Thick Ascending Limb Suspensions Research Articles

Nephron-specific deletion of ALMS1 (Alstrom syndrome 1) increases surface NKCC2 expression and NaCl reabsorption in the Thick Ascending Limb

Mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by progressive metabolic alterations that include obesity, hypertension, and chronic kidney disease (CKD). SNPs in the ALMS1 gene in the general population are associated with lower GFR, hypertension and metabolic syndrome. ALMS1 is widely expressed where it localizes to endosomes, centrosomes and the base of cilia. Our lab has previously shown that ALMS1 is expressed in renal tubules of the Thick Ascending Limb (TAL) where NKCC2 is the primary apical NaCl transporter. Whole animal gene deletion of ALMS1 increases blood pressure, and inhibits NKCC2 endocytosis, increasing its surface expression and renal NaCl reabsorption. However, whole animal ALMS1 KO rats or mice develop obesity, affecting the interpretation of organ-specific data. We hypothesized that ALMS1 is a part of protein complex that binds apical NKCC2 in TALs and promotes its endocytosis. To study the role of ALMS1 in the nephron without influence of obesity we generated a loxP flanked exon 7 ALMS1 transgenic mouse line, that we crossed with nephron specific inducible Pax8-rtTA Cre mice (Pax8-ALMS1 KO). 4 weeks after inducing Cre expression with doxycycline (2mg/ml in 2% sucrose), ALMS1 expression in TAL suspensions decreased by 75% (p<0.01). To study the role of ALMS1 in TAL function, we measured surface to total NKCC2 ratio, which was increased by 53±15% in TALs from Pax8-ALMS1 KO compared to control floxed ALMS1 (p<0.01). Recombinant ALMS1 (GST-Carboxyl terminus-ALMS1), or immunoprecipitation of ALMS1, pulled down NKCC2 from WT TAL lysates (n=3), suggesting that the mechanism by which ALMS1 regulates surface NKCC2 involves protein-protein interactions. 4 weeks after inducing Cre expression with doxycycline we measured bumetanide-induced natriuresis (4h) as an index of NKCC2-mediated NaCl absorption. We found that bumetanide induced UNa excretion was higher in Pax8-ALMS1 KO, compared to control (control: 115±19 vs Pax8-ALMS1 KO: 146±30 μmols Na/4h, p<0.05). Since whole animal ALMS1 deletion induced hypertension, we measured systolic blood pressure (SBP) by tail cuff one month after inducible deletion of ALMS1 or in floxed ALMS1 mice treated with doxycycline as controls. Baseline SBP in normal Na diet (0.4%) was similar between strains. However, SBP increased by 12±3 mmHg in Pax8-ALMS1 KO, but not in control floxed ALMS1 littermates after 9 days of high Na diet (WT: 100±10, Pax8-ALMS1 KO: 115±11 mmHg, p<0.05 from baseline, n=4). We conclude that ALMS1, binds NKCC2 to regulate its surface expression. Depletion of ALMS1 in the nephron enhances surface NKCC2, TAL NaCl reabsorption, and induces salt-sensitivity of blood pressure. Our data indicate that regulation of the ALMS1-NKCC2 interaction in the TAL is important for renal NaCl reabsorption and regulation of blood pressure. Funding: NIH R56DK131114, Henry Ford Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CGMP Stimulates K48‐linked Poly‐ubiquitination of NKCC2 in Rats Thick Ascending Limb

