Necrosis and kidney damage were assessed with H&E-stained kidney tissue 24 h after transplantation. Acute tubular necrosis score (ATN) was decreased significantly in the immunosuppressive treatment group compared with the control group (4 ± 0·63
in control; rapamycin 2·2 ± 0·41; FK506 2 ± 0·63; rapamycin + FK506 1·2 ± 0·41; P < 0·001 versus control; Fig. 2a). Figure 2b PF-01367338 in vivo shows a representative image of H&E stain for the evaluation of renal injury in each treatment group. The use of rapamycin plus tacrolimus (group 4) was associated with a lower level of acute tubular necrosis (ATN) compared with rapamycin alone (P < 0·05), but no statistical difference was observed in comparison with tacrolimus. Also, the number of apoptotic nuclei in renal medulla was determined as evidence of kidney injury. In the control group, the number of TUNEL-positive cells was higher compared with the immunosuppressive treatment groups (control: 138·7 ± 24·8; rapamycin: 22·3 ± 4·5; FK506: 54·8 ± 8·3 and rapamycin + FK506: 17·5 ± 5; P < 0·001 versus control, Fig. 3a and b). As normal kidney control, the number of positive apoptotic nuclei in sham animals was lower than 6/mm2 located only in deep medullary epithelial tubules (data not shown). The use of rapamycin alone or rapamycin plus tacrolimus showed a lower number of apoptotic nuclei cells with respect to
tacrolimus treatment (P < 0·05 and P < 0·01, respectively). Finally, a statistically significant difference in the expression of Bcl2 was detected in selleck chemicals kidney tissue by immunohistochemistry. In accordance with our previous results, Bcl2 levels in the control group were lower than in the immunosuppressive treatment group (control: 1·8 ± 0·5; rapamycin: 16·01 ± 4; FK506: Nutlin-3 molecular weight 9 ± 2·6 and rapamycin + FK506: 6 ± 1·25; P < 0·01 and P < 0·05 versus control, respectively)
(Fig. 3c). These results suggest that preconditioning of the donor with rapamycin and tacrolimus or a combination of both is associated with lower kidney damage after transplantation. In order to determine if the immunosuppressive treatment affected the complement function, the C3 levels in recipient animals were assessed. C3 plasma values in immunosuppressive treatment were significantly lower than control group levels (control: 495 ± 94 pg/ml; rapamycin: 166·7 ± 57·1 pg/ml; FK506: 165 ± 66·3 pg/ml and rapamycin + FK506: 103·3 ± 33·3.; P < 0·001 versus control, Fig. 4a). No differences were found among the various immunosuppressive treatment groups (P > 0·05). In addition, the local expression of C3 within the grafts was analysed. Immunohistochemical analysis of graft tissue 24 h after transplantation revealed that local expression of C3 was higher in the control group compared with the immunosuppressive treatment group (control: 53·98 ± 4·5; rapamycin: 10·62 ± 3·2; FK506: 2·27 ± 0·7 and rapamycin + FK506: 1·58 ± 0·54.