Connective tissue growth factor in tubulointerstitial injury of diabetic nephropathy.

BACKGROUND: Chronic interstitial fibrosis, which follows the onset of glomerular proteinuria, importantly contributes to progressive renal failure in diabetic nephropathy. The present studies examine the potential role of tubular connective tissue growth factor (CTGF). METHODS: The expression of CTGF was examined in rats with diabetic nephropathy. Regulation and actions of CTGF were studied in in vitro cell culture models. RESULTS: CTGF mRNA levels were increased in the renal cortex of rats with streptozotocin-induced diabetes compared with controls. Immunohistology indicated that CTGF was expressed in renal cortex of diabetic rats, in contrast to controls in some tubular cross-sections, particularly dilated-appearing proximal tubules, in which it tended to colocalize with insulin-like growth factor-I (IGF-I). Glomerular ultrafiltrate from diabetic rats, which contained bioactive transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), induced increased CTGF expression in tubular cells. TGF-beta1 and, to a lesser extent, HGF also raised CTGF expression in cultured proximal tubular cells. In contrast, high glucose (25 mmol/L) did not increase the secretion of CTGF. In cultured tubular cells, rhCTGF moderately increased fibronectin but not collagen (Col) type I and type III expression. In NRK-49F renal interstitial fibroblasts, CTGF raised Col alpha1III and thrombospondin-1 levels. CTGF has an IGF-binding domain and binds to IGF-I. In NRK-49F cells, IGF-I increased the activity of CTGF towards the expression of Col alpha1III. CONCLUSIONS: CTGF is expressed and regulated downstream from TGF-beta and HGF in proximal tubular cells, is induced by diabetic rat glomerular ultrafiltrate, and has moderate profibrogenic activity in tubular cells and renal interstitial fibroblasts, where its activity is IGF-I dependent. By these means, CTGF may act downstream of TGF-beta and HGF and may contribute to chronic tubulointerstitial fibrosis in diabetic nephropathy.

In vitro evidence for differential involvement of CTGF, TGFbeta, and PDGF-BB in mesangial response to injury.

BACKGROUND: Connective tissue growth factor (CTGF) is a profibrotic growth factor, which is upregulated in wound healing and renal fibrosis, including anti-Thy-1.1 nephritis. The kinetics of CTGF mRNA expression in anti-Thy-1.1 nephritis suggested that CTGF regulation might contribute to glomerular response to injury downstream of transforming growth factor-beta (TGFbeta). In anti-Thy-1.1 nephritis the initial damage is followed by mesangial repair and limited sclerosis, which involves mesangial cell (MC) activation (alpha-smooth-muscle actin (alphaSMA) expression), proliferation, migration, and extracellular matrix production. The present in vitro study addresses the possible role of CTGF in these different aspects of mesangial response to injury, and how CTGF activity might relate to effects of TGFbeta and platelet-derived growth factor-BB (PDGF-BB). METHODS AND RESULTS: Immunostaining and ELISA showed that alphaSMA expression and transformation of MC into myofibroblast-like cells was induced by TGFbeta, but not affected by PDGF-BB, CTGF, or neutralizing anti-CTGF antibodies. [(3)H]thymidine incorporation and Ki67 staining demonstrated that, unlike PDGF-BB, neither CTGF nor TGFbeta induced the proliferation of MC. In contrast, both CTGF and TGFbeta induced MC migration, as evidenced by approximation of wound edges in scrape-wounded, non-proliferating rat MC monolayers. In addition, fibronectin expression was upregulated by both CTGF and TGFbeta, as measured by dot-blot analysis. Anti-CTGF completely blocked the effect of added CTGF. Moreover, anti-CTGF significantly reduced TGFbeta-induced increase in fibronectin. CONCLUSION: It thus appears that CTGF is specifically involved in a subset of the adaptive changes of MC involved in mesangial repair and sclerosis, which makes it an interesting candidate target for future intervention strategies.

Granulocyte-macrophage colony-stimulating factor is a stimulant of platelet-activating factor and superoxide anion generation by human neutrophils.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied for its ability to stimulate the synthesis and release of the inflammatory mediator platelet-activating factor (PAF) from human neutrophils as measured by bioassay and incorporation of [3H]acetate into PAF. GM-CSF stimulated the synthesis but not the release of PAF from neutrophils. PAF synthesis took place in a time- and concentration-dependent manner, was dependent on a pertussis toxin-sensitive G protein and could be inhibited by antibodies to GM-CSF. On the other hand, pre-incubation of neutrophils with GM-CSF followed by stimulation with the bacterial tripeptide formylmethionylleucylphenylalanine caused PAF synthesis and release. The effect of GM-CSF was qualitative and not simply the result of larger amounts of PAF being synthesized since similar amounts were generated in response to the calcium ionophore A23187 but no released PAF could be detected. In functional studies GM-CSF stimulated superoxide anion generation from neutrophils with a time and dose relationship that paralleled PAF synthesis. In addition, the serine protease inhibitor L-1-tosylamide-2-phenylethyl chloromethyl ketone, which inhibits PAF synthesis, reduced PAF accumulation as well as superoxide generation, raising the possibility of a causal relationship between cell-associated PAF and cell activation. These results identify PAF as a direct product of GM-CSF stimulation in neutrophils where it may play a role in signal transduction and demonstrate that PAF is released only after subsequent neutrophil stimulation. The selective release of PAF may play a role in regulating and amplifying the inflammatory response.