Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: (a) Representative Western blot analysis and (b) bar graph quantification of APOM protein expression in kidney cortices of Col4a3 +/+ and Col4a3 −/− mice (n=3 mice). (c) Representative Western blot analysis and (d) bar graph quantification of S1PR4 protein expression in kidney cortices (n=3 mice). (e) Bar graph quantification of sphingosine-1-phosphate (S1P) levels determined by LC-MS analysis in kidney cortices of Col4a3 +/+ and Col4a3 −/− mice (n=5 mice). (f) Representative Western blot analysis and (g) bar graph quantification of APOM protein expression in isolated glomeruli (n=3 mice). (h) Representative Western blot and (i) bar graph quantification of S1PR4 protein expression in glomeruli of Col4a3 +/+ and Col4a3 −/− mice (n=3). (j) Representative Western blot analysis and (k, l) bar graph quantification of APOM and S1PR4 protein expression in tubules of Col4a3 +/+ and Col4a3 −/− mice (n=4 mice). (m) Representative Western blot analysis and (n) bar graph quantification of APOM protein expression in Col4a3 +/+ and Col4a3 −/− podocytes (n=3). (o) Representative Western blot analysis and (p) bar graph quantification of S1PR4 protein expression in of Col4a3 +/+ and Col4a3 −/− podocytes (n=3). (q) Bar graph quantification of secreted APOM protein in culture media of Col4a3 +/+ and Col4a3 −/− podocytes by ELISA (n=4). (r) Caspase 3 activity (fold change in apoptosis) in normal human podocytes treated with S1P, albumin, or S1P/albumin with or without APOM or CYM. (n=3–7). (s) Caspase 3 activity in Col4a3 +/+ , Col4a3 −/− , and Col4a3 −/− podocytes treated with APOM or CYM. (n=5–6).

Article Snippet: Four-week-old Col4a3 −/− and wildtype (Col4a3 +/+ ) mice were injected with 0.9% saline solution (Teknova, S5819), APOM-Fc (Sino Biologicals, 13495-H02H), IgG-Fc (Sino Biologicals, 10702-HNAH), or CYM50358 (CYM) (Tocris, 4679).

Techniques: Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy, Isolation, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: Four-week-old Col4a3 −/− and wildtype (Col4a3 +/+ ) mice were injected with either saline solution (control), recombinant human APOM-Fc (APOM), or the selective S1PR4 antagonist CYM50358 (CYM). APOM was administered intraperitoneally once per weekly at doses of 100 μg, 100 μg, 75 μg, and 50 μg, respectively. CYM was administered intraperitoneally three times per week at 10 mg/kg. Mice were sacrificed at 8 weeks of age. Bar graph quantification of (a) urinary albumin-to-creatinine ratio, (b) BUN, and (c) plasma creatinine (n = 6–8 mice). (d) Periodic Acid-Schiff (PAS) staining (4 μm paraffin sections, 20x magnification) and (e) bar graph quantification of the glomerulosclerotic index scores (n=6–8 mice). (f) Representative images of Picro-Sirius red staining (4 μm paraffin sections, 10x) and (g) bar graph quantification of the fibrotic area calculated as percentage of total cortical area (n = 5–8 mice). (h) Representative confocal images of WT1 (red), synaptopodin (green), and DAPI (blue) staining in glomeruli (63× magnification), and (i) bar graph quantification of WT1+ podocytes per glomerulus (n = 5–8). (j) Transmission electron microscopy (TEM) images of glomerular basement membrane (GBM) and podocyte foot processes (2500× magnification), and (k) bar graph quantification of foot processes per μm GBM (n = 6).

Article Snippet: Four-week-old Col4a3 −/− and wildtype (Col4a3 +/+ ) mice were injected with 0.9% saline solution (Teknova, S5819), APOM-Fc (Sino Biologicals, 13495-H02H), IgG-Fc (Sino Biologicals, 10702-HNAH), or CYM50358 (CYM) (Tocris, 4679).

