Background: Antigen independent insults to the graft caused by ischemia/reperfusion together with the prevailing baseline inflammatory state of the graft provides the nidus and the cytokine milieu for subsequent adaptive anti-graft immune responses to develop. Assessing this baseline ‘zero hour’ inflammatory milieu in the urine produced intra-operative by the allograft might be of significance in predicting graft outcome.
Materials and methods: Zero-hour intra-operative “first flush” post-reperfusion urine sample was utilized to assess the baseline inflammatory state from 200 prospective renal transplant recipients by looking at the mRNA expression levels for CXCL9, CXCL10 (IP-10), CXCL11, CCL3 (MIP1α), CCL2 (MCP-1), CD3ε, IFNγ, granzyme B, perforin, KIM-1, and clusterin were assessed using customized PCR arrays and the gene expression was calculated using ΔCt.
A panel of 14 cytokines viz. IL-2, IL-6, IL-8, IL1β, IL-10, IFN-γ, TNF, RANTES, IL-1α, Granzyme-B, MCP-1, MIP-1α, IP-10 and IL-12p70 was done using BD™ Cytometric Bead Array (CBA). Levels of Clusterin, KIM1 and NGAL were assessed by ELISA.
Results: Of the 200 recipients analyzed, 38 developed cellular rejection (ACR) and 12 developed antibody mediated rejection (ABMR) over a follow-up period of 24 months whereas remaining 150 were considered as stable allografts. mRNA expression of the IFN-γ responsive CXCR3 ligands CXCL9, CXCL10 and CXCL11 were found to be significantly differentially upregulated in the rejectors, either ABMR and/or ACR, compared to the ones with no rejection (p=0.00004, 0.008 and 0.00001 respectively). This was in concert with mRNA expression of IFN-γ (P=0.008) in view of the fact that CXCL9, -10 and -11 are all Interferon-Inducible CXC Chemokine Receptor 3 Ligands. CCL3 expression was similarly significantly upregulated in the rejectors p=0.002. Expression of TCR associated CD3ε (P=0.001) and the cytotoxic T-cell response associated genes PRF1 (p=0.003) and GZMB (p=0.006) were similarly different. KIM-1 expression showed a p value of 0.01. Levels of clusterin by ELISA (p=0.01) and IL-2, IFN-ϒ and IP-10 by CBA (p=0.001, 0.002 and 0.02 respectively) showed significant difference between the two groups.
Conclusions: A TH-1 cytokine milieu in the zero-hour post-reperfusion first flush urine sample correlated with occurrence of graft rejection. There was a significant difference in mRNA expression levels of CXCL9, CXCL10, CXCL11, IFN-γ, CCL3, CD3ε, PRF1, GZMB and KIM-1 between the rejection group and the stable allografts indicating a parallel role of ischemia reperfusion injury in graft outcome. Use of zero-hour first flush post-reperfusion urine sample to assess the baseline inflammatory state to predict adverse clinical outcomes hasn’t been attempted yet. Assessing this unique sample is likely to have a huge advantage for routine diagnostics.