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Mouse Model for Lowe Syndrome/Dent Disease 2 Renal Tubulopathy

Susan P. Bothwell,*† Emily Chan,*† Isa M. Bernardini,‡ Yien-Ming Kuo,*† William A. Gahl,‡ and Robert L. Nussbaum*†

*Department of Medicine and †Institute for Human Genetics, University of California, San Francisco, California; and ‡Medical Genetics Branch, National Research Institute, Bethesda, Maryland

ABSTRACT The Lowe oculocerebrorenal syndrome is an X-linked disorder characterized by Our previous attempt to create a congenital , cognitive disability, and proximal tubular dysfunction. Both mouse model for OCRL failed when Ϫ this syndrome and Dent Disease 2 result from loss-of-function mutations in the Ocrl mice were shown to have no renal, OCRL , which encodes a type II phosphatidylinositol bisphosphate 5-phos- ophthalmologic, or central nervous sys- phatase. Ocrl-deficient mice are unaffected, however, which we believe reflects a tem abnormalities.28 We hypothesized difference in how humans and mice cope with the enzyme deficiency. Inpp5b and that a difference between how humans INPP5B, paralogous autosomal that encode another type II phosphoinosi- and mice compensate for loss of Ocrl, tide 5-phosphatase in mice and humans, respectively, might explain the distinct rather than a difference between the spe- phenotype in the two species because they are the closest paralogs to Ocrl and cies in Ocrl function itself, is responsible OCRL in their respective genomes yet differ between the two species with regard for the difference in phenotype. We Ϫ Ϫ to expression and splicing. Here, we generated Ocrl / mice that express INPP5B turned our attention to Inpp5b, another

but not Inpp5b. Similar to the human syndromes, all showed reduced postnatal type II PtdIns4,5P2 5-phosphatase, for growth, low molecular weight , and aminoaciduria. Thus, we created an three reasons. First, human INPP5B and animal model for OCRL and Dent Disease 2 tubulopathy by humanizing a modifier mouse Inpp5b are the closest paralogs to paralog in mice already carrying the mutant disease gene . OCRL and Ocrl in the human and mouse genomes, respectively, and share nearly J Am Soc Nephrol 22: 443–448, 2011. doi: 10.1681/ASN.2010050565 all their functional domains.19 Second, Inpp5b and Ocrl overlap in function in vivo in mice; mice defective in Ocrl or The Lowe Oculocerebrorenal syndrome weight (LMW) proteinuria, but may also Inpp5b have little or no phenotype, but (OCRL; MIM #309000) is a rare X-linked include generalized aminoaciduria, hy- mice deficient in both die before implan- disorder of congenital cataracts, mental percalciuria, and bicarbonaturia.1,5 Cat- tation.28,29 Finally, Inpp5b and INPP5B retardation, and proximal tubular dys- aracts develop very early in gestation.21 differ in transcription and splicing.30 We function.1–5 OCRL is caused by loss of Neurologic abnormalities include vari- hypothesized that replacing mouse function of the OCRL gene,6–9 which en- able degrees of cognitive delay and ste- Inpp5b with human INPP5B in Ocrl-de- codes a type II phosphatidylinositol bis- reotypic behavior.1–3 ficient mice would result in mice with a

phosphate (PtdIns4,5P2) 5-phospha- The renal complications of OCRL are phenotype similar to OCRL. Here we tase.10,11 Ocrl also contains a noncatalytic similar to those of Dent disease, a renal disor- show that such mice survive to term and Rho-GTPase activating (GAP) der characterized by low molecular protein- domain with affinity for small G-pro- uria, hypercalciuria, and nephrolithiasis.22,23 Received May 28, 2010. Accepted October 6, 2010. teins,12,13 a clathrin binding domain,14 a Dent disease is genetically heterogeneous.24 pleckstrin-homology domain,16 and an Over 50% of cases of Dent disease are caused Published online ahead of print. Publication date available at www.jasn.org. ASPM, SPD-2, Hydin (ASH) domain17 by mutations in a chloride transporter gene, and interacts with APPL1, a Rab5 effector CLCN5, involved in endosomal acidification Correspondence: Dr. Robert L. Nussbaum, 513 Par- 18,19 nassus Avenue, UCSF Box 0794, San Francisco, CA protein, and the endosomal and are referred to as Dent Disease 1 (DD1), 94143. Phone: 415-476-3200; Fax: 415-502-0720; Ses1 and Ses2.20 whereas approximately 20% of Dent disease E-mail: [email protected]

