Profiling And Initial Validation Of Urinary MicroRNAs As Biomarkers In IgA Nephropathy

BACKGROUND:Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, and also an important cause of end stage renal disease. MicroRNAs (miRNAs) have been found in virtually all body fluids and used successfully as biomarkers for various diseases. Evidence indicates that miRNAs have important roles in IgAN. However, the global urinary profile of miRNAs in patients with IgAN is unknown.METHODS:Patients who received renal biopsy from December 2013 to January 2015 in the Nephrology Department at the Chinese PLA General Hospital were recruited for study. Membranous nephropathy (MN) patients and minimal changes disease (MCD) patients were recruited as disease controls. Early morning urine specimen were collected at the day of renal biopsy. (1) Affymetrix Gene Chip miRNA 4.0 Array was used to profile miRNA expressions. (2) Real-time quantitative polymerase chain reaction (RT-qPCR) was carried out to verify the candidate miRNAs revealed by microarray. Receiver operating characteristic curve (ROC) was constructed for differentially expressed miRNAs to evaluate the efficiency of these miRNAs in diagnosis of IgAN. (3) Target genes of differentially expressed miRNAs were predicted by miRWalk. The GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) database analyses were conducted using a DAVID (The Database for Annotation, Visualization and Integrated Discovery) online analysis tool.RESULTS:The screening cohort included 18 IgAN patients,6 healthy subjects and 8 disease controls (4 MN patients and 4 MCD patients), while the validation cohort included 102 IgAN patients,41 MN patients,27 MCD patients and 34 healthy subjects. (1) At the screening phase, significance analysis of microarrays analysis showed that no miRNA was differentially expressed in the IgAN group compared to all control groups. Subgroup analyses showed that there was an overlap of 4 miRNAs which differentiates IgAN Ⅰ-Ⅱ and all of the control groups (all P< 0.05, fold change> 2 or< 0.5). Two of them were in high levels (miR-223-3p and miR-629-5p), while two of them were in low levels (miR-3613-3p and miR-4668-5p). An overlap of 6 miRNAs differentiated IgAN Ⅲ from all of the controls (all P< 0.05, fold change> 2 or< 0.5). Three of them were in high levels (miR-150-5p, miR-371b-5p and miR-572), while three were in low levels (miR-3613-3p, miR-4668-5p and miR-6750-5p). Seven out of the above-mentioned miRNAs were validated by RT-qPCR in the screening cohort.(2) At the validation phase, RT-qPCR results showed that urinary level of miR-150-5p in IgAN was obviously higher (all P< 0.05) than that in the control groups, while the level of miR-3613-3p was obviously lower (all P< 0.05). Areas under the ROC curve (AUC) were found to be 0.817 and 0.8, respectively. The specificity and the sensitivity of each miRNAs were 64.2% and 84.8%,67.4% and 92.8%, respectively. In IgAN, urinary level of miR-3613-3p was significantly correlated with urine protein, estimated glomerular filtration rate, glomerulosclerosis and Lee’s grades. (3) GO function analysis showed that the functions of predicted target genes of miR-150-5p were enriched in cell proliferation, differentiation and activation of lymphocytes, and regulation of immune response, while the KEGG analysis showed significant enrichment in the Wnt signaling pathway and MAPK pathway. Similarly, the GO function of miR-3613-3p were enriched in the regulation of glucose metabolic process and B cell differentiation, and the KEGG pathways were enriched in the Wnt signaling pathway.CONCLUSIONS:The expression profile of miRNAs was significantly altered in urinary sediments from patients with IgAN. The expression patterns of miR-150-5p and miR-3613-3p in urinary sediments of IgAN patients had certain specificity, and the level of miR-3613-3p were related to the severity of IgAN, indicating these deregulated miRNAs have great potentials of becoming detectable noninvasive biomarkers in IgAN. Further studies are needed to explore the roles of miR-150-5p and miR-3613-3p in the pathogenesis and progression of IgA nephropathy.