A novel CLCN5 mutation associated with FSGS and podocyte injury

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Jun 5, 2018 - implicate a role for the CLCN5 in podocyte function and pathophysiology. ... We identified a novel mutation (L521F) in CLCN5, in two members ...

Accepted Manuscript A novel CLCN5 mutation associated with FSGS and podocyte injury Ashish K. Solanki, Ehtesham Arif, Thomas Morinelli, Robert C. Wilson, Gary Hardiman, Peifeng Deng, John M. Arthur, Juan CQ. Velez, Deepak Nihalani, Michael G. Janech, Milos N. Budisavljevic PII:

S2468-0249(18)30139-6

DOI:

10.1016/j.ekir.2018.06.003

Reference:

EKIR 371

To appear in:

Kidney International Reports

Received Date: 3 May 2018 Revised Date:

5 June 2018

Accepted Date: 9 June 2018

Please cite this article as: Solanki AK, Arif E, Morinelli T, Wilson RC, Hardiman G, Deng P, Arthur JM, Velez JC, Nihalani D, Janech MG, Budisavljevic MN, A novel CLCN5 mutation associated with FSGS and podocyte injury, Kidney International Reports (2018), doi: 10.1016/j.ekir.2018.06.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Department of Medicine, Nephrology Division, Medical University of South Carolina, Charleston, SC, USA ¥ Department of Nephrology, Ochsner Clinic Foundation, New Orleans, LA, USA ¶ Division of Nephrology, Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA § MUSC Bioinformatics, Center for Genomics Medicine, Medical University of South Carolina, Charleston, SC, USA € Division of Transplant Surgery, Department of Surgery, Medical University of South Carolina, Charleston, SC, USA ɫ Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC, USA Ω Department of Public Health Sciences, Medical University of South Carolina, Charleston, SC, USA £ Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA.

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Ashish K. Solanki, # Ehtesham Arif, # Thomas Morinelli, € Robert C. Wilson, £ Gary Hardiman, §, #, Ω, Peifeng Deng, # John M. Arthur, ¶ Juan CQ Velez, ¥, Deepak Nihalani#, *, Michael G. Janech, # Milos N. Budisavljevic #, ɫ, *

*Co-Corresponding authors

Budisavljevic MN Professor of Medicine Medical University of South Carolina Building, DD514, Charleston, SC, USA Email: [email protected]

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A novel CLCN5 mutation associated with FSGS and podocyte injury

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Deepak Nihalani, Ph. D Associate Professor of Medicine, Division of Nephrology, Drug Discovery Medical University of South Carolina, Charleston, SC 29425 Email: [email protected]

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ABSTRACT

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Introduction:

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Tubular-dysfunction is characteristic of Dent disease; however, focal segmental

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glomerulosclerosis (FSGS) can also be present. Glomerulosclerosis could be secondary

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to tubular injury, but it remains uncertain whether the CLCN5 gene, encoding an

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endosomal chloride/hydrogen exchanger, plays a role in podocyte biology. Here, we

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implicate a role for the CLCN5 in podocyte function and pathophysiology.

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Methods:

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Whole exome capture and sequencing of the proband and five maternally-related family

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members was conducted to identify X-linked mutations associated with biopsy-proven-

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FSGS. Human podocyte cultures were utilized to characterize the mutant phenotype on

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podocyte function.

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Results:

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We identified a novel mutation (L521F) in CLCN5, in two members of a Hispanic family

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who presented with a histologic diagnosis of FSGS and low molecular weight

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proteinuria without hypercalciuria. Presence of CLCN5 was confirmed in cultured

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human podocytes. Podocytes transfected with the wild-type or the mutant (L521F)

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CLCN5 constructs showed differential localization. CLCN5 knock-down in podocytes

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resulted in defective transferrin endocytosis and was associated with decreased cell

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proliferation and increased cell migration; hallmarks of podocyte injury.

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Conclusions:

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The CLCN5 mutation, which causes Dent disease, may associate with FSGS without

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hyercalcuria and nepthrolithiasis. The present findings support the hypothesis that

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CLCN5 participates in protein trafficking in podocytes and plays a critical role in

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organizing the components of the podocyte slit diaphragm to help maintain normal cell

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physiology and a functional filtration barrier. In addition to tubular dysfunction, mutations

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in CLCN5 may also lead to podocyte dysfunction resulting in a histologic picture of

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FSGS which may be a primary event and not a consequence of tubular damage.

