Journal: Kidney International

Article Title: Activation of podocyte Notch mediates early Wt1 glomerulopathy

doi: 10.1016/j.kint.2017.11.014

Figure Lengend Snippet: Hairy enhancer of split 1 ( HES1) expression coincides with onset of glomerulosclerosis in CAGG-CreER TM+/− ;Wt1 f/f transgenic 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-CreER TM−/− ;Wt1 f/f (mutant) and CAGG-CreER TM+/− ;Wt1 f/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 .

Article Snippet: The following antibodies were used: cleaved Notch1 (Val1744, rabbit; Cell Signaling Technologies, Danvers, MA) 1:100 immunohistochemistry, 1:50 immunofluorescence; Notch1 (D1E11; Cell Signaling) 1:1,000 Western blot; cleaved Notch2 (cleaved-Ala1734, rabbit; Sigma-Aldrich) 1:150 immunohistochemistry; cleaved Notch2 (07-1234, rabbit; Millipore) 1:200 immunofluorescence, 1:1,000 Western blot; Jagged 1(C-20, goat; Santa Cruz Biotechnology), 1:100 immunofluorescence; Podoplanin (811, hamster; Novus Biologicals, Littleton, CO), 1:100 immunofluorescence; CD31 (0026, rat; BD Pharmingen, BD Biosciences), 1:100; Hes1 (rabbit, kind gift from Professor Ryoichiro Kageyama, University of Kyoto, Japan), 1:1000; Synaptopodin (G1D4, mouse; PROGEN Biotechnik, Heidelberg, Germany); Podocin (P0372; Sigma Aldrich) 1;100; biotinylated Lotus tetragonolobus lectin, LTL (B-1325; Vector Laboratories) 1:100; and cleaved Caspase-3 (Asp175, rabbit; Cell Signaling Technologies), 1:400 immunofluorescence.

Techniques: Expressing, Transgenic Assay, Immunofluorescence, Labeling, Mutagenesis, MANN-WHITNEY, Transduction, Plasmid Preparation, Fluorescence