DeSUMOylation of RBMX regulates exosomal sorting of cargo to promote renal tubulointerstitial fibrosis in diabetic kidney disease

Abstract

Introduction: Diabetic kidney disease (DKD) has become the primary cause of chronic renal failure in China, and renal tubulointerstitial fibrosis plays a central role in DKD progression. Urinary exosomes, which reflect kidney changes, are largely influenced by RNA-binding proteins (RBPs) in their miRNA content.

Objectives: Our research aimed to determine the effect of the RNA-binding protein RBMX on exosomal miRNA in DKD.

Methods: We introduced a higher level of Rbmx into diabetic mice using an adenoassociated virus and isolated exosomes from their kidney tissue through advanced centrifugation techniques and specialized kits. We then conducted a series of tests, including qRT-PCR, Western blot, MitoSOX, ATP luminescence, coimmunoprecipitation, SUMOylation assays, RNA immunoprecipitation, and confocal microscopy.

Results: RBMX is found in higher levels in DKD and contributes to worsening kidney fibrosis, mitochondrial damage, and miRNA mismanagement in exosomes. It specifically binds with miR-26a, miR-23c, and miR-874 within the exosomes. This dysfunction may be linked to changes in RBMX SUMOylation. These miRNAs seem to protect against mitochondrial damage in kidney cells by targeting CERS6.

Conclusion: DeSUMOylation of RBMX plays a crucial role in determining the makeup of miRNAs in kidney cell exosomes, impacting the protective miRNAs which regulate mitochondrial damage through their interaction with CERS6 mRNA, ultimately affecting mitochondrial health in DKD.

Keywords: DeSUMOylation; Diabetic kidney disease; Exosome; RBMX; miRNA.

Graphical abstract

Fig. 1

High expression of RBMX in the renal cortex and tubular epithelial cells in DKD. (A) H&E, PAS, and Masson staining and the localization and levels of Rbmx in the kidney cortex of HFD/STZ-induced DKD mice (n = 6 mice per group). Scale bars: 200 μm. Original magnification × 400. (B) The urine albumin–creatinine ratio (UACR) in HFD/STZ mice (n = 6 mice per group). (C) Semiquantitative analysis of Rbmx in the renal cortex of HFD/STZ-induced DKD mice (n = 3 mice per group). (D) The mRNA expression levels of Rbmx in the renal cortex of HFD/STZ mice (n = 4 mice per group). (E) The mRNA expression levels of FN, TGFβ1, CCN2, and RBMX in HG-induced HK-2 cells (n = 3 per group). (F) The protein expression levels of FN, TGFB1, CCN2 and RBMX in HG-induced HK-2 cells (n = 4 per group). Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed Student’s t test.

Fig. 2

RBMX promotes renal tubulointerstitial fibrosis in DKD. (A) The mRNA expression levels of Rbmx, Fn, Tgfβ1 and Ccn2 in the renal cortex of HFD/STZ mice with renal overexpression of Rbmx (n = 4 mice per group). (B) Immunohistochemical staining for RBMX, FN, TGFβ1 and CCN2 and H&E, PAS and Masson staining in the renal cortex of HFD/STZ mice injected with AAV-Rbmx (n = 6 mice per group). Scale bars: 200 μm. Original magnification × 400. (C) The urine albumin–creatinine ratio (UACR) in HFD/STZ mice with renal overexpression of Rbmx (n = 6 mice per group). (D) The mRNA expression levels of RBMX, FN, TGFβ1 and CCN2 in HG-induced HK-2 cells transfected with CON106-RBMX (n = 3 per group). (E) The protein expression levels of RBMX, FN, TGFβ1 and CCN2 in HG-induced HK-2 cells transfected with CON106-RBMX (n = 4 per group). (F) The mRNA expression levels of RBMX, FN, TGFβ1 and CCN2 in HK-2 cells transfected with si-RBMX and HG (n = 3 per group). (G) The protein expression levels of RBMX, FN, TGFβ1 and CCN2 in HK-2 cells transfected with si-RBMX and HG (n = 4 per group). Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 by one-way ANOVA with Bonferroni post hoc tests.

