Journal: Nature Communications

Article Title: Generation of pluripotent stem cell-derived mouse kidneys in Sall1 -targeted anephric rats

doi: 10.1038/s41467-019-08394-9

Figure Lengend Snippet: Characterization of mouse ESC-derived kidney generated in Sall1 mut/mut rats. a Immunohistochemical staining of neonatal kidneys for GFP (green) with markers for each renal component (red); Nephrin for Podocytes, Aquaporin 1 for proximal convoluted tubule in the renal cortex and thin descending limb/Henle’s loop in the renal medulla, Na + /K + ATPase α-1 for thin ascending limb in the renal medulla and distal convoluted tubule. Calbindin for collecting tubule in the renal medulla. The nuclei were stained with DAPI (blue). Nephrin localizes a linear pattern along the glomerular basement membrane. Aquaporin 1 exists in both apical and basolateral plasma membranes of proximal tubules and descending thin limbs. Na + /K + ATPase α-1 exists in basolateral plasma membranes of thin ascending limbs and distal convoluted tubules. Scale bar: 50 μm. b Immunohistochemical staining of mouse-ESC derived neonatal kidneys in Sall1 mut/mut rats for GFP (green) with vascular endothelium, CD31 (red). The nuclei were stained with DAPI (blue). Scale bar: 25 μm. c Schematics of nephron and collecting tubule in complemented kidney in Sall1 mut/mut rats. Green shows the contribution of mouse ESCs. P: Podocytes; PT: Proximal convoluted tubule; TDL: Thin descending limb of Henle’s loop; HL: Henle’s loop; TAL: Thick ascending limb of Henle’s loop; DT: Distal convoluted tubule; CD: Collecting duct. d Ureter–bladder junction formation confirmed by intraurethral dye infusion in E22.5 Sall1 mut/mut rats with mouse PSC-derived kidneys. Arrowheads represent the lead position of trypan blue solution 1 and 3 s after injection. Ub: urinary bladder

Article Snippet: Primary antibodies used were anit-Sall1 polyclonal antibody (rabbit IgG, 1:100; ab31526, Abcam plc., Cambridge, UK) , anit-Six2 polyclonal antibody (rabbit IgG, 1:100; 11562-1-AP, Proteintech Group, Inc., Rosemont, IL 60018, USA) , anti-GFP polyclonal antibody (Chick IgY, 1:1000 dilution; ab13970, Abcam), anti-DsRed polyclonal antibody (rabbit IgG, 1:200 dilution; 632496, Takara Bio Inc.), anti-Nephrin polyclonal antibody (goat IgG, 1:200 dilution; AF3159-SP, R&D systems Inc., Minneapolis, MN, USA), anti-Podocin polyclonal antibody (rabbit IgG, 1:25 dilution; P0372, Sigma-Aldrich), anti-Calbindin polyclonal antibody (mouse monoclonal IgG, 1:200 dilution; ab82812, Abcam), anti-Aquaporin 1 polyclonal antibody (rabbit IgG, 1:50 dilution; AB2219, Merck Millipore), anti-Na + /K + ATPase α-1 monoclonal antibody (mouse IgG, 1:50 dilution; 05-369, Merck Millipore) and anti-CD31 polyclonal antibody (rabbit IgG, 1:50 dilution; ab28364, Abcam).

Techniques: Derivative Assay, Generated, Immunohistochemical staining, Staining, Membrane, Clinical Proteomics, Injection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy

doi: 10.1681/ASN.2017010030

Figure Lengend Snippet: Generation of anti-THSD7A antibodies in rabbits. (A) Scheme of rabbit immunization with two constructs encoding mTHSD7A and hTHSD7A. (B) Immunofluorescence analysis of reactivity of rabbit sera before and after immunization with membrane-bound mTHSD7A expressed in CHO cells. Bound rabbit IgG was visualized with an AF568 anti-rabbit IgG (red); nuclear GFP (green) was used to indicate transfected cells. (C) Reactivity of purified preimmune IgG and anti-THSD7A IgG with mouse glomerular extracts (MGE), human glomerular extracts (HGE), mTHSD7A, and hTHSD7A in Western blot analysis under nonreducing and reducing conditions. (D) Immunofluorescence staining of rabbit IgG and nephrin 2 hours after intravenous injection of preimmune IgG or anti-THSD7A IgG into male BALB/c mice (Rabbit [AF488], Nephrin [CY3], DNA [Draq5]). Scale bars indicate 50 µm.

