RESUMO
BACKGROUND: Hypomagnesaemia with secondary hypocal-caemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase transient receptor potential melastatin type 6. Patients have very low serum magnesium (Mg2+) levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without a diagnosis. METHODS: In this study, two families with an HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7-identified variants on Mg2+ transport was examined. RESULTS: For the first time, variants in TRPM7 were identified in two families as a potential cause for hereditary HSH. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcaemia. In the first family, a splice site variant caused the incorporation of intron 1 sequences into the TRPM7 messenger RNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit decreased cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. CONCLUSIONS: We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesaemia and secondary hypocalcaemia. Screening of unresolved patients with hypocalcaemia and secondary hypocalcaemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.
Assuntos
Hipocalcemia , Canais de Cátion TRPM , Humanos , Magnésio , Canais de Cátion TRPM/metabolismo , Cãibra Muscular/complicações , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND: Vascular calcification is a key process involved in cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Magnesium supplementation may counteract vascular calcification. In this study we aimed to determine whether increased dietary magnesium intake inhibits vascular calcification in CKD in vivo and explore the mechanisms underlying these effects. METHODS: Sprague Dawley rats were partially nephrectomized and fed a diet with high phosphate and either high or normal magnesium content for 16 weeks. The primary outcome was the tissue calcium content of the aorta in the high versus normal dietary magnesium group. In addition, we analysed plasma mineral concentrations, aortic vascular calcification identified with von Kossa staining, calcium apposition time and aortic expression of genes related to vascular calcification. RESULTS: The number of animals in the highest tissue calcium content tertile was significantly lower in the abdominal aorta [1 (10%) versus 6 (55%); P = .03] in the high versus normal dietary magnesium group, but did not differ in the aortic arch and thoracic aorta. Von Kossa staining and calcium apposition time corresponded to these results. The median tissue calcium content was not significantly different between the groups. Serum phosphate concentrations and expression of osteogenic markers in the aorta did not differ between the groups. CONCLUSIONS: This study demonstrates that increased dietary magnesium inhibits abdominal vascular calcification in an experimental animal model of CKD in vivo. These are promising results for CKD patients and further study is needed to identify the mechanisms involved and to determine the clinical relevance in patients.
Assuntos
Arteriosclerose , Insuficiência Renal Crônica , Calcificação Vascular , Animais , Aorta Abdominal , Cálcio , Suplementos Nutricionais , Modelos Animais de Doenças , Humanos , Magnésio/farmacologia , Magnésio/uso terapêutico , Modelos Animais , Fosfatos , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/tratamento farmacológico , Calcificação Vascular/etiologia , Calcificação Vascular/prevenção & controleRESUMO
Dietary fibers have been shown to increase the intestinal absorption of calcium (Ca2+) and magnesium (Mg2+). However, the mechanisms that explain the enhanced electrolyte absorption remain unknown. Therefore, this study aims to investigate the short-term and long-term effects of 5% (w/w) sodium butyrate (Na-butyrate), an important end-metabolite of bacterial fermentation of dietary fibers, on Ca2+ and Mg2+ homeostasis in mice. Serum Ca2+ levels were only significantly increased in mice treated with Na-butyrate for 1 day. This was associated with a twofold increase in the mRNA expression levels of Trpv6 in the proximal and distal colon. Contrary, Na-butyrate did not affect serum Mg2+ concentrations at either of the intervention periods. However, we observed a reduction in urinary Mg2+ excretion, although not significantly, after 1 day of treatment. A significant reduction of 2.5-fold in urinary Mg2+ excretion was observed after 14 days of treatment. Indeed, 14-day Na-butyrate supplementation increased colonic Trpm7 expression by 1.2-fold compared to control mice. In conclusion, short-term Na-butyrate supplementation increases serum Ca2+ levels in mice. This was associated with increased mRNA expression levels of Trpv6 in the colon, suggesting that Na-butyrate regulates the expression of genes involved in active intestinal Ca2+ absorption.
