The Association Of Serum MicroRNA With The Histological Score For Non-alcoholilc Steatohepatitis And The Risk Assessment Of Concomitant Chronic Kidney Disease

Part 1 The association of serum microRNA with the histological score for non-alcoholilc steatohepatitis Background and aims:Nonalcoholic liver disease(NAFLD)is the most common form of chronic fatty liver diseases in the world,with an estimated prevalence of 19~46%.NAFLD encompasses a wide spectrum of liver diseases,ranging from nonalcoholic fatty liver(NAFL)to nonalcoholic steatohepatitis(NASH).NAFL is usually non-progressive,whereas NASH is associated with an increased risk of progressing to cirrhosis and hepatocellular carcinoma.Liver biopsy remains the gold standard for the diagnosis of NASH.However,potential complication together with sampling and interpretation variability limit its utilization for screening in a high-risk population.Therefore,there is an urgent need for identifying novel biomarkers improving the detection of NASH.Recently,serum microRNAs have been proposed as potential diagnostic tools in different clinical settings.To date,studies of miRNA expression in NAFLD patients have shown inconsistent results.Thus,this study aims to examine the diagnostic performance of serum miR-122,miR-34 a,miR-21,miR-375,CK-18,and multivariate diagnostic panel for the diagnosis of NAFLD and NASH.Method:A total of 73 NAFLD patients and 170 healthy controls were included in this study.The diagnosis of fatty liver was based on biopsy-confirmed histological findings.NAFLD patients were further diagnosed as either NASH(n=41)or without NASH(n=32)in accordance with NASH clinical research network scoring system.Reverse-transcription polymerase chain reaction(RT-qPCR)was used to detect the level of serum miR-122,miR-34 a,miR-21,and miR-375.The serum concentration of CK-18 was measured by the one-step in vitro immunoassay M30-apoptosense ELISA kit.The multivariate diagnostic panel for NAFLD or NASH was developed with a logistic regression model.Receiver operating characteristic(ROC)curves were used to assess the diagnostic accuracy of serum miR-122,miR-34 a,miR-21,miR-375,and multivariate diagnostic panel for NAFLD or NASH.Results:Serum miRNA expression profile is significant different between NAFLD patients and healthy controls(miR-122 a,5.01 fold-change upregulated;miR-34 a,3.62 fold-change upregulated;and miR-21,4.61 fold-change upregulated).Significant fold changes were observed in the level of serum miR-122 between NASH,without NASH and healthy controls(2.08-fold change in NASH vs.without NASH and 3.12-fold change in without NASH vs.healthy controls).Similarly,significant changes were observed in the level of serum miR-34 a between three groups(1.73-fold change in NASH vs.without NASH and 2.57-fold change in without NASH vs.healthy controls).Again,serum miR-21 were significantly upregulated between three groups(2.18-fold change in NASH vs.without NASH and 3.26-fold change in without NASH vs.healthy controls).There is no significant difference in the level of miR-375 between three groups.We developed a multivariate panel(3-miRNA+CK-18 panel)for the diagnosis of NAFLD with an AUROC of 0.916(95%CI: 0.874-0.948).Further,such 3-miRNA+CK-18 panel provided an AUROC of 0.942 for the diagnosis of NASH.Conclusion:Three identified miRNAs were differentially expressed between NAFLD,NASH patients,and healthy controls.Further,a 3-miRNA+CK-18 panel can be used as a useful tool for identifying NAFLD and NASH.In future,large sample studies investigating the specific pattern of altered serum microRNA expression in NAFLD patients are still needed.Part 2 The utility of serum micro RNA-21 in the risk assessment for chronic kidney disease in NAFLD patientsBackground and aims: Nonalcoholic fatty liver disease(NAFLD)is the most common chronic liver disease across the worldwide.NAFLD is not only associated with liver-related morbidity and mortality but also increase the risk of chronic kidney disease(CKD).Increasing evidence suggests that liver fibrosis stage may have the potential to identify subjects at high risk for CKD in NAFLD patients.In clinical practice,liver biopsy is unfeasible because of its