RESUMO
The solute carrier family 12A (SLC12A) superfamily of membrane transporters modulates the movement of cations coupled with chloride across the membrane. In doing so, these cotransporters are involved in numerous aspects of human physiology: cell volume regulation, ion homeostasis, blood pressure regulation, and neurological action potential via intracellular chloride concentration modulation. Their physiological characterization has been largely studied; however, understanding the mechanics of their function and the relevance of structural domains or specific amino acids has been a pending task. In recent years, single-particle cryogenic electron microscopy (cryo-EM) has been successfully applied to members of the SLC12A family including all K+:Cl- cotransporters (KCCs), Na+:K+:2Cl- cotransporter NKCC1, and recently Na+:Cl- cotransporter (NCC); revealing structural elements that play key roles in their function. The present review analyzes the data provided by these cryo-EM reports focusing on structural domains and specific amino acids involved in ion binding, domain interactions, and other important SCL12A structural elements. A comparison of cryo-EM data from NKCC1 and KCCs is presented in the light of the two recent NCC cryo-EM studies, to propose insight into structural elements that might also be found in NCC and are necessary for its proper function. In the final sections, the importance of key coordination residues for substrate specificity and their implication on various pathophysiological conditions and genetic disorders is reviewed, as this could provide the basis to correlate structural elements with the development of novel and selective treatments, as well as mechanistic insight into the function and regulation of cation-coupled chloride cotransporters (CCCs).
Assuntos
Aminoácidos , Cloretos , Humanos , Microscopia Crioeletrônica , Cloretos/metabolismo , Sódio/metabolismo , Cátions , Sítios de LigaçãoRESUMO
The profile of sphingomyelin and its metabolites shows changes in the plasma, organs, and tissues of patients with cardiovascular, renal, and metabolic diseases. The objective of this study was to investigate the effect of empagliflozin on the levels of sphingomyelin and its metabolites, as well as on the activity of acid and neutral sphingomyelinase (aSMase and nSMase) and neutral ceramidase (nCDase) in the plasma, kidney, heart, and liver of streptozotocin-induced diabetic and Angiotensin II (Ang II)-induced hypertension rats. Empagliflozin treatment decreased hyperglycemia in diabetic rats whereas blood pressure remained elevated in hypertensive rats. In diabetic rats, empagliflozin treatment decreased sphingomyelin in the plasma and liver, ceramide in the heart, sphingosine-1-phosphate (S1P) in the kidney, and nCDase activity in the plasma, heart, and liver. In hypertensive rats, empagliflozin treatment decreased sphingomyelin in the plasma, kidney, and liver; S1P in the plasma and kidney; aSMase in the heart, and nCDase activity in the plasma, kidney, and heart. Our results suggest that empagliflozin downregulates the interaction of the de novo pathway and the catabolic pathway of sphingolipid metabolism in the diabetes, whereas in Ang II-dependent hypertension, it only downregulates the sphingolipid catabolic pathway.
Assuntos
Diabetes Mellitus Experimental , Hipertensão , Animais , Compostos Benzidrílicos , Ceramidas/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Glucosídeos , Humanos , Hipertensão/tratamento farmacológico , Ratos , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , EsfingomielinasRESUMO
SIRT1 is an NAD+-dependent class III histone deacetylase that is abundantly expressed in the kidney, where it modulates gene expression, apoptosis, energy homeostasis, autophagy, acute stress responses, and mitochondrial biogenesis. Alterations in SIRT1 activity and NAD+ metabolism are frequently observed in acute and chronic kidney diseases of diverse origins, including obesity and diabetes. Nevertheless, in vitro and in vivo studies and clinical trials with humans show that the SIRT1-activating compounds derived from natural sources, such as polyphenols found in fruits, vegetables, and plants, including resveratrol, quercetin, and isoflavones, can prevent disease and be part of treatments for a wide variety of diseases. Here, we summarize the roles of SIRT1 and NAD+ metabolism in renal pathophysiology and provide an overview of polyphenols that have the potential to restore SIRT1 and NAD+ metabolism in renal diseases.
