RESUMO
Mutations in Cl-/H+ antiporter ClC-5 cause Dent's disease type 1 (DD1), a rare tubulopathy that progresses to renal fibrosis and kidney failure. Here, we have used DD1 human cellular models and renal tissue from DD1 mice to unravel the role of ClC-5 in renal fibrosis. Our results in cell systems have shown that ClC-5 deletion causes an increase in collagen I (Col I) and IV (Col IV) intracellular levels by promoting their transcription through the ß-catenin pathway and impairing their lysosomal-mediated degradation. Increased production of Col I/IV in ClC-5-depleted cells ends up in higher release to the extracellular medium, which may lead to renal fibrosis. Furthermore, our data have revealed that 3-mo-old mice lacking ClC-5 (Clcn5 +/- and Clcn5 -/- ) present higher renal collagen deposition and fibrosis than WT mice. Altogether, we describe a new regulatory mechanism for collagens' production and release by ClC-5, which is altered in DD1 and provides a better understanding of disease progression to renal fibrosis.
Assuntos
Canais de Cloreto , Fibrose , Lisossomos , Camundongos Knockout , beta Catenina , Animais , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Lisossomos/metabolismo , Humanos , Camundongos , beta Catenina/metabolismo , Fibrose/metabolismo , Rim/metabolismo , Rim/patologia , Colágeno Tipo I/metabolismo , Doença de Dent/metabolismo , Doença de Dent/genética , Proteólise , Transdução de SinaisRESUMO
SIGNIFICANCE STATEMENT: Loss of function of the 2Cl - /H + antiporter ClC-5 in Dent disease causes an unknown impairment in endocytic traffic, leading to tubular proteinuria. The authors integrated data from biochemical and quantitative imaging studies in proximal tubule cells into a mathematical model to determine that loss of ClC-5 impairs endosome acidification and delays early endosome maturation in proximal tubule cells, resulting in reduced megalin recycling, surface expression, and half-life. Studies in a Dent mouse model also revealed subsegment-specific differences in the effects of ClC-5 knockout on proximal tubule subsegments. The approach provides a template to dissect the effects of mutations or perturbations that alter tubular recovery of filtered proteins from the level of individual cells to the entire proximal tubule axis. BACKGROUND: Loss of function of the 2Cl - /H + antiporter ClC-5 in Dent disease impairs the uptake of filtered proteins by the kidney proximal tubule, resulting in tubular proteinuria. Reduced posttranslational stability of megalin and cubilin, the receptors that bind to and recover filtered proteins, is believed to underlie the tubular defect. How loss of ClC-5 leads to reduced receptor expression remains unknown. METHODS: We used biochemical and quantitative imaging data to adapt a mathematical model of megalin traffic in ClC-5 knockout and control cells. Studies in ClC-5 knockout mice were performed to describe the effect of ClC-5 knockout on megalin traffic in the S1 segment and along the proximal tubule axis. RESULTS: The model predicts that ClC-5 knockout cells have reduced rates of exit from early endosomes, resulting in decreased megalin recycling, surface expression, and half-life. Early endosomes had lower [Cl - ] and higher pH. We observed more profound effects in ClC-5 knockout cells expressing the pathogenic ClC-5 E211G mutant. Alterations in the cellular distribution of megalin in ClC-5 knockout mice were consistent with delayed endosome maturation and reduced recycling. Greater reductions in megalin expression were observed in the proximal tubule S2 cells compared with S1, with consequences to the profile of protein retrieval along the proximal tubule axis. CONCLUSIONS: Delayed early endosome maturation due to impaired acidification and reduced [Cl - ] accumulation is the primary mediator of reduced proximal tubule receptor expression and tubular proteinuria in Dent disease. Rapid endosome maturation in proximal tubule cells is critical for the efficient recovery of filtered proteins.
