NDUFS4 Regulates Cristae Remodeling in Diabetic Kidney Disease

Abstract

The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generated diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model to investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that these conditional mice exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping proteins in linking NDUFS4 with improved cristae morphology. Taken together, we discover the central role of NDUFS4 as a powerful regulator of cristae remodeling, respiratory supercomplexes assembly, and mitochondrial ultrastructure in vitro and in vivo. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.

Extended Data Fig. 1 |. NDUFS4 expression is downregulated in podocytes, but not in tubules, in diabetic environment.

a, Mitochondria isolation from podocytes or whole kidney using Percoll density gradient centrifugation (left). Immunoblots to validate the purity of mitochondria in different fractions (lower right). India ink staining was used to show all proteins (upper right). b, Proteomic profiling of mitochondrial proteins from primary podocytes of WT and Ins2^Akita/+ mice. Heatmaps of CI subunits (left panel), and CII-CIV subunits (right panel). c,d, qRT-PCR analysis of the relative mRNA expression of selective CI subunits (relative to Actb) in isolated podocytes from 16-week-old WT/ Ins2^Akita/+ (c) and Lepr^db/+/Lepr^db/db mice (d) (n=4, each n represents a pool of RNA samples from 4 different mice). e, mRNA expression of selective CI subunits in glomeruli of human DKD (Nephroseq v5) (Healthy living donor n=21, DKD patients n=12), Median-centered Log₂ values are used for the analysis. f, Western blot analysis of NDUFS4 in primary podocytes and tubular cells isolated from WT and Ins2^Akita/+ mice, Podocin (podocyte marker), PAX8 (tubular marker), and VDAC (mitochondrial marker) were used as cell type specific markers. Exp: exposure. g, Study flowchart of the NDUFS4 staining analysis in kidney biopsies from DKD patients (n=34) and healthy donors (n=9). Data are mean ± SEM. *P < 0.05, **P < 0.01, unpaired two-sided t test, FDR Q< 0.05 (c-e).

Extended Data Fig. 2 |. Podocyte-specific Ndufs4 overexpression mitigates albuminuria in type 2 diabetic mice.

a, Representative images of NADH oxidoreductase (CI) activity in glomeruli from WT and Ndufs4^PodTg mice (left panel). Relative CI activity in glomeruli (right panel) (n=150 from 3 mice/group). Scale bar=20 μm. b, Appearance of a diabetic (Ins2^Akita/+) mouse and a diabetic Ndufs4^PodTg mice at 18 weeks of age. c, Area under the curve (AUC) of blood glucose levels from 4-, 10-, 14-, 18- and 23-week-old mice shown in Fig. 2f. d, Hemoglobin A1C (HbA1c) levels in four different groups of mice. e, Urinary albumin excretion (UAE, μg/24hrs) in 12-, 16-, 20- and 24-week-old mice. f, AUC of UAE shown in Extended Data Fig. 2e. g-k, Body weight (g), blood glucose (h), blood glucose AUC (i), urinary albumin-to-creatinine ratio (UACR) (j), and UAE (k) of control and type 2 diabetic Lepr^db/db mice at different ages (n=7–9/group). Results are presented as median ± IQR (a, bold line: median, and dot line: IQR) or mean ± SEM (c-k). *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Mann-Whitney test (a), One-way ANOVA with post-hoc Tukey’s test (c-k).

Extended Data Fig. 3 |. Ndufs4 overexpression prevents mitochondrial fission in podocytes from type1 and type 2 diabetic mice.

a-e, Representative TEM micrographs of podocyte mitochondria from kidney tissues (a, top) and pseudo-color superimposed images (a, bottom) to assess mitochondrial morphological changes in aspect ratio (b), circularity (c), roundness (d), and feret diameter (e) (n=115–454 from 3 mice/group, Scale bar=200 nm). f-j, Representative immunofluorescent images (f) of primary podocytes stained with mitotracker (white) showing mitochondrial morphological changes in aspect ratio (g), circularity (h), roundness (i), and feret diameter (j) (n=21675–23513/group, Scale bar=40 μm). Data are presented as median ± IQR (b-e, bold line: median, and dot line: IQR) or mean ± SEM (g-j). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Kruskal-Wallis with post-hoc Dunn’s test (b-e) and One-way ANOVA with post-hoc Tukey’s test (g-j).

