Differential Expression And Clinical Research Of MicroRNAs In Cardiac Surgery-associated Acute Kidney Injury

BackgroundAcute kidney injury(AKI)is a common and serious complication in hospitalized patients,which increases the in-hospital mortality.Cardiopulmonary bypass(CPB)is the second common cause of AKI after sepsis.The pathophysiology of cardiac surgery-associated acute kidney injury(CSA-AKI)is very complex and involves many factors,including ischemia-reperfusion injury,exogenous and endogenous toxins,inflammation,oxidative stress and hemodynamic factors.Nowadays it is considered that cardiopulmonary bypass(CPB)is the main pathogenic factor of CSA-AKI.Patients with preoperative high risk factors,such as age,diabetes,heart failure,and heart surgery requiring prolonged CPB,increased the incidence of CSA-AKI by about 30 to 40%.At present,the incidence of CSA-AKI is up with the rising number of preoperative complications and patients requiring complex cardiac surgery.Therefore,CSA-AKI is a clinical syndrome that needs to be elucidated urgently.Patients with CSA-AKI are ideal subjects to study AKI,because the time of injury is known.Until now,the pathogenesis of CSA-AKI is still unclear,and renal injury cannot be judged in time by some biomarkers.Micro RNAs(miRNAs,miRs)are a class of non-coding small RNAs,ranging in length from 20 to 24 nucleotides,that negatively regulate gene expression at the posttranscriptional level by binding to the 3’-untranslated coding regions of targeted messenger RNA(m RNA)to inhibit the translation of targeted proteins.The study of miRNAs on renal diseases has been a hot topic in recent years.Current studies on animal models and some types of AKI,such as AKI of ischemia-reperfusion injury and AKI of kidney transplantation,have accumulated evidence that multiple miRNAs may be involved in the pathogenesis of AKI,showing its potential functions and providing a new way to study the molecular mechanism of CSA-AKI.The miRNAs of CSA-AKI secreted into circulation or urine can be used as biomarkers with a high sensitivity and specificity.miRNAs may be a new way to understand the occurrence,development and outcome of CSA-AKI.ObjectiveScreen and analyze the differentially expressed miRNAs between CSA-AKI and nonCSA-AKI patients.Get the miRNAs involved in the pathogenesis of CSA-AKI.Select some miRNA related to CSA-AKI for futher clinical validation.MethodsSerum samples from patients undergoing cardiac surgery with cardiopulmonary bypass(CPB)were screened by microarray to detect the expression of micro RNAs in CSA-AKI patients and non-CSA-AKI patients.The subject was approved by the medical ethics committee of the First Affiliated Hospital of Anhui Medical University,and all patients have signed the informed consent.Adult patients with cardiac surgery under CPB who underwent heart valve operations with or without coronary artery bypass grafting(CABG)in the First Affiliated Hospital of Anhui Medical University were selected.The CSA-AKI diagnosis was based on the 2012 Kidney Disease Improvement Global Outcome(KDIGO)criteria.When elevated serum creatinine was not less than 0.3 mg/dl(26.5mmol/L)within 48 hours after cardiac surgery or increased 1.5 times above the preoperative baseline within 7 days after cardiac surgery.All patients underwent routine preoperative examination,intraoperative anesthesia and operation,postoperative intensive care and routine examination.All patients had no special circumstances.The clinical data of preoperative(age,sex,weight,height,New York Heart Association functional class),intraoperative(CPB time,aortic cross clamp time,operation time),postoperative(mechanical ventilation time,ICU stay time,hospital stay)were collected.On about 24 hours after the patient enters the ICU,5 ml of blood from the radial artery is extracted and stored in the-80° refrigerator after static,centrifugal and sub-packed,avoiding repeated freezing and thawing.After collection,the miRNAs chip is screened by uniform sampling.Edge R software was used to analyze the differential expression of miRNAs between groups.The screening criteria for differential expression of miRNAs were over 2 times the expression level and the P value was less than 0.05.The miRNAs associated with CSA-AKI were screened by microarray technology and further validated by fluorescence quantitative PCR.The time point between after general anesthesia and before median sternotomy in the surgery(1),the time point after 1 hour(2),3 hours(3),6 hours(4),12 hours(5)and 24 hours(6)in the intensive care unit.5 ml of radial artery blood was collected on above six time points,and finally stored in-80 ° refrigerator(the same experiment operation as above).Indicators included some CSA-AKI-related miRNA(fluorescence quantitative polymerase chain reaction),serum tumor necrosis factor-A(TNF-a),monocyte chemoattractant factor-1(MCP-1)and neutrophil gelatinase-associated lipoprotein(NGAL)(polymerase chain reaction),serum interleukin-6(IL-6)and interleukin-10(IL-10)(chemiluminescence methods).ResultThe serum samples from 7 patients were screened by microarray,including 3 patients in non-AKI group and 4 patients in CSA-AKI group.Four patients with AKI had transient AKI and their renal function recovered to the preoperative baseline before their discharge.The clinical data of the two groups were similar before,during and after operation.The results showed that 38 miRNAs were differentially expressed between the two groups,including 24 up-regulated miRNAs and 14 down-regulated miRNAs.CSA-AKI-related hsa-miR-494-3p increased to a peak at 1 hour after cardiac surgery in AKI group(n=10),and was still significantly different from that in NON-AKI group(n=8)at 3 hours and 6 hours after cardiac surgery(P< 0.05).The NGAL level in AKI group was statistically different from that in NON-AKI group at 3 hours after operation(P<0.05).There was no significant difference in serum IL-6 levels between the two groups at any time point within 24 hours after cardiac surgery and 48 hours and 72 hours after cardiac surgery(P> 0.05).The IL-10,MCP-1 and TNF-a levels were not significantly different between the two groups at any time point within 24 hours after cardiac surgery(P> 0.05).C-reactive protein(CRP)levels continued to rise on the first day,the second day and the third day after cardiac surgery,and CRP levels were higher in AKI group than in NONAKI group,but there was no significant difference in CRP levels between the two groups at any time point(P> 0.05).White blood cell count(WBC)increased gadually after cardiac surgery in both groups and decreased after peaking at about 48 hours.However,there was no significant difference between the two groups at any time point(P> 0.05).The percentage of neutrophils(N(%))increased gradually after cardiac surgery in both groups and decreased slowly after peaking at about 48 hours.However,there was no significant difference in N(%)between the two groups at any time point(P > 0.05).There was no significant difference in albumin(ALB),total bilirubin(TBIL)and alanine aminotransferase(ALT)between the two groups at any time point within seven days after cardiac surgery(P> 0.05).ConclusionHigh-throughput microarray technology was used to screen the differentially expressed miRNAs out between CSA-AKI patients and non-CSA-AKI patients.There were 38 known human miRNAs,of which 24 were up-regulated and 14 were down-regulated.The differential expression of miRNAs may reveal the pathogenesis of CSA-AKI.Among the 38 screened CSA-AKI-related miRNAs,hsa-miR-494-3p was identified as a molecular biomarker for early diagnosis of CSA-AKI,and hsa-miR-494-3p may be involved in the early stage of CSA-AKI and may be as a predictor of prognosis in CSAAKI.