Kidney Tubular Ablation of Ocrl/Inpp5b Phenocopies Lowe Syndrome Tubulopathy

BASIC RESEARCH www.jasn.org

†‡ †‡ | †‡ †‡ | 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 function. 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 proteinuria, 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 observed 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 increase in albuminuria fl fl mice27 were interbred with Inpp5b / 28 and Ocrl KO mice23 relative to controls, which can likely be explained by reduced (Figure 1A). On administration of doxycycline, Pax8rtTATetO-cre reabsorption by proximal tubular endocytosis of the small mice express Cre in most sections of the kidney tubules, in- fraction of this protein that escapes the glomerular filtration cluding the proximal tubules, but not in the glomerulus.27 barrier.8,33 To further characterize proximal tubular func- fl fl Two-month-old Pax8rtTA TetO-cre; Inpp5b / ;OcrlKO mice tion, urine calcium and phosphate levels (normalized to

1400 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1399–1407, 2017 www.jasn.org BASIC RESEARCH

Figure 1. Normal kidney histology and function are observed in cDKO mice within 3 months of doxycycline induction. (A) Ocrl, Inpp5b, Pax8rtTA,andTetO-cre genotypes confirmed by tail genotyping. (B) Expression of OCRL in control, Ocrl KO, and cDKO renal cortex by immunoblot. (C) Inpp5b mRNA expression in control, Ocrl KO, and cDKO renal cortex by RT-PCR. *P,0.05 (n=3). (D) Representative low–power images of H&E–,periodicacid–Schiff (PAS)–, and trichrome–stained kidney sections from control, Ocrl KO, and cDKO mice. Interstitial fibrosis (arrowheads) is observed in cDKO mice kidneys at 8 months after doxycycline induction when stained with trichrome. Scale bars, 50 mm. (E) Quantification of interstitial fibrosis in control, Ocrl KO, and cDKO at 1, 3, and 8 months after doxycycline induction. *P,0.05 (n=3). (F) cDKO mice show elevated plasma creatinine 8 months after doxycycline induction. *P,0.05 (n=4). (G and H) Isolated renal brush border samples from control, Ocrl KO, and cDKO mice kidneys at (G) 1 and (H) 3 months after doxycycline induction immunoblotted with megalin, NaPi2a, and NHE3 antibodies. (I–K) Quantification by densitometry of the immunoblots of proteins in H. *P,0.05 (n=3). creatinine) were analyzed in control, Ocrl KO, and cDKO (Figure 2H). Collectively, these results showed that cDKO mice by ELISA starting at 1 month after doxycycline induc- mice developed the renal Fanconi syndrome. tion. Phosphaturia (Figure 2D) was observed in cDKO mice, which continued to progress as the mice aged, whereas there Endocytic Defects in cDKO Mice was no difference in urine calcium levels between control and Low molecular weight proteins that are freely filtered by the cDKO mice (Figure 2E). Consistent with the metabolic aci- glomerulus are actively reclaimed by endocytosis in proximal dosis typically observed in patients with Lowe syndrome and tubule cells via the megalin and cubulin receptors, primarily patients with Dent 2, reduced plasma bicarbonate levels (Fig- by clathrin-mediated endocytosis.34 Todetermine whether the ure 2G) and acidemia were also detected in cDKO mice loss of low molecular weight proteins in cDKO mice reflects

J Am Soc Nephrol 28: 1399–1407, 2017 Endocytic Defect in Ocrl/Inpp5b Knockout, Inoue et al. 1401 BASIC RESEARCH www.jasn.org

