The Role And Mechanism Of Endothelial EP4 In Regulating Renal Ischemia-reperfusion Injury

Background:Ischemia-repersusion is one of the major factors of acute kidney injury.There is no specific and effective medical therapies for current renal ischemia-reperfusion injury due to a poor understanding of its pathogenesis.Prostaglandin E2(PGE2)is an important lipid mediators and regulates diverse physiological and pathophysiological processes in the kidney.It acts on four specific G-protein-coupled receptor subtypes,namely EP1,EP2,EP3 and EP4.EP4 is distributed in glomerulus,tubule,vasa recta and inflammatory cells of kidney.It is well known that EP4 regulates renal water and sodium excretion,and EP4 in endothelium participates in vasodilatation.But the role of endothelial EP4 in renal ischemia-reperfusion(IR)injury is unknown.In current project,we will utilize vascular endothelial cells specific EP4 knockout mice to testify the function of endothelial EP4 in renal IR injury and prognosis.Methods:1.To investigate the role of endothelial EP4 in renal IR injury,the Cre-loxp gene recombination techniques were used to generate transgenic mice with specific deletion of EP4 in vascular endothelial cells(EP4ΔEC),and the Cre negative mice worked as control(EP4flox/flox).Then renal IR injury mouse model proceeded as follows: The renal arteries of EP4ΔEC and EP4flox/flox mice were clipped for 30 min,then artery clamps were removed for another 24 hours or 14 days.The level of creatinine and urea nitrogen in serum was detected.The renal structure was revealed by PAS staining.2.EP4 deficiency in endothelial cells exacerbated apoptosis and pyroptosis in renal IR injury.Apoptosis and pyroptosis of renal cells was analyzed by using TUNEL assay.The expression of pro-apoptotic factors(bax,Cleaved-caspases3)and pyroptosis-associated protein(caspase1,caspase11,GSDMD)were detected by western blot.To study the mechanism of EP4 in regulating apoptosis and pyroptosis,we tested the MAPKs activation(p-JNK,p-ERK1/2,p-P38)in renal IR injury by western blot.To further confirm whether the apoptosis and pyroptosis of endothelial cells were regulated by a EP4-c AMP-PKA dependent pathway,we conducted HUVECs with cobalt chloride to simulate hypoxia in vivo,and the expression of cleaved-caspase3,caspase1,GSDMD was tested after being treated with EP4 agonist with or without its downstream inhibitors of PKA and Epac1.In addition,EP4 antagonist and c AMP analogue-dbc AMP were also applied to HUVECs as a further proof.3.Knockout EP4 in endothelial cells suppressed angiogenesis and increased Endo MT in the prognosis renal IR injury.Samples were collected at the last 14 days after renal ischemia reperfusion.CD31 positive cells were detected by immunofluorescence to determine the structure and numbers of renal interstitial capillaries.Immunohistochemical technology detected the PCNA positive cells to assess the proliferation of kidney.The degree of renal fibrosis was detected by Sirius scarlet staining and the protein of α-SMA.The possible regulation molecules of renal fibrosis were the phosphorylation levels of smad3 was detected by western blot.To further demonstrate the downstreams of EP4 in regulating the progression of angiogenesis and the transformation of endothelial cell to fibroblast(Endothelial to mesenchymal transition,Endo MT),HUVECs were treated with EP4 agonist,PKA and Epac inhibitors as previous,while western blot detected the expression of α-SMA and p-smad3 in endothelial cells.In addition,EP4 antagonist and c AMP analogue-dbc AMP were also applied as before.Results:1.EP4 deficiency in endothelial cells increased the levels of serum creatinine and urinary protein after renal IR,aggravated the structure of kidney and elevated the expression of KIM1.24 hours after renal ischemia-reperfusion,the levels of serum creatinine,urea nitrogen and urinary protein in EP4ΔEC were higher than those in sham-operated group.After 14 th day of reperfusion,serum creatinine,urea nitrogen and urinary protein were significantly decreased in the control,but not in the deficient mice.In contrast,the PAS stainning showed tubular necrosis appeared in kidney sat 24 hours after renal ischemia-reperfusion,and the pathological changes in the control were basically recovered at 14 days after ischemia-reperfusion,however,tubular necrosis still existed in the kidney of the EP4 defective mice.2.Endothelial EP4 plays a protective role in acute and chronic renal IR injury by inhibiting apoptosis and pyroptosis.The number of TUNEL positive cells increased at 24 hours and 14 days after renal ischemia-reperfusion.However,Bax and Cleaved-caspase3 were only up-regulated at 24 hours after renal ischemia-reperfusion,no significant difference at14 days,but EP4ΔEC mice showed an increasing of N-GSDMD.In vitro,EP4 agonist significantly impressed the expression of apoptosis and pyroptosis related proteins and inhibitedthe phosphorylation of JNK,while EP4 antagonists increase the up regulation of apoptosis and pyroptosis related protein,accompanied by activating of JNK in hopoxic HUVECs.In summary,EP4 showed a protective effect on apoptosis and pyroptosis by inhibiting p-JNK in endothelial cells.3.Endothelial EP4 promoted angiogenesis and inhibited Endo MT in the prognosis of renal IR injury.At 14 day after renal ischemia-reperfusion,the number of renal interstitial capillary in EP4ΔEC decreased.As well as,EP4 agonists can promote endothelial tubes formation in vitro,and EP4 antagonists did the opposite.After 14 days of reperfusion,the expression of fibrosis-associated α-SMA and p-smad3 in EP4ΔEC kidney were significantly higher than that in control.EP4 activation in HUVECs inhibited the Endo MT by a representative increase of smad3 phosphorylation and α-SMA induced by TGF-β.Moreover,EP4 antagonist exercised a relative inhibition in Endo MT.Conclusion:The study gives us more knowledges about the importance of maintaining the homeostasis of endothelial cells by EP4 during the development and progression of renal IR injury.Firstly,EP4 antagonized hypoxia-mediated apoptosis and pyroptosis by suppressing JNK phosphorylation depending on c AMP-PKA activation.Secondly,EP4 improved angiogenesis and inhibited Endo MT by affecting smad3 in the prognosis of renal ischemia-reperfusion injury.