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INTRODUCTION

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Dent Disease, an X-linked inherited disease, is characterized by proximal tubule

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dysfunction which leads to end stage renal disease (ESRD) in more than two-thirds of

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affected males. Mutations in the CLCN5 gene are responsible for 50-60% of cases (1).

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Close to 150 different CLCN5 mutations have been reported in patients with Dent

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disease (2-4). The CLCN5 gene encodes a chloride/proton exchanger playing an

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important role in endosomal acidification and receptor mediated endocytosis. The

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protein has 18 alpha helices (A-R). More than 40% of mutations seen in Dent disease

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have been found in O and P helices (5).

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The clinical presentation of Dent disease may be deceptive with a substantial number of

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patients expressing a partial or atypical phenotype (6) causing difficulty in its diagnosis

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(2). Many patients may not have classical features like rickets, nephrocalcinosis or

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nephrolithiasis, but only severe proteinuria composed mostly of low molecular weight

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proteins without high grade albuminuria. On initial presentation, this high grade

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proteinuria may be confused for nephrotic range proteinuria in patients with primary

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focal segmental glomerulosclerosis (FSGS), when in fact the underlying etiology is Dent

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disease, so careful clinical evaluation is essential. Though Dent disease is largely

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considered a tubular disease (7), focal segmental glomerulosclerosis (FSGS) or more

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commonly focal global glomerulosclerosis (FGGS) may be seen as a dominant feature

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in some patients with Dent Disease (7, 8).

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In kidney, CLCN5 is expressed in proximal tubules, thick ascending limbs and alpha-

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intercalated cells of the collecting duct (9). The protein functions as a 2Cl-/H+ exchanger

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and is involved in the acidification of endosomes, processing, and degradation of

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absorbed proteins, and megalin-dependent absorption of proteins. The expression of

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CLCN5 in glomerular cells has not been well documented. It is therefore intriguing that

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the glomerular pathology is caused by a variant of a tubular protein.

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A key aspect of primary FSGS pathogenesis is podocyte damage and loss (10, 11).

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Mutations in genes that encode glomerular proteins, particularly in visceral epithelial

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cells (podocytes), lead to the development of FSGS (12). Previous reports suggest that

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primary tubular injury may lead to glomerular sclerosis by mechanisms that are not yet

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understood (13, 14). In this study, we show that a variant of CLCN5 is present in a

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family with FSGS and that CLCN5 is expressed in human podocytes and may play a

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role in glomerular physiology and pathology. Based on our results we hypothesize that

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FSGS lesions, observed in patients with Dent disease result from altered localization

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and/or function of CLCN5 in the podocytes, and not purely as a secondary

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consequence of tubular injury. This novel mutation has provided a unique opportunity to

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explore the mechanism by which the 2Cl-/H+ exchanger functions in podocytes.

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Materials and Methods

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The study was approved by the Medical University of South Carolina (MUSC)

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Institutional Review Board and signed informed consent was obtained from all study

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participants. Urine calcium was measured using Abbott Architect analyzer at the MUSC

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central laboratory, and urine β2- macroglobulin at the ARUP Laboratory, Salt lake City,

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UT, using a quantitative chemiluminescent immunoassay. Whole blood was collected

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from affected and unaffected family members in purple top EDTA tubes.

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Whole exome capture and high through-put sequencing

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DNA was extracted using standard protocols from the individuals’ blood. The DNA was

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exome enriched followed by high-throughput sequencing (HTS). Enriched libraries were

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prepared utilizing Agilent’s (Santa Clara, CA) Sure Select XT Human All Exon V5+UTRs

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library kit for the Illumina platform. Adapters were ligated to sheared DNA followed by

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hybridization to baits for a 75 Mb exome capture. Sequencing was performed on the

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captured exomes following the manufacturer’s protocol (Illumina, San Diego, CA) using

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125 bp paired-end sequencing on an Illumina HiSeq2500, utilizing version 4 reagents

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and software. Data for each sample was obtained to ensure an overall average of 100X

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coverage. Fastq file output was used for downstream bioinformatics analysis.

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Bioinformatics analysis of whole exome sequencing data (Data Analysis and

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Statistical Justification

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Paired-end (2×125 bases) DNA sequence reads that passed the Illumina quality control

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step were included in downstream analysis. Alignment and variant calling was

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performed using MiSeq Reporter Software V2.4 (MSR), Illumina San Diego, CA with

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GRCh37 Genome Reference Consortium Human Build 37/HG19 as the reference

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genome.

