MicroRNAs as biomarkers of injury and drug response in renal transplantation

Abstract

Renal transplantation remains the optimal treatment for many patients with end stage renal disease. To meet the shortfall of organs available for transplantation, increasingly organs that have both endured and are more susceptible to ischaemia reperfusion injury (IRI), are being utilised for transplantation. As such, predicting and mitigating the impact of ischaemia reperfusion injury (IRI) remains a key priority in renal transplantation (RT).

The small non-coding RNAs, microRNAs, are increasingly appreciated for their roles as both mechanistic regulators, and biomarkers of disease and drug response. High- throughput approaches such as small RNA-Sequencing (sRNA-seq) have recently been widely used to identify dysregulated microRNAs in renal injury and disease. However, the interpretation of these changes is often hampered by the lack of precision regarding the cellular origin of the microRNA. This limits the application of these miRNAs as either biomarkers or therapeutic targets.

In the treatment of IRI, pre-clinical models have consistently demonstrated the beneficial properties of heme oxygenase-1 (HO-1) upregulation on ischaemia reperfusion injury (IRI). Heme arginate has previously been shown to safely upregulate HO-1 in renal transplant recipients (HOT study) and this formed the basis for the multi-centre trial assessing efficacy of treatment in improving early graft function (HOT2). Increasingly, however, the importance of post-transcriptional regulation is HO-1 is becoming evident, although as yet this is unstudied in humans.

The core purpose of this work was to harness microRNAs as biomarkers to predict both IRI severity and response to heme arginate treatment.

We first employed sRNA-seq separately on tubular cells, endothelial cells, fibroblasts and macrophages isolated from the injured and repairing kidneys in the murine reversible unilateral ureteric obstruction model. We devised an unbiased bioinformatics pipeline to define the miRNA enrichment within these cell populations, constructing a miRNA atlas of injury and repair. We found that a significant proportion of cell-specific miRNAs in healthy animals were no longer specific following injury. We then applied this knowledge of the relative cell specificity of miRNAs to deconvolute bulk miRNA expression changes in murine models and human renal disease cortical kidney samples comprised of mixed cell types. Finally, we used our data-driven approach to rationally select and demonstrate macrophage-enriched miR-16-5p and miR-18a as promising urinary biomarkers of delayed graft function in renal transplant recipients.

In renal transplant recipients treated with heme arginate, we identified a variation in HO-1 protein response between individuals. Using RNA-seq we characterised microRNAs within these patients. We demonstrated a global downregulation of mature microRNAs in high HO-1 responders to HA, but with the relative preservation of microRNA precursors. MicroRNA loss in these patients was evident before treatment administration. This same profile was not observed in a second cohort of cardiac surgery patients also receiving heme arginate. Taken together, this work suggested that a proportion of renal failure patients demonstrate a failure of microRNA biogenesis that significantly affects their ability to produce HO-1 protein in response to HA.

In conclusion, we have identified the likely cellular origin of multiple microRNAs in the kidney, providing a platform for the rational selection of microRNAs as biomarkers in mixed population kidney samples, serum and urine. In a subset of patients with renal failure, we have demonstrated a global loss of microRNA expression. The reason for this loss of global mature microRNA expression and the implications of this on disease and other drug responses remain important unanswered questions.