Analysis of Genetics Markers Associated With Kidney Transplantation Outcome

Abstract

Allograft rejection remains a major hurdle in successful transplantation despite improved immunosuppressive drugs and clinical care. The molecular changes in the renal allograft that lead to graft rejection need to be investigated. In the present work, polymorphisms in chemokine receptors and urinary chemokine levels were investigated for association with rejection. This study includes; (a) gene polymorphisms of chemokine receptors of CCR2 and CCR5 (CCR2V64I and CCR5-59029G>A and CCR5Δ32), (b) urinary levels of interferon induced protein-10 (IP-10), (c) urinary levels of monokine induced by interferon-gamma (MIG) and (d) urinary levels of monocyte chemotactic protein-1 (MCP-1). This is the first study on chemokine receptor polymorphisms and the urinary chemokine levels (IP-10, MIG and MCP-1) in cohorts of Pakistani renal transplant patients. The project was approved by the Institutional Ethical Review Committee and informed consent was taken from all the participants. Briefly, the gene polymorphisms CCR2V64I, CCR5-59029G>A and CCR5Δ32 were investigated in 606 renal transplant patients and their donors by amplified fragments length polymorphisms (RFLP). The results showed that the G/G genotype of CCR2V64I was associated with a high frequency of allograft rejection (P=0.009). The Kaplan-Meier curve also indicated a significant reduction in the overall time to rejection-free allograft survival for patients with the G/G genotype of CCR2V64I as compared to the A/A or G/A genotype (59.2±1.4 vs. 68±2.6 weeks, P=0.008) showing that individuals with the A allele, either in the homozygous or heterozygous state, have a greater chance to accept the graft. ix Human IP-10 is classified as the CXC chemokine sub-family. A total of 206 urine samples of (a) rejection (n=96), (b) non-rejection (n=22) and (c) controls (n=88) were quantified for IP-10 by enzyme-linked immunosorbent assay (ELISA) for association with rejection. The results showed statistically significant differences in the urinary IP-10 levels between the rejection vs. non-rejection groups (P=0.004). The Receiver operating characteristic curve (ROC) of IP-10 showed area under the curve (AUC) of 0.70±0.06 with 72% sensitivity and 64% specificity, at a cut-off value of 27pg/ml. Human MIG also belongs to the CXC chemokine sub-family. A total of 266 urine samples from (a) rejection (n=108), (b) non-rejection (n=70), (c) stable grafts (n=42) and (d) control groups (n=46) were quantified for MIG and analyzed for association with rejection. The results indicated that although urinary MIG levels were higher in patients with rejection the association was not statistically significant (P>0.05). The ROC curve also showed AUC of 0.54±0.04 with low sensitivity (46%) and specificity (55%) at cut-off value of 6pg/ml. Human MCP-1 belongs to the CC chemokine sub-family. A total of 409 urine samples of (a) rejection (n=165), (b) non-rejection (n=93), (c) stable grafts (n=42) and (d) controls (n=109) were quantified for urinary level of MCP-1 by ELISA. The results showed that MCP-1 levels were different between the rejection and other groups (P<0.05). The ROC curve illustrated the area under curve of 0.83±0.04 with a sensitivity and specificity of 84% and 74% respectively, at a cut-off value of 214pg/ml. In conclusion, this work shows the usefulness of chemokine receptor CCR2V64I polymorphism as a marker for the increased possibility of an x immune response against an allograft. Urinary levels of MCP-1, the ligand of CCR2, and IP-10 were increased and show good correlation with rejection. While urinary MIG did not show any association with rejection. These findings may help in developing new therapeutic strategies in renal transplantation based on patient genetic makeup. Additionally, non-invasive screening tests based on urinary levels of IP-10 and MCP-1 would help in the assessment of the immune status of the graft