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Filtering of variant call files (vcf) files was carried out as follows: variants that passed

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filter incorporating all variants types were examined. VariantStudio settings were as

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follows; Quality was required > 100, read depth > 10 and all population frequencies

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were < 5%. X-linked recessive was checked using the family based filtering option. Only

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variants mapping to a single site in all affected family members were selected for further

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Variant analysis was performed with VariantStudio v2.2.174 (Illumina).

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analysis. Initial analysis focused on variants mapping to the X chromosome since the

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pedigree suggested an X linkage. Sequence variants that were homozygous in the two

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affected family members and heterozygous or absent in the three unaffected family

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members were identified and the allele frequency of each variant obtained from the

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dbSNP version 138 reference database. The dbSNP database was then used to assign

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the value of ‘probable-pathogenic’ where appropriate. Those variants present in genes

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previously implicated in FSGS were selected for further study. While the cohort we

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examined is small, the apparent x-linkage improves the chance to identify a pathologic

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variant. All data fastq, vcf and phenotypic information have been submitted to dbGAP

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(accession number pending).

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In silico functional prediction of mutations:

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Functional prediction of the CLCN5 mutation was performed using the online in silico

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prediction software packages, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/);

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SIFT (http://sift.bii.a-star.edu.sg/) and MutationTaster (http://www.mutationtaster.org/).

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Cell culture and histochemistry

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The pCMV6-AC-GFP vector carrying wild-type CLCN5 gene (WT_CLCN5) and L521F

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mutant pCMV6-AC-GFP vector (L521F_CLCN5) were purchased from Origene.

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Sequencing of a constructed vector containing L521F mutation confirmed the veracity of

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the mutation. Both cDNAs were in the frame so that each protein would be fused to

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green fluorescent protein (GFP). HEK293 cells were purchased from ATCC. HK-2 and

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HEK293 cells were grown in DMEM/F12 199/EBSS medium respectively, until cells

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reached 70% confluence. Cells were transiently transfected with vectors using

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Lipofectamine 2000 transfection agent following manufacturer’s instructions. Twenty-

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four hours (HEK293 cells) or 48 hours (HK-2 cells) later images were taken using

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confocal microscopy. Double staining for lysosome and CLCN5 proteins were done. In

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brief, cells were grown as described above and after transfection for 24 hours incubated

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with Lysotracker deep red dye (Life Technologies, Cat. No. L12492) for 2 hours. Images

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of live cells were taken using immunofluorescence microscopy (Leica Microscope, DMI

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4000B). The human podocyte cell line was cultured in RPMI 1640-based medium

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supplemented with 10% fetal bovine serum (FBS) (Corning), insulin-transferrin-selenium

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(ITS) supplement (Sigma-Aldrich), and 200 units/ml penicillin and streptomycin (Roche

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Applied Science), as described previously (15). Human kidney sections were obtained

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from the MUSC tissue bank. In brief, normal renal tissue was obtained from

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nephrectomy specimens and embedded in paraffin. Five-micron sections were cut and

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processed.

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ShRNA based knockdown of CLCN5 gene in human podocytes cell line

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To target CLCN5, we screened Mission Lentiviral Transduction particle shRNAs in

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pLKO.1-puro (CLCN5 MISSION shRNA-particle, commercially purchased from Sigma,

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Catalog

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TRCN0000043904,

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TRCN000414058 and TCRN000427059; each shRNA will be designated as 903, 904,

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905, 906, 907, 058 and 059 respectively in the present study). Transfection of the

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ShRNA plasmids into the human podocyte cell line was performed with Lipofectamine

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2000 (Invitrogen) according to the manufacturer's protocol. Transfected podocytes cells

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SHCLNG-NM_000084; TRCN0000043905,

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number

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TRC

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TRCN0000043903,

TRCN0000043906,

TRCN0000043907,

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were grown in 2.5 µg/ml puromycin containing medium for the selection of stable

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transfectants and the CLCN5 KD was confirmed by western blot.