KO kidneys showed the presence of red fluorescence in vesicles underlying the apical brush border of proximal tubule cells (slightly less prominent in Ocrl KO), which was labeled by the proximal tubule brush border marker Lotus tetragonolobus (LTA) (Figure 3A and quantified in Figure 3D, Supplemental Figure 1A). In contrast, no fluorescence was observed in the proximal tubules of the cDKO mice under identical image acquisition conditions (Figure 3A and quantified in Figure 3D). Fluid-phase endocytosis also seemed to be strongly impaired as revealed by the uptake of fluorescently labeled dextran at 2 weeks and 2 months after doxycycline induction. Ten minutes after injection, dextran fluorescence was abundant in the cortical region of proximal tubule cells under the brush border (Figure 3B and quantified in Figure 3E, Supplemental Fig- ure 1B) but nearly absent in the same cells of cDKO mice with identical acquisition parameters. Likewise, a defect in the uptake of horseradish peroxidase (HRP), also amarkeroffluid-phase endocytosis, was observed by both light and electron microscopy-cytochemistry in proximal tubules of cDKO mice after its injection in the bloodstream (Figure 3, F and G). In proximal – Figure 2. Proximal tubulopathy is observed in cDKO mice. (A) Immunoblots of urine tubules of control mice, abundant electron retinol binding protein, vitamin D binding protein, and albumin normalized to urine dense HRP reaction product could be creatinine concentration. (B and C) Quantification of immunoblots in A standardized observed within 5 minutes after the injection to urine from cDKO at 1 month. *P,0.05 (n=3). (D and E) Quantification of (D) urine in apical tubule–vesicular structures underlying phosphate and (E) calcium normalized to urine creatinine at 0, 1, 2, 3, and 4 months the plasma membrane, whereas no such label- after doxycycline induction (black triangle, control; blue circle, Ocrl KO; and red ing was observed in cDKO mice (Figure 3G). square,cDKOmice).*P,0.05 (n=5). (F) Quantification of urine albumin normalized Collectively, analogous to what has been to urine creatinine at 3 months after doxycycline induction. *P,0.05 (n=5). (G) Blood 2 observed in Clcn5 KO mice and Dent 1 dis- bicarbonate (HCO3 ) concentration and (H) blood pH at 0, 2, and 4 months after ease,11 proximal tubule cells that lack both doxycycline induction (black triangle, control; blue circle, Ocrl KO; and red square, OCRL and INPP5B display a major endo- cDKO mice). *P,0.05 (n=5). cytic defect. an endocytic defect, the uptake of b-lactoglobulin11 by prox- Delayed Parathyroid Hormone–Induced Sodium imal tubule cells after doxycycline induction was assessed. Phosphate Cotransporter Type 2a Internalization in the Red fluorescently labeled b-lactoglobulin was injected intra- cDKO Mice venously into the bloodstream of control, Ocrl KO, and The defect in the internalization of b-lactoglobulin, a receptor- cDKO mice at 2 weeks and 2 months after doxycycline induction. mediated process,11 suggested the occurrence of a defect in In both cases, 15 minutes after injection and kidney retrieval, clathrin-mediated endocytosis in the cDKO mice (Figure macroscopic examination revealed a reddish color of both con- 3A). To further validate the presence of this defect, in addition trol and Ocrl KO kidneys (but slightly less intense in Ocrl KO to a defect in fluid-phase endocytosis, the dynamics of the mice), suggesting retention of labeled b-lactoglobulin in the sodium phosphate cotransporter type 2a (NaPi2a), which is en- parenchyma but not in cDKO kidneys (Figure 3C, Supplemen- docytosed through clathrin-coated pits in response to parathy- tal Figure 1C). roid hormone (PTH) stimulation,29 was examined at 1 month Consistent with these observations, microscopic analysis of after doxycycline induction. Fifteen minutes after intraperitoneal the fluorescence in frozen sections of the same control and Ocrl injection of PTH, a rapid translocation of NaPi2a from the apical

1402 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1399–1407, 2017 www.jasn.org BASIC RESEARCH

of NaPi2a was observed in cDKO mice at the same time point after PTH injection using identical image acquisition conditions (Fig- ure 4C and quantiﬁed in Figure 4D). A mod- est translocation was observed 60 minutes after injection of a higher PTH dose, indicating responsiveness of the PTH receptor to PTH in the cDKO mice (quantiﬁed in Figure 4D, Supplemental Figure 2A). The defective internalization of NaPi2a, the major regulator of phosphate reabsorption in kidney proximal tubules,35 appears in contrast with the observed increased phosphaturia (increased residence of NaPi2a at the apical plasma membrane would be predicted to result in increased phosphate reabsorption). A similar apparent discrepancy, namely robust phosphaturia despite impaired and delayed PTH–dependent internalization of NaPi2a, was reported previously in the Clcn5 KO mice.11 The underlying mecha- nism remains to be precisely understood. As in Clcn5 KO mice, the levels of plasma and urine PTH were increased in cDKO mice relative to controls (Figure 4, E and F). However, no difference in PTH receptor expression (Supplemental Figure 2B) or concentration of FGF-23, a bone-derived factor also known to induce phosphaturia, were found (Supplemental Figure 2C).

Figure 3. Endocytic defects are observed in the cDKO mice proximal tubules. (A) Rep- DISCUSSION resentative images showing uptake of Alexa 546–labeled b-lactoglobulin (red) of control fl and Ocrl KO kidney proximal tubule by immuno uorescence at 15 minutes, but it was not The similar kidney proximal tubule defects observed in the cDKO kidney proximal tubule at identical exposure times. FITC-LTA is observed as a consequence of the loss of shown in green. Scale bars, 10 mm. (B) Representative images showing uptake of Alexa either OCRL (Lowe syndrome and Dent 546–labeled dextran (red) in the control and Ocrl KO kidney proximal tubule by immu- 36 nofluorescence at 10 minutes, but it was not observed in the cDKO kidney proximal tu- 2) or CLC-5 (Dent 1) in human patients bule at identical exposure times. FITC-LTA is shown in green. Scale bars, 10 mm. (C) suggests that the function of two proteins Whole kidneys of control, Ocrl KO, and cDKO mice in A revealing reduced uptake of Alexa may affect a shared physiologic process. 546–labeled b-lactoglobulin in the cDKO mice. (D) Quantification of Alexa 546–labeled Mouse KO studies showed that absence of b-lactoglobulin uptake in A, where the cytoplasmic to tubular brush border lactoglobulin CLC-5 produces a strikingly robust endo- intensity is calculated and expressed as the internalization ratio. *P,0.05 (n=3). (E) Quan- cytic defect at the apical pole of proximal tification of Alexa 546–labeled dextran uptake in B, where the cytoplasmic to tubular brush tubule cells.11 Thus far, whether a similar border dextran intensity is calculated and expressed as the internalization ratio. *P,0.05 dramatic endocytic defect results from loss (n=3). (F) Representative light microscope image of control, Ocrl KO, and cDKO mice of OCRL could not be assessed, because 9 m kidney sections stained with 3,3 -diaminobenzidine. Scale bars, 50 m. (G) Representative Ocrl KO mice did not exhibit obvious electron microscopy micrographs showing the subcellular localization of HRP in kidney pathologic manifestations of disease.23 A proximal tubules at 5 minutes after HRP injectionincontrolandcDKOmice.HRPuptake (arrowheads) is observed in control but not cDKO mice. Scale bars, 500 nm. perturbation of the endocytic pathway (primarily in the endosomal system downstream of the internalization reaction) has surface to the subapical region was observed in control and Ocrl been reported in various OCRL knockdown or KO cell mod- KO mice but not in vehicle (PBS)-injected mice (Figure 4, A and B els.15,19,21 Moderate defects in the internalization reaction of and quantified in Figure 4D). In contrast, no obvious translocation endocytosis have been observed in fibroblasts derived from

