The Effect Of Fluorofenidone On Inflammation In Renal Fibrosis

Part 1 The effect of Fluorofenidone on inflammation in renal fibrosisBackground:Renal fibrosis is the common pathway and main pathogenic basis of end-stage of all kinds of renal disease. Moreover, renal fibrosis strongly correlates with progressive loss of renal function. Therefore, development of novel antifibrotic agent is of great importance. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD) is a novel pyridone agent which is developed recently. Our data showed that AKF-PD exerts a strong antifibrotic effect, as shown in experimental renal fibrosis in vivo and in vitro. However, the mechanism underlying the antifibrotic effect of AKF-PD in renal disease remains to be elucidated. Our recent results showed that AKF-PD significantly reduced the expression and activity of NADPH oxidase, and inhibited ROS generation, the apoptosis of renal tubular epithelial cell, as well as the proliferation of renal fibroblast. In addition, AKF-PD significantly downregulated the expression of pro-fibrotic cytokines, and consequent reduced interstitial matrix deposition. In recent year, inflammation is reported to be essential for the development of renal fibrosis. And The effect of Fluorofenidone on inflammation in renal fibrosis is still unkown.At the stage of renal inflammation, the loss of nephrons is relatively rare. And there is no proliferation of fibroblast cells. Effective repair can restore the impaired cells of normal function; therefore prevent the progress of nephritis. The current concepts of inflammation in renal fibrosis are following:(1) Inflammation is the early event in kidney upon any kind of harmful stimulation (The expression of TNF-a and infiltration of Macrophages can be observed in impaired kidney 4 hours after UUO); the persistence of renal inflammation can induce renal fibrosis finally. (2) The infiltration of inflammatory cells plays an important role in the process of chronic renal inflammation and renal fibrosis. (3) Chemokine and proinflammatory factor is the key factor, which mediates the infiltration of inflammatory cells and maintains the persistent state of chronic inflammation during the early stage of renal fibrosis.Focusing the pathology of renal fibrosis, scholars investigated extensively on the anti-fibrogenic drugs. PFD is a new broad-spectrum anti-fibrogenic compound, which can prevent and even reverse the renal fibrosis. However, the mechanism underlying its anti-fibrogenic effect is yet to be found. And whether PFD has an effect on inflammation of early stage of renal fibrosis is unknown. Angiotensin II is involved in promoting infiltration of inflammatory cells in injured kidney. And it is reported that angiotensin-converting enzyme inhibitor (ACEI) as well as angiotensin receptor blockade (ARB) can ameliorate inflammatory response in tissue of injured kidney.Objective:To investigate whether AKF-PD can ameliorate the infiltration of inflammatory cells and the expression of chemokine and proinflammatory factor after ureteral obstruction; to investigate the effect of AKF-PD on renal inflammation, in order to get the better understandings of the mechanism underlying the anti-fibrogenic effect of AKF-PD.Methods:(1) SD rats were submitted to unilateral ureteral obstruction (UUO) and studied after 3,7 or 14 days. Renal tissue after ureteral obstruction at day 14 was dyed by HE and Masson staining for general histology and collagen detection. For renal tissue after ureteral obstruction at day 3 and 7, immunostaining was used to assess the infiltration of macrophage (CD68) and lymphocyte (CD3). And ELISA and Real-time PCR were done to assess the expression of chemokines (MCP-1, RANTAS, IP-10, MIP-1α, MIP-1β) and proinflammatory factors (TNF-αand IL-1β).Result:(1) HE and MASSON staining revealed that there is significant interstitial pathological change in rat injured kidney, and huge amount of collagen fiber deposited in renal tissue was seen. The Tubulointerstitial damage index and relative area of renal interstitial collagen significantly increased in UUO model 14 days after operation. All of above observations indicate that the establishing of the UUO model is successful. Compared with UUO group, treatment of AKF-PD, PFD and losartan significantly ameliorated renal interstitial fibrosis, and significantly reduced the deposit of collagen fiber, renal interstitial injury index, as well as the relative area of renal interstitial collagen (P<0.05).The therapeutic effects of these three drugs are indistinguishable.(2) AKF-PD, PFD and losartan treatment significantly attenuated macrophage and lymphocyte infiltration in kidney after ureteral obstruction at day 3 and day 7 after ureteral obstruction. All three treatments have the similar inhibitory effect.