BASIC RESEARCH www.jasn.org Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model Megan L. Gliozzi,1 Eugenel B. Espiritu,2 Katherine E. Shipman,1 Youssef Rbaibi,1 Kimberly R. Long,1 Nairita Roy,3 Andrew W. Duncan,3 Matthew J. Lazzara,4 Neil A. Hukriede,2,5 Catherine J. Baty,1 and Ora A. Weisz1 1Renal-Electrolyte Division, Department of Medicine, 2Department of Developmental Biology, and 3Department of Pathology, McGowan Institute for Regenerative Medicine, and Pittsburgh Liver Research Center, Pittsburgh, Pennsylvania; 4Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia; and 5Center for Critical Care Nephrology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania ABSTRACT Background Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL,which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically character- ized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn’tclear. Methods We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish. Results OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment. Conclusions Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients. JASN 31: 67–83, 2020. doi: https://doi.org/10.1681/ASN.2019020125 The X-linked disease Lowe syndrome (LS) is caused by Understanding how the loss of OCRL impairs PT mutations in the gene OCRL that encodes the phos- function has been challenging. Because phosphati- phatidylinositol 59-phosphatase OCRL. Individuals dylinositol 4,5-bisphosphate [PtdIns(4,5)P2], the with LS exhibit congenital cataracts, hypotonia, intel- lectual disability, and renal proximal tubule (PT) dys- Received February 8, 2019. Accepted September 24, 2019. function. Low molecular weight (LMW) proteinuria is observed within the first few months after birth, and E.E. and K.E.S. contributed equally to this work. renal tubular acidosis (RTA), hypercalciuria, and ami- Published online ahead of print. Publication date available at noaciduria are also commonly observed.1 Although www.jasn.org. fi their renal dysfunction has commonly been classi ed Correspondence: Dr. Ora A. Weisz, Renal Electrolyte Division, as Fanconi syndrome, glycosuria is strikingly absent in University of Pittsburgh Medicine, 978.1 Scaife Hall, 3550 Ter- patients with LS.2 Most patients with LS ultimately race Street, Pittsburgh, PA 15261. Email: [email protected] develop ESRD, often within the first 20 years of life.1 Copyright © 2020 by the American Society of Nephrology JASN 31: 67–83, 2020 ISSN : 1046-6673/3101-67 67 BASIC RESEARCH www.jasn.org substrate of OCRL, is a major regulator of endocytosis, it has Significance Statement been anticipated that LMW proteinuria in patients with LS is due to some deficient function along the PT apical endocytic Patients with Lowe syndrome (LS) develop abnormalities in proxi- pathway.3 Consistent with this, a subset of studies in cultured mal tubule function that cause declining kidney function. Mutations in the OCRL gene encoding cause LS. The enzyme encoded by cell models demonstrate that OCRL plays a role in endocytic OCRL fi hydrolyzes phosphatidylinositol 4,5-bisphosphate, a lipid recycling, speci cally by preventing the depolymerization of that regulates myriad cellular functions. The authors created a actin coats that accumulate on endocytic vesicles and/or recy- knockout model of OCRL in human proximal tubule cells and ob- cling compartments.4,5 However, OCRL also has many other served that loss of OCRL impairs cell proliferation and results in the roles in cell homeostasis, including ciliary biogenesis,6–8 cell accumulation of multinucleated cells. Lrp2-positive segments of the ocrl polarity, and autophagy.6,9,10 Moreover, OCRL is recruited to pronephric kidney were shorter in mutant and morphant ze- brafish. The authors also created a mathematic model that explains 11 the site of abscission during cytokinesis. PtdIns(4,5)P2 ac- how loss of functional OCRL can lead to the kidney manifestations cumulation stabilizes the intracellular bridge during cytoki- of LS. The authors hypothesize that impaired cell proliferation re- nesis, and its hydrolysis by OCRL is necessary for abscission.11 sults in a shorter proximal tubule in patients with LS and that this It remains unclear how impairment of these functions con- contributes to proteinuria. tributes to LS pathology. Another unanswered question is 2+ 2 how loss of OCRL impairs PT recovery of Ca , HCO3 , and effects were rescued upon expression of wild-type (WT), but amino acids in patients with