Extended Data Fig. 4 |. DOX-induced NDUFS4 OE in podocytes improves HG-induced mitochondrial remodeling.

a, PiggyBac transposon vector containing a doxycycline (DOX)-inducible Ndufs4 (NDUFS4 OE) expression cassette. b, NDUFS4 expression induced by DOX at different concentrations. c, Procedure of mitochondrial isolation by sucrose step density gradient centrifugation using cultured podocytes (left panel), and immunoblot validation of mitochondrial purity in cytosol, and mitochondrial-enriched fraction (bottom right panel). Protein extracts from whole kidney was used as a control. India ink staining (upper right panel) showed the amounts of proteins loaded. d,e, Relative CI enzymatic activity in isolated mitochondria (d) and ATP production (e) in podocytes cultured under NG (5.5 mM) and HG (25 mM) for 48hrs with (NDUFS4 OE) or without DOX induction (normalized to NG levels; n=6 and n=8 replicates, respectively). f, Immunoblots of BN-PAGE of digitonin-solubilized mitochondria from DOX-induced NDUFS4 OE podocytes showing individual complexes as well as stoichiometry of respiratory supercomplexes (RSC). g, Coomassie staining (left panel) and immunoblot (right panel) of BN-PAGE analysis of digitonin-solubilized mitochondria isolated from podocytes cultured under NG and HG for 48hrs. h, CI in-gel activity of digitonin- and n-Dodecyl β-D-maltoside (DDM)-solubilized mitochondria from the same cells as in Extended Data Fig. 4g. RSC: respirasome supercomplexes. Roman numerals indicate RSC with defined stoichiometry of individual complexes. Data are presented as mean ± SEM (d,e) *P < 0.05, ***P < 0.001, ****P < 0.0001. One-way ANOVA with post-hoc Tukey’s test (d,e).

Extended Data Fig. 5 |. Doxycycline-inducible Ndufs4-APEX construct and NDUFS4-interactome.

a, Immunoblotting of STOML2, IMMT/MIC60, ATAD3, and OPA1 in primary podocytes. β-Actin was used for loading control. b, PiggyBac transposon vector containing a doxycycline (DOX)-inducible NDUFS4-APEX expression cassette. c, The top 46 NDUFS4 associated mitochondrial proteins categorized into various groups according to their biological functions. Mt: mitochondrial. d, Trajectory of histograms for nearest neighboring distance (NND). Histograms show the fraction of the distance between NDUFS4 molecule and nearest neighboring STOML2 molecule.

Extended Data Fig. 6 |. NDUFS4 binds STOML2 at N-terminus domain.

a, Structure of mouse STOML2 WT and deletion mutant constructs engineered with HA-tag at the C-terminus. MTS: mitochondrial targeting sequence. HP: hydrophobic hairpin. STOM: stomatin. CTD: C-terminal coiled-coil domain. ΔCTD: deletion mutant of c-terminus mutant (Δ213–353). ΔN-term1: deletion mutant of N-terminus 1 (Δ59–212). ΔN-term2: deletion mutant of HP and STOM (Δ29212). b, Co-IP assay of NDUFS4 and STOML2 in HEK293T cells transfected with NDUFS4-FLAG and indicated STOML2-HA constructs shown in Extended Data Fig. 6a, using FLAG antibody-conjugated beads. WCL: whole cell lysate.

Fig. 1 |. Reduced Ndufs4 expression in podocytes in DKD.