Assuntos
Doença de Dent , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Animais , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Doença de Dent/genética , Doença de Dent/metabolismo , Endocitose , Proteinúria/patologia , Endossomos/metabolismo , Túbulos Renais Proximais/metabolismo , Modelos Animais de Doenças , Camundongos Knockout , Técnicas de Cultura de Células , AntiportersRESUMO
BACKGROUND AND OBJECTIVES: Dent's disease type 1 (DD1) is a rare X-linked hereditary pathology caused by CLCN5 mutations that is characterized mainly by proximal tubule dysfunction, hypercalciuria, nephrolithiasis/nephrocalcinosis, progressive chronic kidney disease, and low-weight proteinuria, the molecular hallmark of the disease. Currently, there is no specific curative treatment, only symptomatic and does not prevent the progression of the disease. In this study we have isolated and characterized urinary extracellular vesicles (uEVs) enriched in exosomes that will allow us to identify biomarkers associated with DD1 progression and a better understanding of the pathophysiological bases of the disease. MATERIALS AND METHODS: Through a national call from the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP), urine samples were obtained from patients and controls from different Spanish hospitals, which were processed to obtain the uEVS. The data of these patients were provided by the respective nephrologists and/or extracted from the RENALTUBE registry. The uEVs were isolated by ultracentrifugation, morphologically characterized and their protein and microRNA content extracted. RESULTS: 25 patients and 10 controls were recruited, from which the urine was processed to isolate the uEVs. Our results showed that the relative concentration of uEVs/mL is lower in patients compared to controls (0.26 × 106 uEVs/mL vs 1.19 × 106 uEVs/mL, p < 0.01). In addition, the uEVs of the patients were found to be significantly larger than those of the control subjects (mean diameter: 187.8 nm vs 143.6 nm, p < 0.01). Finally, our data demonstrated that RNA had been correctly extracted from both patient and control exosomes. CONCLUSIONS: In this work we describe the isolation and characterization of uEVs from patients with Dent 1 disease and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology.
Assuntos
Doença de Dent , Exossomos , MicroRNAs , Nefrocalcinose , Nefrolitíase , Criança , Humanos , Doença de Dent/genética , Doença de Dent/metabolismo , Exossomos/metabolismo , Nefrocalcinose/genéticaRESUMO
The study aims to explore the clinicopathological features and whether the nonsense mutations of CLCN5 gene have effect on the renal expression of CLC-5 protein and megalin/cubilin complex in children with Dent-1 disease. The clinicopathological features and genetic examination of three patients with Dent-1 disease were investigated. The expression of CLC-5 and megalin/cubilin complex in renal tissues was detected by using immunohistochemistry method. Urinary albumin, α1-microglobulin, ß2-microglobulin, retinol binding protein, and calcium levels were measured by immunonephelometry. Urinary calcium and low molecular weight proteinuria (LMWP) were enhanced in three patients, and two presented with nephrotic range proteinuria. Focal glomerular obsolescence, minor tubulointerstitial injury, and focal calcification in corticomedullary junction were found in one patient. Nonsense mutations of CLCN5 gene from their mothers were identified in all three patients with Dent-1 disease; however, the expression of CLC-5 protein was not decreased in renal tubular cells. As the receptor complex of albumin and LMWP reabsorption, the expression of megalin/cubilin in the brush border of proximal tubules was decreased in Dent-1 patients. Even if the renal CLC-5 protein is expressed normally, the reduced expression of megalin/cubilin in the brush border of renal proximal tubules may be helpful to understand the physiopathology of Dent-1 disease with nonsense mutations of CLCN5 gene.
Assuntos
Canais de Cloreto/metabolismo , Códon sem Sentido , Doença de Dent , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Albuminas/genética , Albuminas/metabolismo , Cálcio/metabolismo , Criança , Códon sem Sentido/metabolismo , Doença de Dent/metabolismo , Humanos , Túbulos Renais Proximais , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteinúria/metabolismo , Receptores de Superfície CelularRESUMO
Mutations in the CLCN5 gene encoding the 2Cl- /1H+ exchanger ClC-5 are associated with Dent disease 1, an inherited renal disorder characterized by low-molecular-weight (LMW) proteinuria and hypercalciuria. In the kidney, ClC-5 is mostly localized in proximal tubule cells, where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC-5 surrounding the "proton glutamate" that serves as a crucial H+ -binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for the second group of mutations in Xenopus laevis oocytes were reduced. Molecular dynamics simulations performed on a ClC-5 homology model demonstrated that such mutations might alter ClC-5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC-5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.
