Journal: Cell Discovery

Article Title: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction

doi: 10.1038/s41421-026-00873-w

Figure Lengend Snippet: a MAM formation in TECs was analyzed via TEM imaging. Scale bars = 2 µm. The ER and mitochondria in the TEM images were graphically reconstructed to visualize MAM formation. Mito: mitochondria (orange), ER (red). b Quantification of the MAM length to the mitochondrial perimeter ratio in different groups of WT mice, n = 15 (150 mitochondria); C5ar2 −/− mice, n = 15 (193 mitochondria); diabetic WT mice, n = 15 (232 mitochondria); and diabetic C5ar2 −/− mice, n = 15 (175 mitochondria) microscopic fields from 5 mice/group. c Representative western blotting images of the specified proteins in the cytoplasm, mitochondrial fractions, ER fractions, and MAM fractions in renal cortex tissue from WT mice. d – f Representative western blotting images and quantitative analysis of PSS1 and PSS2 protein levels in the MAM fraction of renal cortex tissue from different groups of mice ( n = 3 per group). Total proteins stained with Ponceau S served as an internal reference for quantifying MAM proteins. d , g Representative western blotting images and quantitative analysis of calnexin and VDAC1 protein levels in the MAM fraction in renal cortex tissues from different groups of mice ( n = 3 per group). h Representative live-cell confocal microscopy images of ER–mitochondria contacts in TCMK-1 cells under different treatments. The ER was labeled with sec61β-GFP (green), the mitochondria were labeled with Mito Orange (red), and the nuclei were labeled with DAPI (blue). Z-stack images with orthogonal projections of the boxed regions are shown (scale bars = 10 µm). i Quantification of ER–mitochondria colocalization using Pearson’s correlation coefficient ( n = 15 microscopic fields from three independent experiments). j Representative in situ proximity ligation assay (PLA) images of TCMK-1 cells probed with IP3R1 and VDAC1 antibodies. The red dots indicate colocalization sites between IP3R1 and VDAC1. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). k Quantitative analysis of PLA red spot counts in TCMK-1 cells subjected to different treatments. l Surface representation of the molecular docking between truncated MFN2 (Protein Data Bank code 6JFK) and PSS1. MFN2 (orange), PSS1 (blue). m Surface representation of the molecular docking between truncated MFN2 (6JFK) and PSS2. MFN2 (orange), PSS2 (red). n Schematic diagram of the structure of the MFN2 domain and truncation mutants. FL full-length, GTPase GTPase domain, HR1 heptad repeat 1, PR proline-rich domain, TM1/TM2 transmembrane domains 1 and 2, HR2 heptad repeat 2, Δ1/Δ2/Δ3 truncation mutants with the indicated deletions. o , p Co-IP analysis of PSS1–MFN2 and PSS2–MFN2 interactions using full-length and truncation mutants of MFN2 in HEK293T cells. q Representative confocal microscopy images of ER–mitochondria contacts in human kidney cortical proximal tubule epithelial cells (HK-2 cells) transfected with full-length or truncation mutants of MFN2 under HG/HF conditions (scale bars = 10 µm). r Quantification of ER–mitochondria colocalization in HK-2 cells ( n = 15 microscopic fields from three independent experiments). s Representative PLA images of Lv Pss2 ( Flag )-TCMK-1 cells probed with FLAG and MFN2 antibodies. Red spots indicate colocalization sites between FLAG and MFN2. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). t Quantitative analysis of PLA red spot counts in Lv Pss2 -TCMK-1 cells subjected to different treatments. The data in the graphs are presented as the means ± SD. The data were analyzed by two-sided one-way ANOVA with Tukey’s test. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were blocked with 5% skim milk and then incubated with primary antibodies against C5aR2 (sc-515734; Santa Cruz Biotechnology), PSS1 (ab157222; Abcam), PSS2 (ARP49961_P050; Aviva Systems Biology Corporation), MFN2 (12186-1-AP; Proteintech), XBP-1s (143F; BioLegend), p-EIF2α (28740-1-AP; Proteintech), EIF2α (11170-1-AP; Proteintech), CHOP (15204-1-AP; Proteintech), COX IV (11242-1-AP; Proteintech), calnexin (10427-2-AP; Proteintech), ERK1/2 (4695; Cell Signaling Technology), p-ERK1/2 (4370; Cell Signaling Technology), HA (ab9110, Abcam), FLAG (ab213519, Abcam), α-tubulin (HRP-80762; Proteintech), and β-actin (Ac028; ABclonal).