J Am Soc Nephrol 28: 1399–1407, 2017 Endocytic Defect in Ocrl/Inpp5b Knockout, Inoue et al. 1403 BASIC RESEARCH www.jasn.org

patients with Lowe syndrome.19 Additionally, an endocytic defect has been observed in the pronephric tubule of zebrafish, where Ocrl expression was diminished by morpholinos.15 It was hypothesized that the redundancy of OCRL with INPP5B in mouse kidney cells may explain the discrepancy between the mouse Ocrl KO phenotype and the clinical human manifestations observed in Lowe syndrome. Consistent with this possibility, replacement of the Inpp5b gene with the human INPP5B gene in Ocrl KO mice produced defects reminiscent of Lowe syndrome.37 However, the cellular basis of this defect was not explored. In this analysis, we show that the combined loss of Ocrl and Inpp5b in an adult mouse produces a massive endocytic defect at the apical pole of kidney proximal tubule cells. This defect may reflect the strong dependence of apical cell endocytosis on actin 38 dynamics, which in turn, is regulated by PI(4,5)P2,themainsub- strate of OCRL and INPP5B.39 Why the absence of only OCRL is sufficient to produce clinical manifestations in humans but not in mice remains to be explored further, although differences in the regulation of INPP5B expression in the two species or differential alternative splicing are the most likely explanation.26,40 The hetero- geneous phenotypic spectrum of human disease in patients with mutations in OCRL—consistent presence of kidney defects but occurrence of nervous system and/or ophthalmologic defects in only a subset of patients8,14,41—suggests that differences in INPP5B compensation may also occur in humans. We note that, in our study, minor defects at the cellular levels were observed even in mice that lack only OCRL, although clearly insufficient to produce overt impairment of kidney function. Why the endocytic defect begins mildly after doxycycline induction increases in severity over several months remains unclear. One possibility is that early defects in reabsorption may produce progressive tubular cell damage. Although Ocrl KO mice are apparently normal and Inpp5b KO mice only have fertility defects in males,28 germline deletion of both genes results in embryonic lethality.23 Because absence of both proteins in kidney proximal tubule cells does not result in the death of these cells during the first several weeks after doxycycline–induced Inpp5b gene recombination, the presence of OCRL/INPP5B function is not required for cell viability. This was also confirmed by studies in mouse fibroblasts lacking both proteins (D.M.B. and P.D.C., unpublished

of Ocrl KO kidney proximal tubules at 1 month after doxycycline induction stained with NaPi2a 15 minutes after vehicle or PTH injection. FITC-LTA is shown in green. Scale bars, 10 mm. (C) Representative immunofluorescence images of cDKO kidney proximal tubules at 1 month after doxycycline induction stained with NaPi2a 15 minutes after vehicle or PTH injection. FITC- LTA is shown in green. Scale bars, 10 mm. (D) Quantification of Figure 4. Uptake of NaPi2a after PTH treatment is impaired in NaPi2a internalization (A–C) (Supplemental Figure 2A), where the the cDKO mice. (A) Representative immunofluorescence im- cytoplasmic to tubular brush border Napi2a intensity is calculated ages of control kidney proximal tubules at 1 month after and expressed as the internalization ratio (black bar, control; blue doxycycline induction stained with NaPi2a 15 minutes after bar, Ocrl KO;andredbar,cDKOmice).*P,0.05 (n=3). (E) Plasma vehicle or PTH injection. FITC-LTA is shown in green. Scale and (F) urine PTH levels in control, Ocrl KO, and cDKO mice. bars, 10 mm. (B) Representative immunofluorescence images *P,0.05 (n=4).

