Journal: Communications biology

Article Title: Soluble Klotho protects against glomerular injury through regulation of ER stress response.

doi: 10.1038/s42003-023-04563-1

Figure Lengend Snippet: Fig. 4 Hepatocyte-specific overexpression of Klotho ameliorates renal injury and proteinuria in a mouse model of NTS-induced glomerulonephritis. a Left panel: Urinary albumin/creatinine ratio (U-ACR) in WT, Alb-hKL, and Pod-hKL, 0, 1, 3, and 7 days after injection of NTS. $$$$p < 0.0001 vs. Pod-hKL and @@@@p < 0.0001 vs. WT. Right panel: Blood urea nitrogen in the different groups without injury (control mice) or 7 days after injection of NTS (NTS mice). ####p < 0.0001 vs. WT-control; @p < 0.05 vs. WT-NTS. b Representative images of PAS (left panels) and TEM (right panels) of kidney cortex from WT-control and WT, Alb-hKL, Pod-hKL 7 days after NTS injection. c Percentage of affected and crescentic glomeruli were quantified from 30 randomly selected glomeruli in the corresponding PAS staining. Tubular casts were assessed over the whole cross-section giving a score between 0, no cast to 5, >80% of the tubules are affected (n = 11 WT-control; n = 10 WT-NTS; n = 5 Alb-hKL-NTS; n = 11 Pod-hKL-NTS). Quantification of mean glomerular basement membrane (GBM) thickness and the number of slits per μm GBM (n = 5 WT-control; n = 10 WT-NTS; n = 6 Alb-hKL-NTS; n = 8 Pod-hKL-NTS). ###p < 0.001, ####p < 0.0001 vs. WT-control; @p < 0.05, @@p < 0.01 vs. WT-NTS and *p < 0.05, ***p < 0.001 vs. Alb-hKL-NTS. d Representative images of immunofluorescent stainings of Nephrin, WT-1, and α-SMA in the different groups. e Quantification of the number of WT-1 positive podocytes in glomeruli (left panel), glomerular area (middle panel), and WT-1 positive podocytes/glomerular area (right panel) (n = 7 WT-control; n = 9 WT-NTS; n = 5 Alb-hKL- NTS; n = 9 Pod-hKL-NTS). α-SMA positive area was counted per cortical cross section (10 sections per sample; n = 6 WT-control; n = 10 WT-NTS; n = 5 Alb-hKL-NTS; n = 9 Pod-hKL-NTS). ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. WT-control; @p < 0.05 vs. WT vs. WT-NTS and *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. Alb-hKL-NTS. Scale bars: 50 μm, 30 μm, 5 μm (b), 10 μm, 50 μm (d). Statistical significance between groups was determined using one- way or two-way repeated measures ANOVA followed by multiple comparisons.

Article Snippet: Sections were washed in PBST (0.1% Triton-X in 1X PBS, pH 7.4) for 10 min before incubation in a sheep polyclonal antibody against nephrin (AF4269, R&D systems) diluted at 1:100 and a rat monoclonal against Klotho (KM2076, Trans Genic Inc.), diluted at 1:200 in 1X PBS with 0.3% Triton X-100 (PBST) at room temperature overnight.

