ABSTRACT
AIM: Oxidative stress plays an important role in the progression of renal interstitial fibrosis. The nicotinamide adeninedinucleotide phosphate (NADPH) oxidase (Nox) family is considered one of the major sources of reactive oxygen species (ROS). In the present study, we investigated the inhibitory effects of a novel anti-fibrotic agent, Fluorofenidone (AKF-PD), upon Nox-mediated oxidative stress and deposition of extracellular matrix (ECM) in the development of renalinterstitial fibrosis. METHODS: AKF-PD was used to treat renal fibrosis in unilateral ureteral obstruction (UUO) obstructive nephropathy in rats. The expression of Nox homologues, p-Akt, collagen I and III were detected by immunoblotting or immunohistochemistry. Levels of 8-iso prostaglandin F2alpha (8-Iso PGF2a) was measured by enzyme linked immunosorbent assay. In addition, ROS and the expression of collagen I (1a), Nox subunits and p-Akt was measured in angiotensin (Ang) II-stimulated rat proximal tubular epithelial (NRK-52E) cells in culture. RESULTS: AKF-PD treatment significantly attenuated tubulo-interstitial injury, ECM deposition and oxidative stress in fibrotic rat kidneys. In addition, AKF-PD inhibited the expression of ROS, Collagen I (1a), Nox2, p-Akt in Ang II-stimulated NRK-52E cells. CONCLUSION: AKF-PD attenuates the progression of renal interstitial fibrosis partly by suppressing NADPH oxidase and ECM deposition via the PI3K/Akt signalling pathway, suggesting AKF-PD is a potential novel therapeutic agent against renal fibrosis.
Subject(s)
Antioxidants/pharmacology , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Enzyme Inhibitors/pharmacology , Kidney Diseases/prevention & control , Kidney Tubules/drug effects , NADPH Oxidases/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Pyridones/pharmacology , Signal Transduction/drug effects , Angiotensin II/pharmacology , Animals , Cell Line , Class Ia Phosphatidylinositol 3-Kinase/genetics , Collagen Type I/metabolism , Dinoprost/analogs & derivatives , Dinoprost/metabolism , Disease Models, Animal , Fibrosis , Kidney Diseases/enzymology , Kidney Diseases/etiology , Kidney Diseases/pathology , Kidney Tubules/enzymology , Kidney Tubules/pathology , Lipid Peroxidation/drug effects , Losartan/pharmacology , Male , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Oxidative Stress/drug effects , Phosphorylation , Rats , Rats, Sprague-Dawley , Transfection , Ureteral Obstruction/complicationsABSTRACT
Human stem cell factor(hSCF)is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5'flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5x end deletions of hSCF gene. This study indicates that the region of -1190 -853 significantly enhanced the luc gene expression, while the region of -339 -162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both -1190 -853 and -339 -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 -853 and -339 -273 DNA fragments of the hSCF 5'flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.