Assuntos
Doença de Dent , Prótons , Canais de Cloreto/química , Doença de Dent/genética , Doença de Dent/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X , Ácido Glutâmico , Células HEK293 , Humanos , NefrolitíaseRESUMO
This review examines calcium and phosphate transport in the kidney through the lens of the rare X-linked genetic disorder Dent disease. Dent disease type 1 (DD1) is caused by mutations in the CLCN5 gene encoding ClC-5, a Cl- /H+ antiporter localized to early endosomes of the proximal tubule (PT). Phenotypic features commonly include low molecular weight proteinuria (LMWP), hypercalciuria, focal global sclerosis and chronic kidney disease; calcium nephrolithiasis, nephrocalcinosis and hypophosphatemic rickets are less commonly observed. Although it is not surprising that abnormal endosomal function and recycling in the PT could result in LMWP, it is less clear how ClC-5 dysfunction disturbs calcium and phosphate metabolism. It is known that the majority of calcium and phosphate transport occurs in PT cells, and PT endocytosis is essential for calcium and phosphorus reabsorption in this nephron segment. Evidence from ClC-5 KO models suggests that ClC-5 mediates parathormone endocytosis from tubular fluid. In addition, ClC-5 dysfunction alters expression of the sodium/proton exchanger NHE3 on the PT apical surface thus altering transcellular sodium movement and hence paracellular calcium reabsorption. A potential role for NHE3 dysfunction in the DD1 phenotype has never been investigated, either in DD models or in patients with DD1, even though patients with DD1 exhibit renal sodium and potassium wasting, especially when exposed to even a low dose of thiazide diuretic. Thus, insights from the rare disease DD1 may inform possible underlying mechanisms for the phenotype of hypercalciuria and idiopathic calcium stones.
Assuntos
Cálcio/metabolismo , Doença de Dent/patologia , Canais Iônicos/metabolismo , Fosfatos/metabolismo , Animais , Doença de Dent/metabolismo , Humanos , Transporte de ÍonsRESUMO
Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/- mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/- kidneys. The OcrlY/- mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/- mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/- and Clcn5Y/- mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease.
Assuntos
Doença de Dent/genética , Endossomos/metabolismo , Túbulos Renais Proximais/metabolismo , Lisossomos/metabolismo , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/genética , Actinas/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/genética , Doença de Dent/metabolismo , Doença de Dent/fisiopatologia , Modelos Animais de Doenças , Endocitose/genética , Humanos , Rim/fisiopatologia , Túbulos Renais Proximais/fisiopatologia , Locomoção/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/fisiopatologia , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
Dent disease is an X-linked recessive renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure. Inactivating mutations of CLCN5, the gene encoding the 2Cl- /H+ exchanger ClC-5, have been reported in patients with Dent disease 1. In vivo studies in mice harboring an artificial mutation in the "gating glutamate" of ClC-5 (c.632A > C, p.Glu211Ala) and mathematical modeling suggest that endosomal chloride concentration could be an important parameter in endocytosis, rather than acidification as earlier hypothesized. Here, we described a novel pathogenic mutation affecting the "gating glutamate" of ClC-5 (c.632A>G, p.Glu211Gly) and investigated its molecular consequences. In HEK293T cells, the p.Glu211Gly ClC-5 mutant displayed unaltered N-glycosylation and normal plasma membrane and early endosomes localizations. In Xenopus laevis oocytes and HEK293T cells, we found that contrasting with wild-type ClC-5, the mutation abolished the outward rectification, the sensitivity to extracellular H+ and converted ClC-5 into a Cl- channel. Investigation of endosomal acidification in HEK293T cells using the pH-sensitive pHluorin2 probe showed that the luminal pH of cells expressing a wild-type or p.Glu211Gly ClC-5 was not significantly different. Our study further confirms that impaired acidification of endosomes is not the only parameter leading to defective endocytosis in Dent disease 1.
