Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of ECM on the differentiation of kidney organoids in a static culture condition. A Fluorescent images of immunostained kidney organoids cultured with various conditions of ECMs for 6 days. The cells were immunostained by NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue). B Quantitative analysis of kidney antibody-specific markers from kidney organoids (* indicates p <0.05 for experimental group vs. control group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Cell Culture

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of the shear stress on the differentiation of kidney organoids in a kidney organoid-on-a-chip. A Fluorescent images showing hPSCs-derived kidney organoid-related cell differentiation under a shear stress condition. Kidney organoids were cultured within kidney organoid-on-a-chip for 6 days. After culturing for 6 days, the cells were stained for NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue) to identify the kidney-related cells, respectively. B Quantitative analysis of the kidney-related markers from kidney organoids. (* indicates p < 0.05 for LTL expression of Matrigel-coated group vs. Matrigel/VEGF-coated group, *** indicates p < 0.001 for PECAM expression of Matrigel-coated group vs. Matrigel/VEGF-coated group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Derivative Assay, Cell Differentiation, Cell Culture, Staining, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Generation of podocyte-specific Kindlin-2Neph2 cKO mice. (A) The diagram depicts the strategy for generation of Kindlin-2Neph2 cKO (cKO). Mice expressing Neph2-Cre were bred with mice carrying floxed Kindlin-2 locus (exons 5 and 6). (B) Representative PCR analysis of extracted genomic DNA from tail clippings. PCR product bands of floxed (300 bp) and wild-type (200 bp) are shown. Cre PCR product (650 bp) is also indicated. (C) Immunoblotting analysis of Kindlin-2 expression in isolated primary podocytes from WT and Kindlin-2Neph2 cKO mice. (D) Podocytes harvested from WT and Kindlin-2Neph2 cKO mice were plated on laminin-coated glass coverslips and stained for WT1 (red) and for Kindlin-2 (green). Scale bars, 10 μm. (E and F) Double-immunofluorescence detection of Kindlin-2 and WT1 (E) or Kindlin-2 and nephrin (F) on kidney sections of WT and Kindlin-2Neph2 cKO mice. Scale bars, 10 μm. The lower panels of (F) show higher magnification images of the areas outlined with white lines in the upper panels. The experiments in Figure 1, B–F are representative of 4–6 independent experiments. Arrows indicate WT1-positive podocytes. E, Exon; fl, flox; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Expressing, Western Blot, Isolation, Staining, Immunofluorescence

