RESUMO
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-wk-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.
Assuntos
Compostos Benzidrílicos , Glucosídeos , Hipertensão , Ratos Endogâmicos SHR , Ratos Wistar , Inibidores do Transportador 2 de Sódio-Glicose , Trocador 3 de Sódio-Hidrogênio , Regulação para Cima , Animais , Masculino , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Glucosídeos/farmacologia , Compostos Benzidrílicos/farmacologia , Regulação para Cima/efeitos dos fármacos , Ratos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Rim/efeitos dos fármacosRESUMO
Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies have suggested that it could play a role in regulating potassium renal excretion by modulating the activity of the Na+-Cl- cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal-potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low-potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1-/- mice had lower plasma levels of K+ and Cl- while exhibiting higher urinary excretion of Na+, Cl-, and K+ compared with KS-WNK1+/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K+ levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1+/+ mice adapt better to HKD challenge than KS-WNK1-/- mice after a potassium-retaining state. The difference in the phosphorylated NCC-to-NCC ratio between KS-WNK1+/+ and KS-WNK1-/- mice after 0KD and HKD indicates a role for KS-WNK1 in both NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the distal convoluted tubule to respond to extreme changes in potassium intake, such as those occurring in wildlife.NEW & NOTEWORTHY The findings of this study demonstrate that kidney-specific with-no-lysine kinase 1 plays a role in regulating urinary electrolyte excretion during extreme changes in potassium intake, such as those occurring in wildlife. .
Assuntos
Camundongos Knockout , Potássio na Dieta , Proteína Quinase 1 Deficiente de Lisina WNK , Animais , Masculino , Camundongos , Rim/metabolismo , Túbulos Renais Distais/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação , Potássio/urina , Potássio/metabolismo , Potássio/sangue , Potássio na Dieta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Eliminação Renal , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , FemininoRESUMO
The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.
Assuntos
Cloretos , Simportadores de Cloreto de Sódio-Potássio , Animais , Humanos , Cátions/metabolismo , Cloretos/metabolismo , Diuréticos/metabolismo , Túbulos Renais Distais/metabolismo , Sódio/metabolismo , Cloreto de Sódio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Tiazidas/metabolismo , Membro 1 da Família 12 de Carreador de SolutoRESUMO
Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.
Assuntos
Arginina Vasopressina , Proteínas Serina-Treonina Quinases , Camundongos , Humanos , Animais , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Células HEK293 , Arginina Vasopressina/metabolismo , Cotransportadores de K e Cl- , Desamino Arginina Vasopressina , Colforsina , Proteína Fosfatase 1/metabolismo , Rim/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
The primary structure of the thiazide-sensitive NaCl cotransporter (NCC) was resolved 30 years ago by the molecular identification of the cDNA encoding this cotransporter, from the winter's flounder urinary bladder, following a functional expression strategy. This review outlines some aspects of how the knowledge about thiazide diuretics and NCC evolved, the history of the cloning process, and the expansion of the SLC12 family of electroneutral cotransporters. The diseases associated with activation or inactivation of NCC are discussed, as well as the molecular model by which the activity of NCC is regulated. The controversies in the field are discussed as well as recent publication of the three-dimensional model of NCC obtained by cryo-electron microscopy, revealing not only the amino acid residues critical for Na+ and Cl- translocation but also the residues critical for polythiazide binding to the transporter, opening the possibility for a new era in thiazide diuretic therapy.
Assuntos
Proteínas Serina-Treonina Quinases , Cloreto de Sódio , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Cloreto de Sódio/metabolismo , Microscopia Crioeletrônica , Inibidores de Simportadores de Cloreto de Sódio , Clonagem MolecularRESUMO
Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.
Assuntos
Hiperpotassemia , Hipopotassemia , Camundongos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Sódio/metabolismo , Hipopotassemia/metabolismo , Hiperpotassemia/metabolismo , Túbulos Renais Distais/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Camundongos Knockout , Homeostase , Potássio/metabolismoRESUMO
BACKGROUND: The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS: Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.
