Fig 1: Administration of CNO leads to increased Ca2+-levels in podocytes in vivo.(A) Glomeruli of ROSA26hM3D/wt; NPHS2.Cretg/wt; GCaMP3tg/wt mice were visualised in the intact kidney in vivo using a Multi-Photon-microscope. CNO (5 mg/kg) was injected i.a. after 10 sec and caused an immediate increase in intracellular Ca2+ levels (scale bar = 25 µm). (B) Fluorescence is depicted as F/F1–10, where the mean value of the first ten measurements (F1–10) prior to CNO stimulation was used for normalisation. Experiments were performed in three biological replicates. (C) Statistical analysis performed for Multi-Photon-imaging experiments revealed a significant increase of the intracellular Ca2+ after CNO stimulation. N = 18 cells were included in the statistical analysis (t-test p < 0.0001).
Fig 2: Expression of glomerular proteins. Representative laser confocal images of kidney sections from 9-day old Foxc2 fl/fl;Pod-Cre+ and Foxc2 fl/fl mice using antibodies labeling (A) proteins involved in anchoring of podocytes (ITGB1/ß1-integrin, ILK/integrin linked kinase and CYR61/CCN1), (B) basement membrane proteins (COL4A3 and COL4A4), and (C) components of the foot processes (i.e., slit diaphragm proteins (NPHS1/nephrin and NPHS2/podocin), anchoring of slit diaphragm to cytoskeleton (TJP1/ZO-1, ACTN4/a-actinin 4, CD2AP/CD2-associated protein, and NES/nestin), and foot process coating (PODXL/podocalyxin)).
Fig 3: Laser confocal microscopy of kidney sections from 9-day-old Foxc2 fl/fl; Pod-Cre+ and Foxc2 fl/fl mice. (A) Images of whole glomeruli, with separate images of NRP1/neuropilin 1 (green) and NPHS2/podocin (red) signal and a merged image for visualization of co-localization. Orange indicates merged/co-localized signals. (B) Zoomed in on part of the same glomeruli as in (A) to further visualize the localization of green (NRP1) and red (NPHS2) signals.
Fig 4: Podocyte VEGF knockdown (VEGFKD ) prevents glomerular hypertrophy in diabetic mice. (A) VEGFKD Transgenic mouse line carries 4 transgenes: Nphs2-rtTA and tet-0-shVEGF that are activated by doxycycline to synthesize shRNA targeting Vegf-a exon 1, which inhibits expression of all Vegf-a isoforms in podocytes (Veron et al., 2012). (B) VEGFKD mice received STZ (50 mg IP, 5 daily doses) (DM-VEGFKD - dox), STZ + doxycycline (DM-VEGFKD + dox), doxycycline (VEGFKD + dox) or no treatment (VEGFKD - dox). Dox was started a week after STZ, 2 weeks later was considered time 0 (when random blood glucose was steadily elevated) for DM-VEGFKD mice; (C) non-diabetic control glomerulus (VEGFKD - dox) shows normal histology; (D) diabetic control (DM-VEGFKD - dox) glomerulus shows hypertrophy and mesangial expansion; (E) non-diabetic VEGFKD (+ dox) glomerulus is smaller than control (C, - dox); (F) diabetic VEGFKD (+ dox) glomerulus shows diffuse glomerulosclerosis and is smaller than control (D, - dox); Scale bars = 50 µm; (G) quantitation of glomerular size demonstrates significantly smaller glomerular volume in VEGFKD (+ dox) vs. control (-dox) glomeruli from non-diabetic (VEGFKD ) and diabetic (DM-VEGFKD ) mice; unpaired t-test with Welch’s correction was used; asterisk (*) indicates p < 0.05, (***) indicates p < 0.001; control vs. VEGFKD or non-diabetic vs. diabetic, as indicated; non-DM, non-diabetic mice, DM, diabetic mice; dox, uninduced mice; dox, doxycycline- treated mice.
Fig 5: Hairy enhancer of split 1 (HES1) expression coincides with onset of glomerulosclerosis in CAGG-CreERTM+/-;Wt1f/ftransgenic mice. (a,b,b') Shown are representative images of glomeruli following double immunofluorescence labeling of day (D) 5 postinduction (PI) mouse kidney sections with anti-HES1, anti-Synaptopodin, and Lotus tetragonolobus lectin (LTL) of CAGG-CreERTM-/-;Wt1f/f (mutant) and CAGG-CreERTM+/-;Wt1f/f (control) transgenic mice following multichannel labeling with Alexa Fluor 488–conjugated secondary antibody (LTL, demarcates tubules), Alexa Fluor 594–conjugated secondary antibody (Hes1), and Alexa Fluor 647–conjugated secondary (Synaptopodin, demarcates podocytes). Sections are counterstained with 4',6-diamidino-2-phenylindole (DAPI). Bars = 50 µm. (b,b') In mutants, HES1-positive, Synaptopodin-positive glomerular epithelial cells were observed in regions distinct from LTL-positive tubules. Bars = 50 µm. (b') Bars = 10 µm. (c,d,d') Segmental clusters of nuclear HES1 expression in Synaptopodin-positive, platelet–endothelial cell adhesion molecule (PECAM)-negative podocytes are evident in mutant glomeruli and not evident in control glomeruli. Bars = 50 µm. (e) Upregulation of podocyte Snail and Slug transcript at onset of glomerulosclerosis at D6 PI. Median Snail mRNA expression at D6 PI in control versus mutant mice: 1.1 (interquartile range [IQR]: 0.9, 1.2) versus 2.7 (IQR:1.8, 2.8), *P = 0.045, Mann-Whitney test. Median Slug mRNA expression at D6 PI in control versus mutant mice: 1.2 (IQR: 0.6, 1.5) versus 2.8 (IQR: 1.4, 3.7), *P = 0.03, Mann-Whitney test. (f) Increase in Hes1 mRNA in doxycycline-treated primary Nphs2;rtTA podocytes transduced with TetOHes1 plasmid compared with untreated TetOHes1 and treated green fluorescence protein (GFP)–transduced primary Nphs2;rtTA podocytes. Mean Hes1 mRNA expression relative to Gapdh (±SD): untreated control (GFP) versus untreated TetOHes1 versus treated control (doxycycline 2 µg/ml) versus treated TetOHes1 (2 µg/ml) versus treated TetOHes1 (4 µg/ml): 1.15 ± 0.66 versus 1.26 ± 1.13 versus 1.21 ± 0.88 versus 54.56 ± 44.24 (**P < 0.004) versus 42.78 ± 33.07 (**P < 0.008). No significant difference in dose observed, P = .045 (not significant [NS]). (g) Upregulation of Snail and Slug transcripts, genes implicated in epithelial to mesenchyme transition in doxycycline-treated primary Nphs2;rtTA podocytes transduced with TetOHes1 plasmid compared with untreated TetOHes1 and treated GFP-transduced primary Nphs2;rtTA podocytes. Untreated control (GFP) versus untreated TetOHes1 versus treated TetOHes1 (4 µg/ml): mean Snail mRNA expression relative to Gapdh (±SD): 1.76 ± 2.1 versus 1.01 ± 0.31 versus 4.82 ± 3.94, *P < 0.05. Mean Slug mRNA expression relative to Gapdh (±SD): 0.95 ± 1.12 versus 0.31 ± 0.26 versus 5.10 ± 0.3.9 (*P < 0.05). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
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