Novel mechanisms of tubulointerstitial fibrosis

McDowall, Callum (2017) Novel mechanisms of tubulointerstitial fibrosis. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Kidney disease affects 8.8% of people over the age of 18 in the UK and represent a significant public and a financial burden for the National Healthcare Service (NHS). Untreated Chronic kidney disease (CKD) can develop into end-stage renal failure, leading to costly dialysis or kidney transplantation. One of the major causes of CKD is diabetic nephropathy, which is characterised by the progressive degradation of nephrons within the kidney as a result of type 1 and type 2 diabetes. One of the major pathways that drives the progression of CKD is Tubulointerstitial Fibrosis (TIF), which is linked to excessive Extracellular Matrix (ECM) accumulation in the tubules of the kidney nephrons and is considered an irreversible process. Currently there is no treatment for TIF and research needs into new targets and treatment strategies that can lead to prevention or even reversal of TIF. MicroRNAs (miRNAs) regulate protein expression by forming a RNA-induced silencing complex (RISC) and targeting specific mRNAs for degradation, the up or downregulation of miRNAs can heavily influence the progression of diseases making them a crucial area of research, as the inducing or blocking the activity of certain miRNAs could slow or halt disease progression. Therefore, the aim of this project was to identify novel fibrosis-related miRNA targets for treatment of CKD by exposing Renal Proximal Tubular Epithelial Cells (RPTEC) to sera of patients with different stages of diabetic nephropathy. To investigate the effect of serum factors in patients with diabetic nephropathy on initiating TIF, human renal proximal tubular (HK-2) cells were exposed for 24h or 48h to sera from 16 patients at different stages of CKD or sera from age and gender matched healthy volunteers. Then the effects of 1% human serum on cell viability, Plasminogen activator inhibitor-1 (PAI-1) reporter gene assay, PAI-1 and collagen 1 protein expression, and the regulation of 84 fibrosis-related miRNAs were studied. The results demonstrated that progressive stages of CKD reduced cell viability, increased the protein expression of PAI-1 and collagen-1 (in particular by stage 4 CKD sera), and dysregulated the expression of a number of fibrosis related miRNAs at 24h or 48h of exposure to serum. A number of miRNAs were up- or down-regulated by sera from patients with different stages of CKD, particularly those related to signal transduction and transcriptional regulation, as well as pro-fibrotic miRNAs. Of particular interest for further investigations are the following miRNAs that were upregulated both at 24h and 48h: miR-449b-5p (the only miRNA upregulated by all patient sera), miR-122-5p (the only miRNA upregulated by sera of CKD 3a stage patients) and miR-217 (the miRNA down-regulated much more severely than any others). In conclusion, this project demonstrated that the increasing stage of CKD in patients with diabetic nephropathy has a detrimental effect on proximal tubular cells in vitro and dysregulates the expression of fibrosis-related miRNAs. Further research is required to identify the genes regulated by key miRNAs and to determine their suitability as therapeutic targets in treating TIF and CKD.