To assess initially the involvement of calpain/calpastatin balance in allograft rejection, we analyzed by quantitative real-time PCR selleck compound calpain/calpastatin gene expression profiles of nine human transplant kidneys with acute rejection and 12 human transplant kidneys with chronic rejection, comparing with 10 normal human transplant
kidneys, all provided by the European Renal cDNA Bank (ERCB) 14. We found an increased expression of both CAPN 1 and CAPNS 1, encoding μ-calpain and a common small regulatory subunit of μ- and m-calpains, respectively, in transplant kidneys with acute rejection, and an increased expression of CAPN 1 alone in transplant kidneys with chronic rejection (Table 1). By contrast, we observed no significant change in the expression of CAPN 2 and CAST encoding m-calpain and calpastatin, respectively. Immunopathologic examination of kidney biopsies was performed to localize μ-calpain. Only a few tubules showed μ-calpain staining in healthy human kidney (Fig. 1A). In a transplant kidney with chronic rejection, the intensity of the staining and the number of μ-calpain-positive tubules increased markedly (Fig. 1B). Of note, μ-calpain expression was much more pronounced in cells infiltrating the interstitium of rejected kidney, identified as mostly T cells by
the CD3 EX 527 price immunoreactivity in an adjacent area (Fig. 1C). This colocalization was confirmed by double staining on the same section using confocal microscopy (Fig. 1, bottom). Our results suggest a gain of calpain expression in allograft rejection, explained partly by T-cell infiltration. To test the hypothesis that
calpains play a role in allograft rejection, we used a fully allogeneic murine skin allograft model. Donor tail skin from BALB/C mice was transplanted on the dorsal flank of C57BL/6 recipients, either WT or CalpTG. The Kaplan–Meier survival curves showed that allograft rejection was significantly delayed when recipients were CalpTG mice (Fig. 2A). Parallel experiments performed in WT recipients given a calpain inhibitor (PD150606) demonstrated similar prolongation of skin allograft survival (Fig. 2B); thus, confirming the role of calpain/calpastatin balance in rejection process. We characterized the population of cells infiltrating the skin allografts at the buy Paclitaxel onset of acute rejection process, i.e. 8 days after transplantation. In WT recipients, severe infiltration of T cells (CD4+ and CD8+) and to a lesser extent NK cells was noted in both the epidermis and dermis (Fig. 2C). This infiltration was limited in CalpTG recipients. A more precise analysis of infiltrating T-cell populations revealed a ∼50% decreased number of CD3+, CD4+, and CD8+ cells in CalpTG as compared with WT recipients. In contrast, a similar pattern of infiltrating macrophages (F4/80+ cells) was observed in the two groups of skin allograft recipients. Thus, it appears that prolonged allograft acceptance in CalpTG recipients is associated with a selective defect of T cells.