Decreased degradative enzymes in mesangial cells cultured in high glucose media.
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/l 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Leehey
D J
DJ
Veterans Affairs Hospital, Hines, IL 60141, USA.
Song
R H
RH
Alavi
N
N
Singh
A K
AK
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002401:Cathepsin B / Q000096:biosynthesis / Q000378:metabolism; D002402:Cathepsin D / Q000096:biosynthesis / Q000378:metabolism; D002478:Cells, Cultured; D017364:Collagenases / Q000096:biosynthesis / Q000378:metabolism; D003470:Culture Media; D015342:DNA Probes; D010450:Endopeptidases / Q000378:metabolism*; D018093:Gelatinases / Q000096:biosynthesis / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D005920:Glomerular Mesangium / Q000187:drug effects / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence
D003470:Culture Media; D015342:DNA Probes; D007328:Insulin; D012333:RNA, Messenger; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D002401:Cathepsin B; D002402:Cathepsin D; D017364:Collagenases; D018093:Gelatinases; D005947:Glucose