Assuntos
Sódio na Dieta , Canais de Cátion TRPM , Animais , Ácido Butírico/farmacologia , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Colo , Fibras na Dieta/metabolismo , Fibras na Dieta/farmacologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sódio/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Sódio na Dieta/metabolismo , Sódio na Dieta/farmacologia , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
Klotho knock-out mice are an important model for vascular calcification, which is associated with chronic kidney disease. In chronic kidney disease, serum magnesium inversely correlates with vascular calcification. Here we determine the effects of serum magnesium on aortic calcification in Klotho knock-out mice treated with a minimal or a high magnesium diet from birth. After eight weeks, serum biochemistry and aorta and bone tissues were studied. Protective effects of magnesium were characterized by RNA-sequencing of the aorta and micro-CT analysis was performed to study bone integrity. A high magnesium diet prevented vascular calcification and aortic gene expression of Runx2 and matrix Gla protein found in such mice on the minimal magnesium diet. Differential expression of inflammation and extracellular matrix remodeling genes accompanied the beneficial effects of magnesium on calcification. High dietary magnesium did not affect serum parathyroid hormone, 1,25-dihydroxyvitamin D3 or calcium. High magnesium intake prevented vascular calcification despite increased fibroblast growth factor-23 and phosphate concentration in the knock-out mice. Compared to mice on the minimal magnesium diet, the high magnesium diet reduced femoral bone mineral density by 20% and caused excessive osteoid formation indicating osteomalacia. Osteoclast activity was unaffected by the high magnesium diet. In Saos-2 osteoblasts, magnesium supplementation reduced mineralization independent of osteoblast function. Thus, high dietary magnesium prevents calcification in Klotho knock-out mice. These effects are potentially mediated by reduction of inflammatory and extracellular matrix remodeling pathways within the aorta. Hence magnesium treatment may be promising to prevent vascular calcification, but the risk for osteomalacia should be considered.
Assuntos
Glucuronidase/deficiência , Magnésio/farmacologia , Insuficiência Renal Crônica , Calcificação Vascular , Animais , Glucuronidase/genética , Proteínas Klotho , Camundongos , Camundongos Knockout , Hormônio Paratireóideo , Fosfatos , Calcificação Vascular/genética , Calcificação Vascular/prevenção & controleRESUMO
BACKGROUND: Phosphate (Pi) toxicity is a strong determinant of vascular calcification development in chronic kidney disease (CKD). Magnesium (Mg2+) may improve cardiovascular risk via vascular calcification. The mechanism by which Mg2+ counteracts vascular calcification remains incompletely described. Here we investigated the effects of Mg2+ on Pi and secondary crystalline calciprotein particles (CPP2)-induced calcification and crystal maturation. METHODS: Vascular smooth muscle cells (VSMCs) were treated with high Pi or CPP2 and supplemented with Mg2+ to study cellular calcification. The effect of Mg2+ on CPP maturation, morphology and composition was studied by medium absorbance, electron microscopy and energy dispersive spectroscopy. To translate our findings to CKD patients, the effects of Mg2+ on calcification propensity (T50) were measured in sera from CKD patients and healthy controls. RESULTS: Mg2+ supplementation prevented Pi-induced calcification in VSMCs. Mg2+ dose-dependently delayed the maturation of primary CPP1 to CPP2 in vitro. Mg2+ did not prevent calcification and associated gene and protein expression when added to already formed CPP2. Confirmatory experiments in human serum demonstrated that the addition of 0.2 mmol/L Mg2+ increased T50 from healthy controls by 51 ± 15 min (P < 0.05) and CKD patients by 44 ± 13 min (P < 0.05). Each further 0.2 mmol/L addition of Mg2+ led to further increases in both groups. CONCLUSIONS: Our results demonstrate that crystalline CPP2 mediates Pi-induced calcification in VSMCs. In vitro, Mg2+ delays crystalline CPP2 formation and thereby prevents Pi-induced calcification.
