Structured Review

Progen Biotechnik synaptopodin
Effect of MLL3 shRNA on <t>synaptopodin</t> and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P
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Images

1) Product Images from "Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy"

Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

Journal: BMC Nephrology

doi: 10.1186/s12882-016-0390-8

Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P
Figure Legend Snippet: Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P

Techniques Used: shRNA, Expressing, Cell Culture, Derivative Assay, Chromatin Immunoprecipitation

Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P
Figure Legend Snippet: Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P

Techniques Used: Expressing, Electron Microscopy

Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P
Figure Legend Snippet: Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P

Techniques Used: shRNA, Expressing, Immunohistochemistry, Electron Microscopy

Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation
Figure Legend Snippet: Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation

Techniques Used: Expressing

2) Product Images from "Sildenafil Prevents Podocyte Injury via PPAR-γ–Mediated TRPC6 Inhibition"

Article Title: Sildenafil Prevents Podocyte Injury via PPAR-γ–Mediated TRPC6 Inhibition

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2015080885

PDE5A is expressed by podocytes. RNA and protein were isolated from cultured podocytes and renal mouse cortex; subsequently, (A) PCR and (B) Western blot were performed to determine PDE5A expression in these samples. Glomerular PDE5A expression was confirmed by immunofluorescence staining of PDE5A. (C) Importantly, costaining with synaptopodin showed PDE5A expression in the podocytes; merge is a higher magnification of single-channel images. AB, antibody; NTC, no template control.
Figure Legend Snippet: PDE5A is expressed by podocytes. RNA and protein were isolated from cultured podocytes and renal mouse cortex; subsequently, (A) PCR and (B) Western blot were performed to determine PDE5A expression in these samples. Glomerular PDE5A expression was confirmed by immunofluorescence staining of PDE5A. (C) Importantly, costaining with synaptopodin showed PDE5A expression in the podocytes; merge is a higher magnification of single-channel images. AB, antibody; NTC, no template control.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining

3) Product Images from "Interconnected Network Motifs Control Podocyte Morphology and Kidney Function"

Article Title: Interconnected Network Motifs Control Podocyte Morphology and Kidney Function

Journal: Science signaling

doi: 10.1126/scisignal.2004621

Minimally complex directed graph of the cAMP signaling network that controls CREB phosphorylation in the context of podocyte synaptopodin expression and actin bundling
Figure Legend Snippet: Minimally complex directed graph of the cAMP signaling network that controls CREB phosphorylation in the context of podocyte synaptopodin expression and actin bundling

Techniques Used: Expressing

Expression and functional localization of synaptopodin in cultured podocytes is controlled by PKA
Figure Legend Snippet: Expression and functional localization of synaptopodin in cultured podocytes is controlled by PKA

Techniques Used: Expressing, Functional Assay, Cell Culture

4) Product Images from "Mesenchymal Stromal Cells Induce Podocyte Protection in the Puromycin Injury Model"

Article Title: Mesenchymal Stromal Cells Induce Podocyte Protection in the Puromycin Injury Model

Journal: Scientific Reports

doi: 10.1038/s41598-019-55284-7

Comparative analysis of podocyte markers at days 30 and 60 in the different groups. The expression levels of nephrin (A) , podocin (B) , synaptopodin (C) , and podocalyxin (D) were analyzed in renal tissue by qRT-PCR.
Figure Legend Snippet: Comparative analysis of podocyte markers at days 30 and 60 in the different groups. The expression levels of nephrin (A) , podocin (B) , synaptopodin (C) , and podocalyxin (D) were analyzed in renal tissue by qRT-PCR.

Techniques Used: Expressing, Quantitative RT-PCR

Glomerular expression of nephrin and synaptopodin was analyzed by immunohistochemistry at days 30 and 60 in the different groups. Normal expression of these podocyte markers was observed in the illustrative microphotographs (400x magnification) of the Control group (A,D,G,J) . The PAN group (B,E) showed lower nephrin expression than the Control group at days 30 and 60. The mSC administration improved nephrin expression (C,F) compared with the PAN group, particularly at day 60. Similarly, synaptopodin expression was lower in PAN animals compared with the Control group at days 30 and 60 (H,K) . Synaptopodin expression was upregulated by mSC (I,L) . Results of the quantitative histomorphometry by means of an image analysis system is shown in graphs ( M,N ).
Figure Legend Snippet: Glomerular expression of nephrin and synaptopodin was analyzed by immunohistochemistry at days 30 and 60 in the different groups. Normal expression of these podocyte markers was observed in the illustrative microphotographs (400x magnification) of the Control group (A,D,G,J) . The PAN group (B,E) showed lower nephrin expression than the Control group at days 30 and 60. The mSC administration improved nephrin expression (C,F) compared with the PAN group, particularly at day 60. Similarly, synaptopodin expression was lower in PAN animals compared with the Control group at days 30 and 60 (H,K) . Synaptopodin expression was upregulated by mSC (I,L) . Results of the quantitative histomorphometry by means of an image analysis system is shown in graphs ( M,N ).

Techniques Used: Expressing, Immunohistochemistry

5) Product Images from "ARHGEF7 (β-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function"

Article Title: ARHGEF7 (β-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2019090982

β -PIX deficiency in podocytes leads to heavy proteinuria in adult mice. (A) Representative images of the immunofluorescence staining for β -PIX (red), synaptopodin (green), and 4′,6-diamidino-2-phenylindole (blue) of the kidney from 3-week-old control (CTRL) and β -PIX Pod−/− (KO) mice. The right panels show a magnification of the indicated areas (white squares) in the left panels. Bars, 20 μ m. (B) Representative immunoblots for nephrin, β -PIX, and tubulin of glomerular lysates from 5-week-old CTRL and KO mice. (C) Densitometric quantification of the protein levels in (B) ( n =4). (D) Body weight ( n =3–8), (E) appearance of the kidney, (F) kidney weight ( n =3 or 4), and (G) urine ACR ( n =2–8). Statistically significant differences are indicated (* P
Figure Legend Snippet: β -PIX deficiency in podocytes leads to heavy proteinuria in adult mice. (A) Representative images of the immunofluorescence staining for β -PIX (red), synaptopodin (green), and 4′,6-diamidino-2-phenylindole (blue) of the kidney from 3-week-old control (CTRL) and β -PIX Pod−/− (KO) mice. The right panels show a magnification of the indicated areas (white squares) in the left panels. Bars, 20 μ m. (B) Representative immunoblots for nephrin, β -PIX, and tubulin of glomerular lysates from 5-week-old CTRL and KO mice. (C) Densitometric quantification of the protein levels in (B) ( n =4). (D) Body weight ( n =3–8), (E) appearance of the kidney, (F) kidney weight ( n =3 or 4), and (G) urine ACR ( n =2–8). Statistically significant differences are indicated (* P

Techniques Used: Mouse Assay, Immunofluorescence, Staining, Western Blot

β -PIX deficiency causes podocyte apoptosis and loss. (A) Representative images of the immunofluorescence staining for WT1 (red), synaptopodin (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue) of the kidney from 5- to 13-week-old control (CTRL) and β -PIX Pod−/− (KO) mice. (B) Podocyte density in (A) was analyzed. n =3 or 4 in each group. (C) Representative immunoblots for β -PIX and tubulin of cultured mouse podocytes with β -PIX KD and CTRL. (D) Densitometric quantification of β -PIX in (C). (E) Detachment assay using CTRL and β -PIX KD podocytes. Cells were incubated with 250 ng/ml ADR for up to 48 hours on laminin 521–coated plates. The numbers of the attached cells are normalized to 0 hour. n =3 in (D and E) in each group. (F) Representative images of the immunofluorescence staining for cleaved caspase 3 (red) and DAPI (blue) of CTRL and β -PIX KD podocytes at 24 hours after ADR treatment. (G) Quantification of the ratio of cleaved caspase 3–positive cells to total cells at 24 hours after ADR treatment. n =2–5 in each group. Statistically significant differences are indicated (* P
Figure Legend Snippet: β -PIX deficiency causes podocyte apoptosis and loss. (A) Representative images of the immunofluorescence staining for WT1 (red), synaptopodin (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue) of the kidney from 5- to 13-week-old control (CTRL) and β -PIX Pod−/− (KO) mice. (B) Podocyte density in (A) was analyzed. n =3 or 4 in each group. (C) Representative immunoblots for β -PIX and tubulin of cultured mouse podocytes with β -PIX KD and CTRL. (D) Densitometric quantification of β -PIX in (C). (E) Detachment assay using CTRL and β -PIX KD podocytes. Cells were incubated with 250 ng/ml ADR for up to 48 hours on laminin 521–coated plates. The numbers of the attached cells are normalized to 0 hour. n =3 in (D and E) in each group. (F) Representative images of the immunofluorescence staining for cleaved caspase 3 (red) and DAPI (blue) of CTRL and β -PIX KD podocytes at 24 hours after ADR treatment. (G) Quantification of the ratio of cleaved caspase 3–positive cells to total cells at 24 hours after ADR treatment. n =2–5 in each group. Statistically significant differences are indicated (* P

Techniques Used: Immunofluorescence, Staining, Mouse Assay, Western Blot, Cell Culture, Incubation

6) Product Images from "Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes"

Article Title: Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12660

Assessment of in vitro differentiation of human podocytes. Expression of differentiation/podocyte markers podocin and synaptopodin as determined by Western blot analysis ( A ) on human podocytes. To assess equal loading, expression of β‐actin was determined. Pre: pre‐confluent (proliferating) culture; Post: post‐confluent (proliferating) culture; Diff: differentiated culture. Podocin ( B ) and synaptopodin ( C ) immunoreactivity was determined on differentiated human podocytes by immunofluorescence labelling (Alexa‐Fluor ® ‐488, green fluorescence). Nuclei were counterstained by propidium iodide (red fluorescence). Calibration mark: 50 μm. NC : negative control.
Figure Legend Snippet: Assessment of in vitro differentiation of human podocytes. Expression of differentiation/podocyte markers podocin and synaptopodin as determined by Western blot analysis ( A ) on human podocytes. To assess equal loading, expression of β‐actin was determined. Pre: pre‐confluent (proliferating) culture; Post: post‐confluent (proliferating) culture; Diff: differentiated culture. Podocin ( B ) and synaptopodin ( C ) immunoreactivity was determined on differentiated human podocytes by immunofluorescence labelling (Alexa‐Fluor ® ‐488, green fluorescence). Nuclei were counterstained by propidium iodide (red fluorescence). Calibration mark: 50 μm. NC : negative control.

Techniques Used: In Vitro, Expressing, Western Blot, Immunofluorescence, Fluorescence, Negative Control

7) Product Images from "Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy"

Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

Journal: BMC Nephrology

doi: 10.1186/s12882-016-0390-8

Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P
Figure Legend Snippet: Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P

Techniques Used: shRNA, Expressing, Cell Culture, Derivative Assay, Chromatin Immunoprecipitation

Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P
Figure Legend Snippet: Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P

Techniques Used: Expressing, Electron Microscopy

Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P
Figure Legend Snippet: Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P

Techniques Used: shRNA, Expressing, Immunohistochemistry, Electron Microscopy

Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation
Figure Legend Snippet: Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation

Techniques Used: Expressing

8) Product Images from "Activation of mineralocorticoid receptor by ecdysone, an adaptogenic and anabolic ecdysteroid, promotes glomerular injury and proteinuria involving overactive GSK3β pathway signaling"

Article Title: Activation of mineralocorticoid receptor by ecdysone, an adaptogenic and anabolic ecdysteroid, promotes glomerular injury and proteinuria involving overactive GSK3β pathway signaling

Journal: Scientific Reports

doi: 10.1038/s41598-018-29483-7

Ecdysone induces glomerular molecular changes indicative of glomerulopathy. ( A ) Mice were treated as stated in Fig. 1 . Kidney specimens were obtained on day 14 and prepared for Masson trichrome staining, immunohistochemistry staining for fibronectin and cleaved caspase-3 (Cleaved casp-3), and dual color fluorescent immunohistochemistry staining for synaptopodin (Synpo, green signal) and desmin (red signal). Bars = 20 µm. Ecdysone induced podocyte injury, marked by the reduced expression of synaptopodin and augmented expression of desmin in synaptopodin positive podocytes (yellow signal as indicated by the white arrowhead). Apoptotic cells, probed by positive staining for cleaved caspase-3, were predominantly located to the periphery of glomerular tufts that is consistent with podocyte localization (indicated by the black arrowhead). ( B ) Glomeruli were isolated from kidneys by the magnetic beads-based approach and homogenized for immunoblot analysis for indicated molecules, including fibronectin, α-smooth muscle actin (α-SMA), desmin, synaptopodin (Synpo), cleaved caspase-3 (Cleaved casp-3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 1 ).
Figure Legend Snippet: Ecdysone induces glomerular molecular changes indicative of glomerulopathy. ( A ) Mice were treated as stated in Fig. 1 . Kidney specimens were obtained on day 14 and prepared for Masson trichrome staining, immunohistochemistry staining for fibronectin and cleaved caspase-3 (Cleaved casp-3), and dual color fluorescent immunohistochemistry staining for synaptopodin (Synpo, green signal) and desmin (red signal). Bars = 20 µm. Ecdysone induced podocyte injury, marked by the reduced expression of synaptopodin and augmented expression of desmin in synaptopodin positive podocytes (yellow signal as indicated by the white arrowhead). Apoptotic cells, probed by positive staining for cleaved caspase-3, were predominantly located to the periphery of glomerular tufts that is consistent with podocyte localization (indicated by the black arrowhead). ( B ) Glomeruli were isolated from kidneys by the magnetic beads-based approach and homogenized for immunoblot analysis for indicated molecules, including fibronectin, α-smooth muscle actin (α-SMA), desmin, synaptopodin (Synpo), cleaved caspase-3 (Cleaved casp-3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 1 ).