1404 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1399–1407, 2017 www.jasn.org BASIC RESEARCH observations) and fits with the viability of lower organisms at 4500 rpm for 15 minutes, and the supernatant was collected and on loss of the single OCRL/INPP5B protein in Drosophila.42–45 centrifuged at 25,0003g for 30 minutes. The pellet was then resus- After 3 months of disruption of the Inpp5b locus in the Ocrl pended and centrifuged again at 25,0003g for 30 minutes, and the KO mouse (cDKO), a progressive demise of kidney function pellet was stored at 280°C until used for SDS-PAGE. was observed, which may result from indirect effects of abnormal reabsorption. A similar progressive kidney failure is Analyses of Proximal Tubule Endocytosis In Vivo observed in human Lowe syndrome and patients with Dent Bovine b-lactoglobulin (20 mg/kg body wt; Sigma-Aldrich) conjugated disease.8 withAlexaFluor546(Invitrogen)or10kDAlexaFluordextran(20mg/kg Studies of OCRL and INPP5B have shown a localization of body wt; Invitrogen) was injected via tail vein; 15 minutes after the these proteins at various stages of the endocytic pathway (in- injection of b-lactoglobulin or 10 minutes after injection of dextran, cluding clathrin-coated pitsandvesicles, micropinosomes, and mice were briefly perfused with PBS, and the kidney was removed early endosomes and lysosomes) via interactions with a variety and fixedwith4%PFA.Kidneysectionswerethenstainedwith of endocytic proteins, such endocytic Rabs (such as Rab5 and FITC-conjugated LTA, and images were acquired by a Andor Rab35), APPL1, Ses 1 and 2 (IPIP27 A and B), pacsin 2, and in CSU-WDi Spinning Disc Confocal Microscope equipped with a the case of OCRL, clathrin, and endocytic clathrin adap- Nikon Ti-E Cfi Plan Apo Lambda 603 Oil Objective. For quantifi- tors.17,18,22,25,46–49 Various perturbations of the endocytic cation of b-lactoglobulin internalization, the fluorescence overlap- pathway downstream of the endocytic reaction have been re- ping with the brush border (area of the section positive for LTA ported in human cells depleted of OCRL.21 In principle, de- fluorescence) and the fluorescence in the underlying cytosolic region fects in the endocytic reaction itself (the internalization step were calculated. Internalization was then expressed as a ratio be- mediated by fission of a membrane bud from the plasma tween the internal fluorescence and the brush border fluorescence. membrane) produced by the lack of these two closely related For quantification of dextran internalization, the total internalized inositol 5-phosphatases could be explained by the need for fluorescence signal per unit area was calculated. ImageJ (National 19 PI(4,5)P2 dephosphorylation in this process. An additional Institutes of Health [NIH]) was used for the analysis. m but not mutually exclusive possibility is that ectopic PI(4,5)P2 HRP (1.2 gineachmouse;Sigma-Aldrich)wasinjectedvia the accumulation within cells may result in the sequestration of cannulated jugular vein. Mice kidneys were perfusion fixed by periodate- endocytic factors on intracellular vesicles so that they are lysine-paraformaldehyde buffer 5 or 10 minutes after HRP injection. For no longer available for endocytosis.15,21,50 Abnormal dynamics light microscopy, 4-mm-thick frozen sections were cut, processed for of components needed for endocytosis due to the impaired HRP cytochemistry, and mounted in SlowFade (Thermo Fisher Scien- acidification of early endocytic vesicles/endosomes were also tific). For electron microscopy, small pieces of kidney cortex were further hypothesized to explain the kidney proximal tubules endo- fixed in 2.5% glutaldehyde in 0.1 M sodium cacodylate buffer, processed 11,51 fi cytic defect of Clcn5 mutant mice. for HRP cytochemistry, post xed with 2% OsO4 and 1.5% K4Fe in the In conclusion, our results prove the partially overlapping same buffer, en bloc stained with 2% uranyl acetate, embedded in Embed roles of OCRL and INPP5B in kidney proximal tubule cells. 812, and thin sectioned. Images were acquired with a Morada 1k31k They provide in vivo evidence that loss of OCRL/INPP5B function CCD Camera (Olympus) using a Philips CM10 Microscope at 80 kV. results in the pathologic manifestation of disease at least in part by Rat PTH (20 mg; Bachem) was injected intraperitoneally 2 weeks after producing a striking defect in endocytosis at the luminal pole of the end of the doxycycline induction; 15 minutes after the injection, these cells. cDKO mice may represent a useful animal model to mice were briefly perfused with PBS, and kidneys were removed and further study mechanisms of Lowe syndrome and Dent 2 disease fixed by 4% PFA. For the longer– and higher–dose PTH treatment, and test therapeutic approaches. 80 mg rat PTH was administrated intraperitoneally followed (after 30 minutes) by a 20-mg PTH injection into the tail vein. One hour after intraperitoneal injection, kidneys were removed and fixed by CONCISE METHODS 4% PFA. For quantification of internalization, the same method described above for b-lactoglobulin was used. Mice Biochemical analyses of plasma and urine and SDS-PAGE were fl fl 2 2 2 Inpp5b / mice,28 Ocrl / or Ocrly/ mice,23 and doxycycline–inducible carried out by standard procedures (Supplemental Material). Pax8rtTA TetO-cre mice27 were previously described. To induce Inpp5b gene recombination, 2.0 mg/ml doxycycline was added to drinking water Statistical Analyses (also supplemented with 5% sucrose) for 2 weeks starting at postnatal All data are presented as mean6SD. Statistical significance was week 8. determined with P value ,0.05 by two–tailed paired t test.