Techniques: Recombinant, Injection, Saline, Control, Clinical Proteomics, Staining, Transmission Assay, Electron Microscopy, Membrane

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: Bar graph quantification of (a) triglycerides, (b) cholesterol esters, and (c) total cholesterol in kidney cortices of Col4a3 +/+ , Col4a3 −/− , and Col4a3 −/− mice treated with APOM or CYM (n=6–8 mice). (d) Representative Oil Red O staining of frozen kidney sections (6μm; 20x magnification) and (e) bar graph quantification (n = 4 mice). (f) Confocal images of Nile Red-stained lipid droplets in podocytes, and (g) bar graph quantification of lipid droplet number in Col4a3 +/+ , Col4a3 −/− , and treated Col4a3 −/− podocytes (n = 4). Nile Red (green), DAPI (blue), CellMask HCS and phalloidin (red); 20× magnification. Bar graph quantification of (h) triglycerides (i) cholesterol esters, and (j) total cholesterol in podocytes from each treatment group (n=3–6).

Article Snippet: Four-week-old Col4a3 −/− and wildtype (Col4a3 +/+ ) mice were injected with 0.9% saline solution (Teknova, S5819), APOM-Fc (Sino Biologicals, 13495-H02H), IgG-Fc (Sino Biologicals, 10702-HNAH), or CYM50358 (CYM) (Tocris, 4679).

Techniques: Recombinant, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: (a) Venn diagram showing the number of significantly differentially expressed genes (P < 0.05) between Col4a3 +/+ podocytes and Col4a3 −/− podocytes, and between Col4a3 −/− podocytes and Col4a3 −/− podocytes treated with APOM or CYM, as determined by RNA-seq. (b) Volcano plot of RNA-seq results between Col4a3 +/+ podocytes and Col4a3 −/− podocytes (significant genes in red, P < 0.05). (c) KEGG pathway enrichment analysis of differentially expressed genes between Col4a3 +/+ podocytes and Col4a3 −/− podocytes, ranked by significance (d) Volcano plot of RNA-seq results in the APOM treatment group (significant genes in red, P < 0.05). (e) Volcano plot of RNA-seq results for the CYM treatment group (significant genes are shown as red dots (P < 0.05), non-significant genes as black dots) highlighting the most significantly altered gene. (f) Magnified volcano plot of the CYM treatment group excluding Angptl4, which was disproportionately significant (significant genes are shown as red dots (P < 0.05), non-significant genes as black dots). (g) KEGG pathway enrichment analysis of differentially expressed genes in the CYM treatment group, ranked by significance. (h) Heatmap showing Log2 fold change of significantly altered genes (P < 0.05) in the CYM treatment group involved in lysosomal and autophagy pathways. (i) KEGG pathway enrichment analysis of the shared differentially expressed genes between the Col4a3 +/+ podocytes and Col4a3 −/− podocyte group and the CYM treatment group, ranked by significance.

Article Snippet: Four-week-old Col4a3 −/− and wildtype (Col4a3 +/+ ) mice were injected with 0.9% saline solution (Teknova, S5819), APOM-Fc (Sino Biologicals, 13495-H02H), IgG-Fc (Sino Biologicals, 10702-HNAH), or CYM50358 (CYM) (Tocris, 4679).

Techniques: Expressing, RNA Sequencing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: (a) Representative Western blot analysis and (b) bar graph quantification of the LC3-II/LC-I protein expression ratio in Col4a3 −/− podocytes treated with APOM and CYM (n=3). (c) Representative Western blot analysis and (d) bar graph quantification of p62 protein expression in Col4a3 −/− podocytes following treatment (n=5–6). (e) Representative confocal images and bar graph quantification of (f) lysosome number and the (g) co-localization of lysosomes with lipid droplets. Lysotracker (red), Nile red (green), and DAPI (blue) (20x magnification). (h) Representative Western blot analysis and (i) bar graph quantification of ATGL protein expression in Col4a3 −/− podocytes treated with APOM or CYM (n=3). (j) Bar graph quantification of cholesterol efflux in treated Col4a3 −/− podocytes (n=3–8). (k) Representative Western blot of lysosomal acid lipase (LAL) and LC3-II/LC-I protein expression in kidney cortices of Col4a3 −/− mice treated with vehicle or CYM and (l, m) bar graph quantification (n=3–4 mice). (n) Representative Western blot of LAMP1 protein expression in kidney cortices of Col4a3 −/− mice treated with vehicle or CYM and (o) bar graph quantification (n=3–4 mice).