Assuntos
Glicosúria , Simportadores de Cloreto de Sódio , Humanos , Camundongos , Animais , Simportadores de Cloreto de Sódio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Glucose/metabolismo , Células HEK293 , Camundongos Endogâmicos C57BL , Fosforilação , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Túbulos Renais Distais/metabolismo , Camundongos Knockout , Glicosúria/metabolismoRESUMO
The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule, and the inhibition of its function with thiazides is widely used for the treatment of arterial hypertension. In mammals and teleosts, NCC is present as one ortholog that is mainly expressed in the kidney. One exception, however, is the eel, which has two genes encoding NCC. The eNCCα is located in the kidney and eNCCß, which is present in the apical membrane of the rectum. Interestingly, the European eNCCß functions as a Na+-Cl- cotransporter that is nevertheless resistant to thiazides and is not activated by low-chloride hypotonic stress. However, in the Japanese eel rectal sac, a thiazide-sensitive NaCl transport mechanism has been described. The protein sequences between eNCCß and jNCCß are 98% identical. Here, by site-directed mutagenesis, we transformed eNCCß into jNCCß. Our data showed that jNCCß, similar to eNCCß, is resistant to thiazides. In addition, both NCCß proteins have high transport capacity with respect to their renal NCC orthologs and, in contrast to known NCCs, exhibit electrogenic properties that are reduced when residue I172 is substituted by A, G, or M. This is considered a key residue for the chloride ion-binding sites of NKCC and KCC. We conclude that NCCß proteins are not sensitive to thiazides and have electrogenic properties dependent on Cl-, and site I172 is important for the function of NCCß.
Assuntos
Cloretos , Inibidores de Simportadores de Cloreto de Sódio , Animais , Cloretos/metabolismo , Enguias/metabolismo , Mamíferos/metabolismo , Cloreto de Sódio , Inibidores de Simportadores de Cloreto de Sódio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Simportadores de Cloreto de Sódio/genética , Simportadores de Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Tiazidas/farmacologiaRESUMO
The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Rim/enzimologia , Proteínas dos Microfilamentos/metabolismo , Potássio na Dieta/metabolismo , Pseudo-Hipoaldosteronismo/enzimologia , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Culina/metabolismo , Estabilidade Enzimática , Feminino , Rim/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Pseudo-Hipoaldosteronismo/genética , Pseudo-Hipoaldosteronismo/fisiopatologia , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/deficiência , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Xenopus laevisRESUMO
BACKGROUND: Diabetic nephropathy is a global common cause of chronic kidney disease and end-stage renal disease. A lot of research has been conducted in biomedical sciences, which has enhanced understanding of the pathophysiology of diabetic nephropathy and has expanded the potential available therapies. An increasing number of evidence suggests that genetic alterations play a major role in development and progression of diabetic nephropathy. This systematic review was focused on searching an association between Arg913Gln variation in SLC12A3 gene with diabetic nephropathy in individuals with Type 2 Diabetes and Gitelman Syndrome. METHODS: An extensive systematic review of the literature was completed using PubMed, EBSCO and Cochrane Library, from their inception to January 2018. The PRISMA guidelines were followed and the search strategy ensured that all possible studies were identified to compile the review. Inclusion criteria for this review were: 1) Studies that analyzed the SLC12A3 gene in individuals with Type 2 Diabetes and Gitelman Syndrome. 2) Use of at least one analysis investigating the association between the Arg913Gln variation of SLC12A3 gene with diabetic nephropathy. 3) Use of a case-control or follow-up design. 4) Investigation of type 2 diabetes mellitus in individuals with Gitelman's syndrome, with a history of diabetic nephropathy. RESULTS: The included studies comprised 2106 individuals with diabetic nephropathy. This review shows a significant genetic association in most studies in the Arg913Gln variation of SLC12A3 gene with the diabetic nephropathy, pointing out that the mutations of this gene could be a key predictor of end-stage renal disease. CONCLUSIONS: The results showed in this systematic review contribute to better understanding of the association between the Arg913Gln variation of SLC12A3 gene with the pathogenesis of diabetic nephropathy in individuals with T2DM and GS.
Assuntos
Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/genética , Síndrome de Gitelman/genética , Diabetes Mellitus Tipo 2/complicações , Variação Genética , Síndrome de Gitelman/complicações , Humanos , Falência Renal Crônica/genética , Mutação , Simportadores de Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genéticaRESUMO
The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. The functional and structural characterization of NCC from different species has led us to gain insights into the structure-function relationships of the cotransporter. Here we present an overview of different studies that had described these properties. Additionally, we report the cloning and characterization of the NCC from the spiny dogfish (Squalus acanthias) kidney (sNCC). The purpose of the present study was to determine the main functional, pharmacological and regulatory properties of sNCC to make a direct comparison with other NCC orthologous. The sNCC cRNA encodes a 1033 amino acid membrane protein, when expressed in Xenopus oocytes, functions as a thiazide-sensitive Na-Cl cotransporter with NCC regulation and thiazide-inhibition properties similar to mammals, rather than to teleosts. However, the Km values for ion transport kinetics are significantly higher than those observed in the mammal species. In summary, we present a review on NCC structure-function relationships with the addition of the sNCC information in order to enrich the NCC cotransporter knowledge.
