Renal expression of fibrotic matrix proteins and of transforming growth factor-beta (TGF-beta) isoforms in TGF-beta transgenic mice.

Renal pathology in mice that are transgenic for the murine albumin enhancer/promoter linked to a full-length porcine transforming growth factor-beta1 (TGF-beta1) gene has been described previously. In these mice, transgene expression is limited to the liver and the plasma level of TGF-beta is increased. The earliest renal pathologic change is glomerulosclerosis, at 3 wk of age, and this is followed by tubulointerstitial fibrosis. In this study, it was hypothesized that circulating TGF-beta1 increases renal extracellular matrix accumulation and activates local TGF-beta gene expression. Immunostaining at 5 wk revealed increased amounts of collagen I and III within the mesangium, glomerular capillary loops, and interstitium, while the amount of collagen IV was normal. Similarly, Northern analysis showed increased expression of mRNA encoding collagen I and III, as well as biglycan and decorin, while the expression of collagen IV was unchanged. These changes began as early as 1 wk of age, a time before the appearance of glomerulosclerosis. To evaluate matrix degradation, collagenase IV activity was evaluated by gelatin zymography and an increase in matrix metalloproteinase-2 was found. Finally, the production of tissue inhibitors of metalloproteinase was evaluated. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA was increased 18-fold, while TIMP-2 and TIMP-3 were unchanged. In 2-wk-old transgenic kidney, local expression of TGF-beta1, beta2, and beta3 protein was similar to wild-type mice. In 5-wk-old transgenic mice, TGF-beta1 and beta2 protein was present in increased amounts within glomeruli, and renal TGF-beta1 mRNA was increased threefold. It is concluded that elevated levels of circulating TGF-beta1 may act on the kidney to increase matrix protein production and decrease matrix remodeling. Only after glomerulosclerosis is established does local glomerular overproduction of TGF-beta become manifest.

Pentosan polysulfate decreases proliferation and net extracellular matrix production in mouse mesangial cells.

Glomerulosclerosis is characterized by extracellular matrix accumulation and is often associated with mesangial cell proliferation. Heparin-like molecules have been shown to decrease glomerulosclerosis in vivo, although their cellular site and mechanism of action is still unclear. In this study, a line of glomerular mesangial cells derived from normal mice was used to determine whether pentosan polysulfate (PPS) inhibited proliferation and altered extracellular matrix turnover. Cells treated with PPS showed a decrease in cell number beginning 24 h after treatment, which was maintained for 5 d. For matrix accumulation and degradation studies, cells were treated for 5 d and collagen types I and IV protein were measured by enzyme-linked immunosorbent assay as well as matrix metalloproteinases (MMP) measured by zymography. Collagen types 1 and type IV were significantly decreased in the media (P < 0.0001) and cell layer (P < 0.005) after treatment with PPS but not after treatment with heparin. By zymography, MMP-2 was significantly increased after treatment with PPS (P < 0.001) and heparin (P < 0.05). PPS and heparin also decreased MMP-9 (P < 0.001) after treatment. Reverse zymography showed the presence of tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in control mesangial cells. Treatment with PPS and heparin increased TIMP-1. In addition, TIMP-3 was found in the medium of treated but not control cells. In conclusion, PPS alters extracellular matrix turnover through the induction of MMP-2 and alterations in the TIMP profile and may be useful in decreasing progressive glomerulosclerosis.

Glomerular endothelial cells synthesize collagens but little gelatinase A and B.

Mesangial sclerosis is a major feature of progressive renal disease. The mesangium contains mesangial cells and is bounded by the peripheral glomerular basement membrane and endothelial cells. Mesangial cells synthesize and degrade extracellular matrix. Whereas both mesangial and endothelial cells synthesize extracellular matrix components, the degradative pathway, well studied in the former, has not been investigated in endothelial cells. This study examines lines of all three glomerular cell types derived from female B6SJLF1/J mice, as well as mRNA levels for collagens alpha1(I), alpha1(IV), alpha3 (IV), alpha5 (IV), and alpha1 (VI), laminin, tenascin, matrix metalloproteinase-2 (MMP-2), and MMP-9. Type I and IV collagen synthesis was confirmed by enzyme-linked immunosorbent assay. MMP-2 and MMP-9 enzyme activity was measured by zymography. It was found that glomerular endothelial cells are a significant source of collagens, laminin, and tenascin. However, they express only low levels of MMP-2 and no detectable MMP-9. Stimulation with exogenous transforming growth factor-beta1 leads to a significant increase in collagen I, tissue inhibitors of metalloproteinase-1, and MMP-9 in conditioned media. These data suggest that glomerular endothelial cells may play an active role in extracellular matrix remodeling in glomerular disease.