Assuntos
Fosfatos de Cálcio/metabolismo , Suplementos Nutricionais , Magnésio/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Calcificação Vascular/prevenção & controle , alfa-2-Glicoproteína-HS/metabolismo , Células Cultivadas , Humanos , Miócitos de Músculo Liso/metabolismo , Calcificação Vascular/metabolismoRESUMO
Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1α-hydroxylase activity. Vitamin D treatment to reduce proteinuria remains controversial, although there is an inverse correlation between vitamin D levels and proteinuria. Herein, we showed that 1,25-vitamin D3-deficient 25-hydroxy-vitamin-D3-1α-hydroxylase knockout mice and 1,25-vitamin D3-deficient rats develop podocyte injury and renal dysfunction. Glomerular injury was characterized by proteinuria and partial podocyte foot process effacement. Expression of nephrin, podocin, desmin, and transient receptor potential channel C6 in the podocyte was significantly altered in 1,25-vitamin D3-deficient animals. Supplementation with 1,25-vitamin D3 or 1,25-vitamin D2 prevented podocyte effacement or reversed glomerular and tubulointerstitial damage in 1,25-vitamin D3-deficient animals, thereby preserving and restoring renal function, respectively. The effect of 1,25-vitamin D3 deficiency and 1,25-vitamin D3 and 1,25-vitamin D2 repletion on proteinuria could not be explained by hypocalcemia, changes in parathyroid hormone, or fibroblast growth factor 23. This study demonstrates that 1,25-vitamin D3 deficiency directly leads to renal injury in rodents. Translated to human subjects, this would underline the need for early vitamin D supplementation in patients with glomerular disease and chronic renal insufficiency, which might inhibit or potentially reverse renal injury.
Assuntos
Albuminúria/etiologia , Albuminúria/metabolismo , Colecalciferol/deficiência , Nefropatias/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Glomérulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônio Paratireóideo/metabolismo , Ratos , Ratos WistarRESUMO
Over the past decades, hypomagnesemia (serum Mg(2+) <0.7 mmol/L) has been strongly associated with type 2 diabetes mellitus (T2DM). Patients with hypomagnesemia show a more rapid disease progression and have an increased risk for diabetes complications. Clinical studies demonstrate that T2DM patients with hypomagnesemia have reduced pancreatic ß-cell activity and are more insulin resistant. Moreover, dietary Mg(2+) supplementation for patients with T2DM improves glucose metabolism and insulin sensitivity. Intracellular Mg(2+) regulates glucokinase, KATP channels, and L-type Ca(2+) channels in pancreatic ß-cells, preceding insulin secretion. Moreover, insulin receptor autophosphorylation is dependent on intracellular Mg(2+) concentrations, making Mg(2+) a direct factor in the development of insulin resistance. Conversely, insulin is an important regulator of Mg(2+) homeostasis. In the kidney, insulin activates the renal Mg(2+) channel transient receptor potential melastatin type 6 that determines the final urinary Mg(2+) excretion. Consequently, patients with T2DM and hypomagnesemia enter a vicious circle in which hypomagnesemia causes insulin resistance and insulin resistance reduces serum Mg(2+) concentrations. This Perspective provides a systematic overview of the molecular mechanisms underlying the effects of Mg(2+) on insulin secretion and insulin signaling. In addition to providing a review of current knowledge, we provide novel directions for future research and identify previously neglected contributors to hypomagnesemia in T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Deficiência de Magnésio/metabolismo , Magnésio/metabolismo , Desequilíbrio Hidroeletrolítico/metabolismo , Glicemia/metabolismo , Canais de Cálcio Tipo L/metabolismo , Diabetes Mellitus Tipo 2/complicações , Suplementos Nutricionais , Progressão da Doença , Glucoquinase/metabolismo , Glicogênio/biossíntese , Glicólise , Humanos , Inflamação , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Canais KATP/metabolismo , Fígado/metabolismo , Magnésio/uso terapêutico , Deficiência de Magnésio/tratamento farmacológico , Obesidade/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Desequilíbrio Hidroeletrolítico/tratamento farmacológicoRESUMO
BACKGROUND: Proton-pump inhibitor-induced hypomagnesemia (PPIH) is the most recognized side effect of proton-pump inhibitors (PPIs). Additionally, PPIH is associated with hypocalcemia and hypokalemia. It is hypothesized that PPIs reduce epithelial proton secretion and thereby increase the pH in the colon, which may explain the reduced absorption of and Mg2+ and Ca2+. Fermentation of dietary oligofructose-enriched inulin fibers by the microflora leads to acidification of the intestinal lumen and by this enhances mineral uptake. This study aimed, therefore, to improve mineral absorption by application of dietary inulin to counteract PPIH. METHODS: Here, C57BL/J6 mice were supplemented with omeprazole and/or inulin. Subsequently, Mg2+ and Ca2+ homeostasis was assessed by means of serum, urine and fecal electrolyte measurements. Moreover, the mRNA levels of magnesiotropic and calciotropic genes were examined in the large intestine and kidney by real-time PCR. RESULTS: Treatment with omeprazole significantly reduced serum Mg2+ and Ca2+ levels. However, concomitant addition of dietary inulin fibers normalized serum Ca2+ but not serum Mg2+ concentrations. Inulin abolished enhanced expression of Trpv6 and S100g in the colon by omeprazole. Additionally, intestinal and renal mRNA levels of the Trpm6 gene were reduced after inulin intake. CONCLUSIONS: This study suggests that dietary inulin counteracts reduced intestinal Ca2+ absorption upon PPI treatment. In contrast, inulin did not increase intestinal absorption of Mg2+ sufficiently to recover serum Mg2+. The clinical potential of dietary inulin treatment should be the subject of future studies.
