Fig 2: Impact of cold storage alone, combined cold storage plus transplantation, and autotransplantation on mitochondrial fusion protein expression.Renal extracts (30 ug) were resolved on SDS-PAGE gels and immunoblotted. Representative MFN 1 and MFN2 western blots showing distinct protein bands of both MFN1/2 (~75 kDa) in Sham, ATx, and CS/Tx kidneys (A) as well as control and CS alone kidneys (B). Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Representative OPA1 western blot showing long form of OPA1 (L, ~95 kDa) and short form of OPA1 (S, ~75 kDa) in Sham, ATx, and CS/Tx kidneys CS/Tx (C) as well as control and CS alone kidneys (D). Actin was used as a loading control. Densitometry evaluation of each blot (normalized to actin) is shown on the right panel. Values were expressed as Mean ± S.E.M. (n = 4). Unpaired Student’s t test was used to compare the means between control and CS kidneys. One-way ANOVA followed by Tukey’s post-hoc test for multiple group comparisons was used to compare the means between sham, ATx, and CS/Tx kidneys; * indicates means are significantly different (P < 0.05) when compared to control or sham and # indicates means are significantly different (P < 0.05) when compared to ATx.

Fig 3: Haemin pre-treatment suppresses serum deprivation and hypoxia (SD/H)-induced mitochondrial fission and apoptosis of bone marrow-mesenchymal stem cell (BM-MSCs). A, The cell viability of BM-MSCs with or without haemin (1, 5, 10, 20 µmol/L) pre-treatment for 24 hours under normoxia or SD/H was determined by CCK-8 assay (i). The cell viability of BM-MSCs with or without 10 µmol/L haemin pre-treatment for 6, 12, 24 or 48 hours under normoxia or SD/H was determined by CCK-8 assay (ii). B, Representative images of the fragmented mitochondria (magnification of 20x) and quantitative analysis of fragmented mitochondria in BM-MSCs and haemin-pretreated BM-MSCs under normoxia or SD/H. C, Western blotting and quantitative analysis for the expression of Mfn2 and p-Drp1 ser616 in BM-MSCs and haemin-pretreated BM-MSCs under normoxia or SD/H exposure. D, Representative images of TUNEL staining (magnification of 20x) and quantitative analysis of the apoptosis of BM-MSCs or haemin-pretreated BM-MSCs under normoxia or SD/H. Data are expressed as the mean ± SEM. n = 3. Scale bar = 50 µm. *P < .05, **P < .01, ***P < .001. ns, not significant

Fig 4: MitoQ ameliorates mitochondrial fission and ROS production in Ang II-infused mice. (a) Representative image of mitochondrial ultrastructure in podocytes from each group by TEM (original magnification, ×10000), scale bar = 1 µm. (b) Quantitative analysis of the mitochondrial aspect ratio in the different groups. (c) Western blot analysis of Opa1, Mfn2, Drp1, and Fis1 in glomeruli from different groups. (d) Quantification of protein levels from (c). (e) DHE staining to detect oxidative stress in glomeruli from different groups (original magnification, ×600), scale bar = 20 µm. (f) Quantification of fluorescence intensity from (e). *P < 0.05 vs. the normal saline infusion group; #P < 0.05 vs. the Ang II infusion group. n = 5.

Fig 5: Mesenchymal stem cells alleviate mitochondrial activity and high glucose-induced oxidative stress in cultured glomerular endothelial cells. A: Mesenchymal stem cells (MSCs) increased adenosine triphosphate production in high glucose (HG)-induced glomerular endothelial cells (GECs); B: MSCs also attenuated mitochondrial membrane potential (??m) in GECs. Carbonyl cyanide 3-chlorophenylhydrazone as a mitochondrial oxidative phosphorylation uncoupler, was used as the positive control group; C: Effect on mitochondrial fission marker and the fusion marker levels; D: Pretreatment with MSCs attenuated HG-induced upregulation in DCF-DA [intracellular reactive oxygen species (ROS)] and MitoSOX Red (mitochondrial ROS) fluorescent intensity; E: Representative western blot expression levels of ROS-protective enzymes (superoxide dismutase 2 and glutathione peroxidase 3); F: Concentration of interleukin (IL)-6 in the supernatant by enzyme-linked immunosorbent assay; G: Mitochondrial transfer ameliorated the mRNA expression levels of inflammatory markers (tumor necrosis factor-a and IL-1ß), as determined using real-time reverse transcriptase-polymerase chain reaction. Data are presented as the mean ± SD. aP < 0.05 vs normal control group, bP < 0.01 vs normal control group, cP < 0.05 vs high glucose group. MSCs: Mesenchymal stem cells; DRP1: Dynamin-related protein 1; MFN2: Mitofusin 2; ROS: Reactive oxygen species; SOD2: Superoxide dismutase 2; GPx-3: Glutathione peroxidase 3; IL-6: Interleukin-6; CCCP: Carbonyl cyanide 3-chlorophenylhydrazone; NC: Normal control; HG: High glucose; TNF-a: Tumor necrosis factor-a; ATP: Adenosine triphosphate.