Auxiliary Diagnostic Model And Pathogenesis Of Dengue-Associated Acute Kidney Injury

BackgroundDengue fever(DF)is an acute viral infectious disease caused by the dengue virus(DENV),which can cause severe dengue(SD)and has become a serious global public health problem.DF can be accompanied by renal injury and acute kidney injury(AKI)is one of the most important and less-studied complications.In 2013,the International Society of Nephrology launched the 0 by 25 campaign,which aims to improve the diagnosis and treatment of AKI worldwide by 2025 and reduce the preventable deaths from AKI to zero.AKI cases have continue to increase since the reported number of adult patients with DF increases.AKI can lead to an increased fatality rate and prolonged hospital stay in patients with DF,significantly increasing treatment difficulty.Previous studies have demonstrated that some risk factors can increase the risk of DF-associated AKI,such as age,diabetes,nephrotoxic drugs use,SD.The pathogenesis of DF-associated AKI is not fully understood and may be related to direct viral damage,host immune response,hemodynamic changes,and rhabdomyolysis.However,due to limitations in clinical sample size and research methods,there is not much research on DF-associated AKI both domestically and internationally,especially on early diagnosis,risk prediction,and pathogenesis has been limited.The clinical and laboratory characteristics of patients with DF in China differ from those in endemic areas,such as Southeast Asia.Therefore,conducting research on the early identification and pathogenesis of DF-associated AKI in China is necessary.ObjectiveThe study aimed to conduct clinical and basic research,to understand the clinical characteristics and risk factors of DF-associated AKI;to establish an feasible auxiliary diagnostic model;to screen effective diagnostic plasma protein biomarkers;to preliminarily explore whether an AG129 mice model infected with clinical isolates from patients with dengue can provide its research basis,to attempt to reveal the pathogenesis from the perspective of virus and host;to provide a basis for early diagnosis,prevention,and treatment;and to help reduce the high mortality in DF-associated AKI.Methods1.Adult patients(aged≥18 years)with laboratory confirmed DF who were hospitalized in Guangdong Province between 2013 and 2019 were selected as the research object,and the clinical data of the patients were collected and analyzed..The subjects were divided into two groups for analysis,one group included 1460 patients with DF(including dengue and SD)from Guangzhou Eighth People’s Hospital,Guangzhou Medical University,and the other group included 242 patients with SD from multiple centers in Guangdong Province.According to the Kidney Disease:Improving Global Outcomes(KDIGO)guidelines for AKI diagnosis,the patients were divided into the AKI group and non-AKI group.The incidence of AKI in patients with DF was counted,and their clinical data of AKI group and non-AKI group were compared.A Multivariate logistic analysis was used to analyze independent risk factors for AKI,calculate odds ratios(OR)and 95%confidence intervals(CI),and establish an auxiliary diagnostic model.Diagnostic ability was evaluated using the area under curve(AUC)of the receiver operating characteristic(ROC).The Kaplan–Meier survival curve was used to compare the survival of patients with SD with and without AKI.2.Altogether,1501 adult patients with laboratory confirmed DF were enrolled and divided into the AKI and non-AKI groups based on the occurrence of AKI.Serum DENV Ig M/G antibodies,non-structural protein 1(NS1)antigens,viral nucleic acid(RNA)results,DENV-1 viral load,serotype,immune status,virus isolation and sequence were collected or detected 1–2 days after admission.Matching was performed using a 1:5 nearest-neighbor matching method to compare the relationship between different serological and virological indicators and the occurrence of AKI before and after matching.A logistic regression analysis combined with ROC analysis was used to evaluate the ability to optimize the auxiliary diagnostic model.3.Plasma samples from 126 adult patients with dengue admitted to the hospital within1–2 days were included.The exclusion criteria were patients with AKI>7 days of onset and those with chronic kidney disease.Plasma samples from 66 patients with DF(12AKI group,17 SD group,and 37 DF group)and 23 control groups were selected and analyzed using a quantitative data independent acquisition proteomic method.Differentially expressed proteins were screened with a fold change of>1.2 or<0.7times and significance was set at P value<0.05.Gene ontology(GO)and Reactom databases were used to analyze the biological functional annotations of differentially expressed proteins and the enrichment of signaling pathways.Plasma from 60 patients with dengue(20 AKI and 40 non-AKI groups)and 18 controls were used.Plasma differential proteins and previously reported AKI biomarkers such as neutrophil gelatinase-associated lipocalin(NGAL),interleukin 18(IL-18)and kidney injury molecule-1(KIM-1),were detected using an enzyme-linked immunosorbent assay(ELISA).Differences in expression between the AKI groups and other groups were compared,and an ROC analysis was performed to evaluate the diagnostic value of biomarkers for DF-associated AKI.4.Eight-week-old AG129 female mice were intraperitoneally injected by 1×10~6PFU of four DENV-1 clinical isolates were challenging.Post-infection symptoms were observed.Serum DENV RNA,blood urea nitrogen(BUN)and creatinine(SCr)concentrations were measured.DENV RNA in the renal was detected 3 days post-infection.Interleukin 10(IL-10),tumor necrosis factor-α(TNF-α),and IL-18 levels in the renal were measured 9 days post-infection,and a histopathological