Techniques Used: Mouse Assay, Staining, Immunohistochemistry, Expressing, Isolation, Magnetic Beads

Spironolactone, a selective blockade of MR, mitigates molecular changes in glomeruli in ecdysone treated mice. ( A ) Mice were treated as described in Fig. 6C . Kidney tissues were procured on day 14 and processed for peroxidase immunohistochemistry staining for fibronectin and cleaved caspase-3, or for immunofluorescence staining for desmin (red) and synaptopodin (green). Bars = 20 µm. ( B ) Glomeruli were isolated by the magnetic beads-based approach and prepared for immunoblot analysis for fibronectin, desmin, α-smooth muscle actin desmin (α-SMA), synaptopodin (Synpo), cleaved caspase-3 (Cleaved Casp-3), GSK3β, p-GSK3β (S9) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 7 ).
Figure Legend Snippet: Spironolactone, a selective blockade of MR, mitigates molecular changes in glomeruli in ecdysone treated mice. ( A ) Mice were treated as described in Fig. 6C . Kidney tissues were procured on day 14 and processed for peroxidase immunohistochemistry staining for fibronectin and cleaved caspase-3, or for immunofluorescence staining for desmin (red) and synaptopodin (green). Bars = 20 µm. ( B ) Glomeruli were isolated by the magnetic beads-based approach and prepared for immunoblot analysis for fibronectin, desmin, α-smooth muscle actin desmin (α-SMA), synaptopodin (Synpo), cleaved caspase-3 (Cleaved Casp-3), GSK3β, p-GSK3β (S9) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 7 ).

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Immunofluorescence, Isolation, Magnetic Beads

Ecdysone exerts a direct injurious effect on glomerular podocytes. ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (10 −7 M) or vehicle for 24 or 48 h. Representative phase contrast micrographs demonstrate podocyte shape changes. Bar = 20 µm. ( B ) Cells were fixed at 48 h and processed for staining for cytoskeletal F-actin with rhodamine phalloidin, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining or for fluorescent immunocytochemistry staining for synaptopodin (Synpo). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). Bars = 20 µm. ( C ) Podocyte cell lysates were collected at 48 h and processed western blot analysis for synaptopodin (Synpo), cleaved caspase-3 (Cleaved casp-3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 2 ).
Figure Legend Snippet: Ecdysone exerts a direct injurious effect on glomerular podocytes. ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (10 −7 M) or vehicle for 24 or 48 h. Representative phase contrast micrographs demonstrate podocyte shape changes. Bar = 20 µm. ( B ) Cells were fixed at 48 h and processed for staining for cytoskeletal F-actin with rhodamine phalloidin, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining or for fluorescent immunocytochemistry staining for synaptopodin (Synpo). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). Bars = 20 µm. ( C ) Podocyte cell lysates were collected at 48 h and processed western blot analysis for synaptopodin (Synpo), cleaved caspase-3 (Cleaved casp-3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 2 ).

Techniques Used: Staining, TUNEL Assay, Immunocytochemistry, Western Blot

Ecdysone induced mineralocorticoid receptor (MR) nuclear translocation and activation in glomerular podocytes and mesangial cells both in vivo and in vitro . ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (Ecdy, 10 −7 M) or vehicle in the presence or absence of spironolactone (Spiro, 10 −5 M) for 48 h. Cells were fixed and processed for immunofluorescence staining for MR. Bar = 20 µm. Nuclear fractions and cytoplasmic lysates were processed for western blot analysis for indicated molecules (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 4 ). ( B ) Cultured mouse mesangial cells were treated with ecdysone (10 −7 M) or vehicle in the presence or absence of spironolactone (10 −5 M) for 48 h. Cells were fixed and processed for immunofluorescence staining for MR. Bar = 20 µm. Nuclear fractions and cytoplasmic lysates were processed for western blot analysis for indicated molecules (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 5 ). ( C ) Mice were treated with ecdysone (6 µg/g/d) or vehicle in the presence or absence of spironolactone (60 µg/g/d). After 2 weeks, mice were sacrificed and kidney specimens processed for peroxidase immunohistochemistry staining for MR. Representative micrographs of glomerular peroxidase staining for MR were shown. Bar = 20 μm. Nuclear fractions were prepared from isolated glomeruli and then subjected to western immunoblot analysis for MR or histone which served as a loading control for protein normalization (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 6 ). Abbreviations: α-SMA, α-smooth muscle actin; Synpo, synaptopodin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure Legend Snippet: Ecdysone induced mineralocorticoid receptor (MR) nuclear translocation and activation in glomerular podocytes and mesangial cells both in vivo and in vitro . ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (Ecdy, 10 −7 M) or vehicle in the presence or absence of spironolactone (Spiro, 10 −5 M) for 48 h. Cells were fixed and processed for immunofluorescence staining for MR. Bar = 20 µm. Nuclear fractions and cytoplasmic lysates were processed for western blot analysis for indicated molecules (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 4 ). ( B ) Cultured mouse mesangial cells were treated with ecdysone (10 −7 M) or vehicle in the presence or absence of spironolactone (10 −5 M) for 48 h. Cells were fixed and processed for immunofluorescence staining for MR. Bar = 20 µm. Nuclear fractions and cytoplasmic lysates were processed for western blot analysis for indicated molecules (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 5 ). ( C ) Mice were treated with ecdysone (6 µg/g/d) or vehicle in the presence or absence of spironolactone (60 µg/g/d). After 2 weeks, mice were sacrificed and kidney specimens processed for peroxidase immunohistochemistry staining for MR. Representative micrographs of glomerular peroxidase staining for MR were shown. Bar = 20 μm. Nuclear fractions were prepared from isolated glomeruli and then subjected to western immunoblot analysis for MR or histone which served as a loading control for protein normalization (cropped blots are shown as indicated by the lines and full-length blots are included in the Supplementary Figure 6 ). Abbreviations: α-SMA, α-smooth muscle actin; Synpo, synaptopodin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Techniques Used: Translocation Assay, Activation Assay, In Vivo, In Vitro, Immunofluorescence, Staining, Western Blot, Cell Culture, Mouse Assay, Immunohistochemistry, Isolation

9) Product Images from "Novel diagnostic and therapeutic techniques reveal changed metabolic profiles in recurrent focal segmental glomerulosclerosis"

Article Title: Novel diagnostic and therapeutic techniques reveal changed metabolic profiles in recurrent focal segmental glomerulosclerosis

Journal: Scientific Reports

doi: 10.1038/s41598-021-83883-w

Raman spectroscopy gives a molecular fingerprint of recurrent FSGS on tissue level. ( a , b ) PAX8 staining (green) and synaptopodin staining (red) of a glomerulus from the kidney biopsy at transplantation (0-biopsy) ( a , a′ ) and at the time of FSGS recurrence (FSGS recurrence) ( b , b′ ) and from a patient with minimal change disease ( c , c′ ) showing increased PAX8 staining in the Bowman’s capsule (arrow head in b′ ), scale bar = 100 µm. Representative bright field illumination ( a″ , b″ , c″ ) and heat map of Raman signal intensity ( a‴ , b‴ , c‴ ) of a glomerulus from the kidney biopsy at transplantation (0-biopsy) ( a″ , a‴ ), at the time of FSGS recurrence (FSGS recurrence) ( b″ , b‴ ) and of a biopsy with minimal change disease ( c″ , c‴ ) showing increased Raman signal at the region of parietal epithelial cell in the Bowman’s capsule (arrow head in b‴ ). Scale bar = 50 µm. ( d ) Mean Raman spectra of three glomeruli from the kidney biopsy at transplantation (blue line) and three glomeruli at the time of FSGS recurrence (red line). Assignments of the Raman peaks according to the literature are given.
Figure Legend Snippet: Raman spectroscopy gives a molecular fingerprint of recurrent FSGS on tissue level. ( a , b ) PAX8 staining (green) and synaptopodin staining (red) of a glomerulus from the kidney biopsy at transplantation (0-biopsy) ( a , a′ ) and at the time of FSGS recurrence (FSGS recurrence) ( b , b′ ) and from a patient with minimal change disease ( c , c′ ) showing increased PAX8 staining in the Bowman’s capsule (arrow head in b′ ), scale bar = 100 µm. Representative bright field illumination ( a″ , b″ , c″ ) and heat map of Raman signal intensity ( a‴ , b‴ , c‴ ) of a glomerulus from the kidney biopsy at transplantation (0-biopsy) ( a″ , a‴ ), at the time of FSGS recurrence (FSGS recurrence) ( b″ , b‴ ) and of a biopsy with minimal change disease ( c″ , c‴ ) showing increased Raman signal at the region of parietal epithelial cell in the Bowman’s capsule (arrow head in b‴ ). Scale bar = 50 µm. ( d ) Mean Raman spectra of three glomeruli from the kidney biopsy at transplantation (blue line) and three glomeruli at the time of FSGS recurrence (red line). Assignments of the Raman peaks according to the literature are given.

Techniques Used: Raman Spectroscopy, Staining, Transplantation Assay

10) Product Images from "The Role of Palladin in Podocytes"

Article Title: The Role of Palladin in Podocytes

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2017091039

Palladin expression is downregulated in glomeruli of patients suffering from FSGS and DN. Human renal biopsy samples were stained for palladin and synaptopodin. In control kidneys both proteins colocalized in podocytes. In biopsy samples from patients suffering from FSGS or DN, palladin expression showed a significant reduction, whereas synaptopodin expression was unchanged. Scale bar represents 50 µ m.
Figure Legend Snippet: Palladin expression is downregulated in glomeruli of patients suffering from FSGS and DN. Human renal biopsy samples were stained for palladin and synaptopodin. In control kidneys both proteins colocalized in podocytes. In biopsy samples from patients suffering from FSGS or DN, palladin expression showed a significant reduction, whereas synaptopodin expression was unchanged. Scale bar represents 50 µ m.

