

Cellular Stretch Increases Superoxide Production in the Thick Ascending Limb

Superoxide (O2−) is an important regulator of kidney function. We have recently shown that luminal flow stimulates O2− production in the thick ascending limb (TAL), attributable in part to mechanical factors. Stretch, pressure and shear stress all change when flow increases in the TAL. We hypothesized that stretch rather than shear stress or pressure per se stimulates O2− production by TALs. We measured O2− production in isolated perfused rat TALs using fluorescence microscopy and dihydroethidium. Tubules were perfused with a Na-free solution to eliminate the confounding effect of Na transport. Flow induced an increase in O2− production from 29±4 to 90±8 AU/s (P<0.002; n=5). The response to flow is rapidly reversible. O2− production by TALs perfused at 10 nL/min decreased from 113±6 to 25±10 AU/s (P<0.003; n=4) 15 minutes after flow was stopped. Increasing pressure and stretch in the absence of shear stress caused a significant increase in O2− production (40±6 to 118±17 AU/s; P<0.02; n=5). In contrast, eliminating shear stress had no effect (107±9 versus 108±10 AU/s; n=5). Increasing stretch by 27±2% in the presence of flow while reducing pressure stimulated O2− production from 66±7 to 84±9 AU/s (29±8%; P<0.02; n=5). Tempol inhibited this increase (n=5). We conclude that increasing stretch rather than pressure or shear stress accounts for the mechanical aspect of flow-induced O2− production in the TAL. Stretch of the TAL during hypertension, diabetes, and salt loading may contribute to renal damage.

Shear stress increases nitric oxide production in thick ascending limbs

We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min (P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min (P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min (P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O(2)(-) scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm(2) produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively (P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.

PKC-α mediates flow-stimulated superoxide production in thick ascending limbs

We showed that luminal flow increases net superoxide (O(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(2)(-) production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O(2)(-) production from 4 +/- 1 to 61 +/- 12 AU/s (P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O(2)(-) production (2 +/- 1 vs. 1 +/- 1 AU/s; P > 0.05; n = 5). Flow-stimulated O(2)(-) was also blocked in p47(phox)-deficient mice. We measured flow-stimulated 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 +/- 0.02 to 0.96 +/- 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(2)(-) production from 5 +/- 2 to 92 +/- 6 AU/s (P < 0.001; n = 6). The PKC-alpha- and betaI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O(2)(-) production from 54 +/- 15 to 2 +/- 1 AU/s (P < 0.04; n = 5). Flow-induced net O(2)(-) production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-alpha but not dnPKCbetaI or LacZ (Delta = 11 +/- 3 AU/s for dnPKCalpha, 55 +/- 7 AU/s for dnPKCbetaI, and 63 +/- 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O(2)(-) production in thick ascending limbs via PKC-alpha-mediated activation of NADPH oxidase.

High-Salt Diet Increases Sensitivity to NO and eNOS Expression But Not NO Production in THALs

Abstract—l-Arginine inhibits thick ascending limb (THAL) NaCl absorption by activating endothelial NO synthase (eNOS) and increasing NO production. Inhibition of renal NO production combined with a high-salt diet produces hypertension, and the THAL has been implicated in salt-sensitive hypertension. We hypothesized that a high-salt diet enhances the inhibitory action of l-arginine on NaCl absorption by THALs because of increased eNOS expression and NO production. To test this, we used isolated THALs from rats on a normal-salt (NS) or high-salt diet (HS) for 7 to 10 days. l-Arginine (1 mmol/L) decreased chloride absorption by 56±10% in THALs from rats on a HS diet, but only 29±3% in THALs from rats on a NS diet. eNOS expression in isolated THALs from rats on a HS diet was increased by 3.9-fold compared with NS (P <0.03). However, l-arginine increased NO levels to the same extent in THALs from both groups, as measured with DAF-2 DA or a NO-sensitive electrode. To determine whether a HS diet increases the sensitivity of the THAL to NO, we tested the effects of the NO donor spermine NONOate on chloride absorption. In THALs from rats on a HS diet, 1 and 5 &mgr;mol/L spermine NONOate reduced chloride absorption by 35±5% and 58±6%, respectively. In contrast, these same concentrations of spermine NONOate reduced chloride absorption by 4±4% (P <0.03 versus HS diet) and 43±9% in THALs from rats on a NS diet. We conclude that a HS diet enhances the effect of NO in the THAL. l-Arginine–stimulated NO production was not enhanced by a HS diet, despite increased eNOS protein.

Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4.

Angiotensin II (ANG II) stimulates production of superoxide (O(2)(-)) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O(2)(-) production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O(2)(-) production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O(2)(-) production in wild-type and NOX2 knockout mice (KO). ANG II increased O(2)(-) production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O(2)(-) production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O(2)(-) production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O(2)(-) production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O(2)(-) production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O(2)(-) production by TALs.

ATP mediates flow-induced NO production in thick ascending limbs

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane.

Tumor necrosis factor α decreases nitric oxide synthase type 3 expression primarily via Rho/Rho kinase in the thick ascending limb.

Inappropriate Na+ reabsorption by thick ascending limbs (THALs) induces hypertension. NO produced by NO synthase type 3 (NOS3) inhibits NaCl reabsorption by THALs. Tumor necrosis factor &agr; (TNF-&agr;) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-&agr; on THAL NOS3 and the signaling cascade are unknown. TNF-&agr; activates several signaling pathways, including Rho/Rho kinase (ROCK), which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-&agr; decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L of TNF-&agr; for 24 hours, and NOS3 expression was measured by Western blot. TNF-&agr; decreased NOS3 expression by 51±6% (P<0.002) and blunted stimulus-induced NO production. A 10-minute treatment with TNF-&agr; stimulated RhoA activity by 60±23% (P<0.04). Inhibition of Rho GTPase with 0.05 &mgr;g/mL of C3 exoenzyme blocked TNF-&agr;–induced reductions in NOS3 expression by 30±8% (P<0.02). Inhibition of ROCK with 10 &mgr;mol/L of H-1152 blocked TNF-&agr;–induced decreases in NOS3 expression by 66±15% (P<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases, and Rac-1 were also not involved in TNF-&agr;–induced decreases in NOS3 expression. We conclude that TNF-&agr; decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be attributed to the mitigation of TNF-&agr;–induced reduction in NOS3 expression.