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Podocyte-specific Kindlin-2Neph2 cKO mice develop proteinuria and kidney failure. (A) Kindlin-2Neph2 cKO mice fail to gain weight by 6 weeks of age compared with WT mice. ***P<0.001 versus WT; n=6 mice at each time point. (B) Representative picture shows the phenotypic appearance of WT and Kindlin-2Neph2 cKO mice at 8 weeks of age. (C) Survival curve of Kindlin-2Neph2 cKO mice shows 100% mortality by 10 weeks of age. n=20 (WT), n=22 (cKO). (D and E) SDS-PAGE analysis of albumin levels in the urine (D) and plasma (E) at different time points in WT and Kindlin-2Neph2 cKO mice. (F) Quantification of urinary albumin normalized to creatinine at 1, 2, 4, and 8 weeks of age. ***P<0.001 versus WT; n=6 mice at each time point. (G) Kidneys of Kindlin-2Neph2 cKO mice are paler and smaller than those of controls and have an irregular appearance at 8 weeks of age. (H) Elevated plasma creatinine in Kindlin-2Neph2 cKO mice at 2, 4, and 8 weeks of age. ***P<0.001 versus WT; n=6 mice at each time point. cKO, conditional knockout; W, week; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: SDS Page, Clinical Proteomics, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Podocyte-specific Kindlin-2Neph2 cKO mice develop glomerulosclerosis and interstitial fibrosis. (A) Kidney sections from Kindlin-2Neph2 cKO and control littermates at 2, 4, and 8 weeks were subjected to H&E, PAS, or trichrome staining. Arrows indicate diffuse tubular dilatation and proteinaceous casts. Asterisks indicate fibrotic areas. Higher magnification images of representative areas of trichrome staining (indicated by squares) with interstitial fibrosis are shown on the right. Scale bar, 40 μm. (B) Quantification of interstitial fibrosis at 2, 4, and 8 weeks of age. ***P<0.001 versus WT; n=10 mice at each time point. (C) Quantification of glomerulosclerosis at 2, 4, and 8 weeks of age. ***P<0.001 versus WT; n=10 mice at each time point. cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Control, Staining, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Podocyte-specific deletion of Kindlin-2 causes ultrastructural changes of podocytes. (A) Low-magnification (left panel) and high-magnification (right panel) scanning electron microscopy analysis of podocyte ultrastructure at 1 and 8 weeks of age Kindlin-2Neph2 cKO and WT mice. Podocyte FP and microvillous transformation on the apical surface of podocytes can been seen in Kindlin-2Neph2 cKO mice. Scale bar, 5 μm. (B) Low-magnification (left panel) and high-magnification (right panel) TEM analyses identified abnormal FP and GBM structure in Kindlin-2Neph2 cKO at various ages compared with WT mice. Arrow indicates fused FP, arrowhead denotes naked GBM. Scale bar, 5 μm (left panel), 0.5 μm (right panel). (C) Quantification of SD density (i.e., the number of FP per micrometer of GBM) in Kindlin-2Neph2 cKO and WT mice at different time points. ***P<0.001 versus WT; n=3 mice at 1 and 2 weeks of age; n=5 mice at 4 and 8 weeks of age. (D) Quantification of GBM thickness in Kindlin-2Neph2 cKO and WT mice at different time points. ***P<0.001 versus WT; n=3 mice at 2 weeks of age; n=5 mice at 4 and 8 weeks of age. cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Electron Microscopy, Transformation Assay, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Podocyte-specific deletion of Kindlin-2 results in loss of podocytes. (A) Representative images of kidney sections stained with WT1, a podocyte-specific transcription factor, at 1, 2, 4, and 8 weeks Kindlin-2Neph2 cKO and WT mice. Arrow denotes the glomerulus with reduced podocyte number in Kindlin-2Neph2 cKO mice. (B) Quantification of WT1 staining per glomerulus was performed with Kindlin-2Neph2 cKO and WT mice at different time points. ***P<0.001 versus WT; n=4 mice at each time point. At least 30 glomeruli/mouse were counted. (C) Kidney sections were subjected to anti-caspase3 staining to identify apoptotic cells. Arrows indicate apoptotic cells present in both glomerular and tubulointerstitial areas at 4 weeks Kindlin-2Neph2 cKO mice. Caspase3-positive