Techniques Used: Expressing, Staining

Palladin KO in podocytes leads to reduced nephrin expression in vivo and wider foot processes. (A) Immunofluorescence staining of kidney paraffin sections. There is no obvious difference in synaptopodin and α -actinin-4 expression in PodoPalld−/− mice compared with control mice, but a reduction of nephrin was observed. Scale bar represents 20 µ m. (B) The significant downregulation of vinculin, synaptopodin, and nephrin mRNA in PodoPalld−/− mice by qRT-PCR (mean±SEM, n =7 per group, * P
Figure Legend Snippet: Palladin KO in podocytes leads to reduced nephrin expression in vivo and wider foot processes. (A) Immunofluorescence staining of kidney paraffin sections. There is no obvious difference in synaptopodin and α -actinin-4 expression in PodoPalld−/− mice compared with control mice, but a reduction of nephrin was observed. Scale bar represents 20 µ m. (B) The significant downregulation of vinculin, synaptopodin, and nephrin mRNA in PodoPalld−/− mice by qRT-PCR (mean±SEM, n =7 per group, * P

Techniques Used: Expressing, In Vivo, Immunofluorescence, Staining, Mouse Assay, Quantitative RT-PCR

Palladin is colocalized with F-actin, synaptopodin and vinculin in podocytes. (A) Staining of murine kidney sections with anti-palladin antibody indicated a strong expression of palladin in podocytes. Scale bar represents 10 µ m. (B) Additionally, palladin expression in kidney, glomeruli, and CP was confirmed by RT-PCR using palladin primers spanning exons 18–20. (C) Double staining of cultured podocytes showed that palladin is colocalized with F-actin, synaptopodin, and vinculin. Scale bar represents 20 μ m. DAPI, 4′,6-diamidino-2-phenylindole.
Figure Legend Snippet: Palladin is colocalized with F-actin, synaptopodin and vinculin in podocytes. (A) Staining of murine kidney sections with anti-palladin antibody indicated a strong expression of palladin in podocytes. Scale bar represents 10 µ m. (B) Additionally, palladin expression in kidney, glomeruli, and CP was confirmed by RT-PCR using palladin primers spanning exons 18–20. (C) Double staining of cultured podocytes showed that palladin is colocalized with F-actin, synaptopodin, and vinculin. Scale bar represents 20 μ m. DAPI, 4′,6-diamidino-2-phenylindole.

Techniques Used: Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Double Staining, Cell Culture

11) Product Images from "Mfn2 Regulates High Glucose-Induced MAMs Dysfunction and Apoptosis in Podocytes via PERK Pathway"

Article Title: Mfn2 Regulates High Glucose-Induced MAMs Dysfunction and Apoptosis in Podocytes via PERK Pathway

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.769213

Histopathological changes and Mfn2 expression in patients with DKD. (A) Histological changes in patients with DKD were determined by HE and PAS staining (original magnification, ×400). (B) Representative images of immunohistochemical staining of Mfn2 in glomeruli per group (original magnification, ×400). (C,D) Representative images of immunofluorescent staining of Mfn2, WT1 or Synaptopodin and DAPI in glomeruli per group (original magnification, ×600). CTL, control; DKD, diabetic kidney disease.
Figure Legend Snippet: Histopathological changes and Mfn2 expression in patients with DKD. (A) Histological changes in patients with DKD were determined by HE and PAS staining (original magnification, ×400). (B) Representative images of immunohistochemical staining of Mfn2 in glomeruli per group (original magnification, ×400). (C,D) Representative images of immunofluorescent staining of Mfn2, WT1 or Synaptopodin and DAPI in glomeruli per group (original magnification, ×600). CTL, control; DKD, diabetic kidney disease.

Techniques Used: Expressing, Staining, Immunohistochemistry

12) Product Images from "Perinatal Lethality and Endothelial Cell Abnormalities in Several Vessel Compartments of Fibulin-1-Deficient Mice"

Article Title: Perinatal Lethality and Endothelial Cell Abnormalities in Several Vessel Compartments of Fibulin-1-Deficient Mice

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.20.7025-7034.2001

Immunofluorescence staining of kidney glomeruli from heterozygous (+/−) and fibulin-1-deficient (−/−) newborn mice. Protein expression around a single kidney glomerulus was analyzed by using antibodies against fibulin-1 (A and B), fibulin-2 (C and D), nidogen-1 (E and F), laminin α 2 chain (G and H), fibronectin (I and J), and PECAM-1 (K and L) and by double staining for synaptopodin (M and N) and integrin α 8 (O and P). Bar, 25 μm.
Figure Legend Snippet: Immunofluorescence staining of kidney glomeruli from heterozygous (+/−) and fibulin-1-deficient (−/−) newborn mice. Protein expression around a single kidney glomerulus was analyzed by using antibodies against fibulin-1 (A and B), fibulin-2 (C and D), nidogen-1 (E and F), laminin α 2 chain (G and H), fibronectin (I and J), and PECAM-1 (K and L) and by double staining for synaptopodin (M and N) and integrin α 8 (O and P). Bar, 25 μm.

Techniques Used: Immunofluorescence, Staining, Mouse Assay, Expressing, Double Staining

13) Product Images from "Activation of podocyte Notch mediates early Wt1 glomerulopathy"

Article Title: Activation of podocyte Notch mediates early Wt1 glomerulopathy

Journal: Kidney International

doi: 10.1016/j.kint.2017.11.014

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
Figure Legend 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

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

14) Product Images from "Gq-Dependent Signaling Upregulates COX2 in Glomerular Podocytes"

Article Title: Gq-Dependent Signaling Upregulates COX2 in Glomerular Podocytes

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2008010113

Cell-specific expression of the transgene. Tissue sections were stained for expression of the HA epitope and the podocyte marker synaptopodin as described in the Concise Methods S ection. (A) Synaptopodin but not the HA epitope was not detected in this male single-TG GqQ > L mouse treated with doxycycline. (B) In contrast, both the HA epitope and synaptopodin were detected in this male double-TG mouse treated with doxycycline. When the two images are merged, the HA epitope and synaptopodin have a similar cellular distribution in the double-TG mouse.
Figure Legend Snippet: Cell-specific expression of the transgene. Tissue sections were stained for expression of the HA epitope and the podocyte marker synaptopodin as described in the Concise Methods S ection. (A) Synaptopodin but not the HA epitope was not detected in this male single-TG GqQ > L mouse treated with doxycycline. (B) In contrast, both the HA epitope and synaptopodin were detected in this male double-TG mouse treated with doxycycline. When the two images are merged, the HA epitope and synaptopodin have a similar cellular distribution in the double-TG mouse.

Techniques Used: Expressing, Staining, Marker

GqQ > L enhances COX2 protein expression in vivo . Tissue sections were stained for expression of COX2 and the podocyte marker synaptopodin as described in the Concise Methods section. (A) Synaptopodin but not COX2 was detected in this male single-TG GqQ > L mouse treated with doxycycline. (B) In contrast, both COX2 and synaptopodin were detected in this male double-TG mouse treated with doxycycline. When the two images are merged, COX2 and synaptopodin have a similar cellular distribution in the double-TG mouse.
Figure Legend Snippet: GqQ > L enhances COX2 protein expression in vivo . Tissue sections were stained for expression of COX2 and the podocyte marker synaptopodin as described in the Concise Methods section. (A) Synaptopodin but not COX2 was detected in this male single-TG GqQ > L mouse treated with doxycycline. (B) In contrast, both COX2 and synaptopodin were detected in this male double-TG mouse treated with doxycycline. When the two images are merged, COX2 and synaptopodin have a similar cellular distribution in the double-TG mouse.

Techniques Used: Expressing, In Vivo, Staining, Marker

15) Product Images from "Role of calcineurin ( CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm. Role of calcineurin ( CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm"

Article Title: Role of calcineurin ( CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm. Role of calcineurin ( CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm

Journal: Physiological Reports

doi: 10.14814/phy2.12679

Dual‐labeling IF findings of CN ‐A (green) with glomerular cell markers (red). Representative findings of the CN ‐A (green), glomerular cell markers (red), and merge are shown. The staining of CN ‐A has close proximity with the synaptopodin staining. Some portions of the CN ‐A staining were costained with synaptopodin (arrows). The staining of CN ‐A was clearly apart from the stainings of OX ‐7 and RECA ‐1. Major portions of the CN ‐A stainings were costained with nephrin and ZO ‐1, markers of the SD (arrows). The staining was apart from that of podocalyxin, an apical surface marker, and α 3‐integrin, a basal surface marker. These observations indicated that the CN ‐A was mainly localized at the SD area.
Figure Legend Snippet: Dual‐labeling IF findings of CN ‐A (green) with glomerular cell markers (red). Representative findings of the CN ‐A (green), glomerular cell markers (red), and merge are shown. The staining of CN ‐A has close proximity with the synaptopodin staining. Some portions of the CN ‐A staining were costained with synaptopodin (arrows). The staining of CN ‐A was clearly apart from the stainings of OX ‐7 and RECA ‐1. Major portions of the CN ‐A stainings were costained with nephrin and ZO ‐1, markers of the SD (arrows). The staining was apart from that of podocalyxin, an apical surface marker, and α 3‐integrin, a basal surface marker. These observations indicated that the CN ‐A was mainly localized at the SD area.

Techniques Used: Labeling, Staining, Marker

16) Product Images from "Activation of podocyte Notch mediates early Wt1 glomerulopathy"

Article Title: Activation of podocyte Notch mediates early Wt1 glomerulopathy

Journal: Kidney International

doi: 10.1016/j.kint.2017.11.014

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
Figure Legend 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

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

17) Product Images from "Activation of mineralocorticoid receptor by ecdysone, an adaptogenic and anabolic ecdysteroid, promotes glomerular injury and proteinuria involving overactive GSK3β pathway signaling"

Article Title: Activation of mineralocorticoid receptor by ecdysone, an adaptogenic and anabolic ecdysteroid, promotes glomerular injury and proteinuria involving overactive GSK3β pathway signaling

Journal: Scientific Reports

doi: 10.1038/s41598-018-29483-7

Ecdysone exerts a direct injurious effect on glomerular podocytes. ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (10 −7 M) or vehicle for 24 or 48 h. Representative phase contrast micrographs demonstrate podocyte shape changes. Bar = 20 µm. ( B ) Cells were fixed at 48 h and processed for staining for cytoskeletal F-actin with rhodamine phalloidin, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining or for fluorescent immunocytochemistry staining for synaptopodin (Synpo). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). Bars = 20 µm. ( C ).
Figure Legend Snippet: Ecdysone exerts a direct injurious effect on glomerular podocytes. ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (10 −7 M) or vehicle for 24 or 48 h. Representative phase contrast micrographs demonstrate podocyte shape changes. Bar = 20 µm. ( B ) Cells were fixed at 48 h and processed for staining for cytoskeletal F-actin with rhodamine phalloidin, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining or for fluorescent immunocytochemistry staining for synaptopodin (Synpo). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). Bars = 20 µm. ( C ).

Techniques Used: Staining, TUNEL Assay, Immunocytochemistry

Ecdysone induced mineralocorticoid receptor (MR) nuclear translocation and activation in glomerular podocytes and mesangial cells both in vivo and in vitro . ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (Ecdy, 10 −7 M) or vehicle in the presence or absence of spironolactone (Spiro, 10 −5 ). ( B ) Cultured mouse mesangial cells were treated with ecdysone (10 −7 M) or vehicle in the presence or absence of spironolactone (10 −5 ). ( C ). Abbreviations: α-SMA, α-smooth muscle actin; Synpo, synaptopodin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure Legend Snippet: Ecdysone induced mineralocorticoid receptor (MR) nuclear translocation and activation in glomerular podocytes and mesangial cells both in vivo and in vitro . ( A ) Conditionally immortalized mouse podocytes in culture were treated with ecdysone (Ecdy, 10 −7 M) or vehicle in the presence or absence of spironolactone (Spiro, 10 −5 ). ( B ) Cultured mouse mesangial cells were treated with ecdysone (10 −7 M) or vehicle in the presence or absence of spironolactone (10 −5 ). ( C ). Abbreviations: α-SMA, α-smooth muscle actin; Synpo, synaptopodin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Techniques Used: Translocation Assay, Activation Assay, In Vivo, In Vitro, Cell Culture

18) Product Images from "A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction"

Article Title: A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction

Journal: Scientific Reports

doi: 10.1038/srep43934

Gene expression indicates podocyte mechanotransduction can induce a pro-differentiation phenotype. Expression levels of six genes associated with podocyte differentiation and/or function were measured using qRT-PCR to test podocyte phenotype, namely: WT-1, KIRREL, SYNPO, NPHS1, NPHS2 and CD2AP. Colors in the heatmap represent the ratios of differential expression versus control. Four of the six genes showed upregulation on gels with different elastic moduli. Average fold-changes for these genes were 3.2, 7.5, 6.6, and 1.7 on the gels of 0.6 kPa, 2 kPa, 5 kPa, and 13 kPa, respectively.
Figure Legend Snippet: Gene expression indicates podocyte mechanotransduction can induce a pro-differentiation phenotype. Expression levels of six genes associated with podocyte differentiation and/or function were measured using qRT-PCR to test podocyte phenotype, namely: WT-1, KIRREL, SYNPO, NPHS1, NPHS2 and CD2AP. Colors in the heatmap represent the ratios of differential expression versus control. Four of the six genes showed upregulation on gels with different elastic moduli. Average fold-changes for these genes were 3.2, 7.5, 6.6, and 1.7 on the gels of 0.6 kPa, 2 kPa, 5 kPa, and 13 kPa, respectively.

Techniques Used: Expressing, Quantitative RT-PCR

Extent of mechanotransduction is extracellular matrix independent ECM coating with (A) collagen I of reduced density from 5 μg/cm 2 to 1 μg/cm 2 : Upregulation for five protein markers are 2.7 ± 0.3, 1.8 ± 0.1, 1.7 ± 0.2, 1.3 ± 0.1 and 0.7 ± 0.1 respectively, (B) collagen IV (1 μg/cm 2 ): Relative expression levels of WT-1, nephrin, podocin, CD2AP and synaptopodin by Western blots were 3.7 ± 0.4, 1.9 ± 0.1, 1.5 ± 0.1, 1.7 ± 0.1, 0.8 ± 0.1 respectively, (C) fibronectin (1 μg/cm 2 ): Relative expression levels of WT-1, nephrin, podocin, CD2AP and synaptopodin were 3.3 ± 0.4, 2.0 ± 0.1, 1.5 ± 0.1, 1.7 ± 0.1, 1.1 ± 0.1 respectively, (D) laminin (1 μg/cm 2 ): Relative expression levels of WT-1, nephrin, podocin, CD2AP and synaptopodin were 1.8 ± 0.2, 2.4 ± 0.4, 1.4 ± 0.1, 1.8 ± 0.2 and 1.1 ± 0.2 respectively. In all cases, statistical significance indicated changes of the ratios of five protein markers after normalization by those of control (two-tailed t-test, α = 0.05).
Figure Legend Snippet: Extent of mechanotransduction is extracellular matrix independent ECM coating with (A) collagen I of reduced density from 5 μg/cm 2 to 1 μg/cm 2 : Upregulation for five protein markers are 2.7 ± 0.3, 1.8 ± 0.1, 1.7 ± 0.2, 1.3 ± 0.1 and 0.7 ± 0.1 respectively, (B) collagen IV (1 μg/cm 2 ): Relative expression levels of WT-1, nephrin, podocin, CD2AP and synaptopodin by Western blots were 3.7 ± 0.4, 1.9 ± 0.1, 1.5 ± 0.1, 1.7 ± 0.1, 0.8 ± 0.1 respectively, (C) fibronectin (1 μg/cm 2 ): Relative expression levels of WT-1, nephrin, podocin, CD2AP and synaptopodin were 3.3 ± 0.4, 2.0 ± 0.1, 1.5 ± 0.1, 1.7 ± 0.1, 1.1 ± 0.1 respectively, (D) laminin (1 μg/cm 2 ): Relative expression levels of WT-1, nephrin, podocin, CD2AP and synaptopodin were 1.8 ± 0.2, 2.4 ± 0.4, 1.4 ± 0.1, 1.8 ± 0.2 and 1.1 ± 0.2 respectively. In all cases, statistical significance indicated changes of the ratios of five protein markers after normalization by those of control (two-tailed t-test, α = 0.05).

Techniques Used: Expressing, Western Blot, Two Tailed Test

Stiffness-mediated differentiation phenotype induces upregulation of podocyte-specific functional proteins. ( A ) Representative images of Western blots for five proteins critical for physiological function of podocytes. Images of the complete blots are shown in Figure S3 . ( B ) Quantification of kidney podocyte protein markers. Highest upregulation of WT-1 was found on the 2 kPa gel (2.2 ± 0.3). Nephrin, podocin, and CD2AP showed similar trends. Synaptopodin did not show this trend and there was no statistical difference between softer gels and control. T-statistics showed statistically significant changes of the ratios of five protein markers after normalization by control (two-tails, α = 0.05). The average t-statistics were 4.5 for the 2 kPa gel and 4.0 for the 5 kPa gel, which were significantly different relative to the control. Those for the 0.6 kPa and 13 kPa gels were 0.8 and 1.5, and the differences were not significant.
Figure Legend Snippet: Stiffness-mediated differentiation phenotype induces upregulation of podocyte-specific functional proteins. ( A ) Representative images of Western blots for five proteins critical for physiological function of podocytes. Images of the complete blots are shown in Figure S3 . ( B ) Quantification of kidney podocyte protein markers. Highest upregulation of WT-1 was found on the 2 kPa gel (2.2 ± 0.3). Nephrin, podocin, and CD2AP showed similar trends. Synaptopodin did not show this trend and there was no statistical difference between softer gels and control. T-statistics showed statistically significant changes of the ratios of five protein markers after normalization by control (two-tails, α = 0.05). The average t-statistics were 4.5 for the 2 kPa gel and 4.0 for the 5 kPa gel, which were significantly different relative to the control. Those for the 0.6 kPa and 13 kPa gels were 0.8 and 1.5, and the differences were not significant.

Techniques Used: Functional Assay, Western Blot

19) Product Images from "The Role of Palladin in Podocytes"

Article Title: The Role of Palladin in Podocytes

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2017091039

Palladin expression is downregulated in glomeruli of patients suffering from FSGS and DN. Human renal biopsy samples were stained for palladin and synaptopodin. In control kidneys both proteins colocalized in podocytes. In biopsy samples from patients suffering from FSGS or DN, palladin expression showed a significant reduction, whereas synaptopodin expression was unchanged. Scale bar represents 50 µ m.
Figure Legend Snippet: Palladin expression is downregulated in glomeruli of patients suffering from FSGS and DN. Human renal biopsy samples were stained for palladin and synaptopodin. In control kidneys both proteins colocalized in podocytes. In biopsy samples from patients suffering from FSGS or DN, palladin expression showed a significant reduction, whereas synaptopodin expression was unchanged. Scale bar represents 50 µ m.

Techniques Used: Expressing, Staining

Palladin KO in podocytes leads to reduced nephrin expression in vivo and wider foot processes. (A) Immunofluorescence staining of kidney paraffin sections. There is no obvious difference in synaptopodin and α -actinin-4 expression in PodoPalld−/− mice compared with control mice, but a reduction of nephrin was observed. Scale bar represents 20 µ m. (B) The significant downregulation of vinculin, synaptopodin, and nephrin mRNA in PodoPalld−/− mice by qRT-PCR (mean±SEM, n =7 per group, * P
Figure Legend Snippet: Palladin KO in podocytes leads to reduced nephrin expression in vivo and wider foot processes. (A) Immunofluorescence staining of kidney paraffin sections. There is no obvious difference in synaptopodin and α -actinin-4 expression in PodoPalld−/− mice compared with control mice, but a reduction of nephrin was observed. Scale bar represents 20 µ m. (B) The significant downregulation of vinculin, synaptopodin, and nephrin mRNA in PodoPalld−/− mice by qRT-PCR (mean±SEM, n =7 per group, * P

Techniques Used: Expressing, In Vivo, Immunofluorescence, Staining, Mouse Assay, Quantitative RT-PCR

Palladin is colocalized with F-actin, synaptopodin and vinculin in podocytes. (A) Staining of murine kidney sections with anti-palladin antibody indicated a strong expression of palladin in podocytes. Scale bar represents 10 µ m. (B) Additionally, palladin expression in kidney, glomeruli, and CP was confirmed by RT-PCR using palladin primers spanning exons 18–20. (C) Double staining of cultured podocytes showed that palladin is colocalized with F-actin, synaptopodin, and vinculin. Scale bar represents 20 μ m. DAPI, 4′,6-diamidino-2-phenylindole.
Figure Legend Snippet: Palladin is colocalized with F-actin, synaptopodin and vinculin in podocytes. (A) Staining of murine kidney sections with anti-palladin antibody indicated a strong expression of palladin in podocytes. Scale bar represents 10 µ m. (B) Additionally, palladin expression in kidney, glomeruli, and CP was confirmed by RT-PCR using palladin primers spanning exons 18–20. (C) Double staining of cultured podocytes showed that palladin is colocalized with F-actin, synaptopodin, and vinculin. Scale bar represents 20 μ m. DAPI, 4′,6-diamidino-2-phenylindole.

Techniques Used: Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Double Staining, Cell Culture

20) Product Images from "Transplanted organoids empower human preclinical assessment of drug candidate for the clinic"

Article Title: Transplanted organoids empower human preclinical assessment of drug candidate for the clinic

Journal: Science Advances

doi: 10.1126/sciadv.abj5633

In vitro drug evaluation in human kidney organoids. ( A ) Current-voltage relationship, 500-ms voltage ramp from −80 mV to +80 mV before/after GFB-887 (0.1 μM) or ML204 (100 μM). ( B ) Concentration-dependent inhibition of human TRPC5 after GFB-887 at +80 mV. Means ± SEM ( n = 3 to 4 measurements per concentration). IC 50 , median inhibitory concentration. ( C ) Up-regulation of human TRPC5 mRNA expression during organoid differentiation in vitro. Error bars, SD. BLOQ, below limit of quantitation. ( D ) Double labeling of podocytes with synaptopodin (SYNPO, green) and TRPC5 (red). ( E ) CsA and GFB-887 protect podocytes from PS-induced injury, region of interest in blue. Inset: Scale of representative images for injury quantification (top left). Representative images of glomeruli for podocyte injury quantification in organoids treated with DMSO (vehicle), PS, PS + CsA, PS + GFB-887, or PS + CsA + GFB-887. Cyan, synaptopodin; red, phalloidin. ( F ) Quantification of PS-induced actin aggregation. GFB-887 and CsA are nonadditive. DMSO versus PS, P
Figure Legend Snippet: In vitro drug evaluation in human kidney organoids. ( A ) Current-voltage relationship, 500-ms voltage ramp from −80 mV to +80 mV before/after GFB-887 (0.1 μM) or ML204 (100 μM). ( B ) Concentration-dependent inhibition of human TRPC5 after GFB-887 at +80 mV. Means ± SEM ( n = 3 to 4 measurements per concentration). IC 50 , median inhibitory concentration. ( C ) Up-regulation of human TRPC5 mRNA expression during organoid differentiation in vitro. Error bars, SD. BLOQ, below limit of quantitation. ( D ) Double labeling of podocytes with synaptopodin (SYNPO, green) and TRPC5 (red). ( E ) CsA and GFB-887 protect podocytes from PS-induced injury, region of interest in blue. Inset: Scale of representative images for injury quantification (top left). Representative images of glomeruli for podocyte injury quantification in organoids treated with DMSO (vehicle), PS, PS + CsA, PS + GFB-887, or PS + CsA + GFB-887. Cyan, synaptopodin; red, phalloidin. ( F ) Quantification of PS-induced actin aggregation. GFB-887 and CsA are nonadditive. DMSO versus PS, P

Techniques Used: In Vitro, Concentration Assay, Inhibition, Expressing, Quantitation Assay, Labeling

PD studies in rats with transplanted human kidney organoids bolster confidence in GFB-887, an investigational new drug. ( A ) Organoids differentiated for 14 days in vitro were transplanted under the kidney capsule of athymic male rats and followed for 2 (left) or 4 (right) weeks before oral dosing with GFB-887 (10 mg/kg) for three consecutive days. Extracted organoid and kidney samples were normalized to sample weights, and the final drug values were calculated as nanograms per gram tissue; nanograms per milliliter refers to GFB-887 concentration per milliliter of plasma. Oral dosing of GFB-887 resulted in equivalent drug exposure in organoids and rat plasma, thereby showing that the organoids had functional connectivity to the host vasculature at 2 weeks after transplantation. Organoid GFB-887 levels did not further increase at 4 weeks after tr ansplantation. Data show means ± SEM from at least four independent measurements. ( B ) Superresolution imaging reveals PS-induced loss of synaptopodin protein abundance in transplanted organoids, which was prevented by oral dosing of GFB-887; blue, nuclei; green, synaptopodin; red, RECA-1; HBSS, Hank’s balanced salt solution vehicle control. ( C ) Quantification of PS-induced podocyte injury and protection by GFB-887 in transplanted organoids. ( D ) Quantification of PS-induced podocyte injury and protection by GFB-887 in endogenous rat kidney adjacent to transplanted organoids. For both (C) and (D), synaptopodin mean intensity in podocytes was quantified for HBSS vehicle, PS, coperfusion PS + GFB-887, or PS perfusion after oral dosing of GFB-887. Data show means ± SEM; for all treatment conditions versus PS, P
Figure Legend Snippet: PD studies in rats with transplanted human kidney organoids bolster confidence in GFB-887, an investigational new drug. ( A ) Organoids differentiated for 14 days in vitro were transplanted under the kidney capsule of athymic male rats and followed for 2 (left) or 4 (right) weeks before oral dosing with GFB-887 (10 mg/kg) for three consecutive days. Extracted organoid and kidney samples were normalized to sample weights, and the final drug values were calculated as nanograms per gram tissue; nanograms per milliliter refers to GFB-887 concentration per milliliter of plasma. Oral dosing of GFB-887 resulted in equivalent drug exposure in organoids and rat plasma, thereby showing that the organoids had functional connectivity to the host vasculature at 2 weeks after transplantation. Organoid GFB-887 levels did not further increase at 4 weeks after tr ansplantation. Data show means ± SEM from at least four independent measurements. ( B ) Superresolution imaging reveals PS-induced loss of synaptopodin protein abundance in transplanted organoids, which was prevented by oral dosing of GFB-887; blue, nuclei; green, synaptopodin; red, RECA-1; HBSS, Hank’s balanced salt solution vehicle control. ( C ) Quantification of PS-induced podocyte injury and protection by GFB-887 in transplanted organoids. ( D ) Quantification of PS-induced podocyte injury and protection by GFB-887 in endogenous rat kidney adjacent to transplanted organoids. For both (C) and (D), synaptopodin mean intensity in podocytes was quantified for HBSS vehicle, PS, coperfusion PS + GFB-887, or PS perfusion after oral dosing of GFB-887. Data show means ± SEM; for all treatment conditions versus PS, P

Techniques Used: In Vitro, Concentration Assay, Functional Assay, Transplantation Assay, Imaging

21) Product Images from "Sildenafil Prevents Podocyte Injury via PPAR-γ–Mediated TRPC6 Inhibition"

Article Title: Sildenafil Prevents Podocyte Injury via PPAR-γ–Mediated TRPC6 Inhibition

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2015080885

PDE5A is expressed by podocytes. RNA and protein were isolated from cultured podocytes and renal mouse cortex; subsequently, (A) PCR and (B) Western blot were performed to determine PDE5A expression in these samples. Glomerular PDE5A expression was confirmed by immunofluorescence staining of PDE5A. (C) Importantly, costaining with synaptopodin showed PDE5A expression in the podocytes; merge is a higher magnification of single-channel images. AB, antibody; NTC, no template control.
Figure Legend Snippet: PDE5A is expressed by podocytes. RNA and protein were isolated from cultured podocytes and renal mouse cortex; subsequently, (A) PCR and (B) Western blot were performed to determine PDE5A expression in these samples. Glomerular PDE5A expression was confirmed by immunofluorescence staining of PDE5A. (C) Importantly, costaining with synaptopodin showed PDE5A expression in the podocytes; merge is a higher magnification of single-channel images. AB, antibody; NTC, no template control.

Techniques Used: Isolation, Cell Culture, Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining

22) Product Images from "Mitoquinone Protects Podocytes from Angiotensin II-Induced Mitochondrial Dysfunction and Injury via the Keap1-Nrf2 Signaling Pathway"

Article Title: Mitoquinone Protects Podocytes from Angiotensin II-Induced Mitochondrial Dysfunction and Injury via the Keap1-Nrf2 Signaling Pathway

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2021/1394486

Effects of MitoQ on the expression of Keap1 and Nrf2 in podocytes in vivo and in vitro . (a) Western blot analysis of Keap1 and Nrf2 in glomeruli from different groups, n = 3. (b) Representative immunohistochemistry staining of Nrf2 in glomeruli from different groups (original magnification, ×600), scale bar = 20 μ m. (c) Representative fluorescence staining of Nrf2 (green), synaptopodin (podocytes, red), and DAPI (blue) in podocytes from different groups (original magnification, ×1000), scale bar = 10 μ m, n = 5. (d) Quantitative analysis of protein levels from (a). (e) Western blot analysis of Keap1 and Nrf2 in podocytes in vitro , n = 3. (f) Representative fluorescence staining of Nrf2 in podocytes in vitro (original magnification, ×1000), scale bar = 15 μ m, n = 5. (g, h) Quantitative analysis of protein levels from (e). (i) Quantitative analysis of the nuclear translocation of Nrf2. ∗ P
Figure Legend Snippet: Effects of MitoQ on the expression of Keap1 and Nrf2 in podocytes in vivo and in vitro . (a) Western blot analysis of Keap1 and Nrf2 in glomeruli from different groups, n = 3. (b) Representative immunohistochemistry staining of Nrf2 in glomeruli from different groups (original magnification, ×600), scale bar = 20 μ m. (c) Representative fluorescence staining of Nrf2 (green), synaptopodin (podocytes, red), and DAPI (blue) in podocytes from different groups (original magnification, ×1000), scale bar = 10 μ m, n = 5. (d) Quantitative analysis of protein levels from (a). (e) Western blot analysis of Keap1 and Nrf2 in podocytes in vitro , n = 3. (f) Representative fluorescence staining of Nrf2 in podocytes in vitro (original magnification, ×1000), scale bar = 15 μ m, n = 5. (g, h) Quantitative analysis of protein levels from (e). (i) Quantitative analysis of the nuclear translocation of Nrf2. ∗ P

Techniques Used: Expressing, In Vivo, In Vitro, Western Blot, Immunohistochemistry, Staining, Fluorescence, Translocation Assay

23) Product Images from "DNA Aptamer Raised Against AGEs Blocks the Progression of Experimental Diabetic Nephropathy"

Article Title: DNA Aptamer Raised Against AGEs Blocks the Progression of Experimental Diabetic Nephropathy

Journal: Diabetes

doi: 10.2337/db12-1608

Representative photographs of glomerular hypertrophy. Glomerular hypertrophy was evaluated by measuring glomerular area of cross-section in the distal cortex. Ctr-Ctr-aptamer mice ( A ), Ctr-AGEs-aptamer mice ( B ), DM-Ctr-aptamer mice ( C ), DM-AGEs-aptamer mice ( D ). E : Quantitative data of glomerular area. n = 4–5 per group. Magnification ×600. Effect of AGEs-aptamer on glomerular ECM accumulation in each animal. Glomerular ECM accumulation was evaluated by the intensity of Masson trichrome staining in the glomeruli. Representative photographs of the kidney in Ctr-Ctr-aptamer mice ( F ), Ctr-AGEs-aptamer mice ( G ), DM-Ctr-aptamer mice ( H ), and DM-AGEs-aptamer mice ( I ). J : Quantitative data of ECM accumulation. n = 3–5 per group. Synaptopodin levels in the glomeruli. Representative photographs of the kidney in Ctr-Ctr-aptamer mice ( K ), Ctr-AGEs-aptamer mice ( L ), DM-Ctr-aptamer mice ( M ), and DM-AGEs-aptamer mice ( N ). O : Quantitative data of synaptopodin expression. n = 3–5 per group. Magnification ×600. P : Effect of AGEs-aptamer on urinary 8-OHdG levels (ng/day) in each animal group. Urinary 8-OHdG levels were measured by ELISA. n = 8–11 per group.
Figure Legend Snippet: Representative photographs of glomerular hypertrophy. Glomerular hypertrophy was evaluated by measuring glomerular area of cross-section in the distal cortex. Ctr-Ctr-aptamer mice ( A ), Ctr-AGEs-aptamer mice ( B ), DM-Ctr-aptamer mice ( C ), DM-AGEs-aptamer mice ( D ). E : Quantitative data of glomerular area. n = 4–5 per group. Magnification ×600. Effect of AGEs-aptamer on glomerular ECM accumulation in each animal. Glomerular ECM accumulation was evaluated by the intensity of Masson trichrome staining in the glomeruli. Representative photographs of the kidney in Ctr-Ctr-aptamer mice ( F ), Ctr-AGEs-aptamer mice ( G ), DM-Ctr-aptamer mice ( H ), and DM-AGEs-aptamer mice ( I ). J : Quantitative data of ECM accumulation. n = 3–5 per group. Synaptopodin levels in the glomeruli. Representative photographs of the kidney in Ctr-Ctr-aptamer mice ( K ), Ctr-AGEs-aptamer mice ( L ), DM-Ctr-aptamer mice ( M ), and DM-AGEs-aptamer mice ( N ). O : Quantitative data of synaptopodin expression. n = 3–5 per group. Magnification ×600. P : Effect of AGEs-aptamer on urinary 8-OHdG levels (ng/day) in each animal group. Urinary 8-OHdG levels were measured by ELISA. n = 8–11 per group.

Techniques Used: Mouse Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay

24) Product Images from "Ontogeny of the Kidney and Renal Developmental Markers in the Rhesus Monkey (Macaca mulatta)"

Article Title: Ontogeny of the Kidney and Renal Developmental Markers in the Rhesus Monkey (Macaca mulatta)

Journal: Anatomical record (Hoboken, N.J. : 2007)

doi: 10.1002/ar.21242

Quantitative RT-PCR analysis of expression patterns of α-SMA, Gremlin, Nestin, Pax2, Synaptopodin, and WT1 across gestation
Figure Legend Snippet: Quantitative RT-PCR analysis of expression patterns of α-SMA, Gremlin, Nestin, Pax2, Synaptopodin, and WT1 across gestation

Techniques Used: Quantitative RT-PCR, Expressing

Synaptopodin (SYN) and Nestin expression in sequential stages of kidney development in rhesus monkeys
Figure Legend Snippet: Synaptopodin (SYN) and Nestin expression in sequential stages of kidney development in rhesus monkeys

Techniques Used: Expressing

25) Product Images from "Transforming Growth Factor-?1 Is Up-Regulated by Podocytes in Response to Excess Intraglomerular Passage of Proteins "

Article Title: Transforming Growth Factor-?1 Is Up-Regulated by Podocytes in Response to Excess Intraglomerular Passage of Proteins

Journal: The American Journal of Pathology

doi:

Comparison of the glomerular sites of abnormal deposition of protein and altered expression of desmin and synaptopodin. a–d: Dual labeling of desmin and IgG. In a glomerulus of sham control ( a ) desmin (red) is confined to mesangial cells and IgG (green) is not detectable. A glomerulus of RMR kidney at 7 days ( b ) shows granular IgG staining ( arrowheads ) in podocytes, associated with no or very weak desmin staining, consistent with IgG accumulation in advance of high desmin expression. In sections of RMR kidneys at 14 days ( c ) and at 30 days ( d ) both the high expression of desmin and the IgG staining co-localize mainly to peripheral podocytes (yellow). e and f: By dual labeling for synaptopodin and IgG in RMR at 14 days ( e ) and at 30 ( f ), IgG-positive podocytes ( arrowheads ), in contrast to IgG-negative podocytes ( arrows ), show diminution or loss of synaptopodin (red). Severe podocyte damage and possibly detachment and loss are detectable in association with extracellular IgG deposition in areas of segmental adhesion and sclerosis ( asterisks ). Original magnifications, ×250.
Figure Legend Snippet: Comparison of the glomerular sites of abnormal deposition of protein and altered expression of desmin and synaptopodin. a–d: Dual labeling of desmin and IgG. In a glomerulus of sham control ( a ) desmin (red) is confined to mesangial cells and IgG (green) is not detectable. A glomerulus of RMR kidney at 7 days ( b ) shows granular IgG staining ( arrowheads ) in podocytes, associated with no or very weak desmin staining, consistent with IgG accumulation in advance of high desmin expression. In sections of RMR kidneys at 14 days ( c ) and at 30 days ( d ) both the high expression of desmin and the IgG staining co-localize mainly to peripheral podocytes (yellow). e and f: By dual labeling for synaptopodin and IgG in RMR at 14 days ( e ) and at 30 ( f ), IgG-positive podocytes ( arrowheads ), in contrast to IgG-negative podocytes ( arrows ), show diminution or loss of synaptopodin (red). Severe podocyte damage and possibly detachment and loss are detectable in association with extracellular IgG deposition in areas of segmental adhesion and sclerosis ( asterisks ). Original magnifications, ×250.