Assuntos
Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Endossomos/metabolismo , Endossomos/patologia , Mutação/genética , Animais , Doença de Dent/genética , Doença de Dent/metabolismo , Doença de Dent/patologia , Endocitose/genética , Endocitose/fisiologia , Células HEK293 , Humanos , Xenopus laevisRESUMO
Dent's disease is an X-linked inherited renal disease. Patients with Dent's disease often carry mutations in genes encoding the Cl-/H+ exchanger ClC-5 and/or inositol polyphosphate 5-phosphatase (OCRL1). However, the mutations involved and the biochemical effects of these mutations are not fully understood. To characterize genetic changes in Dent's disease patients, in this study, samples from nine Chinese patients were subjected to genetic analysis. Among the nine patients, six were classified as having Dent-1 disease, one had Dent-2 disease, and two could not be classified. Expression of ClC-5 carrying Dent's disease-associated mutations in HEK293 cells had varying effects: (1) no detectable expression of mutant protein; (2) retention of a truncated protein in the endoplasmic reticulum; or (3) diminished protein expression with normal distribution in early endosomes. Dent's disease patients showed genetic heterogeneity and over 20% of patients did not have CLCN5 or OCRL1 mutations, suggesting the existence of other genetic factors. Using next-generation sequencing, we identified possible modifier genes that have not been previously reported in Dent's disease patients. Heterozygous variants in CFTR, SCNN1A, and SCNN1B genes associated with cystic fibrosis (CF) or CF-like disease were detected in four of our nine patients. These results may form the basis for future characterization of Dent's disease and genetic counseling approaches.
Assuntos
Canais de Cloreto/genética , Doença de Dent/genética , Mutação , Criança , Pré-Escolar , Canais de Cloreto/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Doença de Dent/diagnóstico , Doença de Dent/metabolismo , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Células HEK293 , Humanos , Masculino , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nefrolitíase/diagnóstico , Nefrolitíase/genética , Nefrolitíase/metabolismo , Fenótipo , Monoéster Fosfórico Hidrolases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismoRESUMO
Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow-derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5Y/- proximal tubule cells with bone marrow-derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow-derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow-derived cells and diseased tubular cells play a key role in the rescue mechanism.
Assuntos
Transplante de Medula Óssea , Canais de Cloreto/deficiência , Doença de Dent/cirurgia , Túbulos Renais Proximais/fisiopatologia , Animais , Comunicação Celular , Células Cultivadas , Canais de Cloreto/genética , Técnicas de Cocultura , Doença de Dent/genética , Doença de Dent/metabolismo , Doença de Dent/fisiopatologia , Modelos Animais de Doenças , Endocitose , Predisposição Genética para Doença , Glicosúria/genética , Glicosúria/metabolismo , Glicosúria/fisiopatologia , Glicosúria/prevenção & controle , Hipercalciúria/genética , Hipercalciúria/metabolismo , Hipercalciúria/fisiopatologia , Hipercalciúria/prevenção & controle , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Poliúria/genética , Poliúria/metabolismo , Poliúria/fisiopatologia , Poliúria/prevenção & controle , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Proteinúria/prevenção & controle , Recuperação de Função Fisiológica , Quimeras de TransplanteRESUMO
In order to understand the mechanism of albuminuria we have explored how other plasma proteins are processed by the kidney as compared to inert molecules like Ficolls. When fractional clearances are plotted versus protein radius there is a remarkable parallelism between protein (molecular weight range 30-150kDa) clearance in healthy controls, in Dent's disease, in nephrotic states and the clearance of Ficolls. Although there are significant differences in the levels of fractional clearances in these states. Dent's disease results in a 2-fold increase in the fractional clearance of proteins as compared to healthy controls whereas in nephrotic states there is a further 3-fold increase in fractional clearance. Previous thinking that albumin uptake was controlled primarily by the megalin/cubilin receptor does not explain the albumin urinary excretion data and is therefore an incorrect concept. Protein clearance in nephrotic states approach the fractional clearance of inert Ficolls for a given radius. It therefore appears that there are two pathways processing these proteins. A low capacity pathway associated with megalin/cubilin that degrades filtered protein (that is inhibited in Dent's disease) and a high capacity pathway that retrieves the filtered protein and returns it to the blood supply (without retrieval nephrotic protein excretion will occur and this will account for hypoproteinemia). On the other hand low molecular weight proteins (<20kDa) are processed entirely differently by the kidney. They are not retrieved but are comprehensively degraded in the kidney with the degradation products predominantly returned to the blood supply.