cells were only observed in tubulointerstitial areas at 8 weeks Kindlin-2Neph2 cKO mice. No apoptotic cells were found in WT mice. n=4 mice at each time point. (D) Representative immunoblotting of urine samples from Kindlin-2Neph2 cKO and WT mice at 4 weeks analyzed with an antibody for nephrin, a marker for podocytes. Note that nephrin is present in the urine of 4 weeks Kindlin-2Neph2 cKO mice but not that of WT mice, indicating the presence of podocytes in the urine of Kindlin-2Neph2 cKO mice. Original magnification, ×200 (in A and C). cKO, conditional knockout; G, glomerular; IB, immunoblotting; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Staining, Western Blot, Marker, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Podocyte-specific deletion of Kindlin-2 results in dysregulation of SD and actin cytoskeleton. (A) Kidney sections from Kindlin-2Neph2 cKO and WT mice were stained with various antibodies against SD/actin-associated proteins ZO-1, synaptopodin, α-actinin4, and nephrin as well as mesenchymal proteins desmin and α-SMA. Representative micrographs are shown. Scale bar, 10 μm. (B) Immnoblotting analysis reveals that knockout of Kindlin-2 in podocytes reduces ZO-1, α-actinin4, nephrin, and synaptopodin expression. (C and D) Representative photomicrographs of cell morphology and immunofluorescence staining for F-actin (green) (C) or Vinculin (green) (D) in primary podocytes isolated from Kindlin-2Neph2 cKO and WT mice. Podocyte nuclei were visualized with WT1 (red). Scale bars, 50 μm (C) or 10 μm (D). Quantification data are shown in the right panel. ***P<0.001 versus WT (C); **P<0.01 versus WT (D); n=4 independent experiments. cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Staining, Knock-Out, Expressing, Immunofluorescence, Isolation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Podocyte-specific deletion of Kindlin-2 promotes Rac1 activation. (A) KD of Kindlin-2 in immobilized human podocytes. Quantification data are shown in the lower panel. ***P<0.001 versus control; n=8 independent experiments. (B) Representative immunoblotting analysis of Rac1 and RhoA activity in Kindlin-2 KD and control podocytes. (C) Quantification of Rac1 and RhoA activity. *P<0.02 versus control; **P<0.01 versus control; n=4 independent experiments. (D) KD of Kindlin-2 increases podocyte migration. Representative images (upper panel) and quantification analysis (lower panel) of transwell migration assay were shown. *P<0.02 versus control; **P<0.01 versus control; n=4 independent experiments. (E) Representative images (upper panel) and quantification analysis (lower panel) of transwell migration assay were shown. Primary podocytes isolated from Kindlin-2Neph2 cKO and WT mice were used. ***P<0.001 versus WT; n=3 independent experiments. (F) Multiple tracks of individual podocytes (left panel) and quantification of the velocity (right panel) from Kindlin-2 KD and control podocytes were shown. n=3 independent experiments with >160 cell tracks. ***P<0.001 versus control. cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Activation Assay, Control, Western Blot, Activity Assay, Migration, Transwell Migration Assay, Isolation, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Kindlin-2 regulates Rac1 activation through association with RhoGDIα. (A) Immobilized human podocyte lysates were immunoprecipitated with anti–Kindlin-2 antibody or normal IgG followed by immunoblotting with antibodies as indicated. The presence of Kindlin-2 and RhoGDIα in cell lysates is shown as input. (B) Immobilized human podocyte lysates were immunoprecipitated with anti-RhoGDIα antibody or normal IgG followed by immunoblotting with antibodies as indicated. (C) GST fusion proteins containing the full-length, N-terminal (residues 1–67) or C-terminal (residues 68–204) fragment of RhoGDIα were used to pull down Kindlin-2 from human podocytes (lower panel). Upper panel: schematic illustration of RhoGDIα fragments that were used in the GST pull-down assay. Note that Kindlin-2 was pulled down by the N-terminal (residues 1–67) but not the C-terminal (residues 68–204) fragment of