The Moludation Of Augmenter Of Liver Regeneration On TLR4Signal Pathway In Renal Ischemia Reperfusion Injury

Background and objectiveIschemia reper fusion induce d acute kidney inj ury (AKI) is a commonclinical problem with a poor prognosis, and so far,there isn’ t effectiveapproaches to prevent renal ischemia reperfusion injury (IRI). Toll likereceptors (TLRs),which mediate the innate immune response,play a crucialrole during renal IRI physiologic process. The engagement between TLRsand these endogenous ligands results in activation of TLR4signalingpathways,and upregulation of proinflammatory cytokines andchemokines,eventually causes inflammatory reaction during postischemickidney. Increasing number of researches confirm that inhibition of TLR4signal pathway is benefit for ischemia AKI.Augmenter of liver regeneration (ALR)，a hepatocyte growth factorproduced by body，is expressed in multiple organs. ALR was found to playan important role in regulation of the liver immuneesponse recently. Furtherresearches in vivo reveal that ALR act an powerful immunosuppressiveeffect on immune cells,which only for activated immune cells.Our previous studies have demonstrated that ALR expresses highly in renal IRI andprotects both of renal function and structure via inhibition of inflammatoryresponse.However,whether ALR inhibits TLR4signal pathway in order toattenuate inflammation in ischemia kidney and it’s specific mechanismsremain unkown.In this study herein,we firstly investigated the effect of ALR onTLR4/NF-κB signal pathway during renal IRI in vivo.Furthermore,wethroughly explored the specific mechanism of ALR on TLR4signalings invitro.MethodsCells experiment1．The cultured NRK-52E cells were divided into a control group,a IRIgroup,and rrALR preincubated group(25,50ug/ml of rrALR).The IRI groupand polydatin group were incubated in three gas incubators for period ofhypoxia at6h and of reoxygenation at12h,24h,72h to simulate theischemia-reperfusion injury in vitro.2．The mRNA and protein expression of TLR4and NF-κB by RT-PCR andwestern blotting.3．IL-6and IL-1β proteins expressions were detected by ELISA.Animal experiment.1．SD rats randomly divided into thesham-operated group, the I/R+rhALR1 group (100μg/kg, i.p), the I/R+rhALR2group (200μg/kg, i.p) and theI/R+saline group. Both renal pedicles of rats were identified and occludedwith microvascular clamps for60min to induction of ischemic AKI.2．Renal dysfunction and histologic injury were assessed by measurementof serum biochemical markers and histologial grading.Apoptosis wasassessed by terminal deoxynucleotidyl trasferase-mediated dUTP-biotinnick-end labeling (TUNEL).3． Expression of neutrophils and macrophages were measured byimmunohistochemistry.4．The mRNA and proteins expression of inflammatory factors andchemokines were measured by real-time PCR and enzyme linkedimmunosorbent assay (ELISA).5． Expression of the endogenous ligands(HMGB1,biglycan,HAS1,HAS2,HAS3) and TLR4mRNA was determinedby real-time PCR.TLR4protein was detected by immunohistochemistry.6．Phosphorylated-ERK, phosphorylated-JNK and phosphorylated-p38wereassessed by western blot.The activity of NF-κB was assayed by real-timePCR and immunohistochemistry.ResultsCells experiment1．In rrALR intervened H/R cells,TLR4and NF-κB are downregulated atboth mRNA and protein levels compare with those in H/R cells（P<0.05）. They decreased more significantly in H/R+rrALR2group than those inH/R+rrALR1group (P<0.05)2． rrALR appears to downregulate IL-6and IL-1β expression inconcentration-dependent manners (P<0.05).Animal experiment1． Blood urea nitrogen and serum creatinine levels were improvedsignificantly in rats treated with rhALR as compared with saline-treated rats(P <0.05). Renal dysfunction improved more significantly in I/R+rhALR2group than that in I/R+rhALR1group (P <0.05).2．