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Abstract: FR-PO397

High Glucose–Induced Apoptosis and Necroptosis in Podocytes Is Regulated by UCHL1 via the RIPK1/RIPK3 Pathway

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Xu, Yuyin, School of basic medical science, Fudan University, Shanghai, China
  • Qi, Chenyang, Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, China
  • Wu, Huijuan, Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, China
  • Zhang, Zhigang, Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, China
Background

Depletion of podocytes plays a critical role in the pathogenesis of diabetic nephropathy (DN). Many investigations suggested its close association with apoptosis. However, the complex mechanism of podocyte loss in DN remains unclear. Recently, necroptosis has emerged as an important cell death model in cell injury related to many pathological conditions, which is regulated through the receptor interacting kinase 1/3 (RIPK1/RIPK3) pathway. It has been revealed that the molecular pathways to regulate apoptosis and necroptosis are closely related. Therefore, it is speculated that apoptosis and necroptosis may occur simultaneously during the process of podocyte injury in DN.

Methods

Immunofluorescence staining, trypan blue staining, Hoechst staining, TUNEL and ELISA were used to detect apoptosis and necroptosis of podocytes in vitro and in vivo. The ultrastructures of the two cell death patterns were observed under scanning electron microscope (SEM). After stimulation of podocytes with high glucose (HG), the expression of UCHL1 and RIPK1/RIPK3 pathway and the regulation effect of UCHL1 on apoptosis and necroptosis of podocytes were detected by western blot. The endogenous binding of UCHL1 with RIPK1 or RIPK3 and the ubiquitination of RIPK1 and RIPK3 proteins was detected by IP experiment.

Results

The present study demonstrated that necroptosis was involved in HG-induced podocyte injury both in vitro and in vivo. HG could induce both apoptosis and necroptosis in podocytes, which was dependent on HG concentration and treatment duration. This novel study explored the explicitly different morphological characteristics of apoptotic and necroptotic cells using SEM examination with TUNEL and Trypan blue double staining. The inhibition of apoptosis by z-VAD-fmk could enhance the necroptosis process, which was inhibited by Necrostatin-1(Nec-1). The present study also showed that ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) had a central role in regulating the two modes of cell death via mediating the ubiquitination states of the RIPK1/RIPK3 pathway.

Conclusion

These data suggested that necroptosis might be a major contributor to podocyte loss rather than apoptosis in HG treatment. The discovery of the novel function of UCHL1 may assist in developing strategies for treating podocyte damage induced by HG.

Funding

  • Government Support - Non-U.S.