Fig. 3

RBMX aggravates mitochondrial damage in DKD. (A) The protein expression levels of NDUFB8, SDHB, UQCRC2 and MT-CO1 in HG-induced HK-2 cells transfected with CON106-RBMX (n = 4 per group). (B) The levels of mitochondrial ROS in HG-induced HK-2 cells transfected with CON106-RBMX (n = 3 per group). Scale bars: 200 μm. Original magnification × 200. (C) The levels of ATP in HG-induced HK-2 cells transfected with CON106-RBMX (n = 3 per group). (D) The protein expression levels of NDUFB8, SDHB, UQCRC2 and MT-CO1 in HK-2 cells transfected with si-RBMX (n = 4 per group). (E) The levels of mitochondrial ROS in HK-2 cells transfected with si-RBMX in HG conditions (n = 3 per group). (F) The levels of ATP in HK-2 cells transfected with si-RBMX and HG (n = 3 per group). (G, H) Immunohistochemical and semiquantitative analysis of NDUFB8, SDHB, UQCRC2 and MT-CO1 in the renal cortex of HFD/STZ mice with renal overexpression of Rbmx (n = 4 mice per group). Scale bars: 200 μm. Original magnification × 400. Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, ***p < 0.001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 by one-way ANOVA with Bonferroni post hoc tests.

Fig. 4

RBMX regulates miRNA levels and mitochondrial damage through exosomes. (A) The expression of miRNAs in the renal cortex of HFD/STZ mice with renal overexpression of Rbmx (n = 3 mice per group). (B) The expression of miR-26a, miR-23c and miR-874 in the renal cortex of db/db mice with renal overexpression of Rbmx (n = 3 mice per group). (C, D) The levels of miR-26a, miR-23c and miR-874 in exosomes from the renal cortex of HFD/STZ and db/db mice with renal overexpression of Rbmx (n = 3 mice per group). (E) The expression of miR-26a, miR-23c and miR-874 in HK-2 cells transfected with CON106-RBMX in HG conditions (n = 3 per group). (F) The levels of miR-26a, miR-23c and miR-874 in exosomes from HK-2 cells transfected with CON106-RBMX under HG conditions (n = 3 per group). (G) The expression of miR-26a, miR-23c and miR-874 in HK-2 cells transfected with si-RBMX in HG conditions (n = 3 per group). (H) The levels of miR-26a, miR-23c and miR-874 in exosomes from HK-2 cells transfected with si-RBMX under HG conditions (n = 3 per group). (I) The protein expression levels of TSG101 and CD63 in HK-2 cells cotransfected with CON106-RBMX and si-RAB27A in HG (n = 4 per group). (J) The mRNA expression levels of FN, TGFβ1 and CCN2 in HK-2 cells cotransfected with CON106-RBMX and si-RAB27A in HG conditions (n = 3 per group). (K) The protein expression levels of FN, TGFβ1, CCN2, NDUFB8, SDHB, UQCRC2 and MT-CO1 in HG-induced HK-2 cells cotransfected with CON106-RBMX and si-RAB27A (n = 4 per group). (L) The levels of ATP in HK-2 cells cotransfected with CON106-RBMX and si-RAB27A in HG conditions (n = 3 per group). (M) The levels of mitochondrial ROS in HK-2 cells cotransfected with CON106-RBMX and si-RAB27A in HG conditions (n = 3 per group). Scale bars: 200 μm. Original magnification × 200. Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; *p < 0.05, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 by one-way ANOVA with Bonferroni post hoc tests.

Fig. 5

DeSUMOylated RBMX promotes exosomal sorting of miRNAs and renal tubulointerstitial fibrosis. (A) Co-IP assays analyzing the interaction of FLAG-RBMX and SUMO1 or SUMO2/3 in HK-2 cells (n = 3 per group). (B) Co-IP assays assessing which SUMO isoform binds to RBMX in HEK-293T cells transfected with His-UBC9 (n = 3 per group). (C) Co-IP assays analyzing the interaction of FLAG-RBMX and SUMO2/3 in HG-treated HK-2 cells (n = 3 per group). (D, E) Western blotting analysis assessing the SUMOylation of RBMX (n = 3 per group). (F) Co-IP assays analyzing the interaction of FLAG-RBMX and TSG101 in HK-2 cells (n = 3 per group). (G) Representative immunofluorescence images of FLAG-RBMX colocalization with CD63 in HK-2 cells transfected with RBMX^K80R (n = 3 per group). Scale bars: 200 μm. Original magnification × 630. (H, I) The expression of miR-26a, miR-23c and miR-874 in HK-2 cells and their exosomes transfected with RBMX^K80R (n = 3 per group). (J) Western blotting analysis of TGFβ1, CCN2, NDUFB8, SDHB and MT-CO1 expression in HK-2 cells transfected with RBMX^K80R (n = 3 per group). Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01 by two-tailed Student’s t test.

