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Abstract: TH-PO927

The Regulation of MicroRNAs on Galactose-Deficient IgA1 and Its Pathogenic Mechanism in IgA Nephropathy

Session Information

Category: CKD (Non-Dialysis)

  • 1903 CKD (Non-Dialysis): Mechanisms

Authors

  • Liang, Ludan, Zhejiang University, Hangzhou, China
  • Weng, Chunhua, Zhejiang University, Hangzhou, China
Background

Abnormal increase of Gd-IgA1 is a recognized pathogenic factor of IgAN. Glycosylation of IgA1 is dependent on 3 key enzymes (C1GALT1, COSMC, ST6GALNAC2). Deposition of Gd-IgA1 in kidney mesangium causes IgAN, whereas the mechanism is not elucidated yet. This study focuses on how microRNA interfere IgA1 glycosylation by targeting C1GALT1, and futher leading to mesangial cell malfunction, thereby shedding light on the mechanism of IgAN.

Methods

Part 1. 1) Screen candidate miRNAs targeting C1GALT1, verification of targeted regulation by Luciferase Reporter System, RT-PCR and Western Blot; 2) Change expression level of miRNA in DAKIKI cell, detect Gd-IgA1/IgA1 in the culture medium by ELISA; 3) Stimulate Human Mesangial Cells with Gd-IgA1 in vitro, analyse cell cycle by Flow cytometry.
Part 2. 1) Analyse miRNA expression in PBMC, serum Gd-IgA1 level in IgAN and HC; 2) Conduct RNA-seq of glomerulus, analyse DEGs in different kidney cell types, especially in mesangial cells. Calculate DEGs expression change in HMC stimulated by Gd-IgA1.

Results

In Part 1, We screened out 6 candidate miRNAs possibly targeting C1GALT1. miR-152 most effecienty binds to C1GALT1 3’UTR region, inhibits C1GALT1 at both transcriptional and translational level. Overexpression of miR-152 mimics in DAKIKI obviously increases the Gd-IgA1/IgA1 in the culture medium. Stimulation of HMC by Gd-IgA1 drives the cell cycle towards S phase and G2/M phase, which possibly indicates cell proliferation.
In Part 2, expression level of miR-152 was much higher in PBMC of IgAN than of HC. Gd-IgA1 serum concentration was obviously higher in IgAN than in HC. Subgroup analysis revealed that IgA1/IgA ratio is related to S-score in MEST-C Oxford classification system of IgAN. RNA-seq of glomerulus revealed 4 DEGs (SERPINE1, USP2, SOX9, KLF6) closely related to mesangial cells. Stimualting HMC with Gd-IgA1 significantly increased the expression level of those 4 markers, among whom, increasement of SERPINE1 mRNA is the most remarkable, in a dose- and time-dependent manner.

Conclusion

miR-152, which is highly expressed in PBMC of IgAN patients, obstructs the glycosylation process of IgA1 by targeting C1GALT1, resulting in elevation of Gd-IgA1, which will further stimulate kidney mesangial cells, increase the expression level of mesangial cell markers, especially SERPINE1, promote cell proliferation, finally leads to IgAN.