Techniques Used: Expressing, Labeling, Staining

Immunofluorescence detection of desmin ( a–d ) and synaptopodin ( e–h ) in RMR glomeruli. In contrast to normal patterns revealed in sham control ( a and e ) and RMR at 7 days ( b and f ), high desmin expression and loss of synaptopodin in podocytes are visualized in RMR at 14 days ( c and g , arrowheads ) and 30 days ( d and h ) after surgery. In respect to areas in which synaptopodin is preserved ( arrows ), the asterisks indicate well-defined areas in which the foot process-associated differentiation marker is lost. Original magnifications, ×250.
Figure Legend Snippet: Immunofluorescence detection of desmin ( a–d ) and synaptopodin ( e–h ) in RMR glomeruli. In contrast to normal patterns revealed in sham control ( a and e ) and RMR at 7 days ( b and f ), high desmin expression and loss of synaptopodin in podocytes are visualized in RMR at 14 days ( c and g , arrowheads ) and 30 days ( d and h ) after surgery. In respect to areas in which synaptopodin is preserved ( arrows ), the asterisks indicate well-defined areas in which the foot process-associated differentiation marker is lost. Original magnifications, ×250.

Techniques Used: Immunofluorescence, Expressing, Marker

Synthesis of TGF-β1, induction of downstream phenotypic change of mesangial cells, and loss of synaptopodin by podocytes in response to protein load. a–c: Effects of HSA on TGF-β1 mRNA expression by Northern blot analysis ( a and b ) and on TGF-β1 production by enzyme-linked immunosorbent assay ( c ) in cultured podocytes. *, P
Figure Legend Snippet: Synthesis of TGF-β1, induction of downstream phenotypic change of mesangial cells, and loss of synaptopodin by podocytes in response to protein load. a–c: Effects of HSA on TGF-β1 mRNA expression by Northern blot analysis ( a and b ) and on TGF-β1 production by enzyme-linked immunosorbent assay ( c ) in cultured podocytes. *, P

Techniques Used: Expressing, Northern Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

26) Product Images from "Protective effects of PPAR? agonist in acute nephrotic syndrome"

Article Title: Protective effects of PPAR? agonist in acute nephrotic syndrome

Journal: Nephrology Dialysis Transplantation

doi: 10.1093/ndt/gfr240

Podocyte injury. ( A ) Synaptopodin, a podocyte differentiation marker, was markedly and similarly decreased at day 4 and 10 in all PAN groups compared with normal but was significantly restored by simultaneous or delayed Pio treatment (Pio0, PostPio) but not by pretreatment with Pio (prePio) at day 21 (×400) (N, n = 4; PAN, n = 5; Pio, n = 5; prePio, n = 5; PostPio, n = 5; *P
Figure Legend Snippet: Podocyte injury. ( A ) Synaptopodin, a podocyte differentiation marker, was markedly and similarly decreased at day 4 and 10 in all PAN groups compared with normal but was significantly restored by simultaneous or delayed Pio treatment (Pio0, PostPio) but not by pretreatment with Pio (prePio) at day 21 (×400) (N, n = 4; PAN, n = 5; Pio, n = 5; prePio, n = 5; PostPio, n = 5; *P

Techniques Used: Marker

27) Product Images from "CTGF Is Expressed During Cystic Remodeling in the PKD/Mhm (cy/+) Rat Model for Autosomal-Dominant Polycystic Kidney Disease (ADPKD)"

Article Title: CTGF Is Expressed During Cystic Remodeling in the PKD/Mhm (cy/+) Rat Model for Autosomal-Dominant Polycystic Kidney Disease (ADPKD)

Journal: Journal of Histochemistry and Cytochemistry

doi: 10.1369/0022155417735513

Double IHC with anti-CTGF (A, Cy3 red) and anti-synaptopodin (B, Cy2, green) of a glomerulus of a cy/+ rat in the vicinity of a greater cyst (on the right). Merged view and nuclear staining (DAPI, blue) is provided in (C). CTGF protein is exclusively expressed in the cell bodies of podocytes (A), which are clearly identifiable at the border to the urinary space. There is no overlap with synaptopodin-positive foot processes, which are directly connected. Scale bar = 37 µm. Abbreviations: CTGF, connective tissue growth factor; DAPI, 4′,6 diamidino-2-phenylindole dihydrochloride.
Figure Legend Snippet: Double IHC with anti-CTGF (A, Cy3 red) and anti-synaptopodin (B, Cy2, green) of a glomerulus of a cy/+ rat in the vicinity of a greater cyst (on the right). Merged view and nuclear staining (DAPI, blue) is provided in (C). CTGF protein is exclusively expressed in the cell bodies of podocytes (A), which are clearly identifiable at the border to the urinary space. There is no overlap with synaptopodin-positive foot processes, which are directly connected. Scale bar = 37 µm. Abbreviations: CTGF, connective tissue growth factor; DAPI, 4′,6 diamidino-2-phenylindole dihydrochloride.

Techniques Used: Immunohistochemistry, Staining

28) Product Images from "A translational kidney organoid system bolsters human relevance of clinical development candidate"

Article Title: A translational kidney organoid system bolsters human relevance of clinical development candidate

Journal: bioRxiv

doi: 10.1101/2019.12.30.891440

TRPC5 inhibition protects against podocyte injury in human kidney organoids in vivo (A) Oral dosing of GFB-887 in rats results in drug exposure of transplanted, perfused human kidney organoids equivalent to rat plasma levels. Athymic nude, male rats were implanted with day 14 early differentiating organoids under the sub-capsular space. Transplanted rats were grown for 2 ( left panel ) or 4 ( right panel ) weeks and orally dosed with 10 mg/kg GFB-887 for 3 consecutive days prior to plasma, kidney and organoid collection. Data show mean ± SEM of GFB-887 exposure from at least 4 independent measurements suggest that transplanted human kidney organoids achieve maximal vascularization as well as functional connectivity to host vasculature in 2 weeks. (B) Super resolution imaging reveals PS-induced loss of synaptopodin in transplanted human organoids, which can be prevent by orally dosing with GFB-887: HBSS (Hank’s Balanced Salt solution serving as vehicle control), PS, PS + GFB-887. (C) Quantification of PS induced podocyte injury and protection by CsA or GBF-887. Synaptopodin mean intensity in podocytes was quantified from human organoids from transplant experiments for vehicle, PS, co-perfusion GFB-887 + PS, and oral dosing of GFB-887 + PS (Data show mean ± SEM; for all treatment conditions vs. PS p
Figure Legend Snippet: TRPC5 inhibition protects against podocyte injury in human kidney organoids in vivo (A) Oral dosing of GFB-887 in rats results in drug exposure of transplanted, perfused human kidney organoids equivalent to rat plasma levels. Athymic nude, male rats were implanted with day 14 early differentiating organoids under the sub-capsular space. Transplanted rats were grown for 2 ( left panel ) or 4 ( right panel ) weeks and orally dosed with 10 mg/kg GFB-887 for 3 consecutive days prior to plasma, kidney and organoid collection. Data show mean ± SEM of GFB-887 exposure from at least 4 independent measurements suggest that transplanted human kidney organoids achieve maximal vascularization as well as functional connectivity to host vasculature in 2 weeks. (B) Super resolution imaging reveals PS-induced loss of synaptopodin in transplanted human organoids, which can be prevent by orally dosing with GFB-887: HBSS (Hank’s Balanced Salt solution serving as vehicle control), PS, PS + GFB-887. (C) Quantification of PS induced podocyte injury and protection by CsA or GBF-887. Synaptopodin mean intensity in podocytes was quantified from human organoids from transplant experiments for vehicle, PS, co-perfusion GFB-887 + PS, and oral dosing of GFB-887 + PS (Data show mean ± SEM; for all treatment conditions vs. PS p

Techniques Used: Inhibition, In Vivo, Functional Assay, Imaging

Double labeling with synaptopodin reveals podocyte TRPC5 protein expression in human organoids Representative glomerulus ( left panel ) and zoomed in super-resolution images TRPC5 and synaptopodin staining in individual podocytes. Synaptopodin: green, TRPC5: red, Hoechst: blue.
Figure Legend Snippet: Double labeling with synaptopodin reveals podocyte TRPC5 protein expression in human organoids Representative glomerulus ( left panel ) and zoomed in super-resolution images TRPC5 and synaptopodin staining in individual podocytes. Synaptopodin: green, TRPC5: red, Hoechst: blue.

Techniques Used: Labeling, Expressing, Staining

Organoid transplanted promotes differentiation and vascularization (A) Schematic of organoid protocol with rat kidney capsule transplantation and scRNA-Seq timepoints (B) Stacked bar graph containing cell type proportions of in vivo maturation across time points and replicates show an increase in podocytes, tubular cells, endothelial cells, and off-target cell types with a longer period of in vivo maturation. (C) Comparison of cell type proportions across days using boxplot of Jenson-Shannon Divergence (JSD) indices reveals high similarity between different days. (D) Comparison of cell type proportions across weeks using boxplot of Jenson-Shannon Divergence (JSD) indices shows high similarity between different weeks. (E) Hierarchical clustering of Spearman correlations of gene expression profiles of podocytes from adult human samples, fetal human samples, in vitro organoids, and transplanted organoids. (F) Representative transplanted organoid showing CD31/PECAM expressing cells indicating single human endothelial cells and small vessel networks (Top panel) and a zoom representative showing CD31/PECAM cells in a organoid glomerulus (Bottom panels). RECA-1: red; CD31/PECAM1: green. (G)Representative transplanted organoid showing RECA-1 expressing rat derived vascularization of an organoid glomerulus. RECA-1: red; synaptopodin: green; nephrin: blue (H) NanoString gene expression (row z-score) over in vitro organoid maturation (D0 to D28) for selected genes. (I) Average gene expression scaled to row z-score within each scRNA-Seq cluster during in vitro organoid maturation (D0 to D28) for selected genes.
Figure Legend Snippet: Organoid transplanted promotes differentiation and vascularization (A) Schematic of organoid protocol with rat kidney capsule transplantation and scRNA-Seq timepoints (B) Stacked bar graph containing cell type proportions of in vivo maturation across time points and replicates show an increase in podocytes, tubular cells, endothelial cells, and off-target cell types with a longer period of in vivo maturation. (C) Comparison of cell type proportions across days using boxplot of Jenson-Shannon Divergence (JSD) indices reveals high similarity between different days. (D) Comparison of cell type proportions across weeks using boxplot of Jenson-Shannon Divergence (JSD) indices shows high similarity between different weeks. (E) Hierarchical clustering of Spearman correlations of gene expression profiles of podocytes from adult human samples, fetal human samples, in vitro organoids, and transplanted organoids. (F) Representative transplanted organoid showing CD31/PECAM expressing cells indicating single human endothelial cells and small vessel networks (Top panel) and a zoom representative showing CD31/PECAM cells in a organoid glomerulus (Bottom panels). RECA-1: red; CD31/PECAM1: green. (G)Representative transplanted organoid showing RECA-1 expressing rat derived vascularization of an organoid glomerulus. RECA-1: red; synaptopodin: green; nephrin: blue (H) NanoString gene expression (row z-score) over in vitro organoid maturation (D0 to D28) for selected genes. (I) Average gene expression scaled to row z-score within each scRNA-Seq cluster during in vitro organoid maturation (D0 to D28) for selected genes.

