#391 Calpain inhibition enhances autophagy-lysosomal pathway and ameliorates tubulointerstitial fibrosis in Nphp1-targeted models

Abstract

Abstract Background and Aims Nephronophthisis (NPH) belongs to a group of renal ciliopathies and is the most common genetic disease leading to renal failure in children. Mutations in Nphp1 account for the majority of NPH cases, and renal tubulointerstitial fibrosis is one of the most important pathological features. However, the molecular mechanisms of renal tubulointerstitial fibrosis remained unknown. Method Previously, we successfully constructed an Nphp1 knockout mice model with renal phenotype of tubulointerstitial fibrosis by CRISPR-Cas9. In this study, we used lentiviral shRNA vectors to construct Nphp1 knockdown HK2 cells. We carried out RNA sequencing in Nphp1-targeted cells and mice kidneys to explore the molecular mechanisms of renal tubulointerstitial fibrosis in NPH. Calpain (CAPN) was found to be upregulated in both models. Western blot and immunofluorescence (IF) were used to verify the expression of CAPN in Nphp1-targeted HK2 cells and mice kidneys, as well as their respective controls. We also detected autophagy activities in NPH models by western bolt, immunofluorescence, lysotracker and transmission electron microscopy (TEM). Epithelial or mesenchymal-specific markers and Masson's trichrome staining were used to detect the status of tubulointerstitial fibrosis. Furthermore, Nphp1-targeted HK2 cells and mice were treated with a CAPN inhibitor to explore the role of CAPN in autophagy-lysosomal pathway and tubulointerstitial fibrosis. Results CAPN was verified to be upregulated in both Nphp1-targeted HK2 cells and mice kidneys. We also discovered impaired autophagic flux in NPH models. Furthermore, the impaired autophagic flux induced tubulointerstitial fibrosis in NPH models. However, the autophagic flux was improved and the status of tubulointerstitial fibrosis was ameliorated with CAPN inhibition. Conclusion CAPN-mediated autolysosomal pathway disorder may be an important cause of tubulointerstitial fibrosis in NPH. CAPN may be a new target for diagnosis and treatment of NPH.