Introduction: Renal transplantation improves the life expectancy and quality of life of end-stage renal patients. Due to the development of immunosuppressive drugs, the results of short-term renal transplantation have improved significantly, but the long-term survival rate of renal transplantation has hardly changed in the past few decades. Interstitial fibrosis is an important cause of graft loss in chronic allograft renal injury. CD40-CD154 costimulatory pathway block can inhibit T cell activation and prolong the survival of allografts in a variety of transplantation models. In this study, we found that the single use of MR1 (CD154 antibody) can significantly reduce the fibrosis of transplanted kidney.

Method: The kidney of BALB/c mice was transplanted into C57BL6/J recipient by orthotopic transplantation. The contralateral kidney of the recipient was removed 1-3 days after transplantation. The MR1 treatment group was intraperitoneally injected with 500ug of MR1 antibody on the day of operation, and the control group was given normal saline at the same time. After 30 days, serum was collected to measure the blood creatinine. The kidney was obtained after cardiac perfusion. PAS staining was used to detect renal injury. The number of T lymphocytes and macrophages and the expression of a-SMA in the transplanted kidney were analyzed by flow cytometry. Sirius red and gomori trichrome staining were used to detect fibrosis. The colocalization of macrophages and a-SMA was detected by multiple immunofluorescence.

Results: It was found that serum creatinine from MR1 treatment group was significantly lower than control group. Compared with control group, MR1 treated mice also showed milder renal histological lesions that the loss of brush border of proximal tubules, tubular necrosis, tubular atrophy, protein casting and cell infiltration at the cortical medullary junction were much lower. Sirius red and gomori trichrome staining showed that MR1 treatment group significantly reduced the fibrosis of transplanted kidney. RT-PCR showed that the expressions of collagen deposition related proteins such as ColI and FN mRNA were up-regulated in the control group. Further immunohistochemistry analysis showed that the number of infiltrated macrophages in the MR1 treatment group decreased significantly compared to control group. Through flow cytometry analysis, it was found that the number of macrophages secreting a-SMA in control group were significantly higher than those in MR1 treatment group. The FACS results showed that 92.7%± 2.8% of the infiltrating macrophages were M2 type, and they were all derived from the recipients. Immunofluorescence experiment also confirmed this result.

Conclusion: MR1 significantly reduced the fibrosis in kidney graft which may due to less infiltration of M2 macrophages and less a-SMA+ macrophages.