Screening And Verifying The Specific MiRNAs Of Urinary Sediment In IgA Nephropathy And Analyzing The Cell Source

Background and objectives：Immunoglobulin A nephropathy（IgAN）is the most commonchronic kidney disease and the most important cause of end stage renal diseas（eESRD）in China. Formost IgAN is progressive disease，biomarkers reflecting condition timely are required to assesspatient’s condition, help diagnosis, response to therapy or prognosis. Exploring the miRNAs in urinesediment was a new field since2010in IgAN biomarkers. A few beneficial attempts carrying out bysome scholars has found some miRNAs were associated with the pathologic and clinical assessmentin IgAN. Unfortunately, most of the researches were small samples, single center or reflecting thecharacteristic of disease insufficiently. We first used the miRNA microarray technology and screennew nucleic acid biomarkers by high-throughput. Then, some miRNAs were verified by quantitativeReal-time PCR. At last, these miRNAs were confirmed as specific biomarkers for IgAN, and theirroles in the diagnosis and condition assessment of IgAN were illustrated. The source of cells andpossible roles discovered in deeply study may lay a solid foundation for the mechanism research inthe future.Methods: The urine from patients who were first accepted renal biopsy and proven IgAN inChinese PLA General Hospital was collected during Nov.2012to Jan.2014. Urine from the firstbiopsy-proven primary glomerulonephritis (non-IgAN) patients and healthy people were alsocollected as controls. The age and genders among all the groups were matched. Next, a centrifugeseparates urine sediment from the urine sample collected before the renal biopsy. Lee’s classificationand semi-quantitative score were adopted in the renal pathology.1、Urinary sediment miRNAmicroarray：Total miRNAs were extracted from urine sediment and added to miRNA microarrayV19.0, Agilent. Then, all differential expression miRNAs between9IgAN patients (3in LEE’s II，3in LEE’s III and3in LEE’s IV-V) and3normal persons were screened out. A comparative analysiswas carried out between our microarray results and miRNA microarray results in other tissues onIgAN in literatures. Then, some miRNAs having accordant trend were found out.2、Validating thereliability and specificity of these biomarkers in urine sediment：Differential expressed miRNAswere further validated in a large sample of84IgAN patients,29normal persons and21diseasecontrol patients by SYBR Green fluorescence quantitative PCR. Receiver operating characteristiccurve (ROC curve) was plotted for assessing the sensitivity and specificity of these biomarkers inIgAN. Relationships between above miRNAs and clinic, pathology or prognosis were also analyzed.Urine red blood cells in urine sediment were extracted by CD235a magnetic bead. Andmicrovesicles in urine supernatant were extracted by ultracentrifugation. It can reveal the source of cells and the possible roles of miRNA biomarkers.Resμlts:1.214differential expressed miRNAs were screened out by high-throughput miRNAmicroarray technology used in3normal persons and9IgA Nephropathy patients.92differentialexpressed miRNAs were also found out between Lee’s II, III or IV-V grade of IgAN. Some miRNAswith the same expressing trend were picked out by further comparative analysis of our miRNAmicroarray results with IgAN renal tissue or peripheral blood leukocyte miRNA microarray resultsin literatures. Compared with renal tissue microarray results,54.55%of miRNAs was withconsistence expressed and33.78%oppositely. Compared with the peripheral blood leukocyte,48.65%of miRNAs have the same trend and2oppositely. miR-98, miR-148b, let-7d, miR-374b,miR-17and miR-502-3p were all differential expression in all microarray results.2. Compared with normal control group (29samples) and disease control group (21samples),miR-25, miR-144, miR-486in urine sediment were significantly increased in IgAN group (84samples). In contrast, miR-135a and miR-150expression levels were significantly decreased.Specificity and sensitive was100%and89.9%respectively by conjoint analysis of urine sedimentmiR-25, miR-144and miR-486, which were confirmed as specific molecular biomarkers in IgAN. Inaddition, miR-144, miR-486and miR-150expression levels were significantly different