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. The second messenger cyclic guanosine monophosphate (cGMP) decreases NKCC2 activity by decreasing surface NKCC2 levels. We showed that NKCC2 is constitutively ubiquitinated and degraded by the proteasome, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification in which the C‐terminus of the ubiquitin is attached to a substrate protein. Poly‐ubiquitination occurs when the C‐terminus of another ubiquitin, is then linked to the previously added ubiquitin, creating a chain. There are 7 lysine (k) in the ubiquitin molecule where poly‐ubiquitination can occur. K48‐linked poly‐ubiquitination on ubiquitin is associated with proteasomal degradation. Therefore, we hypothesized what cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2 in rats TAL. To test our hypothesis we immunoprecipitated K48‐linked poly‐ubiquitin using Tandem Ubiquitin Binding Entities (TUBEs) and western blot against NKCC2 in Sprague‐Dawley rat TAL suspensions. First, we performed a k48‐TUBEs concentration‐response curve against K48‐linked poly‐ubiquitination of NKCC2 to ensure linear relationship. TALs suspension were aliquoted in 4 samples and placed at 37°C for 30 minutes in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. TALs were lysed at 4°C and excess amount of protein lysate (150 μg) was incubated with increasing concentration of k48‐TUBEs. We obtained a linear increase in K48‐linked poly‐ubiquitination of NKCC2 with increasing concentration of k48‐TUBEs. Next, to study the effect of cGMP on k48‐ubiquitination of NKCC2. The conditions were set to fit within the linear range (80 μg of protein) to ensure complete immunoprecipitation of K48‐linked poly‐ubiquitination of NKCC2. We found that the membrane‐permeant cGMP analogue dibutiryl cGMP (db‐cGMP) stimulates K48‐ubiquitination on NKCC2 in a concentration‐dependent manner (baseline: 100.0%; db‐cGMP 100 μM: 115.3 ± 3.7%; db‐cGMP 250 μM: 127.8 ± 5.2%; db‐cGMP 500 μM: 148.3 ± 18.2% p<0.05). These data indicates that cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2, and suggest a triggering mechanism for NKCC2 degradation by the proteasome. We conclude that cGMP increases K48‐linked poly‐ubiquitination of NKCC2. To our knowledge, this is the first piece of evidence showing that cGMP may trigger a signal for NKCC2 degradation through stimulation of k48‐ubiquitination.Support or Funding InformationAHA Post‐Doctoral (14POST20110009)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs

Angiotensin II (Ang II) causes nitric oxide synthase (NOS) to become a source of superoxide (O2 −) via a protein kinase C (PKC)‐dependent process in endothelial cells. Ang II stimulates both NO and O2 − production in thick ascending limbs. We hypothesized that Ang II causes O2 − production by NOS in thick ascending limbs via a PKC‐dependent mechanism. NO production was measured in isolated rat thick ascending limbs using DAF‐FM, whereas O2 − was measured in thick ascending limb suspensions using the lucigenin assay. Consistent stimulation of NO was observed with 1 nmol/L Ang II (P < 0.001; n = 9). This concentration of Ang II‐stimulated O2 − production by 50% (1.77 ± 0.26 vs. 2.62 ± 0.36 relative lights units (RLU)/s/μg protein; P < 0.04; n = 5). In the presence of the NOS inhibitor L‐NAME, Ang II‐stimulated O2 − decreased from 2.02 ± 0.29 to 1.10 ± 0.11 RLU/s/μg protein (P < 0.01; n = 8). L‐arginine alone did not change Ang II‐stimulated O2 − (2.34 ± 0.22 vs. 2.29 ± 0.29 RLU/s/μg protein; n = 5). In the presence of Ang II plus the PKC α/β 1 inhibitor Gö 6976, L‐NAME had no effect on O2 − production (0.78 ± 0.23 vs. 0.62 ± 0.11 RLU/s/μg protein; n = 7). In the presence of Ang II plus apocynin, a NADPH oxidase inhibitor, L‐NAME did not change O2 − (0.59 ± 0.04 vs. 0.61 ± ×0.08 RLU/s/μg protein; n = 5). We conclude that: (1) Ang II causes NOS to produce O2 − in thick ascending limbs via a PKC‐ and NADPH oxidase‐dependent process; and (2) the effect of Ang II is not due to limited substrate.

Resveratrol increases nitric oxide production in the rat thick ascending limb via Ca2+/calmodulin.

The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca2+ (Cai) and AMP kinase (AMPK) and NAD-dependent deacetylase sirtuin1 (SIRT1) activities, all of which could activate NO production. We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca2+/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Cai was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick ascending limbs via a Ca2+/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects.

Anandamide inhibits transport-related oxygen consumption in the loop of Henle by activating CB1 receptors

