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High glucose decreases matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth
1Department of Medicine, Veterans Affairs Hospital, Hines, Ill 60141, USA.
Abstract:
Diabetic nephropathy is characterized by accumulation of mesangial matrix. Glucose-induced inhibition of matrix-degrading enzymes such as collagenases is believed to contribute to matrix accumulation. We have previously demonstrated that 72 kDa type IV collagenase activity is decreased in the rat mesangial cells cultured in high glucose media [Diabetes 1995;44:929-935]. The present studies were designed to investigate if the cytokine transforming growth factor-beta1 (TGF-beta1) mediates this effect of glucose. Type IV collagenases degrade type IV collagen as well as gelatin (denatured collagen) and are thus also called gelatinases. They belong to the family of matrix metalloproteinases (MMPs); MMP activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). The activity of 72 kDa type IV collagenase, also known as matrix metalloproteinase-2 (MMP-2), was assessed using three methods: (1) fluoresceinated gelatin degradation assay to detect free enzyme activity (activity which is present in excess of TIMP-inhibited activity); (2) zymography to measure total (free + TIMP-bound) enzyme activity; (3) ELISA using specific antibodies to measure MMP-2 levels. TGF-beta1 and TIMP-2 levels were also determined by ELISA. Incubation of primary cultures of rat mesangial cells for 5 days in 30 vs. 5 mM glucose resulted in a 3-fold increase in production of total TGF-beta1, a significant decrease in MMP-2 activity and immunoreactive MMP-2 levels, and an increase in TIMP-2 levels. Addition of exogenous TGF-beta1 to mesangial cells incubated in 5 mM glucose replicated the high glucose effect by producing a significant decrease in MMP-2 levels with a concurrent increase in TIMP-2 levels. Furthermore, glucose-induced inhibition of MMP-2 activity was completely blocked by neutralization of TGF-beta1 with anti-TGF-beta1 antibody. We conclude that the decrease in MMP-2 activity induced by glucose loading is mediated via TGF-beta1.
Insights
High glucose levels in diabetic nephropathy reduce matrix-degrading enzyme MMP-2 activity. This effect is mediated by transforming growth factor-beta1 (TGF-beta1), a key cytokine in kidney disease progression.
Area of Science:
- Nephrology
- Endocrinology
- Molecular Biology
Background:
- Diabetic nephropathy involves mesangial matrix accumulation.
- Glucose may inhibit matrix-degrading enzymes, contributing to this accumulation.
- Previous studies showed decreased 72 kDa type IV collagenase activity in high glucose conditions.
Purpose of the Study:
- Investigate if transforming growth factor-beta1 (TGF-beta1) mediates glucose-induced inhibition of matrix-degrading enzymes.
- Assess the role of TGF-beta1 in the reduction of matrix metalloproteinase-2 (MMP-2) activity in mesangial cells.
Main Methods:
- Rat mesangial cells were cultured in high (30 mM) vs. normal (5 mM) glucose media.
- MMP-2 activity was measured using fluoresceinated gelatin degradation assay and zymography.
- MMP-2, TGF-beta1, and TIMP-2 levels were quantified using ELISA.
- Effects of exogenous TGF-beta1 and anti-TGF-beta1 antibody were examined.
Main Results:
- High glucose increased TGF-beta1 production by 3-fold.
- High glucose significantly decreased MMP-2 activity and levels.
- High glucose increased TIMP-2 levels.
- Exogenous TGF-beta1 mimicked high glucose effects on MMP-2 and TIMP-2.
- Neutralizing TGF-beta1 blocked glucose-induced MMP-2 inhibition.
Conclusions:
- Glucose-induced decrease in MMP-2 activity in mesangial cells is mediated by TGF-beta1.
- TGF-beta1 plays a crucial role in the pathogenesis of diabetic nephropathy by affecting matrix degradation.
- Targeting the TGF-beta1 pathway may offer therapeutic strategies for diabetic nephropathy.
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