Programme(s) to which this project applies:
|☒ MPhil/PhD||☑ MRes[Med]||☒ URIS|
Objective and Significance:
Nephropathy due to diabetes mellitus is the most common cause of end-stage renal failure, and accounts for 40% of new patients who require renal replacement therapy. Glomerular expansion and tubulo-interstitial fibrosis are prominent features of diabetic nephropathy. To date, there is increasing evidence to show that transforming growth factor beta1 (TGF- 1) plays a pivotal role in the pathogenesis of disease. Studies have shown that increased TGF-1 expression in both the glomerular and tubular compartments contribute to increased matrix synthesis and deposition within the kidney. The mechanism by which fibrosis occurs remain to be fully determined. Although numerous studies have investigated renal cells in isolation in the pathogenesis of disease, how glomerular cells influence tubular fibrosis and whether there is glomerular-tubul ar 'cross-talk' remains to be determined, and thus constitutes the theme of this project.
Understanding the molecular mechanism of diabetic nephropathy is prerequisite to better prevention and treatment of this disease.
Research Plan and Methodology:
Human mesangial cells and proximal tubular epithelial cells will be cultured under physiological or elevated concentrations of D-glucose, and assessed for their cell viability, proliferation, transcription and translation of TGF-P1 and matrix proteins [fibronectin, collagen types I, III and IV].
The above parameters will also be investigated in which mesangial cells will be incubated with spent culture media obtained from proximal tubular epithelial cells and vice versa.
Harvesting and primary culture of mesangial and proximal tubular epithelial cells: Both primary cell cultures are well established in our laboratory .
Assessment of cell proliferation: This will be investigated using the MTT assay .
Assessment of cell viability: This will be determined by measuring lactate dehydrogenase release .
Assessment of gene expression of TGF-/31 and matrix proteins: Total RNA will be extracted from cells cultured under basal and experimental conditions, and extracted with TriReagent, transcribed into cDNA, and subjected to PCR amplification . P-actin will be used as the 'house-keeping' gene. After amplification, each PCR reaction mix will be electrophoresced on a flat bed agarose gel (2% w/v) in lx Tris-borate-EDTA buffer containing ethidium bromide (0.5µg/ml). Images of the gels will be taken using a Gel Documentation System and the density of the bands evaluated by densitometry .
Measurement of TGF-/Jl secretion by renal cells: Aliquots of culture supernatant will be obtained from both mesangial and proximal tubular epithelial cells cultured under control and experimental conditions and TGF-P1 secretion assessed using a commercial ELISA according to the manufacturer's instructions.
Analysis of matrix protein synthesis: Cells cultured under control and experimental conditions will be lysed with 4M urea buffer and synthesis of matrix proteins determined in a semi-quantitative manner using Western blot analysis, and also 'in-house' ELISAs .
For more information or to express interest for this project, please email the supervisor or the specified contact point in the project description. Interested candidates are advised to enclose with your email:
Information on the research programme, funding support and admission documentations could be referenced online at the Research Postgraduate Admissions website. General admission enquiries should be directed to firstname.lastname@example.org.
HKUMed MBBS students interested in the Master of Research in Medicine (MRes[Med]) programme may visit the programme website for more information.
HKUMed UG students interested in the Undergraduate Research Internship Scheme (URIS) may visit the scheme’s website for more information.