RhoGDIα. (D) Immunoblotting analysis of RhoGDIα protein levels in primary podocytes isolated from Kindlin-2Neph2 cKO and WT mice (left panel). Quantification data are shown in the right panel. ***P<0.001 versus WT; n=7 independent experiments. (E) RhoGDIα immunoprecipitates from control and Kindlin-2 KD podocytes were analyzed by immunoblotting with anti-Rac1 and anti-RhoGDIα antibodies. Normal IgG is used as negative control. n=4 independent experiments. IB, immunoblotting; IP, immunoprecipitation; cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Pull Down Assay, Isolation, Control, Negative Control, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: Treatment with NSC23766 (NSC) reverses the phenotypes induced by the loss of Kindlin-2 in mice. (A) Kaplan–Meier survival curves of 2-week-old (left panel) and 6-week-old (right panel) Kindlin-2Neph2 cKO mice treated with NSC23766 (cKO+NSC) or PBS (cKO) for 14 days or WT controls. Note that NSC23766 treatment increases the mortality of Kindlin-2Neph2 cKO mice in each age group (P<0.001, log-rank test). For the group of 2 weeks of age, 35 WT, 35 cKO, and 37 cKO+NSC mice were analyzed. For the group of 6 weeks of age, 30 WT, 30 cKO, and 35 cKO+NSC mice were analyzed. (B) Kidneys of WT, cKO, and cKO+NSC mice at 4 weeks of age. Three sets of the mice were analyzed and similar results were obtained. (B) Results from one of the representative sets. (C and D) Coomassie blue staining of SDS-PAGE analyses of urinary proteins (C) and quantification of albumin-to-creatinine levels in the urine (D) from 4 and 8 weeks of age WT, cKO, and cKO+NSC mice. ***P<0.001 versus other groups; n=6 mice for 4 weeks of age group; n=7 mice for 8 weeks of age group. (E) Renal tissues from WT, cKO, and cKO+NSC mice at 4 weeks were subjected to H&E staining (upper panel), and glomerular (middle panel) and tubulointerstitial (lower panel) fibronectin staining. Original magnification, ×200 for the upper and lower panels; ×400 for the middle panel. (F) Quantification of IHC scores for fibronectin staining within the glomeruli and the tubulointerstitial areas. ***P<0.001 versus other groups; n=6 mice for 4 weeks of age group; n=7 mice for 8 weeks of age group. (G and H) Low-magnification (left panel) and high-magnification (right panel) scanning electron microscopy (G) and TEM (H) analyses of podocyte ultrastructure in WT, cKO, and cKO+NSC mice at 4 weeks of age. Note that abnormal podocyte ultrastructure induced by the loss of Kindlin-2 was prevented to a great extent by NSC treatment. Scale bars, 5 μm (scanning electron microscopy and TEM, left panel) and 1 μm (TEM, right panel). (I) Quantification of the number of FP per micrometer of GBM in WT, cKO, and cKO+NSC mice at different time points. ***P<0.001 versus other groups; n=5 mice for each age group. (J) Immunoblotting analysis of RhoGDIα protein levels in primary podocytes isolated from WT, cKO, and cKO+NSC mice (upper panel). Quantification data are shown in the lower panel. *P<0.05 versus other groups; n=5 independent experiments. (K) Activity of Rac1 was determined by G-LISA assay. The primary podocytes isolated from WT, cKO, and cKO+NSC mice were used. ***P<0.001 versus other groups; n=8 independent experiments. (L) Cell migration. The migration of the control and Kindlin-2 KD podocytes treated with or without NSC was analyzed as described in the Concise Methods section. Representative images are shown in the left panel and quantification analyses are shown in the right panel. *P<0.02 versus control; ***P<0.001 versus control; n=4 independent experiments. (M) Cell adhesion. The adhesion of the control and Kindlin-2 KD podocytes treated with or without NSC to laminin was analyzed as described in the Concise Methods section. Representative images are shown in the left panel and quantification analyses are shown in the right panel. ***P<0.001 versus control; n.s. P>0.05 for NSC versus vehicle; n=4 independent experiments. cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Staining, SDS Page, Electron Microscopy, Western Blot, Isolation, Activity Assay, Migration, Control, Knock-Out