Article Snippet: Immunofluorescence Analyses For immunolocalization of nephrin (guinea pig pAB, 1:100; Acris Antibodies), laminin (rabbit pAB, 1:1000; Sigma-Aldrich, St. Louis, MO), THSD7A (goat pAB, 1:200; Santa Cruz Biotechnology), WT-1 (rabbit pAB, 1:600; Santa Cruz Biotechnology), rabbit IgG (F488-rbIgG H+L, 1:200; Jackson ImmunoResearch Laboratories), murine IgG (Cy2-mouse IgG H+L, 1:400; Jackson ImmunoResearch Laboratories), C3 (FITC-goat pAB, 1:100; Cappel), or SOD2 (rabbit pAB, 1:100; Acris Antibodies), 2 μ M paraffin sections of experimental mouse kidneys were deparaffinized and rehydrated to water.

Techniques: Construct, Immunofluorescence, Membrane, Transfection, Purification, Western Blot, Staining, Injection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy

doi: 10.1681/ASN.2017010030

Figure Lengend Snippet: Anti-THSD7A antibodies and podocyte damage. (A) Immunofluorescence staining for rabbit IgG, G-actin, and F-actin (phalloidin) in primary cultured GECs after a 1-hour incubation with 1:100 preimmune or anti-THSD7A IgG (Rabbit IgG [AF488], G-actin [FITC], F-actin [AF568]). Lower panels represent 4× enlargements of the boxed areas in the middle panels. Scale bars indicate 50 µm. (B) Quantification of stress fiber formation after treatment of GECs with preimmune or anti-THSD7A IgG. One hundred images from three independent experiments were analyzed. Data indicate mean±SEM of actin optical density of individually circled cells. ***P<0.001, two-tailed nonparametric Mann–Whitney U test. (C) Immunohistochemical staining for SOD2 in mice 14 days after injection of preimmune or anti-THSD7A IgG (SOD2 [AF488]). The third panel represents a 4× enlargement of the boxed area in the second panel. As the antibody against SOD2 is derived from rabbits, we performed an additional control using the secondary anti-rabbit antibody only on a consecutive section of the diseased mouse (right panel). Scale bars indicate 50 µm. (D) Immunofluorescence staining for nephrin in mice 5 days and 14 days after injection of preimmune IgG or anti-THSD7A IgG. Scale bars indicate 50 µm (Nephrin [C2]). Lower panels represent 4× enlargements of the boxed areas above.

Article Snippet: Immunofluorescence Analyses For immunolocalization of nephrin (guinea pig pAB, 1:100; Acris Antibodies), laminin (rabbit pAB, 1:1000; Sigma-Aldrich, St. Louis, MO), THSD7A (goat pAB, 1:200; Santa Cruz Biotechnology), WT-1 (rabbit pAB, 1:600; Santa Cruz Biotechnology), rabbit IgG (F488-rbIgG H+L, 1:200; Jackson ImmunoResearch Laboratories), murine IgG (Cy2-mouse IgG H+L, 1:400; Jackson ImmunoResearch Laboratories), C3 (FITC-goat pAB, 1:100; Cappel), or SOD2 (rabbit pAB, 1:100; Acris Antibodies), 2 μ M paraffin sections of experimental mouse kidneys were deparaffinized and rehydrated to water.