in differentLEE’s classification subgroup, wherein miR-144and miR-486expression levels were the highest instage III, whereas miR-150level was the highest in the IV-V stage in IgAN group. miR-150showeda positive correlation with global sclerosis. Meanwhile, miR-144, miR-135a, miR-150had a positivecorrelation with segmental sclerosis. miR-25and miR-486had a negative correlation with crescent,and miR-135a had a positive correlation with crescent. The expression level of miR-486wassignificantly higher in IgAN with renal vascular disease than those without vascular disease. Theexpression of miR-150was also significantly different between mild, moderate or severe interstitialfibrosis. It was significantly higher in the severe fibrosis group than in mild, moderate interstitialfibrosis groups. In addition, miR-135a, miR-25miR-486were closely related to segmentalthickening and stratification of Baumann’s capsule wall. miR-25, miR-144, miR-486, miR-150had apositive correlation with serum cystatin. miR-486, miR-150showed a positive correlation withserum creatinine. Whereas miR-486was negatively correlated with eGFR.3. We first found that miR-25, miR-144and miR-486expression were significantly higher infull vision of red blood cells in urine than in the non-full vision group. miRNAs expression levelswere greatly increased in the red blood cell group (positive sorting group) compared with freeerythrocyte group (negative sorting group) by CD235a magnetic bead separation. The abovemiRNAs expression levels were low in urine sediment of normal persons, but significantly increasedwhen added their red blood cells to their own urine sediment. Finally, the major cell components(including peripheral blood mononuclear cells, red blood cells, tubular epithelial cells, etc.)contained in urine sediment were separated and purified. It showed that the baseline miRNA levelsof miR-25, miR-144and miR-486were significantly higher in the red blood cells than in other cells. 4. The expression of miR-144and miR-486had no significant difference between IgAN groupof blood erythrocyte and normal control group of blood erythrocyte, while miR-25level wassignificantly lower in IgAN group of blood erythrocyte. Surgical hematuria model was made byadding IgAN red blood cells to their own urine supernatant. Deeply study showed that miR-25,miR-144and miR-486expression levels were significantly higher in renal hematuria group than insurgical hematuria model group. In addition, the expression levels of miR-144and miR-486inmicrovesicles extracted from urine supernatant were also higher in IgAN group than the normalcontrol group.5. We have found that miR-25and miR-486came from erythrocytes had a strong relation withsegmental thickening and hierarchical of Bowmann capsule wall, and levels in segmental thickeningand hierarchical were higher than those in non-segmental thickening and hierarchical group. At thesame time, segmental thickening and hierarchical of Bowmann capsule wall in IgAN patients is71.43%, which was significant higher than9.52%in control group. It is consistent with theexpression levels of miR-25and miR-486in urine sediment of IgAN group was higher than diseasecontrol group. In the group of macrohematuria with proteinuria, the proportion of diffuse thickeningof the Bowman capsule (72.34%) is significantly higher than the group of microhematuria withproteinuria group (54.5%).Conclusions: Urinary sediment miRNAs were turn out to be good biomarkers in thenon-invasive diagnosis and condition assessment of IgAN. In this study, miRNA microarraybioinformatics technology was used for analyzing miRNA expression profiling of urine sediment ofIgAN, and found similarities and differences of the expression profile derived from IgAN renaltissue and peripheral blood leukocyte. Moreover, these miRNAs can be used to filter out thenon-invasive diagnosis of IgAN, not only has the disease diagnostic specificity, but also has goodcorrelation with clinical and pathological indicators. miR-25, miR-144and miR-486in the urinesediment mainly comes from the urine red blood cells, and has close relationship in thickening andstratification of Bowman capsule wall. Urine red blood cells may play a biological role throughreleasing microvesicles, and involve in patho-physiological changes of IgAN.