The renal tubular dysfunction in patients have mutations in OCRL and are re- Copyright © 2011 by the American Society of OCRL invariably includes low molecular ferred to as Dent Disease 2 (DD2).23,25–27 Nephrology

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have cardinal features of the renal tubu- line C showed significantly reduced post- lopathy seen in OCRL and DD2. natal growth compared with 1M mice by Seven founder mice expressing and 4 weeks of age (Figure 1). This growth transmitting INPP5B were generated by defect was replicated in lines F and G oocyte injection of a bacterial artificial (data not shown). (BAC) containing INPP5B. The single most consistent renal ab- Three of these lines of mice (lines C, F, and normality in Lowe syndrome is LMW Ϫ Ϫ G) were bred with Inpp5b / mice31 and proteinuria.5 1H mice from line C had Ϫ subsequently with Ocrl mice28 to gener- substantial LMW proteinuria detectable ate live-born OcrlϪ/Ϫ;Inpp5bϪ/Ϫ females by Coomassie (Figure 2) and silver (data and OcrlϪ/y;Inpp5bϪ/Ϫ males that were ei- not shown) stain of a sodium dodecyl sul- ther heterozygous or homozygous for hu- fate–polyacrylamide gel electrophoresis in man INPP5B (referred to subsequently as the region between albumin and the major 1H or 2H mice, respectively). The live birth urinary protein (Mup1). By comparison, of 1H and 2H mice demonstrated that hu- 2H mice from line C showed no LMW pro- man INPP5B complements the embryonic teinuria. For comparison, 1M and vari- lethality in OcrlϪ/Ϫ;Inpp5bϪ/Ϫ mice, and ous control mice did not have LMW insertion of the BAC into the genomes of proteinuria. 1H mice made from lines the mice was not deleterious. Controls F and G mice replicated the LMW pro- were OcrlϪ/Ϫ mice that were either teinuria pattern found in line C mice Inpp5bϩ/ϩ or Inpp5bϩ/Ϫ (referred to as (data not shown). 2M or 1M mice, respectively).31 We confirmed and quantified the We measured the expression of generalized LMW proteinuria by West- Figure 1. Early growth retardation in 1H INPP5B in the transgenic mouse line C ern blot analysis of secretoglobin (CC16) mice. Mouse weights (mean ϩ SEM) for 1H relative to endogenous Inpp5b with and vitamin D binding protein (DBP), (n ϭ 14), 2H (n ϭ 10), and 1M (n ϭ 10) mice quantitative reverse transcriptase PCR which are typically seen in the in established from transgenic line C. Mice (Table 1). The amount of INPP5B mes- low molecular weight proteinuria. 1H were weighed weekly from 4 to 16 weeks. sage in 1H mice was 4.4-fold greater and mice from line C had obvious vitamin Growth curves were fitted empirically with the amount in 2H mice was 9.4-fold DBP and CC16 in their urine (Figure 3, A cubic equations centered at 10 weeks and greater than the amount of Inpp5b mes- and B). The CC16 proteinuria was quan- the null hypothesis that the curve for 1M could be described with the same four co- sage in 2M mice. These data suggest that tified by densitometry of the Western efficients as either 1H or 2H could be re- the BAC in this line inserted in multiple blot of urine from the C line (in arbitrary jected at P Ͻ 0.0001, whereas the null tandem copies. Whether this difference units), with 2M arbitrarily set to 1. Urine hypothesis that 1H and 2H could be fitted in mRNA levels translated into greater from 1H mice measured 19 as compared with a curve with the same four coefficients amounts of enzyme could not be deter- with 3 for 2H and 1 for 1M. The 2H line C could not be rejected (P ϭ 0.1335). 2M mined because of the lack of suitable an- and F mice had no DBP, whereas 2 of 8 mice were published previously and had tibody. 2H mice from line G had trace amounts no growth deficit compared with wild-type Patients with Lowe syndrome have a of DBP (data not shown). 1M and 2M mice.24 normal birth weight but are below the mice from all three lines (C, F, and G) 3rd percentile for height by 3 years of showed no DBP or CC16. amino acids.1 DD2 patients also have age.32 At birth, mice of each genotype Generalized aminoaciduria is seen in generalized aminoaciduria with promi- were largely indistinguishable in size, al- both OCRL and DD2, but is significantly nent glycinuria.23 1H mice from line C though occasionally a few of the neonatal more frequent in OCRL (82%) than in had significant increases in urine amino 1H mice, but not the 2H or 1M mice, DD2 (52%) patients.23 There is increased acids compared with 2M mice in a pat- were runted and died by 6 weeks of age. fractional excretion of , , tern similar, but not identical, to what is The majority of the 1H and 2H mice of and cysteine, less so for branched chain seen in OCRL and DD2 patients (Figure 4,