Techniques: Immunofluorescence, Staining, Cell Culture, Incubation, Two Tailed Test, MANN-WHITNEY, Immunohistochemical staining, Injection, Derivative Assay, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Heterologous Model of Thrombospondin Type 1 Domain-Containing 7A-Associated Membranous Nephropathy

doi: 10.1681/ASN.2017010030

Figure Lengend Snippet: Knockdown of thsd7aa in zebrafish disturbs glomerular filtration barrier integrity. (A) Western blot analysis for thsd7aa after injection of a control morpholino (control MO) or a morpholino against thsd7aa (thsd7aa MO) into zebrafish larvae. (B) Phase contrast microscopic images (upper panels) and fluorescence microscopic images (lower panels) of CADE zebrafish larvae expressing the GC-GFP in the bloodstream after injection of either control MO or thsd7aa MO. Arrow indicates the pericardial area. Scale bars indicate 1 mm. ***P<0.001, two-tailed nonparametric Mann–Whitney U Test. (C and D) Immunofluorescence staining for eGFP and F-actin (eGFP, F-actin [AF568]) (C) or nephrin and eGFP (Nephrin [CY3]) (D) in zebrafish injected with control MO or thsd7aa MO. Arrows in (C) indicate Bowman’s space. Scale bars indicate 20 µm. (E) Electron microscopic studies in zebrafish larvae after injection of control MO or thsd7aa MO. Scale bars indicate 1 µm.

Article Snippet: Immunofluorescence Analyses For immunolocalization of nephrin (guinea pig pAB, 1:100; Acris Antibodies), laminin (rabbit pAB, 1:1000; Sigma-Aldrich, St. Louis, MO), THSD7A (goat pAB, 1:200; Santa Cruz Biotechnology), WT-1 (rabbit pAB, 1:600; Santa Cruz Biotechnology), rabbit IgG (F488-rbIgG H+L, 1:200; Jackson ImmunoResearch Laboratories), murine IgG (Cy2-mouse IgG H+L, 1:400; Jackson ImmunoResearch Laboratories), C3 (FITC-goat pAB, 1:100; Cappel), or SOD2 (rabbit pAB, 1:100; Acris Antibodies), 2 μ M paraffin sections of experimental mouse kidneys were deparaffinized and rehydrated to water.

Techniques: Knockdown, Filtration, Western Blot, Injection, Control, Fluorescence, Expressing, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Staining

Journal: Molecular and Cellular Biology

Article Title: The Wt1 +/R394W Mouse Displays Glomerulosclerosis and Early-Onset Renal Failure Characteristic of Human Denys-Drash Syndrome

doi: 10.1128/mcb.24.22.9899-9910.2004

Figure Lengend Snippet: FIG. 1. Generation of a mouse strain carrying the Wt1R394W allele. (A) Targeting construct. (B) Southern blot demonstrating the presence of the Wt1R394W neo-tagged allele in ES cells. (C) Genomic sequence from a Wt1/R394W mouse showing heterozygosity for the C1657T (R394W) mutation (arrow). (D) PCR genotyping to identify Wt1R394W homozygous and heterozygous embryos. (E) Allele-specific TaqMan assays to quantitate expression of wild-type and mutant alleles in kidneys from five Wt1/ and five Wt1/R394W animals.

Article Snippet: Deparaffinized sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature and then incubated for 1 h at room temperature with either anti-Wt1 polyclonal antibody (Santa Cruz) (1:100), which detects both mutant and wild-type proteins, or anti-nephrin polyclonal antibody.

Techniques: Construct, Southern Blot, Sequencing, Mutagenesis, Expressing

Journal: Molecular and Cellular Biology

Article Title: The Wt1 +/R394W Mouse Displays Glomerulosclerosis and Early-Onset Renal Failure Characteristic of Human Denys-Drash Syndrome

doi: 10.1128/mcb.24.22.9899-9910.2004

Figure Lengend Snippet: FIG. 2. (A) Normal kidney development in Wt1/R394W mice. Both regions of fully differentiated kidney (top panels) and of nephrogenic zones of the kidney (middle panels) are shown for newborn animals. Arrowheads indicate normal mature glomeruli; arrows indicate representative normal comma-shaped and S-shaped bodies. (B) Normal Wt1 protein expression in kidneys from Wt1/ and Wt1/R394W mice at different ages: newborn (NB), 3 weeks (3W), 2 months (2M), and 4 months (4M). Sections from mutant kidneys are from presymptomatic animals. Arrows indicate representative podocytes expressing Wt1.