Assuntos
Fibras na Dieta/administração & dosagem , Hipocalcemia/prevenção & controle , Inulina/administração & dosagem , Omeprazol/efeitos adversos , Inibidores da Bomba de Prótons/efeitos adversos , Animais , Cálcio/sangue , Avaliação Pré-Clínica de Medicamentos , Ácidos Graxos/biossíntese , Hipocalcemia/sangue , Hipocalcemia/induzido quimicamente , Absorção Intestinal/efeitos dos fármacos , Magnésio/sangue , Masculino , Camundongos Endogâmicos C57BL , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
The glomerular filtration barrier consists of podocytes, the glomerular basement membrane, and endothelial cells covered with a glycocalyx. Heparan sulphate (HS) in the glomerular filtration barrier is reduced in patients with proteinuria, which is associated with increased expression of the HS-degrading enzyme heparanase. Previously, we showed that heparanase is essential for the development of proteinuria in experimental diabetic nephropathy. Vitamin D supplementation reduces podocyte loss and proteinuria in vitro and in vivo. Therefore, we hypothesize that vitamin D reduces proteinuria by reducing glomerular heparanase. Adriamycin-exposed rats developed proteinuria and showed increased heparanase expression, which was reduced by 1,25-dihydroxyvitamin D3 (1,25-D3) treatment. In vitro, adriamycin increased heparanase mRNA in the podocyte, which could be corrected by 1,25-D3 treatment. In addition, 1,25-D3 treatment reduced transendothelial albumin passage after adriamycin stimulation. In line with these results, we showed direct binding of the vitamin D receptor to the heparanase promoter, and 1,25-D3 dose-dependently reduced heparanase promoter activity. Finally, 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice developed proteinuria and showed increased heparanase, which was normalized by 1,25-D3 treatment. Our data suggest that the protective effect of vitamin D on the development of proteinuria is mediated by inhibiting heparanase expression in the podocyte.
Assuntos
Calcitriol/farmacologia , Glucuronidase/metabolismo , Podócitos/enzimologia , Proteinúria/metabolismo , Animais , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Heparitina Sulfato/metabolismo , Camundongos , Camundongos Knockout , Podócitos/efeitos dos fármacos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Magnesium (Mg(2+)) is an essential ion to the human body, playing an instrumental role in supporting and sustaining health and life. As the second most abundant intracellular cation after potassium, it is involved in over 600 enzymatic reactions including energy metabolism and protein synthesis. Although Mg(2+) availability has been proven to be disturbed during several clinical situations, serum Mg(2+) values are not generally determined in patients. This review aims to provide an overview of the function of Mg(2+) in human health and disease. In short, Mg(2+) plays an important physiological role particularly in the brain, heart, and skeletal muscles. Moreover, Mg(2+) supplementation has been shown to be beneficial in treatment of, among others, preeclampsia, migraine, depression, coronary artery disease, and asthma. Over the last decade, several hereditary forms of hypomagnesemia have been deciphered, including mutations in transient receptor potential melastatin type 6 (TRPM6), claudin 16, and cyclin M2 (CNNM2). Recently, mutations in Mg(2+) transporter 1 (MagT1) were linked to T-cell deficiency underlining the important role of Mg(2+) in cell viability. Moreover, hypomagnesemia can be the consequence of the use of certain types of drugs, such as diuretics, epidermal growth factor receptor inhibitors, calcineurin inhibitors, and proton pump inhibitors. This review provides an extensive and comprehensive overview of Mg(2+) research over the last few decades, focusing on the regulation of Mg(2+) homeostasis in the intestine, kidney, and bone and disturbances which may result in hypomagnesemia.