Effect of glucose on insulin-like growth factor binding protein-4 proteolysis.

The role of hyperglycemia in diabetic changes of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) has not been clearly established. We therefore determined whether glucose modulates IGFBP synthesis and stability in vitro. Porcine vascular smooth muscle cells (pSMC) cultured in low glucose (pSMC-L) had 2.1-fold more IGFBP-4 in the conditioned medium compared with pSMC cultured in high glucose (pSMC-H) (P < 0.01). In contrast, IGFBP-2 levels remained constant. Although pSMC-H and pSMC-L cultures expressed similar levels of IGFBP-4 messenger RNA, in vitro protease assays demonstrated an increase in IGFBP-4 proteolysis in pSMC-H conditioned medium compared with pSMC-L conditioned medium (P < 0.01). The protease had properties similar to a previously characterized IGFBP-4 protease. The addition of 20 mM mannitol to pSMC-L cultures did not decrease IGFBP-4 levels, suggesting that the difference in IGFBP-4 proteolysis was not osmotically induced. The change was not due to selection bias, because cultures that were initially isolated from aortic explants in high and low glucose still exhibited the glucose-dependent difference in IGFBP-4 proteolytic activity. The results suggest that high glucose acts on pSMC to induce a change in IGFBP-4 proteolytic activity, which results in increased IGF-I availability to its receptors thereby enhancing the SMC proliferative response.

Mesangial cells from transgenic mice with progressive glomerulosclerosis exhibit stable, phenotypic changes including undetectable MMP-9 and increased type IV collagen.

Mice transgenic for bovine growth hormone (bGH) develop progressive mesangial sclerosis resulting in uremia. Mesangial cells from bGH mice were isolated to determine whether the cells maintained a stable phenotypic change in the synthesis and degradation of extracellular matrix, which contribute to the glomerular lesions in vivo. The bGH mesangial cells were 1.2-fold larger than cells from control mice. They had a 1.7-fold increase in doubling time, a 7-fold decrease in labeling index (p < 0.0001), and a 2.4- and 2-fold decrease in c-myc (p < 0.05) and insulin-like growth factor I gene expression, respectively. Collagen synthesis and degradation were studied by PCR, ELISA, and gelatin zymography. bGH mesangial cell alpha 1 collagen IV mRNA levels were increased 2.3-fold (0.47 +/- 0.25 versus 0.20 +/- 0.09 attomoles/500 cells, p < 0.01) whereas secreted collagen IV and collagen IV in the cell lysates were increased 1.4-fold (25.1 +/- 5 versus 17.2 +/- 4 ng/ml/10(5) cells) and 1.8-fold (30.5 +/- 3 versus 16.7 +/- 3 ng/ml/10(5), p < 0.05), respectively. There were no differences in collagen I mRNA levels or in the protein content of either the media or cell lysates. We were not able to detect metalloproteinase 9 (MMP-9) mRNA expression or MMP-9 protein in bGH mesangial cell medium, whereas both mRNA and protein were present in controls. MMP-2 mRNA and enzyme activity in bGH cells were, however, elevated 1.5-fold (p < 0.05) and 2.1-fold (p = 0.05) over controls. Transforming growth factor beta 1 mRNA in bGH cells was 1.6-fold higher than that of controls (p < 0.05). The data suggest that (a) mesangial lesions may result from stable, genetically induced, phenotypic changes in mesangial cells, and (b) alterations of MMP-9 and collagen IV expression by mesangial cells may contribute to an imbalance between extracellular matrix synthesis and degradation and play a critical role in the genesis of glomerulosclerosis.