RBBP6-Mediated ERRα Degradation Contributes to Mitochondrial Injury in Renal Tubular Cells in Diabetic Kidney Disease

Abstract

Diabetic Kidney Disease (DKD), a major precursor to end-stage renal disease, involves mitochondrial dysfunction in proximal renal tubular cells (PTCs), contributing to its pathogenesis. Estrogen-related receptor α (ERRα) is essential for mitochondrial integrity in PTCs, yet its regulation in DKD is poorly understood. This study investigates ERRα expression and its regulatory mechanisms in DKD, assessing its therapeutic potential. Using genetic, biochemical, and cellular approaches, ERRα expression Was examined in human DKD specimens and DKD mouse models. We identified the E3 ubiquitin ligase retinoblastoma binding protein 6 (RBBP6) as a regulator of ERRα, promoting its degradation through K48-linked polyubiquitination at the K100 residue. This degradation pathway significantly contributed to mitochondrial injury in PTCs of DKD models. Notably, conditional ERRα overexpression or RBBP6 inhibition markedly reduced mitochondrial damage in diabetic mice, highlighting ERRα's protective role in maintaining mitochondrial integrity. The interaction between RBBP6 and ERRα opens new therapeutic avenues, suggesting that modulating RBBP6-ERRα interactions could be a strategy for preserving mitochondrial function and slowing DKD progression.

Keywords: RBBP6, ubiquitination; diabetic kidney disease; estrogen‐related receptor α; mitochondrial dysfunction; proximal renal tubular cells.

Figure 1

Renal pathological changes and ERRα expression in DKD. A) TEM analyses of immuno‐gold labeling of ERRα in PTCs in DKD (n = 30) and control (n = 10) group patients. B,C) Representative immunofluorescent images and quantification of LTL (gray) and ERRα (red) in PTCs from DKD (n = 30) and control (n = 10) group patients. D) Representative immunohistochemistry images of ERRα in PTCs from control (n = 10), DKD (n = 30), focal segmental glomerular sclerosis (FSGS, n = 6), IgA nephritis (IgAN, n = 6), and minimal change disease (MCD, n = 6) group patients. E) Immunohistochemical semi‐quantitative IOD analysis of ERRα. Correlation between F) ERRα expression and estimated glomerular filtration rate (eGFR) (n = 30), G) urinary creatinine‐protein ratio (ACR) (n = 30), and H) serum albumin (ALB) (n = 30) in patients with DKD. Data are presented as mean ± SEM. ns: p > 0.05; ***p < 0.001.

Figure 2

ERRα is downregulated in diabetic animal models and associated with mitochondrial function. A) Representative immunofluorescent images and quantification of LTL (gray) and ERRα (red) in PTCs from db/m (n = 6) and db/db (n = 6) mice. B) Representative immunofluorescent images and quantification of LTL (green) and ERRα (red) in PTCs from vehicle (n = 6) and STZ (n = 6) mice. C) Representative immunohistochemistry images of ERRα in PTCs from db/m and db/db mice (n = 6). D) Representative immunohistochemistry images of ERRα in PTCs from vehicle and STZ mice (n = 6). E) Representative Western blot and densitometric analysis of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in renal tubules from db/m and db/db mice (n = 6). F) A schematic diagram showing the isolation of primary PTCs. G) Representative Western blot and densitometric analysis of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in primary PTCs from different group cells (n = 4). H) Oxygen consumption rate (OCR) of primary PTCs in both the si‐NC and si‐ERRα group (n = 3). I) Representative plots and statistical graphs for flow cytometry analysis of MitoTracker‐red in primary PTCs infected with si‐NC or si‐ ERRα and treated with 40 × 10⁻³ m glucose for 24 h. J) Mitochondrial‐related genes expression profiles were compared between different group of HK‐2 cells. Data are presented as mean ± SEM. ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Conditional knock‐in of ERRα in PTCs ameliorates mitochondrial injury in DKD. A–C) Serum urea nitrogen (BUN), serum creatinine (SCr) and urine creatinine protein ratio (ACR) levels in different groups of mice (n = 6). D) Representative images of HE, PAS, Masson and SDH stainings from different group of mice (n = 6). E) Mesangial matrix expansion of kidney sections in different groups of mice (n = 6). F) Tubular interstitial damage score of kidney sections indifferent groups of mice (n = 6). G) Mean SDH intensity of kidney sections in different groups of mice (n = 6). H) Representative immunofluorescent images of LTL (gray), PGC1α (red), and TOM20 (green) in PTCs from different groups of mice (n = 6). I) TEM analyses of PTCs ultrastructure and quantitation of aspect ratio, circularity, roundness in different groups of mice (n = 6). The number and area of mitochondria per unit (per ×5000 field of view for mitochondria number). J) Representative Western blot and densitometric analysis of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in renal tubules from different group mice (n = 6). K) Measurement of oxygen consumption rate (OCR) in different groups of primary PTCs (n = 3). L) Representative immunofluorescent images of PGC1α (green) and TOM20 (red) of different groups of primary PTCs. Data are presented as mean ± SEM. ns: P > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

