A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.
ABSTRACT: The expression of chemokines and their receptors is thought to contribute to leukocyte infiltration and progressive renal fibrosis after unilateral ureter obstruction (UUO). We hypothesized that blocking the chemokine receptor CCR1 using the nonpeptide antagonist BX471 could reduce leukocyte infiltration and renal fibrosis after UUO. UUO kidneys from BX471-treated mice (day 0-10 and day 6-10) revealed a 40-60% reduction of interstitial macrophage and lymphocyte infiltrate compared with controls. Treated mice also showed a marked reduction of CCR1 and CCR5 mRNA levels, and FACS analysis showed a comparable reduction of CD8+/CCR5+ T cells. Markers of renal fibrosis, such as interstitial fibroblasts, interstitial volume, mRNA and protein expression for collagen I, were all significantly reduced by BX471-treatment compared with vehicle controls. By contrast treatment was ineffective when the drug was supplied only from days 0 to 5. In summary, blockade of CCR1 substantially reduces cell accumulation and renal fibrosis after UUO. Most interestingly, late onset of treatment is also effective. We therefore conclude that CCR1 blockade may represent a new therapeutic strategy for reducing cellular infiltration and renal fibrosis as major factors in the progression to end-stage renal failure.
Project description:Interstitial fibrosis plays a major role in progression of renal diseases. Oncostatin M (OSM) is a cytokine that regulates cell survival, differentiation, and proliferation. Renal tissue from patients with chronic obstructive nephropathy was examined for OSM expression. The elevated levels in diseased human kidneys suggested possible correlation between OSM level and kidney tissue fibrosis. Indeed, unilateral ureteral obstruction (UUO), a model of renal fibrosis, increased OSM and OSM receptor (OSM-R) expression in a time-dependent manner within hours following UUO. In vitro, OSM overexpression in tubular epithelial cells (TECs) resulted in epithelial-myofibroblast transdifferentiation. cDNA microarray technology identified up-regulated expression of immune modulators in obstructed compared with sham-operated kidneys. In vitro, OSM treatment up-regulated CC chemokine ligand CCL7, and CXC chemokine ligand (CXCL)-14 mRNA in kidney fibroblasts. In vivo, treatment of UUO mice with neutralizing anti-OSM antibody decreased renal chemokines expression. In conclusion, OSM is up-regulated in kidney tissue early after urinary obstruction. Therefore, OSM might play an important role in initiation of renal fibrogenesis, possibly by inducing myofibroblast transdifferentiation of TECs as well as leukocyte infiltration. This process may, in turn, contribute in part to progression of obstructive nephropathy and makes OSM a promising therapeutic target in renal fibrosis.
Project description:Renal interstitial fibrosis is a common pathological feature of chronic kidney disease that may involve changes of metabolism in kidney cells. In the present study, we first showed that blockade of glycolysis with either dichloroacetate (DCA) or shikonin to target different glycolytic enzymes reduced renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). Both inhibitors evidently suppressed the induction of fibronectin and collagen type I in obstructed kidneys, with DCA also showing inhibitory effects on collagen type IV and ?-smooth muscle actin (?-SMA). Histological examination also confirmed less collagen deposition in DCA-treated kidneys. Both DCA and shikonin significantly inhibited renal tubular apoptosis but not interstitial apoptosis in UUO. Macrophage infiltration after UUO injury was also suppressed. Shikonin, but not DCA, caused obvious animal weight loss during UUO. To determine whether shikonin and DCA worked on tubular cells and/or fibroblasts, we tested their effects on cultured renal proximal tubular BUMPT cells and renal NRK-49F fibroblasts during hypoxia or transforming growth factor-?1 treatment. Although both inhibitors reduced fibronectin and ?-SMA production in NRK-49F cells during hypoxia or transforming growth factor-?1 treatment, they did not suppress fibronectin and ?-SMA expression in BUMPT cells. Altogether, these results demonstrate the inhibitory effect of glycolysis inhibitors on renal interstitial fibrosis. In this regard, DCA is more potent for fibrosis inhibition and less toxic to animals than shikonin.
Project description:Renal fibrosis is the final, common pathway of end-stage renal disease. Whether and how autophagy contributes to renal fibrosis remains unclear. Here we first detected persistent autophagy in kidney proximal tubules in the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice. UUO-associated fibrosis was suppressed by pharmacological inhibitors of autophagy and also by kidney proximal tubule-specific knockout of autophagy-related 7 (PT-Atg7 KO). Consistently, proliferation and activation of fibroblasts, as indicated by the expression of ACTA2/?-smooth muscle actin and VIM (vimentin), was inhibited in PT-Atg7 KO mice, so was the accumulation of extracellular matrix components including FN1 (fibronectin 1) and collagen fibrils. Tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration were all inhibited in these mice. Moreover, these mice showed a specific suppression of the expression of a profibrotic factor FGF2 (fibroblast growth factor 2). In vitro, TGFB1 (transforming growth factor ? 1) induced autophagy, apoptosis, and FN1 accumulation in primary proximal tubular cells. Inhibition of autophagy suppressed FN1 accumulation and apoptosis, while enhancement of autophagy increased TGFB1-induced-cell death. These results suggest that persistent activation of autophagy in kidney proximal tubules promotes renal interstitial fibrosis during UUO. The profibrotic function of autophagy is related to the regulation on tubular cell death, interstitial inflammation, and the production of profibrotic factors.
