Guillermo B. Silva

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.

Extracellular ATP Stimulates NO Production in Rat Thick Ascending Limb

NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at ≈200 &mgr;mol/L with an EC50 of 37 &mgr;mol/L. The increase was blunted by 74% by the nonselective NOS inhibitor l-&ohgr;-nitro-arginine-methyl-ester (2 mmol/L; 60±7 versus 16±6 arbitrary fluorescence units; P<0.02; n=5). In the presence of the P2 receptor antagonist suramin (300 &mgr;mol/L), ATP-induced NO production was reduced by 64% (101±11 versus 37±5 arbitrary fluorescence units; P<0.002; n=5). Blocking ATP hydrolysis with a 5′-ectonucleotidase inhibitor, ARL67156 (30 &mgr;mol/L) enhanced the response to ATP and shifted the EC50 to 0.8 &mgr;mol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist β,γ-methylene-adenosine 5′-triphosphate was 4.8 &mgr;mol/L and the EC50 for the P2Y–selective agonist UTP was 40.4 &mgr;mol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.

TRPV4 mediates hypotonicity-induced ATP release by the thick ascending limb

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/kgH2O, using the luciferin-luciferase assay. Decreasing osmolality stimulated ATP release compared with control (38.9+/-7.2 vs. 2.4+/-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+/-5.9 vs. 2.6+/-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+/-19.9 vs. 8.8+/-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+/-3.4 vs. 12.4+/-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 4alpha-PDD increased ATP release (3.6+/-0.9 vs. 25.4+/-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.

Angiotensin II–Dependent Hypertension Increases Na Transport-Related Oxygen Consumption by the Thick Ascending Limb

Renal medullary superoxide (O2−) increases in angiotensin (Ang) II–dependent hypertension. O2− increases thick ascending limb Na transport, but the effect of Ang II–dependent hypertension on the thick ascending limb is unknown. We hypothesized that Ang II–dependent hypertension increases thick ascending limb NaCl transport because of enhanced O2− production and increased protein kinase C (PKC) &agr; activity. We measured the effect of Ang II–dependent hypertension on furosemide-sensitive oxygen consumption (a measure of Na transport), O2− production, and PKC&agr; translocation (a measure of PKC&agr; activity) in thick ascending limb suspensions. Ang II–dependent hypertension increased furosemide-sensitive oxygen consumption (26.2±1.0% versus 36.6±1.2% of total oxygen consumption; P<0.01). O2− was also increased (1.1±0.2 versus 3.2±0.5 nmol of O2−/min per milligram of protein; P<0.03) in thick ascending limbs. Unilateral renal infusion of Tempol decreased O2− (2.4±0.4 versus 1.2±0.2 nmol of O2−/min per milligram of protein; P<0.04) and furosemide-sensitive oxygen consumption (32.8±1.3% versus 24.0±2.1% of total oxygen consumption; P<0.01) in hypertensive rats. Tempol did not affect O2− or furosemide-sensitive oxygen consumption in normotensive controls and did not alter systolic blood pressure. Ang II–dependent hypertension increased PKC&agr; translocation (5.7±0.3 versus 13.8±1.4 AU per milligram of protein; P<0.01). Unilateral renal infusion of Tempol reduced PKC&agr; translocation (5.0±0.9 versus 10.4±2.6 AU per milligram of protein; P<0.04) in hypertensive rats. Unilateral renal infusion of the PKC&agr; inhibitor Gö6976 reduced furosemide-sensitive oxygen consumption (37.4±1.5% versus 25.1±1.0% of total oxygen consumption; P<0.01) in hypertensive rats. We conclude that Ang II–dependent hypertension enhances thick ascending limb Na transport–related oxygen consumption by increasing O2− and PKC&agr; activity.

A High-Salt Diet Dissociates NO Synthase-3 Expression and NO Production by the Thick Ascending Limb

NO produced by endothelial NO synthase (NOS3) decreases sodium transport by the thick ascending limb (THAL). We found previously that 7 days of high salt (HS) increased THAL-NOS3 expression but not NO production. NOS3 phosphorylation regulates enzyme activity. We hypothesized that HS acutely increases NOS3 expression and NO production, and, over time, changes in NOS3 phosphorylation dissociate NO production from expression. NOS3 expression increased by 71±13%, 127±24%, and 69±16% at days 1, 3, and 7 of HS, respectively. At days 14 and 28, expression was back to normal salt. After 1 day of HS, NO production in response to 250 &mgr;mol/L l-arginine was elevated by 146% and, by day 3, returned to normal salt. Similar increases were found in response to endothelin-1. Inhibitors of NOS1/2 did not blunt the salt-induced increase in NO. Phosphorylation at Thr495, an inhibitory site, decreased by 39±8% at day 1 of HS and then increased by 116±18% at day 3. Phosphorylation at Ser633 and Ser1177 (stimulatory sites) decreased by ≈25% at day 1 and remained depressed at day 3. Superoxide production increased by 71% at day 1, decreased by 57% at day 3, and decreased by 55% at day 7. The NOS inhibitor l-NG-nitroarginine methyl ester did not alter superoxide levels at any time point. The addition of reduced nicotinamide-adenine dinucleotide phosphate and tetrahydrobiopterin had no effect on NO release after 3 days of HS. We conclude the following: (1) HS transiently increases NO production and NOS3 expression; (2) NOS3 expression and NO production are dissociated by HS; and (3) changes in phosphorylation explain how THAL NOS3 activity and expression are dissociated by HS.

