Effect Of S1PR3 Induced Vascular Endothelial Dysfunction On Septic Acute Kidney Injury And Its Treatment

Part I The relationship between vascular endothelial dysfunction and acute kidney injury in patients with sepsisObjective: Endothelial activation and injure occured in the early stage of sepsis,and play an important role in the pathophysiology of septic acute kidney injury(S-AKI).Endothelium play an important role on the balance of coagulation system in sepsis.It has been identified that intrinsic and extrinsic coagulation pathways were initiated by activated and injured endothelial cells in sepsis,which resulted in the formation of microthrombus and exacerbated microcirculatory dysfunction in kidney.There is no endothelial biomarkers in routine tests.This study was conducted to investigate whether S-AKI are associated with endothelial dysfuntion by evaluating the relationship between coagulative biomarkers and AKI in sepsis.Methods: An observational retrospective study was conducted in the surgical ICU.We studied patients who met the criteria of septic shock(Sepsis-3)caused by IAI between January 1,2013,and December 31,2016.By adjusting for baseline characteristics,multivariate regression analyses were used to identify independent risk factors for predicting the development of S-AKI and mortality,including activated partial thromboplastin time(APTT),prothrombin time(PT)and D-dimer on admission to the ICU.Results: Of the 138 enrolled patients,65 patients with sepsis developed AKI.The patients who developed AKI exhibited a dramatically higher Sequential Organ Failure Assessment(SOFA)score(median,12),Acute Physiology and Chronic Health Evaluation(APACHE)II score(median,27.5)and mortality rate.In both models,we found that APTT(odds ratio(OR)=1.074,95% confidence interval(CI)1.030–1.120,P=0.001),PT(OR=1.162,95% CI 1.037–1.302,P=0.010)and D-dimer level(OR=1.098,95% CI 1.002–1.202,p=0.045)on admission to the ICU were significant risk factors for AKI.Moreover,Cox regression analysis showed that prolonged APTT(OR=1.065,95% CI 1.025–1.107,P=0.001)was independently associated with high mortality.Conclusion: In patients with sepsis caused by IAI,intrinsic coagulation pathway(APTT)initiated by activated and injured endothelial cells is significantly correlated with S-AKI and mortality,suggesting that endothelial dysfunction plays an important role in S-AKI.Part II Effect of S1PR3 induced vascular endothelial dysfunction on septic acute kidney injury and its molecular mechanismObjective: Endothelial cells(ECs)play an important role in maintaining homeostasis under normal physiological conditions.Circulating endotoxins and inflammatory factors can directly damage or activate endothelial cells in sepsis.Sphingosine-1-phosphate receptor 3(S1PR3),one of G protein coupled receptors,is mainly expressed on ECs.It regulates the signal transduction of ECs through binding to the ligand sphingosine-1-phosphate(S1P).There is evidence that S1PR3 induced endothelial barrier disruption in acute lung injury caued by LPS.However,its effect on septic acute kidney injury(S-AKI)remains unclear.Thus,the study was conducted to explore the relationship between S1PR3 and ECs in S-AKI.Methods: The effects of S1PR3 in S-AKI was evaluated by four aspects.First,we explore the relatiosnship between S-AKI and S1PR3 by Western Blot and immunofluorescence assay in WT mice administered LPS.Second,after S1pr3 +/+(WT)and S1pr3-/-(KO)mice were intraperitoneally injected with LPS to establish the S-AKI model,flow cytometry and Transmission Electron Microscope(TEM)and Evans Blue Dye(EBD) extravasation assay were used to assess the function of renal ECs.Third,ultrastructural structure of mitochondria in renal ECs was observed by TEM in WT and KO mice with sepsis.Four,human umbilical vein endothelial cells(HUVECs)were cultured in ECM to explore the mechanism between S1PR3 and endothelial dysfunction.Results: After adminstration of LPS,the values of creatinine(Crea)and blood urea nitrogen(BUN)in 24 h group were significantly higher than that in the 0h group,6h group and 48 h group(P<0.001).Western Blot showed that,the expression level of S1PR3 in kidney after 24 h of LPS injection was significantly higher than that in 0h group,6h group and 48 h group(P<0.001).Immunofluorescence showed that S1PR3 was mainly expressed in renal ECs.S1pr3 +/+(WT)and S1pr3-/-(KO)mice