Brush Border Membrane Isolation Study Approval Mouse kidney proximal tubular brush border membranes were iso- All animal experiments were approved by the Yale University IACUC. lated as previously described.52 Brieﬂy, mouse kidney cortex was cut All work was carried out in accordance with the principles and and homogenized by a Polytron; 12 mM MgCl2 was added to the procedures outlined in the NIH guidelines for the care and use of lysate and incubated on ice for 15 minutes. The lysate was centrifuged experimental animals.

J Am Soc Nephrol 28: 1399–1407, 2017 Endocytic Defect in Ocrl/Inpp5b Knockout, Inoue et al. 1405 BASIC RESEARCH www.jasn.org

ACKNOWLEDGMENTS 15. Oltrabella F, Pietka G, Ramirez IB, Mironov A, Starborg T, Drummond IA, Hinchliffe KA, Lowe M: The Lowe syndrome protein OCRL1 is required for endocytosis in the zebrafish pronephric tubule. PLoS Genet We thank Dr. K. Sauer (Scripps Research Institute) and Dr. S. Somlo 11: e1005058, 2015 fl/fl – (Yale University) for providing Inpp5b and doxycycline inducible 16. Cauvin C, Rosendale M, Gupta-Rossi N, Rocancourt M, LarraufieP, rtTA Pax8 TetO-cre mice, respectively. Salomon R, Perrais D, Echard A: Rab35 GTPase triggers switch-like This work was supported in part by George O’Brien Kidney Center at recruitment of the Lowe syndrome lipid phosphatase OCRL on new- – Yale grant P30DK079310 (to T.W., P.D.C., and S.I.) and National born endosomes. Curr Biol 26: 120 128, 2016 17. Erdmann KS, Mao Y, McCrea HJ, Zoncu R, Lee S, Paradise S, Institutes of Health grants DK083294 (to S.I.) and DK 093629 (to S.I.). Modregger J, Biemesderfer D, Toomre D, De Camilli P: A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway. Dev Cell 13: 377–390, 2007 DISCLOSURES 18. Mao Y, Balkin DM, Zoncu R, Erdmann KS, Tomasini L, Hu F, Jin MM, None. Hodsdon ME, De Camilli P: A PH domain within OCRL bridges clathrin- mediated membrane trafficking to phosphoinositide metabolism. EMBO J 28: 1831–1842, 2009 REFERENCES 19. Nández R, Balkin DM, Messa M, Liang L, Paradise S, Czapla H, Hein MY, Duncan JS, Mann M, De Camilli P: A role of OCRL in clathrin-coated pit dynamics and uncoating revealed by studies of Lowe syndrome cells. 1. Attree O, Olivos IM, Okabe I, Bailey LC, Nelson DL, Lewis RA, McInnes eLife 3: e02975, 2014 RR, Nussbaum RL: The Lowe’s oculocerebrorenal syndrome gene 20. Pirruccello M, De Camilli P: Inositol 5-phosphatases: Insights from the encodes a protein highly homologous to inositol polyphosphate-5- Lowe syndrome protein OCRL. Trends Biochem Sci 37: 134–143, 2012 phosphatase. Nature 358: 239–242, 1992 21. Vicinanza M, Di Campli A, Polishchuk E, Santoro M, Di Tullio G, Godi A, 2. Bökenkamp A, Ludwig M: The oculocerebrorenal syndrome of Lowe: Levtchenko E, De Leo MG, Polishchuk R, Sandoval L, Marzolo MP, De An update [published online ahead of print March 24, 2016]. Pediatr Matteis MA: OCRL controls trafficking through early endosomes via Nephrol PtdIns4,5P₂-dependent regulation of endosomal actin. EMBO J 30: 3. Hoopes RR Jr., Shrimpton AE, Knohl SJ, Hueber P, Hoppe B, Matyus J, 4970–4985, 2011 Simckes A, Tasic V, Toenshoff B, Suchy SF, Nussbaum RL, Scheinman 22. Hyvola N, Diao A, McKenzie E, Skippen A, Cockcroft S, Lowe M: SJ: Dent Disease with mutations in OCRL1. Am J Hum Genet 76: 260– Membrane targeting and activation of the Lowe syndrome protein 267, 2005 OCRL1 by rab GTPases. EMBO J 25: 3750–3761, 2006 4. Lewis RA, Nussbaum RL, Brewer ED: Lowe syndrome. In: GeneReviews 23. Jänne PA, Suchy SF, Bernard D, MacDonald M, Crawley J, Grinberg A, (R), edited by Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya Wynshaw-Boris A, Westphal H, Nussbaum RL: Functional overlap be- A, Bean LJH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K, tween murine Inpp5b and Ocrl1 may explain why deficiency of the Seattle, WA, University of Washington, 1993 murine ortholog for OCRL1 does not cause Lowe syndrome in mice. 5. Lowe CU, Terrey