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Effects of Proximal Tubule Shortening on 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 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 -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 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 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 , 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. The pHRS plasmid was digested with 5 pM triiodothyronine, 10 ng/ml human EGF, 5.0 mg/ml EcoR1-HF (New England Biolabs) and Bam1H-HF (New m ascorbic acid, 5.0 g/ml insulin, 25 ng/ml PG E1 (EMD Milli- England Biolabs) in CutSmart Buffer (New England Biolabs) pore), 25 ng/ml hydrocortisone, 8.65 ng/ml sodium selenite, at 37°C for 30 minutes and purified after agarose gel electro- 0.1 mg/ml G418 (Corning), and 1.2 g/L sodium bicarbonate phoresis using the Wizard SV Gel and PCR Clean-Up System (Thermo Fisher Scientific). (Promega) according to the manufacturer’s protocol. Annealed oligo duplexes (1:250 dilution) were ligated into the purified Antibodies pHRS vector using T7 DNA ligase at ambient temperature for Mouse monoclonal OCRL antibody (1:5 western blot) was a 30 minutes according to the manufacturer’s protocol. To pre- gift from Drs. Robert Nussbaum and Sharon Suchy.26 Western vent unwanted recombination products, ligation products of blotting primary antibodies also included mouse monoclonal both pX330 and pHRS were treated with PlasmidSafe ATP- actin antibody (1:5000 of approximately 2 mg/ml stock, dependent DNase (Epicentre) at 37°C for 30 minutes. A1978; Sigma) and rabbit polyclonal INPP5B antibody Cleaned ligation products (2.5 ml) were transformed into (1:500 of approximately 0.8 mg/ml stock, 15141–1-AP; One Shot Stbl3 Chemically Competent E. coli (Thermo Fisher ProteinTech). Western blot secondary antibodies used Scientific) according to the manufacturer’s protocol. Transfor- were ECL Mouse IgG, HRP-linked whole Ab (1:5000, mation products were seeded onto ampicillin (pX330) or kana- NA931; GE Healthcare) and ECL Rabbit IgG, HRP-linked mycin (pHRS) LB agar plates and cultured overnight. Plasmids whole Ab (1:5000, NA934; GE Healthcare). Primary cilia were isolated and purified using the QIAprep spin Miniprep were labeled using mouse monoclonal anti-acetylated tubulin Kit (Qiagen) and the QIAGEN Plasmid Maxi Kit (Qiagen) antibody (1:400 of approximately 1 mg/ml stock, T7451; according to the manufacturer’s protocol. Oligonucleotide Sigma) followed by goat anti-mouse Alexa Fluor 568 conju- incorporation was confirmed by Sanger sequencing through gated antibody (1:500 of a 2 mg/ml stock, A11031; the University of Pittsburgh Genomics Research Core using Invitrogen). the hU6 (pX330) and pCMV (pHRS) forward sequencing primers. Sequencing data were analyzed using CLC Genomics CRISPR/Cas9 OCRL KO Clones Workbench (Qiagen). CRISPR/Cas9 KOclones were generated using modifications Approximately 63105 HK-2 cells were seeded in six-well of the approach described previously.27 AsingleguideRNA plates and immediately transfected with 2.5 mgeachofpX330 (sgRNA) targeting exon 13 of OCRL was selected using the and pHRS plasmids per well using 6 ml Lipofectamine 3000 CHOPCHOP design tool28 (Supplemental Table 1). Although (Invitrogen), 10 ml P3000 reagent, and 0.5 ml OptiMEM ac- no off-target effects were found via CHOPCHOP, NCBI nu- cording to the manufacturer’s protocol. Growth medium was cleotide BLASTof our sgRNA sequence against the GRCh38.p2. changed 12 hours after transfection and cells were grown for edition of the was completed for a more con- 48–72 hours with one medium change per day. Cells were servative assessment. Blastn was used to identify any similar then trypsinized with TrypLE Select (Thermo Fisher Scien- sequences with intact PAM sequences. We found no important tific) for 10 minutes, centrifuged at 800 3 g for 3 minutes, potential off-target effects; top hits are listed in Supplemental and resuspended in PBS supplemented with 10% FBS. Sus- Table 2. Standard desalted oligos obtained from Integrated pended cells were passed through a 50-mm Falcon filter (BD DNA Technologies were subcloned into the pX330 CRISPR/Cas9 Biosciences) to ensure single-cell suspension. Cells were an- plasmid (Addgene). The forward and reverse oligos were first alyzed and sorted in a BD FACSAria II (BD Biosciences), with phosphorylated and annealed as follows: 1 mlofeacholigo FACSDiva version 8 software. GFP was detected using the (100 mM), 1 mlof103 T4 ligation buffer (New England 488-nm blue laser with 505LP mirror and the 530/30BP fil- Biolabs), 6.5 mlddH2O, and 0.5 mlT4PNK(NewEngland ter; RFP was detected using the 488-nm blue laser with 595LP Biolabs) with a thermocycler setting at 37°C for 30 minutes, then mirror and 610/20BP filter. RFP/GFP double-positive cells 95°C for 5 minutes and ramping down to 25°C at 5°C/min. The were collected in growth medium in 96-well plates to isolate annealed oligo duplexes were cloned into the pX330 vector clonal populations. in a single-step digestion ligation reaction containing 100 ng Clonal cells were lysed using 23 lysis buffer containing 5% pX330, oligo duplex (1:150 dilution), 1 mlFastDigestBbsI b-mercaptoethanol, and analyzed for OCRL expression by (Thermo Fisher Scientific), 0.5 ml T7 DNA ligase (New Eng- western blot. Clones with no detectable OCRL expression land Biolabs), and ddH2O in a total volume of 20 mlwith were subjected to T7 and sequencing analyses thermocycler settings at 37°C for 5 minutes and 23°C for to confirm genomic editing. Genomic DNA was extracted from 5 minutes (six cycles total). approximately 13105 cells using QuickExtract DNA extraction The same sgRNA sequence with added PAM sequence for solution (Epicentre) according to the manufacturer’sprotocol. binding/activation (Supplemental Table 1) was in- For sequencing analysis, extracted clonal DNA was ampli- serted into the pHRS surrogate reporter plasmid (PNA Bio Inc.). fied by PCR using GoTaq Green Master Mix (Promega) for a

JASN 31: 67–83, 2020 Kidney Pathogenesis in Lowe Syndrome 69 BASIC RESEARCH www.jasn.org