analysis was performed.Results1.In total,1460 adult patients with DF were included in the analysis,and 40 patients(2.74%)had AKI,accounting for 57.50%patients in stage 1,22.50%in stage 2,and20.00%in stage 3.The rates of SD,length of stay,and case fatality were significantly higher in the AKI group than those in the non-AKI group(P<0.05).The multivariate logistic regression analysis revealed that older age(OR=1.049,95%CI=1.024–1.074,P<0.001),high lactate dehydrogenase(LDH)levels(OR=1.001,95%CI=1.000–1.001,P=0.039),proteinuria(OR=3.150,95%CI=1.398–7.097,P=0.006),and SD(OR=15.931,95%CI=6.990–36.31,P<0.001)were independent risk factors of DF-associated AKI.Based on the inclusion of statistically significant baseline data indicators in logistic regression analysis,combined with correlation analysis,an auxiliary diagnostic model was constructed with logit P=0.060×age+0.014×CRP+0.910×hematuria(yes=1,no=0)+1.186×proteinuria(yes=1,no=0)-8.477.In the ROC analysis,the AUC of the auxiliary diagnostic model was 0.854,the sensitivity was75.70%,and the specificity was 85.30%.2.A total of 242 adult patients with SD were included in the analysis.Of the patients,eighty-five(35.12%)had AKI,of which 37(43.53%)were in stage 1,16(18.82%)were in stage 2,and 32(37.65%)were in stage 3.The Kaplan–Meier analysis indicated that the patients with AKI had a significantly higher fatality rate than that of patients without AKI(P<0.001).After adjustment,the mortality risk in patients with stages 1,2,and 3AKI increased by 2.564,2.876,and 10.767 times,respectively.The multivariate logistic regression analysis demonstrated that hypertension(OR=2.031,95%CI=1.098-3.756,P=0.024),nephrotoxic therapy(OR=1.902,95%CI=1.004–3.602,P=0.048),respiratory distress(OR=4.149,95%CI=1.787–9.632,P=0.001),high international normalized ratio(INR)levels(OR=6.439,95%CI=1.889–21.949,P=0.003)and hematuria(OR=2.117,95%CI=1.135–3.949,P=0.018)were independent risk factors for SD-associated AKI.3.A significant difference in the incidence of AKI was observed between the patients with positive and negative DENV Ig M antibodies before matching(2.63%vs.9.61%,χ~2=23.329,P<0.001).The incidence of AKI in patients with secondary infections was significantly different from those with primary infections(6.45%vs.3.11%,χ~2=5.456,P=0.020).After matching,no significant differences in the positivity rate of DENV Ig M antibodies and secondary infections rate was identified between the AKI and non-AKI groups.Before and after matching,no statistically significant differences in DENV Ig G antibodies,NS1 antigen,serotype,RNA positivity,or viral load of DENV-1 were observed between both groups(P>0.05).The incidences of AKI in patients with DENV-1 genotypes I and V were 8.24%and 27.78%,respectively,and the differences were statistically significant(χ~2=7.998,P=0.005).The diagnostic AUC values of the auxiliary diagnostic model combined with DENV Ig M antibodies were higher than those of the auxiliary diagnostic model,with AUC values of 0.822 and 0.787,respectively.4.In the quantitative proteomics analysis,239 different proteins were identified between the AKI and control groups,of which 236 were upregulated and 3 were downregulated in abundance.The Reactom analysis revealed that they were mainly involved in metabolism,the innate immune system,and neutrophil degranulation.Overall,16 upregulated differential proteins were identified between the AKI and SD groups,and Reactom analysis suggested their main involvement in the innate immune system and cytokine signaling.Seven specific proteins were selected from the different proteins of the AKI,control and SD groups as follows:protein S100-A12(S100A12),protein S100-A11(S100A11),6-phosphogluconolactonase(PGLS),6-phosphogluconate dehydrogenase(PGD),carbamoyl-phosphate synthase 1(CPS1),adenosine homocysteine hydrolase(AHCY)and immunoglobulin kappa variable 1-5(IGKV1-5).The ELISA validation analysis revealed that the levels of S100A12,NAGL,and IL-18 were significantly higher in the AKI group than those in the control and non-AKI groups(P<0.05).No significant differences in the levels of S100A11 and KIM-1 were observed between the groups(P>0.05).Moreover,the ROC analysis revealed that the AUC for S100A12,NAGL,and IL-18 were 0.721,0.899,and 0.722,respectively.5.Compared to the control groups,the experimental groups demonstrated partial symptoms of disease,decreased body weight,and increased DENV RNA positivity in the serum.Some experimental mice demonstrated elevated BUN and SCr levels,increased renal index,and positive DENV RNA,as well as increased levels of IL-10and TNF-αexpression levels in the renal.The results of the renal pathological analysis suggested that some experimental groups had lesions that mainly manifested as mild proliferation of connective tissue,and mild inflammatory cell infiltration.ConclusionThis study is the first to focus on the early identification and pathogenesis of DF-associated AKI,from multiple levels including clinical,laboratory,and mice models.The incidence rate of AKI in patients with SD is approximately one third,which is related to hypertension,tachycardia,high INR level,hematuria and nephrotoxic drug use;Based on clinical and laboratory data,the establishment of an auxiliary diagnostic model is helpful for early disease identification;S100A12 and three other plasma biomarkers can be used to potentially diagnose plasma biomarkers;The AG129 mice model infected with clinical isolates from patients with dengue can provide the basis for its research;The excessive host immune response may play an important role in its occurrence.This article provides new evidence and research ideas for the early diagnosis and pathogenesis of DF-associated AKI and provides an important basis for guiding the individualized treatment of DF.