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Kidney Tubular Ablation of Ocrl/Inpp5b Phenocopies Lowe Syndrome Tubulopathy

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Kidney Tubular Ablation of Ocrl/Inpp5b Phenocopies Lowe Syndrome Tubulopathy BASIC RESEARCH www.jasn.org Kidney Tubular Ablation of Ocrl/Inpp5b Phenocopies Lowe Syndrome Tubulopathy †‡ †‡ | †‡ †‡ | Kazunori Inoue,* Daniel M. Balkin, § Lijuan Liu, § Ramiro Nandez, § Yumei Wu, § ††‡‡ †‡ | Xuefei Tian,* Tong Wang,¶ Robert Nussbaum,** Pietro De Camilli, § and Shuta Ishibe*¶ Departments of *Internal Medicine, †Cell Biology, |Neuroscience, and ¶Cellular and Molecular Physiology, ‡Howard Hughes Medical Institute, and §Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale School of Medicine, New Haven, Connecticut; **Department of Medicine and ††Institute of Human Genetics, University of California, San Francisco, California; and ‡‡Invitae Corporation, San Francisco, California ABSTRACT Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule func- tion. Genetic ablation of Ocrl in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule–specific BASIC RESEARCH inactivation of Inpp5b on a global Ocrl–knockout mouse background resulted in low molecular weight pro- teinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin- dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis. J Am Soc Nephrol 28: 1399–1407, 2017. doi: https://doi.org/10.1681/ASN.2016080913 Human mutations in the inositol 5-phosphatase CLC-5 is localized on early endocytic vesicles and oculocerebrorenal syndrome of Lowe (OCRL)cause endosomes, its absence is thought to produce a de- the X–linked conditions OCRL and Dent disease.1–6 fect on endocytosis indirectly via an effect on the Clinical manifestations of these illnesses include ionic composition of the endosome lumen that may urinary loss of low molecular weight proteins, phos- affect the dynamics of the endocytic pathway.13 phate, and bicarbonate.7,8 These defects (Fanconi Given that mutations in CLCN5 and OCRL pro- syndrome) result from dysfunction of the kidney duce very similar kidney defects in human pa- proximal tubule, a segment of the nephron that tients,14 one might expect that CLC-5 and OCRL plays a vital role in the reabsorption of the vast ma- jority of the daily filtered load via endocytic mem- Received August 27, 2016. Accepted October 5, 2016. brane traffic as well as transporters and channels.9,10 Similar kidney defects are observed in Dent 1 dis- K.I. and D.M.B. contributed equally to this work. ease, another X–linked condition, which is due to Published online ahead of print. Publication date available at human mutations in the endosomal proton- www.jasn.org. 11,12 chloride exchanger CLCN5. On the basis of Correspondence: Dr. Pietro De Camilli, Department of Cell studies of Clcn5 knockout (KO) mice, the patho- Biology, Yale University School of Medicine, PO Box 208002, logic phenotype due to the loss of CLC-5 was attrib- 333 Cedar Street, New Haven, CT 06520-8029 or Dr. Shuta Ishibe, Section of Nephrology, Yale University School of uted to an impairment of the endocytosis of low Medicine, PO Box 208029, 333 Cedar Street, New Haven, molecular weight proteins that escape the glomeru- CT 06520-8029. Email: [email protected] or shuta. lar filter and the abnormal endocytic trafficking of [email protected] proximal tubule receptors and channels.11 Because Copyright © 2017 by the American Society of Nephrology J Am Soc Nephrol 28: 1399–1407, 2017 ISSN : 1046-6673/2805-1399 1399 BASIC RESEARCH www.jasn.org fl fl cooperate in a similar or shared cellular process. OCRL is resulting from the crossing of Pax8rtTA TetO-cre; Inpp5b / ; 2 localized at various stations of the endocytic pathway and Ocrl +/ mice were treated with doxycycline to induce loss thought to play a role in the progression of membranes along of INPP5B expression in kidney tubules, thus generating mice the pathway by coupling the flux of endocytic membrane to that lack both OCRL and INPP5B selectively in kidney tubules 15–22 the dephosphorylation of PI(4,5)P2. This reaction is re- (conditional double knockout [cDKO]). Littermate mice fl fl quired to facilitate the shedding of endocytic factors and actin lacking the TetO-cre gene (Pax8rtTA; Inpp5b / ; Ocrl +/+ lacking from endocytic membranes.18,19 Supporting an endocytic role TetO-cre) or lacking both the TetO-cre gene and a functional fl fl for OCRL, dysfunction of endocytic traffic, including partial im- Ocrl gene (Pax8rtTA Inpp5b / ; Ocrl KO lacking TetO-cre)were