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

a OCRs in primary podocytes. Dotted lines denote injections of oligomycin (2 μM), FCCP (2 μM), rotenone, and antimycin A (both 0.5 μM). Basal respiration (b), maximal respiration (c), ATP-linked OCR (d), and spare OCR (e) (a–e, n = 6 (WT), n = 7 (Ins2^Akita/+ and Ndufs4^PodTg;Ins2^Akita/+), n = 8 (Ndufs4^PodTg), replicates/group). 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 assessed by NADH oxidase staining from kidney sections (left panel) in different group of mice (n = 60 from 3 mice/group, scale bar = 50 μm), and quantitative analysis using Image J software normalized to the median intensity of the WT (right panel). 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 = 312 (WT), n = 167 (Ins2^Akita/+), n = 141 (Ndufs4^PodTg) and n = 284 (Ndufs4^PodTg;Ins2^Akita/+), measurements from 3 mice/group). TEM micrographs from primary podocytes isolated from experimental mice (bottom row). Scale bars = 200 nm. Quantitative analyses of cristae number (m) and junction (n) normalized by mitochondrial area using TEM micrographs of primary podocytes isolated from mice (n = 45 mitochondria/group). o Representative images of mROS in primary podocytes assessed by mito-roGFP (Left panel). The heatmap shows the ratio of the oxidized to reduced roGFP signals in mitochondrial matrix. Quantification of mitochondrial redox states (right panel). (n = 20–22 cells/group). Scale bar = 10 μm. Total (p) and mitochondrial (q) ATP production rates in primary podocytes assessed by Seahorse Analyzer (n = 9 (WT), n = 8 (Ndufs4^PodTg, Ins2^Akita/+, and Ndufs4^PodTg;Ins2^Akita/+), replicates/group). Data are presented as mean ± SEM (a–f, p, q) or median ± IQR (g, i–o, bold line: median, and dot line: IQR). ****P < 0.0001. One-way ANOVA with post-hoc Tukey–Kramer test (b–f, p, q), or Kruskal–Wallis with post-hoc Dunn’s test (g, i–o). Source data are provided as a Source Data file.