a, Experimental workflow of quantification and comparison of mitoproteomes in primary mouse podocytes from diabetic Ins2^Akita/+ vs. WT mice (n=8/group). b, Heatmap illustrating log₂ fold change in mitochondrial ETC protein abundance in diabetic Ins2^Akita/+ vs. WT mice. c, Aggregated abundance of ETC proteins in podocytes of WT vs. diabetic Ins2^Akita/+ mice. Data are presented as median ± inter quartile range (IQR). d, CI activity in mitochondrial enriched samples isolated from primary podocytes (n=3–4 where each sample represents a pool of mitochondrial enriched samples from 4–6 mice). e, qRT-PCR validation of selected reduced CI subunits from Fig. 1b. Expression levels of mRNA subunits in diabetic subjects with DKD was obtained from Nephroseq v5 dataset (nephroseq.org; Ju CKD glom database). ns: not significant and n/a: not available. f, Human CI structure highlighting the location of NDUFS4 (blue) in the N module of the matrix arm of CI. The structure visualization was rendered through UCSF ChimeraX software (www.rbvi.ucsf.edu/chimerax). g, Left panel, representative immunoblots of NDUFS4 using mitochondria-enriched samples from primary podocytes of WT, Ins2^Akita/+, Lepr^db/+, and Lepr^db/db mice. VDAC was used as a loading control. Right panel, densitometric analysis of western blots normalized to control levels (n=4, each n represents a pool of mitochondria from 4 different mice). h, Pearson’s correlation analysis of human NDUFS4 mRNA expression. Data obtained from Nephroseq v5 (n=12). Median-centered Log₂ values are used for the analysis. i,j, Pearson’s correlation of glomerular NDUFS4 immunostaining with eGFR (i) and UACR (j) from diabetic subjects with DKD (n=34). k, Representative NDUFS4 immunostaining in glomeruli obtained from biopsies from healthy donors (n=9) and DKD patients with various degrees of albuminuria, including DKD with normoalbuminuria (n=4), DKD with microalbuminuria (n=2), DKD with macroalbuminuria (UACR >300, ≤1000 mg/gCr, n=8), and DKD with macroalbuminuria (UACR >1000mg/gCr, n=19). l, Glomerular NDUFS4 immunostaining in biopsies from healthy donors and DKD individuals with different stages of glomerular involvement: Donors (n=9), Glomerular (G)-class I DKD (n=3), G-class IIa DKD (n=5), G-class IIb DKD (n=5), G-class III DKD (n=14), G-class IV DKD (n=6), Scale bars= 50 μm. Data are presented as mean ± SEM except for (c). *P< 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Mann-Whitney test, FDR Q< 0.01 (c), unpaired two-sided t test (d,g), one-way ANOVA with post-hoc Tukey’s test (k,l), test for trend analysis for different classifications of DKD (l)

Fig. 2 |. Podocyte-specific Ndufs4 overexpression ameliorates DKD progression.

a, Schematic depiction of the construct used to engineer podocyte-specific Ndufs4-transgenic mice (Ndufs4^PodTg) (top), representative images of WT and transgenic mice (lower left), and PCR genotyping (lower right). b, qRT-PCR of Ndufs4 mRNA in primary podocytes and podocytes-depleted samples isolated from 8-week-old WT and Ndufs4^PodTg mice. (n=4). c, A representative NDUFS4 immunoblot (left), and quantification of NDUFS4 protein expression (right) (n=3). d, CI enzymatic activity in podocytes isolated from 8-week-old WT and Ndufs4^PodTg mice (n=3). e-g, Body weight (BW) (e), blood glucose (f), and urinary albumin-to-creatinine ratio (UACR) (g) in mice (n=8–12/group). h, Area under the curve (AUC) of UACR shown in Figure 2G (n=8–12/group). i, Representative micrographs of Periodic acid-Schiff (PAS) stained kidney sections, podocyte morphology in transmission electron microscopy (TEM), scanning electron microscopy (SEM), and immunostaining of Wilms’ tumor 1 (WT1) (pink), Synaptopodin (green), and DAPI (blue). Scale bars=50 μm in row 1, 500 nm in rows 2 and 3, and 50 μm in row 4. j, Kidney weight (KW) per body weight (BW) of 26-week-old mice (n=8–10/group). k, PAS positive area in glomeruli (n=60 from 3 mice/group). l, Glomerular basement membrane thickness (n=99–105 areas of TEM images from 3 mice/group). m, WT1 positive nuclei in glomeruli (n=60 from 3 mice/group). Data are presented as mean ± SEM (b-h,j) or median ± IQR (k-m, bold line: median, and dot line: IQR). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; unpaired two-sided Welch’s t test (b-d), one-way ANOVA with post-hoc Tukey’s test (e-h,j), or Kruskal-Wallis with post-hoc Dunn’s test (k-m).

Fig. 3 |. Ndufs4 overexpression improves mitochondrial morphology in the diabetic environment.

a, OCRs in primary podocytes (n=6–8 replicates/group). Dotted lines denote injections of oligomycin (2 μM), FCCP (2 μM), rotenone, and antimycin A (both 0.5 μM). b-e, Basal respiration (b), maximal respiration (c), ATP-linked OCR (d), and spare OCR (e). f, Primary podocytes susceptibility to rotenone (left panel), and rotenone half maximal inhibition (IC₅₀) (right panel, n=6 replicates/group). g, Representative glomerular CI activity in different group of mice (n=60 from 3 mice/group, scale bar=50 μm). h, Representative podocyte mitochondria from kidney tissues (top row) and pseudo-color superimposed images (middle row) to assess mitochondrial morphological changes, including aspect ratio (i), circularity (j), roundness (k), and feret diameter (l) (n=141–312 from 3 mice/group). TEM micrographs from primary podocytes isolated from experimental mice (bottom row). Scale bars=200 nm. m,n, Mitochondrial ROS assessed by Mitosox Red staining (n=4 replicates/group) (m), and relative ATP production (n=10 replicates/group) (n). Data are presented as mean ± SEM (a-f,m,n) or median ± IQR (g,i-l, bold line: median, and dot line: IQR). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with post-hoc Tukey’s test (b-f,m,n), or Kruskal-Wallis with post-hoc Dunn’s test (g,i-l).

Fig. 4 |. Cristae integrity and RSCs formation are restored by Ndufs4 overexpression.

a-d, Representative TEM micrographs of cristae morphology (a) and quantitative analyses of cristae density (b-d) in podocytes cultured under NG (5.5 mM) or HG (25 mM) for 48hrs with DOX induction (NDUFS4 OE) or without DOX induction (n=60/group, scale bars: 200 nm). IMM: inner mitochondrial membrane, OMM: outer mitochondrial membrane. e, Cryo-ET analysis of purified mitochondria in DOX-inducible Ndufs4 transfected podocytes cultured under NG without DOX, HG without DOX, and HG with DOX induction (NDUFS4 OE). Slices through representative Cryo-ET tomograms are shown in upper panels and corresponding segmentations of characteristic features in lower panels (Scale bars: 200 nm). f, BN-PAGE of digitonin-solubilized mitochondria isolated from HG-DOX or DOX-inducible NDUFS4 (NDUFS4 OE). Left panel: Coomassie staining; right panel: Immunoblots with OXPHOS cocktail antibodies. RSC: respiratory supercomplexes. g, Representative CI in-gel activity of n-Dodecyl β-D-maltoside (DDM)-solubilized mitochondria isolated from the same cells as in Fig. 4f. h, Immunodetection of NDUFS4 after SDS-PAGE of mitochondria proteins in cells from Fig. 4f. VDAC was used as a loading control. Data are presented as median ± IQR (Bold line: median, and dot line: IQR). ***P < 0.001, ****P < 0.0001. Kruskal-Wallis with post-hoc Dunn’s test (b-d).

Fig. 5 |. STOML2 interacts with NDUFS4.

