OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease.

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.

Genetic Analysis of Dent's Disease and Functional Research of CLCN5 Mutations.

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.

Bone marrow transplantation improves proximal tubule dysfunction in a mouse model of Dent disease.

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.

[Renal hypophosphatemia:pathophysiology and treatment].

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.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1.

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.

Inositol 5-phosphatases: insights from the Lowe syndrome protein OCRL.

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.

Vitamin A deficiency associated with urinary retinol binding protein wasting in Dent's disease.

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.

ATP induces conformational changes in the carboxyl-terminal region of ClC-5.

ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation.