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Abstract

INTRODUCTION: Diabetic kidney disease (DKD), mediated by both hyperglycaemia and transforming growth factor beta 1 (TGF-b1), is the principal cause of end stage renal disease (ESRD). Tubulointerstitial fibrosis is the best prognostic indicator of renal function in diabetic patients. Drugs targeting the sodium glucose co-transporter 2 (SGLT2) at the proximal tubule are successful anti-diabetic therapeutics. This thesis aims to establish a cellular DKD model for studying the novel glucose/TGF-b1 pathway and its regulation by SGLT2 inhibitors.

METHODS: Human primary proximal tubule epithelial cells (PTECs) were cultured and treated with D-glucose at either 7mM (control), 25mM (hyperglycaemia), or 7mM Dglucose+ 18mM L-glucose (osmotic control), with or without TGF-b1 (0.75-5ng/ml). SGLT2 was inhibited by dapagliflozin (0.1-10nM), SGLT1 by phlorizin (500μM), facilitative glucose transporter 1 (GLUT1) by phloretin (1mM) and extracellular regulated signal kinase (ERK) by U0126 (10μM). PTECs were transfected with a Smad3 serine 204 (S204) dominant negative (DN) construct (0.5-2μg). Proteins were examined by western blotting, gene transcription by qPCR and dapagliflozin breakdown by high performance liquid chromatography (HPLC).

RESULTS: High D-glucose (25mM) and TGF-b1 (0.75ng/ml) induced connective tissue growth factor (CCN2), fibronectin and phosphorylated Smad3 S204. These were all attenuated in the presence of dapagliflozin. Smad3 S204 and ERK phosphorylation was attenuated by U0126. The DN S204 construct prevented CCN2 but not fibronectin transcription. HPLC showed a time dependent decrease in dapagliflozin concentration in the medium. Urinary CCN2 and fibronectin protein were detected in the DKD patient group with high albumin to creatinine ratios (ACR) (median 336) and low estimated glomerular filtration rates (eGFR) (<60ml/min/1.73m2).

CONCLUSION: This work describes a role for SGLT2 in regulating select pro-fibrotic signalling outcomes and provides evidence for a novel disease pathway in DKD PTECs. The reduction of fibronectin and CCN2 by dapagliflozin provides strong support for the renoprotective benefits of SGLT2 inhibition in DKD.

Publication Type:	Thesis (Doctoral)
Subjects:	Q Science > Q Science (General) R Medicine > RB Pathology
Departments:	School of Health & Medical Sciences > Infection and Immunity Research Institute School of Health & Medical Sciences > School of Health & Medical Sciences Doctoral Theses Doctoral Theses

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