Sympathetic stimulation of thiazide-sensitive sodium chloride cotransport in the generation of salt-sensitive hypertension

Abstract

Excessive renal efferent sympathetic nerve activity contributes to hypertension in many circumstances. Although both hemodynamic and tubular effects likely participate, most evidence supports a major role for α-adrenergic receptors in mediating the direct epithelial stimulation of sodium retention. Recently, it was reported, however, that norepinephrine activates the thiazide-sensitive NaCl cotransporter (NCC) by stimulating β-adrenergic receptors. Here, we confirmed this effect and developed an acute adrenergic stimulation model to study the signaling cascade. The results show that norepinephrine increases the abundance of phosphorylated NCC rapidly (161% increase), an effect largely dependent on β-adrenergic receptors. This effect is not mediated by the activation of angiotensin II receptors. We used immunodissected mouse distal convoluted tubule to show that distal convoluted tubule cells are especially enriched for β₁-adrenergic receptors, and that the effects of adrenergic stimulation can occur ex vivo (79% increase), suggesting they are direct. Because the 2 protein kinases, STE20p-related proline- and alanine-rich kinase (encoded by STK39) and oxidative stress-response kinase 1, phosphorylate and activate NCC, we examined their roles in norepinephrine effects. Surprisingly, norepinephrine did not affect STE20p-related proline- and alanine-rich kinase abundance or its localization in the distal convoluted tubule; instead, we observed a striking activation of oxidative stress-response kinase 1. We confirmed that STE20p-related proline- and alanine-rich kinase is not required for NCC activation, using STK39 knockout mice. Together, the data provide strong support for a signaling system involving β₁-receptors in the distal convoluted tubule that activates NCC, at least in part via oxidative stress-response kinase 1. The results have implications about device- and drug-based treatment of hypertension.

Keywords: diuretics; hypertension; ion transport; sodium-potassium-chloride symporters; sympathetic nervous system.

Figure 1. Confirmation that chronic norepinephrine (NE) infusion causes salt-sensitive hypertension and increases NCC and pNCC

Panel A: NE infusion caused salt-induced hypertension. During week one, all animals were maintained on a normal salt diet without infusion. NE or control infusion was started during week two while the normal salt diet was continued. During week three, both groups were switched to a high salt diet. n=6 per group. Differences were determined by two-way ANOVA with repeated measures, where p-values are 0.0018 for Time, 0.0073 for Treatment (NE vs Control), and 0.0119 for Interaction. Panel B: NE infusion caused an increase in NCC abundance compared with control mice. n=4 per group. p<0.05 by unpaired t-test. Panel C: NE infusion caused an increase in pNCC-T53 abundance compared with control mice (p<0.05 by unpaired t-test); however, WNK4 abundance remained unchanged. n=5 per group. Representative images are shown. See online supplement for densiometry.

Figure 2. NE rapidly increases pNCC independent of angiotensin II signaling

Panel A: Treatment with NE for 30 minutes increased pNCC-T53 abundance, but did not alter total NCC. n=8 per group. p<0.05 by unpaired t-test. Panel B: Increased pNCC-T53 abundance could also be observed by immunofluorescence staining. Staining was performed on 3 mice per group. Panel C: Genetic deletion of the angiotensin II receptor type 1a (AT_1a) does not alter NCC or pNCC-T53 abundance compared with wild-type (WT) controls. n=7 per group. Panel D: Treatment with NE for 30 minutes in AT_1a^−/− mice increased pNCC-T53 compared with control animals. p<0.05 by unpaired t-test. Total NCC abundance remained unchanged. n=5 per group. Representative images are shown. See online supplement for densiometry.

Figure 3. β-receptors mediate the effects of NE on NCC

Panel A: Treatment with the α-receptor agonist, phenylephrine (PE), for 30 minutes did not significantly increase pNCC-T53. Total NCC abundance also remained unchanged. n=5 per group. Panel B: Treatment with the β-receptors agonist, isoproterenol (Iso), for 30 minutes significantly increased pNCC-T53 abundance. p<0.05 by unpaired t-test. Total NCC abundance remained unchanged. n=4 per group. Panel C: Treatment with both agonists together increased pNCC-T53 abundance greater than either agonist alone. p<0.016 by unpaired t-test. Total NCC abundance remained unchanged. n= 5 for the Control and Iso groups and 4 for the Iso+PE group. See online supplement for densiometry.

Figure 4. DCT-specific RT-PCR for β-adrenergic receptor subtypes

Panel A: RT-PCR on COPAS-sorted DCT cells indicated that both β₁- and β₂-adrenergic receptors are expressed in the DCT, but the β₁-receptor subtype is highly enriched. The template used for RT-PCR was either 5 ng cDNA produced from RNA extracted from total kidney (TK) or COPAS-sorted DCT cells (DCT). In place of template cDNA, negative control reactions contained either the product from a cDNA reaction using DCT RNA that lacked reverse transcriptase (-RT) or water (H₂0). β₁AR, β₁-adrenergic receptor; β₂AR, β₂-adrenergic receptor; NCC, Na⁺-Cl⁻ cotransporter; AQP2, aquaporin-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Panel B: Ex vivo treatment of COPAS-sorted DCT cells with isoproterenol (Iso) for 10 minutes increased pNCC-T53 abundance, but did not alter total NCC. n=4 per group. Graphs depict mean ± s.e.m. * p<0.05 by unpaired t-test.

Figure 5. SPAK is not essential for the rapid effects of NE on NCC

Panel A: Treatment with NE for 30 minutes did not alter SPAK cellular localization within the DCT in wild-type animals. Staining was performed on 3 mice per group. Panel B: Treatment with NE for 30 minutes did not alter SPAK abundance in wild-type animals. n=3 per group. Panel C: Treatment with NE for 30 minutes in SPAK^−/− animals significantly increased pNCC-T53 abundance. p<0.05 by unpaired t-test. Total NCC protein remained unchanged. n=5 per group. Panel D: Increased pNCC-T53 abundance in SPAK^−/− animals could also be observed by immunofluorescence staining. Staining was performed on 3 mice per group. See online supplement for densiometry.

Figure 6. NE increased DCT OxSR1 apical localization in both wild-type and SPAK^−/− mice

Treatment with NE for 30 minutes increased the apical localization of OxSR1 in the DCT of both wild-type and SPAK^−/− mice. Insets are high magnification images of the represented tubule sections. Staining was performed on 3 mice per group.

Figure 7. KS OxSR1^−/− mice exhibited a diminished response to NE

Panel A: KS OxSR1^−/− mice had significantly decreased abundance of OxSR1 in their kidneys compared with wild-type (WT) controls. n=3 per group. p<0.05 by unpaired t-test. Panel B: The abundance of NCC protein and phosphorylation at threonine-53 was unaltered in KS OxSR1^−/− mice at baseline. n=3 per group. Panel C: KS OxSR1^−/− mice exhibited a blunted response to a 30-minute treatment with NE. NCC phosphorylation at threonine-53 increased in these mice, but to a lesser degree than that observed in wild-type controls. p<0.05 by unpaired t-test. n=3 for WT per group mice and 4 for KS OxSR1^−/− mice per group. C, Con, control. Representative images are shown. See online supplement for densiometry.

Figure 8. Model of NE effects on SPAK and OxSR1 in the DCT

Cartoon shows effects of NE on SPAK (left panels) and OxSR1 (middle panels) distribution in wild type mice. Note that OxSR1, but not SPAK, shifts to an apical location. The right panels show effects of NE on OxSR1 in SPAK knockout mice. Notice that SPAK knockout causes OxSR1 to shift to a more punctate appearance. Upon NE stimulation, OxSR1 shifts substantially toward the apical membrane. The NCC is phosphorylated (P), upon activation.