Table 1. Quantitative PCR of INPP5B, Inpp5b, and Hprt transcripts in kidney tissue of 2M, 1H, and 2H mice INPP5B or Inpp5b expression Genotype N Ct INPP5B ؎ SEM Ct Inpp5B ؎ SEM Ct Hprt ؎ SEM as%ofHprt expression ؎ 2 SEM 2M 5 28.06 Ϯ 0.24 21.99 Ϯ 0.05 1.7 Ϯ 0.4% 1H 4 25.53 Ϯ 0.15 22.11 Ϯ 0.11 7.5 Ϯ 0.6% 2H 5 24.50 Ϯ 0.13 22.29 Ϯ 0.08 16 Ϯ 2%

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Figure 2. Low molecular weight protein- uria. Coomassie stain of urine from 2-month-old mice with genotypes as fol- ϩ Ϫ ϩ lows: Lane 1, male Inpp5b / ;Ocrl /y; ϩ Ϫ Ϫ BAC . Lanes 2 and 5, female Inpp5b / ; ϩ ϩ Ϫ Ocrl / ;BAC . Lane 3, male 1M. Lane 4, male 2H. Lanes 6 and 7, 1H. Lane 8, FVB wild-type mouse. Solid line rectangle out- lines the LMW proteinuria in two indepen- dent 1H mice, whereas the dotted line rectangle indicates the absence of LMW proteinuria in a 1M and a 2H mouse. Equal amounts of total urine protein were loaded per lane.