Assuntos
Nefropatias , Sirtuína 1 , Humanos , Rim/metabolismo , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , NAD/metabolismo , Polifenóis/farmacologia , Sirtuína 1/metabolismoRESUMO
Foods and beverages provide a source of fluoride exposure in Mexico. While high fluoride concentrations are neurotoxic, recent research suggests that exposures within the optimal range may also pose a risk to the developing brain. This prospective study examined whether dietary fluoride intake during pregnancy is associated with toddlers' neurodevelopment in 103 mother-child pairs from the PROGRESS cohort in Mexico City. Food and beverage fluoride intake was assessed in trimesters 2 and 3 using a food frequency questionnaire and Mexican tables of fluoride content. We used the Bayley-III to evaluate cognitive, motor, and language outcomes at 12 and 24 months of age. Adjusted linear regression models were generated for each neurodevelopment assessment time point (12 and 24 months). Mixed-effects models were used to consider a repeated measurement approach. Interactions between maternal fluoride intake and child sex on neurodevelopmental outcomes were tested. Median (IQR) dietary fluoride intake during pregnancy was 1.01 mg/d (0.73, 1.32). Maternal fluoride intake was not associated with cognitive, language, or motor outcomes collapsing across boys and girls. However, child sex modified the association between maternal fluoride intake and cognitive outcome (p interaction term = 0.06). A 0.5 mg/day increase in overall dietary fluoride intake was associated with a 3.50-point lower cognitive outcome in 24-month old boys (95 % CI: -6.58, -0.42); there was no statistical association with girls (ß = 0.07, 95 % CI: -2.37, 2.51), nor on the cognitive outcome at 12-months of age. Averaging across the 12- and 24-month cognitive outcomes using mixed-effects models revealed a similar association: a 0.5 mg/day increase in overall dietary fluoride intake was associated with a 3.46-point lower cognitive outcome in boys (95 % CI: -6.23, -0.70). These findings suggest that the development of nonverbal abilities in males may be more vulnerable to prenatal fluoride exposure than language or motor abilities, even at levels within the recommended intake range.
Assuntos
Fluoretos/efeitos adversos , Transtornos do Neurodesenvolvimento/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Adulto , Pré-Escolar , Dieta/efeitos adversos , Feminino , Fluoretos/administração & dosagem , Humanos , Lactente , Masculino , Testes Neuropsicológicos , Gravidez , Estudos ProspectivosRESUMO
SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.
Assuntos
Sirtuínas , Simportadores , Acetilação , Animais , Células HEK293 , Humanos , Rim , Camundongos , Sirtuínas/genética , Sirtuínas/metabolismo , Simportadores/genética , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.
Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
BACKGROUND: Sources of fluoride exposure for Mexicans include foods, beverages, fluoridated salt, and naturally fluoridated water. There are no available data describing fluoride content of foods and beverages consumed in Mexico. OBJECTIVE: To measure the content of fluoride in foods and beverages typically consumed and to compare their content to that of those from the United States and the United Kingdom. METHODS: Foods and beverages reported as part of the Mexican Health and Nutrition Survey (n = 182) were purchased in the largest supermarket chains and local markets in Mexico City. Samples were analyzed for fluoride, at least in duplicate, using a modification of the hexamethyldisiloxane microdiffusion method. Value contents were compared to those from the US Department of Agriculture and UK fluoride content tables. RESULTS: The food groups with the lowest and highest fluoride content were eggs (2.32 µg/100 g) and seafood (371 µg/100 g), respectively. When estimating the amount of fluoride per portion size, the lowest content corresponded to eggs and the highest to fast foods. Meats and sausages, cereals, fast food, sweets and cakes, fruits, dairy products, legumes, and seafood from Mexico presented higher fluoride contents than similar foods from the United States or the United Kingdom. Drinks and eggs from the United States exhibited the highest contents, while this was the case for pasta, soups, and vegetables from the United Kingdom. CONCLUSION: The majority of items analyzed contained higher fluoride contents than their US and UK counterparts. Data generated provide the first and largest table on fluoride content, which will be useful for future comparisons and estimations.
Assuntos
Bebidas/análise , Comércio/estatística & dados numéricos , Fluoretos/análise , Análise de Alimentos/estatística & dados numéricos , Humanos , México , Inquéritos Nutricionais , Reino Unido , Estados UnidosRESUMO
Fructose intake has been associated with non-alcoholic fatty liver disease (NAFLD). The objective of this study was to assess the consumption of dietary fructose according to: 1) classification of hepatic steatosis by two indexes and 2) diagnosis of NAFLD by MRI. We conducted a cross-sectional analysis among 100 young adults from Mexico City. The Hepatic Steatosis Index (HSI) and the Fatty Liver Index (FLI) were estimated using Body Mass Index (BMI), waist circumference, and fasting concentrations of glucose, triglycerides, and hepatic enzymes (ALT, AST, GGT). A semi-quantitative food frequency questionnaire was administered to obtain dietary sources of fructose. We estimated the concordance between the hepatic indices and NAFLD and the correlation between the index scores and the percentage of liver fat. Eighteen percent presented NAFLD; 44% and 46% were classified with hepatic steatosis according to HSI and FLI, respectively. We compared dietary intake of fructose by each outcome: HSI, FLI, and NAFLD. Sugar-sweetened beverages (SSB) and juices were consumed significantly more by those with steatosis by FLI and NAFLD suggesting that SSB intake is linked to metabolic alterations that predict the risk of having NAFLD at a young age.
