INTRODUCTION: Diagnosing antibody-mediated rejection (AMR) can be difficult without an invasive graft biopsy. A specific, non-invasive biomarker to monitor immunological graft status would facilitate diagnosis and treatment of common transplantation-related complications. Murine recipients lacking the chemokine receptor CCR5 reject renal allografts with marked C4d deposition in peritubular capillaries and high serum donor-reactive antibody titers, features consistent with AMR. The current studies were conducted to identify biomarkers of AMR using gene expression in mRNA isolated from urine sediments in this renal allograft model.

METHODS: CCR5−/− and CD8−/− mice were crossed to generate the CCR5−/−/CD8−/− C57BL/6 mice. Complete MHC-mismatched A/J kidneys were transplanted into CCR5−/−/CD8−/− C57BL/6 or wild-type C57BL/6 recipients. Renal allograft rejection was considered imminent when the mouse showed signs of illness or the serum creatinine level was elevated to >1.0 mg/dL at which time allografts, and serum and urine samples were collected for analysis. RNA was extracted from prepared urine pellets and a preamplification step was included in order to improve gene detection by quantitative RT-PCR. Serum antibody titers were assessed by staining graft donor cells with dilutions of serum followed by flow cytometry analysis.

RESULTS: While A/J renal allografts were accepted long-term in all wild-type C57BL/6 recipients, CCR5−/−/CD8−/− recipients rejected the renal allografts with a median survival of 18.5 days. CCR5−/−/CD8−/− recipients showed significantly higher titers of donor-reactive antibody and serum creatinine levels at the time of rejection than those of wild-type C57BL/6 recipients at day 30 post-transplant. Histological analysis revealed diffuse C4d deposition of the rejecting renal allografts. At the time of AMR, high levels of FasL and perforin mRNA expression was detected in urine sediments from CCR5−/−/CD8−/− recipients of A/J allografts. This expression was low/undetectable in urine from naÏve CCR5−/−/CD8−/− mice and from the urine of CCR5−/−/CD8−/− recipients of isografts and from wild-type C57BL/6 recipients of A/J allografts on day 30 post-transplant.

CONCLUSIONS: CCR5−/−/CD8−/− recipients are useful as a murine model of AMR of renal allografts. Urinary FasL and Perforin mRNA levels may be a diagnostic biomarker to monitor the presence of AMR.

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