Figure 4. Aminoaciduria. concentrations (mM) relative to (mM) from 4-month-old 2M (n ϭ 8),1M(n ϭ 6),2H(n ϭ 5), and 1H (n ϭ 10) mice. (A) Amino acids showing highly significant differences (P Ͻ 0.001) between 2M and 1H mice and a strong correlation of aminoaciduria versus genotype (increasing from 2M to 1M to 2H to 1H). (B) Amino acids showing significant differences (P Ͻ 0.05) between 2M and 1H mice and a strong correlation of aminoaciduria versus genotype. (C) Amino acids showing little or no significant differences between 2M and 1H mice but with 7 of 10 Figure 3. High urinary levels of two pro- demonstrating significant (P Ͻ 0.05) correlation of aminoaciduria versus genotype. teins characteristic of low molecular weight proteinuria. (A) Western blot analysis of urine with anti-secretoglobin (CC16) from four mice of each genotype: 1H mice There were no histologic differences at the apical surface of renal proximal tu- (lanes 1, 2, 9, and 10), 2H mice (lanes 3, 4, between 1H and 2M mice in H&E bules.34–36 Megalin and cubilin recycle 11, and 12), 1M mice (lanes 5, 6, 13, and 14), and 2M mice (lanes 7, 8, 15, and 16). stained sections of the kidney examined between the apical plasma membrane Also shown is the signal from the Mup1 by light microscopy (data not shown), and early/recycling endosomes. Reduced protein due to nonspecific antibody bind- similar to what is seen in young OCRL amounts of megalin at the brush border ing caused by the massive amount of patients.33 There were also no cataracts were observed in a kidney sample from Mup1 normally present in the urine of male by light microscopy. This may be either one of two patients with DD1.37 DD1 mice. (B) Western blot analysis of urine because overexpression of INPP5B in and OCRL patients both have reduced with anti–vitamin D binding protein of the our mouse lines was adequate to allow urine levels of the proteolytic cleavage same urine samples, loaded in the same normal lens development or lens devel- products of megalin that are normally order, as was used in (A). opment is different enough between hu- generated in the apical brush border, mans and mice to make mouse lens de- suggesting they may have reduced A through C). Nine amino acids were sig- velopment independent of both Ocrl and amounts of megalin at the apical sur- nificantly elevated in the urine of 1H versus Inpp5b. face.38 The Clcn5 knockout mouse model 2M mice (Table 2). For 16 of 19 amino ac- Low molecular weight proteins are of DD1 suggested decreased megalin at ids (panels A and B), urinary amino acids absorbed from the proximal tubule by the apical surface was due to defective correlated significantly with genotype, in- clathrin-mediated endocytosis by two megalin recycling, caused by a failure to creasing from 2M to 1M to 2H to 1H mice. receptors, megalin and cubilin, present acidify endosomes.39 Given the intracel-