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Immunoblot Analysis

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Immunoblotting experiments were done as described previously (15). A Human

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podocyte cell line with CLCN5 knockdown were plated and allowed to grow at 33̊ C. The

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cells were harvested 48 h later and rinsed twice with PBS. Cell lysate was made using

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Radio immunoprecipitation assay buffer (RIPA buffer/lysis buffer). The membrane was

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immunoblotted with Anti-Chloride Channel CLC-5 antibody produced in rabbit

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(C1116, Sigma-Aldrich), 1:500 dilution in TBST with 3% BSA. To assure equivalent

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protein loading, the membranes were simultaneously incubated with GAPDH

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monoclonal antibody from Sigma (1:10,000) at 4˙C overnight. Membranes were washed

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three times with TBST, incubated with HRP-conjugated secondary antibodies (Pierce)

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at 1:10000 dilutions for 1 h at room temperature, and washed extensively before

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detection. The membranes were subsequently developed using Super Signal West

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Femto Maximum Sensitivity Substrate (Pierce, Cat. No. 34095) substrate and images

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were collected with a LICOR Image analyzer.

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Endocytosis

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Internalization assays using Transferrin from Human Serum, Alexa Fluorconjugated

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(Molecular Probes, Cat. No. T23365) were performed as described (16, 17) with some

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modifications. Citrate buffer (pH: 2.5) was used to remove surface-bound transferrin.

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Images were collected at 10, 15 and 30 minutes post-incubation using a Leica confocal

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microscope (TCS SP5). For the analysis of transferrin uptake in cells where CLCN5 was

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knocked down, images of at least 50 surface-bound transferrin cells were collected for

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two clones and a corresponding control. Cells that exhibited punctate transferrin

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labeling were counted as positive and cells that did not contain distinct transferrin

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puncta were counted as negative.

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Migration/Wound assay

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A migration assay was done as described previously with minor modification (18, 19).

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Control and CLCN5 knockdown podocytes were grown in 35-mm glass-bottom culture

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dishes (MatTek Corporation, USA) until a confluent cell monolayer was achieved. The

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cells were serum-starved in RPMI 1640 medium for 8 to 12 h. A scratch wound was

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created using a 1- to 10-µl pipette tip. Wounds were created with 2 strokes at a 90°

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angle and washed twice with PBS to remove all the suspended cells in the medium. The

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cells were then cultured in RPMI 1640 medium supplemented with 10% fetal bovine

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serum (FBS) (Corning), insulin-transferrin-selenium (ITS) supplement (Sigma-Aldrich),

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and 200 units/ml penicillin and streptomycin (Roche Applied Science) at 33°C for 12

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hours. Images were taken at different time points (0, 6 and 10 hours). The experiment

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was performed 3 times, and the rate of migration was calculated using ImageJ software.

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Cell proliferation assay

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An equal number (50000 cells) of control and CLCN5 knockdown podocytes were

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plated and allowed to grow for 24, 48 or 72 h. To measure the difference in the rate of

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proliferation, the cells were trypsinized and cells in suspension were counted using a

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hemocytometer. The number of cells at different time points was plotted and differences

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between the control and CLCN5 knockdown clones were calculated.

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Immunofluorescence Microscopy

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Cultured podocytes were grown on coverslips as described previously (20). Each

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experiment was carried out in three independent sets and the images were collected

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using a Leica immunofluorescence Microscope (Lieca DMI 400B).

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Statistical analysis

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Statistical analysis was performed using GraphPad PRISM 7.01 software. Distribution of

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cell wound closure data normality was tested through the D'Agostino & Pearson

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normality test and passed this test; whereas, the Shapiro-Wilk normality test showed all

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the data used for the cell proliferation analysis had a normal distribution. Statistical

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significance was determined by two way ANOVA and p-value were adjusted using

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Tukey’s multiple comparison and p-values less than 0.05 was considered statistically

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significant. Mean fluorescence intensity was measured by immunofluorescence images

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using Image-J software(National Institutes of Health, Bethesda, USA) and more than 50

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images were used from three different experiments. Mean pixel intensity differences

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between control and CLCN5 knockdown podocytes was analyzed through the

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nonparametric Mann Whitney test and p< 0.05 is considered as statistically significant.

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RESULTS Pedigree analysis and whole exome sequencing

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The proband, a 37 years old Hispanic man, was evaluated by a local nephrologist in

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Guadalajara, Mexico for proteinuria and an increase in serum creatinine. Based on the

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kidney biopsy, shown in Figure 1A, he was diagnosed with FSGS in 2012. Two years

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later, in January 2014 he was presented to our hospital for a kidney transplant. During a

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donor evaluation at that time, it was revealed that his 32-year-old brother had similar

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symptoms, and based on a kidney biopsy (Figure 1B) was also diagnosed with FSGS.

