Monday March 28, 2022 - 19:20 to 20:30
Mehak Malhotra, India has been granted the IPTA Scientific Congress Award
Cytokine profile of allograft ‘post-reperfusion first-flush’ urine sample as a predictor of adverse graft outcome in renal transplantation
Mehak Malhotra1, Biman Saikia1, Ashish Sharma2, Ranjana W. Minz1, Ritambhra Nada3, HS Kohli4, Adil Karim1.
1Immunopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India., CHANDIGARH, India; 2Renal Transplant Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India., CHANDIGARH, India; 3Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India., CHANDIGARH, India; 4Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh, India., CHANDIGARH, India
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.