Project description:Objective and Design:Allergic rhinitis (AR) is an immunoglobulin E (IgE)-mediated inflammatory respiratory hypersensitivity characterized by elevated Th2 cytokines and infiltration of inflammatory cells to nasal tissues. BX471 is a small-molecule C-C chemokine receptor type 1 (CCR1) antagonist involved in suppression of inflammation via blocking of primary ligands. In this study, we examined the anti-inflammatory effect of BX471 on ovalbumin (OVA)-induced AR mice model. Materials and Methods:Levels of OVA-specific IgE and Th1 cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Nasal expression of proinflammatory mediators was assessed by real-time polymerase chain reaction (RT-qPCR). Nasal-cavity sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to study eosinophil infiltration and goblet cell metaplasia. Relative protein levels of Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB), Toll-like Receptor 4 (TLR4) and Toll-like-receptor 2 (TLR2) were assessed by Western Blot. Percentage of CD4+CD25+Foxp3+ T regulatory cells (Treg) was measured by flow cytometry. Results:Mice treated with BX471 showed significantly relieved sneezing and nasal-rubbing behaviors. The expression of nasal proinflammatory factors was significantly downregulated by BX471, and protein levels of tumor necrosis factor alpha (TNF- ?) and NF-kB were suppressed. Blockade of CCR1 ligands inhibited eosinophil recruitment in nasal cavity. In addition, Treg cells population were upregulated in BX471-treated mice. Conclusion:BX471 exerts anti-inflammatory effects in a mouse model of AR by inhibiting CCR1-mediated TNF-? production, which subsequently suppresses NF-kB activation in inflammatory cells, leading to a decrease in Th2 cytokines, IL-1?, VCAM-1, GM-CSF, RANTES, and MIP-1? expression levels, thus inhibiting eosinophil recruitment to nasal mucosa. In addition, BX-471 exhibits anti-allergic effect by increasing Treg cell population. Overall, BX471 represents a promising therapeutic strategy against AR.
Project description:Hepatic fibrosis develops as a response to chronic liver injury and almost exclusively occurs in a proinflammatory environment. However, the role of inflammatory mediators in fibrogenic responses of the liver is only poorly understood. We therefore investigated the role of CC chemokines and their receptors in hepatic fibrogenesis. The CC chemokines MIP-1alpha, MIP-1beta, and RANTES and their receptors CCR1 and CCR5 were strongly upregulated in 2 experimental mouse models of fibrogenesis. Neutralization of CC chemokines by the broad-spectrum CC chemokine inhibitor 35k efficiently reduced hepatic fibrosis, and CCR1- and CCR5-deficient mice displayed substantially reduced hepatic fibrosis and macrophage infiltration. Analysis of fibrogenesis in CCR1- and CCR5-chimeric mice revealed that CCR1 mediates its profibrogenic effects in BM-derived cells, whereas CCR5 mediates its profibrogenic effects in resident liver cells. CCR5 promoted hepatic stellate cell (HSC) migration through a redox-sensitive, PI3K-dependent pathway. Both CCR5-deficient HSCs and CCR1- and CCR5-deficient Kupffer cells displayed strong suppression of CC chemokine-induced migration. Finally, we detected marked upregulation of RANTES, CCR1, and CCR5 in patients with hepatic cirrhosis, confirming activation of the CC chemokine system in human fibrogenesis. Our data therefore support a role for the CC chemokine system in hepatic fibrogenesis and suggest distinct roles for CCR1 and CCR5 in Kupffer cells and HSCs.
Project description:<h4>Background</h4>Congenital obstructive nephropathy is the main cause of end-stage renal disease in infants and children. Renal insufficiency is due to impaired growth and maturation in the developing kidney with obstruction. Congenital obstructive nephropathy leads to cytokine mediated inflammation and the development of interstitial fibrosis. The Janus kinase-2 (JAK-2) and Signal Transducer and Activator of Transcription'-3 (STAT3) are involved in cytokine production, inflammation, and interstitial fibrosis.<h4>Methods</h4>We studied the role of JAK2/STAT3 in a model of congenital obstructive nephropathy using unilateral ureteral obstruction (UUO) in neonatal mice at the second day of life. Cytokine production, inflammation, and interstitial fibrosis were analyzed in obstructed and sham operated kidneys of neonatal mice treated with or without JAK2/STAT3 inhibitor Tyrphostin AG490. To mimic obstruction and distension, proximal tubular cells were stretched in vitro.<h4>Results</h4>We show that STAT3 is highly activated in the developing kidney with obstruction and in proximal tubular cells following stretch. JAK2/STAT3 activation mediates cytokine release and leukocyte recruitment into neonatal kidneys after UUO. Pharmacological blockade of JAK2/STAT3 by Tyrphostin AG490 reduced inflammation, tubular apoptosis, and interstitial fibrosis. JAK2/STAT3 blockade decreased pro-inflammatory and profibrotic mediators in tubular cells.<h4>Conclusion</h4>Our findings provide evidence that JAK2/STAT3 mediates inflammation and fibrosis in the developing kidney with obstruction. Blocking JAK2/STAT3 may prove beneficial in congenital obstructive nephropathy in children.
