It has been demonstrated that allo-antibodies mediate a substantial proportion of graft rejection episodes, contributing to both early and late graft loss. In particular, it has been shown that the chronic interaction of donor anti-HLA antibodies with graft endothelial cells triggers biological processes that lead to the development of tissue lesions typical of antibody-mediated chronic rejection (AMCR) (Colvin RB – 2005 ; Naemi FM- 2011 ; Zhang X-2012 . Despite endothelial cells are considered the main target of antibodies in AMCR, their effects on different types of renal cells, localized within sites of tissue injury, are unknown. Basing on these considerations the aim of this study was the evaluation of biological mechanisms triggered by anti-HLA class I antibodies on proximal tubular epithelial cells (PTEC).
We studied 6 patients of the Torino Renal Transplant Center with AMCR (C4d-positive biopsies), presence of donor specific anti-HLA antibodies (DSA) in sera and proteinuria > 1 g/die. For detection of DSA in patients’ urine, 50 ml samples were collected and centrifuged at 3300 x g for 10 minutes to obtain the complete removal of the sediment. Then, 50 ml of clarified urine were concentrated in Amicon Ultra Centrifugal Filter Unit devices with a 50 kDa cellulose membrane (Millipore Corporation) by centrifugation at 3300 x g for 45 minutes. After concentration samples were analyzed by Luminex assay to detect the presence of anti-HLA antibodies. These data were compared with those obtained by serum analysis.
Primary cultures of human proximal tubular epithelial cells (PTEC) were obtained from renal cortical tissues collected from healthy parts of tumor-nephrectomies Conaldi PG- 1998 . The biological effects of anti-HLA class I antibodies were evaluated by stimulation of PTEC with 10 μg/ml of monoclonal antibody W6/32 for different time points (24, 48 and 96 hours) in serum-free medium. The following assays were then performed on stimulated cells: – XTT assay for cytotoxicity evaluation; – Measurement of transepithelial electrical resistance (TER) by Voltohmmeter to detect cell polarity alterations; – Analysis of JAK2 phosphorylation by western blot; -Quantification of IL-6 secretion by ELISA immunoassay.
We evaluated the presence of donor anti-HLA antibodies in 6 recipients with AMCR and proteinuria to investigate the dynamics of interaction between antibodies and tubular cells: in all patients enrolled in this study, the anti-donor antibodies were present both in serum and urine suggesting their endo-luminal interaction with tubular cells (Figure 1). In the second part of the study we evaluated the effects of an anti-HLA class I monoclonal antibody (W6/32) on proximal tubular epithelial cells (Figure 2-4). FACS analysis showed that PTEC expressed constitutively high levels of HLA-I (Figure 2A ).The incubation of cells with W6/32 induced an increase of mitochondrial activity (Figure 2B) and a reduction of transepithelial resistance (TER), sign of loss of cell polarity (Figure 2C). In addition, basing on recent studies that described an increased phosphorylation of the tyrosine kinase JAK2 in biopsies of patients with acute antibody-mediated rejection Loverre A-2011  , we analyzed this effect on PTEC stimulated with W6/32 observing an induced JAK2 phosphorylation, that reached maximum levels after 96 hours (Figure 3 A-B). Finally, we evaluated levels of PTEC cytokine secretion after stimulation with W6/32 observing increased levels of the proinflammatory IL-6 at 24 and 48 hours (Figure 4).
The interaction between anti-HLA antibodies and PTEC may occur intraluminally in presence of a proteinuric state. Indeed, we observed the presence of anti-DSA antibodies in urine of kidney transplanted patients affected by AMCR. In vitro experiments showed that stimulation of PTEC with anti-HLA I antibodies may induce a low increase of the proliferation rate, associated to a reduction of cell polarity that may potentially affect the function of this specific cell type. In selected experiments, we studied the molecular mechanisms triggered by anti-HLA I antibodies observing an increased phosphorilation of the cytoplasmic tyrosine kinase JAK2. As recently described by Loverre et al. in the setting of acute rejection, the activation of JAK2 may trigger a signaling pathway involved in tubular expression of IL-17, a proinflammatory cytokine that may contribute along with the activation of the complement cascade in the recruitment of neutrophils within the peritubular capillaries in acute antibody-mediated rejection. Finally, our results showed that PTEC, stimulated with anti-HLA I antibodies actively participate in the local inflammatory response by producing IL-6 that may contribute in vivo to the regulation of interstitial infiltration.
In conclusion, the preliminary results of this study showed that the interaction of anti-HLA class I antibodies with proximal tubular cells are able to induce biological modifications, suggesting that tubulointerstitial alterations associated with anti-donor antibodies may directly contribute to the pathogenesis of AMCR.