Assuntos
Carboidratos da Dieta/administração & dosagem , Carboidratos da Dieta/efeitos adversos , Frutose/administração & dosagem , Frutose/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Feminino , Humanos , Masculino , México/epidemiologia , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Adulto JovemRESUMO
Childhood diet has been implicated in timing of sexual maturation. A key limitation of published studies is the focus on individual foods rather than patterns. We hypothesized that dietary patterns characterized by fruits and vegetables during early childhood (age 3 years) would be associated with delayed pubertal timing, whereas energy-dense and meat-based dietary patterns would relate to earlier puberty. The study population included 496 participants of a Mexico City birth cohort. The exposures of interest were dietary patterns derived from principal component analysis of dietary data collected via a semiquantitative food frequency questionnaire when the children were 3 years of age, and the outcomes were physician-assessed Tanner stages for pubic hair, breast (girls), genitalia, and testicular volume (boys) between 9 and 18 years, and initiation of menarche (girls). In regression analyses, we estimated adjusted hazard ratios and 95% confidence intervals for having reached Tanner stage ≥4 or initiation of menarche in girls and testicular volume ≥15â¯mL in boys. Among girls, those in the highest vs lowest tertile of vegetables and lean proteins pattern had a 35% (95% confidence interval 3%-67%) lower adjusted probability of having reached breast stage ≥4. Among boys, the processed meats and refined grain pattern score was associated with more advanced testicular development (adjusted hazard ratioâ¯=â¯3.58 [0.62-6.53]). Early childhood dietary patterns may play a role in the tempo of sexual maturation, which could ultimately carry implications for chronic disease susceptibility.
Assuntos
Dieta , Comportamento Alimentar , Maturidade Sexual , Adolescente , Mama , Criança , Pré-Escolar , Cidades , Inquéritos sobre Dietas , Carboidratos da Dieta , Gorduras na Dieta , Proteínas Alimentares , Grão Comestível , Feminino , Genitália , Humanos , Masculino , Carne , Menarca , México , Estudos Prospectivos , Testículo , VerdurasRESUMO
Geranium seemannii Peyr is a perennial plant endemic to central Mexico that has been widely used for its diuretic effect, but the responsible compound of this effect is unknown as well as the mechanism by which the diuretic effect is achieved. Geraniin is one of the compounds isolated from this kind of geranium. This study was designed to determinate whether geraniin possesses diuretic activity and to elucidate the mechanism of action. Geraniin was extracted and purified from Geranium seemannii Peyr. Male Wistar rats were divided into four groups: 1) Control, 2) 75 mg/kg of geraniin, 3) 20 mg/kg of furosemide, and 4) 10 mg/kg of hydrochlorothiazide. Each treatment was administered by gavage every 24 h for 7 days. The urinary excretion of electrolytes and the fractional excretion of sodium (FENa) were determined. To uncover the molecular target of geraniin, Xenopus laevis oocytes were microinjected with cRNAs encoding the Na+-Cl- cotransporter (NCC) and the Na+-K+-2Cl- cotransporter NKCC2 to functionally express these cotransporters. Geraniin significantly increased diuresis, natriuresis, and calciuresis to a similar extent as was observed in the furosemide-treated rats. Consistent with the furosemide-like effect, in X. laevis oocytes, geraniin significantly reduced the activity of NKCC2, with no effect on NCC activity. In contrast to furosemide, the effect of geraniin on NKCC2 was irreversible, apparently due to its inhibitory effect on heat shock protein 90. Our observations suggest that geraniin could have a potential role in the treatment of hypertension or edematous states.
