| Objectives:IgA nephropathy (IgAN) is not only the most common primary glomerular disease, but also the major cause of end-stage renal failure all over the world. Approximately 45% of primary glomerulonephritis presented IgAN in China. O-linked glycans in the hinge region of IgAl molecules are incompletely galactosylated which induce IgAl deposition in the glomerular mesangium and cause mesangial activation and renal injury. However, pathoginesis of galactose-deficient IgAl synthesis in patients with IgAN has not yet been fully elucidated to date. Accumulating evidences indicate that miRNAs involve in post-transcriptional regulation and play an important role in the pathogenesis of many human diseases, including renal diseases. MiR-374b was revealed to be increased in peripheral B lymphocytes of patients with IgAN in our previous miRNA chip array analysis.In this study, we validated the change of miR-374b level and explored its clinical significance. Moreover, pathogenesis of miR-374b induced was lucubrated.Methods:Thirty cases of IgAN patients who were hospitalized and diagnosed by renal biopsy as primary IgAN in National Clinical Research Center of Kidney Diseases of Jinling hospital. Renal biopsy includes examination of light microscopy and immune pathology, and some cases were examined by electron microscopic to eliminate thin-basement membrane nephropathy. Secondary IgA nephropathy such as purpura, cirrhosis were clinically excluded. The normal control group was comprised of 15 healthy volunteers, and peripheral blood was collected. Peripheral B lymphocytes were isolated with anti-CD 19 beads, analyzed by RT-PCR, western blot and ISH, or cultured primarily in vitro. Aberrant glycosylation level of IgAl in patients’serum and supernatant was analyzed by HA A binding assay. MiRNA targeting site was evaluated by luciferase reporter assay. Expression of Cosmc and PTEN as well as cell proliferation were explored in B cells transfected with miR-374b mimics. The study was carried out in accordance with the principles of the Declaration of Helsinki and was approved by the ethics committees of Jinling Hospital.Results:The average age of enrolled patients with IgAN was 34.8+12.1 years old, and male/female ratio was 16/14. Level of proteinuria, urinary sediment red blood cells and serum creatinine was 1.53±1.19g/d,157.0(63.0～290.0)×104/ml,1.09± 0.49mg/dl respectively. Protein and miRNA level of Cosmc and PTEN were significantly decreased in B cells, and were associated with the aberrant glycosylation level of IgAl and B lymphocyte counts, respectively. Recently study reported 37 miRNAs differentially expressed in PBMCs of IgAN, among the list miR-374b was increased. We analyzed miRNAs expression profile of peripheral blood B lymphocytes isolated by anti-CD 19 beads from patients with IgAN, and found that level of miR-374b was significant increase compared to healthy controls. We also found that upregulation of miR-374b potentially regulated Cosmc and PTEN according to related analysis. Moreover, the level of miR-374b is positively related with pathological MEST score and urine protein level according to correlation analysis. MiR-374b was validated to target Cosmc by luciferase assays and western blot analysis. Transfection of primarily cultured B cells from healthy controls with miR-374b mimics reduced endogenous Cosmc and PTEN levels and lead to an abnormal glycosylation of IgAl and B lymphocytes proliferation. Conversely, inhibition of miR-374b in primarily cultured B cells of IgAN increased Cosmc and PTEN levels and suppressed the abnormal glycosylation of IgAl and B lymphocyte proliferation.Conclusions:miR-374b up expression promotes aberrant glycosylation of IgAl by targeting Cosmc in B cells of IgA nephropathy. Inhibition of miR-374b prevent the aberrant glycosylation of IgAl, thus may represent a new therapeutic approach for IgAN. |
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