Association of crumbs homolog-2 with mTORC1 in developing podocyte

Hamano, Sho; Nishibori, Yukino; Hada, Ichiro; Mikami, Naoaki; Ito-Nitta, Noriko; Fukuhara, Daisuke; Kudo, Akihiko; Xiao, Zhijie; Nukui, Masatoshi; Patrakka, Jaakko; Tryggvason, Karl; Kou, Yan

doi:10.1371/journal.pone.0202400

Cited by 6 publications

(19 citation statements)

References 40 publications

(44 reference statements)

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“…Crb2 showed a different expression profile compared with Nephrin, 15 not only during embryo- and organogenesis 28,32 but also, in developing podocytes during glomeruli maturation. 27 Moreover, during S-shaped and capillary loop stages, Crb2 is also expressed in the parietal epithelium of glomeruli, whereas Nephrin expression was restricted to podocytes. 8, 27…”

Section: Discussionmentioning

confidence: 99%

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

Möller-Kerutt

Rodriguez-Gatica

Wacker

et al. 2021

JASN

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BackgroundCrumbs2 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).MethodsTo 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.ResultsCrb2podKO 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.ConclusionsCrb2 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.

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Section: Discussionmentioning

confidence: 99%

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

Möller-Kerutt

Rodriguez-Gatica

Wacker

et al. 2021

JASN

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“…A monoclonal rat antibody against full-length mouse Crb2 (anti–Crb2-r Ab) was generated as reported previously 31 . Briefly, the lysate of HEK-293 cells expressing mouse Crb2 32 conjugated to Freund’s incomplete adjuvant (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) was injected into the hind footpads of Wistar Kyoto rat (Charles River Laboratories, Yokohama, Japan) and enlarged medial iliac lymph nodes were used for cell fusion to produce hybridomas. Rabbit antibody against the intracellular domain of human Crb2 (amino acid 1248–1285, anti-Crb2-int Ab) was generated in New Zealand white rabbits using a standard protocol (National Veterinary Institute, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

“…Immortalized mouse podocyte line (kindly provided by Dr. Katsuhiko Asanuma) was previously described 38 . Because this cell line does not express endogenous Crb2, 32 we generated a mouse podocyte cell line stably expressing mouse Crb2 (hereinafter referred to as mPodo-Crb2). Immortalized mouse podocytes cultured in a 10-cm dish were transfected with 10 μ g of mouse Crb2 plasmid DNA 32 using FuGENE 6 (Roche) according to the manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

“…38 Because this cell line does not express endogenous Crb2, 32 we generated a mouse podocyte cell line stably expressing mouse Crb2 (hereinafter referred to as mPodo-Crb2). Immortalized mouse podocytes cultured in a 10-cm dish were transfected with 10 mg of mouse Crb2 plasmid DNA 32 using FuGENE 6 (Roche) according to the manufacturer's protocol. Cells were selected and cultured in RPMI 1640 medium with 10% FCS containing 20 mg/ml Zeocin (Thermo Fisher Scientific).…”

Section: Crb2-expressing Mouse Podocyte Cell Linementioning

confidence: 99%

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A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2

Hada

Shimizu

Takematsu

et al. 2022

JASN

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Background: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. Methods: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. Results: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which co-localized with Crb2 at the podocyte foot process. A subset of mice developed features resembling focal segmental glomerulosclerosis after 18 weeks. In glomeruli of immunized mice as well as in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. Conclusion: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunological pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

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“…In the kidney Crb2, Crb3 and polarity components Pals1, Patj and Lin7c are expressed ( Kamberov et al, 2000 ; Makarova et al, 2003 ; Olsen et al, 2007 ; Duning et al, 2008 ; Yin et al, 2014 ; Hochapfel et al, 2017 ). Crb3 isoforms are the main isoforms of the renal tubules (especially Crb3a), whereas parietal cells and podocytes of renal glomeruli express high levels of Crb2 ( Hamano et al, 2018 ; Martin et al, 2021 ; Möller-Kerutt et al, 2021 ). Of note, Pals1 is the only protein that binds to all core components of the Crumbs complex ( Kamberov et al, 2000 ; Roh et al, 2002 ; Roh et al, 2003 ).…”

Section: Introductionmentioning

confidence: 99%

Impact of Pals1 on Expression and Localization of Transporters Belonging to the Solute Carrier Family

Berghaus

Groh

Breljak

et al. 2022

Front. Mol. Biosci.

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Pals1 is part of the evolutionary conserved Crumbs polarity complex and plays a key role in two processes, the formation of apicobasal polarity and the establishment of cell-cell contacts. In the human kidney, up to 1.5 million nephrons control blood filtration, as well as resorption and recycling of inorganic and organic ions, sugars, amino acids, peptides, vitamins, water and further metabolites of endogenous and exogenous origin. All nephron segments consist of polarized cells and express high levels of Pals1. Mice that are functionally haploid for Pals1 develop a lethal phenotype, accompanied by heavy proteinuria and the formation of renal cysts. However, on a cellular level, it is still unclear if reduced cell polarization, incomplete cell-cell contact formation, or an altered Pals1-dependent gene expression accounts for the renal phenotype. To address this, we analyzed the transcriptomes of Pals1-haploinsufficient kidneys and the littermate controls by gene set enrichment analysis. Our data elucidated a direct correlation between TGFβ pathway activation and the downregulation of more than 100 members of the solute carrier (SLC) gene family. Surprisingly, Pals1-depleted nephrons keep the SLC’s segment-specific expression and subcellular distribution, demonstrating that the phenotype is not mainly due to dysfunctional apicobasal cell polarization of renal epithelia. Our data may provide first hints that SLCs may act as modulating factors for renal cyst formation.

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Association of crumbs homolog-2 with mTORC1 in developing podocyte

Cited by 6 publications

References 40 publications

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2

Impact of Pals1 on Expression and Localization of Transporters Belonging to the Solute Carrier Family

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