various limitations.Against this,several invasive scores for liver fibrosis stage(FIB-4 and AST/ALT score as serum biomarkers,and liver stiffness by transient elastography)have been proposed and shown to serve as biomarkers for detecting CKD among NAFLD patients.However,given the fact that the accuracy of current serum biomarkers is moderate,it is imperative to develop novel highly accurate biomarkers.Recently,serum micro RNA-21 has been suggested to play a vital role in the pathogenesis of CKD and could serve as a biomarker for diagnosing CKD,while their accuracy in detecting CKD among NAFLD patients remains unclear.Thus,this study aims to assess the diagnostic performance of serum micro RNA-21,liver stiffness,and serum biomarker in identifying CKD in NAFLD patients.Methods: A total of 1415 NAFLD patients were included in this study.The diagnosis of fatty liver was on the basis of the criteria proposed by the Asian Pacific Association for Study of the Liver.Diabetes mellitus was diagnosed according to the American Diabetes Association criteria.Estimated glomerular filtration rate(e GFR)was calculated by using the four-variable Modification of Diet in Renal Disease(MDRD)study equation as follows: e GFR =175×(serum creatinine-1.154)×(age-0.203)×1.212(if black)×0.742(if female).CKD was defined as the presence of e GFR?60 ml/min/1.73 m2.The FIB-4 score and AST/ALT score were calculated from blood tests,and liver stiffness was measured using transient elastography.Reverse-transcription polymerase chain reaction(RT-q PCR)was used to detect the level of serum micro RNA-21.The multivariate diagnostic panel for CKD was developed with a logistic regression model.Receiver operating characteristic(ROC)curves were used to assess the diagnostic accuracy of serum micro RNA-21,liver stiffness,serum biomarkers and multivariate diagnostic panel for CKD among NAFLD patients.Results: The mean age for the entire cohort was 52.1±4.9 years.There were 787(55.6%)men.The main characteristics were as follows: CKD in 136 patients(9.6%);current smokers in 141 patients(10%);diabetes mellitus in 234 patients(16.5%);hypertension in 466 patients(32.9%).The patients with CKD were older than those without CKD(55.7± 4.7 vs.51.7± 4.8,p<0.001),and had higher levels of BMI,AST,ALT,serum uric acid(p<0.001).They also had a higher prevalence of diabetes mellitus(25% vs.15.6%,p<0.01).There were no significant differences in gender,waist circumference,and current smokers,diastolic blood pressure(DBP),GGT,PLT,TG and LDL between these two groups.Further,serum mi RNA-21,liver stiffness,FIB-4 score,and ALT/AST score were significantly elevated in CKD patients,compared with those without CKD(P<0.001).The diagnostic performance(AUROC)of serum mi RNA-21,liver stiffness,FIB-4 score and AST/ALT score was 0.724(95%CI: 0.700-0.747),0.694(95%CI: 0.670-0.718),0.707(95%CI: 0.682-0.730),and 0.712(95%CI: 0.688-0.736).Multivariate logistic regression identified four independent indicators of CKD: age(OR=1.28;95%CI: 1.20-1.38;p<0.05),diabetes mellitus(OR=1.83;95%CI: 1.12-2.97;p<0.05),serum uric acid(OR=1.03;95%CI: 1.01-1.04;p<0.05),serum mi RNA-21(OR=1.35;95%CI: 1.25-1.50;p<0.05).Moreover,a four variables diagnostic panel for CKD was established on the basis of the logistic regression model with the equation as follows: logit(P)= 0.30*serum mi RNA-21 + 0.25*age + 0.60*diabetes(1 or 0)+ 0.03*serum uric acid-23.79.The diagnostic performance of the four variables diagnostic panel for identifying CKD among NAFLD patients was 0.853(AUROC;95%CI: 0.843-0.871),which is significantly higher than that of a single application of serum mi RNA-21,liver stiffness,FIB-4 score,and AST/ALT score.Conclusions: Serum mi RNA-21 and liver stiffness could serve as biomarkers for CKD in ultrasonography-diagnosed NAFLD patients.Further,a four variables panel(serum mi RNA-21,age,diabetes mellitus,and serum uric acid)could be a useful tool for identifying subjects at high risk for CKD in NAFLD patients.Patients with NAFLD should be closely monitored for the development and progression of incident CKD.Further prospective studies are warranted to establish the causal relationship between the severity of NAFLD and CKD.