Techniques: Over Expression, Injection, Control, Staining, Membrane

Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: MRL.lpr mice were injected i.p. with free KN93 (10 μg/wk), anti-podocin or anti-nephrin antibody–coated KN93-loaded nlg (10 μg of KN93/week), or empty nlg at from 8 to 16 weeks of age (n = 5–7 mice in each group). (A) Urine albumin/creatinine (Alb/Cre) ratio. Urine samples were obtained biweekly, and albumin and creatinine levels were determined by ELISA. Error bars represent mean ± SEM. *P < 0.05, 2-way ANOVA with Bonferroni’s post test. (B and C) Representative images of glomeruli from 16-week- old MRL.lpr mice treated with anti-nephrin antibody–coated empty nlg or KN93-loaded nlg. PAS (B) and Masson’s trichrome staining (C) are shown. The arrows point to the crescent. Scale bars: 50 μm. (D) Mean histological scores in the kidneys of mice from the indicated treatment groups (n = 5 in each group). *P < 0.05; **P < 0.01, Student’s t test. (E and F) Electron microscopic images of a glomerulus from a mouse treated with anti-nephrin empty nlg (left) showing diffuse podocyte foot process (FP) effacement with slit diaphragm occlusion (arrows) and subepithelial dense deposits (asterisks) and a mouse treated with anti-nephrin KN93-loaded nlg (right), which shows podocytes with normal foot processes and slit diaphragms and no deposits in the basement membrane. Original magnification, ×8,000 (E); ×30,000 (F). Three glomeruli were evaluated in each of 3 mice in each experimental condition. (G) C3 and IgG deposition was significantly suppressed in the glomeruli of MRL.lpr mice treated with KN93 targeted to podocytes, as indicated. Kidney sections from MRL/MpJ (16 weeks of age) mice are shown as controls (n = 5 mice in each group). (H) Nephrin (green) and synaptopodin (red) expression detected by immunofluorescence. Scale bars: 50 μm. (I) The fluorescence intensity of nephrin and synaptopodin quantified in glomeruli from mice subjected to the indicated treatments. ****P < 0.0001, Student’s t test. (J) Nphs1, Nphs2, and Synpo expression in glomeruli from mice treated with anti-nephrin–coated empty or KN93-loaded nlg. Results were normalized to the expression of GAPDH (n = 5 in each group). ****P < 0.0001, Student’s t test.

Article Snippet: Isolated cells were stained for flow cytometry with antibodies against nephrin (bs-10233R-A488, Bioss Antibodies) and podocin (bs-6597R-A647, Bioss Antibodies).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Immunofluorescence, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: Mice treated with KN93-loaded nlg targeted to podocytes and CaMK4-deficient mice develop proteinuria after exposure to LPS. Each B6 or B6 Camk4–/– mouse was injected i.p. with LPS on day 0. (A) Urine albumin/creatinine ratio of mice treated with anti-nephrin antibody–coated nlg either empty or loaded with KN93. Nlg were injected i.p. on day –1 (n = 10 mice in each group). (B) Urine albumin/creatinine ratio of B6 or B6 Camk4–/– mice (n = 6 in each group). Error bars represent mean ± SEM. *P < 0.05; **P< 0.01, 2-way ANOVA with Bonferroni’s post test. (C) Representative immunofluorescent images of nephrin (upper panels) and synaptopodin (lower panels) in the kidneys of mice injected with LPS or PBS. Scale bar: 50 μm. (D) Nphs1 expression in human podocytes after stimulation with LPS with or without KN93. Cells were treated with KN93 1 hour before stimulation. Results were normalized by the expression of GAPDH. Four independent experiments were performed. *P < 0.05, 1-way ANOVA with Tukey’s post test. (E and F) Mean percentage of nephrin-positive human podocytes evaluated by flow cytometry after stimulation with LPS for 72 hours with KN93 (E) or CAMK4 siRNA (F). Four independent experiments were performed. *P < 0.05, 1-way ANOVA with Tukey’s post test.

Article Snippet: Isolated cells were stained for flow cytometry with antibodies against nephrin (bs-10233R-A488, Bioss Antibodies) and podocin (bs-6597R-A647, Bioss Antibodies).

Techniques: Injection, Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: Each mouse was injected i.v. with adriamycin (ADM) on day 0. (A–C) Anti-nephrin antibody–coated empty or KN93-loaded nlg were injected i.p. into BALB/c mice on day –1 and day 3 (n = 7 in each group). (A) Mean urine albumin/creatinine ratio from mice subjected to the indicated treatment. Error bars represent mean ± SEM. ****P < 0.0001, 2-way ANOVA with Bonferroni’s post test (B) Representative images showing PAS staining of kidney from BALB/c mice treated with anti-nephrin antibody–coated empty nlg or KN93-loaded nlg. Scale bar: 50 μm. (C) Representative immunofluorescence images of nephrin and synaptopodin expression in glomeruli. Scale bar: 100 μm. (D) Free KN93 (10 μg/wk), anti-nephrin antibody–coated empty, or KN93-loaded nlg (10 μg of KN93/wk) were injected i.p. into BALB/c mice on day 7 (n = 5 in each group). **P < 0.01; ***P < 0.001, 2-way ANOVA with Bonferroni’s post test. (E) Mean urine albumin/creatinine ratio of B6 or B6 Camk4–/– mice treated with adriamycin. (n = 5 in each group; 2 independent experiments were performed). ****P < 0.0001, 2-way ANOVA with Bonferroni’s post-test. (F) Representative electron microscopy images of glomeruli from BALB/c mice at 7 days after exposure to adriamycin, treated with anti-nephrin antibody–coated empty nlg (left) or KN93-loaded nlg (right). Original magnification, ×8,000.

