The Roles Of Glutamine Metabolism On Macrophage Activation In Renal Fibrosis Via Akt-mTORC1 Pathway

Object:This study will investigate the effects and mechanisms of glutamine metabolism on macrophage activation in renal fibrosis.Methods : In vitro,human myeloid leukemia cell line U937 was induced to rest macrophages(M0).Subsequently,macrophages can be further differentiated into classically activated macrophages(M1)with IFN-γ/LPS and alternatively activated macrophages(M2)with IL-4/IL-13.Macrophages activation and the expression of key proteins and phosphorylation of Akt-m TORC1 signaling were detected in the presence or absence of glutamine or with the inhibitor of glutamine metabolism.After the pretreatment with Akt inhibitor MK2206 and m TORC1 inhibitor Rapamycin,the effect on macrophage polarization was examined.In vivo,Mice fibrosis model was constructed by unilateral ureteral obstruction(UUO).Intraperitoneal injection of BPTES(12.5mg/Kg)at the 3rd,5th and 7th day after the UUO model was constructed and the effect of BPTES on renal inflammatory infiltration,macrophage activation and renal fibrosis were observed.Results : In vitro,glutamine deprivation and the treatment with BPTES promoted the activation of M1 but impaired the M2 phenotype.Meanwhile,macrophage deprived of glutamine and BPTES treatment suppressed IL-4/IL-13-induced phosphorylation and boosted IFN-γ/LPS-induced phosphorylation of Akt-m TORC1 signaling.Treatment with Akt inhibitor and m TORC1 inhibitor promoted M1 activation but suppressed the expression of M2-specific genes.In vivo,BPTES group was showed to inhibit inflammatory infiltration and M2 polarization.Indeed,BPTES group reduced renal fibrosis.Conclusion : Glutamine metabolism can regulate macrophage activation through the Akt-m TORC1 pathway.Inhibition of glutamine metabolism can impair M2 activation and improve renal fibrosis.Part1 Glutamine metabolism regulate macrophage activation through the Akt-m TORC1 pathwayObject:To investigate the effects and mechanisms of glutamine metabolism on macrophage activation.Methods: Human myeloid leukemia cell line U937 was induced to rest macrophages(M0)by PMA(100ng/ml).Subsequently,macrophages can be further differentiated into M1 with20ng/ml IFN-γ and 100ng/ml LPS,M2 with 20ng/ml IL-4 and 20ng/ml IL-13.Flow cytometry was used to detect the expression of CD86 and CD206,Realtime PCR was conducted for TNFα and TGFβ1 expression,Western Blot was used to test the expression of GBP5 and Arg-1 in culture conditions with or without glutamine or with the inhibitor of glutamine metabolism.Detected the expression of key proteins(Akt,m TOR,S6 k,S6)and phosphorylation of Akt-m TORC1 signaling.The cells were pretreated with Akt inhibitor MK2206(5μM)and m TORC1 inhibitor Rapamycin(100μM)for 1h.The samples were tested by Flow cytometry(CD86,CD206)and Western Blot(GBP5,Arg-1).Results: The glutamine-deprived media and BPTES treatment showed higher expression of M1-specific marker,including CD86,TNFα and GBP5.In contrast,glutamine deprivation and BPTES treatment impaired expression of M2-specific marker,including CD206,TGFβ1,Arg-1.Western blot showed that both IFN-γ/LPS stimulation and IL-4/IL-13 stimulation increased the expression of p Akt,pm TOR,p S6 k,p S6.Notably,glutamine deprivation and BPTES treatment suppressed IL-4/IL-13-induced phosphorylation and boosted IFN-γ/LPS-induced phosphorylation of Akt-m TORC1 signaling.Treatment with MK2206 and Rapamycin impaired the expression of CD206 and Arg-1,but augmented the expression of CD86 and GBP5.Conclusion : Glutamine metabolism can regulate macrophage polarization through the Akt-m TORC1 pathway.Inhibition of the Akt-m TORC1 pathway can impair M2 activation and promote M1 polarization.Part2 Inhibition of glutamine metabolism can improve UUO-induced renal fibrosisObject:To verify whether glutamine metabolism can improve UUO-induced renal fibrosis through its effect on macrophage activation.Methods : Mice fibrosis model was constructed by unilateral ureteral obstruction(UUO).Intraperitoneal injection of BPTES at the 3rd,5th and 7th day after the UUO model was constructed,and the effect of BPTES on inflammatory infiltration was detected by immunohistochemical expression of CD3,CD11 c,F4/80 and B220;the effect of BPTES on macrophage activation was tested by immunohistochemical expression of CD86 and CD206;the effect of BPTES on renal fibrosis was determined by Masson staining,Sirius red staining and detected by immunohistochemical expression of α-SMA.Results:In the UUO model,the expressions of CD3,CD11 c,F4/80,B220 and CD206 were significantly reduced after BPTES intervention,but the expression of CD86 was unaffected.Otherwise,Masson staining and Sirius red staining showed that renal interstitial fibrosis was significantly reduced in the BPTES group.What’s more,the expression of α-SMA was obviously decreased.Conclusion:Inhibition of glutamine metabolism can suppress M2 activation and improve renal fibrosis.