Assuntos
Diurese/efeitos dos fármacos , Diuréticos/farmacologia , Glucosídeos/farmacologia , Taninos Hidrolisáveis/farmacologia , Rim/efeitos dos fármacos , Membro 1 da Família 12 de Carreador de Soluto/antagonistas & inibidores , Animais , Biomarcadores/urina , Cálcio/urina , Relação Dose-Resposta a Droga , Furosemida/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Rim/metabolismo , Masculino , Natriurese/efeitos dos fármacos , Ratos Wistar , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Fatores de Tempo , Xenopus laevisRESUMO
The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCß is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCß from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCß is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCß is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCß exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Enguias/metabolismo , Proteínas de Peixes/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Simportadores de Cloreto de Sódio/metabolismo , Animais , Enguias/genética , Proteínas de Peixes/genética , Humanos , Oócitos , Ratos , Simportadores de Cloreto de Sódio/genética , Xenopus laevisRESUMO
BACKGROUND: Mercury is a global contaminant of concern though little is known about exposures in México. OBJECTIVES: To characterize mercury levels in pregnant women, children, and commonly consumed seafood samples. METHODS: Use resources of the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) birth cohorts to measure total mercury levels in archived samples from 348 pregnant women (blood from three trimesters and cord blood), 825 offspring (blood, hair, and urine) and their mothers (hair), and 91 seafood and canned tuna samples from Mexico City. RESULTS: Maternal blood mercury levels correlated across three trimesters and averaged 3.4 µg/L. Cord blood mercury averaged 4.7 µg/L and correlated with maternal blood from trimester 3 (but not trimesters 1 and 2). In children, blood, hair and urine mercury levels correlated and averaged 1.8 µg/L, 0.6 µg/g, and 0.9 µg/L, respectively. Hair mercury was 0.5 µg/g in mothers and correlated with child's hair. Mean consumption of canned tuna, fresh fish, canned sardine, and shellfish was 3.1, 2.2, 0.5, and 1.0 times per month respectively in pregnant women. Mean mercury content in 7 of 23 seafood species and 5 of 9 canned tuna brands purchased exceeded the U.S. EPA guidance value of 0.3 µg/g. CONCLUSIONS: Mercury exposures in pregnant women and children from Mexico City, via biomarker studies, are generally 3-5 times greater than values reported in population surveys from the U.S., Canada, and elsewhere. In particular, mercury levels in 29-39% of the maternal participants exceeded the biomonitoring guideline associated with the U.S. EPA reference dose for mercury.
Assuntos
Cidades , Exposição Ambiental/análise , Poluentes Ambientais/análise , Contaminação de Alimentos/análise , Mercúrio/análise , Alimentos Marinhos/análise , Animais , Criança , Estudos de Coortes , Monitoramento Ambiental/estatística & dados numéricos , Feminino , Sangue Fetal/química , Cabelo/química , Humanos , Mercúrio/sangue , Mercúrio/urina , México/epidemiologia , Gravidez , Refratometria , Atum/metabolismoRESUMO
The K+:Cl- cotransporters or KCCs are membrane proteins that move K+ and Cl- ions across the membrane without changing the transmembrane potential. KCCs belong to the SLC12 (Solute Carrier Family 12) family of electroneutral cation-chloride cotransporters (CCC), and they are secondary active ion transporters because use the established gradients from the primary active transporter through the Na+/K+- ATPase. Although there are nine members identify in this family, up today only seven genes had been characterized. Among them are two loop diuretics-sensitive Na+:K+:2Clcotransporters (NKCC1/NKCC2), the thiazide-sensitive Na+:Cl- cotransporter (NCC), and finally the K+:Cl- cotransporters (KCC), encoded for at least four homologous genes (KCC1-KCC4), and from which there are many isoforms due to alternative splicing. KCC1 is a ubiquitous isoform, KCC3 and KCC4 isoforms are widely expressed, particularly in epithelial cells, while KCC2 is restricted to the central nervous system (CNS). All these cotransporters play an essential role in many physiological processes such as cell volume regulation, transepithelial salt transport and regulation of the intraneuronal chloride concentration. This review has the purpose to show briefly the molecular characteristics as well as the physiological importance and roles of the KCCs in several pathologies.
