c‐Cbl induced podocin ubiquitination contributes to the podocytes injury in diabetic nephropathy

Abstract The ubiquitination function in diabetic nephropathy (DN) has attracted much attention, but there is a lack of information on its ubiquitylome profile. To examine the differences in protein content and ubiquitination in the kidney between db/db mice and db/m mice, we deployed liquid chromatography‐mass spectrometry (LC–MS/MS) to conduct analysis. We determined 145 sites in 86 upregulated modified proteins and 66 sites in 49 downregulated modified proteins at the ubiquitinated level. Moreover, 347 sites among the 319 modified proteins were present only in the db/db mouse kidneys, while 213 sites among the 199 modified proteins were present only in the db/m mouse kidneys. The subcellular localization study indicated that the cytoplasm had the highest proportion of ubiquitinated proteins (31.87%), followed by the nucleus (30.24%) and the plasma membrane (20.33%). The enrichment analysis revealed that the ubiquitinated proteins are mostly linked to tight junctions, oxidative phosphorylation, and thermogenesis. Podocin, as a typical protein of slit diaphragm, whose loss is a crucial cause of proteinuria in DN. Consistent with the results of ubiquitination omics, the K261R mutant of podocin induced the weakest ubiquitination compared with the K301R and K370R mutants. As an E3 ligase, c‐Cbl binds to podocin, and the regulation of c‐Cbl can impact the ubiquitination of podocin. In conclusion, in DN, podocin ubiquitination contributes to podocyte injury, and K261R is the most significant site. c‐Cbl participates in podocin ubiquitination and may be a direct target for preserving the integrity of the slit diaphragm structure, hence reducing proteinuria in DN.