Techniques Used: Transplantation Assay, In Vivo, Expressing, In Vitro, Derivative Assay

Transplantation promotes organoid vascularization and collecting duct maturation (A) Representative transplanted organoid showing RECA-1 expressing rat derived vascularization of an organoid glomerulus. G: glomerulus; V: RECA-1 positive rat vessels. RECA-1: red; synaptopodin: green; nephrin: blue. (B) NanoString quantification of CD31/PECAM1 induction over time for in vitro and transplanted organoids for d10, d12, and d14 plus 2wk or 4wk development in vivo . These data show that the there is significant endothelial CD31/PECAM1 expression with transplantation. The most CD31/PECAM1 expression occurs during D12 + 2-week, D12 + 4-week, and D14 + 4-week transplantation with no significant difference among these conditions (p > 0.1). (C) NanoString quantification of AQP2 induction over time for in vitro and transplanted organoids for D10, D12, and D14 plus 2-week or 4-week development in vivo . These data show that the there is significant endothelial A QP2 expression with transplantation. The highest AQP2 expression occurs during D10 + 2-week, D10 + 4-week, D12 + 2-week, D12 + 4-week, and D14+ 4-week transplantation.
Figure Legend Snippet: Transplantation promotes organoid vascularization and collecting duct maturation (A) Representative transplanted organoid showing RECA-1 expressing rat derived vascularization of an organoid glomerulus. G: glomerulus; V: RECA-1 positive rat vessels. RECA-1: red; synaptopodin: green; nephrin: blue. (B) NanoString quantification of CD31/PECAM1 induction over time for in vitro and transplanted organoids for d10, d12, and d14 plus 2wk or 4wk development in vivo . These data show that the there is significant endothelial CD31/PECAM1 expression with transplantation. The most CD31/PECAM1 expression occurs during D12 + 2-week, D12 + 4-week, and D14 + 4-week transplantation with no significant difference among these conditions (p > 0.1). (C) NanoString quantification of AQP2 induction over time for in vitro and transplanted organoids for D10, D12, and D14 plus 2-week or 4-week development in vivo . These data show that the there is significant endothelial A QP2 expression with transplantation. The highest AQP2 expression occurs during D10 + 2-week, D10 + 4-week, D12 + 2-week, D12 + 4-week, and D14+ 4-week transplantation.

Techniques Used: Transplantation Assay, Expressing, Derivative Assay, In Vitro, In Vivo

A high-throughput platform for human kidney organoid generation (A) Schematic of organoid protocol with scRNA-Seq timepoints (B) Representative planar cross-section of a D28 in vitro organoid that expressing markers of podocytes, tubules, and endothelial cells ( left panel ). A zoomed in region of the cross-section showing endogenous human CD31/PECAM positive vessels developing in vitro amongst podocytes and tubules ( right panel ). Synaptopodin: magenta; CD31/PECAM1: red; E-cadherin: green; Hoechst: blue. (C) Representative cross-section of a D28 in vitro organoid demonstrating apical and basal nephron distribution of podocyte-containing glomeruli, proximal tubules, and distal tubules. Synaptopodin: magenta; LTL: red; E-cadherin: green. (D) Stacked bar graph of cell type proportions of in vitro organoid differentiation across time points and replicates reveal an increase in podocytes and tubular cells at later time points. (E) Comparison of cell type proportions across days using boxplots of Jenson-Shannon Divergence (JSD) indices show high similarity between time points. (F) Comparison of cell type proportions across replicates using boxplots of Jenson-Shannon Divergence (JSD) indices show high similarity between replicates of same time point.
Figure Legend Snippet: A high-throughput platform for human kidney organoid generation (A) Schematic of organoid protocol with scRNA-Seq timepoints (B) Representative planar cross-section of a D28 in vitro organoid that expressing markers of podocytes, tubules, and endothelial cells ( left panel ). A zoomed in region of the cross-section showing endogenous human CD31/PECAM positive vessels developing in vitro amongst podocytes and tubules ( right panel ). Synaptopodin: magenta; CD31/PECAM1: red; E-cadherin: green; Hoechst: blue. (C) Representative cross-section of a D28 in vitro organoid demonstrating apical and basal nephron distribution of podocyte-containing glomeruli, proximal tubules, and distal tubules. Synaptopodin: magenta; LTL: red; E-cadherin: green. (D) Stacked bar graph of cell type proportions of in vitro organoid differentiation across time points and replicates reveal an increase in podocytes and tubular cells at later time points. (E) Comparison of cell type proportions across days using boxplots of Jenson-Shannon Divergence (JSD) indices show high similarity between time points. (F) Comparison of cell type proportions across replicates using boxplots of Jenson-Shannon Divergence (JSD) indices show high similarity between replicates of same time point.

Techniques Used: High Throughput Screening Assay, In Vitro, Expressing

CsA and TRPC5 inhibition protects against podocyte injury in kidney organoids in vitro (A) Representative current-voltage relationship recording during a 500 ms voltage ramp from -80 mV to +80 mV in the absence and presence of 0.1 μM GFB-887 or the reference TRPC5 inhibitor, 100 μM ML-204. (B) Concentration dependence of inhibition of human TRPC5 currents after a 3 min application of GFB-887 at +80 mV. Data points are mean ± SEM of 3-4 observations at each concentration. (C) Upregulation of TRPC5 mRNA expression during organoid differentiation in vitro (D) Double labeling with synaptopodin reveals podocyte TRPC5 protein expression in human organoids (E) CsA and GFB-887 protects against PS induced podocyte injury in in vitro D28 organoids. Representative PS injury mask for an organoid is depicted in blue and the inset box indicates and area in the region that can be used for identification of injured podocytes for quantitation ( top left panel ). Representative glomeruli identified for podocyte injury quantitation of aggregated actin for treatment with vehicle DMSO, PS alone, CsA + PS, GFB-887 + PS, and the combination of CsA and GFB-887 + PS. Synaptopodin: green; Phalloidin: red. (F) Quantification of PS induced actin aggregation. GFB-887 and CsA are non-additive, consistent with shared mechanism of action. DMSO vs. PS, p
Figure Legend Snippet: CsA and TRPC5 inhibition protects against podocyte injury in kidney organoids in vitro (A) Representative current-voltage relationship recording during a 500 ms voltage ramp from -80 mV to +80 mV in the absence and presence of 0.1 μM GFB-887 or the reference TRPC5 inhibitor, 100 μM ML-204. (B) Concentration dependence of inhibition of human TRPC5 currents after a 3 min application of GFB-887 at +80 mV. Data points are mean ± SEM of 3-4 observations at each concentration. (C) Upregulation of TRPC5 mRNA expression during organoid differentiation in vitro (D) Double labeling with synaptopodin reveals podocyte TRPC5 protein expression in human organoids (E) CsA and GFB-887 protects against PS induced podocyte injury in in vitro D28 organoids. Representative PS injury mask for an organoid is depicted in blue and the inset box indicates and area in the region that can be used for identification of injured podocytes for quantitation ( top left panel ). Representative glomeruli identified for podocyte injury quantitation of aggregated actin for treatment with vehicle DMSO, PS alone, CsA + PS, GFB-887 + PS, and the combination of CsA and GFB-887 + PS. Synaptopodin: green; Phalloidin: red. (F) Quantification of PS induced actin aggregation. GFB-887 and CsA are non-additive, consistent with shared mechanism of action. DMSO vs. PS, p

Techniques Used: Inhibition, In Vitro, Concentration Assay, Expressing, Labeling, Quantitation Assay

29) Product Images from "JAK inhibitor blocks COVID-19 cytokine–induced JAK/STAT/APOL1 signaling in glomerular cells and podocytopathy in human kidney organoids"

Article Title: JAK inhibitor blocks COVID-19 cytokine–induced JAK/STAT/APOL1 signaling in glomerular cells and podocytopathy in human kidney organoids

Journal: JCI Insight

doi: 10.1172/jci.insight.157432

APOL1 expression is upregulated in podocytes and GECs of patients with COVAN. ( A – L ) IHC for APOL1 in biopsy tissue from ( A – F ) patient 1 and ( G – L ) patient 6. Original magnification, 40×. ( B , E , H , and K ) Co-staining of APOL1 with podocyte marker synaptopodin. ( C , F , I , and L ) Co-staining of APOL1 with endothelial marker CD31. Arrow represents a podocyte. Arrowhead represents a GEC.
Figure Legend Snippet: APOL1 expression is upregulated in podocytes and GECs of patients with COVAN. ( A – L ) IHC for APOL1 in biopsy tissue from ( A – F ) patient 1 and ( G – L ) patient 6. Original magnification, 40×. ( B , E , H , and K ) Co-staining of APOL1 with podocyte marker synaptopodin. ( C , F , I , and L ) Co-staining of APOL1 with endothelial marker CD31. Arrow represents a podocyte. Arrowhead represents a GEC.

Techniques Used: Expressing, Immunohistochemistry, Staining, Marker

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    Progen Biotechnik synaptopodin
    Effect of MLL3 shRNA on <t>synaptopodin</t> and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P
    Synaptopodin, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Progen Biotechnik primary mouse monoclonal anti synaptopodin
    Double immunofluorescence staining of kidney sections from Wistar rats. Immunofluorescence staining of glomeruli: (A) anti-meprinβ antibody, (B) <t>anti-synaptopodin</t> antibody, (C) merged image meprin/synaptopodin, (D) anti-meprinβ antibody, (E) anti-Thy 1.1 antibody, (F) merged image meprin/Thy 1.1. Nuclei are marked by staining with 4,6-diamidino-2-phenylindole (DAPI).
    Primary Mouse Monoclonal Anti Synaptopodin, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Progen Biotechnik mouse anti synaptopodin
    Dual color 3D-SIM of rodent (A) in mouse and (B) in rat) paraffin kidney sections shows the interdigitating foot process pattern of podocytes. Foot processes and the slit diaphragm were labeled using <t>anti-synaptopodin</t> and anti-nephrin antibodies, respectively. Although both proteins are in close structural vicinity the individual staining patterns do not overlap and therefore demonstrates the high resolution accomplished by 3D-SIM. All scale bars represent 1 μm.
    Mouse Anti Synaptopodin, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P

    Journal: BMC Nephrology

    Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

    doi: 10.1186/s12882-016-0390-8

    Figure Lengend Snippet: Effect of MLL3 shRNA on synaptopodin and cathepsin L expression and histone H3K4 me3 levels and the determination of H3K4 me3 level on the promoter region of cathepsin L in cultured mouse podocytes co-cultured with LPS-stimulated peritoneal macrophages. In podocytes co-cultured with peritoneal macrophages stimulated with LPS, increased H3K4 me3 was found compared to untreated podocytes (Veh) ( a ). MLL3 shRNA administration decreased H3K4 me3 compared to that of cells administered with control shRNA ( a ). MLL3 shRNA administration also decreased the level of cathepsin L protein expression compared to that of cells administered the control shRNA ( b ). MLL3 shRNA increased the expression of synaptopodin compared to that of the cells administered control shRNA in cultures stimulated with cytokines derived from co-cultured macrophages ( c ). In chromatin immunoprecipitation (ChIP) assays with E11 cells stimulated with cytokines derived from co-cultured macrophages, the stimulated levels of H3K4 me3 at the cathepsin L promoters were significantly lower in E11 cells treated with MLL3 shRNA 72 h post-administration compared with the control shRNA ( d ). * P

    Article Snippet: Histological analysis The kidneys were fixed in a solution of buffered paraformaldehyde, embedded in paraffin, and sectioned (4 μm) in an LPS-induced proteinuria model. For immunohistochemistry, paraffin-embedded kidney sections from human subjects and mice were incubated with antibodies against nephrin (NOVUS BIOLOGICALS, Littleton, CO, USA), histone H3K4 me3 (EPIGENETEK, Farmingdale, NY, USA), cathepsin L (R & D Systems, Inc., Minneapolis, MN, USA), Wilm’s tumor-1 protein (WT-1, DAKO, USA), synaptopodin (PROGEN Biotechnik, Heidelberg, German), and anti-GFP (anti-GFP-tag, rabbit polyclonal antibody; AnaSpec, Inc., Fremont, CA, USA), NEPH-1 (KIRREL1, BIOSS ANTIBODIES, Woburn, MA, USA), phospholipase A2 receptor 1 (Anti-PLA2R1, ATLAS ANTIBODIES, Stockholm, Sweden), followed by an incubation with a biotinylated secondary antibody and streptavidin-HRP.

