TY - JOUR
T1 - Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier
AU - Möller-Kerutt, Annika
AU - Rodriguez-Gatica, Juan E
AU - Wacker, Karin
AU - Bhatia, Rohan
AU - Siebrasse, Jan-Peter
AU - Boon, Nanda
AU - Van Marck, Veerle
AU - Boor, Peter
AU - Kubitscheck, Ulrich
AU - Wijnholds, Jan
AU - Pavenstädt, Hermann
AU - Weide, Thomas
N1 - Copyright © 2021 by the American Society of Nephrology.
PY - 2021
Y1 - 2021
N2 - BACKGROUND: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).METHODS: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants.RESULTS: Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response.CONCLUSIONS: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.
AB - BACKGROUND: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).METHODS: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants.RESULTS: Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response.CONCLUSIONS: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.
U2 - 10.1681/ASN.2020040501
DO - 10.1681/ASN.2020040501
M3 - Article
C2 - 33687977
VL - 32
SP - 1053
EP - 1070
JO - Journal of the American Society of Nephrology : JASN
JF - Journal of the American Society of Nephrology : JASN
SN - 1046-6673
ER -