The Trafficking and Degradation of R138Q-Podocin and Nephrotic Syndrome

Abstract

Podocin, a crucial component of the podocyte slit diaphragm (SD) protein complex in the kidneys, plays a pivotal role in preventing plasma proteins from entering the urine. Mutations in Podocin, such as the severe R138Q mutation, disrupt its function and lead to nephrotic syndrome, characterized by proteinuria, hypoalbuminemia, and edema. Wild Type Podocin is located within detergent resistant microdomains (DRMS) at the plasma membrane (PM) of the podocyte. Podocin mutations are generally categorized into two major types based on their subcellular localization: those that are trafficked correctly to the PM and those that are retained in the Endoplasmic Reticulum (ER). To investigate the pathological mechanisms of these two types, lentiviral transduction was employed to create immortalized podocytes containing different disease-causing Podocin mutations (G92C, R138Q, V180M, R238S and R291W). The use of immortalized podocytes facilitates the direct comparison of the Podocin mutant proteins, making it possible to investigate the underlying mechanisms of disease.
Using these cells, differences in pathogenic mechanisms between the ER- retained R138Q-Podocin and the PM-bound mutations were identified, specifically alterations in the rate of degradation of the ER-retained R138Q mutant. Additionally, it was observed that the various PM-bound mutations (G92C, V180M, R238S and R291W) show significant differences in abundance at the PM and in the DRMS. This implies that these mutants may affect cholesterol binding capacity, hinder Podocin access to lipid rafts, cause lipid raft instability or alter binding to other key SD proteins.
Compared to other mutations, the R138Q mutant has a specific trafficking issue: it is retained in the ER. To investigate this trafficking defect, proteomics was employed to identify and compare the biological pathways involving the protein interactors of the R138Q mutant with the corresponding wild type pathways. These findings reveals that, compared to the wild type podocin, the R138Q-Podocin exhibits significant interactions with proteins involved in the processes of ER glycoprotein folding quality control (ERQC) and proteasomal degradation. These interactions were validated using Western Blot (WB) and immunofluorescence (IF) and revealed the unique profile in this R138Q mutation which is retained in the ER as a result of misfolding which triggers ER stress and the subsequent proteasomal degradation pathway.
Excitingly, following treatment of proteasome inhibitors, the R138Q mutant was seen to traffic to the PM and localized to DRMS. However, this treatment did not lead to correct interactions with other SD proteins such as Nephrin.
Therefore, these results demonstrated the complexity of mechanisms underlying Podocin mutant pathogenicity. To rescue the function of R138Q-Podocin, it will be essential not only to increase the intracellular abundance the protein but also to ensure it can fold correctly to interact properly with other PM proteins.

Date of Award	17 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Gavin I Welsh (Supervisor) & Moin Saleem (Supervisor)