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Based on a survey of both affected brothers, we created a pedigree and identified 9

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family members in total that had kidney disease. The pedigree presented in Figure 1C

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is suggestive of an X-linked mode of inheritance. We obtained whole blood and

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performed whole exome sequencing in 6 individuals; the proband, one affected brother,

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one non-affected brother, an affected uncle and the proband’s non-affected mother and

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sister. Urine was obtained from the proband’s sister, mother, clinically non-affected

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brother and affected brother not yet with ESRD. Total Calcium and β2 microglobulin

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excretion values are presented in Table 1. The excretion of β2 microglobulin (corrected

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for urine Cr) was more than 2000 times greater for the affected brother compared to the

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non-affected brother and sister; whereas, in the unaffected, carrier mother β2

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microglobulin excretion (corrected for Cr) was only about 64 times greater.

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Whole exome sequencing identified 6 gene variants in 5 genes on the X chromosome in

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the proband: CLCN5 (NM_001127898.1:c.1771C>T), HDX (NM_144657.4:c.1190T>C),

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KIAA2022 (two variants: NM_001008537.2:c.3001G>C, NM_001008537.2:c.2851G>A),

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SLC16A2 (NM_006517.4:c.538G>A), and SSX5 (NM_021015.3:c.337C>T). Of the six

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candidate gene variants, only one was present in all affected members, demonstrating

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heterozygosity in the proband’s mother and sister (i.e. only one X chromosome

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affected), and was not present in the non-affected brother. Based on this rationale the

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most likely candidate responsible for the development of FSGS in this family is the

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mutation found in the CLCN5 gene. The mutation involves replacement of

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phenylalanine for leucine at position 521 (L/F521, CLCN5 variant 3 NM_000084.4) in

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the shorter variant or position 591(L/F591, CLCN5 variant 1 NM_001127899.3) in the

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longer variant.

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In silico prediction of the effect of amino acid change on CLCN5

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Polyphen-2, SIFT and MutationTaster servers were used to predict the effect of

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mutation L521F in CLCN5 gene. Based on the output obtained using server Polyphen-

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2, the mutation was predicted to be damaging with a score of 0.884 (sensitivity: 0.82;

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specificity: 0.94). Interestingly, the server SIFT substitution at position 521 from L to F

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was predicted to be intolerant and may affect protein function with a score of 0.04.

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Mutation Taster server also predicted the mutation to affect protein physiology leading

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to a disease phenotype.

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Cellular localization of wild-type and mutant CLCN5-L521F proteins

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To further analyze if the L521F mutation results in defective cellular localization of

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CLCN5 protein, we overexpressed these genes in cultured HEK and HK-2 cells. Cells

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were transiently transfected with the pCMV6-AC-GFP vector carrying wild-type CLCN5

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cDNA (WT_CLCN5) and the L521F mutant pCMV6-AC-GFP vector (L521F_CLCN5).

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As seen in Figure 2A, left panel, HEK cells transfected with WT_CLCN5 showed

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significant localization of CLCN5 protein at the cell membrane. In contrast, HEK cells

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transfected with L521F-CLCN5 showed expression of the mutant L521F CLCN5 protein

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primarily in the cytoplasm [Figure 2A, right panel]. Similar findings were noted in the

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HK-2 transfected cells as demonstrated in Figure 2B (left and right panel for wild-type

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CLCN5 and L521F mutant respectively).

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To further investigate if L521F CLCN5 was entrapped in lysosomal structures, we used

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a lysosome specific stain Lysotracker deep red. Figure 2C shows co-localization of

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GFP labeled CLCN5 protein (green) with the lysosomal specific marker (red). As

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demonstrated in Figure 2C, compared to the wild-type CLCN5 protein (left panel), the

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mutant L521F CLCN5 protein showed increased colocalization with lysosomes (right

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panel). These findings are consistent with previous reports showing that the CLCN5

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(L521F) mutation results in the formation of defective protein that is aberrantly

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processed and transferred to lysosomes for degradation.