Fig. 6

The mechanisms of RBMX-mediated exosomal sorting of miRNAs. (A) The region of RBMX that binds to miR-26a, miR-23c or miR-874. (B) The expression of miR-26a, miR-23c and miR-874 bound to the RBMX antibody in HG- or BSA-induced HK-2 cells (n = 3 per group). (C) The protein expression levels of FN, TGFβ1, CCN2, NDUFB8, SDHB, UQCRC2 and MT-CO1 in HG-induced HK-2 cells cotransfected with CON106-RBMX and miRNA mimic (n = 3 per group). (D) The mRNA expression levels of FN, TGFβ1 and CCN2 in HK-2 cells cotransfected with CON106-RBMX and miRNA mimic in HG (n = 3 per group). (E) The levels of ATP in HK-2 cells cotransfected with CON106-RBMX and the miRNA mimic in HG conditions (n = 3 per group). (F) The levels of mitochondrial ROS in HK-2 cells cotransfected with CON106-RBMX and the miRNA mimic in HG conditions (n = 3 per group). Scale bars: 200 μm. Original magnification × 200. Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; ^##p < 0.01, ^###p < 0.001 by two-tailed Student’s t test or one-way ANOVA with Bonferroni post hoc tests.

Fig. 7

The role of miR-26a, miR-23c and miR-874 in DKD. (A) The mRNA expression levels of FN, TGFβ1 and CCN2 in HK-2 cells transfected with miR-26a, miR-23c or miR-874 mimic in HG (n = 3 per group). (B) The protein expression levels of FN, TGFβ1, CCN2, NDUFB8, SDHB, UQCRC2 and MT-CO1 in HG-induced HK-2 cells transfected with miR-26a, miR-23c or miR-874 mimic (n = 3 per group). (C) The levels of ATP in HK-2 cells transfected with miR-26a, miR-23c or miR-874 mimic in HG (n = 3 per group). (D) The levels of mitochondrial ROS in HK-2 cells transfected with miR-26a, miR-23c or miR-874 mimic in HG conditions (n = 3 per group). Scale bars: 200 μm. Original magnification × 200. (E) The mRNA expression levels of FN, TGFβ1 and CCN2 in HK-2 cells transfected with miR-26a, miR-23c or miR-874 inhibitor in HG (n = 3 per group). (F) The protein expression levels of FN, TGFβ1, CCN2, NDUFB8, SDHB, UQCRC2 and MT-CO1 in HG-induced HK-2 cells transfected with miR-26a, miR-23c or miR-874 inhibitor (n = 3 per group). (G) The levels of ATP in HK-2 cells transfected with miR-26a, miR-23c or miR-874 inhibitor in HG (n = 3 per group). (H) The levels of mitochondrial ROS in HK-2 cells transfected with miR-26a, miR-23c or miR-874 inhibitor in HG conditions (n = 3 per group). Scale bars: 200 μm. Original magnification × 200. Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01 by two-tailed Student’s t test.

Fig. 8

CERS6 is a common target gene of miR-26a, miR-23c and miR-874. (A) Analysis of common target genes of miR-26a, miR-23c and miR-874. (B) The mRNA expression levels of mitochondria-related factors in HG-treated HK-2 cells (n = 3 per group). (C) miR-26a, miR-23c and miR-874 binding sites within the 3′-UTRs of CERS6. (D-F) Relative luciferase activity in HEK-293T cells cotransfected with mimic-miR-26a (D), mimic-miR-23c (E) or mimic-miR-874 (F) and pmirGLO containing the CERS6 3′-UTR site (n = 3 per group). (G, H) The expression levels of CERS6 in HK-2 cells transfected with miR-26a, miR-23c or miR-874 mimic in HG conditions (n = 3 per group). (I, J) The expression of CERS6 in HK-2 cells transfected with si-RBMX in HG conditions (n = 3 per group). (K, L) Expression of CERS6 in HG-induced HK-2 cells transfected with CON106-RBMX (n = 3 per group). (M, N) The expression of CERS6 in the renal cortex of HFD/STZ mice with renal overexpression of Rbmx (n = 3 mice per group). Scale bars: 100 μm. Original magnification × 400. Horizontal and error bars represent the mean and SEM, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; ^##p < 0.01, ^###p < 0.001 by two-tailed Student’s t test or one-way ANOVA with Bonferroni post hoc tests.