RESUMEN
Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.
Asunto(s)
Huesos/metabolismo , Calcio/sangre , Estudio de Asociación del Genoma Completo , Homeostasis/genética , Animales , Densidad Ósea/genética , Regulación de la Expresión Génica , Humanos , Riñón/metabolismo , Ratones , Polimorfismo de Nucleótido Simple , Población Blanca/genéticaRESUMEN
Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e., distal convoluted tubule (DCT) and connecting tubule (CNT) and the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf, and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, alphaENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms, and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function.
Asunto(s)
Relojes Biológicos/genética , Ritmo Circadiano/genética , Perfilación de la Expresión Génica , Túbulos Renales/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 4/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Presión Sanguínea , Proteínas CLOCK , Proteínas de Unión al ADN/genética , Electrólitos/sangre , Capacidad de Concentración Renal , Túbulos Renales/fisiología , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores Citoplasmáticos y Nucleares/genética , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción/genética , UrodinámicaRESUMEN
Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.
Asunto(s)
Diabetes Insípida Nefrogénica/inducido químicamente , Canales Epiteliales de Sodio/fisiología , Cloruro de Litio/toxicidad , Absorción , Animales , Acuaporina 2/análisis , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/patología , Cloruro de Litio/farmacocinética , Ratones , Ratones Noqueados , ATPasas de Translocación de Protón/análisisRESUMEN
Mutations in α, ß, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Aldosterona/sangre , Animales , Acuaporina 2/metabolismo , Canales Epiteliales de Sodio/genética , Femenino , Homeostasis/fisiología , Corteza Renal/citología , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Sodio en la Dieta/farmacologíaRESUMEN
Mutations in the WNK kinases WNK1 and WNK4 cause a rare familial form of hypertension (Gordon syndrome) by increasing expression of the thiazide-sensitive co-transporter NCCT in the kidney. Regulation of NCCT expression involves a scaffold of proteins composed of several kinases, including the third member of the WNK kinase family, WNK3. This protein, expressed in several tissues including kidney and brain, displays splice variation around exons 18 and 22. We expressed these proteins in Xenopus oocytes and found that the renal isoform of WNK3 increased but the brain isoform decreased NCCT expression and activity. Introduction of a kinase-inactivating mutation into renal WNK3 reversed its action on NCCT, and the same mutation in the brain isoforms led to loss of function. We also studied the effect of phosphorylation of a key NCCT threonine (T58) on the effects of WNK3/4 coexpression; NCCT mutants with a T58A or T58D substitution had the same surface expression as T58 but had significantly altered transporter activity; however, both isoforms of WNK3 as well as WNK4 still modulated expression of these NCCT mutants. Finally, experiments using kinase-dead STE20/SPS1-related proline/alanine-rich kinase (SPAK), a putative downstream target for WNKs, revealed that brain WNK3 acts in tandem with SPAK, whereas renal WNK3 seems to upregulate NCCT through a SPAK-independent pathway. Taken together, these results suggest that the C-terminal motifs contributed by exons 18 and 22 play an important role in the actions of WNK3 isoforms on NCCT.
