The Role And Mechanism Of Platelet In Acute Kidney Injury

Acute kidney injury（AKI）is a common clinical critical illness with no effective treatment currently.There are many pathogenic factors leading to AKI.From the way of kidney injury,it is mainly divided into ischemia-reperfusion injury（I/R）and nephrotoxic substances（mainly by toxic drugs,rhabdomyolysis,etc.）.Claudia Schwarzenberger reported that in mice with glomerular endothelial cell injury induced by concanavalin/anti-concanavalin antibody,removal of platelets or the use of the platelet inhibitor clopidogrel can alleviate kidney damage.In glomerular diseases,platelet-secreting active substances（pro-inflammatory mediators,growth factors,etc.）alter the permeability of the glomerular basement membrane,leading to deposition of immune complexes that disrupt kidney structure and function.Takaharu Tanaka’s study demonstrated that human platelets aggravate glomerular damage by stimulating mesangial cells to produce monocyte chemoattractant protein-1（MCP-1）,a process associated with the CD40/CD40L pathway.However,the role and the specific mechanism of platelet in acute kidney injury are still unclear.In this study,the rat model of ischemia-reperfusion-induced acute kidney injury（I/R AKI）and the mouse model of rhabdomyolysis-induced acute kidney injury（RM-AKI）were used to elucidate the role and mechanism of platelet and its receptors in acute kidney injury induced by different causes,and provide a new strategy for prevention and treatment of acute kidney injury.Part 1 The role of platelets in ischemia-reperfusion-induced acute kidney injuryAims:Ischemia reperfusion is a common cause of acute kidney injury,which in turn causes coagulation activation and inflammation,leading to necrosis of renal tubular epithelial cells（TECs）.Platelets play an important role in coagulation and inflammation,and platelet activation has been shown to exacerbate acute kidney injury.However,the activation mechanism of platelets in ischemia-reperfusion injury and how activated platelets damage kidney tissue are not clear.Finding out the reason of platelet activation and the relationship between activated platelets and renal tubular epithelial cell damage to elaborate the role of platelet played in ischemia-reperfusion-induced acute kidney injury.Method:SD rats were used to prepare ischemia-reperfusion-induced acute kidney injury model after left renal pedicle was clamped for 60 min.Blood and kidney were harvested by drawing from the inferior vena cava and body perfusion respectively.Serum creatine（Cr）and blood urea nitrogen（BUN）were detected for renal function;platelet aggregation and release were valued by the aggregometer（Chrono-log）;GPVI,P2Y₁₂ andαIIb protein expression of platelet was detected by Western blot and kidney injury was observed by renal tissue hematoxylin and eosin（HE）stain.Platelet-poor plasma（PPP）of I/R AKI rats was used to incubate washed platelets of Sham rats to value platelet aggregation.In vitro,human renal tubular epithelial cells（HK-2）and rat renal tubular epithelial cells（NRK）were hypoxic for 9 h and 12 h respectively and then reoxygenated for 2 h to simulate I/R injury.Then the effect of cell supernatant on platelet aggregation and release function was examined by incubating with normal washed platelets.The supernatant of activated platelets was used to incubate with the hypoxic-treated TECs during reoxygenation period,then the cell viability was detected by CCK-8 kit.Results:The platelet functions as aggregation and release of I/R AKI rats were significantly enhanced compared with that of the Sham group,and the platelet-poor plasma of I/R AKI rats increased the platelet aggregation of Sham rats.The culture supernatant of TECs treated with hypoxia-reoxygenation（H/R）enhanced normal platelet aggregation,and the supernatant of platelet release promoted the apoptosis of TECs treated by hypoxia-reoxygenation.While the expression of platelet GPVI,P2Y₁₂ andαIIb in I/R AKI rats was unchanged.Conclusion:In the I/R AKI rat model,platelet activity was significantly enhanced,while the expression of membrane receptors GPVI,P2Y₁₂ andαIIb was unchanged.The substances released by damaged renal tubular epithelial cells activated platelets,conversely,substances released by platelet activation also aggravated renal tubular epithelial cell damage,forming a vicious circle of"tubule epithelial cell damage-platelet activation".Part 2 The role and underlying mechanism of platelet GPVI in rhabdomyolysis-induced acute kidney injury miceAims: Rhabdomyolysis causes muscle cell damage to release large amounts of intracellular substances into the blood,which is an important factor in causing acute kidney injury.However,the relationship between heme and platelets in RM-AKI has not been reported,and the specific role of platelets and their receptors in AKI and its mechanism are still unclear.Studying the causes of platelet activation and changes in platelet receptor expression to reveal its role and working mechanism in RM-AKI mice.Method: 50% glycerol（10 m L/kg）was injected into the thigh muscle of mice to prepare RM-AKI mouse model.Blood and kidneys were harvested at 48 h after glycerol injection.For the platelet GPVI depletion RM-AKI mouse model,mice were injected with JAQ1（2 μg/g）through caudal vein,while the control group mice were given the same dose of Ig G2 a,and on the fourth day,50% glycerol（5 m L/kg）was applied to thigh muscle to prepare RM-AKI model.After 24 h,blood and kidneys were collected.Mouse platelets and human megakaryocytes（Meg-01）were incubated with Heme 50/100 μM for 30 min or 24 h respectively to detect platelet function and the protein expression of platelet and Meg-01 cells.Resting platelets,thrombin（0.2 U/m L）activated platelets,and thrombin-activated platelets with pre-incubated JAQ1 for 5 min were incubated with mouse macrophages for 3 h to induce METs formation.Biochemical kit was used to detect BUN and Cr;HE staining was used to observe renal tissue pathological changes;immunohistochemical technique was used to detect platelet infiltration of renal tissue;fluorescence immunoassay was used to detect METs in renal tissue and in vitro experiment;the aggregometer（Chrono-log）was used to value platelet aggregation and release;flow cytometry was used to detect platelet P-selectin,JON/A and GPVI expression;Western blot was used to detect GPVI,P2Y12 and αIIb protein expression of platelets and Meg-01 cells.Results: The platelet aggregation,release and plaque retraction function of RM-AKI mice were higher than those of the control mice,and the expression of GPVI was significantly increased,while the expression of P2Y12 and αIIb was unchanged.Heme increased platelet surface P-selectin,JON/A and GPVI expression,and directly induce platelet aggregation and release.However,blocking GPVI leaded to Heme-induced platelet aggregation to be blocked.Western blot showed that heme enhanced PLCγ2,Syk phosphorylation in GPVI downstream signaling pathway and increased platelet GPVI expression without affecting the expression of P2Y12 and αIIb.Heme did not affect the expression of GPVI,P2Y12 and αIIb in Meg-01 cells.The degree of renal injury and METs formation in renal tissue of RM-AKI mice given JAQ1 were significantly reduced compared with these of control mice.Blocking platelet GPVI with JAQ1 reduced platelet-induced METs formation in vitro experiment.Conclusion: RM-AKI mice have enhanced platelet activity and increased GPVI expression;heme activates platelets through GPVI,directly induces platelet aggregation and specifically increases platelet GPVI expression;METs formation and renal injury are reduced by blocking platelet GPVI in RM-AKI mice.Therefore,platelet GPVI may be a candidate target for RM-AKI,suggesting a novel alternative treatment of AKI.