Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 microg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.
BackgroundConnective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.ResultsIntraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.ConclusionCooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.
Angiotensin II upregulates tumor necrosis factor-alpha (TNF-alpha) in the rat kidney with unilateral ureteral obstruction (UUO). In a mouse model of UUO, we found that tubulointerstitial fibrosis is blunted when the TNF-alpha receptor, TNFR1, is functionally knocked out. In this study, we used mutant mice with UUO in which the angiotensin II receptor AT(1a) or the TNF-alpha receptors TNFR1 and TNFR2 were knocked out to elucidate interactions between the two systems. The contribution of both systems to renal fibrosis was assessed by treating TNFR1/TNFR2-double knockout (KO) mice with an angiotensin-converting enzyme inhibitor, enalapril. The increased interstitial volume (Vv(int)) in the C57BI/6 wild-type mouse was decreased in the AT(1a) KO from 32.8 +/- 4.0 to 21.0 +/- 3.7% (P < 0.005) or in the TNFR1/TNFR2 KO to 22.3 +/- 2.1% (P < 0.005). The Vv(int) of the TNFR1/TNFR2 KO was further decreased to 15.2 +/- 3.7% (P < 0.01) by enalapril compared with no treatment. The induction of TNF-alpha mRNA and transforming growth factor-beta1 (TGF-beta1) mRNA in the kidney with UUO was significantly blunted in the AT(1a) or TNFR1/TNFR2 KO mice compared with the wild-type mice. Treatment of the TNFR1/TNFR2 KO mouse with enalapril reduced both TNF-alpha and TGF-beta1 mRNA and their proteins to near normal levels. Also, alpha-smooth muscle actin expression and myofibroblast proliferation were significantly inhibited in the AT(1a) or TNFR1/TNFR2 KO mice, and they were further inhibited in enalapril-treated TNFR1/TNFR2 KO mice. Incapacitating the angiotensin II or the TNF-alpha systems individually leads to partial blunting of fibrosis. Incapacitating both systems, by using a combination of genetic and pharmacological means, further inhibited interstitial fibrosis and tubule atrophy in obstructive nephropathy.
Unilateral ureteral obstruction (UUO) results in tubulointerstitial fibrosis of the obstructed kidney. In this study, we report the contribution of tumor necrosis factor-alpha (TNF-alpha) to the fibrosis that develops after ureteral obstruction. Mice in which individual TNF-alpha receptors TNFR1 or TNFR2 had been genetically knocked out were used, and results were compared with mice of C57Bl/6 background after 5 days UUO. Both kidneys were removed and examined histologically for changes in interstitial volume (Vv(int)), collagen IV deposition, alpha-smooth muscle actin (alpha-SMA) matrix score, nuclear factor-kappaB (NF-kappaB) activity, and TNF-alpha mRNA levels. We found that the Vv(int) of contralateral unobstructed kidneys averaged approximately 7% and was indistinguishable among the three genotypes of mice. Vv(int) of ureteral obstructed kidney of C57Bl/6 mice averaged 33 +/- 3.9% after 5 days of UUO. Vv(int) of obstructed kidneys of TNFR1 mice was significantly reduced to 19.4 +/- 3.1%, whereas that of TNFR2 mice was significantly decreased to 25.4% +/- 4.8%. There was a modest but significant difference between Vv(int) of TNFR1 and TNFR2 (P < 0. 047). Both collagen IV and alpha-SMA matrix scores were decreased significantly in obstructed kidney of TNFR1 mouse compared with that of C57Bl/6 and TNFR2 mice. Nuclear extracts prepared from kidney cortex were found to have a significant increase in NF-kappaB binding activity in obstructed kidney compared with contralateral kidney. Individual knockout of the TNFR1 or TNFR2 genes resulted in significantly less NF-kappaB activation compared with the wild type, with TNFR1 being less than TNFR2 knockout. There was a significant increase in TNF-alpha mRNA in the kidney with ureteral obstruction in all three genotypes. TNFR1 knockout displayed a significant reduction in amount of TNF-alpha mRNA induced compared with wild-type or TNFR2 knockout mice. Treatment of TNFR1 knockout mice with an angiotensin converting enzyme inhibitor further decreased Vv(int) and TNF-alpha mRNA induction, suggesting an interaction of ANG II and TNF-alpha systems. These results suggest that TNF-alpha contributes, in part, to changes in interstitial volume, myofibroblast differentiation, and NF-kappaB activation in the kidney during ureteral obstruction. These changes appear to be mediated through both TNFR1 and TNFR2 gene products with effects through the TNFR1 receptor predominating. Furthermore, ANG II appears to stimulate TNF-alpha pathophysiological events leading to renal fibrosis.
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