The purpose of this study was to compare the expression of BSC-1 (bumetanide-sensitive Na ϩ -K ϩ -2Cl Ϫ cotransporter) in kidneys of spontaneously hypertensive rats (SHR) versus Wistar-Kyoto (WKY) rats by immunoblotting and reverse transcription-polymerase chain reaction. To determine the specificity of any observed changes in BSC-1 expression, we also compared expression of the thiazide sensitive Na ϩ -Cl Ϫ cotransporter (TSC), the type-3 Na ϩ -H ϩ exchanger (NHE-3), Na ϩ -K ϩ -ATPase-␣ 1 , the inwardly rectifying K ϩ channel (ROMK-1), the type-1 Na ϩ -HCO 3 Ϫ cotransporter (NBC-1), aquaporin-1, and aquaporin-2. Analyses were performed on outer cortex, outer medulla, and inner medulla. BSC-1 protein was detected in outer medulla and was markedly (6-fold) higher in SHR. TSC protein was detected in the cortex and was not overexpressed in SHR. Aquaporin-1 protein was detected in all three regions and was not overexpressed in SHR. Aquaporin-2 and ROMK-1 proteins were detected in all three regions, but were moderately elevated (2-fold) only in the SHR inner medulla. Na ϩ -K ϩ -ATPase and NHE-3 proteins were detected in all three regions. Na ϩ -K ϩ -ATPase-␣ 1 was modestly (25%) increased in SHR outer and inner medulla, whereas NHE-3 was moderately (2-fold) increased in the SHR cortex and inner medulla. NBC-1 protein was detected only in the cortex and was higher (2-fold) in SHR. mRNA levels of BSC-1, aquaporin-2, and ROMK-1 were not elevated in SHR, indicating a post-translational mechanism of protein overexpression. High-dose furosemide increased fractional sodium excretion more in SHR than WKY (3-fold). We conclude that increased expression of BSC-1, and to a lesser extent, aquaporin-2, ROMK-1, NHE-3, and NBC-1 may contribute to the pathogenesis of hypertension in the SHR.
1. Because chronic activation of the renal sympathetic nervous system promotes sodium and water retention, it is conceivable that long-term exposure of the kidney to the sympathetic neurotransmitter noradrenaline upregulates the expression of key renal epithelial transport systems. 2. To test this hypothesis, we used immunoblotting of renal cortical and medullary tissue to investigate the abundance of major transport systems expressed along the renal tubule in response to long-term (15 days) infusions of noradrenaline (600 ng/min) in rats. 3. Mean arterial blood pressure and heart rate were significantly elevated in rats receiving chronic infusions of noradrenaline (128 +/- 10 mmHg and 492 +/- 16 b.p.m., respectively) compared with animals treated with saline only (89 +/- 3 mmHg and 376 +/- 14 b.p.m., respectively). 4. Chronic infusions of noradrenaline also increased the protein abundance of the cortical Na(+)/H(+) exchanger isoform 3 (NHE-3; 2.5-fold; P = 0.0142), the cortical sodium-bicarbonate cotransporter NBC-1 (2.5-fold; P = 0.0067), the bumetanide-sensitive sodium-potassium-chloride cotransporter BSC-1/NKCC2 in the inner stripe of outer medulla (threefold; P = 0.0020) and aquaporin-2 in the inner medulla (twofold; P = 0.0039). 5. In contrast, noradrenaline did not significantly affect expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter in the cortex, Na(+)/K(+)-ATPase-alpha(1) in the cortex and inner stripe of the outer or inner medulla, the inwardly rectifying K(+) channel (ROMK-1) in the inner stripe of the outer medulla or aquaporin-1 in the cortex or inner medulla. Noradrenaline did significantly, but modestly (less than twofold), increase aquaporin-1 in the inner stripe of the outer medulla. 6. We conclude that noradrenaline-induced increases in the expression of NHE-3, NBC-1, BSC-1 and aquaporin-2 are likely to play an important role in the regulation of salt and water transport by noradrenaline in the kidney and may explain, at least in part, the altered renal sodium and water handling associated with overactivation of the sympathetic system.
1. The renal bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) is expressed only in the thick ascending limb and selectively traffics from intracellular vesicles (IVs) to apical plasma membranes (PMs), where BSC-1 regulates sodium reabsorption. We showed previously that in kidneys from adult spontaneously hypertensive rats (SHR; model of essential hypertension) total protein expression of BSC-1 was higher compared with kidneys from normotensive Wistar-Kyoto (WKY) rats. However, whether this change is associated with an increased trafficking of BSC-1 from IVs to PMs is unknown. The goal of the present study was to test the hypothesis that the increase in total renal BSC-1 protein expression in SHR is accompanied by an augmented distribution of BSC-1 from IVs to PMs. 2. To test the hypothesis, we obtained renal tissue from the inner stripe of the outer medulla (ISOM; enriched in thick ascending limbs) and isolated IVs and PMs from this tissue by differential centrifugation. Total BSC-1 protein expression in ISOM and BSC-1 protein expression in ISOM IVs and PMs were measured by semiquantitative western blotting in SHR and aged-matched WKY rats at different ages and stages of hypertension. 3. At 5 weeks of age, SHR were prehypertensive (mean arterial blood pressure (MABP) 97 mmHg). At this age, both the total abundance and cellular distribution of BSC-1 were similar in ISOM from SHR and WKY rats. 4. As SHR aged, their hypertension progressed (MABP 137 and 195 mmHg at 8 and 14 weeks of age, respectively). Associated with the increase in MABP was an increase in both steady state protein levels of ISOM BSC-1 and the distribution of ISOM BSC-1 to PMs (four- and sixfold increases at 8 and 14 weeks of age, respectively, compared with age-matched WKY rats; P < 0.001). 5. Using semiquantitative reverse transcription-polymerase chain reaction, BSC-1 mRNA was measured and was found not to differ between SHR and WKY rat ISOM at any age or level of MABP. 6. We conclude that as SHR transition from prehypertensive to established hypertension, there is a marked increase in the total expression of BSC-1 in ISOM that is not related to increases in steady state levels of BSC-1 mRNA and therefore unlikely to be due to changes in either the rate of BSC-1 gene transcription or the stability of BSC-1 mRNA. This suggests changes in either translational efficiency or BSC-1 protein stability in SHR. 7. We also conclude that the age/hypertension-related increase in BSC-1 protein levels in ISOM is accompanied by an equally marked increased trafficking of BSC-1 to PMs in SHR ISOM.
