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Original Articles

Decreased Degradative Enzymes in Mesangial Cells Cultured in High Glucose Media

  1. David J Leehey,
  2. Ruo Hua Song,
  3. Nahid Alavi and
  4. Ashok K Singh
  1. Hines Veterans Affairs Hospital, Hines; Loyola University Stritch School of Medicine Maywood; and Section of Renal Diseases, Department of Medicine, University of Illinois at Chicago Chicago, Illinois (N.A.)
  1. Address correspondence and reprint requests to Dr. David J. Leehey, 111-L, Veterans Affairs Hospital, Hines, IL 60141.
Diabetes 1995 Aug; 44(8): 929-935. https://doi.org/10.2337/diab.44.8.929
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Abstract

Abnormalities in extracellular matrix degradation may play a pathogenetic role in diabetic nephropathy. Cultured renal mesangial cells are known to synthesize increased amounts of matrix proteins when incubated in high glucose media (e.g., 30 mmol/l). However, the effect of glucose loading on degradative enzymes is unknown. Primary cultures of rat mesangial cells were grown until confluent in the presence of fetal calf serum (FCS) and insulin (0.67 U/ml). Cells were then cultured for 7 days in plastic wells in either 10 or 30 mmol/1 glucose media containing neither FCS nor insulin. Collagenase activity in media were determined by zymography and quantitative spectrofluorometry. Cathepsin B and D activities in cell extracts were measured by spectrofluorometry (using the fluorescent substrate Z-Arg-Arg-7-amido-4-methylcoumarin) and 125I-labeled hemoglobin digestion, respectively. Gelatin-degrading activity of live mesangial cells was also determined. mRNA levels for collagenase IV, cathepsin B, and cathepsin D were determined by Northern analysis. A major band of collagenase activity with a molecular size of 72 kDa was observed in all mesangial cell media. Exposure of cells to high glucose media resulted in significant reductions in collagenase and cathepsin B activities as well as impairment in gelatin-degrading activity. Collagenase IV and cathepsin B and D mRNA levels were also decreased by glucose loading. To exclude the possibility that glucose loading was injurious to cells, 3H-leucine uptake (as a measure of protein synthesis) and membrane alkaline phosphatase activity (as a biochemical marker of viability) were not affected by the high glucose condition. We conclude that proteinase activity is decreased in mesangial cells incubated in high glucose media. This may be due to changes in levels of gene expression (mRNA) of these enzymes.

  • Received October 6, 1994.
  • Revision received April 20, 1995.
  • Accepted April 20, 1995.
  • Copyright © 1995 by the American Diabetes Association

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August 1995, 44(8)
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Decreased Degradative Enzymes in Mesangial Cells Cultured in High Glucose Media
David J Leehey, Ruo Hua Song, Nahid Alavi, Ashok K Singh
Diabetes Aug 1995, 44 (8) 929-935; DOI: 10.2337/diab.44.8.929

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Decreased Degradative Enzymes in Mesangial Cells Cultured in High Glucose Media
David J Leehey, Ruo Hua Song, Nahid Alavi, Ashok K Singh
Diabetes Aug 1995, 44 (8) 929-935; DOI: 10.2337/diab.44.8.929
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