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Identification of CLCN5 protein in cultured human podocytes and kidney sections

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A single laboratory group has demonstrated that CLCN5 is expressed in glomerular

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podocytes, where CLCN5 belongs to a complex of proteins involved in albumin

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reabsorption in the podocytes (21, 22); however, these data have not been

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independently verified and the effect of CLCN5 mutations in podocytes have not been

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studied. Figure 3A shows a western blot of homogenized human cultured podocyte

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lysate probed with anti CLCN5 antibody. We identified a single band corresponding to

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the approximate molecular weight of 80 KDa most likely representing the shorter, 746

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amino acid CLCN5 proteoform. Immunofluorescence studies presented in Figure 3B

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further supported the western blot findings and demonstrate the expression of

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endogenous CLCN5 in human cultured podocytes. We further tested glomerular

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expression in sections of normal mouse and human kidney and found significant

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expression of the CLCN5 protein in mice kidney glomeruli and parietal epithelial cells.

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Cellular localization of wild-type and L521F mutant CLCN5 protein in cultured

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human podocytes

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A cultured human podocyte cell line was transfected with wild-type CLCN5

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(WT_CLCN5) and mutant CLCN5 (L521F_CLCN5) constructs. As demonstrated in

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Figure 3C, cells transfected with a GFP-tagged WT construct demonstrated both

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cytoplasmic and cell surface distribution of the protein; in contrast, the cultured human

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podocytes transfected with GFP tagged L521F CLCN5 demonstrated predominantly

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intracellular distribution. Importantly, the results obtained with human podocytes are

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similar to the observations noted in the transfected human renal proximal tubule cells.

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Effect of Knockdown of CLCN5 Expression on Cell Proliferation-Cell Migration in

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Podocyte Cells

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To further examine the involvement of CLCN5 in trafficking mechanisms in podocytes,

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especially in endocytosis, we generated a stable human podocyte cell line where the

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CLCN5 gene was knocked down using the CLCN5 specific shRNA. The CLCN5

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knockdown in podocyte cell lines was confirmed by immunoblotting (Figure 4A, upper

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panel). The cell proliferation assay (Figure 4A (lower panel). showed a reduced rate of

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proliferation in the CLCN5 knock down human podocytes when compared to the control

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podocytes. Interestingly, the rate of cell migration, as assessed by a scratch assay, in

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CLCN5 knockdown cells was increased when compared to the control podocyte cells

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(Figure 4B). The increased rate of cell migration is reported as abnormal and is a sign

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of podocytes injury.

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Endocytosis

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CLCN5 has been shown to play a role in the uptake of low molecular mass proteins

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through receptor-mediated endocytosis in proximal tubules (23). It has been reported

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that CLCN5 channel disruption impairs endocytosis in a mouse model for Dent disease

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(24). To examine whether CLCN5 is involved in endocytosis in human podocytes, we

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monitored uptake of Transferrin (Alexa Fluor conjugated) in CLCN5 knockdown human

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podocyte cells. As shown in Figure 4C(left and right panel) and 4D, the fluorescence

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intensity of endocytosed transferrin was much higher in wild-type human podocytes as

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compared to the CLCN5 knockdown cells after 10, 20 and 30 minutes of incubation.

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These results suggest that CLCN5 plays a key role in trafficking mechanisms in

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podocytes.

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DISCUSSION

In this report, we have identified and characterized a novel mutation in the CLCN5 gene

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found in a Hispanic family whose two members were presented with overt proteinuria

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and were diagnosed with FSGS. None of the family members were aware of or had

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classical features of Dent disease e.g. nephrocalcinosis, nephrolithiasis, rickets, and

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hypercalciuria. Proteinuria on the initial evaluation was not characterized as consisting

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of only low molecular weight proteins. According to the literature (3, 7, 25), erroneous

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diagnosis and even treatment of presumptive FSGS is not a rare event because of a

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considerable number of patients with Dent disease, like in our case, have an

361

oligosymptomatic phenotype.

