This dissertation, "The Effect of Elevated Glucose Concentration on the Expression of -ACTININ-1 and F-ACTIN in Human Mesangial Cells" by Qing, Zhang, 張凊, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled The effect of elevated glucose concentration on the expression of α-actinin-1 and F-actin in human mesangial cells Submitted by Zhang Qing for the degree of Master of Philosophy at The University of Hong Kong in August 2004 Diabetic nephropathy is a leading cause of renal failure, and is characterized by mesangial expansion and thickening of the glomerular basement membrane, both consequent to hyperglycemia. Mesangial cells play a key role in the normal physiology of glomerulus and intra-renal homeostasis. The cytoskeleton is essential for maintaining the structural and functional integrity of the mesangium. Altered F- actin assembly in diabetic nephropathy is attributed to protein kinase C (PKC) activation. α-actinin-1 is an actin-binding protein that regulates cell receptor activity, and it serves as a scaffold connecting the cytoskeleton to diverse signaling pathways. The mechanisms by which elevated glucose concentrations alter α-actinin-1 and F- actin expression have not been investigated in human mesangial cells (HMC), and constitute the theme of this study. Confluent HMC were pre-conditioned in physiological (5 mM) or elevated (10 mM or 30 mM) D-glucose for up to 8 weeks, with mannitol at identical concentrations as the hexose control. The following parameters were investigated: (i) cell proliferation and viability by MTT assay and lactate dehydrogenase (LDH) release respectively; (ii) expression of α-actinin-1 by RT-PCR, Western Blot analysis, and immunohistochemistry; (iii) F-actin expression by immunohistochemical staining with phalloidin-FITC and semi-quantitated by fluorography; (iv) expression of integrins by Western Blot analysis; (v) expression of TGF-β1 by RT-PCR and ELISA; (vi) PKC isoforms by Western Blot analysis. To determine whether TGF-β1 induction and PKC-α activation contributed to changes in the cytoskeleton, in separate studies HMC were incubated with PMA (100 nM), exogenous TGF-β1 (0-5ng/ml) for up to 24 h, or TGF-β1 neutralizing antibody (1 g/ml) for 1 week. We demonstrated that HMC became hypertrophic under elevated glucose concentration. This was associated with reduced cell proliferation (maximum reduction occurring after 8 weeks stimulation: 45.45.2% and 68.04.1% for 10 mM and 30 mM D-glucose respectively compared to control, P