The energy required for active Na chloride reabsorption in the thick ascending limb (TAL) depends on oxygen consumption and oxidative phosphorylation (OXP). In other cells, Na transport is inhibited by the endogenous cannabinoid anandamide through the activation of the cannabinoid receptors (CB) type 1 and 2. However, it is unclear whether anandamide alters TAL transport and the mechanisms that could be involved. We hypothesized that anandamide inhibits TAL transport via activation of CB1 receptors and NO. For this, we measured oxygen consumption (Q(O(2))) in TAL suspensions to monitor the anandamide effects on transport and OXP. Anandamide reduced Q(O(2)) in a concentration-dependent manner. During Na-K-2Cl cotransport and Na/H exchange inhibition, anandamide did not inhibit TAL Q(O(2)). To test the role of the cannabinoid receptors, we used specific agonists and antagonists of CB1 and CB2 receptors. The CB1-selective agonist WIN55212-2 reduced Q(O(2)) in a concentration-dependent manner. Also, the CB1 receptor antagonist rimonabant blocked the effect of anandamide on Q(O(2)). In contrast, the CB2-selective agonist JHW-133 had no effect on Q(O(2)), while the CB2 receptor antagonist AM-630 failed to block the anandamide effects on Q(O(2)). To confirm these results, we measured CB1 and CB2 receptor expression and only CB1 expression was detected. Because CB1 receptors are strong nitric oxide synthase (NOS) stimulators and NO inhibits transport in TALs, we evaluated the role of NO. Anandamide stimulated NO production and the NOS inhibitor N(G)-nitro-L-arginine methyl ester blocked the anandamide effects on Q(O(2)). We conclude that anandamide inhibits TAL Na transport-related Q(O(2)) via activation of CB1 receptor and NOS.

SUPEROXIDE INCREASES SURFACE NKCC2 EXPRESSION IN RAT THICK ASCENDING LIMBS

The apical Na/K/2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL). We found that the free radical superoxide stimulates NaCl absorption by TALs by stimulating NKCC2 activity. NKCC2 activity is a product of the number of transporters in the apical membrane plus the rate of turnover of each transporter. We hypothesized that superoxide stimulates apical surface NKCC2 expression. Apical surface NKCC2 expression was measured in rat TAL suspensions by surface biotinylation and Western blot. We found that increasing superoxide production with 1 μM xanthine oxidase and 500 μM hypoxanthine stimulated surface NKCC2 expression by 18±5% (p<0.05). This was blocked by scavenging superoxide with 50 μM tempol. However scavenging hydrogen peroxide with 100 U/mL catalase failed to block superoxide stimulated expression (22±8% of control, p<0.05). Inhibiting endogenous nitric oxide production with L-NAME enhanced the superoxide stimulated surface NKCC2 expression from 21±6 to 41±10% (p<0.05). This effect was completely blocked by scavenging superoxide with 300 U/mL superoxide dismutase. Total NKCC2 expression was not changed by any treatment. We conclude that superoxide increases apical surface NKCC2 expression and this effect is blunted by endogenous nitric oxide. Our data suggest that superoxide stimulates NaCl absorption in part by enhanced trafficking of NKCC2 which may be involved in salt-sensitive hypertension.

Rac1 mediates NaCl‐induced superoxide production by the thick ascending limb

NADPH oxidase‐derived superoxide (O2−) regulates Na absorption and renal hemodynamics. Increasing NaCl in the thick ascending limb stimulates O2− generation. NaCl activates the NADPH oxidase regulatory subunit Rac1 in other cells. However, it is unknown whether physiological changes in luminal NaCl augment O2−, nor the mediators involved in the thick ascending limb. We hypothesized that increasing luminal NaCl activates Rac1 and thereby stimulates O2− production in this segment. To test our hypothesis we increased NaCl from 10 to 57 mM in medullary thick ascending limb suspensions and measured O2− using lucigenin and Rac1 activity by translocation. Increasing NaCl stimulated O2− from 1.41±0.16 to 2.71±0.30 nmol O2−/min/mg protein (p&lt;0.05). This was blocked by the Na/K/2Cl cotransporter inhibitor furosemide and the NADPH oxidase inhibitor apocynin. NaCl increased Rac1 in the particulate fraction from 6.8±0.8 to 11.7±2.4%, indicating Rac1 activation (p&lt;0.05). Increasing intracellular Na with Nystatin increased Rac1 in the particulate fraction by 481±110% (p&lt;0.01). In the presence of the Rac1 inhibitor, NaCl did not increase O2− (0.49±0.16 vs. 0.55±0.25 nmol O2−/min/mg protein). Dominant‐negative Rac1 decreased NaCl‐induced O2− from 0.81±0.17 to 0.33±0.04nmol O2−/min/mg protein (p&lt;0.05), a 60% inhibition. We conclude that NaCl stimulates thick ascending limb O2− generation by activating Rac1.

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.

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.