RBBP6 mediates the degradation of ERRα via UPS. A,B) Representative images and quantification of fluorescent in situ hybridization (FISH) for ERRα and Hnf4a in PTCs of db/m and db/db mice (n = 6). C,D) Representative immunohistochemistry images and quantitative analysis of ubiquitination in PTCs from db/m and db/db mice (n = 6). E) Representative Western blot and densitometric analysis of ERRα in different groups of HK‐2 cells (n = 3). F,G) Representative Western blot and densitometric analysis of ERRα in different groups of HK‐2 cells (n = 3). H) Representative Western blot and densitometric analysis of ERRα in different groups of HK‐2 cells (n = 3). I) SDS‐PAGE gel of proteins bound to IgG (left lane) or ERRα (right lane). Protein marker stands stand for (from top to bottom): 180, 130, 100, 70, 55, 45, 35, 25, 15/10 kDa. J) Gene Ontology (GO) enrichment of proteins bound to ERRα. K) Protein secondary spectrum of peptide segments of ERRα. L,M) HK‐2 cell lysates were subjected to immunoprecipitation (IP) with IgG, Myc, and ERRα antibody, respectively, followed by RBBP6 immunoblotting (IB). N) Representative confocal microscopic images of colocalization of RBBP6 and ERRα in HK‐2 cells. O) Proposed interaction model of RBBP6 binding to ERRα. 3D illustrations of the interaction between RBBP6 and ERRα. Data are presented as mean ± SEM. ns: P > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

The expression of RBBP6 is upregulated in DKD. A) Spatial transcriptomics for DKD patient (Kidney Tissue Atlas, https://atlas.kpmp.org/, Kidney Tissue Atlas Spatial Transcriptomics for sample S‐2109‐011418, participant 32‐10346). B,C) RBBP6 gene expression in healthy and DKD patients from single‐nucleus RNA‐seq. RBBP6 Expression Comparison across Clusters in healthy and DKD group. (GEO ID: GSE131882). D) Representative immunohistochemistry images and quantification of RBBP6 in PTCs from control (n = 10) and DKD (n = 30) patients. E) Representative immunofluorescent images of ERRα (green) and RBBP6 (red) of PTCs in control (n = 10) and DKD (n = 30) patients. F) Representative immunohistochemistry images and quantification of RBBP6 in PTCs of db/m and db/db mice (n = 6). G) Representative immunohistochemistry images and quantification of RBBP6 in PTCs of Vehicle and STZ group mice (n = 6). Data are presented as mean ± SEM. ***p < 0.001.

Figure 6

Downregulation of RBBP6 can alleviate pathological damage and ameliorate mitochondrial injury. A–C) Levels of BUN, SCr, and ACR in different groups of mice (n = 6). D) Representative images of HE, PAS, Masson and SDH stainings from different groups of mice (n = 6). E) Mesangial matrix expansion of kidney sections in different group of mice (n = 6). F) Tubular interstitial damage score of kidney sections in different groups of mice (n = 6). G) Mean SDH intensity of kidney sections in different groups of mice (n = 6). H) Representative immunofluorescent images of LTL (gray), PGC1α (red), and TOM20 (CY5) in PTCs from different groups of mice (n = 6). I) TEM of PTCs ultrastructure and quantitation of aspect ratio, circularity, roundness in different groups of mice (n = 6). The number and area of mitochondria per unit (per ×5000 field of view for mitochondria number). J) Representative Western blot and densitometric analysis of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in renal tubules from different groups of mice (n = 6). Data are presented as mean ± SEM. ns: P > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