50-ml reaction using 5 ml genomic DNA. The same primer se- mono-, bi-, and multinucleated cells with clearly visible cell quences and touchdown PCR thermocycler settings were used as borders were counted in each image. Studies in siRNA-transfected above in the T7 assay methods. Products were electrophoresed mRPTC and RPTEC/TERT1 cells and in fibroblasts from pa- on a 1% agarose gel and purified using the Wizard SV Gel tients with LS and control fibroblasts were conducted and and PCR Clean-Up System, then ligated into the pGEM-T analyzed in the same manner, except that 23105 cells were Easy Vector Systems plasmid (Promega). The ligation prod- plated per coverslip. uct was transformed into MAX Efficiency DH5a Competent Cells (Invitrogen) according to the manufacturer’s protocol. OCRL Rescue Experiments Transformation products were seeded onto ampicillin LB agar Approximately 13105 HK-2 cells were transfected with 1 mg plates (100 ml). Plasmid isolation and purification were performed plasmid DNA containing GFP-tagged empty vector (pGFP-N1), using the QIAprep spin Miniprep Kit. Fifteen separate isolated WT OCRL-GFP (pEGFP-C1), or G304E mutant OCRL-GFP plasmids for each clone were sequenced using Sanger Sequenc- (pEGFP-C1) constructs22 and plated on 12-mm coverslips. ing performed by the University of Pittsburgh Genomics Re- Lipofectamine 2000 was used for transfection according to search Core using the T7 forward sequencing primer. Mutation the manufacturer’s protocol. Medium was changed daily. analysis was completed using CLC Genomics Workbench. HK-2 cells were treated with siRNA for 2 days after the ini- Although HK-2 cells were isolated from a male human tial transfection. CRISPR/Cas9 clonal cells were cultured for kidney, two different OCRL mutations were found in the 3 days, whereas siRNA treated HK-2 cells were cultured for genomic sequence of each of our two isolated clonal cell 4 days. Coverslips were incubated with 10 mg/ml Alexa Fluor populations, leading to frameshifts and early termination 647–conjugated wheat germ agglutinin on ice; fixed with 4% of the protein. CRISPR/Cas9 editing of another X-linked paraformaldehyde; mounted onto glass slides using ProLong gene in this cell line also required mutagenesis of two in- Gold antifade with DAPI; imaged using a Leica DM6000 B dependent alleles, suggesting that HK-2 cells may have a microscope using a 340 objective, 1.25–0.75 NA; and fields duplicated X- (A. Labilloy, unpublished re- containing GFP-positive cells acquired. For each condition, sults). Consistent with this, DAPI staining of chromosome the numbers of mono-, bi-, and multinucleated GFP-positive spreads revealed a range of 53–56 in parental cells with clearly visible cell borders were counted in each image. HK-2 cells (M. Gliozzi, unpublished results). Time-Lapse Imaging of Cell Division siRNA Transfection HK-2 CRISPR/Cas9 OCRL KO cells were plated sparsely on a Cells were transfected using Lipofectamine RNAiMAX with 35310-mm2 glass-bottomed dish (MatTek) and cultured for siRNA constructs targeting OCRL or luciferase as a control. approximately 12 hours. Random fields were selected and A new siRNA oligonucleotide (construct 1) targeting OCRL then imaged every 30 minutes overnight using a Leica TCS was used in all studies except in rescue experiments (Supple- SP8 confocal microscope using a DIC setting with a 340 mental Table 1). For rescue experiments, a previously char- objective, 1.30 NA. Additional experiments were conducted acterized oligonucleotide and appropriate siRNA-resistant where control and OCRL KO cells were incubated with 5 mM OCRL construct (construct 2) were used22 (Supplemental Draq5 (Thermo Fisher Scientific) for 10 minutes according Table 1). Both OCRL target sequences were purchased from to manufacturer’s instructions to elucidate nuclei. These ex- GE Dharmacon. The control luciferase siRNA was purchased periments were imaged every 15 minutes for 5.75–11.25 hours from Sigma. Cells were transfected twice for each experiment using a Leica TCS SP8 confocal microscope using a combina- with 1 mg siRNA and 1.5 ml Lipofectamine RNAiMAX reagent tion of confocal and transmitted light settings with a 340 in OptiMEM: first during plating and again approximately objective, 1.10 NA. Metaphase length was assigned on the 24 hours later. Experiments were performed up to 4 days after basis of the presence of chromosomes aligned in a single initial transfection. Duplicate samples of transfected cells were column. The initiation of anaphase was classified by a sep- solubilized to assess the efficiency of knockdown by western aration of chromosomes or, in cases where visualizing DNA blotting. content was difficult, the furrowing of cell membrane. The end of cytokinesis was determined by the formation of a nuclear Quantitation of Cell Multinucleation structure in daughter cells, the complete shift in cell morphol- Approximately 13105 HK-2 cells transfected with siRNA or ogy from rounded to spread, and the absence of any notice- HK-2 CRISPR/Cas9 clonal cells, were plated on 12-mm cov- able cytokinetic bridge. When possible, cells were followed erslips, cultured for 3–4 days, then incubated with 10 mg/ml beyond the determined end of division to further ensure that Alexa Fluor 647–conjugated wheat germ agglutinin (Thermo cell division was completed. Images were analyzed in FIJI. Fisher Scientific) and fixed with 4% paraformaldehyde. Cov- For time-lapse imaging of apoptotic cells, control and OCRL erslips were mounted onto glass slides using ProLong Gold KO cells were plated at equal cell counts (10,000–25,000 cells antifade with DAPI (Thermo Fisher Scientific), imaged us- per quadrant) on two quadrants of a split glass-bottom dish ing a Leica DM6000 B microscope, and random fields ac- and cultured for approximately 12 hours. Cells were incu- quired using a 340 objective, 1.25–0.75 NA. The numbers of bated with 5 mM NucView 488 Caspase-3 substrate (Biotium)

70 JASN JASN 31: 67–83, 2020 www.jasn.org BASIC RESEARCH for 30 minutes and 5 mM Draq5 for 3 minutes in PBS at 37°C. Therefore, Cells were washed twice in PBS and 250 ml culture medium was À Á fi 2 ln 1 2 fw added per quadrant for imaging. Random elds from approx- w ¼ : imately equally dense locations in control and OCRL KO L quadrants were selected and imaged every 20 minutes for 4.7–11.3 hours using a Leica TCS SP8 confocal microscope The average axial velocity can be defined as: using a combination of confocal and DIC setting with a 340 QðzÞ objective, 1.30 NA. Only cells that developed positive NucView uðzÞ¼ : p 2 staining during the imaging process were counted. Images were R analyzed in FIJI. Therefore, FACS Sorting of CRISPR/Cas9 OCRL KO Cells 2 du 2 uðzÞ¼u e wz and ¼ 2 u we wz FACS sorting on the basis of cell nuclear ploidy was performed 0 dz 0 using modifications of the approach described by Duncan 29 et al. Cells recovered from a stock plate were resuspended with (23106 cells per ml) in phenol red–free culture medium and incubated with 15 mg/ml Hoechst 33342 (Sigma) and 5 mM SNGFR u ¼ : reserpine (Invitrogen) for 30 minutes at 37°C. Cells were then 0 pR2 incubated on ice with 5 mg/ml propidium iodide (Invitrogen) before FACS analysis and sorting in a FACS Aria II-SORP (BD We have previously demonstrated that endocytic uptake in PT cells is Biosciences) using a 130-mm nozzle and DiVa v8.0.2 software. flow dependent.23,24 We assume that the reabsorptive capacity of the PT, Deadandaggregatedcellswereremovedonthebasisofpropidium Vmax, varies with axial velocity. The simplest form in which to include a fl iodide incorporation and pulse width analysis, respectively. ow component in Vmax, such that Vmax is not zero in the absence of fl Cells were sorted into 2C and .2C ploidy populations on the ow,iswiththeadditionofalineartermsothatVmax has the form: basis of DNA content measured using a violet 405-nm laser ¼ ð þ a 3 Þ; and 450–50-nm bandpass filter. An unsorted population Vmax Vmax0 1 u was collected as a control. Cells were collected in medium fl a containing 50% FBS and the cultures were supplemented where Vmax0 is the basal uptake capacity in the absence of ow, is 2 2 for the next 5 days with penicillin-streptomycin (Gibco). 1 sz mm 1, and u has the units mmzs 1. It is important to note, however, that the correlation be- Mathematic Modeling of Protein Reabsorption tween shear stress and uptake is unknown. We would expect A mathematic model describing protein reabsorption along the uptake capacity of the PT to decrease with decreasing the human PT was created in MATLAB (MATLAB R2017a flow, but without a quantitative assessment of the endocytic with ode45 solver). This model represents an adaptation of capacity and flow, we cannot conclusively define the relation- fl Lazzara and Deen’s published model of albumin reabsorption ship between Vmax and uid velocity. that used experimental parameters obtained from rat studies.30 The equation that describes uptake in a straight tubule with Parameter Values Used to Model Albumin and LMWP no differences in uptake capacity along the length and ne- Uptake along the Human PT glecting any resistance of radial mass transfer is: Default values for the parameters for human tubule length (L)   and luminal radius were obtained from a published model 2 dC ¼ 2 VmaxC 2 C du; of transport along the human nephron.33 Values for fractional dz uR K þ C u dz m water reabsorption (fw) and single nephron GFR (SNGFR) have also been previously determined.30 The creatinine con- where C is the concentration of protein in the tubule and u is the centration in the tubule lumen increases according to the axial velocity of the filtrate. exponential water loss. We assume that the starting concen- The definition of axial velocity is on the basis of an expo- trationofcreatinineinthetubulelumenisequivalenttose- nential flow profile31,32: rum creatinine. Serum creatinine was estimated on the basis of reported values for children.34 The protein concentration ð Þ¼ ð 2 wzÞ; Q z Q0e was divided by the creatinine concentration along the length of the tubule to give the protein-to-creatinine ratio (mg/mmol). where Q is the volume flow in the PT. The change in volume flow The specific trafficking rates of megalin trafficking in PT per unit length, w, can be estimated on the basis of tubule length, L, cells are unknown, so we relied on the published half-saturation 35 and the normal fractional reabsorption of water, fw: concentration for albumin reabsorption (Km,A). Our esti- À Á mate of the concentration of albumin entering the tubule ð 2 wLÞ QðLÞ¼ 1 2 f Q ¼ Q e : 36 w 0 0 (A0) was on the basis of a sieving coefficient of 0.00062 and