Aquaporin‐1 is expressed in the thick ascending limb.

The thick ascending limb (TAL) is the water‐impermeable segment of the nephron. Isolated TALs exhibit low luminal water permeability even in the presence of AVP, a characteristic that allows this segment to dilute the forming urine. Thus it is assumed that TALs do not express water channels. However, TALs rapidly shrink in response to increased basolateral osmolality suggesting the presence of water channels. We hypothesized that the TAL expresses AQP1. We measured AQP1 by: 1) Western blot, 2) RTPCR, and 3) immunofluorescence. By Western blot, homogenates of rat TAL suspensions exhibited two bands characteristic of glycosylated (0.8 ± 0.5 OD units) and unglycosylated (3.4 ± 0.5 OD units) AQP1 (n=5). To ensure that the signal was not due to other cell types in our preparation, we used microdissected TALs. AQP1 was detected in microdisected rat and mice TALs. No bands were detected in TALs from AQP1 ‐/‐ mice. By RTPCR, microdissected rat TALs, showed the predicted 430 bp and 603 bp products for AQP1 and the NaK2Cl co‐transporter (specific for TALs). Double immunofluorescence labeling showed both, basolateral localization of AQP1 and luminal localization for Tham Horsfall protein (specific for TALs). We conclude that AQP1 is expressed in the basolateral side of the TAL. Since the TAL is water impermeable, these data suggest AQP1 could be involved in transport of molecules other than water in this nephron segment.

Constitutive endocytosis of NKCC2 in thick ascending limbs is dynamin dependent

The Na/K/2Cl cotransporter (NKCC2) mediates NaCl absorption by the epithelial cells of the thick ascending limb (TAL). Trafficking into (exocytosis) and out (endocytosis) of the apical membrane regulates surface NKCC2 levels. Dynamin‐2 is a ubiquitous protein that mediates endocytosis in many epithelial cells. It is not known whether NKCC2 undergoes constitutive endocytosis and whether dynamin‐2 is involved. We hypothesized that constitutive endocytosis of NKCC2 is mediated by dynamin‐2. We measured surface NKCC2 and the rate of NKCC2 endocytosis by biotinylation and Western blot. First, we studied whether dynamin‐2 is expressed in TAL. We found that dynamin‐2 is expressed in rat medullary TAL suspensions as a single band at the predicted molecular weight of 100 kDa. In control TALs we observed constitutive NKCC2 endocytosis over 30 min that averaged 10.1 ± 0.8 a.u/min. We tested whether the dynamin inhibitor (Dynasore) inhibits constitutive NKCC2 endocytosis. In the presence of Dynasore (100 μM), the rate of constitutive NKCC2 endocytosis averaged 2.8 ± 0.6 a.u/min ( n = 6, p &lt; 0.01 vs control), a 72% inhibition. Acute (20 min) blockade of endocytosis with Dynasore increased surface NKCC2 by 66 ± 26% (p&lt;0.05). The total pool of NKCC2 was not changed by Dynasore. We concluded that constitutive NKCC2 endocytosis is mediated by dynamin‐2. These are the first data showing NKCC2 endocytosis and a role for dynamin in TALs.

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.

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

Renal medullary superoxide (O(2)(-)) increases in angiotensin (Ang) II-dependent hypertension. O(2)(-) 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 O(2)(-) production and increased protein kinase C (PKC) alpha activity. We measured the effect of Ang II-dependent hypertension on furosemide-sensitive oxygen consumption (a measure of Na transport), O(2)(-) production, and PKCalpha translocation (a measure of PKCalpha 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). O(2)(-) was also increased (1.1+/-0.2 versus 3.2+/-0.5 nmol of O(2)(-)/min per milligram of protein; P<0.03) in thick ascending limbs. Unilateral renal infusion of Tempol decreased O(2)(-) (2.4+/-0.4 versus 1.2+/-0.2 nmol of O(2)(-)/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 O(2)(-) or furosemide-sensitive oxygen consumption in normotensive controls and did not alter systolic blood pressure. Ang II-dependent hypertension increased PKCalpha translocation (5.7+/-0.3 versus 13.8+/-1.4 AU per milligram of protein; P<0.01). Unilateral renal infusion of Tempol reduced PKCalpha 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 PKCalpha 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 O(2)(-) and PKCalpha activity.

