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Polycystins regulate ezrin function and cleavage to control renal cell and tubular morphology
Abstract
Background: Renal cyst formation in ADPKD requires altered cell and tubular morphology. Previously, we used inducible inactivation of Pkd2 in a 3D-tubuloid model to measure acute changes in tubuloid shape after Pkd2 loss and observed alterations in tubule morphology before changes in overall volume, arguing that morphological changes are among the first consequences of polycystin loss. We hypothesized a direct role for the polycystin complex in regulating the apical cytoskeleton protein, ezrin (EZR), to control cell and tubular morphology. Methods: We used clonal cell lines derived from the kidneys of Pkd1 or Pkd2 Pax8rtTA TetOCre mice crossed with the SV40 immorto-mouse: Pkd1-iKO (#312) and Pkd2-iKO (#125). Pkd1/2 can be efficiently deleted with addition of Doxycyline (DOX) to the cell media. Results: We observed a strong correlation between the loss of Pkd1/2 and increased apical cell area, as defined by ZO1, in 2D Pkd1/2-iKO cells. The acute loss of Pkd1/2 also resulted in decreased ezrin abundance and altered localization. Although Pkd1/2 deletion resulted in decreased ezrin we found upregulation of Ezr mRNA in both the DOX treated Pkd2-iKO cells and in human ADPKD cystic tissue, leading us to hypothesize that PC1/2 regulates ezrin protein more directly. Immunoprecipitation experiments of HEK293 cells transfected with ezrin, Myc-PC2 or Flag-PC1 showed both PC1 and PC2 successfully pulled down ezrin suggesting a protein interaction. Interestingly, the pull down efficiency was greater with the full length PC1 than the CTF- PC1 fragment. To confirm an endogenous interaction between ezrin and PC1 we isolated primary renal epithelial cells from a transgenic Pkd1-HA mouse and found again HA-PC1 successfully pulled down ezrin. Mechanistically, ezrin activity is regulated by cystine proteases calpain 1 and 2. We observed a substantial increase in the N-terminal 55KDa ezrin cleavage product after acute Pkd2 loss in the DOX treated Pkd2- iKO cells as well in human APDKD cystic tissue. Chemical inhibition of either calpain 1 or calpain 2 led to increased ezrin cleavage in control cells mimicking Pkd2 loss. However, inhibition of both calpain 1 and 2 reduced ezrin cleavage in DOX treated Pkd2-iKO cells to levels comparable to control cells. Conclusion: The polycystin proteins regulate ezrin function and cleavage to control renal cell and tubular morphology.Description
Kidney Week 2024: American Society of Nephrology (ASN). October 23, 2024.Rights/Terms
Attribution-NonCommercial-NoDerivatives 4.0 InternationalIdentifier to cite or link to this item
http://hdl.handle.net/10713/22981Collections
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- Creative Commons
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International