Significance Of Urine Sediment MiR-330-3p In Non-invasive Diagnosis Of Sepsis Induced Acute Kidney Injury

BackgroundAcute kidney injury（AKI）is a clinical syndrome characterized by rapid decline of renal function caused by a variety of causes and mechanisms.Sepsis is the most common clinical factor.About 47.9%of patients in the intensive care unit（ICU）were diagnosed with septic acute kidney injury（SA-AKI）,and this trend is increasing.And the mortality of SA-AKI is higher than that of patients with sepsis.At present,the clinical diagnosis of SA-AKI is mainly based on serum creatinine and urine volume.The results are easily affected by diet,infection and other factors.New biological indicators such as kidney injury molecule-1（Kim-1）and neutrophil gelatase associated lipocalin（NGAL）have low specificity,and there is no effective diagnostic methods at present.Mi RNA is 19-25 nucleotide length non-coding RNA that is involved in the post-transcriptional regulation of multiple genes and play key roles in the physiological and pathological processes of a variety of diseases,including acute kidney injury diseases.A variety of miRNAs information can be detected in excludeable cells in urinary sediment,and some miRNAs are expected to be non-invasive biomarkers for the diagnosis of SA-AKI.ObjectiveIn this study,miRNA high-throughput sequencing technology was used to find miRNAs with significantly different expression levels in clinical urinary sediment specimens and SA-AKI cell model among the screening cohorts.SA-AKI specific miRNA was screened by combining bioinformatics techniques.Real-time Quantitative Polymerase Chain Reaction（RT-q PCR）was used to validate the sequencing results and screen out SA-AKI specific miRNA biomarkers.To evaluate the role of the miRNA in the diagnosis of SA-AKI,and to provide some guiding significance for the discovery of biomarkers for non-invasive diagnosis of SA-AKI.Methods1 Determination of miRNA expression profile in urine sediment from septic acute kidney injury.1.1 Urine specimens of hospitalized patients in the ICU of Henan People’s Hospital from October 2020 to January 2021 were collected.The experimental group was the urine sediment of SA-AKI,the healthy control group was the urine sediment of healthy people who underwent physical examination in the same hospital and the same time,the disease control group were septic patients.Urine samples were collected in the early morning within 24 hours after the onset of sepsis and AKI,the urine sediment was obtained after centrifugation,then store samples in-80℃refrigerator.1.2 Using the miRNA high-throughput sequencing analysis technology provided by Lianchuan Biological Company and following the standard procedures provided by Illumina,including preparation of the library and sequencing experiments.4 SA-AKI patients,4 sepsis patients and 4 HC patients were selected for the determination of miRNA expression profile in urine sediment.Analyze micro RNAs’expression levels comprehensively which have statistically significant differences in each group,GO（Gene Ontology,GO）database,KEGG（Kyoto Encyclopedia of Genes and Genomes）and biological information analysis techniques were used to the further screen.2 Detection of miRNA expression profile and screening of common differential miRNA in HK-2 cell model induced by LPS.2.1HK-2 cells were stimulated by LPS to establish a SA-AKI cell model.Cell experiments were generally divided into:blank control group（NC group）and LPS（10ug/m L）treatment groups for 2h,6h,12h and 24h.The m RNA expression of inflammatory cytokines IL-1βand IL-6 in cell specimens of each group was detected by RT-q PCR.2.2 In this study,HK-2 cells were stimulated by LPS（10ug/ml）to establish a cell model.The experimental group（LPS group）was treated with LPS,and the blank control group（NC group）was treated without LPS.The sample size of each group was n=4.After 24h culture,the cells were collected by centrifugation,and1ml Trizol was added to every 10⁶cells.The extraction of RNA.After passing the RNA quality inspection,the miRNA high-throughput sequencing technology of Lianchuan Biological Company was used.Analyze miRNA’s expression levels comprehensively