LS. not disease-causing mutants, of OCRL. Consistent with our fi Mouse and zebra sh models for LS have been developed previous studies, we found no defect in constitutive or flow- in recent years, but a link between the molecular and cellular regulated endocytosis in these cells.22–24 Careful examination defects observed in cell culture and the phenotype of patients of ocrl mutant zebrafish revealed that the length of the megalin fi and animal models remains elusive. Transgenic zebra sh (lrp2)-expressing segment of the pronephric kidney was re- lacking OCRL exhibit reduced levels of megalin, reduced up- duced. This effect was phenocopied by morpholino fl take of a uid phase marker, and fewer subapical vesicles in (MO)-based knockdown of ocrl. We propose that impaired the pronephric kidney PT, in addition to eye and facial defects cell division during development and/or repair after injury 8,12 consistent with those observed in patients with LS. Devel- lead to shorter PTs in patients with LS, resulting in reduced opment of a mouse LS model has been more complicated. recovery of filtered proteins, ions, and other molecules by the OCRL knockout (KO) mice have no overt phenotype, osten- PT. Mathematic modeling suggests that a reduction in the sibly because they express high levels of INPP5B, another lengthofhumanPTscanaccountfortheurinaryexcretion 9 phosphatidylinositol 5 -phosphatase that apparently com- profile of albumin and LMW proteins (LMWPs) of patients 13–15 pensates for some OCRL functions. INPP5B in the PT with LS. Our data suggest a comprehensive new model for of mice is expressed at higher levels and with different splice how impaired development and/or repair of the PT contrib- 16 variants compared with humans. Because global KO of both ute to LMW proteinuria and transport defects in LS. More- 17,18 Ocrl and Inpp5b in mice is lethal, an LS mouse model was over, dynamic remodeling of nephron segment length by generated by crossing Ocrl KO mice that overexpress human physiologic, pharmacologic, or dietary maneuvers provides INPP5B with a mouse Inpp5b KO: the resulting male mice a possible approach to slow kidney disease progression in pa- exhibited modest proteinuria and aminoaciduria starting at tients with LS. 8 weeks of age.19,20 A more recent mouse model has been described where Inpp5b was conditionally inactivated in the kidney of Ocrl KO mice.21 PT cells in these mice expressed METHODS reduced levels of megalin and showed profoundly impaired endocytosis. Surprisingly, proteinuria was not observed im- Cell Culture mediately after KO, but rather required several months to All cell culture reagents were from Sigma unless otherwise develop. This temporal lag is inconsistent with a direct effect specified. HK-2 cells (RRID:CVCL_0302; Homo sapiens, of OCRL on endocytic pathway function and suggests that adult male, cortex/PTs, papilloma immortalized) were obtained additional changes that occur over longer time periods con- from ATCC. The cells were cultured in DMEM/F12 with tribute to the LS phenotype. Additionally, the need to target 5 mg/ml insulin, 0.02 mg/ml dexamethasone, 0.01 mg/ml sele- both Ocrl and Inpp5b to observe any renal phenotype com- nium, 5 mg/ml transferrin, 2 mM L-glutamine, and 10% FBS plicates the effort to pinpoint the specific role(s) of OCRL (Atlanta Biologicals). Human control fibroblasts and fibro- in PT function. blasts from patients with LS were obtained from Dr. Robert To investigate how loss of OCRL affects PT function, we NussbaumandculturedinDMEMwith2mML-glutamine generated chronic CRISPR/Cas9 OCRL KO and acute siRNA and 10% FBS. Conditionally immortalized mRPTC cells knockdown models of LS in PT cells. Strikingly, in all of our (Mus musculus)wereprovidedbyDr.RoyZentandcul- models,aswellasinpatientfibroblasts, we observed that loss tured in DMEM/F12 with 5 mg/ml insulin, 50 ng/ml hy- of functional OCRL lengthened the duration of cell division drocortisone, 5 mg/ml selenium, 5 mg/ml transferrin, 6.5 ng/ml and caused the accumulation of multinucleated cells. These triiodothyronine, 2 mM L-glutamine, 2.5% FBS, and 92 mg/ml 68 JASN JASN 31: 67–83, 2020 www.jasn.org BASIC RESEARCH D-valine, as described.25 RPTEC/TERT1 cells (RRID:CVCL_K278; The forward and reverse oligos were phosphorylated and an- H. sapiens, adult male, cortex/PTs, hTERT immortalized) nealed as described in the previous paragraph for pX330 were obtained from ATCC and cultured in DMEM/F12, plasmid subcloning.