Article Snippet: Deparaffinized sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature and then incubated for 1 h at room temperature with either anti-Wt1 polyclonal antibody (Santa Cruz) (1:100), which detects both mutant and wild-type proteins, or anti-nephrin polyclonal antibody.

Techniques: Expressing, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: The Wt1 +/R394W Mouse Displays Glomerulosclerosis and Early-Onset Renal Failure Characteristic of Human Denys-Drash Syndrome

doi: 10.1128/mcb.24.22.9899-9910.2004

Figure Lengend Snippet: FIG. 4. Electron micrographs of kidneys from 4-month-old Wt1/ and Wt1/R394W mice. Arrows, normal GBM in wild-type kidney and thickened GBM in mutant kidney. Arrowheads, normal podocyte foot processes in wild-type and effaced processes in mutant kidney.

Article Snippet: Deparaffinized sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature and then incubated for 1 h at room temperature with either anti-Wt1 polyclonal antibody (Santa Cruz) (1:100), which detects both mutant and wild-type proteins, or anti-nephrin polyclonal antibody.

Techniques: Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: The Wt1 +/R394W Mouse Displays Glomerulosclerosis and Early-Onset Renal Failure Characteristic of Human Denys-Drash Syndrome

doi: 10.1128/mcb.24.22.9899-9910.2004

Figure Lengend Snippet: FIG. 5. Genetic background-dependent development of protein- uria in Wt1/R394W mice at 4 months of age. Arrows indicate animals with proteinuria.

Article Snippet: Deparaffinized sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature and then incubated for 1 h at room temperature with either anti-Wt1 polyclonal antibody (Santa Cruz) (1:100), which detects both mutant and wild-type proteins, or anti-nephrin polyclonal antibody.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: The Wt1 +/R394W Mouse Displays Glomerulosclerosis and Early-Onset Renal Failure Characteristic of Human Denys-Drash Syndrome

doi: 10.1128/mcb.24.22.9899-9910.2004

Figure Lengend Snippet: FIG. 6. (A) Immunohistochemical analysis of nephrin protein expression in Wt1/R394W mice and control littermates at 2 months (2M) and 4 months (4M) of age (MF1 background). From mutant animals (/R394W) are shown glomeruli (GM) from presymptomatic animals (/R394W, top panels), histologically normal glomeruli from affected animals (/R394W, middle panels), and sclerotic glomeruli from affected animals (/R394W, bottom panels). (B) Cell proliferation assayed by PCNA immunohistochemical analysis of kidneys from Wt1/ and Wt1/R394W mice at 3 weeks (3W), 2 months (2M), and 4 months (4M) of age. Arrows indicate representative PCNA-positive podocytes.

Article Snippet: Deparaffinized sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature and then incubated for 1 h at room temperature with either anti-Wt1 polyclonal antibody (Santa Cruz) (1:100), which detects both mutant and wild-type proteins, or anti-nephrin polyclonal antibody.

Techniques: Immunohistochemical staining, Expressing, Control, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

doi: 10.1172/JCI171237

Figure Lengend Snippet: ( A ) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1 -KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). ( B ) Representative immunofluorescence images of podocytes in control -FUCCI and Pfn1- KO- FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. ( C ) Quantification of the distribution of podocytes in cell cycle phase in B . Total of 100 glomeruli in 5 different mice. ( D ) Primary podocytes isolated from control and Pfn1 -KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. ( E ) Representative immunofluorescence images of podocytes in control and Pfn1- KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. ( F ) Quantification of podocyte density in glomeruli in E . Total of 40 glomeruli from 5 different mice. ( G ) Representative immunofluorescence images of urinary podocytes from control and Pfn1 -KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G 1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G 2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

Techniques: Immunofluorescence, Control, Staining, Isolation