Preconditioning donor with a combination of tacrolimus and rapamacyn to decrease ischaemia-reperfusion injury in a rat syngenic kidney transplantation model

Abstract

Reperfusion injury remains one of the major problems in transplantation. Repair from ischaemic acute renal failure (ARF) involves stimulation of tubular epithelial cell proliferation. The aim of this exploratory study was to evaluate the effects of preconditioning donor animals with rapamycin and tacrolimus to prevent ischaemia-reperfusion (I/R) injury. Twelve hours before nephrectomy, the donor animals received immunosuppressive drugs. The animals were divided into four groups, as follows: group 1 control: no treatment; group 2: rapamycin (2 mg/kg); group 3 FK506 (0, 3 mg/kg); and group 4: FK506 (0, 3 mg/kg) plus rapamycin (2 mg/kg). The left kidney was removed and after 3 h of cold ischaemia, the graft was transplanted. Twenty-four hours after transplant, the kidney was recovered for histological analysis and cytokine expression. Preconditioning treatment with rapamycin or tacrolimus significantly reduced blood urea nitrogen and creatinine compared with control [blood urea nitrogen (BUN): P < 0·001 versus control and creatinine: P < 0·001 versus control]. A further decrease was observed when rapamycin was combined with tacrolimus. Acute tubular necrosis was decreased significantly in donors treated with immunosuppressants compared with the control group (P < 0·001 versus control). Moreover, the number of apoptotic nuclei in the control group was higher compared with the treated groups (P < 0·001 versus control). Surprisingly, only rapamycin preconditioning treatment increased anti-apoptotic Bcl2 levels (P < 0·001). Finally, inflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, showed lower levels in the graft of those animals that had been pretreated with rapamycin or tacrolimus. This exploratory study demonstrates that preconditioning donor animals with rapamycin or tacrolimus improves clinical outcomes and reduce necrosis and apoptosis in kidney I/R injury.

Fig. 1

Plasma blood urea nitrogen (BUN) and creatinine after transplantation. Twenty-four hours after transplantation blood samples were obtained and serum parameters (a) BUN and (b) creatinine were determined. Values are expressed as the difference between post-transplantation minus pretransplantation for each group. Values are mean ± standard deviation (n = 6). *P < 0·05, **P < 0·01 and ***P < 0·001 for comparison between groups. No differences among sham animals were observed.

Fig. 2

Kidney tubular damage after transplantation. (a) The acute tubular necrosis score was graded in haematoxylin and eosin (H&E) staining cortex according to particular histological findings from 1 to 5. Values are mean ± standard deviation (n = 6). *P < 0·05 and ***P < 0·001 for comparison between groups. (b) Representative H&E stain for the evaluation of renal injury after 24 h of ischaemia–reperfusion.

Fig. 3

Apoptosis and Bcl2 expression. (a) Apoptotic cells using the terminal deoxynucleotidyl transferase deoxuridine triphosphate (dUTP) nick end labelling (TUNEL) method in outer and inner medulla. The total number of labelled nuclei was counted in 10 fields of 1 mm². 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. (b) Representative TUNEL staining of kidney sample after 24 h of reperfusion. Positive apoptotic cells appear with brown stained nuclei in outer medulla and cortex (A × 100, scale bar = 100 µ). (c) Immunohistochemical score of Bcl2 in renal tissue from transplanted rats. Quantitative analysis of Bcl2 expression was assessed in 10 consecutive cortex and medulla fields. Values are mean ± standard deviation (n = 6). *P < 0·05, **P < 0·01 and ***P < 0·001 for comparison between groups.

Fig. 4

C3 determination after transplantation. (a) Plasma C3 complement fraction (C3) was measured by radial immunodiffusion. Values are expressed as the difference between post-transplantation minus pretransplantation for each group. (b) Presence of C3 was assessed in 10 consecutive cortex and medulla fields. The extension rather than intensity of these markers was evaluated using a semiquantitative score. (c) Immunohistochemical demonstration of C3 expression in kidney sections from transplanted animals without treatment or treated with rapamycin, tacrolimus or the combination of both (A × 100). Values are mean ± standard deviation (n = 6). ***P < 0·001 for comparison to control group.

Fig. 5

Score of tumour necrosis factor (TNF)-α and interleukin (IL)-6 after transplantation. The score of TNF-α and IL-6 were graded on a scale from 1 to 5 by using microscopic criteria as described in Materials and methods. Values are mean ± standard deviation (n = 6). ***P < 0·001 for comparison to control group.