MicroRNA-23b-3p Deletion Induces an IgA Nephropathy-like Disease Associated with Dysregulated Mucosal IgA Synthesis

Abstract

Background: IgA nephropathy (IgAN) is the most common primary GN worldwide. Circulating immune complexes form that are prone to deposition in the mesangium, where they trigger glomerular inflammation. A growing body of evidence suggests that dysregulated expression of microRNAs in IgAN may play a significant role in establishing the disease phenotype.

Methods: We generated single miR-23b-3p(miR-23b) knockout mice using CRISPR-Cas9.

Results: In humans, miR-23b levels are downregulated in kidney biopsies and sera of patients with IgAN, and serum miR-23b levels are negatively correlated with serum IgA1 levels. We show that miR-23b^-/- mice develop an IgAN-like phenotype of mesangial IgA and C3 deposition associated with development of albuminuria, hypertension, an elevated serum creatinine, and dysregulated mucosal IgA synthesis. Dysregulation of IgA production is likely mediated by the loss of miR-23b-mediated suppression of activation-induced cytidine deaminase in mucosal B cells. In addition, we show that loss of miR-23b increases the susceptibility of the kidney to progressive fibrosis through loss of regulation of expression of gremlin 2 and IgA accumulation through downregulation of the transferrin receptor.

Conclusions: Our findings suggest an indispensable role for miR-23b in kidney disease, and in particular, IgAN. miR-23b may in the future offer a novel therapeutic target for the treatment of IgAN.

Keywords: IgA; chronic kidney disease; immunology.

Graphical abstract

Figure 1.

miR-23b is dysregulated in human IgAN. Next-generation sequencing of kidney biopsy tissue showed that miR-23b was downregulated in kidneys from (A) Chinese and (B, upper panel) UK patients with IgAN, compared with healthy subjects and patients with MN and TMN. RT-qPCR studies (B, lower panel) confirmed downregulation of miR-23b in the kidneys of UK patients with IgAN (n=13) compared with MN (n=6) and TMN (n=7). Furthermore, serum miR-23b levels were significantly lower in patients with IgAN compared with (C) healthy subjects and (D) negatively correlated with serum IgA1 levels in an independent Chinese IgAN cohort. *P<0.05. Data are shown as the mean±SEM.

Figure 2.

Characterization of the miR-23b^−/− mouse. (A) Changes in the body weight of miR-23b^−/− and WT mice over the first 3 months. (B) Quantitative analysis of miR-23b in miR-23b^−/− mice (n=3) in different tissues by RT-qPCR. (C) Whole-genome sequencing confirmed deletion of the miR-23b sequence. (D) Quantitative analysis of miR-23a in miR-23b^−/− mice (n=3) in different tissues by RT-qPCR indicated deletion of miR-23b did not result in cluster compensation. (E) Quantitative analysis of miR-24 and -27 in kidney, two miRs closely located (<1000 bp) to miR-23b on chromosome 13, again indicated an absence of cluster compensation in miR-23b^−/− mice (n=3). *P<0.05. Data are shown as the mean±SEM.

Figure 3.

miR-23b^−/− mice develop a mesangioproliferative GN. (A) Quantification of 24-hour albumin excretion in 5-month-old miR-23b^−/− (n=8) and WT (n=4) mice. (B) Quantification of serum creatinine in 5-month-old miR-23b^−/− (n=8) and WT (n=5) mice. (C) Representative images of periodic acid–Schiff stained kidney biopsy sections from 5-month-old miR-23b^−/− and WT mice. (D) Quantification of the ratio of the mesangial matrix to total glomerular area in miR-23b^−/− and WT mice. (E) Representative images of transmission and scanning electron microscope (TEM and SEM) images from 5-month-old miR-23b^−/− and WT mice. Red arrows indicate mesangial cells, white arrows indicate foot process fusion, asterisk indicates basement membrane, the bars indicate 2 μm. (F) Quantification of mesangial cell number using the TEM images in miR-23b^−/− and WT mice. (G) and (H) Quantification of “tight” pores in podocyte foot processes and glomerular basement membrane thickness in 3-month-old miR-23b^−/− and WT mice. **P<0.01 and ***P<0.001. Data are shown as mean±SEM.

Figure 4.

miR-23b^−/− mice develop an IgAN-like disease. (A) Representative images of IgA, C3, IgG, and IgM immunofluorescence staining of kidney sections and TEM. TEM images demonstrate the presence of electron-dense mesangial deposits (red arrows) consistent with mesangial IgA immune complex deposition in miR-23b^−/− mice. Quantification of (B) serum IgA, (C) IgG, and (D) IgM levels in 5-month-old WT (n=5) and miR-23b^−/− (n=8) mice. SDS-PAGE and IgA western blotting of serum IgA from (E) 5-month-old WT (n=11) and miR-23b^−/− (n=11) mice with (F) densitometric analysis of polymeric IgA: total serum IgA and monomeric IgA: (G) total serum IgA. (H) Levels of IgA-IgG immune complexes in the sera of 5-month-old WT (n=9) and miR-23b^−/− (n=10) mice.

