ha-1100 and Fibrosis

Renal fibrosis is the major cause of chronic kidney disease, and the Rho/Rho-associated coiled-coil kinase (ROCK) signaling cascade is involved in the renal fibrotic processes. Several studies have reported that ROCK inhibitors attenuate renal fibrosis. However, the mechanism of this process remains to be fully elucidated. The present study assessed the inhibitory effect of fasudil, a ROCK inhibitor using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blot analyses, in vivo and in vitro, to elucidate the mechanisms underlying renal interstitial fibrosis. In mice induced with unilateral ureteral obstruction (UUO), collagen accumulation, the expression of fibrosis‑associated genes and the content of hydroxyproline in the kidney increased 3, 7, and 14 days following UUO. Fasudil attenuated the histological changes, and the production of collagen and extracellular matrix in the UUO kidney. The expression of α‑smooth muscle actin (α‑SMA) and the transforming growth factor‑β (TGFβ)‑Smad signaling pathway, and macrophage infiltration were suppressed by fasudil in the kidneys of the UUO mice. The present study also evaluated the role of intrinsic renal cells and infiltrated macrophages using NRK‑52E, NRK‑49F and RAW264.7 cells. The mRNA and protein expression levels of collagen I and α‑SMA increased in the NRK‑52E and NRK‑49F cells stimulated by TGF‑β1. Hydroxyfasudil, a bioactive metabolite of fasudil, attenuated the increase in the mRNA and protein expression levles of α‑SMA in the two cell types. However, the reduction in the mRNA expression of collagen I was observed in the NRK‑49F cells only. Hydroxyfasudil decreased the mRNA expression of monocyte chemoattractant protein‑1 (MCP‑1) induced by TGF‑β1 in the NRK‑52E cells, but not in the NRK‑49F cells. In the RAW264.7 cells, the mRNA expression levels of MCP‑1, interleukin (IL)‑1β, IL‑6 and tumor necrosis factor α were increased significantly following lipopolysaccharide stimulation, and were not suppressed by hydroxyfasudil. These data suggested that the inhibition of ROCK activity by fasudil suppressed the transformation of renal intrinsic cells into the myofibroblast cells, and attenuated the infiltration of macrophages, without inhibiting the expression or the activation of cytokine/chemokines, in the progression of renal interstitial fibrosis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Actins; Animals; Cell Line; Cell Movement; Collagen Type I; Disease Models, Animal; Fibrosis; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Protein Kinase Inhibitors; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Up-Regulation; Ureteral Obstruction

Tubulointerstitial fibrosis in diabetic nephropathy (DN) was investigated using an in vitro tissue model of remodeling, to determine the pathogenic mechanism of fibrosis that leads to renal atrophy, i.e., renal failure. The remodeling model consisted of a renal fibroblast-populated collagen lattice (FPCL). The overexpression of transforming growth factor (TGF)-beta1 in the diabetic kidney gave rise to FPCL contraction. FPCL relaxation was induced by the subsequent addition of cytochalasin D. The FPCL failed to contract when exposed to TGF-beta1 plus Y27632, a Rho kinase inhibitor. TGF-beta1 induced the phosphorylation of myosin light chains, and Y27632 blocked this activity. TGF-beta1-induced FPCL contraction was suppressed by the addition of 2,3-butanedione monoxime, a myosin ATPase inhibitor. As shown in the video, the contraction rate of the projections of the cells in the FPCL was significantly greater in the TGF-beta1 group than in the control group. Collectively, these results indicate that TGF-beta1-induced FPCL contraction is attributable to actin-myosin interactions in the fibroblasts through the activation of Rho kinase, the phosphorylation of myosin light chains, and the subsequent activation of myosin ATPase. We propose that via these mechanisms, tubulointerstitial fibrosis generates tissue contraction that leads to renal atrophy and renal failure in DN.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Animals; Cells, Cultured; Collagen; Cytochalasin D; Diabetic Nephropathies; Diacetyl; Fibroblasts; Fibrosis; Kidney Tubules; Microscopy, Electron, Transmission; Models, Biological; Myosin Light Chains; Phosphorylation; Pyridines; Rats; rho-Associated Kinases; Transforming Growth Factor beta1; Wound Healing

Rho-kinase has been implicated as an important regulator of inflammatory responses mediated by cytokines and chemokines. Because proinflammatory cytokines play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), we examined whether long-term blockade of Rho-kinase suppresses LV remodeling in a mouse model of MI in vivo.. Mice underwent ligation of the left coronary artery and were treated with a Rho-kinase inhibitor, fasudil (100 mg x kg(-1) x d(-1) in tap water), for 4 weeks, starting 1 day after the surgery. At 4 weeks, LV infarct size was histologically comparable between the 2 groups. LV cavity dilatation and dysfunction evaluated by echocardiography were significantly suppressed in the fasudil group (P<0.05, n=15 to 28). The beneficial effects of fasudil were accompanied by suppression of cardiomyocyte hypertrophy and interstitial fibrosis (both P<0.01, n=6). The expression of inflammatory cytokines, including transforming growth factor (TGF)-beta2, TGF-beta3, and macrophage migration inhibitory factor, was upregulated in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (both P<0.05, n=10 to 11). Rho-kinase activity as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, was significantly increased in the noninfarcted LV in the control group and was significantly suppressed in the fasudil group (P<0.05, n=5).. These results indicate that Rho-kinase is substantially involved in the pathogenesis of LV remodeling after MI associated with upregulation of proinflammatory cytokines, suggesting a therapeutic importance of the molecule for the prevention of post-MI heart failure.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; DNA-Binding Proteins; Drug Administration Schedule; Enzyme Inhibitors; Fibrosis; Gene Expression Regulation; Hypertrophy, Left Ventricular; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Male; Mice; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Organ Size; Phosphorylation; Protein Processing, Post-Translational; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Ultrasonography; Ventricular Dysfunction, Left; Ventricular Remodeling

This study was designed to investigate possible effects of the Rho-kinase inhibitor, fasudil, on the progression of renal failure in rats with unilateral ureteral obstruction. The renal failure markers monitored were the extent of renal interstitial fibrosis and that of macrophage infiltration. In kidneys with unilateral ureteral obstruction, interstitial fibrosis was observed, using Sirius-Red staining, on day 16 after unilateral ureteral obstruction. Macrophage infiltration was observed by immunohistochemistry, using the antibody, ED1. Interstitial fibrosis and macrophage infiltration were significantly attenuated in fasudil-treated animals. The migration of monocytes in vitro elicited by N-formyl-methionyl-leucyl-phenylalanine was potently inhibited by fasudil and its active metabolite, hydroxyfasudil. These results suggest that inhibition of Rho-kinase produces a reduction of macrophage infiltration and represents a new therapeutic strategy for renal fibrosis, a major factor in the progression to end-stage renal failure.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Chemotaxis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibrosis; Kidney; Macrophages; Male; Monocytes; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Ureteral Obstruction