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Table 2. Statistical significance of comparisons of urine amino acid intron boundaries of INPP5B were per- concentration relative to creatinine in 2M, 1M, 2H, and 1H mice formed to confirm location and integrity of Tukey Multiple Comparison INPP5B. Amino Acid ANOVA Correlationa Test for 1H versus 2M Ala Ͻ0.0001 Ͻ0.001 Ͻ0.0001 BAC Transgenic Mice Gly 0.0009 Ͻ0.001 Ͻ0.0001 BAC RP11-431J04 was introduced into fertil- His Ͻ0.0001 Ͻ0.001 Ͻ0.0001 ized FVB/N oocytes by pronuclear injection Lys 0.0002 Ͻ0.001 0.0012 as described previously.40 Thr 0.0008 Ͻ0.001 Ͻ0.0001 Ͻ Ͻ Cys 0.0001 0.05 0.0007 Genotyping, Interphase FISH, and Ͻ Met 0.0182 0.05 0.0004 Expression Analysis in Transgenic Ser 0.0165 Ͻ0.05 Ͻ0.0001 Mice Val 0.0064 Ͻ0.05 0.0009 Whole BAC was labeled and used in inter- Arg 0.189 NS 0.048 phase FISH as described previously.41 For Asn 0.241 NS NS Asp 0.198 NS NS INPP5B expression studies in offspring of the Glu 0.0421 NS 0.0011 various founder mouse lines, brain and kid- Gln 0.0574 NS 0.010 neys were dissected from mice and stored at Ile 0.012 NS 0.023 4°C overnight in RNAlater (Ambion). RNA Leu 0.090 NS 0.0058 was isolated from tissues using Trizol (Invit- Orn 0.077 NS NS rogen) and converted to cDNA using a First Phe 0.080 NS 0.0047 Strand cDNA Synthesis kit with random Tyr 0.130 NS 0.0289 primers (GE Healthcare) according to the ϭ Ն NS P 0.05. manufacturer’s instructions and then sub- aSpearman correlation of urine amino acid concentration relative to creatinine versus arbitrary numerical genotype ranking, with 2M ϭ 1, 1M ϭ 2, 2H ϭ 3, and 1H ϭ 4. jected to PCR. lular location of Ocrl, its PtIns 5-phos- clinical manifestations of Lowe syn- Quantitative Reverse-Transcriptase phatase activity, and its binding partners, drome, created by ablating the mouse or- PCR loss of Ocrl might interfere with megalin tholog of the disease gene in combina- Quantitative PCR of INPP5B, Inpp5b, and recycling by a different mechanism, that tion with humanizing a compensatory Hprt were performed by TaqMan assay (Ap- is, by impairing the progression of mega- mouse paralog of the disease-causing plied Biosystems) according to the manufac- lin-containing endosomes through the gene. These mice are a model system in turer’s instructions using Mm01182224_m1 early endocytic compartment. which to study how a defect in the Ocrl for Inpp5b, Hs00413235_m1 for INPP5B, and Except for the absence of a clathrin phosphatase leads to the tubular defects as control Mm01318743_m1 for Hprt. Equiv- binding domain in Inpp5b present in seen in OCRL and DD2 and provides an alent amplification efficiencies for INPP5B, Ocrl, the two enzymes are very similar. animal model to test potential therapies Inpp5b, and Hprt were confirmed by the serial Human INPP5B can complement the for these disorders. dilution method.42 embryonic lethality in Inpp5bϪ;OcrlϪ mice, but compensation is not complete Urine Protein Analysis Ϫ Ϫ because Inpp5b ;Ocrl mice hemizy- CONCISE METHODS Spot urine samples were collected for protein gous for INPP5B insertion develop a re- stain, Western blot analysis, and amino acid nal phenotype typical of OCRL and DD2. Mouse Strains analysis. Protein concentration was mea- The degree of compensation is, however, Mice with disruption of Inpp5b are a 129/Sv sured by BCA Protein Assay (Pierce) accord- dose-dependent because 2H mice, which and FVB/N mixture.31 Ocrl-deficient mice are ing to the manufacturer’s instructions. have double the gene dosage of INPP5B in a 129/Sv and C57BL/6 mixed background. Twenty micrograms of protein were boiled in compared with 1H mice, have markedly Both strains have been previously described.27 loading buffer and separated by electropho- reduced LMW proteinuria and amino- Animals were housed and studied according resis ona4to20%(Coomassie blue or silver aciduria. to NIH Guidelines for the Care and Use of stain), 12% (DBP Western blot), or 10 to 20% We have shown that mice expressing Laboratory Animals under UCSF Protocols (CC16 Western blot) polyacrylamide gel. Sil- human INPP5B on an Inpp5b- and Ocrl- AN076327 and AN81551. ver and Coomassie stains were performed ac- deficient background consistently dem- cording to the manufacturer’s instructions (In- onstrate the most common tubular ab- Analysis of BAC RP11؊431J04 vitrogen). Gels for Western blots were normalities seen in Lowe syndrome and BAC RP11Ϫ431J04 was obtained from Chil- transferred to a polyvinylidene fluoride mem- Dent Disease 2: LMW proteinuria and dren’s Hospital of Oakland Research Institute brane (Bio-Rad) and probed with rabbit poly- generalized aminoaciduria. This is the (CHORI). Restriction mapping and sequenc- clonal antibodies against DBP (Dako, 1:5000) first animal model of one of the major ing of both BAC ends and all exons and exon/ or CC16 (Abcam, 1:1000).43 A horseradish per-