Project description:The epithelial-to-mesenchymal transition (EMT) is one of mechanisms that induce renal interstitial fibrosis. Understanding EMT in renal fibrosis has important therapeutic implications for patients with kidney disease. Alpha-lipoic acid (ALA) is a natural compound with antioxidant properties. Studies for ALA are performed in acute kidney injury with renal tubular apoptosis, renal inflammation, and oxidative stress. We investigated the effects of ALA on EMT-mediated renal interstitial fibrosis in mice with unilateral ureteral obstruction (UUO). UUO mice developed severe tubular atrophy and tubulointerstitial fibrosis, with a robust EMT response and ECM deposition after 7 postoperative days. In contrast, ALA-treated UUO mice showed only moderate injury and minimal fibrosis and also larger reductions in the expression of ECM proteins, inflammatory factors, and EMT markers. ALA was shown to be involved in the suppression of infiltrating macrophages associated with EMT and the progression of interstitial fibrosis. It also lessened the destruction of the tubular basement membrane, by reducing the expression of matrix metalloproteinases. This is the first study to show that ALA modulates EMT in a UUO mouse model. Our results suggest that ALA merits further exploration as a therapeutic agent in the prevention and treatment of chronic kidney disease.
Project description:Renal interstitial fibrosis is an inevitable consequence of virtually every type of chronic kidney disease. The underlying mechanisms, however, are not completely understood. In the present study, we identified surfactant protein A (SP-A) as a novel protein factor involved in the renal fibrosis induced by unilateral ureter obstruction (UUO). UUO induced SP-A expression in mouse kidney epithelium, likely due to the increased acidic stress and inflammation. Interestingly, SP-A deficiency aggravated UUO-prompted kidney structural damage, macrophage accumulation, and tubulointerstitial fibrosis. SP-A deficiency appeared to worsen the fibrosis by enhancing interstitial myofibroblast accumulation. Moreover, SP-A deficiency increased the expression of TGF-?1, the major regulator of kidney fibrosis, particularly in the interstitial cells. Mechanistically, SP-A deficiency increased the expression and release of high mobility group box 1 (HMGB1), a factor regulating TGF-? expression/signaling and implicated in renal fibrosis. SP-A also blocked HMGB1 activities in inducing TGF-?1 expression and myofibroblast transdifferentiation from kidney fibroblasts, demonstrating that SP-A protected kidney by impeding both the expression and fibrogenic function of HMGB1. Since SP-A physically interacted with HMGB1 both in vitro and in kidney tissue in vivo, SP-A may exert its protective role by binding to HMGB1 and thus titrating its activity during UUO-induced renal fibrosis.
Project description:Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.
Project description:Farnesoid X receptor (FXR) is a nuclear receptor that has emerged as a key regulator in the maintenance of hepatic steatosis, inflammation, and fibrosis. However, the role of FXR in renal fibrosis remains to be established. Here, we investigate the effects of the FXR agonist EDP-305 in a mouse model of tubulointerstitial fibrosis <i>via</i> unilateral ureteral obstruction (UUO). Male C57Bl/6 mice received a UUO on their left kidney. On postoperative d 4, mice received daily treatment by oral gavage with either vehicle control (0.5% methylcellulose) or 10 or 30 mg/kg EDP-305. All animals were euthanized on postoperative d 12. EDP-305 dose-dependently decreased macrophage infiltration as measured by the F4/80 staining area and proinflammatory cytokine gene expression. EDP-305 also dose-dependently reduced interstitial fibrosis as assessed by morphometric quantification of the collagen proportional area and kidney hydroxyproline levels. Finally, yes-associated protein (YAP) activation, a major driver of fibrosis, increased after UUO injury and was diminished by EDP-305 treatment. Consistently, EDP-305 decreased TGF-β1-induced YAP nuclear localization in human kidney 2 cells by increasing inhibitory YAP phosphorylation. YAP inhibition may be a novel antifibrotic mechanism of FXR agonism, and EDP-305 could be used to treat renal fibrosis.-Li, S., Ghoshal, S., Sojoodi, M., Arora, G., Masia, R., Erstad, D. J., Ferriera, D. S., Li, Y., Wang, G., Lanuti, M., Caravan, P., Or, Y. S., Jiang, L.-J., Tanabe, K. K., Fuchs, B. C. The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.