The Mechanism Of Effect Of Augmenter Of Liver Regeneration On Renal Tubular Interstitial Fibrosis In Vitro And In Vivo

Part1Augmenter of Liver Regeneration Inhibits TGF-β-Induced Renal Tubular-to-Epithelial TransitionObjective：To explore whether augmenter of liver regeneration (ALR)is involved in the pathological process of renal tubular epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor-β1(TGF-β1). To investigate whether administration of recombinant humanALR (rhALR) affected the expression of TGF-β1and the process of EMTinduced by TGF-β1. Further, to elucidate the molecular mechanismunderlying the antifibrotic capacity of ALR.Methods：Using the cells model that renal tubular EMT was inducedby TGF-β1in renal tubular epithelial cells(NRK-52E), the protein andmRNA expression of ALR were observed by Western blot and real timeRT-PCR.Then, NRK-52E cells were divided into five groups:①controlgroup;②rhALR alone treated group；③TGF-β1alone treated group；④ TGF-β1+20μg/ml rhALR treated group；⑤TGF-β1+80μg/ml rhALRtreated group. TGF-β1levels in conditioned medium and cell lysates inNRK-52E cells treated with rhALR alone were measured with enzymelinked immunosorbent assay(ELISA); The expression of Vimentin, α-SMAand E-cadherin in NRK-52E cells stimulated with TGF-β1or/and rhALRwere measured using Western blot and Laser confocal immunofluorescencestaining. The conventional TGF-β1/Smad signaling pathway(Smad-2,Smad-4, Smad-6, Smad-7, phosphorylated Smad-2and NF-κB) was furtherinvestigated to elucidate the potential molecular mechanism.Results：TGF-β1activated the protein and mRNA expression ofrhALR in NRK-52E cells; There was no effect of rhALR alone on thelevels of TGF-β1in NRK-52E cells; rhALR significantly inhibitedTGF-β1-induced downregulated E-cadherein expression and upregulatedVimentin and α-SMA expression in NRK-52E cells; And, rhALRsuppressed the expression of TGF-β receptor type Ⅱ(TβRⅡ) and had noeffect on TGF-β receptor typeⅠ(TβR-Ⅰ), Smad-2, Smad-4, Smad-6,Smad-7, but significantly alleviated TGF-β1-induced phosphorylation ofSmad2and nuclear transcription of activated NF-κB in tubular epithelialcells.Conclusions：ALR may play a role in the progression of renal tubularEMT. RhALR inhibits TGF-β1-induced EMT via suppressing theexpression of TβRⅡ, and then reducing the phosphorylation of Smad2and NF-κB in NRK-52E cells, which may be contributed to further investigateALR as an effective antifibrotic strategy to reverse renal interstitial fibrosisfor CKD. Part2Augmenter of Liver Regeneration Ameliorates RenalInterstitial Fibrosis In RatsObjective：To explore the effects of Augmenter of liver regeneration(ALR) on the pathological progression of renal interstitial fibrosis inunilateral ureteral obstruction(UUO) rat model and it’s molecularmechanism.Methods：Sprague-Dawley adult male rats were randomly dividedinto four groups:①sham control;②UUO+phosphate-buffered saline(PBS);③UUO+recombinant human ALR (rhALR)100μg/kg.d;④UUO+rhALR200μg/kg.d. RhALR was dissolved in PBS and administered on rats viadaily intraperitoneal injection after UUO surgery according to the indicateddose. The sections of kidney tissues were stained with hematoxylineosin(HE) and Masson’s trichrome. The expression of proteins and genesinvolved in pathological progression of renal fibrosis(Vimentin, α-SMA, Fibronectin, CollagenⅠ, TGF-β1, Smad-2, Smad-3and phosphorylatedSmad-2, Smad-3) were observed using Western blot analysis or Real-TimePolymerase Chain Reaction.Results： Endogenous ALR was involved in the pathologicalprogression of renal interstitial fibrosis in unilateral ureteralobstruction(UUO) rat model. The administration of rhALR significantlyalleviated the tubulointerstitial fibrosis and reduced related proteins inUUO rats with renal fibrosis(P＜0.05, P＜0.01). Moreover, rhALRsuppressed the protein expression of TGF-β1in the obstructed kidney, andinhibited the phosphorylation of Smad2and Smad3activated by the UUOinduced injury in the animal model.Conclusions：ALR is involved in the progression of renal interstitialfibrosis and administration of rhALR protects the kidney against renalfibrosis by inhibition of TGF-β/Smad activity.