a, Schematic depiction of APEX2 proximity labeling followed by the LC-MS/MS analysis in podocytes transduced with a DOX-inducible NDUFS4-APEX2 chimeric construct. B: biotin, IMM: inner mitochondrial membrane, OMM: outer mitochondrial membrane, RSC: respiratory supercomplexes. b, Volcano plots of NDUFS4-associated mitochondrial proteins identified by LC-MS/MS analysis. The average values of NDUFS4-APEX2 transfected podocytes without H₂O₂ and those without DOX induction are used as controls. The dotted line indicates a cut off for significant change based on FDR Q< 0.1. c, NDUFS4 interactomes identified by the LC-MS/MS following APEX2 labeling. d, Immunoblotting of STOML2, ATAD3, and IMMT in whole cell lysates (Input) and streptavidin pulldown samples. OPA1 was used as a negative control. e, RSCs were excised (Band 1 to 5) from BN-PAGE, followed by LC/MS-MS analysis. f, Heatmap of cristae organizing proteins in the RSCs from podocytes cultured in HG with DOX induction (HG+DOX) or without (HG-DOX). g, Immunoblotting of OXPHOS, STOML2, ATAD3 and IMMT following BN-PAGE analysis as described in Fig. 5e. h, Co-IP of NDUFS4 and STOML2. HEK 293T cells were transiently transfected with Ndufs4-FLAG expression construct. Input was used as a positive control while IgG was used as a negative control (Ctrl). i, GST affinity pulldown assay in HEK293T cells overexpressing a STOML2-HA fusion protein. j, Representative images of NDUFS4 (red) and STOML2 (green) by STED analysis in cultured podocytes under the NG, HG, and HG plus NDUFS4 OE conditions (Scale bar: 5 μm). k, Intensity-based colocalization expressed by Mander’s coefficient. M1 and M2 indicate the coefficient for NDUFS4 overlapping STOML2 and STOML2 overlapping NDUFS4, respectively (n=6). M2 coefficient was substantially lower than M1 since STOML2 as compared to NDUFS4 is more widely distributed both in cytoplasm and mitochondria. l, Object-based colocalization expressed by percent colocalization (n=6). m, Representative maximal intensity projection (MIP) images and detailed molecular images of NDUFS4 (red) and STOML2 (green) by STORM in cultured podocytes under the same conditions as shown in Fig. 5j. Scale bars: 10μm (left panel) and 500 nm (right panel). n, Percent colocalization based on the nearest neighboring distance (NND) <40 nm (n=4). o, Median NND (n=4). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with post-hoc Tukey’s test (k,l,n,o).

Fig. 6 |. STOML2 is essential for NDUFS4-mediated improvement of RSCs formation and cristae remodeling.

a, Immunoblot analysis of indicated proteins in NDUFS4 overexpression stable podocytes cultured in high glucose (HG) without (HG+DOX) or with (HG+DOX+STOML2KO) STOML2 CRISPR targeting; parental podocyte without Ndufs4 transduction was used as a control (HG). NDUFS4 was blotted with V5 tag antibody. b, BN-PAGE of digitonin-solubilized mitochondrial proteins from the same cells as shown in Fig. 5A stained with Coomassie or immunoblotted (IB) with OXPHOS cocktail or STOML2 antibodies. RSC: respiratory supercomplexes, DOX: doxycycline. c, Representative TEM micrographs of mitochondria in cells described in Fig. 6a, and cristae density measurements by IMM/OMM ratio (d), total cristae length (e) and cristae junction (f), (n=60 mitochondria, scale bars: 200 nm). IMM: inner mitochondrial membrane, OMM: outer mitochondrial membrane. g, Structure of mouse STOML2 wild type (WT) and deletion mutant constructs engineered with HA-tag at the C-terminus. MTS: mitochondrial targeting sequence. HP: hydrophobic hairpin. STOM: stomatin. CTD: C-terminal coiled-coil domain. β1–4: β1–4 sheets in STOM domain. α1–5: α1–5 helices in STOM domain. Protein-protein docking simulation of human STOML2 (blue) and NDUFS4 (green; in the context of human respirasome). h, GST pull-down assay from HEK293T cells overexpressing indicated STOML2-HA fusion proteins or deletion mutants. WT: full length wild type, ΔCTD: deletion mutant of C-terminal domain, ΔHP: deletion mutant of hydrophobic hairpin domain (Δ29–78), ΔSTOM: deletion mutant of stomatin domain (Δ79–212), Δ70–109: β1–3 sheets mutant, Δ110–168: α1–4 helices mutant, Δ169–212: β4 sheet and α5 helix mutant. Data are presented as median ± IQR (Bold line: median, and dot line: IQR). *P < 0.05, **** P < 0.0001. Kruskal-Wallis with post-hoc Dunn’s test (d-f).

Fig. 7 |. Schematics of NDUFS4-mediated cristae remodeling in DKD.

Normal crista structure (top) and proposed model depicting the remodeling of ETC in podocytes from diabetic condition (lower left), and how NDUFS4 OE restored the mitochondrial morphology and function (lower right). DKD: diabetic kidney disease, IMM: inner mitochondrial membrane, OMM: outer mitochondrial membrane, RSC: respiratory supercomplexes.