Assuntos
Deficiência de Magnésio/prevenção & controle , Magnésio/administração & dosagem , Magnésio/metabolismo , Osso e Ossos/metabolismo , Encéfalo/metabolismo , Sistema Cardiovascular/metabolismo , Comunicação Celular , Proliferação de Células , Sistema Digestório/metabolismo , Humanos , Rim/metabolismo , Pulmão/metabolismo , Deficiência de Magnésio/tratamento farmacológico , Músculos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de SinaisRESUMO
The transient receptor potential cation channel C6 (TRPC6) is a slit diaphragm protein expressed by podocytes. TRPC6 gain-of-function mutations cause autosomal dominant focal segmental glomerulosclerosis. In acquired proteinuric renal disease, glomerular TRPC6 expression is increased. We previously demonstrated that acquired increased TRPC6 expression is ameliorated by antiproteinuric angiotensin receptor blockers and angiotensin-converting enzyme inhibitors. Vitamin D also has an antiproteinuric effect. We hypothesized that vitamin D reduces proteinuria by affecting TRPC6 expression in podocytes. Adriamycin-induced nephropathy increased TRPC6 mRNA and protein expression and induced proteinuria in rats. Treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) normalized TRPC6 expression and reduced proteinuria. In vitro, podocyte injury induced by adriamycin exposure in cultured podocytes increased TRPC6 expression. Treatment of injured podocytes with 1,25-D3 dose dependently reduced adriamycin-induced TRPC6 expression. Chromatin immunoprecipitation analysis demonstrated that the vitamin D receptor directly binds to the TRPC6 promoter. Moreover, 1,25-D3 reduced TRPC6 promoter activity in a luciferase reporter assay. In 1,25-D3-deficient 25-hydroxy-1α-hydroxylase knockout mice, TRPC6 expression was increased, accompanied by podocyte foot process effacement and proteinuria. 1,25-D3 supplementation normalized TRPC6 expression, podocyte morphology, and proteinuria in these mice. These results demonstrate that vitamin D down-regulates the enhanced TRPC6 expression in in vivo and in vitro podocyte injury, possibly through a direct effect on TRPC6 promoter activity. This TRPC6 down-regulation could contribute to the antiproteinuric effect of vitamin D.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Nefropatias/patologia , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/patologia , Canais de Cátion TRPC/genética , Vitamina D/farmacologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Imunoprecipitação da Cromatina , Regulação para Baixo/genética , Glomerulosclerose Segmentar e Focal/complicações , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Nefropatias/complicações , Nefropatias/genética , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Camundongos , Podócitos/efeitos dos fármacos , Podócitos/ultraestrutura , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteinúria/complicações , Proteinúria/genética , Ratos , Ratos Wistar , Receptores de Calcitriol/metabolismo , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6 , Vitamina D/análogos & derivadosRESUMO
BACKGROUND: The kidney is one of the affected organs involved in the clinical symptoms of parathyroid hormone (PTH)-related disorders, like primary hyperparathyroidism and familial hypocalciuric hypercalcemia. The molecular mechanism(s) underlying alterations in renal Ca(2+) handling in these disorders is poorly understood. METHODS: Parathyroidectomized and PTH-supplemented rats and mice infused with the calcimimetic compound NPS R-467 were used to study the in vivo effect of PTH on the expression of renal transcellular Ca(2+) transport proteins, including the epithelial Ca(2+) channel transient receptor potential, vanilloid, member 5 (TRPV5), calbindins, and the Na(+)/Ca(2+)-exchanger (NCX1). In addition, the effect of PTH on transepithelial Ca(2+) transport in rabbit connecting tubule/cortical collecting duct (CNT/CCD) primary cultures was determined. RESULTS: Decreased PTH levels in parathyroidectomized rats or NPS R-467-infused mice, resulted in reduced expression of these proteins, which is consistent with diminished Ca(2+) reabsorption, causing the development of the observed hypocalcemia. PTH supplementation of parathyroidectomized rats restored the expression