MicroRNA profile and function in kidney ischaemia and reperfusion injury

Abstract

Ischaemia and reperfusion injury (IRI) in renal allografts is an important contributing factor to chronic allograft dysfunction. MicroRNAs (miRNA) have been shown to play important roles in cellular adaptation to pathological conditions, including IRI. This study aimed to evaluate changes in miRNA profile following IRI, and how the changes in particular miRNAs may influence renal proximal tubular epithelial cell (PTEC) morphology and function, potentially contributing to the development of chronic allograft dysfunction. To achieve this, several objectives were set. These included: (1) isolation and culture of primary human PTECs, (2) miRNA expression profiling following IRI and selection of candidate miRNA, and (3) in vitro and human pathology validation to explore the molecular mechanism of the candidate miRNA. Primary PTECs were isolated from normal renal tissue. These cells showed features of epithelial cells under light microscope and electron microscope. The cells were also characterised using immunofluorescent staining, which showed positive expression of epithelial cell markers, and negative expression for mesenchymal cell markers. MiRNA profiling using NanoString platform was performed on cell lines (HKC-8 and HK-2) and primary PTECs, which were exposed to either hypoxia or free radicals. Results revealed distinct miRNA signature changes following IRI in cells. However, only a small proportion of microRNAs were found to be significantly up/down-regulated in either cell lines or primary cells, which included miR-21, miR-376, miR-190b, miR-34a, miR-210, miR-363, miR-142 and miR-130b. MiR-21 was shown to be up-regulated in all cells following both type of injuries. Online target prediction analysis also showed miR-21 to be involved in pathways relevant to cellular response to IRI and the development of fibrosis. The role of miR-21 was studied in detail. Up-regulation of miR-21 following ischaemia was shown to suppress SMAD7 and facilitate intra-nuclear localisation of SMAD3. In the presence of exogenous TGF-b1 or hypoxia, over-expression of miR-21 in cells led to an increase in SMAD3 activity. Over-expression of miR-21 also led to phenotypic shift in HKC- 8 cells, characterised as a decrease in E-cadherin and an increase in a-SMA and Collagen-1 expression. Human pathology evaluation confirmed high expression of miR-21 in the tubular cells of severely ischaemic kidneys compared to non-ischaemic kidneys. In conclusion, changes in miRNA profile were observed in acute IRI in the kidney. One of the significantly affected miRNAs was miR-21. MiR-21 up-regulation resulted in sensitisation of tubular cells to TGF-b1, which may be essential in cellular repair processes, but may also contribute to deterioration of long-term organ function.