Article Snippet: Isolated cells were stained for flow cytometry with antibodies against nephrin (bs-10233R-A488, Bioss Antibodies) and podocin (bs-6597R-A647, Bioss Antibodies).

Techniques: Injection, Staining, Immunofluorescence, Expressing, Electron Microscopy

Journal: Nature Communications

Article Title: A slit-diaphragm-associated protein network for dynamic control of renal filtration

doi: 10.1038/s41467-022-33748-1

Figure Lengend Snippet: A Epitope-localization of the anti-Podocin ABs used for meAP-MS analysis (further details in Supplementary Fig. ). B Electron micrographs showing the distribution of Podocin proteins labeled by anti-Podocin AB a1 (and visualized by gold-particle coupled anti-IgG ABs, black dots) on the cytoplasmic side (P-face) of the plasma membrane in freeze-fracture replicas prepared from isolated glomeruli. Image on the right is the frame on the left at an expanded scale. Note that immuno-gold particles for Podocin (arrows) are observed close to the slit-facing membrane, as well as on the sole of the podocyte foot processes (FPs). Scale bars are 500 nm. Micrographs are representative of at least four experiments. C t-SNE plot of tnR-values determined for all proteins identified in anti-Podocin APs from CL-47 solubilized membrane fractions of isolated mouse glomeruli (using KO source materials as negative controls). Podocin and all proteins clustered by t-SNE analysis close to the target are indicated by colored squares and shown at an extended scale. D Table summarizing the results of the finally annotated constituents of the Podocin interactome in all APs with the indicated ABs (Supplementary Fig. ). Each column illustrates the results of the indicated anti-Podocin AP controlled by the target KO. Color-coding symbolizing evaluation by target-normalized ratios (tnR, AP versus control) as follows: yellow/green: tnR against KO control >0.25, white: tnR below the threshold of 0.25, crossed: protein was not identified in the respective target AP. MW of all proteins is indicated on the right. ID refers to the Swiss-Prot entry of the respective protein.

Article Snippet: All other rabbit polyclonal antibodies against Nephrin, Neph1, and Podocin were generated by AbFrontier (Seoul, Korea).

Techniques: Labeling, Membrane, Isolation, Control

Journal: Nature Communications

Article Title: A slit-diaphragm-associated protein network for dynamic control of renal filtration

doi: 10.1038/s41467-022-33748-1

Figure Lengend Snippet: Constituents of the Podocin interactome determined by meAP-MS, together with annotation of subcellular localization and primary function(s) indicated in literature and public databases

Article Snippet: All other rabbit polyclonal antibodies against Nephrin, Neph1, and Podocin were generated by AbFrontier (Seoul, Korea).

Techniques: Membrane

Journal: Nature Communications

Article Title: A slit-diaphragm-associated protein network for dynamic control of renal filtration

doi: 10.1038/s41467-022-33748-1

Figure Lengend Snippet: A – C Quantitative analysis of mRNAs coding for Nephrin, Neph1, and Podocin three weeks after induction of the target-gene knockout (left panel) and of the albumin/creatinine ratio determined post-induction (right panel) in control and knockout animals. Data are mean (±SEM) of 5 (WT, Nephrin KO), 4 (WT, Neph1 KO), 7 (WT, Podocin KO) (mRNA), and 10, 10, 9, and 6 WT, Nephrin KO, Neph1 KO, and Podocin KO animals (albumin/creatinine ratio), respectively. D Immunofluorescence was determined with anti-Nephrin (green) and anti-Podocin (red) ABs before and at the indicated periods after doxycycline-triggered knockout of Podocin in mouse glomeruli. Blue color originates from DAPI staining. Scaling as indicated; experiments were repeated three times with similar results. E Albuminuria quantified as albumin/creatinine ratio as a function of time after induction of the target-gene knockout. Data points are the mean values shown in (A)-(C). Note the largely different development of albuminuria after induction of the targeted knockout.

Article Snippet: All other rabbit polyclonal antibodies against Nephrin, Neph1, and Podocin were generated by AbFrontier (Seoul, Korea).

Techniques: Gene Knockout, Control, Knock-Out, Immunofluorescence, Staining