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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.

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ABSTRACT. Previous studies have shown that glucose increases angiotensin II (AngII) levels in rat glomerular mesangial cells and that AngII mediates the inhibitory effects of high glucose on matrix degradation in these cells. The present study addresses the following questions: (1) What are the mechanisms for the generation of AngII in mesangial cells? (2) What are the effects of glucose on AngII generation by these mechanisms? Experiments employed primary mesangial cells from normal Sprague-Dawley rats. The levels of immunoreactive angiotensinogen (AGT), angiotensin I (AngI), and angiotensin II (AngII) were measured by ELISA. AGT mRNA expression was determined by Northern blot analysis. Incubation of cells for 24 h in high glucose (30 mM) increased AGT levels by 1.5-fold and increased AGT mRNA expression; this was accompanied by a 1.5-fold increment in AngI and 1.7-fold increment in AngII levels. Renin activity (measured as AngI generation in the presence of excess AGT) and ACE levels and activity were not altered by high glucose. In further experiments, the effect of high glucose on formation of Ang peptides from exogenous AngI in mesangial cell extracts was examined using HPLC. Exogenous AngI was converted into various Ang peptides, including AngII, Ang(1-9), Ang(1-7), and Ang(3-8). A significant increase in formation of AngII from AngI was observed in cells incubated in high glucose. In addition, AngII production from exogenous Ang(1-9) in cell extracts was also stimulated by high glucose. These findings demonstrate that glucose increases mesangial AngII levels via an increase in AGT and AngI. In addition, this study provides new information that Ang(1-9) is produced by mesangial cells, can be converted to AngII, and that this conversion is also stimulated under high-glucose conditions. E-mail: dleehey@lumc.edu

Relaxin may have a useful application in decreasing interstitial fibrosis and thereby slowing the progression of renal disease.

This study quantified the uptake of five antibiotics (chlortetracycline, monensin, sulfamethazine, tylosin, and virginiamycin) by 11 vegetable crops in two different soils that were fertilized with raw versus composted turkey and hog manures or inorganic fertilizer. Almost all vegetables showed some uptake of antibiotics from manure treatments. However, statistical testing showed that except for a few isolated treatments the concentrations of all antibiotics in vegetable tissues were generally less than the limits of quantification. Further testing of the significant treatments showed that antibiotic concentrations in vegetables from many of these treatments were not significantly different than the corresponding concentrations from the fertilizer treatment (matrix effect). All five antibiotic concentrations in the studied vegetables were <10 μg kg(-1). On the basis of the standards for maximum residue levels in animal tissues and suggested maximum daily intake based on body weight, this concentration would not pose any health risk unless one is allergic to that particular antibiotic.