The characteristic histopathological features of ischemic injury wereevident at24h and were milder at72h after reperfusion in kidneys obtainedfrom I/R+saline treated rats. Specifically, the most severe and pronouncedinjuries were observed in the cortex and the outer stripe of the outer medulla,and showed a typical tubular necrosis pattern. This pattern includedwidespread degeneration of tubular architecture, detachment of epithelialcells from the basement membrane, tubular cell necrosis, intratubular castformation, and luminal congestion, infiltration of inflammatory cells. In therhALR-treated group, however, the injury of kidney was more focal andmilder. Renal proliferating index inflecting the proliferation of renal tubularcells improved more significantly in I/R+rhALR2group than that in I/R+rhALR1group (P<0.05). 3．Few TUNEL-positive cells were detected in the renal cortex or the outerstripe of the outer medulla in kidneys from sham-operated rats. The kidneysfrom animals that were subjected to renal ischemia/reperfusion exhibited amarked increase in the number of TUNEL-positive cells, and the number ofapoptotic cells peaked at24h after reperfusion. In the rhALR-treated group,the mean number of apoptotic cells was significantly lower than that in thesaline-treated group at the same time point (P<0.05).4．Immunochemistry exhibited that few neutrophils and macrophages weredetected in sham-operated rats.Increasing number of neutrophils andmacrophages infiltrated renal interstitial during renal IRI.The number ofeutrophils peaked at24h after IRI,whereas macrophages arrived to the topat72h. In the rhALR-treated group, the mean number of inflammtory cellswas significantly lower than that in the saline-treated group (P<0.05)5．The mRNA and protein levels of inflammatory cytokines (TNF-α、IL-1β、IL-6) andchemokines (MCP-1and MIP-2) in kidneys from thesham-operated rats was low. T hey all increased after renalischemia/reperfusion injury. The expression of proinflammtory cytokineswere lower in kidneys obtained from the rats treated with rhALR thanthose obtained from the saline-treated rats (P<0.05). They decreased moresignificantly in I/R+rhALR2group than those in I/R+rhALR group(P<0.05).6．The mRNA levels of TLR4and endogenous ligands (HMGB1、biglycan、 HAS1、HAS2、HAS3) in kidneys from the sham-operated rats was low.They all increased after renal ischemia reperfusion injury24h. Theexpression of TLR4and endogenous ligands were lower in kidneys obtainedfrom the rats treated with rhALR than those obtained from the saline-treatedrats with a dosage manner (P<0.05).Immunochemistry showed that TLR4mainly expressed on renal tubular epithelial cells and infiltrativeinflammatory cells after IRI.TLR4expression decreased more significantlyin I/R+rhALR2group than those in I/R+rhALR group (P<0.05).7．The protein ratio of pERK/ERK,pJNK/JNK,pp38/p38was distinctlylower in rhALR-treated groups than in I/R+saline groups.The activity ofNF-κB in kidney was lower in kidneys obtained from the rats treated withrhALR than in those obtained from the saline-treated rats during IRI(P<0.05).ConclusionCell experimentrrALR protects NRK-52E cells from H/R injury possibly by relievingthe inflammatory response through regulation of TLR4-NF-κB signalingpathway.Animal experiment1．Renal dysfunctio n and histological injury was improved significantly,andapoptosis of tubular epithelia cells were inhibited distinctly in rats treated with rhALR comparedwith saline-treated rats. This indicates that rhALR canprotect kidneys from acute kidney injury.2．The protection of rhALR on acute kidney injury is associated with itsinhibition of renal inflammatory cells infiltration and expression ofinflammatory cytokines and chemokines in kidney.3. The inhibition effect of rhALR on inflammation is associated withdownregulation of TLR4and its endogenous liands expression,andsuppression of activation of MAPKs and NF-κB.In conclusion,the present study exhibited that a role of ALR as aimmunoregulator in renal IRI,most likely due to suppression ofTLR4-triggered innate immune reactions.These results encourage ALR inthe development of novel molecular therapies for renal IRI.