Role of angiotensin II in glucose-induced inhibition of mesangial matrix degradation.
Accumulation of mesangial matrix in diabetic nephropathy is caused by increased synthesis and decreased degradation. We have previously demonstrated that incubation in high-glucose medium decreases mesangial cell collagenase activity (Diabetes 44:929-935, 1995). Because angiotensin II (AII) is involved in the pathogenesis of diabetic nephropathy, the present studies were performed to determine if AII mediates glucose-induced 1) inhibition of mesangial collagenase activity, 2) mesangial matrix accumulation, and 3) in-crease in transforming growth factor (TGF)-beta1 secretion in mesangial cells. The direct effect of high glucose on AII generation in mesangial cells was also determined. Primary mesangial cells from normal Sprague-Dawley rats were used in all studies. Collagenase activity in cell medium was determined using three methods: 1) zymography; 2) quantitative assay using fluoresceinated gelatin as substrate; and 3) a new enzyme-linked immunosorbent assay (ELISA) that specifically measures 72-kDa collagenase (MMP-2), the principal collagenase synthesized by mesangial cells. Matrix accumulation was estimated by immunoperoxidase assay on cell layers using anti-glomerular basement membrane (GBM) antibodies. TGF-beta1 and AII levels were determined by ELISA. Exposure of mesangial cells to 30 mmol/l glucose (high glucose) vs. 5 mmol/glucose (normal glucose) for 5 days resulted in a significant decrease in collagenase activity (25%) that was normalized by 10(-4) mol/l losartan, a type 1 angiotensin II (AT1) receptor antagonist. High glucose increased anti-GBM binding compared with normal glucose; this effect of glucose was reversed by losartan. Incubation of cells with 30 mmol/l glucose increased total TGF-beta1 secretion, which was also normalized by losartan. Addition of AII (10(-6) mol/l) for 24 h to the culture medium inhibited collagenase activity by 33%; losartan (10(-4) mol/l) blocked this inhibition of enzyme activity. Also, AII decreased collagenase (MMP-2) levels but stimulated TGF-beta1 secretion in mesangial cells. Finally, glucose increased mesangial AII generation in a concentration-dependent manner, with incubation in 30 mmol/l glucose increasing AII by 25% compared with 5 mmol/l glucose. We conclude that glucose increases AII production by mesangial cells, which results in stimulation of TGF-beta1 secretion, decreased matrix degradation, and increased matrix accumulation. These effects of AII are mediated by the AT1 receptor.
Singh
R
R
Hines Veterans Affairs Hospital, Illinois 60141, USA.
Alavi
N
N
Singh
A K
AK
Leehey
D J
DJ
D000804:Angiotensin II / Q000096:biosynthesis*; D000818:Animals; D017364:Collagenases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D019808:Losartan / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats; D016212:Transforming Growth Factor beta / Q000378:metabolism
D016212:Transforming Growth Factor beta; D000804:Angiotensin II; D017364:Collagenases; D005947:Glucose; D019808:Losartan