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.

Extracellular ATP inhibits transport in medullary thick ascending limbs: role of P2X receptors

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 microM) blocked the effect of ATP on TAL oxygen consumption (147 +/- 15 vs. 146 +/- 16 nmol O2 x min(-1) x mg protein(-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 microM) plus dimethyl amiloride (100 microM), ATP did not inhibit TAL oxygen consumption (from 78 +/- 13 to 98 +/- 5 nmol O2 x min(-1) x mg protein(-1)). The Na ionophore nystatin (200 U/ml) increased TAL oxygen consumption to a similar extent in both ATP- and vehicle-treated samples (368 +/- 41 vs. 397 +/- 47 nmol O2 x min(-1) x mg protein(-1)). The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (3 mM) blocked the ATP effects on TAL oxygen consumption (157 +/- 10 vs. 165 +/- 15 nmol O2 x min(-1) x mg protein(-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.

Rac1 mediates NaCl-induced superoxide generation in the thick ascending limb

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

Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs

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 microM) stimulated NO in wild-type mice [26 +/- 4 arbitrary units (AU)], but not in NOS3 -/- mice (2 +/- 2 AU; P < 0.04). In the presence of the NOS1- and NOS2-selective inhibitors 7-NI and 1400W, ATP stimulated NO by 30 +/- 2 and 33 +/- 3 AU, respectively (not significant vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5 +/- 2 vs. 28 +/- 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 +/- 4%. ATP significantly stimulated Akt1 phosphorylation at Ser(473) by 91 +/- 13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominant-negative Akt1 significantly decreased ATP-induced NO by 88 +/- 6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL.

8-iso-prostaglandin-F2α stimulates chloride transport in thick ascending limbs: role of cAMP and protein kinase A

Salt reabsorption by the loop of Henle controls NaCl handling and blood pressure regulation. Increased oxidative stress stimulates NaCl transport in one specific segment of the loop of Henle called the thick ascending limb (TAL). The isoprostane 8-iso-prostaglandin-F2α (8-iso-PGF2α) is one of the most abundant nonenzymatic lipid oxidation products and has been implicated in the development of hypertension. However, it is not known whether 8-iso-PGF2α regulates transport or the mechanisms involved. Because protein kinase A (PKA) stimulates NaCl transport in several nephron segments, we hypothesized that 8-iso-PGF2α increases NaCl transport in the cortical TAL (cTAL) via a PKA-dependent mechanism. We examined the effect of luminal 8-iso-PGF2α on NaCl transport by measuring chloride absorption (J(Cl)) in isolated microperfused cTALs. Adding 8-iso-PGF2α to the lumen increased J(Cl) by 54% (from 288.7 ± 30.6 to 446.5 ± 44.3 pmol·min(-1)·mm(-1); P < 0.01), while adding it to the bath enhanced J(Cl) by 35% (from 236.3 ± 35.3 to 319.2 ± 39.8 pmol·min(-1)·mm(-1); P < 0.05). This stimulation was blocked by Na-K-2Cl cotransporter inhibition. Next, we tested the role of cAMP. Basal cAMP in the cTAL was 18.6 ± 1.6 fmol·min(-1)·mm(-1), and 8-iso-PGF2α raised it to 35.1 ± 1.4 fmol·min(-1)·mm(-1), an increase of 94% (P < 0.01). Because cAMP stimulates PKA, we measured J(Cl) using the PKA-selective inhibitor H89. In the presence of H89 (10 μM), 8-iso-PGF2α failed to increase transport regardless of whether it was added to the lumen (216.1 ± 16.7 vs. 209.7 ± 23.8 pmol·min(-1)·mm(-1); NS) or the bath (150.4 ± 32.9 vs. 127.1 ± 28.6 pmol·min(-1)·mm(-1); NS). We concluded that 8-iso-PGF2α stimulates cAMP and increases Cl transport in cTALs via a PKA-dependent mechanism.