Therapeutic Effects Of Fluorofenidone On Kidney Fibrosis In Db/Db Mice With Diabetic Nephropathy And Its Potential Mechanisms Of Action

BackgroundDiabetic nephropathy (DN) is the common and serious complication of diabetes that often progresses to end-stage renal disease (ESRD). Available therapies only slow but do not halt the progression of renal dysfunction in diabetic nephropathy in clinical settings. It is therefore necessary to develop new therapeutic agents that target major pathological mechanisms of diabetic nephropathy. The histological injury of diabetic nephropathy is mainly characterized by the progressive accumulation of extracellular matrix (ECM) components such as collagen and fibronectin in the glomerular mesangium and tubulointerstitium, which ultimately results in the irreversible fibrosis of kidneys. Considerable efforts are being made to explore therapeutic agents effectively inhibiting ECM accumulation to treat diabetic nephropathy, and promising results have been obtained in recent years. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD) is a novel low-molecular-weight pyridone agent. We recently reported that AKF-PD exerts a strong antifibrotic effect and inhibits TGF-β-stimulated production of ECM in mouse mesangial cells and rat proximal tubular epithelial cells in culture.ObjectiveThe present study was designed to evaluate the potential therapeutic efficacy of AKF-PD on kidney fibrosis and to obtain an insight into its underlying mechanisms of action in db/db mice with diabetic nephropathy and cultured murine mesangial-derived MES13cells exposed to high glucose, in order that this pyridone agent can be developed into a potent antifibrotic drug for the therapy of diabetic kidney disease. Method1. Male db/db mice and age-matched dblm mice were used for this study. Mice at10weeks of age were divided into five groups:a dblm mice group, a dbldb mice group, and three AKF-PD treatment groups in which dbldb mice were administered with AKF-PD (dissolved in CMCNa solution) at doses of125mg,500mg or1000mg·kg body weight-1·day-1respectively. The mice of dblm and dbldb groups were administered with CMCNa solution alone. The administration of AKF-PD or CMCNa solution was performed by gavage daily for a period of12weeks.Mice were individually housed in metabolic cages and the urine samples were collected over24hours. Levels of urinary albumin were quantified using a mouse albumin ELISA Kit.Mice were anesthetized with ether, and then blood samples were obtained from the tail vein. Levels of blood glucose were determined with an Automatic Analyzer.The ether-anesthetized mice were sacrificed at the end of12-wk treatment. The kidneys were rapidly dissected out, weighed and separate the cortices. Sections of renal tissues were prepared and stained with periodic acid-Schiff (PAS).20glomeruli were evaluated for each mouse. The degree of damage in each glomerulus was assessed using a semi-quantitative (grade0-4) scoring method. The glomerular matrix expansion index (GMI) was calculated according to the number of glomeruli in each grade of mesangial expansion.TGF-β1protein levels in homogenates of renal cortices were determined by using a TGF-β1ELISA kit.Total RNA was isolated from kidney tissues. The cDNA was synthesized from total RNA. RT-PCR primers for α1(I)collagen, α1(IV)collagen, fibronectin, TGF-(31, tissue inhibitors-1of metalloproteinases (TIMP-1), α-smooth muscle actin (a-SMA) and β-actin were ordered from Sangon Inc. Amplification reactions were performed in duplicate with SYBR Green PCR reagent kit. The relative abundance of mRNA was standardized with β-actin mRNA as the invariant control.The lysates of kidney cortex tissues were subjected to SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Following the blocking step, the electroblotted membranes were immunoblotted with the primary antibodies against a-SMA, TIMP1, a-tublin respectively. The membranes immunoblotted were incubated with horseradish peroxidase-labelled secondary antibodies. After washing, the membranes were exposed to X-ray film. The bands of resulting autoradiographs were quantified densitometrically using Bandscan software.2. The synchronized MES-13cells were assigned to six groups:(1) NG, medium containing normal glucose concentration (-5.6mmol/L);(2) HG, medium containing25mmol/L glucose;(3) AKF-PD, medium containing HG and400μg/mL AKF-PD;(4) PFD, medium containing HG and400μg/mL pirfenidone;(5) Los, medium containing HG and2μmol/L losartan;(6) OC, medium containing NG and19.4mmol/L mannitol as an osmotic control. The cells in all six groups were cultured for72h, and then cells and culture supernatants were collected for RT-PCR and Western blotting.TGF-β1protein levels in supernatants from MES-13cell cultures were determined by using a TGF-β1ELISA kit.Total RNA was isolated from MES-13cells. The cDNA was synthesized from total RNA. RT-PCR primers for α1(Ⅰ)collagen, al(IV)collagen, fibronectin, TGF-β1, tissue inhibitors-1of metalloproteinases (TIMP-1), cc-smooth muscle actin (a-SMA) and β-actin were ordered from Sangon Inc. Amplification reactions were performed in duplicate with SYBR Green PCR reagent kit. The relative abundance of mRNA was standardized with β-actin mRNA as the invariant control.The lysates of MES-13cells were subjected to SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Following the blocking step, the electroblotted membranes were immunoblotted with the primary antibodies against a-SMA, TIMP1, a-tublin respectively. The membranes immunoblotted