M, MacLACHLAN EA: Organic-aciduria, decreased JClinInvest101: 2042–2053, 1998 renal ammonia production, hydrophthalmos, and mental retardation; a 24. Jefferson AB, Majerus PW: Properties of type II inositol polyphosphate clinical entity. AMA Am J Dis Child 83: 164–184, 1952 5-phosphatase. JBiolChem270: 9370–9377, 1995 6. Staiano L, De Leo MG, Persico M, De Matteis MA: Mendelian disorders 25. Pirruccello M, Swan LE, Folta-Stogniew E, De Camilli P: Recognition of of PI metabolizing enzymes. Biochim Biophys Acta 1851: 867–881, 2015 the F&H motif by the Lowe syndrome protein OCRL. Nat Struct Mol Biol 7. Charnas LR, Bernardini I, Rader D, Hoeg JM, Gahl WA: Clinical and 18: 789–795, 2011 laboratory findings in the oculocerebrorenal syndrome of Lowe, with 26. Bothwell SP, Farber LW, Hoagland A, Nussbaum RL: Species-specific special reference to growth and renal function. N Engl J Med 324: difference in expression and splice-site choice in Inpp5b, an inositol 1318–1325, 1991 polyphosphate 5-phosphatase paralogous to the enzyme deficient in 8. Bockenhauer D, Bokenkamp A, van’t Hoff W, Levtchenko E, Kist-van Lowe Syndrome. Mamm Genome 21: 458–466, 2010 Holthe JE, Tasic V, Ludwig M: Renal phenotype in Lowe syndrome: A 27. Traykova-Brauch M, Schönig K, Greiner O, Miloud T, Jauch A, Bode M, selective proximal tubular dysfunction. Clin J Am Soc Nephrol 3: 1430– Felsher DW, Glick AB, Kwiatkowski DJ, Bujard H, Horst J, von Knebel 1436, 2008 Doeberitz M, Niggli FK, Kriz W, Gröne HJ, Koesters R: An efficient and 9. Marshansky V, Ausiello DA, Brown D: Physiological importance of versatile system for acute and chronic modulation of renal tubular endosomal acidification: Potential role in proximal tubulopathies. Curr function in transgenic mice. Nat Med 14: 979–984, 2008 Opin Nephrol Hypertens 11: 527–537, 2002 28. Hellsten E, Evans JP, Bernard DJ, Jänne PA, Nussbaum RL: Disrupted 10. Sirac C, Bridoux F, Essig M, Devuyst O, Touchard G, Cogné M: Toward sperm function and fertilin beta processing in mice deficient in the understanding renal Fanconi syndrome: Step by step advances inositol polyphosphate 5-phosphatase Inpp5b. Dev Biol 240: 641–653, 2001 through experimental models. Contrib Nephrol 169: 247–261, 2011 29. Bacic D, Lehir M, Biber J, Kaissling B, Murer H, Wagner CA: The renal 11. Piwon N, Günther W, Schwake M, Bösl MR, Jentsch TJ: ClC-5 Na+/phosphate cotransporter NaPi-IIa is internalized via the receptor- Cl- -channel disruption impairs endocytosis in a mouse model for mediated endocytic route in response to parathyroid hormone. Kidney Dent’s disease. Nature 408: 369–373, 2000 Int 69: 495–503, 2006 12. Wrong OM, Norden AG, Feest TG: Dent’s disease; a familial proximal 30. Biemesderfer D: Regulated intramembrane proteolysis of megalin: renal tubular syndrome with low-molecular-weight proteinuria, hyper- Linking urinary protein and gene regulation in proximal tubule? Kidney calciuria, nephrocalcinosis, metabolic bone disease, progressive renal Int 69: 1717–1721, 2006 failure and a marked male predominance. QJM 87: 473–493, 1994 31. Curthoys NP, Moe OW: Proximal tubule function and response to aci- 13. Novarino G, Weinert S, Rickheit G, Jentsch TJ: Endosomal chloride- dosis. Clin J Am Soc Nephrol 9: 1627–1638, 2014 proton exchange rather than chloride conductance is crucial for renal 32. Soda K, Balkin DM, Ferguson SM, Paradise S, Milosevic I, Giovedi S, endocytosis. Science 328: 1398–1401, 2010 Volpicelli-Daley L, Tian X, Wu Y, Ma H, Son SH, Zheng R, Moeckel G, 14. Hoopes RR Jr., Raja KM, Koich A, Hueber P, Reid R, Knohl SJ, Cremona O, Holzman LB, De Camilli P, Ishibe S: Role of dynamin, Scheinman SJ: Evidence for genetic heterogeneity in Dent’s disease. synaptojanin, and endophilin in podocyte foot processes. JClinInvest Kidney Int 65: 1615–1620, 2004 122: 4401–4411, 2012

1406 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1399–1407, 2017 www.jasn.org BASIC RESEARCH