Techniques: Imaging, Western Blot, Staining, Confocal Microscopy, Labeling, In Situ, Proximity Ligation Assay, Co-Immunoprecipitation Assay, Transfection

Journal: Cell Discovery

Article Title: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction

doi: 10.1038/s41421-026-00873-w

Figure Lengend Snippet: a Experimental scheme for assessing PSS2 overexpression. AAV9-Ksp- Pss2 was administered via tail vein (i.v.) injection at week 5, and the mice were sacrificed at week 20 for analysis ( n = 6 per group). b uACRs in different groups of mice ( n = 6 per group). c Representative images of PAS staining of the glomeruli and tubulointerstitium (scale bars = 50 µm); representative TEM micrographs (scale bars = 2 µm). MAM formation was analyzed via TEM imaging in TECs (scale bars = 5 µm). The ER and mitochondria in the TEM images were graphically reconstructed to visualize MAM formation. Mito (orange) and ER (red). d , e Quantitative analysis of mesangial matrix expansion ( d ) and the tubulointerstitial injury index ( e ) in different groups of mice. f , g Quantification of the GBM thickness ( f ) and foot process width ( g ) in different groups of mice ( n = 6 per group). h Quantification of the MAM length to mitochondrial perimeter ratio in different groups of mice: STZ/HFD-WT + OE Ctrl mice, n = 15 (with 167 mitochondria); STZ/HFD- C5ar2 −/− + OE Ctrl mice, n = 15 (with 272 mitochondria) microscopic fields from 5 mice/group; STZ/HFD-WT + OE PSS2 mice, n = 15 (with 193 mitochondria); STZ/HFD- C5ar2 −/− + OE PSS2 mice, n = 15 (with 220 mitochondria) microscopic fields from 6 mice/group. i The mitochondrial OCRs of LvNC and Lv Pss2 -TCMK-1 cells were analyzed in the presence or absence of HG/HF or C5ar2 knockdown. j , k Representative OCR tracings (from three independent experiments) and quantified OCR values are presented. l Representative PLA images of TCMK-1 cells probed with IP3R1 and VDAC1 antibodies. The red spots indicate sites of IP3R1 and VDAC1 colocalization. The cells were counterstained with DAPI (blue) (scale bars = 50 μm). m Quantitative analysis of PLA red spot counts in TCMK-1 cells subjected to different treatments. n Representative live-cell confocal microscopy images of ER–mitochondria contacts in TCMK-1 cells under different treatments. The ER was labeled with sec61β-GFP (green), the mitochondria were labeled with Mito Orange (red), and the nuclei were labeled with DAPI (blue). Z-stack images with orthogonal projections of the boxed regions are shown (scale bars = 5 µm). o Working model of how PSS interacts with MFN2 at the mitochondria–ER interface to maintain MAM formation and PS homeostasis. The data in the graphs are presented as the means ± SDs. The data were analyzed by two-sided one-way ANOVA with Tukey’s test. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were blocked with 5% skim milk and then incubated with primary antibodies against C5aR2 (sc-515734; Santa Cruz Biotechnology), PSS1 (ab157222; Abcam), PSS2 (ARP49961_P050; Aviva Systems Biology Corporation), MFN2 (12186-1-AP; Proteintech), XBP-1s (143F; BioLegend), p-EIF2α (28740-1-AP; Proteintech), EIF2α (11170-1-AP; Proteintech), CHOP (15204-1-AP; Proteintech), COX IV (11242-1-AP; Proteintech), calnexin (10427-2-AP; Proteintech), ERK1/2 (4695; Cell Signaling Technology), p-ERK1/2 (4370; Cell Signaling Technology), HA (ab9110, Abcam), FLAG (ab213519, Abcam), α-tubulin (HRP-80762; Proteintech), and β-actin (Ac028; ABclonal).

