A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury Solanki, A. K., Arif, E., Morinelli, T., Wilson, R. C., Hardiman, G., Deng, P., Arthur, J. M., Velez, J. CQ., Nihalani, D., Janech, M. G., & Budisavljevic, M. N. (2018). A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury. Kidney International Reports, 3(6), 1443-1453. https://doi.org/10.1016/j.ekir.2018.06.003 Published in: Kidney International Reports Document Version: Publisher's PDF, also known as Version of record Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights Copyright 2018 the authors. 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Download date:25. Sep. 2021 TRANSLATIONAL RESEARCH A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury Ashish K. Solanki1, Ehtesham Arif1, Thomas Morinelli5, Robert C. Wilson8, Gary Hardiman1,4,7, Peifeng Deng1, John M. Arthur3, Juan CQ Velez2, Deepak Nihalani1, Michael G. Janech1 and Milos N. Budisavljevic1,6 1Department of Medicine, Nephrology Division, Medical University of South Carolina, Charleston, South Carolina, USA; 2Department of Nephrology, Ochsner Clinic Foundation, New Orleans, Louisiana, USA; 3Division of Nephrology, Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA; 4MUSC Bioinformatics, Center for Geno- mics Medicine, Medical University of South Carolina, Charleston, South Carolina, USA; 5Division of Transplant Surgery, Department of Surgery, Medical University of South Carolina, Charleston, South Carolina, USA; 6Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina, USA; 7Department of Public Health Sciences, Medical University of South Carolina, Charleston, South Carolina, USA; and 8Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina, USA Introduction: Tubular dysfunction is characteristic of Dent’s disease; however, focal segmental glomer- ulosclerosis (FSGS) can also be present. Glomerulosclerosis could be secondary to tubular injury, but it remains uncertain whether the CLCN5 gene, which encodes an endosomal chloride and/or hydrogen exchanger, plays a role in podocyte biology. Here, we implicate a role for CLCN5 in podocyte function and pathophysiology. Methods: Whole exome capture and sequencing of the proband and 5 maternally-related family members was conducted to identify X-linked mutations associated with biopsy-proven FSGS. Human podocyte cultures were used to characterize the mutant phenotype on podocyte function. Results: We identified a novel mutation (L521F) in CLCN5 in 2 members of a Hispanic family who pre- sented with a histologic diagnosis of FSGS and low-molecular-weight proteinuria without hypercalciuria. Presence of CLCN5 was confirmed in cultured human podocytes. Podocytes transfected with the wild-type or the mutant (L521F) CLCN5 constructs showed differential localization. CLCN5 knockdown in podocytes resulted in defective transferrin endocytosis and was associated with decreased cell proliferation and increased cell migration, which are hallmarks of podocyte injury. Conclusions: The CLCN5 mutation, which causes Dent’s disease, may be associated with FSGS without hyercalcuria and nepthrolithiasis. The present findings supported the hypothesis that CLCN5 participates in protein trafficking in podocytes and plays a critical role in organizing the components of the podocyte slit diaphragm to help maintain normal cell physiology and a functional filtration barrier. In addition to tubular dysfunction, mutations in CLCN5 may also lead to podocyte dysfunction, which results in a his- tologic picture of FSGS that may be a primary event and not a consequence of tubular damage. Kidney Int Rep (2018) 3, 1443–1453; https://doi.org/10.1016/j.ekir.2018.06.003 KEYWORDS: CLCN5; Dent’s disease; FSGS; podocytes; renal biopsy ª 2018 International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY- NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ent’s disease, an X-linked inherited disease, is two-thirds of affected males. Mutations in the CLCN5 D characterized by proximal tubule dysfunction that gene are responsible for 50% to 60% of cases.1 Close leads to end-stage renal disease (ESRD) in more than to 150 different CLCN5 