33. Gekle M: Renal tubule albumin transport. Annu Rev Physiol 67: 573– 45. Abbasi K, DuBois KN, Leung KF, Dacks JB, Field MC: A novel Rho-like 594, 2005 protein TbRHP is involved in spindle formation and mitosis in try- 34. Nielsen R, Christensen EI, Birn H: Megalin and cubilin in proximal tu- panosomes. PLoS One 6: e26890, 2011 bule protein reabsorption: From experimental models to human dis- 46. Billcliff PG, Noakes CJ, Mehta ZB, Yan G, Mak L, Woscholski R, Lowe M: ease. Kidney Int 89: 58–67, 2016 OCRL1 engages with the F-BAR protein pacsin 2 to promote bio- 35. Forster IC, Hernando N, Biber J, Murer H: Proximal tubular handling of genesis of membrane-trafficking intermediates. Mol Biol Cell 27: 90– phosphate: A molecular perspective. Kidney Int 70: 1548–1559, 2006 107, 2016 36. Claverie-Martín F, Ramos-Trujillo E, García-Nieto V: Dent’s disease: 47. Dambournet D, Machicoane M, Chesneau L, Sachse M, Rocancourt M, Clinical features and molecular basis. Pediatr Nephrol 26: 693–704, El Marjou A, Formstecher E, Salomon R, Goud B, Echard A: Rab35 2011 GTPase and OCRL phosphatase remodel lipids and F-actin for suc- 37. Bothwell SP, Chan E, Bernardini IM, Kuo YM, Gahl WA, Nussbaum RL: cessful cytokinesis. Nat Cell Biol 13: 981–988, 2011 Mouse model for Lowe syndrome/dent disease 2 renal tubulopathy. 48. Noakes CJ, Lee G, Lowe M: The PH domain proteins IPIP27A and B link JAmSocNephrol22: 443–448, 2011 OCRL1 to receptor recycling in the endocytic pathway. MolBiolCell 38. Gottlieb TA, Ivanov IE, Adesnik M, Sabatini DD: Actin microfilaments 22: 606–623, 2011 play a critical role in endocytosis at the apical but not the basolateral 49. Swan LE, Tomasini L, Pirruccello M, Lunardi J, De Camilli P: Two closely surface of polarized epithelial cells. J Cell Biol 120: 695–710, 1993 related endocytic proteins that share a common OCRL-binding motif 39. Di Paolo G, De Camilli P: Phosphoinositides in cell regulation and with APPL1. Proc Natl Acad Sci USA 107: 3511–3516, 2010 membrane dynamics. Nature 443: 651–657, 2006 50. Messa M, Fernández-Busnadiego R, Sun EW, Chen H, Czapla H, 40. Bernard DJ, Nussbaum RL: X-inactivation analysis of embryonic le- Wrasman K, Wu Y, Ko G, Ross T, Wendland B, De Camilli P: Epsin de- thality in Ocrl wt/-; Inpp5b-/- mice. Mamm Genome 21: 186–194, 2010 ficiency impairs endocytosis by stalling the actin-dependent in- 41. Bökenkamp A, Böckenhauer D, Cheong HI, Hoppe B, Tasic V, Unwin R, vagination of endocytic clathrin-coated pits. eLife 3: e03311, 2014 Ludwig M: Dent-2 disease: A mild variant of Lowe syndrome. JPediatr 51. Schwake M, Friedrich T, Jentsch TJ: An internalization signal in ClC-5, 155: 94–99, 2009 an endosomal Cl-channel mutated in dent’s disease. JBiolChem276: 42. Ben El Kadhi K, Roubinet C, Solinet S, Emery G, Carréno S: The inositol 12049–12054, 2001 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis and is necessary 52. Biber J, Stieger B, Stange G, Murer H: Isolation of renal proximal tubular for cytokinesis. Curr Biol 21: 1074–1079, 2011 brush-border membranes. Nat Protoc 2: 1356–1359, 2007 43. Loovers HM, Kortholt A, de Groote H, Whitty L, Nussbaum RL, van Haastert PJ: Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4. Traffic 8: 618–628, 2007 44. Lowe M: Structure and function of the Lowe syndrome protein OCRL1. This article contains supplemental material online at http://jasn.asnjournals. Traffic 6: 711–719, 2005 org/lookup/suppl/doi:10.1681/ASN.2016080913/-/DCSupplemental.

J Am Soc Nephrol 28: 1399–1407, 2017 Endocytic Defect in Ocrl/Inpp5b Knockout, Inoue et al. 1407 Mouse kidney tubular ablation of Ocrl/Inpp5b phenocopies Lowe syndrome tubulopathy

Supplemental experimental procedure

Antibodies and plasmids. Rabbit anti-OCRL (Sigma-Aldrich); Mouse anti-β-actin (Sangene

Biotech); Rabbit anti-retinol binding protein (DAKO); Rabbit anti-vitamin D binding protein

(Novus Biological Inc); FITC-conjugated Lotus tetragonolobus agglutinin (Vectors

Laboratories); Rabbit anti-bovine serum albumin (BSA) (EMD Millipore Corporation); Rabbit anti-parathyroid hormone (PTH) receptor 1 (Abcam); Rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell signaling technology); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen) ; and Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen) were purchased commercially. Rabbit anti-type 2a sodium phosphate cotransporter was a kind gift Dr.

