Identification of Podocyte Cargo Proteins by Proteomic Analysis of Clathrin-Coated Vesicles

Abstract

Background: Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME have been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis. Methods: Glomeruli isolated from Podocin-Cre Rosa-DTR flox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification. Results: Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs amongst other small vesicles was observed on electron microscopy. Proteomics yielded a total of over 1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed. Conclusions: Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.

Podocyte Endocytosis in Regulating the Glomerular Filtration Barrier

“…This aims to decrease or avoid the systemic side effects caused by the clinical glucocorticoids administration ( 138 ). Moreover, with some of the most abundant podocyte-specific cargoes identified through mass spectrometry ( 139 ), future studies should enlighten critical pathways regulated by podocytes. In conclusion, these current cutting-edge findings on the relationship between endocytosis and podocyte biology will help us to further understand the precise mechanism of podocyte injury and provide impetus to find novel and useful therapeutic approaches to target abnormal endocytic machinery.…”

Steroid-Resistant Nephrotic Syndrome–Associated MYO1E Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity

“…59 Thus, the interaction of Myo1e and dynamin may assist in maintaining a functional slit diaphragm and prevent filter clogging by removing defective nephrin. 17,18,59 Additional insight into Myo1e functions in CME may be gained in the future by using specific endocytic cargos in podocytes 60 to develop new endocytosis assays.…”

Murine Epsins Play an Integral Role in Podocyte Function

“…This is corroborated by the absence of epsin 1, 2, and 3 in clathrin-coated vesicles of podocytes on mass spectrometry. 63 Thus, we focused on examining the small Rho-GTPase signaling pathway, because it has been shown previously that mammalian epsin binds RalBP1/RLIP76, a GAP that acts on Cdc42 and Rac1 through epsin's ENTH domain. 24,26,62 In podocytes.…”