Assuntos
Rim/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/química , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Síndrome de Gitelman/genética , Humanos , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Relação Estrutura-AtividadeRESUMO
The renal thiazide-sensitive NaCl cotransporter (NCC) is the major salt transport pathway in the distal convoluted tubule of the mammalian nephron. NCC activity is critical for modulation of arterial blood pressure and serum potassium levels. Reduced activity of NCC in genetic diseases results in arterial hypotension and hypokalemia, while increased activity results in genetic diseases featuring hypertension and hyperkalemia. Several hormones and physiological conditions modulate NCC activity through a final intracellular complex pathway involving kinases and ubiquitin ligases. A substantial amount of work has been conducted to understand this pathway in the last 15 yr, but advances over the last 3 yr have helped to begin to understand how these regulatory proteins interact with each other and modulate the activity of this important cotransporter. In this review, we present the current model of NCC regulation by the Cullin 3 protein/Kelch-like 3 protein/with no lysine kinase/STE20-serine-proline alanine-rich kinase (CUL3/KELCH3-WNK-SPAK) pathway. We present a review of all genetically altered mice that have been used to translate most of the proposals made from in vitro experiments into in vivo observations that have helped to elucidate the model at the physiological level. Many questions have been resolved, but some others will require further models to be constructed. In addition, unexpected observations in mice have raised new questions and identified regulatory pathways that were previously unknown.
Assuntos
Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Culina/genética , Proteínas Culina/metabolismo , Modelos Animais de Doenças , Predisposição Genética para Doença , Síndrome de Gitelman/enzimologia , Síndrome de Gitelman/genética , Humanos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Pseudo-Hipoaldosteronismo/enzimologia , Pseudo-Hipoaldosteronismo/genética , Transdução de Sinais , Membro 3 da Família 12 de Carreador de Soluto/genética , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismoRESUMO
Background Hypercalciuria can result from activation of the basolateral calcium-sensing receptor (CaSR), which in the thick ascending limb of Henle's loop controls Ca2+ excretion and NaCl reabsorption in response to extracellular Ca2+ However, the function of CaSR in the regulation of NaCl reabsorption in the distal convoluted tubule (DCT) is unknown. We hypothesized that CaSR in this location is involved in activating the thiazide-sensitive NaCl cotransporter (NCC) to prevent NaCl loss.Methods We used a combination of in vitro and in vivo models to examine the effects of CaSR on NCC activity. Because the KLHL3-WNK4-SPAK pathway is involved in regulating NaCl reabsorption in the DCT, we assessed the involvement of this pathway as well.Results Thiazide-sensitive 22Na+ uptake assays in Xenopus laevis oocytes revealed that NCC activity increased in a WNK4-dependent manner upon activation of CaSR with Gd3+ In HEK293 cells, treatment with the calcimimetic R-568 stimulated SPAK phosphorylation only in the presence of WNK4. The WNK4 inhibitor WNK463 also prevented this effect. Furthermore, CaSR activation in HEK293 cells led to phosphorylation of KLHL3 and WNK4 and increased WNK4 abundance and activity. Finally, acute oral administration of R-568 in mice led to the phosphorylation of NCC.Conclusions Activation of CaSR can increase NCC activity via the WNK4-SPAK pathway. It is possible that activation of CaSR by Ca2+ in the apical membrane of the DCT increases NaCl reabsorption by NCC, with the consequent, well known decrease of Ca2+ reabsorption, further promoting hypercalciuria.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sódio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ativação Enzimática/genética , Células HEK293 , Humanos , Imidazóis/farmacologia , Masculino , Camundongos , Proteínas dos Microfilamentos , Oócitos , Fenetilaminas/farmacologia , Fosforilação/efeitos dos fármacos , Propilaminas/farmacologia , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Pirrolidinas/farmacologia , Receptores de Detecção de Cálcio/genética , Transdução de Sinais , Membro 1 da Família 12 de Carreador de Soluto/antagonistas & inibidores , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Transfecção , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Familial hyperkalemic hypertension (FHHt) can be mainly attributed to increased activity of the renal Na+:Cl- cotransporter (NCC), which is caused by altered expression and regulation of the with-no-lysine (K) 1 (WNK1) or WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/STE20-proline-alanine rich kinase (SPAK)/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK-interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylated. In contrast, this serine becomes phosphorylated when the intracellular chloride concentration ([Cl-]i) is reduced or when KS-WNK1 is coexpressed with WNK4. KS-WNK1-mediated activation of WNK4 is not due to a decrease of the [Cl-]i. Coimmunoprecipitation analysis revealed that KS-WNK1 and WNK4 interact with each other and that WNK4 becomes autophosphorylated at serine 335 when it is associated with KS-WNK1. Together, these observations suggest that WNK4 becomes active in the presence of KS-WNK1, despite a constant [Cl-]i.