which have statistically significant differences in each group,GO（Gene Ontology,GO）database,KEGG（Kyoto Encyclopedia of Genes and Genomes）and biological information analysis techniques were used to the further screen.2.3 By comparing the sequencing results of Method 1.2 and Method 2.2,miRNAs of common differences of HK-2 stimulated by LPS and urinary sediment in SA-AKI patients were screened out.3 Verification of miR-330-3p expression and its significance in the early diagnosis of SA-AKI.3.1 RT-q PCR technique was used to enlarge the sample size of urine samples,and the expression level of miR-330-3p was detected in sepsis patients and SA-AKI patients.3.2 The expression level of miR-330-3p was detected by RT-q PCR at 0h and 2h,6h,12h and 24h after LPS（10ug/ml）stimulation of HK-2 cells.3.3 The correlation between miR-330-3p and the clinical indicators of SA-AKI patients was analyzed.3.4Receiver Operating Characteristic（ROC）curves were drawn to evaluate and calculate the sensitivity and specificity of miR-330-3p in the diagnosis of SA-AKI.Results1 We constructed micro RNA expression profiles of urine sediment cells in patients with SA-AKI,sepsis and HC.1.1The results showed that there were statistically significant differences in the expression levels of 71 miRNAs between the sepsis group and the HC group.There were statistically significant differences in the expression levels of a total of76 miRNAs between the SA-AKI group and the HC group.Compared with the sepsis group,a total of 43 miRNAs in the SA-AKI group had statistically significant differences（P<0.05）.1.2 Through the comparison of different miRNAs among SA-AKI group,sepsis group and HC group,it was found that 18 miRNAs were expressed in the comparison between the SA-AKI group and the sepsis group,and between the SA-AKI group and HC group,but not expressed in the comparison between the sepsis group and HC group.2 We constructed micro RNA expression profiles of NC group and HK-2 cells stimulated by LPS for 24h group.2.1 HK-2 cells were stimulated by LPS for 24 hours with higher expression of inflammatory cytokines.LPS（24h）group was selected as SA-AKI cell model experimental group.2.2 The results showed that there were 4 miRNAs expression levels in the LPS（24h）group compared with the NC group with statistical significance（P<0.05）.Compared with the NC group,the expression levels of 2 miRNAs in the LPS（24h）group were increased and 2 miRNAs were decreased.The difference between the two groups were statistically significant（P<0.05）.2.3 After screening results 1.2 and 2.2,it was found that miR-330-3p was differentially expressed in both of them.In this study,miR-330-3p was considered as a specific miRNA derived from renal tubular epithelial cells in SA-AKI urinary sediment.3 In this study,RT-PCR technology was used to verify the results of miR-330-3p and its significance for the early diagnosis of SA-AKI.3.1 Results showed that the expression levels of miR-330-3p in urine sediment of patients with sepsis and SA-AKI had statistically significant differences（P=0.0058）.The expression level of miRNA was expressed by 2^-△△Ct.The expression level of miR-330-3p in SA-AKI group was 4.641（0.733,12.90）higher than sepsis group,which was 0.689（0.482,0.997）.3.2 Results showed that the relative expression levels of HK-2 cells stimulated by LPS（10ug/ml）for 0h and 2h,6h,12h.LPS（24h）group was higher than LPS（0h）group（P<0.05）.3.3 Correlation analysis between miR-330-3p and clinical indicators was found that miR-330-3p level was correlated with white blood cells,C-reactive protein,hemoglobin,platelet,creatinine,urea and SOFA score,but the correlation was not obvious.3.4 ROC curve was drawn for miR-330-3p differentially expressed in the sepsis group and the SA-AKI group from the validation cohort.AUC represents the area under the curve.The areas under the miR-330-3p curves was 0.737.The sensitivity were 0.654 and the specificity was 0.995,95%CI was 0.573-0.899.P was 0.0064.ConclusionsThe expression level of miR-330-3p was increased in the urine sediment of SA-AKI patients and in the HK-2 cell model stimulated by LPS.Mi R-330-3p is expected to be a novel non-invasive diagnostic biomarker for SA-AKI.