Abstract: TH-PO1101
Loss of Ubiquitin-Specific Protease 40 in Podocyte Enhances Kidney Injury
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Mikami, Naoaki, Kyorin University, Tokyo, Japan
- Matsusaka, Taiji, Tokai University School of Medicine, Isehara, KANAGAWA, Japan
- Tanaka, Eriko, Kyorin University School of Medicine, Tokyo, Japan
- Kawanishi, Kunio, University of Tsukuba, Tsukuba, IBARAKI, Japan
- Nagata, Michio, University of Tsukuba, Tsukuba, IBARAKI, Japan
- Yan, Kunimasa, Kyorin University School of Medicine, Tokyo, Japan
Background
Although an indispensable role of ubiquitin specific protease-40 (USP40) in podocyte development of zebrafish was revealed, downstream of USP40 function is still unknown. Since there is a close relationship between ubiquitin system and endoplasmic reticulum function, we aimed to explore the protein function of USP40 in the podocyte injury and to determine the interacting partner by focusing on ER resident chaperone.
Methods
USP40-knockout (USP40KO) mice were generated and showed no apparent kidney abnormality. To explore the role of USP40 in podocyte injury, we crossed USP40KO with NEP25 mice, in which selective podocyte injury can be induced by injection with an immunotoxin, LMB2. Urinary protein was analyzed until day 9 after LMB2 injection, and then kidney tissues were subjected to light microscopy, immunohistochemistry of p57 and immunofluorescence microscopy of the ER stress marker BIP and calreticulin (CRT). The glomeruli were isolated and subjected to immunoblot analysis of BIP and CRT. The effect of USP40 knockdown on BIP and CRT expression was studied in cultured mouse podocytes. Finally, the protein binding of BIP and CRT with USP40 was determined by immunoprecipitation using cultured mouse podocytes.
Results
NEP-USP40KO mice developed higher levels of proteinuria compared with NEP mice. Light microscopy and immunohistochemistry showed higher levels of kidney injury in NEP-USP40KO mice compared with NEP mice in terms of hyalinosis (p<0.01), crescent formation (p<0.01), and cell proliferation (p<0.01) in the glomeruli, podocyte loss (p<0.05), and tubulointerstitial injury (p<0.01). Immunoblot analysis and immunofluorescence revealed increased expression of CRT, but not of BIP, in podocytes of NEP-USP40KO mice compared with NEP mice. Gene knockdown of USP40 in cultured podocytes increased the protein expression of CRT, but not of BIP. Finally, apparent protein binding of USP40 with CRT, but not with BIP, was observed.
Conclusion
USP40 may be involved in a self-defense mechanism in podocyte injury probably through interacting with CRT.