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Osmotic and metabolic effects of hyperglycaemia on glycolysis, mitochondrial function and fibrosis-related microRNA expression in immortalised human proximal tubule cells

Alsehli, Fahad (2019) Osmotic and metabolic effects of hyperglycaemia on glycolysis, mitochondrial function and fibrosis-related microRNA expression in immortalised human proximal tubule cells. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The incidence of chronic kidney disease (CKD) is increasing and CKD can progress to end-stage renal disease (ESRD) which requires costly renal replacement therapies. Diabetes is one of the major factors for CKD and ESRD. Around 40% of the diabetic patients develop kidney damage known as diabetic nephropathy. Renal tubulointerstitial fibrosis (TIF) is irreversible and leads to the excessive deposition of extracellular matrix, disruption of cell-to-cell interactions and loss of tissue elasticity, thus leading to the destruction of the renal tubules. However, at present there are no successful therapies that can block or reverse TIF. Therefore, new approaches are required to identify suitable therapeutic targets for the prevention or reversal of TIF. This thesis evaluated the metabolic and osmotic effects of hyperglycaemia on the glycolytic and mitochondrial function, the expression of fibrosis-related cytokines, markers of inflammation, ECM accumulation, and the expression of a panel of fibrosis-related miRNA in immortalised human proximal tubule (HK-2) cells. The work demonstrated impairment of the two energy pathways, oxidative phosphorylation and glycolysis, in response to 30mM D-glucose, and that was linked to the osmotic effect of hyperglycaemia. In contrast, the reductions of cell viability and glucose consumption, and impaired wound healing were linked to its metabolic effect. The study also showed that the osmolarity component of hyperglycaemia activated Smad3 and upregulated proximal tubular injury and inflammation markers (e.g. IL-6, KIM-1, MCP-1 and endostatin), suggesting a role of osmolarity in the pathogenesis of DN. Finally, the thesis evaluated the expression of 84 fibrosis-related miRNAs in HK-2 cells exposed to different concentrations of glucose. The study showed that 16 miRNAs were dysregulated over a 72h exposure to hyperglycaemia. Furthermore, the pro-fibrotic miR-216a-5p was downregulated in response to hyperglycaemia. As one of the potential targets of miR-216a-5p is the TGF-β1 Receptor 2, hyperglycaemia may exacerbate fibrosis by facilitating the signalling of the pro-fibrotic cytokine TGF-β1. Future work will investigate which of the 16 dysregulated miRNAs is a suitable target for treating TIF in diabetic nephropathy.

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