Assuntos
Albuminúria/metabolismo , Proteínas Sanguíneas/metabolismo , Doença de Dent/metabolismo , Rim/metabolismo , Animais , Humanos , Peso Molecular , RatosRESUMO
Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H(+) exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has recently come into question. To clarify the mechanism of pathogenesis for Dent's disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomal acidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl(-) channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl(-) channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomal acidification and hypotonicity-inducible vacuolar-type H(+)-ATPase (V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPase and CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl(-)/H(+) exchanger into a Cl(-) channel induces Dent's disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.
Assuntos
Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Doença de Dent/metabolismo , Mutação/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Criança , Doença de Dent/genética , Endocitose/fisiologia , Feminino , Células HEK293 , Homeostase/fisiologia , Humanos , Transporte de Íons/fisiologia , Túbulos Renais Proximais/metabolismo , Masculino , Oócitos/metabolismo , Xenopus laevis/metabolismoRESUMO
Serum level of phosphate is regulated by the kidney, especially proximal tubule. The transcellular transport of phosphate in the proximal tubule is mediated via Na dependent transporters, i.e., NPT2a and NPT2b at the luminal membrane, and unknown channel at the basolateral side. The transport of phosphate via NPT2a and NPT2b is further regulated by factors, such as PTH, FGF23, and 1,25(OH)(2)D. Several hereditary diseases that cause hypophoshatemia specically are known. In addition, dysfunction of proximal tubule may develop Fanconi syndrome, which also causes hypherphosphaturia. In this section, I describe the renal mechanisms of phosphate handling and the causes of hypophosphatemia along with its treatment.
Assuntos
Hipofosfatemia/etiologia , Hipofosfatemia/metabolismo , Túbulos Renais Proximais/metabolismo , Fosfatos/metabolismo , Administração Oral , Calcitriol/fisiologia , Canais de Cloreto , Doença de Dent/etiologia , Doença de Dent/genética , Doença de Dent/metabolismo , Síndrome de Fanconi/etiologia , Síndrome de Fanconi/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/fisiologia , Humanos , Hipofosfatemia/terapia , Doenças Mitocondriais , Síndrome Oculocerebrorrenal , Hormônio Paratireóideo/fisiologia , Monoéster Fosfórico Hidrolases , Compostos de Fósforo/administração & dosagem , Compostos de Fósforo/uso terapêutico , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/fisiologia , Vitamina D/administração & dosagemRESUMO
Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.
Assuntos
Canais de Cloreto/genética , Doença de Dent/genética , Mutação , Animais , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Estudos de Coortes , Doença de Dent/metabolismo , Estudos de Associação Genética , Humanos , Masculino , Camundongos , Camundongos Knockout , LinhagemRESUMO
Cl(-) transport in animal cells has fundamental physiological roles and it is mediated by a variety of protein families, one of them being the CLC family of ion channels and transporters. Besides their physiological relevance, CLC proteins show peculiar biophysical properties. This review will focus on a member of the CLC protein family, the endosomal Cl(-)/H(+) antiporter ClC-5. ClC-5 mutations cause Dent's disease, a renal syndrome due to defective protein reabsorption in the proximal tubule. This established the critical function of ClC-5 for endocytosis. However, our understanding of ClC-5's molecular role in endosomes and of its biophysical properties has proved elusive in spite of important progress achieved in the last two decades. Early models in which ClC-5 would provide a shunt conductance to enable efficient endosomal acidification conflicted with the antiport activity of ClC-5 that has more recently emerged. Currently, the physiological role of ClC-5 is hotly debated and its biophysical properties are still not fully understood.