of the renal Ca(2+) transport machinery and serum Ca(2+) levels, independent of serum 1,25-dihydroxyvitamin D(3) levels and renal vitamin D or Ca(2+)-sensing receptor mRNA abundance. Inhibition of the PTH-stimulated transepithelial Ca(2+) transport by the TRPV5-specific inhibitor ruthenium red reduced the PTH-stimulated expression of calbindin-D(28K) and NCX1 in rabbit CNT/CCD primary cultures. CONCLUSION: PTH stimulates renal Ca(2+) reabsorption through the coordinated expression of renal transcellular Ca(2+) transport proteins. Moreover, the PTH-induced stimulation is enhanced by the magnitude of the Ca(2+) influx through the gatekeeper TRPV5, which in turn facilitates the expression of the downstream Ca(2+) transport proteins. Therefore, the renal transcellular Ca(2+) transport proteins, including TRPV5, could contribute to the pathogenesis of PTH-related disorders.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Rim/fisiologia , Hormônio Paratireóideo/fisiologia , Canais de Cátion TRPV/metabolismo , Compostos de Anilina/farmacologia , Animais , Calbindinas , Cálcio/agonistas , Canais de Cálcio/genética , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Hipocalcemia/tratamento farmacológico , Hipocalcemia/metabolismo , Hipocalcemia/fisiopatologia , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/farmacologia , Paratireoidectomia , Coelhos , Ratos , Ratos Endogâmicos , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
The epithelial Ca(2+) channels TRPV5 and TRPV6 mediate the Ca(2+) influx in 1,25-dihydroxyvitamin D(3)-responsive epithelia and are therefore essential in the maintenance of the body Ca(2+) balance. These Ca(2+) channels assemble in (hetero)tetrameric channel complexes with different functional characteristics regarding Ca(2+)-dependent inactivation, ion selectivity, and pharmacological block. Glutathione S-transferase pull-downs and co-immunoprecipitations demonstrated an essential role of the intracellular N- and C-tails in TRPV5 channel assembly by physical interactions between N-N tails, C-C tails, and N-C-tails. Patch clamp analysis in human embryonic kidney (HEK293) cells and (45)Ca(2+) uptake experiments in Xenopus laevis oocytes co-expressing TRPV5 wild-type and truncated proteins indicated that TRPV5 Delta N (deleted N-tail) and TRPV5 Delta C (deleted C-tail) decreased channel activity of wild-type TRPV5 in a dominant-negative manner, whereas TRPV5 Delta N Delta C (deleted N-tail/C-tail) did not affect TRPV5 activity. Oocytes co-expressing wild-type TRPV5 and TRPV5 Delta N or TRPV5 Delta C showed virtually no wild-type TRPV5 expression on the plasma membrane, whereas co-expression of wild-type TRPV5 and TRPV5 Delta N Delta C displayed normal channel surface expression. This indicates that TRPV5 trafficking toward the plasma membrane was disturbed by assembly with TRPV5 Delta N or TRPV5 Delta C but not with TRPV5 Delta N Delta C. TRPV5 channel assembly signals were refined between amino acid positions 64-77 and 596-601 in the N-tail and C-tail, respectively. Pull-down assays and co-immunoprecipitations demonstrated that N- or C-tail mutants lacking these critical assembly domains were unable to interact with tails of TRPV5. In conclusion, two domains in the N-tail (residues 64-77) and C-tail (residues 596-601) of TRPV5 are important for channel subunit assembly, subsequent trafficking of the TRPV5 channel complex to the plasma membrane, and channel activity.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Linhagem Celular , Membrana Celular , Eletrofisiologia , Embrião de Mamíferos , Embrião não Mamífero , Escherichia coli/genética , Expressão Gênica , Glutationa Transferase/genética , Humanos , Técnicas de Imunoadsorção , Rim , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , RNA Complementar/genética , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Relação Estrutura-Atividade , Canais de Cátion TRPV , Transfecção , Xenopus laevisRESUMO