Assuntos
Simportadores de Cloreto de Sódio-Potássio/fisiologia , Acidose Tubular Renal/genética , Acidose Tubular Renal/fisiopatologia , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/fisiopatologia , Epilepsia/genética , Epilepsia/fisiopatologia , Hemoglobinopatias/genética , Hemoglobinopatias/fisiopatologia , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Neoplasias/genética , Neoplasias/fisiopatologia , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/fisiopatologia , Simportadores de Cloreto de Sódio-Potássio/genéticaRESUMO
K(+)-Cl(-) cotransporter (KCC) isoforms 3 (KCC3) and 4 (KCC4) are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low-salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low- or high-K(+) diets. Both KCC3 mRNA and protein expression were increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not with a low-salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low-sodium diet in the whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low-salt diet was also observed in WNK4 knockout mice, suggesting that upregulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low- or high-K(+) diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the thick ascending loop of Henle's loop and acid secretion of the collecting duct.
Assuntos
Rim/metabolismo , Simportadores/metabolismo , Acidose/induzido quimicamente , Acidose/metabolismo , Cloreto de Amônio , Animais , Transporte Biológico , Glicemia/metabolismo , Dieta Hipossódica , Modelos Animais de Doenças , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potássio na Dieta/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Cloreto de Sódio na Dieta/metabolismo , Estreptozocina , Simportadores/genéticaRESUMO
The K(+):Cl(-) cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.
Assuntos
Pressão Osmótica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Oócitos/citologia , Oócitos/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Serina/genética , Serina/metabolismo , Simportadores/genética , Xenopus laevisRESUMO
With-no-lysine kinase 3 (WNK3) is a member of a subfamily of serine/threonine kinases that modulate the activity of the electroneutral cation-coupled chloride cotransporters. WNK3 activates NKCC1/2 and NCC and inhibits the KCCs. Four splice variants are generated from the WNK3 gene. Our previous studies focused on the WNK3-18a variant. However, it has been suggested that other variants could have different effects on the cotransporters. Thus, the present study was designed to define the effects of all WNK3 variants on members of the SLC12 family. By RT-PCR from a fetal brain library, exons 18b and 22 were separately amplified and subcloned into the original WNK3-18a or catalytically inactive WNK3-D294A to obtain all four potential combinations with and without catalytic activity (18a, 18a+22, 18b, and 18b+22). The basal activity of the cotransporters and the effects of WNK3 isoforms were assessed in Xenopus laevis oocytes coinjected with each of the WNK3 variant cRNAs. In isotonic conditions, the basal activity of NCC and NKCC1/2 were increased by coinjection with any of the WNK3. The positive effects occurred even in hypotonic conditions, in which the basal activity of NKCC1 is completely prevented. Consistent with these observations, when expressed in hypotonicity, all KCCs were active, but in the presence of any of the WNK3 variants, KCC activity was completely reduced. That is, NKCC1/2 and NCC were inhibited, even in hypertonicity, while KCCs were activated, even in isotonic conditions. We conclude that the effects of all WNK3 variants toward SLC12 proteins are similar.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Simportadores/metabolismo , Substituição de Aminoácidos/fisiologia , Animais , Biocatálise , Domínio Catalítico/genética , Humanos , Oócitos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Complementar/administração & dosagem , RNA Complementar/genética , Receptores de Droga/genética , Receptores de Droga/metabolismo , Rubídio/metabolismo , Sódio/metabolismo , Simportadores de Cloreto de Sódio/genética , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto , Membro 2 da Família 12 de Carreador de Soluto , Membro 3 da Família 12 de Carreador de Soluto , Simportadores/genética , Xenopus laevis , Cotransportadores de K e Cl-RESUMO
The authors tested the hypotheses that maternal bone lead burden is associated with increasing maternal whole blood and plasma lead levels over the course of pregnancy and that this association is modified by rates of maternal bone resorption. A total of 193 Mexican women were evaluated (1997-1999) in the first, second, and third trimesters of pregnancy. Whole blood lead and plasma lead levels were measured in each trimester. Urine was analyzed for cross-linked N-telopeptides (NTx) of type I collagen, a biomarker of bone resorption. Patella and tibia lead levels were measured at 4 weeks postpartum. The relation between whole blood, plasma, and bone lead and NTx was assessed using mixed models. Plasma lead concentrations followed a U-shape, while NTx levels increased significantly during pregnancy. In a multivariate model, the authors observed a significant and positive interaction between NTx and bone lead when plasma lead was used as the outcome variable. Dietary calcium intake was inversely associated with plasma lead. Results for whole blood lead were similar but less pronounced. These results confirm previous evidence that bone resorption increases during pregnancy, with a consequential significant release of lead from bone, constituting an endogenous source of prenatal exposure. They also provide a rationale for testing strategies (e.g., nutritional supplementation with calcium) aimed at decreasing prenatal lead exposure.