were intraperitoneally injected with LPS to establish the S-AKI model.After 24 h of LPS injection,the values of Crea and BUN in S1pr3-/-mice with spesis were significantly lower than S1pr3 +/+ mice in sepsis(P<0.01).And the leukocyte infiltration in S1pr3-/-mice with spesis were significantly lower than S1pr3 +/+ mice in sepsis(P<0.01).Meanwhile,the activation of S1PR3 induced the endothelial disruption(P<0.01).After 24 h of LPS injection,the ultrastructural changes of mitochondria were observed in vascular endothelial cells of S1pr3 +/+ mice with sepsis via TEM,including mitochondrial swelling and fracture of mitochondrial cristae.In addition,the ATP production in S1pr3 +/+ mice with sepsis was significantly lower than S1pr3-/-mice in sepsis(P<0.01).In vitro,we found that S1PR3 inhibitor TY-52156 was effective in reducing mitochondrial Ca2+ influx in endothelial cells(P<0.05)and increasing ATP production(P<0.01)compared with the LPS+S1P group.Moreover,immunofluorescence showed that,compared with the LPS+S1P group,TY-52156 protected tight and adherens junction,and maintained the normal morphological structure of cytoskeletal.Conclusion: The present study demonstrates that blocking the S1P-S1PR3 signaling pathway can effectively prevent activation of endothelial cell to maintain the integrity of renal endothelial barrier and protect renal function via inhibiting mitochondrial calcium overload in S-AKI model.Part III The design and synthesis and application of TY-52156 liposomeObjective: S1PR3 specific antagonist TY-52156 can effectively inhibit extracellular Ca2+ influx by preventing S1P[3H] binding to extracellular S1PR3-CHO,which is a potential drug for targeted therapy of S1P-S1PR3 signaling pathway.However,TY-52156 only dissolve in dimethylsulfoxide or ethanol due to strong hydrophobicity,which has toxicity on tissues.Liposome is a nanostructured drug delivery carrier,which composed of phospholipid and cholesterol.In order to reduce toxicity of organic solvents and improve solubility and biocompatibility,we designed and synthesized the TY-52156 liposomes.Then we explore the biological effect of TY-52156 liposomes in mice with S-AKI.Methods: We designed Liposome-Negative-TY-52156(LP-Neg-TY-52156)and Liposome-Neutral-TY-52156(LP-Neu-TY-52156)to improve water solubility and membrane biocompatibility.Liposomes were synthesized by film dispersion method.Two kinds of liposome were dissolved in water,the nanostructure of liposomes was observed by Transmission Electron Microscope(TEM).The particle size and Zeta potential of nanoparticles were detected by Grainsize analyzer.Cell experiments were carried out to detect the phosphorylation levels of p44/p42 MAPK in the cells after 10 min or 30 min pretreatment with liposomes.We further explored the biological effects of the liposomes by vivo experiments.Results: The nanostructures of the liposomes were observed by TEM after dissolving in water.The particle size of LP-Neg-TY-52156 was 615.7±131nm,and the Zeta potential of LP-Neg-TY-52156 was-33.14±5.515 m V.Meanwhile,the particle size of LP-NeuTY-5215 was 1793±212.6nm,and the Zeta potential of LP-Neu-TY-5215 was-1.601± 3.552 m V.Western Blot showed that the phosphorylation levels of LP-Neg-TY-52156 group and TY-52156 group were significantly lower than that of non-administered group after antagonist pretreatment for 10min(P<0.0001).Otherwise,there was no statistical difference btween LP-Neu-TY-52156 group and S1 P group.In additon,the phosphorylation level of LP-Neg-TY-52156 group,LP-Neu-TY-52156 group and TY-52156 group was significantly lower than that of non-administered group after antagonist pretreatment for 30min(P<0.0001).Moreover,the phosphorylation level of LP-Neg-TY-52156 group was lower than that of TY-52156 group(P < 0.05).Furthermore,the values of Crea in TY-52156 group(P<0.05)and LP-Neg-TY-52156 group(P<0.01)was significantly lower than that of LPS group after intraperitoneal injection of LPS for 24 h.The value of BUN showed a downtrend,although there was no statistical difference in BUN level.Conclusion: TY-52156 liposomes can effectively improve the water solubility and biocompatibility.Moreover,LP-Neg-TY-52156 has a better pharmacodynamic effect due to better biocompatibility.We further proved that the specific inhibitior of S1PR3 alleviated acute kidney injury by inhibiting S1P-S1PR3 signaling in sepsis.