Targeting miR-21 to inhibit the inflammatory response following ischaemic kidney injury

Abstract

Targeting ischaemia-reperfusion injury (IRI) in kidney transplantation has the potential to improve transplant survival, thereby benefiting recipients. MicroRNAs (miR) are potential targets through which to modulate these injury pathways and rodent studies suggested a protective effect from blocking miR-21-5p in kidney IRI. I have delivered a miR-21-5p inhibitor (antimiR-21) to human kidney in vitro and ex vivo models of IRI to explore the potential of antimiR-21 as a therapeutic target in this context. Proximal tubule epithelial cells (PTEC) were isolated from human kidneys declined for transplantation and characterised with immunofluorescence, light microscopy and electron microscopy. Endothelial and mesenchymal cell contamination was determined with flow cytometry. PTEC were shown to express miR-21 and HIF1α stabilisation was confirmed on incubation in 1% oxygen to model ischaemia. PTEC took up antimiR without the use of transfection reagents. Macropinocytosis was a dominant uptake mechanism as uptake was reduced by a macropinocytosis inhibitor but unaffected by blocking megalin-mediated uptake. The response of PTEC to antimiR-21 treatment from reoxygenation was explored with bulk RNA sequencing and liquid chromatography mass spectrometry-based (LCMS-based) proteomics. 24 hours after antimiR-21 treatment, miR-21 targets were upregulated relative to control treated PTEC. Ingenuity pathway analysis predicts antimiR-21 treatment activates cell survival and blood vessel formation pathways during reoxygenation. A 24-hour normothermic machine perfusion model was optimised for the circuit used at Newcastle University and used to deliver antimiR-21 or control antimiR to pairs of human kidneys. The response was assessed with TUNEL stain for apoptosis, perfusate cytokine concentrations and LCMS-based proteomics on biopsies. In summary, PTEC isolated from human kidneys are a valuable in vitro model for exploring the effects of antimiR-21 treatment without transfection reagents. In vitro, antimiR-21 treatment during reoxygenation appears to be beneficial and in the whole organ model, antimiR-21 could effectively reduce miR-21 expression by 24 hours.