#1978 Proteomic analysis of exosomes derived from mesangial cells treated with galactose-deficient IgA1

Abstract

Abstract Background and Aims IgA nephropathy (IgAN) is an autoimmune disease characterized by IgA deposits that build up in the kidneys, inflame and damage glomeruli causing hematuria and proteinuria. It is a leading cause of chronic kidney disease, which often results in end-stage kidney disease. There is no disease specific treatment for IgAN. The glomerulus, in which IgA1 lacking galactose residues (gdIgA1) are deposited, consists of mesangial cells, podocytes, and endothelial cells. Crosstalk between the cell types allows the glomerulus to act as a unit and react to its dynamic environment. There is very little evidence on mesangial cell derived exosomes and their cargo to understand the pathological crosstalk signaling in the glomerulus of IgAN. We isolated exosomes from mesangial cells that were treated with patient-derived gdIgA1 and IgA1 complexes. Proteomic analysis was performed to identify the exosome cargo and delineate the signaling pathways leading to maladaptive paracrine crosstalk. From our study, we aim to understand the molecular targets for therapeutic strategies in IgAN. Method Human mesangial cells were treated with/without IgA1, gdIgA1 or IgG for 24 h. Cell culture supernatant was collected for exosome isolations. Exosomes were analyzed by size, morphology and number using TEM and NanoFCM. Exosome protein extracts were processed for LC-Tandem mass spectrometry analysis using filter-aided sample preparation protocol. Proteomic analysis was performed using Qlucore omics explorer and pathway analysis using Ingenuity Pathway Analysis (IPA). Exosome marker expressions such as CD63, CD81 and FLOT1 and proteome validation was performed using Western blotting. Statistical analysis was performed using GraphPad Prism and values <0.05 were considered significant. Results We confirmed the release of exosomes by mesangial cells in response to IgA1/gdIgA1 complexes. Exosomes were fewer in unstimulated cells as compared to IgA1/gdIgA1 treated cells. They showed round morphology with a dense core and uniform size distribution. Exosomes from treated cells showed a mean size of about 75 nm and concentration in the range of 1-2 × 10^10 particles/ml. While untreated cells showed a mean size of about 100 nm and concentration of about 1 × 10^9 particles/ml. We confirmed the protein expression of exosome markers such as CD63, CD81 and FLOT1. For the first time, we demonstrated global proteomics on exosomes from mesangial cells followed by pathway analysis. We have identified 89 unique proteins and pathways in exosomes derived from gdIgA1 treated mesangial cells that could mediate pathological signaling in the glomerulus in comparison to exosomes derived from IgA1 treated mesangial cells. Conclusion Our study provides an understanding of the pathological crosstalk signaling through exosomes and key molecular targets for IgAN.