H. Murer (University of Zurich, Zurich, Switzerland). Rabbit anti-megalin was a kind gift Dr. D.

Biemesderfer (Yale University, Connecticut USA). Mouse anti-NHE3 was a kind gift Dr. Peter

S. Aronson (Yale University, Connecticut USA).

Biochemical analysis of plasma and urine. Urine samples were collected and analyzed at 0, 1, 2,

3, 4 months after the two weeks of doxycycline induction. The following parameters were measured in duplicate by ELISA kits: urine calcium (Cayman Chemical), urine phosphate

(Abcam), urine albumin (Bethyl Laboratories Inc), plasma and urine creatinine, and PTH,

(Bioassay Systems, Hayward, CA and My BioSource Inc, respectively), and plasma C-terminal fibroblast growth factor 23 (cFGF23) (Immunotopics Inc). Plasma bicarbonate and pH were measured by using VetScan i-STAT 1 analyzer (ABAXIS).

Quantitative PCR analysis. Total RNA was extracted from the kidney cortex using Trizol

(Thermo Fisher scientific). Its concentration was measured by spectrophotometry (Nanodrop Technologies, Montchanin, DE). One microgram of total RNA was used for reverse-transcription

by a high-capacity cDNA Synthesis Kit according to the manufacturer’s instructions (Applied

Biosystems, Foster City, CA). Quantitative PCR amplifications were performed using power

SYBR green mix (Applied Biosystems) with a 7300 AB real-time PCR machine (Applied

Biosystems).

Immunoblotting. Freshly isolated kidney cortex were lysed in lysis buffer containing 50 mM

Tris-HCl (pH 7.6), 500 mM NaCl, 0.1 % SDS, 0.5 % deoxycholate, 1 % Triton X-100, 0.5 mM

MgCl2, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF and protease inhibitor cocktail (Roche

Diagnostics). Protein concentrations were quantified using the Bio-Rad protein assay. Equal

amount of kidney cortex proteins, renal brush border proteins, or urine proteins normalized to

urine creatinine were separated by SDS-PAGE and transferred on PVDF membranes (EMD

Millipore). Membranes were incubated with 5% nonfat milk in Tris-buffered saline and Tween

20 (TBST), incubated with the appropriate primary antibody at 4°C overnight, washed with

TBST, and incubated with the appropriate HRP-labeled anti-IgG secondary antibody (Bio-Rad).

HRP signal was developed using enhanced chemiluminescence reagents (Bio-Rad) and detected with Odyssey (LI-COR bioscience). For quantification, densitometry was performed using Image

J software (NIH).

Histology and immunofluorescence. Mice were anesthetized by intraperitoneal injection of

ketamine and xylazine followed by perfusion fixation with 4% paraformaldehyde with or without

glutaldehyde through the left ventricle. Histology [H & E, periodic acid-Schiff (PAS), and

Masson’s trichrome staining] was performed by the Yale Pathology Core Tissue Services. To

evaluate interstitial fibrosis, Masson stained kidney sections were assessed as previously

described 1. For immunofluorescence, kidney cryosections (4 μm) were exposed to 0.01% sodium borohydride (Sigma) for 30 minutes, permeablized with 0.2% Triton X-100 in TBS for

30 minutes and incubated with 3% BSA in TBS for 1 hour. Immunostaining was performed with primary antibodies overnight at 4°C, followed by Alexa Fluor 488– and/or Alexa Fluor 594– conjugated secondary antibodies and/or FITC conjugated LTA, washed, and mounted with

Fluoro Gel with Tris buffer (Electron Microscopy Science). Images were acquired by confocal microscopy. For quantification, immunofluorescence intensity in regions of interest was measured by Image J software (NIH).

References

1. Hassan, H, Tian, X, Inoue, K, Chai, N, Liu, C, Soda, K, Moeckel, G, Tufro, A, Lee, AH, Somlo, S, Fedeles, S, Ishibe, S: Essential Role of X-Box Binding Protein-1 during Endoplasmic Reticulum Stress in Podocytes. J Am Soc Nephrol, 2015.

Mouse kidney tubular ablation of Ocrl/Inpp5b phenocopies Lowe syndrome tubulopathy

Supplemental figure legend

Supplemental Figure 1. Endocytic defect in proximal tubules at 2 weeks following

doxycycline induction. (A) Representative images showing uptake of Alexa 546-labeled β-

lactoglobulin (red) of control and Ocrl KO kidney proximal tubule by immunofluorescence at 15

minutes, but was not observed in the cDKO at identical exposure time. FITC-LTA (green). Scale

bars: 10 μm. (B) Representative images showing uptake of Alexa 546–labeled Dextran (red) in

the control and Ocrl KO kidney proximal tubule by immunofluorescence at 10 minutes, but was

not observed in the cDKO at identical exposure time. FITC-LTA (green). Scale bars: 10 μm. (C)

Whole kidneys of control, Ocrl KO, and cDKO mice in (A) revealing reduced uptake of Alexa

546-labeled β-lactoglobulin in the cDKO mice

Supplemental Figure 2. Uptake of NaPi2a after PTH treatment is markedly delayed in the

cDKO mice. (A) Representative immunofluorescence images of kidney proximal tubules stained with NaPi2a (red) 60 minutes after PTH injection in control, Ocrl KO, and cDKO mice, FITC-

LTA (green) Scale bars: 10 μm. (B) Expression of PTH receptor 1 (PTHR1) in renal brush border membrane detected by immunoblotting. (C) Plasma C-terminal FGF23 (cFGF23) levels in control, Ocrl KO, and cDKO mice. *p<0.05 N=4.