JASN 31: 67–83, 2020 Kidney Pathogenesis in Lowe Syndrome 71 BASIC RESEARCH www.jasn.org plasma levels of 3.5 g/dl.37 We believe that this is a more accu- Table 1. Parameter values used to model albumin uptake rate estimate than the human albumin sieving coefficient along the human PT calculated by Norden and colleagues on the basis of measure- Parameter Value Reference 38 ments of urinary albumin in patients with Dent disease. That Tubule length, L 17 mm 33 study assumed that no albumin is reabsorbed by the PT, Tubule radius, R 18.5 mm33 whereas our data suggest that endocytic capacity may not SNGFR 71 nl/min 30 2a be impaired in LS PT cells. The Vmax was fitonthebasisof Basal reabsorptive 0.039 ng/s per mm the reported fractional reabsorption values for albumin, so capacity, Vmax0 Albumin concentration at 31 mg/ml 35 that the fraction of A0 that is remaining in the lumen at the end of the tubule is (12f ).30 Specifically, V was ad- half saturation, Km,A A max0 fi m justed to minimize the difference between the model at Initial ltrate albumin 21.7 g/ml 36,37 z=17 mm and the normal excretion ([12f ]3A ). Using these concentration, A0 A 0 LMWP concentration at 10.4 mg/ml a parameters, the resulting basal uptake capacity, Vmax0,is 2 half saturation, Km,LMWP 0.039 ng/s per mm , and the average value of Vmax over Initial filtrate LMWP 67.3 mg/ml 36 the length of the tubule with a normal SNGFR is 0.065 ng/s concentration, LMWP 2 0 per mm , which is similar to that in the rat model (0.086 ng/s Initial filtrate creatinine 0.5 mg/dl 34 2 per mm ).30 concentration The concentration of LWMP entering the tubule (LMWP0) Fractional water 0.70 30 was estimated on the basis of the finding that approximately reabsorption, fw 3.1 times as much LMWP enters the PT as albumin.36 Frac- Fractional albumin 0.986 30 reabsorption, fA tional LWMP reabsorption (fLMWP) was estimated on the ba- sisofnormalfractionalreabsorptionofRBP.39 On the basis Fractional LMWP 0.9997 39 of our assumption that megalin trafficking rates do not de- reabsorption, fLMWP a fi pend on binding of ligands to the receptor, we can use the Indicates t parameter. same Vmax for both albumin and LMWP. This Vmax was used in the LMWP model and the reabsorption Km,LMWP was fit Amegalin/lrp2a fragment was PCR amplified (500 bp) from because we lack an estimate for the reabsorption half-saturation genomic DNA with the following primers adding a T3 promoter concentration for LMWPs. Again, the model was fitsothat in antisense: forward 59-CGAGGGCCACGTTTACTGGAC- the fraction of LMWP0 thatisremaininginthelumenatthe GACT-39 and reverse 59-GGATCCAATTAACCCTCACTAAAG end of the tubule is (12fLMWP). The resulting Km,LMWP is 10.4 GGTGTCAATGAAGGACACCCCG-39.Theclcnk cDNA se- mg/ml. The fA in patients with LS (0.86) was set such that quence (approximately 2.4 kb) was cloned into pBluescript albumin excretion was ten-fold higher than normal and the antisense to a T7 promoter. A cdh17 clone (synthetically gen- fLMWP in LS (0.7) was set such that the LMWP excretion was erated by Eurofins) containing the cdh17 sequence, 255 bp 1000-fold higher than normal, as determined by urinary ex- of the end of the coding sequence, and 195 bp of 39UTR; cretion measurements.2,40 Table 1 provides the parameters the reverse cdh17 sequence was cloned with a SP6 prior and used in the final model. NheI restriction site afterwards into a pCR2.1 vector. DIG antisense probes for lrp2a, clcnk,andcdh17 were generated Zebrafish Husbandry using the appropriate RNA polymerase and DIG RNA Label- Zebrafish were reared according to established protocols,41 ing Mix (Roche). Whole mount in situ hybridization with which were approved by the University of Pittsburgh Institu- DIG antisense RNA probes was conducted as described pre- tional Animal Care and Use Committee. viously.42 Animals probed for lrp2a and cdh17 were deyolked mechanically with forceps. In situ images were captured on a Zebrafish Injections, Crosses, and Whole Mount In Situ Leica MZ16 Stereomicroscope with a QImaging Retiga 1300 Hybridization camera and QCapture Suite 2.98.2 software. After imaging, 2 The validated zebrafish ocrl MO, which translationally ocrl+/ progeny were genotyped for ocrl mutant alleles by blocks ocrl transcripts, was synthesized by Gene Tools LLC PCR amplification.8 (Philomath, OR) as previously described.6 AB strain zebrafish one-cell embryos were injected with 6 ng or 8 ng ocrl MO into Quantitation of Albumin Endocytosis 2 the yolk. Ocrl+/ heterozygous animals were gifted to us by Approximately 33105 CRISPR/Cas9 control and OCRL KO Martin Lowe; generation of the mutant allele was previously clonal cells were plated in m-slide VI six-well ibi-treated cham- 2 2 2 described.12 Ocrl / larvae were acquired by incrossing ocrl+/ bers (Ibidi). Cells were cultured for 3 days with medium animals. All embryos developed at 28°C for 3 days in E3 em- changes twice per day. For endocytosis assays, cells were incu- bryo medium containing 30 mg/ml phenylthiourea to inhibit bated with 40 mg/ml Alexa Fluor 647-BSA (Thermo Fisher pigment deposition. Larvae were fixed in 4% paraformaldehyde/ Scientific) for 1 hour at 37°C under static conditions or at phosphate buffer solution overnight at 4°C. 0.1 dyne/cm2 FSS as previously described.23 Cells were

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A OCRL Genomic Mutation

Wild Type

Allele 1

cl. 1 Allele 2

Allele 1

cl. 2 Allele 2

OCRL1 Protein Mutation PH 5-ptase ASH Rho-GAP B Control OCRL KO CB CB cl.1 cl.2 cl.2cl.1 Wild Type 130kDa OCRL Allele 1 100kDa 100kDa

cl. 1 Allele 2 INPP5B 70kDa Allele 1 55kDa actin cl. 2 Allele 2 35kDa

Figure 1. CRISPR/Cas9-generated OCRL KO clones in HK-2 cells. A CRISPR/Cas9 guide sequence was generated to target exon 13 (within the 59-phosphatase domain) of OCRL in HK-2 cells. (A) Two separate KO clones (cl.1 and cl.2) were collected, each with insertion/ deletion mutations causing a frameshift and protein truncation (asterisk) within the 59-phosphatase domain. WT genomic DNA section correlates to nucleotides 129,565,848–129,565,883 in GRCh38.p7. WT protein sequence section correlates to amino acids 441–464. (B) Blotting with antibodies against OCRL, INPP5B, and b-actin confirms the absence of OCRL expression, with no compensatory increase in INPP5B expression in either CRISPR/Cas9 KO clone. washed, fixed in 4% paraformaldehyde, and imaged using coverslips (Fisher Scientific) in a 12-well plate. Cells were a Leica TCS SP5 confocal microscope using a 340 objective, cultured for 3 days with daily medium changes, serum- 1.25 NA. Five confocal stacks were acquired per condition starved overnight in HK-2 medium without FBS, and then and the intensity of maximum projections was quantified in fixed in 4% paraformaldehyde. Cells were processed for in- FIJI after background subtraction.23 direct immunofluorescence using mouse anti-acetylated tu- Because of technical challenges in generating efficient bulin (1:400 of approximately 1 mg/ml stock, T7451) and siRNA-mediated knockdown in Ibidi chambers, we measured Alexa Fluor 568–conjugated goat anti-mouse secondary an- endocytosis in siRNA-treated cells plated on Transwell filter tibody (1:500 of a 2 mg/ml stock, A11031). Coverslips were supports. HK-2 cells (53105) were plated on 12-mm Transwell mounted onto glass slides with ProLong Gold antifade with 0.4-mm pore polycarbonate membrane inserts (Corning) with DAPI. Random fields for each condition were imaged in a 12-well plate, with 0.5 ml apical medium and 1.0 ml baso- using a Leica DM6000 B microscope using a 340 objective, lateral medium. Cells were treated with siRNA as described in 1.25–0.75 NA, and analyzed in FIJI. Cilia were measured by the siRNA transfection section. Cells were cultured for 4 days tracing using the freehand drawing tool. The same drawing on an orbital shaker at approximately 74 rpm with daily me- tool was used to measure 10 mm from a scaled image to nor- dium changes. We have previously demonstrated that chronic malize measurements. exposure to orbital shear stress enhances cell proliferation and differentiation.24 To measure endocytosis under static condi- Statistical Analysis tions, filters were transferred to static conditions the night be- Statistical significances of all data were assessed using Prism fore the experiment. All cells were incubated with 40 mg/ml 7(GraphPad). Alexa Fluor 647-BSA for 1 hour at 37°C under static conditions or orbital shear stress as indicated. Cells were fixed in 4% parafor- maldehyde and mounted using ProLong Gold antifade with DAPI. RESULTS Imaging and analysis were completed in the same manner as de- scribed for the CRISPR/Cas9 clones above. In both CRISPR/Cas9 OCRL Depletion Leads to Accumulation of clonal and siRNA experiments, albumin uptake under static con- Multinucleated Cells ditions was normalized to 1 for each cell population and the fold We used CRISPR/Cas9 genome editing to isolate two HK-2 increase in albumin uptake upon exposure to FSS was calculated. OCRL KO clones containing indels within the 59-phosphatase domain of OCRL (Figure 1A). As controls, we isolated two Measurement of Cilia Length HK-2 clones after transfection with empty vector. OCRL Approximately 53105 HK-2 cells transfected with siRNA was readily detected by western blotting of control HK-2 cell or HK-2 CRISPR/Cas9 clonal cells, were plated on 12-mm clones using an antibody directed against the amino terminus