were incubated with horseradish peroxidase-labelled secondary antibodies. After washing, the membranes were exposed to X-ray film. The bands of resulting autoradiographs were quantified densitometrically using Bandscan software.ResultsKidney weights of diabetic db/db mice were markedly greater than those of dblm mice(P<0.05). Treatment with AKF-PD reduced the kidney hypertrophy of diabetic dbldb mice, as indicated by decreased kidney weight and kidney/body weight ratio in treated dbldb mice compared with untreated dbldb mice. The diminished effect of AKF-PD on the renal hypertrophy was most obvious at a dosage of500mg/kg body weight per day(P<0.01).Diabetic db/db mice had heavy albuminuria. The urinary albumin excretion rate was higher by8-fold in dbldb mice than that in dblm mice group(P<0.01). The administration of AKF-PD significantly attenuated albuminuria of diabetic db/db mice and a dose of500mg/kg body weight was the peak-effective dose at which24-h urinary albumin excretion declined by58.5percent(P<0.01).The dbldb mice remained hyperglycemic throughout the experimental period, and their mean blood glucose levels were about5times as high as in dblm mice(P<0.01). No significant differences of blood glucose were observed between AKF-PD treated and untreated dbldb mice groups.In comparison with dblm mice, PAS-positive mesangial matrix areas were substantially enlarged and the glomerular mesangial expansion index (GMI) i.e., the ratio of mesangial matrix area divided by the tuft area, was significantly increased in dbldb mice(P<0.01). Compared with untreated dbldb mice group, the PAS-positive mesangial matrix areas and GMIs decreased obviously in AKF-PD-treated dbldb mice groups(P<0.05) and GMI was reduced by approximately55.8%at the dose of500mg/kg body weight(P<0.01).The mRNA levels of α1(Ⅰ)collagen, α1(Ⅳ)collagen and fibronectin in renal cortex of db/db mice were3-4times higher than those in dblm mice(P<0.01). The up-regulated expressions of the three ECM components at the transcriptional level were dose-dependently reduced in renal cortex of diabetic dbldb mice subjected to AKF-PD treatment(P<0.05).Diabetic db/db mice had the marked upregulation of expression TGF-β1mRNA and protein in renal cortex compared with dblm mice(P<0.01). AKF-PD treatment reduced significantly the enhanced expression of TGF-(31mRNA and protein in dbldb mice(P<0.05).Compared with dblm mice, expression of renal cortex TIMP-1was markedly upregulated in diabetic dbldb mice as indicated by real-time RT-PCR and western blotting(P<0.01). The upregulated renal expressions of TIMP-1mRNA and protein in dbldb mice were significantly inhibited by AKF-PD treatment(P<0.01).The mRNA and protein expressions of a-SMA in renal cortex of diabetic dbldb mice were markedly upregulated compared with dblm mice(P<0.01). AKF-PD administration to dbldb mice inhibited dramatically the upregulated expressions of a-SMA mRNA and protein in renal cortex(P<0.05), and the maximally effective doses were about500mg/kg body weight per day.The expression of α1(Ⅰ) and α1(Ⅳ) collagen mRNAs in HG group of MES-13cells was obviously increased compared with NG group(P<0.01). The addition of AKF-PD, PFD or losartan to culture media inhibited significantly the high glucose-induced upregulation of α1(Ⅰ) and α1(Ⅳ) collagen mRNA expression in mesangial cells(P<0.05), and there was no significant difference among the three agents.Each of AKF-PD, PFD and losartan can inhibit significantly the up-regulated expression of TGF-β1mRNA induced by high glucose in MES-13cells in vitro(P<0.01), and differences between the three agents were not statistically significant. AKF-PD, PFD and losartan also inhibit significantly the high glucose-stimulated upregulation of TGF-β1protein expression in the cultured mesangial cells(P<0.05). However, both AKF-PD and PFD can reverse the expression of TGF-β1protein approximately to the level of NG group, but this inhibitory effect of losartan seemed to be slightly weaker.Both mRNA and protein expression of TIMP-1by cultured mesangial cells were remarkably increased in HG group compared with the NG group (P<0.01). The levels of TIMP-1mRNA and protein in the AKF-PD-, PFD-and losartan-treatment groups were obviously lower than those in HG group(P<0.01). No significant difference in TIMP-1was observed among these three treatment groups.In comparison with NG group, the expression level of a-SMA protein in HG group was significantly higher(P<0.01). The addition of AKF-PD, PFD or losartan to the culture media significantly inhibited the expression of a-SMA protein by cultured mesangial cells under high glucose conditions(P<0.05). The differences in a-SMA protein expression among these three agents were not significant. ConclusionThis study demonstrates that long-term administration of AKF-PD attenuates renal hypertrophy, mesangial matrix expansion and albuminuria in the diabetic db/db mice despite continued hyperglycemia. Upregulated expressions of several ECM components, TIMP-1, TGF-β1and a-SMA at the transcriptional and protein levels are significantly inhibited by AKF-PD treatment in renal cortex of db/db mice and cultured mesangial cells under high glucose conditions. The results presented here suggest that AKF-PD is able to ameliorate the ECM production and degradation imbalance via the inhibition of TGF-β1pathway, resulting in attenuation of renal fibrosis associated with diabetic state. Therefore, use of AKF-PD as a potent antifibrotic agent holds great promise for the therapy of diabetic nephropathy.