Figure 5.

miR-23b^−/− mice develop dysregulated mucosal immunity in the gut. (A) Representative images of CD19 and IgA immunofluorescence and AID immunohistochemistry staining of the intestinal mucosa from 5-month-old WT and miR-23b^−/− mice. (B) Representative images of AID, CD19, and IgA immunofluorescence staining of the intestinal mucosal of 5-month-old WT and miR-23b^−/− mice. (C) Luciferase activity in HEK 293A cells transfected with an AID 3′-UTR reporter construct demonstrates binding of miR-23b with the 3′-UTR of each reporter. *P<0.05, **P<0.01. Data are shown as mean±SEM.

Figure 6.

Deletion of miR-23b induces structural kidney changes. (A) Representative images of B-mode ultrasonography of the kidney (first panel, bars estimating the renal parenchyma distance) in WT (n=5) and miR-23b^−/− (n=7); MRI T1/2 images (second and third panels) in WT (n=10), and miR-23b^−/− (n=12) 5-month-old male mice. (B) Quantification of the renal parenchymal thickness measured from kidney ultrasound images. (C) Quantification of the renal cortical thickness measured from MRI T2 images. (D) Quantification of the ratio of MRI T2 cortex to medulla thickness. (E) Quantification of MRI T2 medulla thickness; *P<0.05, **P<0.01, and ***P<0.001. Data are shown as the mean±SEM.

Figure 7.

miR-23b^−/− mice develop hypertension. (A) Representative Doppler flow images of the intrarenal artery in 5-month-old WT (left image) and miR-23b^−/− (right image) mice. (B) Quantification of the kidney artery resistance index (KRI, the peak systolic and end-diastolic blood velocities divided by the peak systolic velocity) in 5-month-old male WT (n=18) and miR-23b^−/− (n=21) mice. (C) Quantification of mean arterial blood pressure in WT (n=10) and miR-23b^−/− (n=10) mice. (D) KEGG analysis of the 3-month miR-23b^−/− RNA-seq data highlighted the complement and coagulation cascades and renin angiotensin system as significantly enriched terms. (E) Western blot analyses for renin, angiotensin converting enzyme 2, and angiotensin II receptor type 1, in 3- and 5-month-old miR-23b^−/− and WT mice. **P<0.01, ***P<0.001. Data are shown as the mean±SEM.

Figure 8.

Analysis of the 3- and 5-month miR-23b ^−/− kidney RNA-seq data revealed distinct patterns of transcript expression. (A) Quantification of dysregulated genes in the kidneys of 3- and 5-month-old miR-23b^−/− mice. (B) Venn diagram showing the number of genes in the kidneys with significant changes in both 3- and 5-month-old miR-23b^−/− mice and those identified as potential miR-23b targets using the Targetscan database. (C) and (D) Heatmaps of differentially expressed genes and potential miR-23b targets in the kidneys of 3- and 5-month-old miR-23b^−/− mice for all genes (C) and only those potential miR-23b targets (D), (the color gradation indicates a log2 fold change in gene expression). (E) GO term analysis for those genes dysregulated in the kidneys of both 3- and 5-month-old miR-23b^−/− mice and potential miR-23b targets.

Figure 9.

miR-23b regulates the BMP pathway through Grem2. (A) Luciferase activity in HEK 293A cells transfected with GREM2, TFRC, and JARID2 3′-UTR reporter constructs demonstrated binding of miR-23b to the 3′-UTR of each reporter. (B) Western blot analysis for Grem2 and BMP pathway related proteins in kidneys of 3- and 5-month-old WT and miR-23b^−/− mice. (C) Western blot analysis of BMP pathway–related proteins and TFRC in HMC driven to overexpress or downregulate Grem2 and miR-23b. (D) Western blot analysis of SMAD4, Grem2, and TFRC in HMC driven to overexpress SMAD4; RT-qPCR quantification of BMP2 (E) and miR-23b (F) expression in HMC treated with Grem2 siRNA and protein, miR-23b agomir, and antagomir, miR-23b agomir combined with Grem2 siRNA or protein, SMAD4-activated plasmid, SMAD4-activated plasmid combined with miR-23b agomir, and miRNA or siRNA-negative control. (G) A schematic model of potential regulation and function of miR-23b. *P<0.05, **P<0.01, ***P<0.001. Data are shown as the mean±SEM.