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Kindlin-2 Association with Rho GDP-Dissociation Inhibitor α Suppresses Rac1 Activation and Podocyte Injury

doi: 10.1681/ASN.2016091021

Figure Lengend Snippet: A working model of Kindlin-2–RhoGDIα–Rac1 signaling in podocytes. In WT mouse podocytes, Kindlin-2 interacts with RhoGDIα, and sequesters Rac1 in its inactive form. In Kindlin-2Neph2 cKO mice, loss of Kindlin-2 stimulates release of Rac1 from RhoGDIα for Rac1-GEF-effector interaction which leads to actin stress fiber disruption and SD protein reorganization, thereby resulting in podocyte foot effacement, enhanced motility, podocyte dysfunction, and ultimately proteinuria and renal failure. cKO, conditional knockout; WT, wild-type.

Article Snippet: Human immortalized podocytes were lysed following the manufacturer’s protocol (product code v4xc-2012; Thermo Fisher Scientific) and incubated with GST-PAK-agarose (which binds only to Rac-GTP) or GST-Rhotekin-agarose (which binds only Rho-GTP) for 2 hours, followed by separation by SDS-PAGE and immunoblotting with the appropriate primary and secondary antibodies.

Techniques: Disruption, Knock-Out

Journal: Scientific Reports

Article Title: Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

doi: 10.1038/s41598-019-52872-5

Figure Lengend Snippet: Ischemic-hypoxia elicits podocyte injury. ( A ) Staining for WT1 in sham and MCAO rat glomerular sections. The scale bar represents images of 10 µm and images were captured with a 100x objective of Leica trinocular microscope. ( B ) TEM images of podocyte foot processes in sham and MCAO rat kidney sections. In ischemic-stroke rats, podocyte foot-processes were small and the thickness of GBM was increased compared to sham-operated rats.

Article Snippet: Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592.

Techniques: Staining, Microscopy

Journal: Scientific Reports

Article Title: Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

doi: 10.1038/s41598-019-52872-5

Figure Lengend Snippet: Ischemic-hypoxia induces ZEB2 and its target genes in glomerular podocytes: ( A ) Lysate from primary podocyte isolated from sham and MCAO rat kidney was used to assess the expression of HIF1α, ZEB2, E-cadherin, and TRPC6. Fold change values are mentioned under each blot. ( B ) Differentiated human podocytes treated with or without FG-4592 and analyzed the expression of HIF1α, ZEB2, E-cadherin and TRPC6. Fold change values are mentioned under each blot. The steady-state abundance of mRNA levels of HIF1α, ZEB2, and TRPC6 from podocytes isolated from sham and MCAO rats ( C ) and human podocytes treated with or without FG-4592 ( D ). Error bars indicate mean ± SE; n = 6. ****p < 0.0001. ( E ) Immunohistochemical analysis of HIF1α, ZEB2, and TRPC6 in glomerular sections from sham and MCAO rats. The scale bar represents images of 10 µm and images were captured with a 100x objective of Leica trinocular microscope. ( F ) Co-localization of HIF1α and ZEB2 in human podocytes treated with or without FG-4592. Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592. Images were acquired with a Leica trinocular 63x objective and ( H ) fluorescence intensity of ZEB2 and TRPC6 expression in podocytes treated with or without FG-4592. Error bars indicate mean ± SE; n = 20. ****p < 0.0001.

Article Snippet: Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592.

Techniques: Isolation, Expressing, Immunohistochemical staining, Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

doi: 10.1038/s41598-019-52872-5

Figure Lengend Snippet: Hypoxia induces HIF1α-ZEB2-TRPC6 axis: ( A ) ChIP analysis with chromatin fractions from podocytes exposed to FG-4592 was performed as described in methods. Input DNA and DNA from each of the immunoprecipitated samples were PCR amplified for hypoxia response element (HRE) in VEGF promoter and ZEB2 promoter and E2-box region in both TRPC6 promoter and E-cadherin promoter. HRE in VEGF promoter fragment and E2-box in E-cadherin promoter serve as positive controls for HIF1α binding and ZEB2 binding respectively. Error bars indicate mean ± SE; n = 6. **p < 0.008 and ***p < 0.0004. ( B ) TRPC6 promoter activity was measured in HEK293T cells that ectopically expressing ZEB2 and exposed to FG-4592. Renilla luciferase was used as an internal control to normalize transfection efficiency. Error bars indicate mean ± SE; N = 6. ****p < 0.0001. ( C ) Intracellular free calcium levels in podocytes were measured by Fluo3-AM following treatment with FG-4592 in the presence or absence of 2-APB. Calcium levels were measured in podocytes that ectopically express ZEB2 and treated with or without 2-APB. Error bars indicate mean ± SE; n = 6. ****p < 0.0001. ( D ) Immunoblot analysis of ZEB2, TRPC6, and pFAK in podocytes treated with FG-4592 or ectopically expressing ZEB2 (ZEB2 OE). ( E ) Quantification of band intensities of ZEB2, TRPC6, pFAK was ImageJ analysis (NIH). Error bars indicate mean ± SE; n = 3. *p < 0.01,**p < 0.008, and ***p < 0.0002.