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*** A Control OCRL KO B 15 *** *** *** 10 Exp.1 Exp.2 Exp.3 5 CRISPR/Cas9 Multinucleated cells (%) 0 cl.1 cl.2 cl.1 cl.2 Control KO C Control OCRL KD D 15 **

10 Exp.1

siRNA Exp.2 Exp.3 5 Multinucleated cells (%) 0 siRNA: Control OCRL Lowe E Control Syndrome F 10 *

8 Exp.1 6 Exp.2 fibroblasts 4 Exp.3 Exp.4 2 Multinucleated cells (%) 0 Control Lowe Syndrome G Control siRNA OCRL siRNA cDNA: GFP OCRL G304E GFP OCRL G304E 250kDa

* 130kDa

OCRL1 I 40 * 100kDa *** **** 55kDa * *** 30 * Actin * 35kDa ** Exp.1 * Exp.2 Exp.3 H +GFP +OCRL +G304E 20 cDNA: GFP OCRL-GFP G304E-GFP

Multinucleated cells (%) 10 siRNA

0 siRNA: Control OCRL

Figure 2. Cells lacking OCRL exhibit increased multinucleation. Control and OCRL-depleted cells were stained with fluorescently conjugated wheat germ agglutinin (red) and DAPI (blue) to visualize cell borders and nuclei, respectively. (A) Representative images of mononuclear cells in control CRISPR/Cas9 clones and multinucleated cells in CRISPR/Cas9 OCRL KO clones. (B) The percentage of HK-2 cells with multiple nuclei is plotted (mean and individual points of three independent experiments; 69–394 cells per condition scored in each experiment). (C) Representative images of mononuclear cells in cells transfected with control siRNA and multinucleated cells in cells transfected with OCRL siRNA. (D) The percentage of cells with multiple nuclei is plotted (mean and individual points of

74 JASN JASN 31: 67–83, 2020 www.jasn.org BASIC RESEARCH of the protein (Figure 1B). No band was observed at the pre- caused by OCRL depletion can be prevented, but that re- dicted location corresponding either to the full length (ap- expression of OCRL does not resolve preexisting multinu- proximately 97 kDa; Figure 1B) or to the expected truncated cleation in KO cells. protein (approximately 51 kDa) in the OCRL KO clones (Sup- plemental Figure 1). There was no compensatory increase Impaired Cell Cycle Transit Leads to Accumulation of in INPP5B levels in our OCRL KO clones (Figure 1B). Multinucleated Cells In characterizing our CRISPR/Cas9 clones, we noticed Time-lapse microscopy confirmed the defect in cytokinesis in that our OCRL KO clones exhibited reduced proliferation an OCRL KO clone. Figure 3A shows frames taken 30 minutes rates compared with control clones. Quantitation of cell nu- apart documenting a tripolar cell division that resolved into clei in OCRL KO cells or in siRNA-transfected cells revealed a two daughter cells, suggesting that one daughter cell retained 22%–31% decrease in nuclei per field in OCRL-depleted cells two nuclei. Quantitation of the time spent in later stages of the compared with controls plated at identical densities (Supple- cell cycle (metaphase through cytokinesis) revealed that OCRL mental Figure 2, A–D). There was no significant difference in KO cells had markedly prolonged stages of metaphase and/or the fraction of apoptotic cells in KO clones at steady state anaphase/cytokinesis compared with control cells (Figure 3B, (Supplemental Figure 2E). To test whether cell division was Supplemental Videos 1–3). Moreover, a delay in cell cycle impaired, we quantified the frequency of cells with multiple transit time correlated with impaired mitotic outcomes, be- nuclei in our siRNA-transfected or OCRL KO models (Figure 2). cause multinucleated cells preferentially resulted from longer Whereas ,2% of cells in the control cell populations had cell divisions (Figure 3C). To examine how proliferation, di- more than one nucleus, approximately 10% of the OCRL vision, and death establish the equilibrium between cells hav- KO clones (Figure 2, A and B) and OCRL siRNA transfected ing one versus more than one nucleus, CRISPR/Cas9 cell clones cells (Figure 2, C and D) were multinucleated. We attempted were sorted by FACS for nuclear content to isolate popula- to quantify the frequency of PT cell multinucleation in hema- tions enriched for mononuclear (2C) or multinucleated toxylin and eosin–stained sections of kidney samples obtained (.2C) cells and followed over multiple passages (Figure 3D). from deceased patients with LS and age-matched controls, but The mononuclear-enriched cell population became progres- thetissuequalitywasinsufficient to reliably identify multi- sively multinucleated, suggesting that a fraction of cells lacking nucleated cells (not shown). However, we did observe an in- OCRL exhibit defective cell division. By contrast, the fraction creased fraction of multinucleated cells in primary fibroblasts of multinucleated cells declined slightly in the .2C fraction from patients with LS (Figure 2, E and F) as well as upon siRNA (Figure 3D). By passage 10, the percentage of multinucleated knockdown of OCRL in two other PT cell lines derived from cells in both populations had returned to that of the unsorted mice and humans (Supplemental Figure 3). Multinucleated parental cells (approximately 12%). cells contained up to four nuclei per cell (Figure 2, A and C), suggesting that errors in division can occur repeatedly in some Mathematic Model for the Effect of PT Length and cells. Heterologous expression of siRNA-resistant, GFP-tagged Diameter on Albumin Uptake WT OCRL prevented the accumulation of multinucleated cells Our data in CRISPR/Cas9 clones and siRNA-depleted PT cells in OCRL siRNA-transfected cells (Figure 2, G–I). By contrast, suggest that an impairment in cell division results in reduced transfection with a disease-causing mutant of OCRL (G304E)22 proliferation and an increased proportion of multinucleated increased the frequency of multinucleation in both control and cells in cell populations lacking functional OCRL. We hy- OCRL-knockdown cells, likely reflecting a dominant-negative pothesize that changes in these set points during kidney de- effect of overexpressing the mutant construct (Figure 2I). Ex- velopment affect PT length and/or diameter and lead to the pression of G304E in control and OCRL KO clones also in- incomplete reabsorption of filtered proteins, ions, and me- creased multinucleation, but WT OCRL was unable to resolve tabolites characteristically observed in LS. Analysis of urinary preexisting multinucleation in cells (data not shown). To- protein profiles in patients with LS reveals a ten-fold increase gether, these data suggest that the impairment of cell division in albumin excretion and an approximately 1000-fold increase three independent experiments; 111–481 cells per condition scored in each experiment). (E) Wheat germ agglutinin and DAPI were used to visualize cell borders and nuclei in control and LS fibroblast primary cultures. (F) The percentage of cells with multiple nuclei is plotted (mean and individual points in three independent experiments; 99–171 cells per condition scored in each experiment). (G) OCRL-depleted cells were transfected with cDNAs encoding GFP, or with siRNA-resistant GFP-tagged WT OCRL (OCRL) or LS-causing mutant OCRL-G304E (G304E). Efficient knockdown of OCRL (90%–95%) in these cells and heterologous expression of GFP-tagged OCRL and G304E (red asterisk) were confirmed by western blotting of cell lysates. (H) Representative expression patterns of each construct are shown (green). (I) The percentage of cells with multiple nuclei in cells treated with siRNA and expressing GFP, OCRL, and G304E is plotted (mean and individual points of three experiments for GFP and OCRL, two experiments for G304E; 11–146 cells per condition scored in each experiment). Scale bar (A, C, E, and G), 10 mm. *P,0.05, **P,0.01, ***P,0.001, and ****P,0.001 on the basis of a one-way ANOVA Tukey’s multiple comparisons test in (B), unpaired t test in (D) and (F), and two-way ANOVA Tukey’s multiple comparisons test in (I). cl, clone; Exp, experiment.

