Endoplasmic reticulum–associated degradation is required for nephrin maturation and kidney glomerular filtration function
Sei Yoshida, Xiaoqiong Wei, Gensheng Zhang, Christopher L. O’Connor, Mauricio Torres, Zhangsen Zhou, Liangguang Lin, Rajasree Menon, Xiaoxi Xu, Wenyue Zheng, Yi Xiong, Edgar Otto, Chih-Hang Anthony Tang, Rui Hua, Rakesh Verma, Hiroyuki Mori, Yang Zhang, Chih-Chi Andrew Hu, Ming Liu, Puneet Garg, Jeffrey B. Hodgin, Shengyi Sun, Markus Bitzer, Ling Qi
Sei Yoshida, Xiaoqiong Wei, Gensheng Zhang, Christopher L. O’Connor, Mauricio Torres, Zhangsen Zhou, Liangguang Lin, Rajasree Menon, Xiaoxi Xu, Wenyue Zheng, Yi Xiong, Edgar Otto, Chih-Hang Anthony Tang, Rui Hua, Rakesh Verma, Hiroyuki Mori, Yang Zhang, Chih-Chi Andrew Hu, Ming Liu, Puneet Garg, Jeffrey B. Hodgin, Shengyi Sun, Markus Bitzer, Ling Qi
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Research Article Cell biology Nephrology

Endoplasmic reticulum–associated degradation is required for nephrin maturation and kidney glomerular filtration function

Abstract

Podocytes are key to the glomerular filtration barrier by forming a slit diaphragm between interdigitating foot processes; however, the molecular details and functional importance of protein folding and degradation in the ER remain unknown. Here, we show that the SEL1L-HRD1 protein complex of ER-associated degradation (ERAD) is required for slit diaphragm formation and glomerular filtration function. SEL1L-HRD1 ERAD is highly expressed in podocytes of both mouse and human kidneys. Mice with podocyte-specific Sel1L deficiency develop podocytopathy and severe congenital nephrotic syndrome with an impaired slit diaphragm shortly after weaning and die prematurely, with a median lifespan of approximately 3 months. We show mechanistically that nephrin, a type 1 membrane protein causally linked to congenital nephrotic syndrome, is an endogenous ERAD substrate. ERAD deficiency attenuated the maturation of nascent nephrin, leading to its retention in the ER. We also show that various autosomal-recessive nephrin disease mutants were highly unstable and broken down by SEL1L-HRD1 ERAD, which attenuated the pathogenicity of the mutants toward the WT allele. This study uncovers a critical role of SEL1L-HRD1 ERAD in glomerular filtration barrier function and provides insights into the pathogenesis associated with autosomal-recessive disease mutants.

Authors

Sei Yoshida, Xiaoqiong Wei, Gensheng Zhang, Christopher L. O’Connor, Mauricio Torres, Zhangsen Zhou, Liangguang Lin, Rajasree Menon, Xiaoxi Xu, Wenyue Zheng, Yi Xiong, Edgar Otto, Chih-Hang Anthony Tang, Rui Hua, Rakesh Verma, Hiroyuki Mori, Yang Zhang, Chih-Chi Andrew Hu, Ming Liu, Puneet Garg, Jeffrey B. Hodgin, Shengyi Sun, Markus Bitzer, Ling Qi

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Figure 8

ERAD of disease mutants attenuates their pathogenicity toward the WT allele.

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ERAD of disease mutants attenuates their pathogenicity toward the WT all...
(A) Western blot analysis of WT and mutant HMW and monomeric nephrin in transfected WT and HRD1–/– HEK293T cells under nonreducing and reducing conditions. (B and C) Western blot analysis of NP40-soluble (B) and NP40-insoluble fractions (C) in transfected WT and HRD1–/– HEK293T cells, showing increased formation of HMW and insoluble nephrin aggregates in HRD1–/– HEK293T cells for both WT and mutant nephrin. (D) Western blot analysis of nephrin HMW aggregation in HEK293T cells transfected with different combinations of Myc-WT nephrin and nephrin mutants under nonreducing conditions, with the level of HMW nephrin from 1 representative experiment shown below the blot. (E and F) Western blot analysis of Myc-WT and Flag-mutant nephrin in HEK293T cells transfected with different combinations of Myc-WT nephrin and Flag-tagged mutant nephrin at a 1:1 or 1:3 ratio. Quantitation of the percentage of a form WT nephrin in total WT nephrin is shown in F, indicating a decrease in the percentage of a form WT nephrin in HRD1–/– HEK293T cells (upon cotransfection of an increased amount of mutant nephrin) when compared with that in WT HEK293T cells. Values represent the mean ± SEM. Data are representative of at least 2 independent experiments.