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‐induced hypertension stimulates thick ascending limb NaCl absorption by increasing O2−

Renal medullary O2− increases in angiotensin‐induced hypertensive (Ang‐IH) rats. We have shown that O2− increases thick ascending limb (TAL) Na transport, but the effect of Ang‐IH on the TAL is unknown. We hypothesized that TAL Na absorption in Ang‐IH rats is enhanced due to elevated O2− levels and protein kinase C (PKC) α activity. We measured O2− levels, furosemide‐sensitive oxygen consumption (FSQO2) as a measure of Na transport and PKCα activity in TAL suspensions from control and Ang‐IH rats. In TALs from Ang‐IH rats O2− levels were 2.5±0.5 compared to 0.8±0.3 arbitrary units (AU)/min/mg protein for controls (p&lt;0.05). FSQO2 also increased (25±3 to 36±2 % of total QO2; p&lt;0.05). Tempol, a O2− scavenger, infused into 1 kidney of Ang‐IH rats decreased O2− levels from 2.3±0.4 to 1.3±0.2 AU/min/mg protein (p&lt;0.05) and FSQO2 from 33±2 to 24±2 % of total QO2 (p&lt;0.05). Tempol had no effect on O2− or FSQO2 in controls. Tempol did not alter blood pressure of Ang‐IH rats. In Ang‐IH rats PKCα activity was 3.7±0.9 compared to 1.1±0.2 AU/mg protein (p&lt;0.05) in controls. Tempol infusion into 1 kidney of Ang‐IH rats reduced PKCα activity from 3.0±0.6 to 0.6±0.1 AU/mg protein (p&lt;0.05). Infusion of the PKCα inhibitor Gö6976 into one kidney of Ang‐IH rats reduced FSQO2 from 38±3 to 26±3 % of total QO2 (p&lt;0.05). We conclude that in Ang‐IH rats TAL NaCl absorption is enhanced due to elevated O2− and PKCα activity rather than increased blood pressure.

DDAVP increases surface NKCC2 levels via activation of PKA in rat thick ascending limbs

cAMP stimulates NaCl absorption in the thick ascending limb (THAL) by increasing surface NKCC2. Arginine vasopressin (AVP) stimulates NaCl absorption and increases cAMP in the THAL via V2 receptors (V2R). It is not known whether AVP increases surface NKCC2 by activation of cAMP‐dependent protein kinase A (PKA). We hypothesize that stimulation of V2R in the THAL increases surface NKCC2 levels via PKA. We used surface biotinylation and Western blot to measure NKCC2 in the plasma membrane of rat medullary THAL suspensions. We found that 30 minutes stimulation of V2R with dDAVP (1 μM) increased surface NKCC2 by 23 ± 9 % (p &lt;0.05). In the presence of the phosphodiesterase inhibitor IBMX (0.5 mM), dDAVP increased surface NKCC2 by 44 ± 11 % (p &lt;0.05). However, in the presence of the PKA inhibitor H‐89 (10 μM), addition of dDAVP/IBMX did not significantly change surface NKCC2 (basal: 100%, dDAVP/IBMX +H‐89: 108 ± 16%, n = 5). In control experiments, H‐89 alone did not significantly affect surface NKCC2 (basal: 100%, H‐89: 92 ± 4%, n = 6). The total NKCC2 pool was not affected by dDAVP/IBMX (basal: 100%, dDAVP/IBMX: 109 ± 11%, n = 5) or by 10 μM H‐89 (basal: 100%, H‐89: 107 ± 10%, n =5). The intracellular protein GAPDH was not detected in the surface fraction, indicating that intracellular proteins are not biotinylated. We concluded that acute V2R stimulation increases surface NKCC2 levels in the THAL via PKA activation.

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

Abnormal production of superoxide (O(2)(-)) 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. O(2)(-) stimulates NaCl absorption by the thick ascending limb by enhancing Na(+)/K(+)/2Cl(-) cotransporter activity; however, the signaling mechanism is unknown. We hypothesized that O(2)(-) stimulates NaCl absorption by activating protein kinase C (PKC). To test this, we measured the effect of O(2)(-) 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 O(2)(-) generated by xanthine oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L). O(2)(-) 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), O(2)(-) did not stimulate Cl(-) absorption (Delta-5.7+/-8.6%; n=6). In thick ascending limb suspensions, O(2)(-) increased total PKC activity by 33% (from 66+/-11 to 88+/-12 mU/mg protein; n=5; P<0.05) and increased PKC-alpha and PKC-delta activity by 1.75- and 0.37-fold, respectively. The PKC-alpha/beta-selective inhibitor Gö976 (100 nmol/L) blocked the ability of O(2)(-) to stimulate Cl(-) absorption by isolated perfused medullary thick ascending limbs (Delta4.5+/-15.0%; n=5). The role of PKC-delta could not be studied because of cell necrosis caused by the selective inhibitor rottlerin. We conclude that PKC-alpha is required for O(2)(-)-stimulated NaCl absorption in the thick ascending limb.

Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb

Superoxide (O2-) 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 O2- stimulates NaCl absorption primarily by enhancing Na-K-2Cl cotransport. First, we measured steady-state intracellular Na (Nai) and chloride (Cli). Xanthine oxidase (XO; 0.75 mU/ml) and hypoxanthine (HX; 0.125 mM) were added to the bath to increase O2-. During the control period, Nai was 12.2 +/- 1.9 mM. After treatment with O2-, it rose to 20.9 +/- 3.3 mM, a 71% increase (P < 0.01). Cli also increased (P < 0.01). Neither XO nor HX alone had a significant effect on Nai or Cli. Next, we tested cotransport activity by measuring the initial rate of increase in Nai 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 +/- 0.02 arbitrary units (AU)/min. After treatment with O2-, it was 0.22 +/- 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 O2- 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 +/- 0.77 AU. After O2- treatment, it was 6.18 +/- 0.90 AU (P < 0.05), a 15% decrease. Finally, we assessed the effects of O2- on Na-K-ATPase activity in THAL suspensions by measuring ATP hydrolysis. Vmax and K1/2 for Na were not affected by O2-. We concluded that O2- stimulates THAL NaCl absorption primarily by enhancing Na entry via Na-K-2Cl cotransport.

Inhibition of Na-K-ATPase in thick ascending limbs by NO depends on O2- and is diminished by a high-salt diet.

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% NaCl for 7-10 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 microM) reduced Na-K-ATPase activity from 0.44 +/- 0.03 to 0.30 +/- 0.04 nmol P(i).microg protein(-1).min(-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 +/- 0.05 nmol P(i).microg protein(-1).min(-1)). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONOO(-) formed by NO reacting with O(2)(-) was involved, we measured O(2)(-) production. THALs from rats on normal and high salt produced 35.8 +/- 0.3 and 23.7 +/- 0.8 nmol O(2)(-).min(-1).mg protein(-1), respectively (P < 0.01). Because O(2)(-) production differed, we studied the effects of the O(2)(-) scavenger tempol. In the presence of 50 microM tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 +/- 0.05 vs. 0.52 +/- 0.07 nmol P(i).microg protein(-1).min(-1)). Propyl gallate, another O(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(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(2)(-). NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased O(2)(-) production.

NO decreases thick ascending limb chloride absorption by reducing Na+-K+-2Cl−cotransporter activity

First published August 9, 2001; 10.1152/ajprenal.0075.2001.—We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption ( JCl−). NaCl transport in the THAL depends on apical Na+-K+-2Cl−cotransporters, apical K+channels, and basolateral Na+-K+-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL JCl−by inhibiting the Na+-K+-2Cl−cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na+]i) and chloride concentrations ([Cl−]i) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na+]ifrom 13.5 ± 1.2 to 9.6 ± 1.6 mM ( P &lt; 0.05) and also decreased [Cl−]i( P &lt; 0.01). We next tested whether NO decreases Na+-K+-2Cl−cotransporter activity by measuring the initial rate of Na+transport. In the presence of SPM in the bath, initial rates of Na+entry were 49.6 ± 6.0% slower compared with control rates ( P &lt; 0.05). To determine whether NO inhibits apical K+channel activity, we measured the change in membrane potential caused by an increase in luminal K+from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K+concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K+permeability could affect NO-induced inhibition of THAL JCl−. In the presence of luminal valinomycin, which increases K+permeability, addition of SPM decreased THAL JCl−by 41.2 ± 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na+-K+-ATPase by measuring oxygen consumption rate (Qo2) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased Qo2to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2· mg protein−1· min−1in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased Qo2by 104 ± 7 and 94 ± 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL JCl−by inhibiting Na+-K+-2Cl−cotransport rather than inhibiting apical K+channels or the sodium pump.