mutations have been reported – in patients with Dent’s disease.2 4 The CLCN5 gene en- codes a chloride and/or proton exchanger that plays an Correspondence: Milos N. Budisavljevic, Medical University of fi South Carolina, Building, DD514, Charleston, South Carolina important role in endosomal acidi cation and receptor- 29425, USA. E-mail: [email protected]; and Deepak Nihalani, mediated endocytosis. The protein has 18 a-helices Division of Nephrology, Drug Discovery, Division of Nephrology, (AÀR). More than 40% of mutations seen in Dent’s dis- Drug Discovery, Charleston, South Carolina 29425, USA. E-mail: 5 [email protected] ease have been found in O and P helices. ’ Received 3 May 2018; revised 5 June 2018; accepted 9 June 2018; The clinical presentation of Dent s disease may be published online 18 June 2018 deceptive, with a substantial number of patients Kidney International Reports (2018) 3, 1443–1453 1443 TRANSLATIONAL RESEARCH AK Solanki et al.: FSGS and Podocytes expressing a partial or atypical phenotype,6 which Whole Exome Capture and High-Throughput causes difficulty in its diagnosis.2 Many patients may Sequencing not have classical features (e.g., rickets, nephrocalci- DNA was extracted from the blood of the individuals nosis, or nephrolithiasis) but may only have severe using standard protocols. The DNA was exome-enriched, proteinuria, which consists of mostly low-molecular- followed by high-throughput sequencing. Enriched li- weight proteins without high-grade albuminuria. On braries were prepared using Agilent’s(SantaClara,CA) initial presentation, this high-grade proteinuria may be Sure Select XT Human All Exon V5þUTRs library kit for confused for nephrotic range proteinuria in patients the Illumina platform (Illumina, San Diego, CA). Adapters with primary FSGS, when in fact, the underlying etiol- were ligated to sheared DNA followed by hybridization ogy is Dent’s disease; therefore, careful clinical evalua- to baits for a 75-Mb exome capture. Sequencing was tion is essential. Although Dent’s disease is largely performed on the captured exomes following the manu- considered a tubular disease,7 FSGS, or more commonly, facturer’s protocol using 125 bp paired-end sequencing focal global glomerulosclerosis (FGGS), may be seen as a on an Illumina HiSeq2500, using version 4 reagents and dominant feature in some patients with Dent’s disease.7,8 software. Data for each sample was obtained to ensure an In the kidney, CLCN5 is expressed in proximal tubules, overall average of 100Â coverage. Fastq file output was thick ascending limbs, and a-intercalated cells of the used for downstream bioinformatics analysis. À þ collecting duct.9 Theproteinfunctionsasa2Cl/H exchanger and is involved in the acidification of endo- Bioinformatics Analysis of Whole Exome somes, processing, and degradation of absorbed proteins, Sequencing Data (Data Analysis and Statistical and megalin-dependent absorption of proteins. The Justification) expression of CLCN5 in glomerular cells has not been Paired-end (2 Â 125 bases) DNA sequence reads that well-documented. Therefore, it is intriguing that the passed the Illumina quality control step were included glomerular pathology is caused by a variant of a tubular in downstream analysis. Alignment and variant calling protein. was performed using MiSeq Reporter Software version A key aspect of primary FSGS pathogenesis is podo- 2.4 (MSR) (Illumina) with GRCh37 Genome Reference cyte damage and loss.10,11 Mutations in genes that encode Consortium Human Build 37/HG19 as the reference glomerular proteins, particularly in visceral epithelial genome. Variant analysis was performed with Var- cells (podocytes), lead to the development of FSGS.12 iantStudio version 2.2.174 (Illumina). Filtering of Previous reports have suggested that primary tubular variant call files (vcf) files was carried out as follows: injury may lead to glomerular sclerosis by mechanisms variants that passed the filter that incorporated all that are not yet understood.13,14 In this study, we show variants types were examined. VariantStudio settings that a variant of CLCN5 is present in a family