(3) The expression of all the chemokines and proinflammatory factors is also significantly reduced by the treatment of AKF-PD, PFD and losartan at day 3 (P<0.05). All three treatments have the similar inhibitory effect.However, expression of chemokines and proinflammatory factors in rat kidney at day 7 after ureteral obstruction is lower than that of at day 3; AKF-PD, PFD and losartan differentially affect the expression of chemokines and proinflammatory factors in rat kidney at day 7 after ureteral obstruction:there is no significant difference in the mRNA lever of IP-10 and MIP-1βbetween the groups (P>0.05); AKF-PD treatment significantly reduced the mRNA lever of RANTES,MCP-1,MIP-1α,TNF-a(P<0.05); PFD treatment significantly reduced the mRNA lever of MCP-1,MIP-1α(P<0.05); whereas losartan treatment significantly reduced the mRNA lever of MCP-1 (P<0.05); all three drugs did not have significant effect on the mRNA expression of IL-1β(P>0.05). However, AKF-PD and PFD treatment decrease the protein lever of all kinds of chemokines and proinflammatory factors (P<0.05); whereas losartan treatment selectively decrease the protein lever of RANTES,MCP-1 IP-10,MIP-1α(P<0.05).Conclusion:(1) AKF-PD ameliorats renal fibrosis. (2) AKF-PD significantly attenuated macrophage and lymphocyte infiltration in kidney as well as the expression of chemokines and proinflammatory factors in unilateral ureteral obstruction (UUO) model, indicating that the beneficial effect of AKF-PD in renal interstitial fibrosis is attributable, at least in part, to an anti-inflammatory action.(3) AKF-PD, PFD and losartan have the similar anti-inflammation effect in renal fibrosis, but the effect on chemokines and proinflammatory factors in rat kidney at day 7 after ureteral obstruction is bit of different.Part 2 The effect of Fluorofenidone on lethal endotoxemia and inflammatory factorBackground:Fluorofenidone(1-(3-fluorophenyl)-5-methyl-2-(1 H)-pyridone, AKF-PD) is a novel pyridone agent which is developed recently. Our data showed that AKF-PD exerts a strong antifibrotic effect, as shown in experimental renal fibrosis in vivo and in vitro. However, the mechanism underlying the antifibrotic effect of AKF-PD in renal disease remains to be elucidated. Our results showed that AKF-PD significantly attenuated macrophage and lymphocyte infiltration in kidney as well as the expression of chemokines and proinflammatory factors in unilateral ureteral obstruction (UUO) model. However, the inflammation which results in renal fibrosis, is different from that of in sepsis.And The effect of Fluorofenidone on sepsis and inflammatory factor is unkown.Sepsis is the systemic inflammatory response syndrome (SIRS) induced by infection. The host defence system will response immediately and correctly to the presence of micro-organisms or tissue invasion by microorganisms. Immunodeficiency, immunological response too strong or two weak can result in the genesis and progress of sepsis. The most common cause of sepsis in clinic is the infection of gram negative bacteria. And It is the common concept that gram negative bacterial endotoxin (also referred to lipopolysaccharide (LPS)) plays an important role in initiation of immune response and pathogenesis of septic shock. LPS can stimulate monocyte/macrophage to express and release all kinds of proinflammatory factors (such as TNF-a and IL-1β). These proinflammatory factors can promote adhesion of neutrophils and endothelial cells, activate coagulant system, induce systemic inflammatory response syndrome, and even result in shock, multiple organ failure and death.At the same time, the anti-inflammatory response is induced, which result in the anti-inflammatory factors such as IL-10. IL-10 can inhibit macrophage/lymphocyte adhesion and infiltration as well as the expression of proinflammatory factors, which facilitate sepsis.Objective:To investigate whether AKF-PD can promote survival in animal model of established endotoxemia, and whether it can reduce the level of TNF-a, IL-1βand IL-10 in vivo and in vitro, which we can get a better understanding of the anti-inflammation effect of AKF-PD.Methods:Balb/c mice were injected i.p. with LPS (15mg/kg); Different dose of AKF-PD (100mg/kg or 200mg/kg) was administered i.p. concurrently with LPS injection or 2 hours after LPS injection. And animal survival was monitored for up to 7 days. Then mice were injected i.p. with LPS (15mg/kg), AKF-PD (200mg/kg) was administered i.p. concurrently with LPS injection; At dedicated time points(2h,4h,8h), serum was obtained and was assayed for TNF-a and IL-1βproduction by ELISA.In vitro experiments, primary mouse peritoneal macrophage were stimulated with LPS (0.5μg/ml) in the presence or absence of AKF-PD with a concentration range of 400-800μg/ml.24h after stimulation, supernatant was collected and the levels of TNF-α, IL-1βand IL-10 were determined by ELISA. In another set of experiment, primary mouse peritoneal macrophage were stimulated with LPS (0.5μg/ml) in the presence or absence of AKF-PD (800μg/ml). The supernatant was collected at the indicated time(3,6,12,16,24h) and the levels of TNF-a and IL-1βwere determined by ELISA.Result:In the model of murine endotoxemia, treatment of AKF-PD (200mg/kg) both at the beginning of LPS infusion and after the onset of endotoxemia(2h after LPS infusion) prevented the lethality of endotoxemia (P<0.05). A lower dose of AKF-PD (100mg/kg, i.p.) provided partial protection (P>0.05). Besides, AKF-PD treatment (200mg/kg, i.p.) reduced circulating levels of TNF-a and IL-1βin a time-dependent manner (P<0.05).In vitro, AKF-PD significantly inhibited TNF-a and IL-1βrelease in a dose-and time-dependent manner (P<0.05), with the maximal effect at concentration of 800μg/ml. However, AKF-PD did not affect IL-10 release (P>0.05).Conclusion:AKF-PD significantly inhibited the release of the proinflammatory cytokine (TNF-a and IL-1β) but not anti inflammatory cytokine (IL-10), and improves survival during lethal endotoxemia, indicating that this new pyridone agent may be a novel candidate for the treatment of sepsis.