Assuntos
Canais de Cloreto/metabolismo , Animais , Canais de Cloreto/química , Canais de Cloreto/genética , Doença de Dent/metabolismo , Doença de Dent/patologia , Endossomos/metabolismo , Humanos , Transporte de Íons , Estrutura Terciária de Proteína , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
UNLABELLED: We report the use of three dimensional computational analysis of chloride channel 5 (ClC-5) based on a novel mutation, L266V, identified in a 15-year-old Japanese boy with Dent's disease. Since both leucine and valine are branched-chain amino acids, it has not been proved conclusively whether L266V mutation is actually responsible for the development of Dent's disease. In the present study using molecular analysis, we investigated the mechanism for loss of function of the ClC-5 protein resulting from the L266V mutation. Structural analysis of the normal ClC-5 transmembrane region using molecular modeling showed that the two respective Leu266 residues were located at the interface of the dimer formed by the aligned ClC-5 monomers. The Leu266 side-chains were positioned close to each other through hydrophobic interaction, resembling two interconnecting hooks. When Leu266 was replaced by a valine residue, the hydrophobic interaction between the CLC-5 monomers was reduced, and dimer formation was impaired. This computer simulation analysis has thus provided strong evidence for the important role of Leu266 in the dimerization of human ClC-5 in membranes. CONCLUSION: The finding of the present study suggest that computational modeling and molecular analysis could be an alternative to labor-intensive in vitro functional studies.
Assuntos
Canais de Cloreto/genética , Doença de Dent/genética , Mutação de Sentido Incorreto , Adolescente , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Simulação por Computador , Análise Mutacional de DNA , Doença de Dent/diagnóstico , Doença de Dent/metabolismo , Predisposição Genética para Doença , Testes Genéticos , Humanos , Japão , Masculino , Modelos Moleculares , Estrutura Molecular , Fenótipo , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
Mutations in the CLCN5 (chloride channel, voltage-sensitive 5) gene cause Dent's disease because they reduce the functional expression of the ClC-5 chloride/proton transporter in the recycling endosomes of proximal tubule epithelial cells. The majority (60%) of these disease-causing mutations in ClC-5 are misprocessed and retained in the ER (endoplasmic reticulum). Importantly, the structural basis for misprocessing and the cellular destiny of such ClC-5 mutants have yet to be defined. A ClC-5 monomer comprises a short N-terminal region, an extensive membrane domain and a large C-terminal domain. The recent crystal structure of a eukaryotic ClC (chloride channel) transporter revealed the intimate interaction between the membrane domain and the C-terminal region. Therefore we hypothesized that intramolecular interactions may be perturbed in certain mutants. In the present study we examined two misprocessed mutants: C221R located in the membrane domain and R718X, which truncates the C-terminal domain. Both mutants exhibited enhanced protease susceptibility relative to the normal protein in limited proteolysis studies, providing direct evidence that they are misfolded. Interestingly, the membrane-localized mutation C221R led to enhanced protease susceptibility of the cytosolic N-terminal region, and the C-terminal truncation mutation R718X led to enhanced protease susceptibility of both the cytosolic C-terminal and the membrane domain. Together, these studies support the idea that certain misprocessing mutations alter intramolecular interactions within the full-length ClC-5 protein. Further, we found that these misfolded mutants are polyubiquitinated and targeted for proteasomal degradation in the OK (opossum kidney) renal epithelial cells, thereby ensuring that they do not elicit the unfolded protein response.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/genética , Códon sem Sentido/genética , Doença de Dent/genética , Mutação de Sentido Incorreto/genética , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Animais , Doença de Dent/enzimologia , Doença de Dent/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Gambás , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional/genética , Deficiências na Proteostase/enzimologia , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismoRESUMO
Oculocerebrorenal syndrome of Lowe (OCRL) gene product is a phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P(2)] 5-phosphatase, and mutations of OCRL cause Lowe syndrome and Dent disease, both of which are frequently associated with hypercalciuria. Transient receptor potential, vanilloid subfamily, subtype 6 (TRPV6) is an intestinal epithelial Ca(2+) channel mediating active Ca(2+) absorption. Hyperabsorption of Ca(2+) was found in patients of Dent disease with increased Ca(2+) excretion. In this study, we tested whether TRPV6 is regulated by OCRL and, if so, to what extent it is altered by Dent-causing OCRL mutations using Xenopus laevis oocyte expression system. Exogenous OCRL decreased TRPV6-mediated Ca(2+) uptake by regulating the function and trafficking of TRPV6 through different domains of OCRL. The PI(4,5)P(2) 5-phosphatase domain suppressed the TRPV6-mediated Ca(2+) transport likely through regulating the PI(4,5)P(2) level needed for TRPV6 function without affecting TRPV6 protein abundance of TRPV6 at the cell surface. The forward trafficking of TRPV6 was decreased by OCRL. The Rab binding domain in OCRL was involved in regulating the trafficking of TRPV6. Knocking down endogenous X. laevis OCRL by antisense approach increased TRPV6-mediated Ca(2+) transport and TRPV6 forward trafficking. All seven Dent-causing OCRL mutations examined exhibited alleviation of the inhibitory effect on TRPV6-mediated Ca(2+) transport together with decreased overall PI(4,5)P(2) 5-phosphatase activity. In conclusion, OCRL suppresses TRPV6 via two separate mechanisms. The disruption of PI(4,5)P(2) 5-phosphatase activity by Dent-causing mutations of OCRL may lead to increased intestinal Ca(2+) absorption and, in turn, hypercalciuria.
