Background: Acute rejection (AR) occurs in 10-20% of renal allograft recipients despite combination immunosuppression, suggesting there are pathways that are not adequately inhibited by current therapy. Large scale gene expression profiling using microarray has enabled the characterisation of the molecular changes in AR. In recent years there has been an increased focus on identifying the most highly connected genes in various disease states, rather than the most highly expressed, as these ‘key driver’ genes are more likely to assume key regulatory roles. This has not been studied in the context of AR in renal transplantation, and may serve as potential targets for novel therapeutics.
Aims: 1.To derive key driver genes from microarray data of human renal allograft biopsy tissue with AR 2.Identify drugs that can inhibit the key driver genes 3. Experimental validation.
Methods: A meta-analysis of microarray data of 735 human kidney allograft biopsy samples was performed to identify the differentially expressed genes, and the key drivers were further derived using bioinformatics methods. Drug repurposing tools were used to identify candidate therapeutics and Minocycline was selected for further evaluation in a full allo-mismatch murine heterotopic cardiac allograft model. The impact of minocycline on the allograft when used alone, or when administered with immunosuppression (CTLA4Ig), was evaluated. Allograft histology was assessed and quantitative RT-PCR was performed to measure mRNA expression of key driver genes and cytokines/chemokines. A competing risk analysis was used to assess cumulative incidence of graft rejection, with graft loss due to other causes regarded as a competing risk.
Results: 14 key driver genes were identified, with Caspase 1 identified as the central key driver gene. Minocycline, a tetracycline antibiotic, is a well described inhibitor of Caspase 1, the central key driver gene. Minocycline (50mg/kg IP BD) compared with saline did not prolong allograft survival, butwas able to reduce the inflammatory profile of acute rejection as demonstrated by the reduction in the mRNA expression in the allograft tissue of a number of key drivers and inflammatory cytokines and chemokines (eg IFNγ, CXCL9) at day 4 post transplant. However, when minocycline was administered in conjunction with immunosuppression (CTLA4Ig 250ug IP Day 2 alone with minocycline 50mg/kg IP BD up to 27 days post transplant), an extension in allograft survival was observed compared with the control group (CTLA4Ig Day 2 with saline) (Hazard ratio 0.27 CI 0.099-0.736 p-value <0.01). 3 treatment animals were euthanased prior to cessation of allograft heart beat for other reasons.
Conclusion: Minocycline alters the inflammatory profile of AR, and prolongs murine cardiac allograft survival when given in conjunction with immunosuppressive therapy. Further evaluation as to the precise mechanisms by which this occurs is required.