The Experimental Study Of Telbivudine Attenuates Renal Inflammation And Fibrosis Through TLR4/NF-κB Signaling Pathway

Objective:Chronic kidney disease(CKD)is defined as when there is the presence of kidney damage or a decrease in kidney function(glomerular filtration rate(glomerular filtration rate,GFR)<60 ml/min/1.73 m2,proteinuria with albumin to creatinine ratio>30 mg/g)for 3 or more months,regardless of cause.Because the onset is always insidious and the intervention is belated,the majority of CKD patients eventually develop into end stage renal disease(ESRD)and have to sustain life by dialysis.CKD can not only bring pain to the patients,but also cause great pressure to the society.Renal fibrosis is regarded as the pathologic manifestation of a wide variety of CKD,the underlying mechanism of renal fibrosis is an imbalance of components of the extracellular matrix(ECM)owing to their excessive synthesis and reduced breakdown.In the end,normal renal tissue is replaced and renal function is lost.As the key effector cells in renal fibrosis,fibroblasts and myofibroblasts are believed to be the major cell types associated with synthesis and deposition of ECM.Myofibroblasts that harbor the α-smooth muscle actin(α-sma)marker are considered to have the activated fibroblast phenotype.In addition,renal tubular epithelial cells can be transformed into ECM synthesis cells through epithelial mesenchymal transformation(EMT),which has great significance in renal fibrosis.Transforming growth factor(TGF)-β plays a central role in renal fibrosis;its profibrotic effect results from imbalanced regulation of ECM through Smad,so TGF-β/Smad signaling was regarded as the canonical pathway in renal fibrosis.The development of fibrosis is an extremely complex process,in which inflammation plays a really important role.Chronic inflammation leads to the synthesis of growth factors,angiogenic factors and fibrogenic cytokines,then the collagen-producing cells are activated,which in turn generate excessive ECM.Of these molecules,nuclear factor-κB(NF-κB)is a central regulator of the inflammatory response.NF-κB can stimulate not only the expression of inflammatory mediators but also renal fibrosis through interacting with TGF-β/Smad signaling.It is worth noting that Toll like receptor4(TLR4)can activate NF-κB,recent researches have found that TLR4 has an important role in both renal inflammation/fibrosis and the induction of HBV infection injury to cells.Although several preclinical studies aimed at inhibiting renal fibrosis and inflammation have yielded promising findings regarding treatment of CKD,limited advances have been made in translating this research to patients.Results of clinical trials generally have indicated limited efficacies of these anti-fibrotic agents.So CKD treatment is always the focus problem.Telbivudine(LdT;β-L-2’-deoxythymidine)has been used widely to treat chronic HBV infection(CHB)in recent 10 years.It is a synthetic thymine nucleotide analogue,which can be combined with the thymine of HBV to prevent HBV DNA synthesis and antagonise viral replication effectively.Clinical findings indicate that telbivudine therapy also improves renal function(eGFR).Not only that,the decreased eGFR caused by nephrotoxic adefovir or cisplatin in patients with CHB could be rescued by adding telbivudine.The mechanism by which telbivudine improves eGFR is not clear but involves a directly beneficial effect on the kidney—rather than an indirect effect of HBV suppression.Several hypotheses exist to explain this phenomenon:(1)Telbivudine improves renal blood flow or tubular function;(2)Telbivudine may act directly on kidney structures or on inflammatory/fibrotic pathways;(3)Telbivudine is excreted by passive diffusion via renal tubular cells,unlike other nucleos(t)ide analogues that are actively pumped into the glomerular filtrate.(4)Telbivudine can decrease the activity of angiotensin converting enzyme(ACE).Up to now,there is no research explain the mechanism of Telbivudine’s function on renal function,only Kader et al found that Telbivudine significantly improved renal function(Cystatin C)and pathologic change on a model of gentamycin-induced acute nephrotoxicity.At the same time,Telbivudine inhibits the expression of TGF-β and α-sma.Unilateral ureteral obstruction(UUO)results in renal inflammation and fibrosis followed by tubular injury and changes in renal hemodynamics and metabolism.In the current study,we applied a rat model of UUO to evaluate the influence of renal function by Telbivudine,and examined components of the canonical TGF-β and NF-κB signaling pathway to observe the effect of Telbivudine on renal inflammation and fibrosis.In addition,we cultured the human renal tubular epithelial cells(HK-2),on the