Assuntos
Cálcio/metabolismo , Doença de Dent/metabolismo , Mucosa Intestinal/metabolismo , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Doença de Dent/enzimologia , Doença de Dent/genética , Feminino , Técnicas de Silenciamento de Genes/métodos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Síndrome Oculocerebrorrenal/enzimologia , Síndrome Oculocerebrorrenal/genética , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/fisiologia , Monoéster Fosfórico Hidrolases/genética , Ligação Proteica/genética , Transporte Proteico/genética , Ratos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Xenopus laevisRESUMO
The precise regulation of phosphoinositide lipids in cellular membranes is crucial for cellular survival and function. Inositol 5-phosphatases have been implicated in a variety of disorders, including various cancers, obesity, type 2 diabetes, neurodegenerative diseases and rare genetic conditions. Despite the obvious impact on human health, relatively little structural and biochemical information is available for this family. Here, we review recent structural and mechanistic work on the 5-phosphatases with a focus on OCRL, whose loss of function results in oculocerebrorenal syndrome of Lowe and Dent 2 disease. Studies of OCRL emphasize how the actions of 5-phosphatases rely on both intrinsic and extrinsic membrane recognition properties for full catalytic function. Additionally, structural analysis of missense mutations in the catalytic domain of OCRL provides insight into the phenotypic heterogeneity observed in Lowe syndrome and Dent disease.
Assuntos
Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Doença de Dent/genética , Doença de Dent/metabolismo , Humanos , Inositol Polifosfato 5-Fosfatases , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genéticaRESUMO
BACKGROUND: Three patients with Dent's disease presented with complaints of impaired night vision or xerophthalmia and were found to have severely decreased serum retinol concentrations. Retinol, bound to its carrier retinol-binding protein (RBP), is filtered at the glomerulus and reabsorbed at the proximal tubule. We hypothesized that urinary loss of retinol-RBP complex is responsible for decreased serum retinol. OBJECTIVE AND METHODS: The study aim was to investigate vitamin A status and RBP in serum and urine of patients with genetically confirmed Dent's disease. RESULTS: Eight patients were studied, three boys had clinical vitamin A deficiency, three had asymptomatic deficiency, and two young men with Dent's disease and impaired renal function had normal retinol values. Serum RBP concentrations were low in patients with vitamin A deficiency and were correlated with vitamin A levels. Urinary RBP concentrations were increased in all patients (2,000-fold), regardless of vitamin A status. This was in contrast to patients with glomerular proteinuria who had only mildly increased urinary RBP with normal serum RBP and vitamin A, and patients with cystinosis with impaired renal function who had massive urinary RBP losses but without a decrease in serum RBP or vitamin A levels. Treatment with vitamin A supplements in patients with retinol deficiency resulted in rapid resolution of ocular symptoms and an increase in serum retinol concentrations. CONCLUSIONS: Vitamin A deficiency is common in patients with Dent's disease and preserved renal function. We therefore recommend screening these patients for retinol deficiency and treating them before visual symptoms develop.