P0061LOSS OF UBIQUITIN SPECIFIC PROTEASE-40 CAUSES PODOCYTE DETACHMENT AND CONTRIBUTES TO THE DISEASE PROGRESSION OF GLOMERULOSCLEROSIS

Abstract

Abstract Background and Aims We reported that the ubiquitin specific protease-40 (USP40) is expressed in podocytes beyond the species and plays a crucial role in glomerulogenesis in zebrafish (Takagi H, Am J Physiol Renal Physiol 2017). However, the role of USP40 in podocytes and its functional downstream after birth is largely unknown. Recent report indicated that other USP family: USP10 regulates integrin beta1 and fibrotic wound healing in myofibroblasts (Gillespie SR, J Cell Sci 2017). The present study aimed to explore the biological role of USP40 in the acquired podocyte injury and to determine the interacting partner by focusing on podocyte detachment. Method We generated USP40-knockout mice (USP40KO); however, they showed no apparent kidney abnormality. Therefore, to explore the biological role of USP40 in acquired 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 integrin beta1. The glomeruli were isolated and subjected to immunoblot analysis of integrin beta1. The effect of USP40 knockdown on integrin beta1 expression was studied in cultured mouse podocytes. Finally, the protein binding of integrin beta1 with USP40 was determined by immunoprecipitation using cultured mouse podocytes. Results NEP-USP40KO mice developed earlier and 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 decreased expression of integrin beta1 in podocytes of NEP-USP40KO mice compared with NEP mice. Gene knockdown of USP40 in cultured podocytes decreased the protein expression of integrin beta1. Finally, apparent protein binding of USP40 with integrin beta1 was observed. Conclusion Integrin beta1 is known to play a crucial role as the adhesion molecule that connects the podocytes with the extracellular matrix of the glomerular basement membrane. It is also known that the lack of integrin beta1 causes podocyte detachment, which is involved in the disease progression of glomerulosclerosis. Therefore, the present study suggests that USP40 may be involved in a self-defense mechanism in podocyte injury probably through interacting with the regulatory machinery of integrin beta1 in podocytes.