one hand,TGF-β was added to observe the effect of Telbivudine on EMT,on the other hand,lipopolysaccharide(LPS)and TLR4 inhibitors were added to observe the effect of Telbivudine on cell inflammation and related mechanisms.Methods:In the first chapter of the study,the UUO model was chosen to investigate the drug action on renal function,30 Sprague-Dawley(SD)rats(male,7-8 weeks)were randomly divided into five groups,including control group,UUO group and 3 Telbivudine treatment groups,they were low-dose group(60mg/kg),middle-dose group(180mg/kg),high-dose group(540 mg/kg)respectively.Urea nitrogen,blood and urinary creatinine were measured before operation and on the 10th,20th,40th day after operation respectively,endogenous creatinine clearance(eCcr)was calculated,the differences of renal function among different groups at each time point were observed.In the second part,30 male SD rats aged 7-8 weeks were selected and grouped into 5 groups as before.According to the results of the first part,the intragastric dose was increased to 1,1.5,2 g/kg daily for 35 days.H&E staining and Masson staining were used to investigate the pathological feature of kidney,Collagen I and III were detected to observe the production of ECM,the expression levels of MMP-2 and TIMP-1 were detected to observe the effect on the regulation of ECM metabolism,the marker α-sma was detected to observe the effect on the activity of myofibroblasts,the mRNA and protein level of TGF-β/Smad pathway were detected to observe the effect on canonical fibrotic signaling,IL-1 and TNF-α were detected to observe the effect on inflammation,the expressions of NF-κB and its upstream protein TLR4,IKKa,regulatory enzymes IkBa were detected to observe the effect on NF-κB and its signaling pathway.In the final part,we gave TGF-β to HK-2 cell to induce EMT,and then Telbivudine treatment(the dose was 0.01,0.05,0.1mg/ml)was given,the key proteins in epithelial-mesenchymal transition were detected,including the marker of epithelial cells E-cadherin and the marker of myofibroblasts α-sma,to observe the drug action on EMT.Besides,the major regulation enzymes MMP-2 and TIMP-1 were detected to investigate the mechanism of Telbivudine on EMT,inflammatory factors were also investigated.Then the TLR4 inhibitor(C34),Telbivudine(LdT)and LdT+C34 were given respectively to the HK-2 cells stimulated by LPS,the inflammatory factors and the proteins in TLR4/NF-κB and TGF-β/Smad signaling pathway were detected to investigate the molecular mechanism of Telbivudine on HK-2cells.Results:1.At baseline,there is no difference in eCcr,creatinine or urea nitrogen between each group.At each time point after surgery,renal function in UUO group was higher than control group,among them,eCcr(20th day,40th day)and creatinine(40th day)had statistical difference.Renal function in LdT-treated groups were lower than UUO group,but the difference were all not statistically significant.2.Renal pathological feature of UUO rats could be improved by Telbivudine,including tubular atrophy,tubular necrosis,the infiltration of inflammatory cells and the fibrosis of renal interstitial part.Telbivudine could inhibit the activity of TGF-β/Smad pathway,downregulate MMP-2 and TIMP-1,decrease the expression of α-sma,namely the number of myofibroblasts was decreased,and reduce the synthesis and accumulation of COL I and III in the interstitium.Not only that,Telbivudine could reduce the activation of TLR4/NF-κB signaling pathway and the release of IL-β and TNF-α.3.Telbivudine reduced the activity of MMP-2/TIMP-1 induced by TGF-β,decrease the expression of α-sma but increase the expression of E-cadherin to inhibit EMT,prevent the expression of TNF-α and NF-κB in EMT.4.Telbivudine inhibits the activation of TLR4 induced by LPS and the expression of its downstream proteins,such as IKKα,NF-κB and thus reduces the release of IL-1β and TNF-α.At the same time,LPS could activate while Telbivudine could inhibit TGF-β/Smad signaling pathway,LdT+C34 didn’t attenuate the above results more.Conclusion:To sum up,we found that Telbivudine can inhibit the activity of TLR4 and its downstream NF-κB,suppress the expression and release of inflammatory cytokines IL-1 and TNF-α.At the same time,Telbivudine inhibits the activation of TGF-β/Smad signaling pathway,and reduces the activity of MMP-2/TIMP-1,inhibite the EMT of the HK-2 cells,the number of myofibroblasts were decreased and the synthesis of ECM in the interstitial part were reduced,ultimately the pathological manifestations of renal interstitial fibrosis were improved.In conclusion,Telbivudine has a renal protective effect which is unrelated to the decrease of viral replication,this effect is manifested in the improvement of renal fibrosis and inflammation.