A role for genetic susceptibility in sporadic focal segmental glomerulosclerosis
Haiyang Yu, … , Mark J. Daly, Andrey S. Shaw
Haiyang Yu, … , Mark J. Daly, Andrey S. Shaw
Published February 22, 2016
Citation Information: J Clin Invest. 2016;126(3):1067-1078. https://doi.org/10.1172/JCI82592.
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Research Article Nephrology

A role for genetic susceptibility in sporadic focal segmental glomerulosclerosis

Abstract

Focal segmental glomerulosclerosis (FSGS) is a syndrome that involves kidney podocyte dysfunction and causes chronic kidney disease. Multiple factors including chemical toxicity, inflammation, and infection underlie FSGS; however, highly penetrant disease genes have been identified in a small fraction of patients with a family history of FSGS. Variants of apolipoprotein L1 (APOL1) have been linked to FSGS in African Americans with HIV or hypertension, supporting the proposal that genetic factors enhance FSGS susceptibility. Here, we used sequencing to investigate whether genetics plays a role in the majority of FSGS cases that are identified as primary or sporadic FSGS and have no known cause. Given the limited number of biopsy-proven cases with ethnically matched controls, we devised an analytic strategy to identify and rank potential candidate genes and used an animal model for validation. Nine candidate FSGS susceptibility genes were identified in our patient cohort, and three were validated using a high-throughput mouse method that we developed. Specifically, we introduced a podocyte-specific, doxycycline-inducible transactivator into a murine embryonic stem cell line with an FSGS-susceptible genetic background that allows shRNA-mediated targeting of candidate genes in the adult kidney. Our analysis supports a broader role for genetic susceptibility of both sporadic and familial cases of FSGS and provides a tool to rapidly evaluate candidate FSGS-associated genes.

Authors

Haiyang Yu, Mykyta Artomov, Sebastian Brähler, M. Christine Stander, Ghaidan Shamsan, Matthew G. Sampson, J. Michael White, Matthias Kretzler, Jeffrey H. Miner, Sanjay Jain, Cheryl A. Winkler, Robi D. Mitra, Jeffrey B. Kopp, Mark J. Daly, Andrey S. Shaw

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Figure 4

Validation of 5 candidate FSGS disease genes.

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Validation of 5 candidate FSGS disease genes. (A) Validation of shRNAs f...
(A) Validation of shRNAs for Arhgef17, Dlg5, Kank1, Kank2, Wnk4, and Kat2b. As described in Methods, shRNAs were tested for the ability to inhibit a target sequence fused to GFP in 293 cells. GFP immunoblotting was used to measure the degree of inhibition. Each immunoblot is representative of at least 3 independent experiments measuring RNAi efficiency. (B–G) Mouse validation screening for candidate FSGS genes. ES cells were generated with the specific RNAis targeted to the Hgprt locus. Essentially pure chimeric mice were generated by laser-assisted microinjection of ES cells into C57BL6 8-cell embryos. Injections generally resulted in cohorts of 14 to 30 animals; smaller cohorts of animals were not used. Mice were divided into 2 groups and treated with or without DOX to induce expression of the RNAi transgene. Urine albumin/creatinine ratios were measured 4 and 8 weeks after DOX treatment. Albumin/creatinine ratios are shown for each cohort of mice at the indicated time points. A 2-tailed Mann-Whitney U test was used to calculate the P values for B–G. A P value of less than 0.0083 was considered statistically significant (multitest penalty was used).