Techniques: Over Expression, Injection, Staining, Imaging, Knockdown, Confocal Microscopy, Labeling

Journal: Cell Discovery

Article Title: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction

doi: 10.1038/s41421-026-00873-w

Figure Lengend Snippet: a UMAP plot showing five subpopulations of PT cells in vehicle-treated m/m mice, vehicle-treated db/db mice, and P59-treated db/db mice ( n = 3 per group). Each dot corresponds to a single cell and is colored according to the cell type. b , c Violin plots showing Pss1 and Pss2 transcript expression levels of the indicated genes in PT cells from vehicle-treated m/m mice, vehicle-treated db/db mice, and P59-treated db/db mice. d Expression levels of Pss2 in each PT cluster revealed by scRNA-seq. e Representative western blotting images and quantitative analysis of PSS1 and PSS2 expression in renal cortex tissues from different groups of mice ( n = 6 per group). f MAM formation in TECs was analyzed via TEM imaging (scale bars = 2 µm). The ER and mitochondria in TEM images were graphically reconstructed to visualize MAM formation. Mito (orange) and ER (yellow). g Quantification of the MAM length to mitochondrial perimeter ratio in different groups of mice; m/m + vehicle group, n = 15 (with 261 mitochondria); m/m + P59 (3 mg/kg) group, n = 15 (with 270 mitochondria); db/db + vehicle group, n = 15 (with 192 mitochondria); db/db + P59 (3 mg/kg) group, n = 15 (with 235 mitochondria) microscopic fields from 6 mice/group. h – j Representative western blotting images and quantitative analysis of PSS1 and PSS2 protein levels in the MAM fraction in renal cortex tissues from different groups of mice ( n = 3 per group). Total proteins stained with Ponceau S served as an internal reference for quantifying MAM proteins. h , k Representative western blotting images and quantitative analysis of calnexin and VDAC1 protein levels in the MAM fraction in renal cortex tissues from different groups of mice ( n = 4 in the db/db + P59-3 mg/kg group; n = 3 in the other groups). l ELISA analysis of PS levels in the MAM fraction of renal tubules in different groups of mice ( n = 6 per group). m Representative live-cell confocal microscopy images of ER–mitochondria contacts in TCMK-1 cells under different treatments. The ER was labeled with sec61β-GFP (green), the mitochondria were labeled with Mito Orange (red), and the nuclei were labeled with DAPI (blue). Z-stack images with orthogonal projections of the boxed regions are shown (scale bars = 10 µm). n Representative PLA images of TCMK-1 cells probed with IP3R1 and VDAC1 antibodies. The red spots indicate sites of IP3R1 and VDAC1 colocalization. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). o Quantitative analysis of PLA red spot counts in TCMK-1 cells subjected to different treatments. p Representative PLA images of Lv Pss2 ( Flag )-TCMK-1 cells probed with FLAG and MFN2 antibodies. Red spots indicate colocalization sites between FLAG and MFN2. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). q Quantitative analysis of PLA red spot counts in Lv Pss2 ( Flag )-TCMK-1 cells subjected to different treatments. The data in the graphs are presented as the means ± SD. The data were analyzed by two-sided one-way ANOVA with Tukey’s test. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were blocked with 5% skim milk and then incubated with primary antibodies against C5aR2 (sc-515734; Santa Cruz Biotechnology), PSS1 (ab157222; Abcam), PSS2 (ARP49961_P050; Aviva Systems Biology Corporation), MFN2 (12186-1-AP; Proteintech), XBP-1s (143F; BioLegend), p-EIF2α (28740-1-AP; Proteintech), EIF2α (11170-1-AP; Proteintech), CHOP (15204-1-AP; Proteintech), COX IV (11242-1-AP; Proteintech), calnexin (10427-2-AP; Proteintech), ERK1/2 (4695; Cell Signaling Technology), p-ERK1/2 (4370; Cell Signaling Technology), HA (ab9110, Abcam), FLAG (ab213519, Abcam), α-tubulin (HRP-80762; Proteintech), and β-actin (Ac028; ABclonal).

Techniques: Single Cell, Expressing, Western Blot, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Labeling