Assuntos
Cloretos/metabolismo , Rim/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sódio/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Ativação Enzimática , Feminino , Humanos , Oócitos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Ratos , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Loss of function of mutated solute carrier family 12 member 3 (SLC12A3) gene is the most frequent etiology for Gitelman syndrome (GS), which is mainly manifested by hypokalemia, hypomagnesemia and hypocalciuria. We report the genetic characteristics of one suspicious Chinese GS pedigree by gene sequencing. Complete sequencing analysis of the SLC12A3 gene revealed that both the proband and his elder sister had a novel homozygous SLC12A3 mutation: c.2099T>C and p.Leu700Pro. Moreover, the SLC12A3 genes of his mother and daughter encoded the same mutated heterozygote. It was noted that in this pedigree, only the proband complained about recurrent episodes of bilateral lower limb weakness over 8 years, while his elder sister, mother and daughter did not present symptoms. The inconsistent clinical features of this pedigree implied that besides diverse phenotypes possibly originated from the same genotype, gender difference may also dominate the variant GS phenotypes. Further genetic and proteomic research are needed to investigate the precise mechanisms of GS, including the study of specific ethnicities.
Assuntos
Síndrome de Gitelman/genética , Homozigoto , Mutação/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Povo Asiático , Feminino , Síndrome de Gitelman/diagnóstico , Humanos , Masculino , Linhagem , Fenótipo , Adulto JovemRESUMO
PURPOSE OF REVIEW: Abundant evidence supports that the NaCl cotransporter (NCC) activity is tightly regulated by the with-no-lysine (WNK) kinases. Here, we summarize the data regarding NCC regulation by WNKs, with a particular emphasis on WNK4. RECENT FINDINGS: Several studies involving in-vivo and in-vitro models have provided paradoxical data regarding WNK4 regulation of the NCC. Although some studies show that WNK4 can activate the NCC, other equally compelling studies show that WNK4 inhibits the NCC. Recent studies have shown that WNK4 is regulated by the intracellular chloride concentration ([Cl]i), which could account for these paradoxical results. In conditions of high [Cl]i, WNK4 could act as an inhibitor via heterodimer formation with other WNKs. In contrast, when [Cl]i is low, WNK4 can activate Ste20-related, proline-alanine-rich kinase (SPAK)/oxidative stress responsive kinase 1 (OSR1) and thus the NCC. Modulation of WNK4 by [Cl]i has been shown to account for the potassium-sensing properties of the distal convoluted tubule. Other regulators of WNK4 include hormones and ubiquitination. SUMMARY: Modulation of WNK4 activity by [Cl]i can account for its dual role on the NCC, and this has important physiological implications regarding the regulation of extracellular potassium concentration. Defective regulation of WNKs by ubiquitination explains most cases of familial hyperkalemic hypertension.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Cloretos/metabolismo , Humanos , Túbulos Renais Distais/metabolismo , Potássio/metabolismo , Pseudo-Hipoaldosteronismo/fisiopatologiaRESUMO
To evaluate the genotype-phenotype relationship of Gitelman syndrome in Chinese patients. We selected patients with Gitelman syndrome presenting hypokalemia. Medical history, clinical manifestations, laboratory test results, and imaging data of these patients were collected for analysis. Target gene sequencing was performed to evaluate the genotype-phenotype relationship. Gitelman syndrome was diagnosed based on medical history, clinical manifestations, laboratory test results, and imaging data. The causative gene for Gitelman syndrome, SLC12A3, and the causative gene for the classic Bartter syndrome, CLCNKB, were screened for disease-causing mutations by direct sequencing. Clinical diagnoses of ten patients were consistent with Gitelman syndrome. Disease-causing mutations in the SLC12A3 gene were found in six patients. Among the variants, T60M in exon 1 was the hot spot in Chinese patients. Additionally, we found a small deletion of ACGG in exon 3 and L671P in exon 16; these have not been reported in previous studies. No disease-causing mutations were observed in the other four patients. Since mutations in the SLC12A3 and CLCNKB genes are not present in all patients with clinical manifestations of Gitelman syndrome, genetic screening after clinical diagnosis is essential.