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A B 8 Control cl.1 KO cl.1 *** 6

4

Duration (hours) 2

0 Total Total Anaphase- Anaphase- Metaphase Metaphase Cytokinesis Cytokinesis

C 8 Mononuclear Multinucleated **** 6 ****

4

Duration (hours) 2

0 Total Total Anaphase- Anaphase- Metaphase Metaphase Cytokinesis Cytokinesis

D 20 passage 1 passage 5 passage 10 15

10

5 Multinucleated cells (%)

0 unsorted 2C >2C KO cl.2 populations

Figure 3. Multinucleated CRISPR/Cas9 OCRL KO cells result from prolonged cell cycle transit. (A) DIC imaging time course showing a tripolar division that resolves into two cells (borders highlighted in red, dashed lines) Scale bar, 25 mm. (B) CRISPR/Cas9 control clone 1 (cl.1) and KO cl.1 cells were imaged live every 15 minutes for 5.75–11.25 hours to capture cell divisions in randomly selected fields. The plot shows the durations of the metaphase and anaphase-cytokinesis stages of mitosis, and the total time spent in metaphase-cytokinesis measured in individual cells that underwent mitosis during the imaging period (control cl.1, 10 cells and KO cl.1, 23 cells). (C) Data from KO cl.1 cells in (B) were segregated on the basis of the outcome of mitosis (13 mononuclear versus nine multinucleated cells), and the durations of metaphase, anaphase-cytokinesis, and the total time spent in metaphase- cytokinesis for each are plotted. ***P,0.001 and ****P,0.001 on the basis of two-way ANOVA Holm–Sidak multiple comparisons

76 JASN JASN 31: 67–83, 2020 www.jasn.org BASIC RESEARCH in LMWP excretion, on the basis of measurements of RBP.2 To length in ocrl mutant and morphant zebrafish using segment- assess how changes in PT length might affect the uptake of specific markers. Megalin/lrp2a is selectively expressed in the filtered ligands, we adapted a published mathematic model PT segment,44 whereas cdh17 is expressed along the entire on the basis of experimentally determined parameters in ro- length of the pronephric kidney.45,46 Using in situ hybridiza- dents30 to incorporate best estimated values of tubule size, tion, we measured the lengths of lrp2a-andcdh17-expressing 2 2 plasma albumin levels, and fractional reabsorption of albumin segments in ocrl mutant (ocrl / ) and morphant zebrafish. 2 2 and LMW proteinuria in humans (Figure 4). Details of the Ocrl transgenic (ocrl / )zebrafish exhibited a significant re- model parameters and assumptions are provided in the ductioninthelrp2a-expressing segment compared with 2 Methods. The model predicts rapid initial albumin uptake WT (ocrl+/+) and heterozygote (ocrl+/ )controlfish (Figure in the tubule because concomitant water reabsorption main- 5, A–D). By contrast, there was no difference in the length of tains the albumin concentration above the estimated Km.The the entire pronephric tubule quantified by cdh17 expression fractional reabsorbance values in LS are estimated to be 0.86 (Figure 5, B–E). Overall, this resulted in the selective short- 2 2 for albumin and 0.7 for LMWP versus 0.986 and 0.9997 for ening of the PT of ocrl / fish by 17% relative to controls normal, respectively. The model predicts that an approxi- (Figure 5G). Injection of ocrl MO also resulted in a dose- mately 30% decrease in PT length can account for the excess dependent reduction in lrp2a-expressing segment lengths in urinary albumin and LMWP excretion in patients with LS (Supplemental Figure 5, A and B), as well as in the entire (28.5% for albumin and 33.8% for LMWP; black arrows in cdh17-expressing tubule (Supplemental Figure 5, C and D). Figure 4A). Reduced GFR has been reported in patients with The effect on lrp2a-expressing segments was more pro- LS, so we tested the effect of reducing the SNGFR in our model nounced, because the overall fraction of the pronephric tubule (Figure 4B). Reduced GFR is likely due in part to nephron that was lrp2a-positive decreased in a dose-dependent manner dropout, and studies in rats demonstrate that SNGFR drops (Supplemental Figure 5E). by only approximately 30% when whole kidney GFR is reduced To assess whether reduced PT length was compensated by by approximately 70%.43 Our model predicts that a greater elongation of other pronephric kidney segments, we measured reduction in length would be required to result in the levels the length of distal tubule (clcnk-expressing) segments in ocrl of urinary albumin and LMWP excretion observed in patients transgenic fish versus controls (Figure 5, C–F). We observed a with LS as SNGFR is reduced. For example, a 20% reduction in slight increase in overall and fractional clcnk expression length SNGFR would necessitate a 36.2% reduction in PT length for in ocrl mutants, although this increase did not reach statistical albumin and 42.6% for LMWP (Figure 4B). Interestingly, significance (Figure 5, F–H). Together, our data suggest that changes in PT diameter had little effect on urinary excretion OCRL plays a major role in development of the PT, and that profiles under normal conditions or upon reduction in ocrl depletion in zebrafish models of LS results in a reduction SNGFR, because this resulted in opposing effects on the in PT length consistent with that predicted to result in tubular flow-dependent reabsorptive capacity and residence time of proteinuria. filtered proteins in the PT lumen that tended to cancel out. Because an endocytic defect has been observed in disease Flow-Dependent Uptake in PT Cells Is Unaffected by models of LS, we also modeled the effect of fractionally reduced OCRL KD PTreabsorptive capacity on albumin and LMWP clearance. As We previously found that PT cells rapidly adjust their endocytic showninSupplementalFigure4,AandB,anapproximately capacity in response to acute changes in fluid shear stress.23,24 30% reduction in Vmax due to a decreased number of free Defective responses to changes in tubular flow resulting from megalin receptors in the PT could result in the urinary excre- defects in endocytic trafficking and/or in cilia-dependent flow tion profile observed in patients with LS. Similar to the pre- sensing could contribute to the tubular proteinuria character- dicted effects on changes in PT length, a 20% reduction in istic of LS. We quantified constitutive and flow-stimulated endo- SNGFR would necessitate a 40%–45% reduction in Vmax (Sup- cytic uptake of AlexaFluor 647 albumin in control and OCRL plemental Figure 4, C and D). Because Vmax in our model has a KO CRISPR/Cas9 clones, and in cells treated with control or synergistic effect with shortened PT length, smaller reductions OCRL siRNA (Supplemental Figure 6). Although individual in each could also result in the proteinuria observed in LS. CRISPR/Cas9 clones had different baseline levels of endocytic uptake, we found no consistent effect on constitutive endocy- Loss of OCRL Shortens Megalin/lrp2a-Expressing tosis upon KO of OCRL by CRISPR/Cas9. Similarly, constitu- Segments in the Zebrafish Pronephric Kidney tive endocytosis in OCRL-depleted cells was similar to control, To assess whether there is a functional consequence of im- consistent with our previously published studies using a dif- paired cell division on kidney segmentation, we examined PT ferent siRNA oligonucleotide.22 In all cases, exposure to FSS

test. (D) CRISPR KO clone 2 (cl.2) cells were sorted by FACS on the basis of nuclear content. Mononuclear (2C) and multinucleated (.2C) cells were isolated and cultured separately for ten passages. The percentage of cells with multiple nuclei was assessed at passages 1, 5, and 10 by fluorescence staining. cl, clone.

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A resulted in an approximately 1.3-fold increase in albumin up-

1500 albumin take, regardless of whether OCRLwas expressed (Supplemental LMWP Figure 6). 1250 Both shorter and longer cilia have been reported upon 1000 OCRLdepletionincellsandzebrafish, and an emerging con- sensus is that effects on cilia length are irrelevant to LS pa- 750 thology.6,8,47 Similar to our previous observation in other cell 500 types, acute knockdown of OCRL in HK-2 cells using siRNA increased mean cilia length (Supplemental Figure 7, A and B). 250 However, cilia length was variably affected in individual protein/creatinine ratio (mg/mmol) 0 CRISPR/Cas9 OCRL KO clones compared with control clones 0246810121416 (Supplemental Figure 7, C and D). We conclude that loss of distance from origin of tubule (mm) functional OCRL does not affect flow-dependent endocytic responses in PT cells. B 500 100% SNGFR 90% SNGFR DISCUSSION 400 80% SNGFR 70% SNGFR Despite considerable study, the direct causes of tubular protein- 300 60% SNGFR uria and transport defects in patients with LS have remained elusive. Most studies to date have focused on a potential 200 role for OCRL in maintaining efficient endocytosis of albu- min and other filtered megalin/cubilin ligands by the PT. Our 100 studies here suggest that reduced cell proliferation resulting

albumin/creatinine ratio (mg/mmol) from defective cytokinesis leads to selective shortening of 0 megalin-expressing nephron segments. Mathematic modeling 0246810121416 distance from origin of tubule (mm) demonstrates that shortened PT lengths, within the range we measured in zebrafish, could account for the incomplete re- fi C sorption of ltered ligands observed in patients with LS. Im- portantly, shorter PTs can also explain the other renal transport 100% SNGFR 1500 fi 90% SNGFR de ciencies commonly observed in these patients. 1250 80% SNGFR We found a consistent decrease in cell proliferation when 70% SNGFR we used either siRNA or gene-editing to deplete OCRL. Ad- 1000 60% SNGFR ditionally, we observed a striking accumulation of multinucle-