Asunto(s)
Encéfalo/metabolismo , Riñón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Droga/metabolismo , Simportadores/metabolismo , Empalme Alternativo , Animales , Exones , Humanos , Isoenzimas/metabolismo , Ratones , Mutación , Oocitos/metabolismo , Receptores de Droga/genética , Miembro 3 de la Familia de Transportadores de Soluto 12 , Simportadores/genética , Xenopus laevisRESUMEN
A high salt intake in industrialized countries is an important cardiovascular risk factor. It remains at typically twice the maximum recommended levels of 5-6 g/day, and halving this would have enormous public health benefit in preventing stroke and cardiovascular disease. Salt homeostasis can also be affected pharmacologically by diuretic drugs that target mechanisms within the distal kidney nephron to cause salt wasting. Indeed, thiazide-type diuretics are among the most widely used agents in the management of hypertension and work by blocking NCCT, the NaCl-transporter in the distal nephron. The biology of this membrane transporter was not previously well understood until the discovery of the molecular basis of a rare familial form of hypertension called Gordon syndrome (pseudohypoaldosteronism type 2, PHAII). This has established that the NCCT transporter is dynamically regulated in the kidney by WNK kinases and a signaling cascade using a second kinase called SPAK. Common polymorphisms in the SPAK gene have recently been shown to affect blood pressure in human cohorts and removing its function lowers blood pressure in mice. The SPAK-deficient mouse actually has a phenotype reminiscent of Gitelman syndrome. This suggests that specific inhibitors of SPAK kinase may provide a novel class of diuretic drugs to lower blood pressure through salt wasting. The expectation is that SPAK inhibitors would mimic the on-target effects of thiazides but not their adverse off-target effects. An antihypertensive drug that could lower blood pressure with the efficacy of a thiazide without producing metabolic side effects such as hyperuricaemia or impaired glucose tolerance is therapeutically very attractive. It also exemplifies how data coming from the rare monogenic hypertension syndromes can together with genome-wide association studies in hypertension deliver novel druggable targets.
Asunto(s)
Hipertensión/etiología , Receptores de Droga/fisiología , Cloruro de Sodio Dietético/administración & dosificación , Simportadores/fisiología , Presión Sanguínea , Transporte Iónico , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Sodio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12RESUMEN
Mutations within the with-no-K(Lys) (WNK) kinases cause Gordon's syndrome characterized by hypertension and hyperkalaemia. WNK kinases phosphorylate and activate the STE20/SPS1-related proline/alanine-rich kinase (SPAK) protein kinase, which phosphorylates and stimulates the key Na(+):Cl(-) cotransporter (NCC) and Na(+):K(+):2Cl(-) cotransporters (NKCC2) cotransporters that control salt reabsorption in the kidney. To define the importance of this pathway in regulating blood pressure, we generated knock-in mice in which SPAK cannot be activated by WNKs. The SPAK knock-in animals are viable, but display significantly reduced blood pressure that was salt-dependent. These animals also have markedly reduced phosphorylation of NCC and NKCC2 cotransporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in messenger RNA (mRNA) levels. On a normal Na(+)-diet, the SPAK knock-in mice were normokalaemic, but developed mild hypokalaemia when the renin-angiotensin system was activated by a low Na(+)-diet. These observations establish that SPAK plays an important role in controlling blood pressure in mammals. Our results imply that SPAK inhibitors would be effective at reducing blood pressure by lowering phosphorylation as well as expression of NCC and NKCC2. See accompanying Closeup by Maria Castañeda-Bueno and Gerald Gamba (DOI 10.1002/emmm.200900059).
Asunto(s)
Presión Sanguínea/fisiología , Homeostasis , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Técnicas de Sustitución del Gen , Riñón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Receptores de Droga/metabolismo , Sales (Química)/metabolismo , Simportadores de Cloruro de Sodio-Potasio , Miembro 1 de la Familia de Transportadores de Soluto 12 , Miembro 3 de la Familia de Transportadores de Soluto 12 , Análisis de Supervivencia , Simportadores/metabolismoRESUMEN
The antidiuretic effect of vasopressin is mediated by V2 receptors (V2R) that are located in kidney connecting tubules and collecting ducts. This study provides evidence that V2R signaling is negatively regulated by regulator of G protein signaling 2 (RGS2), a member of the family of RGS proteins. This study demonstrates that (1) RGS2 expression in the kidney is restricted to the vasopressin-sensitive part of the nephron (thick ascending limb, connecting tubule, and collecting duct); (2) expression of RGS2 is rapidly upregulated by vasopressin; (3) the vasopressin-dependent accumulation of cAMP, the principal messenger of V2R signaling, is significantly higher in collecting ducts that are microdissected from the RGS2(-/-) mice compared with their wild-type littermates; and (4) analysis of urine output of mice that were exposed to water restriction followed by acute water loading revealed that RGS2(-/-) mice exhibit an increased renal responsiveness to vasopressin. It is proposed that RGS2 is involved in negative feedback regulation of V2R signaling.