Adrenergic stimulation of HK-2 proximal tubular cells in hyperglycaemic conditions
One of the most commonly associated complications of Diabetes Mellitus is nephropathy, with one in five diabetics sustaining renal injury over their lives. Renal denervation has been shown to decrease systemic blood pressure and increase renal function, by removing sympathetic innervation to the kidneys. Our lab investigated the effects of renal denervation on diabetic nephropathy in hypertensive rats and found it decreased a range of injury markers, such as TGF-β1. It is possible that part of the renoprotection afforded by renal denervation in hypertensive diabetics is due to decreased adrenergic signalling. This study aimed to determine the effects of combined noradrenaline and glucose on proximal tubule cells and their profibrotic signalling, by measuring the excretion of the prosclerotic cytokine, TGF-β1. HK-2 cells were treated with a range of glucose concentrations (Control: None, Normoglycaemic: 6.1 mmol/L D-glucose, Hyperglycaemic: 25 mmol/L D-glucose, Osmotic Control: 6.1 mmol/L D-glucose + 18.9 mmol/L D-mannitol) in the presence of noradrenaline (1 nM) or a control (PBS or 0.1 nM ascorbic acid). After 48 hours the samples were harvested and levels of TGF-β1 measured via western blot. However, the cells were severely damaged by washing. The primary aim of investigation subsequently became the optimisation of plating protocols. Seeding densities, growth time and well size were increased, extensions to growth arrest and low serum (2%) growth arrest media used, as well as a range of different wash solutions and culture surfaces. By increasing the seeding density to 2x105 cells/mL and surface area up to a 100 mm culture dish a monolayer was formed that could repeatedly survive the wash phases (P<0.001). The initial study recommenced and western blotting was unable to find any trace of TGF-β1 in either the cell lysate or conditioned media. These findings suggest that neither alone nor together noradrenaline and glucose do not cause an upregulation of the production and excretion of TGF-β1.
Advisor: Sammut, Ivan
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Pharmacology
Publisher: University of Otago
Keywords: TGF-β1; Adrenergic stimulation; HK-2; proximal tubular cells; hyperglycaemic; diabetes mellitus; renal function; renal denervaton; plating protocol; profibrotic signalling
Research Type: Thesis