446 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 443–448, 2011 www.jasn.org BRIEF COMMUNICATION oxidase–linked sheep anti-mouse antibody (GE Author contributions: S.B. wrote the manu- 8. Lin T, Orrison BM, Suchy SF, Lewis RA, Nuss- Healthcare, 1:2000) was used as the secondary script, performed the majority of the experi- baum RL: Mutations are not uniformly dis- tributed throughout the OCRL1 gene in antibody. Bands were visualized using chemilu- ments, and analyzed much of the data; E.C. Lowe Syndrome patients. Mol Genet Metab minescence (GE Healthcare). performed and analyzed the three crosses 64: 58–61, 1998 used to create the 1H, 2H, 1M, and 2M mouse 9. Monnier N, Satre V, Lerouge E, Berthoin F, Amino Acid Measurements lines from transgenic lines C, F, and G; Y.- Lunardi J: OCRL1 mutation analysis in French Spot urine was pooled from collections over 3 M.K. obtained all tissues from animals, pro- Lowe syndrome patients: Implications for mo- lecular diagnosis strategy and genetic coun- consecutive days for amino acid measure- vided S.B. with advice on laboratory work, ϭ seling. Hum Mutat 16: 157–165, 2000 ments in 4-month-old M2 mice (n 8), M1 contributed to preparation of the manu- 10. Suchy SF, Olivos-Glander IM, Nussbaum RL: mice (n ϭ 6), H2 mice (n ϭ 5), and H1 mice script, and provided critical reading of the Lowe Syndrome, a deficiency of a phospha- (n ϭ 10). Amino acids were measured on a manuscript; I.B. and W.G. performed and tidylinositol 4,5-bisphosphate 5-phospha- Biochrom 30 amino acid analyzer (Cam- analyzed amino acid and creatinine quantita- tase in the Golgi apparatus. Hum Mol Genet 4: 2245–2250, 1995 bridge, U.K.), which uses an ion-exchange tion in urine and provided critical reading of 11. Zhang X, Jefferson AB, Auethavekiat V, Maje- column with postcolumn ninhydrin derivati- the manuscript; R.N. designed the overall ex- rus PW: The protein deficient in Lowe syn- zation. Creatinine was measured by the Jaffe periments, joined with S.B. in analyzing the drome is a phosphatidylinositol-4,5-bisphos- method using a kit from TECO Diagnostics data, and wrote a substantial part of the man- phate 5-phosphatase. Proc Natl Acad Sci USA according to manufacturer’s instructions uscript. 92: 4853–4856, 1995 (Anaheim, CA). The amino acid concentra- 12. Faucherre A, Desbois P, Satre V, Lunardi J, Dorseuil O, Gacon G: Lowe syndrome pro- tions were calculated as mM amino acid/ mM tein OCRL1 interacts with Rac GTPase in the creatinine. DISCLOSURES trans-Golgi network. Hum Mol Genet 12: None. 2449–2456, 2003 Histologic Analysis 13. Lichter-Konecki U, Farber LW, Cronin JS, Tissues were dissected from mice and fixed in Suchy SF, Nussbaum RL: The effect of mis- sense mutations in the RhoGAP-homology fresh 4% paraformaldehyde overnight. They REFERENCES domain on ocrl1 function. Mol Genet Metab were dehydrated and embedded for section- 89: 121–128, 2006 ing followed by staining with hematoxylin 1. Charnas L, Bernardini I, Rader D, Hoeg J, Gahl 14. Choudhury R, Noakes CJ, McKenzie E, Kox and eosin. WA: Clinical and laboratory findings in the C, Lowe M: Differential clathrin binding and oculocerebrorenal syndrome of lowe, with subcellular localization of OCRL1 splice iso- special reference to growth and renal function. forms. J Biol Chem 284: 9965–9973, 2009 Statistical Analysis New Engl J Med 324: 1318–1325, 1991 15. Choudhury R, Diao A, Zhang F, Eisenberg E, Statistical analysis was performed in Prism 2. 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McCrea HJ, Paradise S, Tomasini L, Addis drome: A selective proximal tubular dys- M, Melis MA, De Matteis MA, De Camilli P: ACKNOWLEDGMENTS function. Clin J Am Soc Nephrol 3: 1430– All known patient mutations in the ASH- 1436, 2008 RhoGAP domains of OCRL affect targeting This study was generously supported by The 6. Attree O, Olivos IM, Okabe I, Bailey LC, and APPL1 binding. Biochem Biophys Res Nelson DL, Lewis RA, McInnes RR, Nuss- Commun 369: 493–499, 2008 Lowe Syndrome Trust, U.K. (S.P.B., E.C., and baum RL: The Lowe oculocerebrorenal syn- 19. Erdmann KS, Mao Y, McCrea HJ, Zoncu R, R.L.N.), intramural NHGRI/NIH (I.M.B. drome gene encodes a novel protein highly Lee S, Paradise S, Modregger J, Biemesder- and W.A.G.), and institutional support from homologous to inositol polyphosphate-5- fer D, Toomre D, De Camilli P: A role of the the Department of Medicine and the Institute phosphatase. 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