The epithelial Ca(2+) channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca(2+) entry pathway in the process of active Ca(2+) reabsorption. Ca(2+) influx through TRPV5 is tightly controlled by modulators of Ca(2+) homeostasis, including 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). However, little is known about intracellular proteins that interact with TRPV5 and directly regulate the activation of this channel. By the use of cDNA microarrays, the present study identified 80K-H as the first protein involved in the Ca(2+)-dependent control of the epithelial Ca(2+) channel TRPV5. 80K-H was initially identified as a protein kinase C substrate, but its biological function remains to be established. We demonstrated a specific interaction between 80K-H and TRPV5, co-localization of both proteins in the kidney, and similar transcriptional regulation by 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). Furthermore, 80K-H directly bound Ca(2+), and inactivation of its two EF-hand structures totally abolished Ca(2+) binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EF-hand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EF-hand pair reduced the TRPV5-mediated Ca(2+) current and increased the TRPV5 sensitivity to intracellular Ca(2+), accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca(2+) sensor controlling TRPV5 channel activity.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Fosfoproteínas/fisiologia , Absorção , Animais , Biotinilação , Calcitriol/metabolismo , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Eletrofisiologia , Glucosidases , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação , Substrato Quinase C Rico em Alanina Miristoilada , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/metabolismo , Testes de Precipitina , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV , Distribuição Tecidual , XenopusRESUMO
BACKGROUND: Pseudovitamin D deficiency rickets (PDDR) is an autosomal disease, characterized by undetectable levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), rickets and secondary hyperparathyroidism. Mice in which the 25-hydroxyvitamin D3-1 alpha-hydroxylase (1 alpha-OHase) gene was inactivated, presented the same clinical phenotype as patients with PDDR. METHODS: cDNA Microarray technology was used on kidneys of 1 alpha-OHase knockout mice to study the expression profile of renal genes in this Ca2+-related disorder. Genome wide molecular events that occur during the rescue of these mice by high dietary Ca2+ intake were studied by the use of 15K cDNA microarray chips. RESULTS: 1 alpha-OHase knockout mice fed a normal Ca2+ diet developed severe hypocalcemia, rickets and died with an average life span of 12 +/- 2 weeks. Intriguingly, 1 alpha-OHase-/- mice supplemented with an enriched Ca2+ diet were normocalcemic and not significantly different from wild-type mice. Inactivation of the 1 alpha-OHase gene resulted in a significant regulation of +/- 1000 genes, whereas dietary Ca2+ supplementation of the 1 alpha-OHase-/- mice revealed +/- 2000 controlled genes. Interestingly, 557 transcripts were regulated in both situations implicating the involvement in the dietary Ca2+-mediated rescue mechanism of the 1 alpha-OHase-/- mice. Conspicuous regulated genes encoded for signaling molecules like the PDZ-domain containing protein channel interacting protein, FK binding protein type 4, kinases, and importantly Ca2+ transporting proteins including the Na+-Ca2+ exchanger, calbindin-D28K and the Ca2+ sensor calmodulin. CONCLUSION: Dietary Ca2+ intake normalized disturbances in the Ca2+ homeostasis due to vitamin D deficiency that were accompanied by the regulation of a subset of renal genes, including well-known renal Ca2+ transport protein genes, but also genes not previously identified as playing a role in renal Ca2+ handling.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Cálcio da Dieta/farmacologia , Rim/fisiologia , Raquitismo/fisiopatologia , Animais , Regulação Enzimológica da Expressão Gênica , Homeostase/fisiologia , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Raquitismo/mortalidadeRESUMO
The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17beta-estradiol (17beta-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17beta-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17beta-E2. Treatment with 17beta-E2 upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 +/- 0.10 to 2.03 +/- 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1alpha-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.