750 ated cells under these conditions. We also observed increased numbers of multinucleated cells in mouse PT cells depleted of 500 OCRL and in primary cultures of human LS fibroblasts. Het- erologous expression of functional OCRL prevented the ac- 250 cumulation of new multinucleated cells in siRNA experiments, LMWP/creatinine ratio (mg/mmol) 0 suggesting that multinucleated cell formation is dependent on 0246810121416 the absence of functioning OCRL. Interestingly, heterologous distance from origin of tubule (mm) expression of a disease-causing mutant of OCRL resulted in Figure 4. Mathematic model of albumin uptake along the PT. (A) accumulation of increased numbers of multinucleated cells. Albumin-to-creatinine ratio (blue curve) and LMWP-to-creatinine We hypothesize that a subset of OCRL mutants have dominant ratio (red curve) along the human PT were modeled as described in negative effects on cytokinesis that contribute to phenotypic the Methods. The models were fitted to the measured fractional variation in patients with LS. reabsorbance in humans (0.986 for albumin and 0.9997 for LMWP) We found that megalin/lrp2a-positive segment lengths in at the end of the tubule. In patients with LS, the estimated fractional ocrl mutant and morphant zebrafish were shortened (by 17% reabsorptions for albumin and LMWP are 0.86 and 0.70, respec- in transgenic, 16% in 6 ng MO, and 42% in 8 ng MO) com- tively. As denoted by the black arrows, an approximately 30% re- pared with the overall length of the pronephric kidney. Con- duction in PT length is predicted to result in the tubular proteinuria phenotype characteristic of LS. The effects of reduced SNGFR on sistent with the decrease in PTsegment length that we observed fi urinary excretion profiles of albumin (B) and LMWP (C) are plotted. in transgenic and morphant zebra sh, our mathematic model A progressively greater reduction in PT length would be needed to for albumin uptake in humans30 suggests that an approxi- produce the predicted urinary excretion profile as SNGFR declines mately 30% decrease in PT length could account for the from 100% to 60% of control. urinary protein excretion profile of patients with LS. Although

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A ocrl +/- ocrl -/- B ocrl +/- ocrl -/- C ocrl +/- ocrl -/-

D E F 400 800 450 **** m) m) µ 750 µ 400 300 700 350 650 200 300 600

250 100 550 cdh17 expression (length, clcnk expression (length, lrp2a expression (length, µ m)

0 ocrl +/+, ocrl +/- ocrl -/- ocrl +/+, ocrl +/- ocrl -/- ocrl +/+, ocrl +/- ocrl -/-

G H 0.4 0.6

0.3 0.4

0.2

0.2 0.1 ratio of lrp2a / cdh17 length ratio of clcnk/ cdh17 length

0.0 0.0 ocrl +/+, ocrl +/- ocrl -/- ocrl +/+, ocrl +/- ocrl -/-

Figure 5. LS transgenic zebrafish have selectively shortened megalin/lrp2a-positive pronephric segments. In situ hybridization of megalin/lrp2a (PT marker) (A and D), cdh17 (pronephric tubule marker) (B and E), and clcnk (distal tubule marker) (C and F) 2 2 2 was performed in WT (ocrl+/+), heterozygous mutant (ocrl+/ ), and homozygous mutant (ocrl / ) transgenic zebrafish at 72 hpf. The distributions of lrp2a (A), cdh17 (B), and clcnk (B), and expression in representative zebrafish, are shown. Scatter plots show 2 the lengths of lrp2a-(D),cdh17-(E),andclcnk- (E) positive segments measured in ocrl+/+ and ocrl+/ zebrafish compared with 2 2 ocrl / zebrafish (mean6SD; lrp2a, n=40–92; cdh17, n=33–56; clcnk, n=17–47). Ratios of average lrp2a (G) and clcnk (H) lengths 2 2 2 over average cdh17 length were compared among ocrl+/+, ocrl+/ ,andocrl / zebrafish. ****P,0.001 on the basis of an unpaired t test in (B).

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Normal PT

Lowe Syndrome PT

LMW Proteinuria Aminoaciduria Hypercalciuria Tubular Acidosis

2+ - = LMW protein = Amino acid = Ca = Glucose = HCO3

Figure 6. Model for how shortened PTs may explain kidney pathogenesis in LS. We hypothesize that defective cell division caused by lack of functional OCRL alters PT development or regeneration after injury, resulting in shorter (and/or narrower) PTs. Decreased PT reabsorption area can lead to incomplete reabsorption of LMWPs, and can also account for the aminoaciduria, hypercalciuria, and RTA characteristically observed in patients (see text for details). PTs have a high excess capacity for glucose uptake, so shorter PTs do not necessarily lead to glycosuria. we detected potentially multinucleated cells in samples from ligands in human PT cells.22 This was true both for constitutive patients with LS and in morphant zebrafish, definitive confir- endocytosis under static conditions and for the higher capac- mation was technically very challenging. Using less rigorous ity pathway that is stimulated by acute exposure to fluid shear criteria, the frequency of false positives made it impossible to stress. However, HK-2 cells are a poor model for PT apical reliably determine whether multinucleated cells are more preva- endocytosis, and OCRL may modulate endocytic efficiency lent in OCRL-deficient tissues. It is also possible that impaired in more highly differentiated cells. Studies in transgenic zebra- cytokinesis within the geometric constraints of the proneph- fish and in the Ocrl/Inpp5b KO/replacement mouse models re- ric kidney results simply in slower proliferation that affects port defects in PTendocytic uptake.8,20,21 Reduced endocytosis PT growth. may reflect a defect in the uncoating of actin-coated endocytic Defective elongation of the PT due to impaired cytokinesis structures as has been suggested4,5 and/or could be secondary is consistent with the development of renal malfunction in to the lower levels of PT megalin expression noted in both the patients with LS and in mice, and may contribute to the im- mouse and zebrafish models.8,21,20 Reduced levels of shed megalin paired proliferation of tubule-producing mesenchymal cells have also been reported in urine from patients with LS.38 described in kidney-differentiated iPSCs derived from patients An attractive feature of our model is that decreased PT with LS.48 Patients with LS typically develop LMW proteinuria length and/or diameter can also account for the other renal during the first several months after birth. Studies of micro- hallmarks of LS, including RTA, hypercalciuria, and amino- dissected human PTs suggest that PTs grow rapidly in length aciduria (Figure 6). RTA is thought to be due to reduced Na+/H+ for the first several months after birth.49 At about 6 months exchange via NHE3 in the PTand can lead to impaired amino of age, which roughly coincides with the onset of LMW pro- acid transport. A mathematic model for acid reabsorption in teinuria, PT diameters also begin to increase. Similarly, in a the kidney suggests that shorter proximal convoluted tubules recently described mouse model for LS, LMW proteinuria would also result in decreased urinary acid excretion.31 Sim- was not immediately observed upon conditional KO of ilarly, Ca2+ is thought to be reabsorbed passively along the 2 2 Inpp5b in the kidneys of 2-week-old Ocrl / mice, but rather PT via the paracellular pathway51; thus, decreased PT length took 1–3 months to develop.21 This period occurs during an would be expected to result in increased levels of Ca2+ reach- active stage of PT development, and, indeed, the PT is the only ing distal tubule segments. By contrast, clinical and mathe- nephron segment in mice that increases in size between 2 and matic modeling studies reveal an excess capacity for glucose 12 months of age.50 The slow onset of LMW proteinuria after uptake by the PT.31,52,53 For example, loss of 90% of SGLT2 is acute KO of Inpp5b seems more consistent with a gradually predicted to lead to only a 40% increase in glucose excretion.52 forming defect in kidney architecture than with the sudden Thus, it is likely that a significant reduction in PT length could loss of a specific PT cell function. be tolerated without changes to urinary glucose excretion In agreement with our previous studies, we found no ev- levels. Of note, although their renal phenotype has been fre- idence for a role of OCRL in endocytosis of megalin/cubilin quently described as Fanconi syndrome, many patients with

