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Expression of extracellular matrix molecules in human mesangial cells in response to prolonged hyperglycaemia
N A Wahab1, K Harper, R M Mason
1Department of Biochemistry, Charing Cross and Westminster Medical School, London, U.K.
Abstract:
Post-mitotic cultures of human mesangial cells were maintained in media containing 4-30 mM D-glucose for up to 28 days. Changes in mRNA and protein levels for specific macromolecules occurred between 7 and 14 days after initiating hyperglycaemic conditions. Slot blot analysis showed 2-3-fold increases in mRNAs for collagen type I, fibronectin, versican and perlecan, whereas mRNA for decorin was increased by up to 20-fold. Levels of mRNAs for biglycan and syndecan were unaffected by hyperglycaemic culture. Reverse transcriptase PCR (RT-PCR) confirmed that decorin mRNA levels are greatly elevated and also showed increased transcription of the TGF-beta 1 gene in hyperglycaemic cultures. Western analysis and ELISA indicated accumulations of collagen types I and III, laminin and fibronectin in the cell layers and media of hyperglycaemic cultures with increasing time. Type IV collagen did not accumulate in either compartment of hyperglycaemic mesangial cell cultures. Collagen types I, III, and fibronectin did not accumulate in the cell layers of hyperglycaemic human dermal fibroblasts, indicating a cell-specific response in mesangial cultures. Decorin and versican, but not biglycan, were increased in the hyperglycaemic mesangial cell culture media. There were no apparent changes in core proteins for decorin and biglycan in fibroblast media. Transforming growth factor beta 1 (TGF-beta 1) in hyperglycaemic mesangial cell cultures increased 5-fold after 7 days, but decreased thereafter to only approx. 2-fold after 28 days. The changes in TGF-beta 1 mRNA, as detected by RT-PCR, and protein followed one another closely.
Insights
High glucose levels in human mesangial cells significantly increase specific extracellular matrix proteins like decorin and fibronectin, and transforming growth factor beta 1 (TGF-beta 1), indicating a cell-specific response to hyperglycemia.
Area of Science:
- Cell Biology
- Biochemistry
- Nephrology
Background:
- Hyperglycemia is a hallmark of diabetes mellitus and is associated with diabetic nephropathy.
- Mesangial cells play a crucial role in the glomerular structure and function.
- Alterations in extracellular matrix (ECM) production by mesangial cells contribute to kidney damage in diabetes.
Purpose of the Study:
- To investigate the impact of sustained hyperglycaemic conditions on human mesangial cells.
- To quantify changes in mRNA and protein levels of specific ECM macromolecules and related growth factors.
- To determine if these changes are specific to mesangial cells compared to dermal fibroblasts.
Main Methods:
- Human mesangial cells cultured in media with varying D-glucose concentrations (4-30 mM) for up to 28 days.
- Analysis of mRNA levels using slot blot and reverse transcriptase PCR (RT-PCR).
- Assessment of protein accumulation via Western analysis and ELISA.
Main Results:
- Hyperglycaemia significantly increased mRNA for decorin (up to 20-fold), collagen type I, fibronectin, versican, and perlecan.
- Protein accumulations of collagen types I and III, laminin, and fibronectin were observed in hyperglycaemic mesangial cell cultures.
- Transforming growth factor beta 1 (TGF-beta 1) mRNA and protein levels showed a transient increase in hyperglycaemic mesangial cells.
Conclusions:
- Sustained hyperglycaemia induces significant alterations in ECM composition in human mesangial cells.
- The observed changes, particularly the marked increase in decorin and TGF-beta 1, are specific to mesangial cells.
- These findings highlight potential molecular mechanisms underlying mesangial cell dysfunction in diabetic nephropathy.