362

CLCN5 protein has two predominant splice proteoforms, a shorter form consisting of

363

746 amino acids (MW 83 kDa; Uniprot accession number P51795-1 ), and a longer form

364

that includes an additional 70 amino acids at the N-terminal end thereby creating an 816

365

amino acid protein (MW 90 kDa; Uniprot accession number P51795-2). The mutation

366

described herein which results in the replacement of leucine with phenylalanine at

367

position 521 (short variant, Uniprot accession number P51795-1) or position 591 (long

368

variant, Uniprot accession number P51795-2). Based on our findings, we postulate that

369

this mutation has pathogenic significance. First, the mutation on this specific site

370

involving replacement of leucine with arginine (L521R) was previously reported in a

371

pediatric patient with Dent disease (26) but there has been no study with respect to the

372

consequence of CLCN5 mutations on the function of human podocytes that may

373

underlie the observed glomerular involvement. Secondly, the experimental replacement

374

of leucine with arginine at position 521 resulted in defective trafficking to the surface of

375

Xenopus oocytes compared to wild-type CLCN5 suggesting mutations at this site can

376

impair localization to the plasma membrane (27). Thirdly, the mutation is located in the

377

P helix of the CLCN5 protein, a region that is highly conserved across the members of

378

the CLCN family (5). The CLCN5 exchanger functions as a homodimer and integrity of

379

the P helix is essential for homodimer formation (33). Our results are consistent with the

380

previous report, where mutations at the leucine 521 position results in improper

381

trafficking of the mutant protein and may be directed to lysosomes for degradation as

382

observed from the Lysotracker staining experiments in our study (Figure 2C).

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The lesions of FSGS and FGGS have increasingly been reported in patients with Dent

384

disease. In the most extensive study published so far involving 30 kidney biopsies,

385

FGGS was found in 83.3% of cases, FSGS in 6.6%, and segmental capillary collapse in

386

6.6% of cases. Segmental foot process effacement was found in all cases, but was

387

analyzed

388

glomerulosclerosis in a patient with Dent disease has generally been regarded as a

389

consequence of tubular damage; however, no plausible mechanism has been proposed

390

(2, 3, 26). Based on data presented within this study, we postulate that a mutation in

391

CLCN5 can lead to glomerulosclerosis in part through primary podocyte injury.

392

To evaluate the role of CLCN5 in podocyte damage, we first demonstrated that CLCN5

393

is

394

immunohistochemistry. The physiologic role of CLCN5 in podocytes has not been

395

previously investigated though its expression has been shown in podocytes(29).

396

Evidence suggests that similar to proximal tubular cells, endocytic mechanisms operate

397

in podocytes and play an important role in the maintenance of the glomerular filtration

398

barrier (30). Furthermore, it appears that glomerular expression of CLCN5 is increased

399

in some proteinuric patients (21).

400

Our experiments demonstrated that the identified human mutation (L521F) leads to

401

altered subcellular localization of the CLCN5 protein in podocytes in the same manner

402

described for cultured human proximal tubular cells (31). It is thus conceivable that

403

CLCN5 may have a similar role in podocytes as in proximal tubular cells. Data from

404

immortalized proximal tubule cell lines derived from patients with Dent disease indicate

405

that similar abnormalities resulting from CLCN5 mutations may be derived through

less

than

half

of

biopsy

samples

(28).

The

appearance

of

cultured

human

podocytes

both

by Western

blotting and

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expressed in

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differing cellular phenotypes affecting either endosomal acidification and/or receptor

407

mediated endocytosis (31). We demonstrated impaired endocytosis in cultured

408

podocytes with genetic knock-down of CLCN5 genes.

409

Previous studies have demonstrated that in response to direct cell injury, cultured

410

podocytes change from limited motility to enhanced motility and loss of stress fibers

411

(18). Upon insult, stationary podocytes upregulate cytosolic cathepsin L expression and

412

activity and develop motile podocyte foot processes. This migratory response leads to

413

foot processes effacement, slit diaphragm remodeling, and proteinuria (32-34). This

414

observation suggests that stress-induced podocyte motility associated with actin

415

dynamics results in dysfunction of slit molecules and the integrin network. We

416

demonstrated that deletion of CLCN5 gene in cultured podocytes resulted in increased

417

cell migration, an established phenotype of injured podocytes. These data along with

418

our finding of impaired endocytosis suggests the presence of CLCN5 is necessary for

419

proper podocyte function. Recognition of the crucial role of CLCN5 in podocyte protein

420

trafficking holds promise for the identification of important molecular events that regulate

421

podocyte injury.