    Techniques: shRNA, Expressing, Cell Culture, Derivative Assay, Chromatin Immunoprecipitation

    Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P

    Journal: BMC Nephrology

    Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

    doi: 10.1186/s12882-016-0390-8

    Figure Lengend Snippet: Podocyte swelling and the expression of H3K4 me3 and synaptopodin in the LPS model. Electron microscopy reveals podocyte swelling induced by LPS administration ( right side in a and d ) compared with the controls (vehicle alone, left side in a and d ). The expression of histone H3K4 me3 was significantly higher following LPS administration ( right side in b and e ) than in the controls (vehicle alone, left side in b and e ). Synaptopodin expression was significantly lower following LPS administration ( right side in c and f ) compared to that of the controls (vehicle alone, left side in c and f ). * P

    Article Snippet: Histological analysis The kidneys were fixed in a solution of buffered paraformaldehyde, embedded in paraffin, and sectioned (4 μm) in an LPS-induced proteinuria model. For immunohistochemistry, paraffin-embedded kidney sections from human subjects and mice were incubated with antibodies against nephrin (NOVUS BIOLOGICALS, Littleton, CO, USA), histone H3K4 me3 (EPIGENETEK, Farmingdale, NY, USA), cathepsin L (R & D Systems, Inc., Minneapolis, MN, USA), Wilm’s tumor-1 protein (WT-1, DAKO, USA), synaptopodin (PROGEN Biotechnik, Heidelberg, German), and anti-GFP (anti-GFP-tag, rabbit polyclonal antibody; AnaSpec, Inc., Fremont, CA, USA), NEPH-1 (KIRREL1, BIOSS ANTIBODIES, Woburn, MA, USA), phospholipase A2 receptor 1 (Anti-PLA2R1, ATLAS ANTIBODIES, Stockholm, Sweden), followed by an incubation with a biotinylated secondary antibody and streptavidin-HRP.

    Techniques: Expressing, Electron Microscopy

    Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P

    Journal: BMC Nephrology

    Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

    doi: 10.1186/s12882-016-0390-8

    Figure Lengend Snippet: Effect of MLL3 shRNA on synaptopodin expression and podocyte swelling in the LPS model. LPS-induced decreased synaptopodin expression was significantly restored by MLL3 shRNA compared with control shRNA as detected by immunohistochemistry ( a and b ). Electron microscopy showed the suppressive effect of MLL3 shRNA on LPS-induced podocyte swelling ( c and d ). * P

    Article Snippet: Histological analysis The kidneys were fixed in a solution of buffered paraformaldehyde, embedded in paraffin, and sectioned (4 μm) in an LPS-induced proteinuria model. For immunohistochemistry, paraffin-embedded kidney sections from human subjects and mice were incubated with antibodies against nephrin (NOVUS BIOLOGICALS, Littleton, CO, USA), histone H3K4 me3 (EPIGENETEK, Farmingdale, NY, USA), cathepsin L (R & D Systems, Inc., Minneapolis, MN, USA), Wilm’s tumor-1 protein (WT-1, DAKO, USA), synaptopodin (PROGEN Biotechnik, Heidelberg, German), and anti-GFP (anti-GFP-tag, rabbit polyclonal antibody; AnaSpec, Inc., Fremont, CA, USA), NEPH-1 (KIRREL1, BIOSS ANTIBODIES, Woburn, MA, USA), phospholipase A2 receptor 1 (Anti-PLA2R1, ATLAS ANTIBODIES, Stockholm, Sweden), followed by an incubation with a biotinylated secondary antibody and streptavidin-HRP.

    Techniques: shRNA, Expressing, Immunohistochemistry, Electron Microscopy

    Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation

    Journal: BMC Nephrology

    Article Title: Alteration of histone H3K4 methylation in glomerular podocytes associated with proteinuria in patients with membranous nephropathy

    doi: 10.1186/s12882-016-0390-8

    Figure Lengend Snippet: Association between histone H3K4 me3 and the expression of synaptopodin and proteinuria in patients with MN. Expression of H3K4 me3 ( left side in a ), synaptopodin ( center in a ), and the merged image ( right side in a ) are shown. The level of H3K4 me3 expression was positively correlated with albuminuria ( b ), and the level of H3K4 me3 expression was inversely correlated with the level of synaptopodin expression in the podocytes of patients with MN ( c ). The level of synaptopodin expression was inversely correlated with the level of albuminuria ( d ). MN, membranous nephropathy; H3K4 me3, histone H3K4 trimethylation

    Article Snippet: Histological analysis The kidneys were fixed in a solution of buffered paraformaldehyde, embedded in paraffin, and sectioned (4 μm) in an LPS-induced proteinuria model. For immunohistochemistry, paraffin-embedded kidney sections from human subjects and mice were incubated with antibodies against nephrin (NOVUS BIOLOGICALS, Littleton, CO, USA), histone H3K4 me3 (EPIGENETEK, Farmingdale, NY, USA), cathepsin L (R & D Systems, Inc., Minneapolis, MN, USA), Wilm’s tumor-1 protein (WT-1, DAKO, USA), synaptopodin (PROGEN Biotechnik, Heidelberg, German), and anti-GFP (anti-GFP-tag, rabbit polyclonal antibody; AnaSpec, Inc., Fremont, CA, USA), NEPH-1 (KIRREL1, BIOSS ANTIBODIES, Woburn, MA, USA), phospholipase A2 receptor 1 (Anti-PLA2R1, ATLAS ANTIBODIES, Stockholm, Sweden), followed by an incubation with a biotinylated secondary antibody and streptavidin-HRP.

    Techniques: Expressing

    PDE5A is expressed by podocytes. RNA and protein were isolated from cultured podocytes and renal mouse cortex; subsequently, (A) PCR and (B) Western blot were performed to determine PDE5A expression in these samples. Glomerular PDE5A expression was confirmed by immunofluorescence staining of PDE5A. (C) Importantly, costaining with synaptopodin showed PDE5A expression in the podocytes; merge is a higher magnification of single-channel images. AB, antibody; NTC, no template control.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Sildenafil Prevents Podocyte Injury via PPAR-γ–Mediated TRPC6 Inhibition

    doi: 10.1681/ASN.2015080885

    Figure Lengend Snippet: PDE5A is expressed by podocytes. RNA and protein were isolated from cultured podocytes and renal mouse cortex; subsequently, (A) PCR and (B) Western blot were performed to determine PDE5A expression in these samples. Glomerular PDE5A expression was confirmed by immunofluorescence staining of PDE5A. (C) Importantly, costaining with synaptopodin showed PDE5A expression in the podocytes; merge is a higher magnification of single-channel images. AB, antibody; NTC, no template control.

    Article Snippet: Frozen kidney cortex was cut and stained for TRPC6 (rat;: Abcam, Cambridge, United Kingdom; mice: Alomone, Jerusalem, Israel), nephrin (R & D, Minneapolis, MN), synaptopodin (Progen Biotechnik GmbH, Heidelberg, Germany), desmin, and PDE5A (Santa Cruz).

    Techniques: Isolation, Cell Culture, Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Staining

    Double immunofluorescence staining of kidney sections from Wistar rats. Immunofluorescence staining of glomeruli: (A) anti-meprinβ antibody, (B) anti-synaptopodin antibody, (C) merged image meprin/synaptopodin, (D) anti-meprinβ antibody, (E) anti-Thy 1.1 antibody, (F) merged image meprin/Thy 1.1. Nuclei are marked by staining with 4,6-diamidino-2-phenylindole (DAPI).

    Journal: PLoS ONE

    Article Title: Metalloprotease Meprin? in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

    doi: 10.1371/journal.pone.0002278

    Figure Lengend Snippet: Double immunofluorescence staining of kidney sections from Wistar rats. Immunofluorescence staining of glomeruli: (A) anti-meprinβ antibody, (B) anti-synaptopodin antibody, (C) merged image meprin/synaptopodin, (D) anti-meprinβ antibody, (E) anti-Thy 1.1 antibody, (F) merged image meprin/Thy 1.1. Nuclei are marked by staining with 4,6-diamidino-2-phenylindole (DAPI).

    Article Snippet: We used the following antibodies: primary rabbit polyclonal anti-meprinβ, targeting amino acids 468–612 of meprinβ, as previously described , primary mouse monoclonal anti-synaptopodin (Progen, Heidelberg, Germany), primary monoclonal mouse anti-Thy 1.1 (Oxford Biotechology, UK), secondary goat anti-rabbit and goat anti-mouse (Dako Cytomation, Denmark), diluted 1:500, 1:4, 1:20, 1:1000 and 1:400 respectively.

    Techniques: Double Immunofluorescence Staining, Immunofluorescence, Staining

    Meprinβ expression in rat glomeruli after induction of Passive Heyman nephritis (PHN). Immunofluorescence staining (A–C) of meprinβ and (D–F) synaptopodin in kidney sections from Sprague-Dawley rats. (G–I) Merged images of meprin/synaptopodin. (J–L) Immunohistochemical staining of meprinβ in glomeruli and in proximal tubules. Staining (A, D, G and J) in control rats, (B, E, H and K) in rats at day 3 and (C, F, I and L) in rats at day 6 after induction of PHN. A change in distribution of the meprinβ in PHN kidneys from a linear to a granular appearance was observed concomitantly associated with an overall reduction in signal intensity. The distribution of meprinβ and synaptopodin in glomeruli appeared increasingly divergent following progression of PHN.

    Journal: PLoS ONE

    Article Title: Metalloprotease Meprin? in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

    doi: 10.1371/journal.pone.0002278

    Figure Lengend Snippet: Meprinβ expression in rat glomeruli after induction of Passive Heyman nephritis (PHN). Immunofluorescence staining (A–C) of meprinβ and (D–F) synaptopodin in kidney sections from Sprague-Dawley rats. (G–I) Merged images of meprin/synaptopodin. (J–L) Immunohistochemical staining of meprinβ in glomeruli and in proximal tubules. Staining (A, D, G and J) in control rats, (B, E, H and K) in rats at day 3 and (C, F, I and L) in rats at day 6 after induction of PHN. A change in distribution of the meprinβ in PHN kidneys from a linear to a granular appearance was observed concomitantly associated with an overall reduction in signal intensity. The distribution of meprinβ and synaptopodin in glomeruli appeared increasingly divergent following progression of PHN.

    Article Snippet: We used the following antibodies: primary rabbit polyclonal anti-meprinβ, targeting amino acids 468–612 of meprinβ, as previously described , primary mouse monoclonal anti-synaptopodin (Progen, Heidelberg, Germany), primary monoclonal mouse anti-Thy 1.1 (Oxford Biotechology, UK), secondary goat anti-rabbit and goat anti-mouse (Dako Cytomation, Denmark), diluted 1:500, 1:4, 1:20, 1:1000 and 1:400 respectively.

    Techniques: Expressing, Immunofluorescence, Staining, Immunohistochemistry

    Dual color 3D-SIM of rodent (A) in mouse and (B) in rat) paraffin kidney sections shows the interdigitating foot process pattern of podocytes. Foot processes and the slit diaphragm were labeled using anti-synaptopodin and anti-nephrin antibodies, respectively. Although both proteins are in close structural vicinity the individual staining patterns do not overlap and therefore demonstrates the high resolution accomplished by 3D-SIM. All scale bars represent 1 μm.

    Journal: Frontiers in Endocrinology

    Article Title: Novel Microscopic Techniques for Podocyte Research

    doi: 10.3389/fendo.2018.00379

    Figure Lengend Snippet: Dual color 3D-SIM of rodent (A) in mouse and (B) in rat) paraffin kidney sections shows the interdigitating foot process pattern of podocytes. Foot processes and the slit diaphragm were labeled using anti-synaptopodin and anti-nephrin antibodies, respectively. Although both proteins are in close structural vicinity the individual staining patterns do not overlap and therefore demonstrates the high resolution accomplished by 3D-SIM. All scale bars represent 1 μm.

    Article Snippet: Sections where dehydrated, antigen retrieved by pressure-cooking in citrate buffer, blocked in 1% FBS, 1% BSA, 1% NGS, 0.1% cold fish gelatin in PBS and co-labeled with guinea pig anti-nephrin (GP-N2, 1:300) and mouse anti-synaptopodin (GP94-C, 1:50, both Progen, Heidelberg, Germany) overnight at 4°C.

    Techniques: Labeling, Staining