Article Snippet: Four-week-old Col4a3 −/− and wildtype (Col4a3 +/+ ) mice were injected with 0.9% saline solution (Teknova, S5819), APOM-Fc (Sino Biologicals, 13495-H02H), IgG-Fc (Sino Biologicals, 10702-HNAH), or CYM50358 (CYM) (Tocris, 4679).

Techniques: Western Blot, Expressing

Journal: Pediatric Nephrology (Berlin, Germany)

Article Title: Re-evaluating the MYH9 p.I1816V variant in a patient with atypical clinical presentation

doi: 10.1007/s00467-025-07059-8

Figure Lengend Snippet: Peripheral blood smear and kidney biopsy findings. a – c Light microscopy of kidney tissue stained with periodic acid–methenamine silver, original magnification × 400, scale bar = 50 μm. a Segmental spikes and bubbling of the glomerular basement membrane are observed. b Enlarged view of the boxed area in a . c Focal segmental sclerotic lesions are observed in two glomeruli. d Immunofluorescence staining for IgG shows capillary wall deposition, whereas C3 deposition was absent. Original magnification × 400. e , f Electron microscopy of kidney tissue, original magnification × 3000, scale bar = 2 μm. e Thickening of the glomerular basement membrane with intramembranous lucent deposits (arrow). f Focal foot process effacement (arrowheads) is observed. g – i Immunofluorescence staining for NMMHC-IIA in kidney tissue, original magnification × 400, scale bar = 20 μm. Red: NMMHC-IIA; green: COL4A3 (Supplementary Fig. ). Primary antibodies: rabbit polyclonal anti–NMMHC-IIA (PRB-440P, BioLegend; 1:100) and mouse monoclonal anti–COL4A3 (sc-52317, Santa Cruz; 1:50). Secondary antibodies: Alexa Fluor 555–conjugated anti-rabbit IgG (1:500) and Alexa Fluor 488–conjugated anti-mouse IgG (1:1000). g Control sample from a time-zero kidney transplant biopsy. h Markedly reduced NMMHC-IIA expression in podocytes from a patient with the severe p.R702C variant, compared with control. i No appreciable reduction in NMMHC-IIA expression in the current patient with the p.I1816V variant. j , k Peripheral blood smear stained with May–Giemsa, original magnification × 1000, scale bar = 10 μm. j Giant platelets (arrows) are observed, although only two were identified across all high-power fields (< 5%). k Döhle-like inclusion bodies are absent. l Immunofluorescence staining for NMMHC-IIA in neutrophils shows no abnormal cytoplasmic accumulation, original magnification × 1000

Article Snippet: Primary antibodies: rabbit polyclonal anti–NMMHC-IIA (PRB-440P, BioLegend; 1:100) and mouse monoclonal anti–COL4A3 (sc-52317, Santa Cruz; 1:50).

Techniques: Light Microscopy, Staining, Membrane, Immunofluorescence, Electron Microscopy, Control, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Deep Learning Enables Automated Segmentation and Quantification of Ultrastructure from Transmission Electron Microscopy Images

doi: 10.1101/2025.11.05.686793

Figure Lengend Snippet: (A) Schematic illustrating two mouse models of glomerular disease: knocking out Col4a3 or specific podocyte ILK knocking out. (B) Schematic of a rat model of glomerular disease (PHN) induced by anti-Fx1a antibody injection. (C) Representative TEM images analyzed by the TEAMKidney model for WT, Col4a3 KO, and ILK cKO mice. (D) Violin plots showing quantitative measurements of FP and GBM widths across WT, Col4a3 KO, and ILK cKO mice. (E) Representative TEAMKidney predicted images for WT and PHN rat samples. (F) Violin plots comparing FP and GBM widths between WT and PHN groups.

Article Snippet: Col4a3 cKO mice were purchased from the Jackson laboratory (RRID:IMSR_JAX:002908).

Techniques: Injection