pairment of clathrin-mediated endocytosis, was detected in fibro- used as control or Ocrl KO, respectively. Absence of Ocrl (Fig- blasts derived from patients with Lowe syndrome lacking OCRL ure 1B) in Ocrl KO and cDKO mice was confirmed by Western expression.19 However, surprisingly, the global Ocrl KO in mice blotting. Loss of INPP5B could not be assessed by Western did not produce an obvious phenotype.23 OCRL has a paralog, the blotting, because INPP5B immunoreactivity was below the protein type 2 inositol polyphosphate-5-phosphatase (INPP5B), detection limit of available antibodies. Thus, Inpp5B gene dis- in both mice and humans.18,24,25 It is, therefore, possible that, due ruption after 2 weeks of doxycycline induction was confirmed to different expression and/or splicing of its gene in mice and by analysis of Inpp5b mRNA expression (Figure 1C). cDKO humans,26 the INPP5B protein may compensate for OCRL loss mice were viable and appeared normal. of function in mice but not in humans. Supporting this Histologic examination of the kidney cortex on the basis possibility, a proximal tubular phenotype characterized by low of H&E, periodic acid–Schiff, and trichrome staining did not molecular weight proteinuria was observed in transgenic mice show obvious changes at early time points (1–3 months) after that overexpress human INPP5B (from a BAC transgene) and doxycycline induction (Figure 1D). This indicates that the lack harbor germline disruptions of both the Ocrl and the Inpp5b of both Inpp5b and Ocrl doesnotresultincelldeath, genes.23 although a decrease in the levels of megalin, NaPi2a, and The goal of this study was to determine whether the com- NHE3, three proteins expressed at the apical surface of prox- bined absence of both OCRL and INPP5B in mouse kidney imal tubule cells,29–31 was observed at 3 months but not at proximal tubules results in a defect similar to that observed in 1 month (Figure 1, G and H and quantified in Figure 1, I–K). patients with Lowe syndrome and whether these changes are, at Furthermore, there was no evidence of kidney failure in these least in part, due to an impairment of endocytosis. Because mice within the first few months after doxycycline induction germline KO of both Ocrl and Inpp5b results in embryonic (Figure 1F). However, by 8 months, a general demise of kidney lethality,23 the Inpp5b gene was conditionally deleted in kidney function was observed with the onset of interstitial fibrosis proximal tubules of germline Ocrl global KO mice. These mu- (Figure 1D and quantified in Figure 1E) and elevation of tant mice exhibited dramatic defects in proximal tubule reab- plasma creatinine (Figure 1F). sorption that mimicked what has been observed in patients Assays assessing proximal tubule function were performed with Lowe syndrome.4 After induction of genetic recombina- at 2 weeks and 2 months before these changes occurred. tion of the Inpp5b locus in germline Ocrl global KO mice, microscopic analysis of their kidneys revealed a striking defect cDKO Mice Develop Fanconi Syndrome of both clathrin-dependent and -independent endocytosis at One month after doxycycline induction, the urine of cDKO the apical pole of kidney proximal tubule cells. These results mice showed elevated levels of the retinol and vitamin D provide support to the hypotheses that Clcn5 and Ocrl are binding proteins (Figure 2A), two proteins that are filtered functional partners and that the different effects of Ocrl mu- by the glomerulus and reabsorbed in the proximal tubule, tations in humans and mice are likely due to the different with elevation in the urine that is characteristic of Lowe syn- contributions of INPP5B to kidney function in the different drome and Dent 1 and 2 disease.8 Such phenotype worsened species. over the ensuing months (Figure 2A and quantified in Figure 2, B and C). Significant albuminuria, however, was not ob- served in cDKO mice (Figure 2A), indicating that the kidney RESULTS filtration barrier was intact in the cDKO mice. For compar- ison, there was massive albuminuria present in the urine of rtTA fl/fl Generation of Pax8 TetO-cre Inpp5b /Ocrl KO podocyte–specific dynamin 1 and 2 double–KO mice, where Mice (Conditional Double–KO Mice) the glomerular filtration barrier is drastically perturbed (Fig- To achieve kidney tubule excision of the Inpp5b gene in germ- ure 2A).32 However, on quantification of urinary albumin by line Ocrl KO mice, doxycycline–inducible Pax8rtTA TetO-cre ELISA (Figure 2F), there was a mild
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