Abstract-The sympathetic nervous system, via norepinephrine, regulates renal sodium transport, and chronic sympathetic activation causes sustained increases in blood pressure by reducing sodium excretion. Our previous studies show that chronic norepinephrine infusion increases the abundance of the bumetanide-sensitive cotransporter type 1, the apical sodium transporter of the thick ascending limb of Henle's loop. The present study was initiated to elucidate the mechanisms by which norepinephrine regulates the protein levels of this transporter in an immortalized thick ascending limb epithelial cell line. Treatment with norepinephrine, either alone or in the presence of actinomycin D or cycloheximide, had no effect on cotransporter mRNA levels. Treatment with norepinephrine, however, increased bumetanide-sensitive cotransporter type 1 protein levels (70% increase versus control; Pϭ0.012), and pretreatment with cycloheximide blocked the effect of norepinephrine on bumetanide-sensitive cotransporter type 1 protein levels. To further elucidate the mechanism, thick ascending limb cells were treated with norepinephrine in the presence of phentolamine (␣-adrenoceptor blocker), propranolol (-adrenoceptor blocker), SQ22536 (adenylyl cyclase inhibitor), PD098059 (mitogen-activated protein kinase pathway inhibitor), H-89 (protein kinase A inhibitor), or staurosporine (protein kinase C inhibitor). Treatment with propranolol, SQ22536, and H-89 abolished the effects of norepinephrine on bumetanide-sensitive cotransporter type 1 protein levels, whereas staurosporine had no effect. Treatment with PD098059 partially inhibited the effects of norepinephrine (40% decrease versus norepinephrine; Pϭ0.03), and treatment with phentolamine potentiated the effects of norepinephrine (30% increase versus norepinephrine; Pϭ0.02) on bumetanide-sensitive cotransporter type 1 protein levels. We conclude that regulation of bumetanide-sensitive cotransporter type 1 by norepinephrine proceeds via the -adrenoceptor receptor-cAMP-protein kinase A pathway that involves in part mitogen-activated protein kinases and that ␣-adrenoceptor activation negatively regulates bumetanidesensitive cotransporter type 1 protein levels. Key Words: BSC-1/NKCC2 Ⅲ thick ascending limb Ⅲ norepinephrine Ⅲ ␣-adrenoceptor Ⅲ -adrenoceptor Ⅲ cAMP Ⅲ protein kinase A Ⅲ cAMP T he bumetanide-sensitive cotransporter type 1 (BSC-1) Na-K-2Cl cotransporter is the principal apical Na ϩ entry pathway in the thick ascending limb (TAL). 1,2 Therefore long-term dysregulation of BSC-1 may contribute to longterm dysregulation of arterial blood pressure. In support of this concept, recent studies demonstrate that enhanced expression of BSC-1 in the TAL causes sodium retention in rats with congestive heart failure. 3 Moreover, BSC-1 is upregulated in rats with small-to-moderate myocardial infarctions, 4 dehydration, and cardiac failure 5 and in an animal model of liver cirrhosis. 6 Moreover, our results in the spontaneously hypertensive rat (SHR) show that expression of BSC-1 is also ele...
Summary We developed methods for prolonged (12 h), sterile, normothermic perfusion of rat kidneys and screened compounds for renal preservation including: mitochondrial transition pore inhibitor (decylubiquinone); caspase inhibitor (Z‐VAD); peroxisome proliferator‐activated receptor‐alpha (PPARα) agonists (gemfibrozil, WY‐14643); antioxidants (trolox, luteolin, quercetin); growth factors (HGF, PDGF, EGF, IGF‐1, VEGF, transferrin); calpain inhibitor (Z‐Val‐Phe‐CHO); calmodulin inhibitor (W7); KATP opener (minoxidil, minoxidil sulfate); PARP inhibitor (3‐aminobenzamide); calcium channel blocker (verapamil); V2 agonist (DDAVP); diuretics (acetazolamide, hydrochlorothiazide, furosemide, mannitol); peroxisome proliferator‐activated receptor‐beta agonist (L‐165041); dopamine agonist (dopamine); essential fatty acid (linolenic acid); β‐NAD; urea; uric acid; and aldosterone. In pilot studies, only PPARα agonists and mannitol provided promising results. Accordingly, these agents were investigated further. Fifteen rat kidneys were perfused for 12 h with L‐15 media at 37 °C in the absence or presence of mannitol, gemfibrozil, gemfibrozil + mannitol or WY‐14643. Chronic perfusion in untreated kidneys caused destruction of glomerular and tubular architecture (light and electron microscopy), disappearance of Na+‐K+‐ATPase‐α1 (Western blotting), and apoptosis (Apoptag staining). Gemfibrozil and WY‐14643 marginally improved some biomarkers of renal preservation. However, the combination of gemfibrozil with mannitol markedly improved all parameters of renal preservation. We conclude that PPARα agonists, particularly when combined with mannitol, protect organs from normothermic, perfusion‐induced damage.
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