422

Collectively, the results presented in this study suggest that CLCN5 is expressed in

423

podocytes and plays a critical role in podocyte function by participating in protein

424

endocytosis. We provide some evidence that FSGS or FGGS observed in Dent disease

425

may be the result of primary podocyte injury independent of tubular injury. However, at

426

this time we cannot conclude that the mutation presented here has pathological

427

consequences for podocytes. The concept that a condition, which has historically been

428

considered primarily tubular in origin can lead to FSGS does not appear to be limited to

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CLCN5 mutations. Recently, lesions of FSGS were seen in patients with a mutation in

430

the TTC21B gene that encodes a ciliary protein and causes nephronophthisis, a

431

classical tubular disease. Similar to CLCN5, the expression of TTC21B was detected in

432

podocytes (35, 36). The probability that Dents disease is also podocyte disease raises

433

intriguing questions regarding the use of angiotensin converting enzyme inhibitors or

434

angiotensin receptor blockers in this condition. The use of enalapril and candesartan

435

have been reported in isolated case reports (37, 38) but never tested systematically.

436

Based on our observations, we believe placebo control trials using angiotensin

437

antagonists would be highly recommended to assess their impact on the progression of

438

Dent disease.” Additionally, CLCN5 knock out mice that replicate Dent disease have

439

been generated and may serve as a useful animal model to assess the effect of

440

angiotensin blockade on disease progression.

441

For physicians treating patients with a pathologic diagnosis of FSGS and proteinuria, it

442

is of utmost importance to make sure that low molecular weight proteinuria and Dent

443

disease are excluded before subjecting patients to toxic immunosuppressive treatment,

444

as Dent disease with glomerular involvement is no longer considered a rare event (3, 7,

445

25).

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Acknowledgements:

448

The authors thank the American Society of Nephrology for the Ben J. Lipps Research

449

Fellowship to A.K.S. This work was supported in whole or in part by an NIH/NIDDK

450

Grant, 2R01DK087956-06A1 to D. N, DCI (Dialysis Clinics Incorporated) research funds

451

to M.B. and M.J.

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Disclosures:

455

None

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TABLES AND FIGURE LEGENDS:

556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599

Figure 1 Identification of FSGS patients and pedigree analysis: We identified two brothers, the proband (A) and the affected brother (B) who presented to a local nephrologist, in Mexico, with proteinuria and altered renal function. The silver stained biopsy image showed a segmentally sclerotic glomerulus. Other areas of the glomeruli appear relatively normal. Both were diagnosed with FSGS based on kidney biopsies presented here. (C) Pedigree of the Family with X-linked Inheritance of FSGS. The red arrow indicates proband. Blood was collected from the proband and five additional family members indicated by asterisks. The pedigree suggested an X-linked pattern of inheritance. The open boxes and circles represent the normal male and female respectively. The closed boxes indicate affected male and the crossed-out box indicate the deceased ones.

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Figure 2 Transient expression of wild-type and L521F mutant CLCN5-GFR constructs in HEK cells and HK-2 cells: (A) In HEK cells: WT CLCN5 protein predominantly expressed on cell membrane (left panel), while L591F mutant displayed cytoplasmic localization (right panel). (B) In HK-2 cells, WT CLCN5 protein was predominantly expressed on the cell membrane (left panel), while L591F mutant displayed cytoplasmic localization (right panel). (C) Intracellular localization of wildtype and mutant CLCN5 in human podocytes: Right panel shows mutant CLCN5 protein displays greater co-localization within lysosomes and disperse cytoplasmic distribution compared to a primarily membrane distribution in the wild-type CLCN5 podocytes (left panel).

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Figure 3 Expression of CLCN5 constructs in human podocyte cell lines (A) Western blot showed a single band consistent with the short 83 KDa CLCN5 proteoform. Neph1 expression which was used a positive control was confirmed using an anti-Neph1 antibody. (B) Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes. (C) Upper panel: Human podocytes transfected with GFP tagged WT CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. Lower panel: Cultured human podocytes transfected with GFP tagged L521F mutant CLCN5 protein demonstrated predominantly intracellular distribution. Figure 4: shRNA based knockdown of CLCN5 in human podocytes: (A) Western blot showing CLCN5 knockdown in human podocytes using seven different shRNA clones. (B) Cell proliferation assay showed that CLCN5 knockdown podocytes have a reduced cell proliferation rate compared to control. The rate of clone 1 was depressed at 24 hours and the rate of clone 2 was depressed at 72 hours (p-value=0.01 and 0.004, respectively). All data are presented as Mean±SD (n = 4). (C) Wound closure assay showed that both clone 1 and clone 2 have a reduction in wound closure at 6 hours (p-value=0.002 and 0.05, respectively) and 10hours (p-value

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