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LS do not exhibit glycosuria. Moreover, urinary excretion of FUNDING filtered proteins in patients with LS was considerably higher than that measured in patients with Dent disease or autosomal- This study was supported by grants from the Lowe Syndrome Association; dominant idiopathic Fanconi syndrome.40 Such differences in by National Institutes of Health (NIH) R01 DK101484, R01 DK118726, and the characteristic tubular dysfunction observed in genetic dis- S10 OD021627 to Dr. Weisz; NIH R01 DK069403 to Dr. Hukriede; and by fl the Pittsburgh Center for Kidney Research (P30 DK079307). Dr. Gliozzi was eases that manifest with tubular proteinuria likely re ect the supported by NIH grants T32 DK061296 and TL1 TR001858. Dr. Espiritu was effects of their causative mutations on different pathways. supported by NIH grant T32 DK061296. Ms. Shipman was supported by an Increasing appreciation that kidney segment lengths can American Society of Nephrology predoctoral fellowship award and by NIH be dynamically remodeled has significant implications for F31 DK121394. Dr. Duncan was supported by NIH R01 DK103645. therapeutic approaches to treat LS. A recent publication demonstrates that pharmacologic activation of an atypical C rescues the cytokinesis defect in cells from SUPPLEMENTAL MATERIAL patients with LS.54 PT length in rats can be reversibly regu- lated by .20% in response to prolactin and other hormonal This article contains the following supplemental material online at triggers that accompany pregnancy, lactation, and diabe- http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019020125/-/ tes.55–59 Such changes are not exclusive to the PT: the length DCSupplemental. of the distal tubule was observed to elongate in mice fed a high- Supplemental Table 1. Oligonucleotides and primers used in this salt diet.50 Similarly, Welling and colleagues60 found that study. expression of constitutively active SPAK leads to a reduction Supplemental Table 2. Potential off-targets of CRISPR/Cas9 OCRL in connecting tubule mass. Hormonal or pharmacologic inter- guide sequence. ventions may thus be beneficial in treating LS early after diag- Supplemental Figure 1. CRISPR/Cas9 OCRL clones have no OCRL nosis and could also help re-establish PTs after injury to slow expression. the deterioration of kidney function in patients with LS. Supplemental Figure 2. OCRL depletion slows proliferation of HK-2 cells without increasing apoptosis. Supplemental Figure 3. OCRL knockdown increases multinucleation in mRPTC and hRPTC cell lines. ACKNOWLEDGMENTS Supplemental Figure 4. Effect of reduced Vmax on PT uptake of albumin and LMWP. fi WeareverygratefultoMartinLoweforgenerouslyprovidingthe Supplemental Figure 5. Ocrl morphant zebra sh injected with ocrl 2 2 transgenic ocrl / zebrafish line (via R. Claudio Aguilar) for these morpholino have shorter pronephric kidney segments. fi studies, and to tissue donors and the National Institutes of Health Supplemental Figure 5. Ocrl morphant zebra sh injected with ocrl NeuroBioBank/University of Maryland School of Medicine Brain morpholino have shorter pronephric kidney segments. and Tissue Bank for access to samples of human kidney. We thank Supplemental Figure 6. Acute or chronic depletion of OCRL does Anatália Labilloy for assistance in generating the CRISPR/Cas9 not impair endocytic uptake in HK-2 cells. OCRL knockout cells, Francisco Carattino for help with quantifying Supplemental Figure 7. Cilia length in siRNA treated and cilia lengths, Allison Marciszyn for scoring blinded human kidney CRISPR/Cas9 OCRL knockout clones. sections, Roy Zent for providing conditionally immortalized mouse Supplemental Video 1. Cell division in CRISPR/Cas9 control RPTC cells, Polly Mattila for cell culture of mouse RPTCs, Lynda clone 1. Guzik (McGowan Institute Flow Cytometry Core at University of Supplemental Video 2. Cell division in CRISPR/Cas9 KO clone 2 Pittsburgh) and Frances Alencastro for assistance with FACS sorting, that resolves into two cells. and Sheldon Bastacky for help with assessing human tissue pathology. Supplemental Video 3. Cell division in CRISPR/Cas9 KO clone 2 We appreciate the thoughtful suggestions from a dedicated reviewer that resolves into one multinucleated cell. for improving our mathematic model of urinary protein excretion. Dr. Gliozzi and Dr. Weisz designed the study. Dr. Gliozzi, Dr.Espiritu,Ms.Shipman,Mr.Rbaibi,Dr.Long,Dr.Roy,and REFERENCES Dr. Baty carried out experiments. Dr. Gliozzi, Dr. Espiritu, Ms. Shipman, Dr. Duncan, Dr. Hukriede, Dr. Baty, and Dr. Weisz analyzed the data. 1. Loi M: Lowe syndrome. Orphanet J Rare Dis 1: 16, 2006 ’ fi 2. 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JASN 31: 67–83, 2020 Kidney Pathogenesis in Lowe Syndrome 83 Supplementary Material Table of Contents

Table S1. Oligonucleotides and primers used in this study Table S2. Potential off-targets of CRISPR/Cas9 OCRL guide sequence

Figure S1. CRISPR/Cas9 OCRL clones have no OCRL expression. Figure S2. OCRL depletion slows proliferation of HK-2 cells without increasing apoptosis. Figure S3. OCRL knockdown increases multinucleation in mRPTC and hRPTC cell lines. Figure S4. Effect of reduced Vmax on PT uptake of albumin and LMWP. Figure S5. Ocrl morphant zebrafish injected with ocrl morpholino have shorter pronephric kidney segments. Figure S5. Ocrl morphant zebrafish injected with ocrl morpholino have shorter pronephric kidney segments. Figure S6. Acute or chronic depletion of OCRL does not impair endocytic uptake in HK-2 cells. Figure S7. Cilia length in siRNA treated and CRISPR/Cas9 OCRL knockout clones.

Video S1. Cell division in CRISPR/Cas9 control clone 1. Video S2. Cell division in CRISPR/Cas9 KO clone 2 that resolves into two cells. Video S3. Cell division in CRISPR/Cas9 KO clone 2 that resolves into one multinucleated cell.

Table S1. Oligonucleotides and primers used in this study. OCRL exon Plasmid/siRNA Sequence (5’-3’) target F: AAACCGAATTTCAAGAGTCTCTGAT pX330 13 R: TAAAATCAGAGACTCTTGAAATTCG F: AATTATGCTTAAAGTTCTCAGAGACTTCCG pHRS 13 R: GATCCGGAAGTCTCTGAGAACTTTAAGCAT F: TCCTTAGGGTTGTCATTTGGTT PCR 13 R: TAAGACGTTTCCATCACTCCCT siRNA construct 1 17 GGTTCCCTGCCATTTTTCA siRNA construct 2 15/16 GGGUGAAGGUUGUGGAUGUUU

Table S2. Potential off-targets of CRISPR/Cas9 OCRL guide sequence. % Chrom. Expressed Gene Protein Match Loc. Function in Kidney

TMEM117 Transmembrane Low - (isoform 82.6 12 Unknown protein 117 Moderate X3)

Involved in tight junction ARHGAP17 Rho-GTPase- maintance by (isoform activating protein 65.2 16 Moderate regulating activity X3) 17 of CDC42. GTPase activator Methylates carpoxyl group of Leucine carboxyl the C-terminal LCMT1 Moderate - methyltransferase 65.2 16 leucine residue of (isoform a) High 1 2A catalytic subunits May be a Zinc finger protein ZNF717 60.9 22 transcription Yes 717-like factor

Olfactory receptor OR11H1 60.9 22 Odorant receptor None 11H1

No off-target events were reported on CHOPCHOP during guide selection. The guide sequence was blasted against the GRCh38.p2. edition of the human genome for a more conservative analysis of off- target effects. Top hits that contained the required PAM sequence are listed. UniProt and Expression Atlas were used to interpret protein function and expression. As none of the top hits match 100% to the guide and few have importance to our system, off-target effects are considered to be unlikely.

Supplementary Figure Legends Figure S1. CRISPR/Cas9 OCRL clones have no OCRL expression. CRISPR/Cas9 OCRL knockout clone lysates were western blotted to detect OCRL and β-actin. No expression of either truncated or full length OCRL was evident.

Figure S2. OCRL depletion slows proliferation of HK-2 cells without increasing apoptosis. Equal numbers (400,000 cells) of CRISPR/Cas9 control cl.1, KO cl.1, or KO cl.2 cells (A,B) or HK2 cells transfected with control or OCRL siRNA (C,D) were plated on Transwell filters, and cultured for four days, fixed, and stained with DAPI to visualize nuclei. Random fields were acquired and representative images are shown in (A and C). Nuclei were quantitated using the MatLab program described in Long et al. (2017) Mol. Biol. Cell 28:2508-2517. Nuclei/field (mean and individual points; five fields per condition in two or three independent experiments) is plotted in (B and D). (E) CRISPR/Cas9 Control cl.1, KO cl.1 and KO cl.2 were incubated with Biotium NucView 488 Caspase-3 substrate and imaged live every 20 min for 4.7-11.3 h. Cells undergoing apoptosis were identified by an increase in fluorescence signal and the percentage of apoptotic cells in each condition is plotted [mean and individual points of five independent experiments (four with KO cl.2 and one with KO cl.1)]. (F) Knockdown efficiency in HK-2 cells transfected with control or OCRL siRNA was confirmed by western blotting lysates with anti-OCRL antibodies and normalized to β-actin. OCRL depletion ranged from 70-95% and was depleted by 92% in the representative experiment shown. Scale bar (A and C): 10 µm. *p<0.05 by one-way ANOVA, Dunnett’s multiple comparisons test in (B).

Figure S3. OCRL knockdown increases multinucleation in mRPTC and hRPTC cell lines. Mouse (mRPTC, A-C) and human (hRPTC, D-F) renal proximal tubule cells were transfected with control or OCRL siRNA. Knockdown efficiency was quantified by western blotting and was typically 40-50% for mRPTCs (43% in the experiment shown in A) and 90-97% in hRPTCs (97% in the experiment shown in D). (B and E) Wheat germ agglutinin and DAPI were used to visualize cell borders and nuclei, respectively. Representative images of control siRNA mononuclear cells in samples transfected with control siRNA and multinucleated cells in samples treated with OCRL siRNA are shown. (C and F) The percentage (mean and individual points) of cells with multiple nuclei is plotted (panel C, three independent experiments; 261-539 cells per condition scored in each experiment; panel F, two independent experiments; 114-295 cells per condition scored in each experiment). Scale bar (B and E): 10 µm. * p<0.05 and *** p<0.001 based on unpaired t test in (C) and (F).

