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Sun, Yingyu, Kronenberg, Nils M. ORCID: 0000-0001-6386-3848, Sethi, Sidharth K., Dash, Surjya N. ORCID: 0000-0003-1232-2923, Kovalik, Maria E., Sempowski, Benjamin ORCID: 0000-0003-1494-8540, Strickland, Shelby, Raina, Rupesh ORCID: 0000-0003-3892-8376, Sperati, C. John ORCID: 0000-0002-8409-0714, Tian, Xuefei ORCID: 0000-0003-4235-0896, Ishibe, Shuta, Hall, Gentzon and Gather, Malte C. ORCID: 0000-0002-4857-5562 (2025). CRB2 depletion induces YAP signaling and disrupts mechanosensing in podocytes. American Journal of Physiology-Renal Physiology, 328 (4). F578-F595. American Physiological Society. ISSN 1931-857X

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Abstract

Focal segmental glomerulosclerosis (FSGS) is a histologic lesion caused by a variety of injurious stimuli that lead to dysfunction/loss of glomerular visceral epithelial cells (i.e., podocytes). Pathogenic mutations in crumbs homolog-2 (CRB2), encoding the type 1 transmembrane protein crumbs homolog-2, have been shown to cause early-onset corticosteroid-resistant nephrotic syndrome (SRNS)/FSGS. Here, we identified a two-generation Indian kindred (DUK40595) with biopsy-proven SRNS/FSGS caused by a compound heterozygous mutation in CRB2 comprised of the previously described truncating mutation p.Gly1036_Alafs*43 and a rare 9-bp deletion mutation p.Leu1074_Asp1076del. Because compound heterozygous mutations involving the truncating p.Gly1036_Alafs*43 variant have been associated with reduced CRB2 expression in podocytes and autosomal recessive SRNS/FSGS, we sought to define the pathogenic effects of CRB2 deficiency in podocytes. We show that CRB2 knockdown induces yes-associated protein (YAP) activity and target gene expression in podocytes. It upregulates YAP-mediated mechanosignaling and increases the density of focal adhesion and F-actin. Using elastic resonator interference stress microscopy (ERISM), we demonstrate that CRB2 knockdown also enhances podocyte contractility in a substrate stiffness-dependent manner. The knockdown effect decreases with increasing substrate stiffness, indicating impaired mechanosensing in CRB2 knockdown cells at low substrate stiffness. Although the mechanical activation of CRB2 knockdown cells is associated with increased YAP activity, the enhanced cell contractility is not significantly reduced by the selective YAP inhibitors K-975 and verteporfin, suggesting that multiple pathways may be involved in mechanosignaling downstream of CRB2. Taken together, these studies provide the first evidence that CRB2 deficiency may impair podocyte mechanotransduction via disruption of YAP signaling in podocytes. NEW & NOTEWORTHY We identified a rare compound heterozygous CRB2 mutation as the cause of familial SRNS/FSGS in a two-generation East Asian kindred. Modeling the effect of the mutation, we show that CRB2 knockdown in podocytes induces YAP transcriptional activity and upregulates YAP-mediated mechanosignaling. Using elastic resonator interference stress microscopy (ERISM), we demonstrate that CRB2 knockdown enhances podocyte contractility in a substrate stiffness-dependent manner. The knockdown effect decreases with increasing substrate stiffness, indicating impaired mechanosensing in CRB2-deficient podocytes.

Item Type:	Article
Creators:	Creators Email ORCID ORCID Put Code Sun, Yingyu UNSPECIFIED UNSPECIFIED UNSPECIFIED Kronenberg, Nils M. UNSPECIFIED https://orcid.org/0000-0001-6386-3848 UNSPECIFIED Sethi, Sidharth K. UNSPECIFIED UNSPECIFIED UNSPECIFIED Dash, Surjya N. UNSPECIFIED https://orcid.org/0000-0003-1232-2923 UNSPECIFIED Kovalik, Maria E. UNSPECIFIED UNSPECIFIED UNSPECIFIED Sempowski, Benjamin UNSPECIFIED https://orcid.org/0000-0003-1494-8540 UNSPECIFIED Strickland, Shelby UNSPECIFIED UNSPECIFIED UNSPECIFIED Raina, Rupesh UNSPECIFIED https://orcid.org/0000-0003-3892-8376 UNSPECIFIED Sperati, C. John UNSPECIFIED https://orcid.org/0000-0002-8409-0714 UNSPECIFIED Tian, Xuefei UNSPECIFIED https://orcid.org/0000-0003-4235-0896 UNSPECIFIED Ishibe, Shuta UNSPECIFIED UNSPECIFIED UNSPECIFIED Hall, Gentzon UNSPECIFIED UNSPECIFIED UNSPECIFIED Gather, Malte C. UNSPECIFIED https://orcid.org/0000-0002-4857-5562 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-812011
Identification Number:	10.1152/ajprenal.00318.2024
Journal or Publication Title:	American Journal of Physiology-Renal Physiology
Volume:	328
Number:	4
Page Range:	F578-F595
Number of Pages:	18
Date:	1 April 2025
Publisher:	American Physiological Society
ISSN:	1931-857X
Language:	English
Faculty:	Faculty of Medicine Faculty of Mathematics and Natural Sciences
Divisions:	CECAD - Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	Chemistry and allied sciences Life sciences
Uncontrolled Keywords:	Keywords Language Cell mechanics; CRB2 ; FSGS ; podocytes ; YAP signaling English
['eprint_fieldname_oa_funders' not defined]:	Publikationsfonds UzK
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/81201