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1.
Kidney Int ; 100(4): 850-869, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34252449

RESUMEN

Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT.


Asunto(s)
Acidosis , Magnesio , Animales , Calcineurina/genética , Túbulos Renales Distales , Ratones , Proteoma/genética , Transcriptoma
2.
Nephrol Dial Transplant ; 35(3): 411-432, 2020 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-31436795

RESUMEN

BACKGROUND: Loss-of-function mutations in the sodium chloride (NaCl) co-transporter (NCC) of the renal distal convoluted tubule (DCT) cause Gitelman syndrome with hypokalemic alkalosis, hypomagnesemia and hypocalciuria. Since Gitelman patients are usually diagnosed around adolescence, we tested the idea that a progressive regression of the DCT explains the late clinical onset of the syndrome. METHODS: NCC wild-type and knockout (ko) mice were studied at Days 1, 4 and 10 and 6 weeks after birth using blood plasma analysis and morphological and biochemical methods. RESULTS: Plasma aldosterone levels and renal renin messenger RNA expression were elevated in NCC ko mice during the first days of life. In contrast, plasma ion levels did not differ between genotypes at age 10 days, but a significant hypomagnesemia was observed in NCC ko mice at 6 weeks. Immunofluorescent detection of parvalbumin (an early DCT marker) revealed that the fractional cortical volume of the early DCT is similar for mice of both genotypes at Day 4, but is significantly lower at Day 10 and is almost zero at 6 weeks in NCC ko mice. The DCT atrophy correlates with a marked reduction in the abundance of the DCT-specific Mg2+ channel TRPM6 (transient receptor potential cation channel subfamily M member 6) and an increased proteolytic activation of the epithelial Na+ channel (ENaC). CONCLUSION: After an initial outgrowth, DCT development lags behind in NCC ko mice. The impaired DCT development associates at Day 1 and Day 10 with elevated renal renin and plasma aldosterone levels and activation of ENaC, respectively, suggesting that Gitelman syndrome might be present much earlier in life than is usually expected. Despite an early downregulation of TRPM6, hypomagnesemia is a rather late symptom.


Asunto(s)
Síndrome de Bartter/patología , Síndrome de Gitelman/patología , Hiperaldosteronismo/patología , Túbulos Renales Distales/patología , Magnesio/metabolismo , Simportadores del Cloruro de Sodio/fisiología , Sodio/metabolismo , Animales , Síndrome de Bartter/etiología , Síndrome de Bartter/metabolismo , Síndrome de Gitelman/etiología , Síndrome de Gitelman/metabolismo , Hiperaldosteronismo/etiología , Hiperaldosteronismo/metabolismo , Túbulos Renales Distales/metabolismo , Ratones , Ratones Noqueados , Renina/metabolismo , Canales Catiónicos TRPM/metabolismo
3.
Physiol Rep ; 6(20): e13899, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30350402

RESUMEN

In the kidney, purinergic (P2) receptor-mediated ATP signaling has been shown to be an important local regulator of epithelial sodium transport. Appropriate sodium regulation is crucial for blood pressure (BP) control and disturbances in sodium balance can lead to hypo- or hypertension. Links have already been established between P2 receptor signaling and the development of hypertension, attributed mainly to vascular and/or inflammatory effects. A transgenic mouse model with deletion of the P2X4 receptor (P2X4-/- ) is known to have hypertension, which is thought to reflect endothelial dysfunction and impaired nitric oxide (NO) release. However, renal function in this model has not been characterized; moreover, studies in vitro have shown that the P2X4 receptor can regulate renal epithelial Na+ channel (ENaC) activity. Therefore, in the present study we investigated renal function and sodium handling in P2X4-/- mice, focusing on ENaC-mediated Na+ reabsorption. We confirmed an elevated BP in P2X4-/- mice compared with wild-type mice, but found that ENaC-mediated Na+ reabsorption is no different from wild-type and does not contribute to the raised BP observed in the knockout. However, when P2X4-/- mice were placed on a low sodium diet, BP normalized. Plasma aldosterone concentration tended to increase according to sodium restriction status in both genotypes; in contrast to wild-types, P2X4-/- mice did not show an increase in functional ENaC activity. Thus, although the increased BP in P2X4-/- mice has been attributed to endothelial dysfunction and impaired NO release, there is also a sodium-sensitive component.


Asunto(s)
Presión Sanguínea , Dieta Hiposódica , Hipertensión Renal/metabolismo , Receptores Purinérgicos P2X4/genética , Reabsorción Renal , Animales , Canales Epiteliales de Sodio/metabolismo , Hipertensión Renal/dietoterapia , Hipertensión Renal/genética , Riñón/metabolismo , Riñón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Receptores Purinérgicos P2X4/metabolismo , Sodio/metabolismo
4.
J Physiol ; 594(21): 6319-6331, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27457700

RESUMEN

KEY POINTS: High dietary potassium (K+ ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K+ , similar to those occurring after a K+ rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K+ appears to depend on cellular Cl- fluxes, the rapid NCC dephosphorylation in response to increased extracellular K+ is not Cl- -dependent. The Cl- -dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl- independent pathway may include additional signalling cascades. ABSTRACT: A high dietary potassium (K+ ) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K+ concentration ([K+ ]ex ) modulate NCC phosphorylation via a Cl- -dependent modulation of the with no lysine (K) kinases (WNK)-STE20/SPS-1-44 related proline-alanine-rich protein kinase (SPAK)/oxidative stress-related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K+ ]ex , with the most prominent effects occurring around physiological plasma [K+ ]. Cellular Cl- conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K+ ]ex . However, NCC dephosphorylation triggered by high [K+ ]ex is neither blocked by removing extracellular Cl- , nor by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid. The response to [K+ ]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K+ ]ex directly and rapidly controls NCC phosphorylation by Cl- -dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.


