Proteomic analysis of urinary extracellular vesicles of kidney transplant recipients with BKV viruria and viremia: A pilot study

INTRODUCTION: To better define the biological machinery associated with BK virus (BKV) infection, in kidney transplantation, we performed a proteomics analysis of urinary extracellular vesicles (EVs). METHODS: Twenty-nine adult kidney transplant recipients (KTRs) with normal allograft function affected by BKV infection (15 with only viremia, 14 with viruria and viremia) and 15 controls (CTR, KTRs without BKV infection) were enrolled and randomly divided in a training cohort (12 BKV and 6 CTR) used for the mass spectrometry analysis of the EVs (microvesicles and exosomes) protein content and a testing cohort (17 BKV and 9 CTR) used for the biological validation of the proteomic results by ELISA. Bioinformatics and functional analysis revealed that several biological processes were enriched in BKV (including immunity, complement activation, renal fibrosis) and were able to discriminate BKV vs. CTR. Kinase was the only gene ontology annotation term including proteins less abundant in BKV (with SLK being the most significantly down-regulated protein). Non-linear support vector machine (SVM) learning and partial least squares discriminant analysis (PLS-DA) identified 36 proteins (including DNASE2, F12, AGT, CTSH, C4A, C7, FABP4, and BPNT1) able to discriminate the two study groups. The proteomic profile of KTRs with BKV viruria alone vs. viremia and viruria was quite similar. Enzyme-linked immunosorbent assay (ELISA) for SLK, BPNT1 and DNASE2, performed on testing cohort, validated proteomics results. DISCUSSIONS: Our pilot study demonstrated, for the first time, that BKV infection, also in the viruric state, can have a negative impact on the allograft and it suggested that, whether possible, an early preventive therapeutic strategy should be undertaken also in KTRs with viruria only. Our results, then, revealed new mechanistic insights into BKV infection and they selected potential biomarkers that should be tested in future studies with larger patients’ cohorts.