ALTERED ANTIGEN HANDLING AND INVOLVEMENT OF THE CX3CR1-FKN AXIS IN PROMOTING HEMATURIA IN IGA NEPHROPATHY PATIENTS

Introduction and Aims: The hallmark of IgA nephropathy (IgAN) is gross hematuria (GH) coinciding with or immediately following an upper respiratory or gastrointestinal tract infection; thus a dysregulation of innate immunity has been extensively postulated in IgAN. Oral immunization with a variety of common pathogens and alimentary components is able to reproduce IgAN in experimental mice models. Furthermore, some of these exogenous antigens can be detected in the renal tissue of IgAN patients. Since GH can represent a disease triggering event, wedecided to study this important clinical time point in order to unravel the link between mucosal encountered antigens and the occurrence of glomerular hematuria.Methods: The first step of the study was to perform an exploratory/hypothesis-generating analysis using microarray technology in order to identify genes/pathways modulated in the acute phase of the disease. For this aim we analyzed the genomic profile of PBMCs from 3 IgAN patients at two different clinical time points, the first sample during the GH episode and the second during the remission phase of the disease characterized by persistent microscopic hematuria (MH). Identified genes and pathways were then validated with classical biomoleculartools on a large cohort of 79 biopsy-proven IgAN patients, 5 membranous nephropathy (MN), 3 membranoproliferative glomerulonephritis (MPGN) patients and 10 Healthy blood donors (HBD).Results: The modulated genes during GH showed a clear involvement of the interferon signalling (e.g. STAT1), antigen presenting pathway (HLA-E, TAPBP) and the immuno-proteasome (e.g. PSMB8, PSMB9, PSMB10). The gene characterizing cytotoxic effector lymphocytes (CX3CR1), implicated in vascular endothelial damage, was found up-regulated at both mRNA and protein level. Furthermore, a significant increase of CX3CR1 surface expression was found in cell subsets with well knowncytotoxic effector functions. In vitro antigenic stimulation of PBMCs on an independent set of IgAN patients demonstrated that patients up- regulate specifically CX3CR1 and STAT1 in an enhanced and dose dependant manner, while an expecteddown-regulation occurred in HBD and in other glomerulonephritis control groups. This enhanced activation not only occurred in patients characterized by recurrent GH or by permanent microscopic hematuria (MH) but also in IgAN patients without hematuria. These results confirm that the deregulation of the interferon signaling pathway through STAT1 and the enhanced CX3CR1 expression during endotoxin challenge is specific to IgAN patients, however, the enhanced receptor expression is not sufficient to cause GH. Then we analyzed glomerular fractalkine(FKN) expression, since this ligand, is involved in the vascular gateway for CX3CR1+ cells towards the inflamed tissues and induces vascular injury. A significantly higher glomerular and urinary FKN level was found in IgAN patients with recurrent GHepisodes compared to permanent MH patients and MPGN and MN patients, suggesting a predisposition for cytotoxic cell extravasation only in recurrent GH patients.Conclusions: Taken together, our findings demonstrate, for the first time, a defect in antigen handling in PBMCs of IgAN patients with a specific up-regulation of CX3CR1 and STAT-1. Furthermore, the constitutive up regulation of glomerular FKN, suggests an involvement of the CX3CR1-FKN axis in the exacerbation of GH.