Hyp0xia-inducible Factor 2a Regulates Macrophage Function In Renal Ischemia-reperfusion Injury

Objective: To illuminate the effect of HIF-2? knockout macrophages in renal ischemia-reperfusion injury and its molecular mechanism.Method: The Lyz-Cre+HIF-2?loxp/loxp and Lyz-Cre-HIF-2?loxp/loxp mice are randomly divided into the sham operation group and experimental group,and mouse renal ischemia-reperfusion model is constructed.The mice are killed in 24 h after reperfusion to measure the content of Cr and BUN in serum.In addition,the content of TNF-?,IFN-?,IL-1?,IL-6 and IL-10 in serum and kidney are also determined.Furthermore,the histomorphological changes of kidney are observed by HE staining.Finally,the survival rates of mice are observed.Mouse bone marrow–derived macrophages are assigned to the following groups:A:HIF-2?+/+?LPS 100ng/ml?21%O2 24h;B:HIF-2?-/-?LPS100ng/ml?21%O2 24h;C:HIF-2?+/+?LPS 100ng/ml?15?M PFT-??21%O224h;D:HIF-2?-/-?LPS 100ng/ml?15?M PFT-??21%O2 24h;E:HIF-2?+/+?LPS100ng/ml?1%O2 24h;F:HIF-2?-/-?LPS 100ng/ml?1%O2 24h;G:HIF-2?+/+?LPS 100ng/ml?15?M PFT-??1%O2 24h;H:HIF-2?-/-?LPS 100ng/ml?15?M PFT-??1%O2 24 h.ELISA is used to measure the content of TNF-a,IFN-?,IL-1?,IL-6 and IL-10 in cell culture supernatants.The apoptosis of macrophages in each group is analyzed by FACS;and the expression level of HIF-2?,P53,Phospho-p53(Ser15),Puma,Bax and cleaved caspase-3 in each group of macrophages is determined by Western blot.Results: Contrast with Lyz-HIF-2?+/+ mice,the contents of Cr and BUN in serum are lower in Lyz-HIF-2?-/-mice after renal IR.The levels of TNF-?and IFN-? in kidney of Lyz-HIF-2?-/-mice are decreased while IL-10 is increased.Consistent with this,Lyz-HIF-2?-/-mice have higher 48 h survival rate and lighter renal histopathologic damage.Further study found that,BMDM extracted from Lyz-HIF-2?-/-mice have higher apoptosis rates and expression levels of P53,Phospho-p53(Ser15),Puma,Bax and cleaved caspase-3 in hypoxia compared with Lyz-HIF-2?+/+ mice,which can be eliminated by PFT-?(the inhibitor of P53).Moreover,BMDM obtained from Lyz-HIF-2?-/-mice have higher contents of TNF-??IL-6 and lower content of L-10 in cell culture supernatants when cultured in hypoxia and it can not be affected by PFT-?.Conclusion: Knockout of HIF-2? in macrophages can reduce the secretion of proinflammatory factors and increase the secretion of anti-inflammatory factors,and enhance the apoptosis rate of macrophages by promoting the activation of P53 pathway to alleviate the renal ischemia-reperfusion injury.