Asunto(s)
Cloruros/metabolismo , Túbulos Renales Distales/metabolismo , Potasio/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Canales de Cloruro/metabolismo , Túbulos Renales Distales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Potasio/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Transcripción/metabolismo
5.
J Am Soc Nephrol ; 25(3): 511-22, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24231659

RESUMEN

The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and arterial BP. Loss-of-function mutations of NCC cause renal salt wasting with arterial hypotension (Gitelman syndrome). Conversely, mutations in the NCC-regulating WNK kinases or kelch-like 3 protein cause familial hyperkalemic hypertension. Here, we performed automated sorting of mouse DCTs and microarray analysis for comprehensive identification of novel DCT-enriched gene products, which may potentially regulate DCT and NCC function. This approach identified protein phosphatase 1 inhibitor-1 (I-1) as a DCT-enriched transcript, and immunohistochemistry revealed I-1 expression in mouse and human DCTs and thick ascending limbs. In heterologous expression systems, coexpression of NCC with I-1 increased thiazide-dependent Na(+) uptake, whereas RNAi-mediated knockdown of endogenous I-1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I-1 (I-1(-/-)) knockout mice without changes in total NCC expression. The abundance and phosphorylation of other renal sodium-transporting proteins, including NaPi-IIa, NKCC2, and ENaC, did not change, although the abundance of pendrin increased in these mice. The abundance, phosphorylation, and subcellular localization of SPAK were similar in wild-type (WT) and I-1(-/-) mice. Compared with WT mice, I-1(-/-) mice exhibited significantly lower arterial BP but did not display other metabolic features of NCC dysregulation. Thus, I-1 is a DCT-enriched gene product that controls arterial BP, possibly through regulation of NCC activity.


Asunto(s)
Hipotensión/enzimología , Túbulos Renales Distales/enzimología , Proteínas/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Presión Sanguínea , Femenino , Humanos , Asa de la Nefrona/enzimología , Masculino , Ratones , Ratones Transgénicos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Transportadores de Sulfato , Regulación hacia Arriba , Xenopus
6.
Am J Physiol Renal Physiol ; 299(6): F1473-85, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20861076

RESUMEN

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) that inactivates 11ß-hydroxy glucocorticoids. High expression levels of 11ß-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11ß-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11ß-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.


Asunto(s)
Túbulos Renales Distales/efectos de los fármacos , Transporte de Proteínas , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Adrenalectomía , Aldosterona/metabolismo , Aldosterona/farmacología , Animales , Especificidad de Anticuerpos , Corticosterona/administración & dosificación , Corticosterona/metabolismo , Corticosterona/farmacología , Túbulos Renales Distales/metabolismo , Masculino , Ratones , Nefronas/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de Glucocorticoides/inmunología , Receptores de Mineralocorticoides/inmunología , Sodio en la Dieta/administración & dosificación , Sodio en la Dieta/farmacología
7.
Am J Physiol Renal Physiol ; 295(2): F556-67, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18480177

RESUMEN

We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.


Asunto(s)
Canales de Cloruro/metabolismo , Hipertensión/metabolismo , Corteza Renal/metabolismo , Médula Renal/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Regulación hacia Arriba , Aldosterona/metabolismo , Animales , Canales de Cloruro/genética , Antiportadores de Cloruro-Bicarbonato/metabolismo , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/metabolismo , Hipertensión/genética , Hipertensión/fisiopatología , Corteza Renal/fisiopatología , Médula Renal/fisiopatología , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Simportadores del Cloruro de Sodio/genética , Cloruro de Sodio Dietético/efectos adversos , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Transportadores de Sulfato
8.
Am J Physiol Renal Physiol ; 294(6): F1373-80, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18322017

RESUMEN

Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of Na(+)-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient (Romk(-/-)) mice. Plasma aldosterone was 40% higher and urinary PGE(2) excretion was 1.5-fold higher in Romk(-/-) compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule Na(+)/H(+) exchanger (NHE3) and Na(+)-P(i) cotransporter (NaPi-IIa) and TAL-specific Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2/BSC1) in Romk(-/-) mice, while the distal convoluted tubule (DCT)-specific Na(+)-Cl(-) cotransporter (NCC/TSC) was markedly increased. The abundance of the alpha-,beta-, and gamma-subunits of the epithelial Na(+) channel (ENaC) was slightly increased, although only differences for gamma-ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of Romk(-/-) mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent Na(+) absorption in the TAL is compensated predominately by upregulation of Na(+) transport in downstream DCT cells. These adaptive changes in Romk(-/-) mice may help to limit renal Na(+) loss, and thereby, contribute to survival of these mice.


Asunto(s)
Síndrome de Bartter/metabolismo , Síndrome de Bartter/fisiopatología , Proteínas Portadoras/metabolismo , Asa de la Nefrona/metabolismo , Canales de Potasio de Rectificación Interna/genética , Sodio/metabolismo , Adaptación Fisiológica/fisiología , Animales , Síndrome de Bartter/genética , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Dinoprostona/sangre , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Ratones Mutantes , Canales de Potasio de Rectificación Interna/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Regulación hacia Arriba/fisiología , Agua/metabolismo
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