Downregulation of RBBP6 ameliorates mitochondrial injury in HK‐2 cells. A) Representative Western blot and densitometric analysis of ERRα and RBBP6 in HK‐2 cells transfected with si‐RBBP6 or si‐NC (n = 3). B,C) Representative Western blot and densitometric analysis of ERRα in HK‐2 cells treated with cycloheximide (CHX) of different concentrations and transfected with si‐RBBP6/si‐NC (n = 3). D) Representative Western blot and densitometric analysis of ERRα and RBBP6 in HK‐2 cells treated with MG132/BafA1 and transfected with pcDNA‐RBBP6/pcDNA (n = 3). E,F) Representative Western blot and densitometric analysis of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in HK‐2 cells treated with XCT790/PROTAC‐1 and transfected with si‐RBBP6/si‐NC (n = 3). G) Representative Western blot and densitometric analysis of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in HK‐2 cells treated with DK3 and transfected with pcDNA‐RBBP6/pcDNA (n = 3). H) Representative immunofluorescent images of ERRα (green) and TOM20 (red) in HK‐2 cells after transfecting with si‐NC or si‐RBBP6. Cells were either unstimulated or treated with XCT790 (n = 3). I) TEM analyses of HK‐2 cell ultrastructure and quantitation of IMM/OMM ratio, total cristae length/mitochondrial area, cristae junction/mitochondrial area in different group. The number and area of mitochondria per unit (per ×5000 field of view for mitochondria number) (n = 3). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8

RBBP6 promotes K48‐linked ubiquitination at the K100 residue of ERRα. A,B) HK‐2 cells were transfected with si‐RBBP6 or pcDNA‐RBBP6, and HA‐ubiquitin for 6 hours and incubated with antibiotic‐free medium. Cell lysates were subjected to IP with ERRα antibody, followed by HA, RBBP6, and ERRα (IB) (n = 3). C,D) HK‐2 cells were transfected with si‐RBBP6 for 6 h and incubated with antibiotic‐free medium and 40 × 10⁻³ m HG stimulation for an additional 24 h. Cell lysates were subjected to IP with ERRα antibody, followed by K48‐ubiquitin/K63‐ubiquitin, RBBP6, ERRα (IB) (n = 3). E) HK‐2 cells were transfected with HA‐ubiquitin, Myc‐RBBP6 plasmid and different mutant ERRα plasmids, and the immunoprecipitation with His affinity gels and immunoblotting were performed (n = 3). F) Representative immunofluorescent images of ERRα (green) and TOM20 (red) in HK‐2 cells transfected with Myc‐RBBP6 plasmid, different mutant ERRα plasmids (K68 and K100 residues) (n = 3). G) HK‐2 cells were transfected with Myc‐RBBP6 plasmid and different mutant ERRα plasmids (K68 and K100 residues). TEM analyses of HK‐2 cell ultrastructure and quantitation of IMM/OMM ratio, total cristae length/mitochondrial area, cristae junction/mitochondrial area in different groups. The number and area of mitochondria per unit (per ×5000 field of view for mitochondria number) (n = 3). H) Representative Western blot of ERRα, TOM20, PGC1α, and OXPHOS (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in HK‐2 cells from different group (n = 3). I) Pattern map and Sanger sequencing results of ERRα K100 site mutated to arginine (R) in HK‐2 cells. J) Representative Western blot of RBBP6 and ERRα in HK‐2 cells from different group (n = 3). K) Representative immunofluorescent images of TOM20 and TEM analyses of HK‐2 cell ultrastructure and quantitation. The number and area of mitochondria per unit (per ×5000 field of view for mitochondria number) (n = 3). Data are presented as mean ± SEM. ns: p > 0.05; **p < 0.01; ***p < 0.001.

Figure 9

Downregulation of ERRα and mitochondrial impairment in diabetic kidney disease.