Assuntos
Canais de Cloreto/genética , Síndrome de Gitelman/genética , Fenótipo , Adolescente , Adulto , Idoso , Éxons , Feminino , Genótipo , Síndrome de Gitelman/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Membro 3 da Família 12 de Carreador de Soluto/genéticaRESUMO
The renal thiazide-sensitive NaCl cotransporter, NCC, is the major pathway for salt reabsorption in the distal convoluted tubule. The activity of this cotransporter is critical for regulation of several physiological variables such as blood pressure, serum potassium, acid base metabolism, and urinary calcium excretion. Therefore, it is not surprising that numerous hormone-signaling pathways regulate NCC activity to maintain homeostasis. In this review, we will provide an overview of the most recent evidence on NCC modulation by aldosterone, angiotensin II, vasopressin, glucocorticoids, insulin, norepinephrine, estradiol, progesterone, prolactin, and parathyroid hormone.
Assuntos
Hormônios/metabolismo , Túbulos Renais Distais/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Hormônios Esteroides Gonadais/metabolismo , Hormônios/farmacologia , Humanos , Túbulos Renais Distais/efeitos dos fármacos , Receptores de Peptídeos/efeitos dos fármacos , Receptores de Peptídeos/metabolismo , Sistema Renina-Angiotensina , Membro 3 da Família 12 de Carreador de Soluto/efeitos dos fármacosRESUMO
Loss of function of mutated solute carrier family 12 member 3 (SLC12A3) gene is the most frequent etiology for Gitelman syndrome (GS), which is mainly manifested by hypokalemia, hypomagnesemia and hypocalciuria. We report the genetic characteristics of one suspicious Chinese GS pedigree by gene sequencing. Complete sequencing analysis of the SLC12A3 gene revealed that both the proband and his elder sister had a novel homozygous SLC12A3 mutation: c.2099T>C and p.Leu700Pro. Moreover, the SLC12A3 genes of his mother and daughter encoded the same mutated heterozygote. It was noted that in this pedigree, only the proband complained about recurrent episodes of bilateral lower limb weakness over 8 years, while his elder sister, mother and daughter did not present symptoms. The inconsistent clinical features of this pedigree implied that besides diverse phenotypes possibly originated from the same genotype, gender difference may also dominate the variant GS phenotypes. Further genetic and proteomic research are needed to investigate the precise mechanisms of GS, including the study of specific ethnicities.
Assuntos
Humanos , Masculino , Feminino , Adulto Jovem , Síndrome de Gitelman/genética , Homozigoto , Mutação/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Povo Asiático , Síndrome de Gitelman/diagnóstico , Linhagem , FenótipoRESUMO
Mounting evidence has implicated the SLC12A3 gene in essential hypertension. Here, we examined the potential associations of common variants of the SLC12A3 gene with blood pressure traits in Mongolians in China. Genomic DNA was extracted from 508 unrelated Mongolian patients with essential hypertension and 246 normotensive Mongolian subjects for genotyping. The genotype distributions of all selected polymorphisms were consistent with Hardy-Weinberg equilibrium. The presence of the G allele in the rs7187932 polymorphism was found to be associated with an increased risk of hypertension (OR: 1.30; 95%CI = 1.00-1.38; P = 0.048), whereas the rs2399594 G allele was associated with a reduced risk for hypertension (OR: 0.76; 95%CI = 0.60-0.97; P = 0.030). No significant difference was observed for other alleles. Haplotype analysis revealed an association of the rs2399594 and rs711746 GG haplotype with a reduced risk for hypertension (OR: 0.76; 95%CI = 0.60-0.97; P = 0.029). No significant association was observed between other haplotypes and hypertension. These results suggest that the SLC12A3 gene is a susceptibility gene for hypertension in the Mongolian population.