Figure S4. Effect of reduced Vmax on PT uptake of albumin and LMWP. (A) Albumin/creatinine ratio and (B) LMWP/creatinine ratio models with reductions in Vmax. Each line corresponds to a model solution using a Vmax that is 100-50% of the fit value. A 33% and 32% reduction in Vmax in the absence of proximal tubule shortening is predicted to result in a similar urinary excretion profile of albumin and LMWP, respectively, similar to that observed in LS, although it cannot explain the additional PT dysfunction in the disease. Changes in Vmax could also be relevant to tubular proteinuria in other genetic diseases, such as Dent disease. The effect of varying Vmax on urinary protein excretion of albumin (C) and LMWP (D) when SNGFR is reduced by 20% is plotted. Under this condition, a 41% and 43% reduction in Vmax would produce the predicted urinary excretion profiles of albumin and LMWP, respectively. In each graph, the black arrow denotes the expected urinary protein excretion ratio (mg/mmol creatinine) at the distal end of the PT of LS patients.

Figure S5. Ocrl morphant zebrafish injected with ocrl morpholino have shorter pronephric kidney segments. In situ hybridization of megalin/lrp2a, a proximal tubule marker, and cdh17, a pronephric tubule marker, was performed in uninjected controls and in zebrafish injected with 6ng or 8 ng of ocrl morpholino (MO) at 72 hpf. The distributions of lrp2a (A) and cdh17 (C) expression in representative zebrafish are shown. Scatter plots show the length of lrp2a (B) and cdh17 (D) positive segments (mean +/- SD; lrp2a, n=22-38; cdh17, n=48-80). (E) Ratio of average lrp2a length over average cdh17 length was compared across conditions. ** p<0.01 *** p<0.001 **** p<0.0001 based on one-way ANOVA via Tukey’s multiple comparisons test in (B) and (D).

Figure S6. Acute or chronic depletion of OCRL does not impair endocytic uptake in HK-2 cells. Control or OCRL knockout clones (A and B) or cells transfected with control or OCRL siRNA (C and D) were incubated under static conditions or exposed to orbital fluid shear stress (FSS) for 1 h in the presence of Alexa Fluor 647-albumin, then fixed, imaged, and albumin uptake quantified as described in Methods. Representative fields are shown in (A) and (C). (B) Albumin uptake was normalized to that under static conditions in each clone, and the fold change (mean +/- SE) of three independent experiments is plotted. (D) Data were normalized to uptake under static conditions and the mean fold change +/- SE of three independent experiments is plotted. Scale bars (A and C): 25 µm. * p<0.05 ** p<0.001 *** p<0.005 based on a two-way ANOVA via Sidak’s multiple comparisons test in (B).

Figure S7. Cilia length in siRNA treated and CRISPR/Cas9 OCRL knockout clones. HK-2 cells treated with either control or OCRL siRNA (A, B) or CRISPR/Cas9 control and OCRL KO clones (C, D) were fixed and processed for immunofluorescence to visualize acetylated tubulin and nuclei. Representative images of cells with cilia of approximately median length for each condition are shown in (A) and (C). (B) and (D) Cilia lengths were quantified as described in Methods and plotted in order of increasing length. Mean cilia length was approximately 6.5 µm and 7.9 µm in control and OCRL siRNA treated cells respectively, and 4.7 µm, 4.5 µm, 3.9 µm, and 5.1 µm in Control cl.1, Control cl.2, KO cl.1, and KO cl.2 cells, respectively. Cilia lengths were measured for 67-88 cells per condition in two independent experiments using CRISPR/Cas9 clones and 95 cells per condition in one experiment using siRNA treated cells. Scale bars (A and C): 10 µm.

Video S1. Cell division in CRISPR/Cas9 control clone 1. Control cl.1 cells, plated sparsely, were stained with Draq5 and imaged every 15 min using a combination of confocal and transmitted light to visualize DNA content and cell morphology. At the beginning of imaging, one cell in the selected field (arrow) started in interphase and successfully divided into two cells (~120 min from the initiation of metaphase to completion of cytokinesis).

Video S2. Cell division in CRISPR/Cas9 KO clone 2 that resolves into two cells. OCRL KO cl.2 cells, plated sparsely, were stained with Draq5 and imaged every 15 min using a combination of confocal and transmitted light to visualize DNA content and cell morphology. One cell in the selected field (arrow) was followed throughout cell division. This cell successfully divides into two cells. The period from initiation of metaphase to completion of cytokinesis was ~180 min.

Video S3. Cell division in CRISPR/Cas9 KO clone 2 that resolves into one multinucleated cell. OCRL KO cl.2 cells, plated sparsely, were stained with Draq5 and imaged every 15 min using a combination of confocal and transmitted light to visualize DNA content and cell morphology. One cell in the selected field (arrow) was followed throughout cell division. This cell does not properly complete cell division as the cytokinetic bridge is never cleaved. Eventually, abscission is aborted and the cell resolves into a single multinucleated cell. The period from metaphase to aborted cell division is ~300 min, and is longer than the average division time for OCRL KO cells that successfully divided into two daughter cells.

Gliozzi et al. Fig. S1

Control OCRL KO cl.1cl.2 cl.1 cl.2 130kDa

100kDa

70kDa OCRL 55kDa

35kDa

55kDa

Actin 35kDa Gliozzi et al. Fig. S2

A Control cl.1 OCRL KO cl.1 OCRL KO cl.2 C Control siRNA OCRL siRNA

B D 140 * 120 * 120 100

100 80

80 Exp.1 60 Exp.2 Exp.1 Nuclei/field (average count) Nuclei/field (average count) Exp.3 Exp.2 60 40 Control cl.1 KO cl.1 KO cl.2 siRNA: Control OCRL E 6 F

4 OCRL

2 Apoptosis (%)

0 Control KO Gliozzi et al. Fig. S3

A mRPTCD hRPTC

OCRL OCRL

B E

C 8 F 10 * *** 8 6

6 4 4

2 Exp.1 Exp.2 2 Exp.1 Multinucleated cells (%) Exp.3 Multinucleated cells (%) Exp.2 0 0 siRNA: Control OCRL siRNA: Control OCRL Gliozzi et al. Fig. S4

A B 100% Vmax 500 100% Vmax 1500 90% Vmax 90% Vmax 80% Vmax 400 80% Vmax 1250 70% Vmax 70% Vmax 60% Vmax 1000 300 60% Vmax 50% Vmax 50% Vmax 750 200 (mg/mmol) (mg/mmol) 500 100 250 LMWP/Creatinine Ratio LMWP/Creatinine Albumin/Creatinine Ratio 0 0 0246810121416 0246810121416 distance from origin of tubule (mm) distance from origin of tubule (mm)

C 80% SNGFRD 80% SNGFR 500 100% Vmax 100% Vmax 1500 90% Vmax 90% Vmax 400 80% Vmax 1250 80% Vmax 70% Vmax 70% Vmax 300 60% Vmax 1000 60% Vmax 50% Vmax 50% Vmax 750 200 (mg/mmol) (mg/mmol) 500 100 250 LMWP/Creatinine Ratio LMWP/Creatinine Albumin/Creatinine Ratio 0 0 0 2 4 6 8 10121416 0246810121416 distance from origin of tubule (mm) distance from origin of tubule (mm) Gliozzi et al. Fig. S5

A C uninjected 6ng MO 8ng MO uninjected 6ng MO 8ng MO

lrp2a cdh17 BDE **** **** 800 *** ** 400 *** **** 0.5 700 m)

 0.4 m)  300 600 length 0.3

200 500 0.2 expression length ( lrp2a/cdh17

expression length ( 400 100 ratio cdh17 0.1 lrp2a

0 0.0 O -M -MO -MO -MO -MO -MO crl crl o o ocrl ocrl ocrl ocrl g g g g ninjected g g uninjected uninjected u 6n 8n 6n 8n 6n 8n Gliozzi et al. Fig. S6

A Static FSS B 2.0 * * * *

1.5 Control cl.1

1.0

Exp.1 Albumin Uptake Exp.2 0.5 Exp.3 Fold Change (FSS/static) Static Control cl.2 FSS 0.0 cl.1 cl.2 cl.1 cl.2 Control OCRL KO KO cl.1 OCRL KO cl.2 OCRL

C Static FSS D 2.0

1.5

1.0 Control siRNA

Exp.1 Albumin Uptake 0.5 Exp.2 siRNA

Fold Change (FSS/static) Exp.3 static

OCRL FSS 0.0 siRNA: ControlControl OCRLOCRL Gliozzi et al. Fig. S7

A DAPI Tubulin Merge B 18 Control siRNA 16 OCRL siRNA 14 M)

 12 Control siRNA 10 8

siRNA 6 Cilia Length ( 4

OCRL 2 0 0 20406080100 Percentile C DAPI Tubulin Merge D

14 Control cl.1 Control cl.2 12 KO cl.1 KO cl.2 Control cl.1 10 M)  8

6 Control cl.2 Cilia Length ( 4

KO cl.1 2

0

OCRL 0 20406080100 Percentile KO cl.2 OCRL