Article Snippet: Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592.

Techniques: Immunoprecipitation, Amplification, Binding Assay, Activity Assay, Expressing, Luciferase, Control, Transfection, Western Blot

Journal: Scientific Reports

Article Title: Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

doi: 10.1038/s41598-019-52872-5

Figure Lengend Snippet: Essential role of ZEB2 in regulating TRPC6 expression: ( A ) Immunoblotting analysis of ZEB2, TRPC6, and pFAK expression in podocytes expressing siZEB2 and treated with or without FG-4592. ( B ) Quantification of band intensities of ZEB2, TRPC6, pFAK was performed with ImageJ software. Error bars indicate mean ± SE; n = 3, *p < 0.01, **p < 0.008, and ***p < 0.0002. ( C ) Immunoblotting analysis of TRPC6 and pFAK expression in podocytes in which TRPC6 expression was knocked-down and treated with or without FG-4592. ( D ) Quantification of band intensities of western blots was performed with ImageJ. Error bars indicate mean ± SE; n = 3, **p < 0.008 and ****p < 0.0001. ( E ) Immunoblotting analysis of TRPC6, pFAK, and RhoA expression in podocytes exposed to FG-4592 and treated with or without 2APB and FAKI14. ( F ) Quantification of band intensities of western blots was performed with ImageJ. Error bars indicate mean ± SE; n = 3, *p < 0.01 and ****p < 0.0001.

Article Snippet: Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592.

Techniques: Expressing, Western Blot, Software

Journal: Scientific Reports

Article Title: Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

doi: 10.1038/s41598-019-52872-5

Figure Lengend Snippet: HIF1α-ZEB2-TRPC6 axis regulates podocyte cytoskeleton reorganization: ( A ) Phalloidin staining was performed to visualize intracellular F-actin stress fibers (SFs) in podocytes. Podocytes treated with FG-4592 (ii) compromised their morphology and SFs arrangement compared with cells naïve to FG-4592 (i). Ectopic expression of ZEB2 with and without FG-4592 treatment also resulted in an altered SFs arrangement (iii & iv). Calcium channel blocker; 2APB or FAK inhibitor; FAKI14 (v & vi) ameliorated FG-4592 induced SFs reorganization. The scale bar represents images of 50 µm and images were captured with a 63x objective of Leica trinocular microscope. ( B ) The percentage of stress fibers containing podocyte cells were quantified and analyzed by Image-J (NIH). Error bars indicate mean ± SE; n = 10 podocyte cells in each condition. ****p < 0.0001. ( C ) Quantification of albumin influx across podocyte monolayer. Podocytes transduced with or without ZEB2 were grown as a monolayer on cell culture insert and allowed to differentiate. Differentiated podocytes were exposed to FG-4592 for 24 h and treated with FAKI14 or 2APB and albumin influx assay was performed as described in methods. Error bars indicate mean ± SE; n = 3. ****p < 0.0001.

Article Snippet: Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592.

Techniques: Staining, Expressing, Microscopy, Transduction, Cell Culture

Journal: Scientific Reports

Article Title: Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

doi: 10.1038/s41598-019-52872-5

Figure Lengend Snippet: Proposed model for ischemic-hypoxia mediated podocyte injury. Ischemia-stroke rats develop systemic hypoxia that induces HIF1α accumulation in several susceptible sites including glomerular podocytes. HIF1α drives ZEB2 expression, which in turn induces TRPC6 expression. Elevated TRPC6 increases intracellular calcium levels and calcium-dependent phosphorylation of FAK elicits cytoskeletal rearrangements. These cytoskeletal rearrangements eventually manifest in the effacement of podocyte foot-processes and increased permeability to proteins and large molecules. The overactivity of the HIF1α/ZEB2/TRPC6 axis in podocytes elicits cytoskeletal abnormalities and proteinuria.

Article Snippet: Images were acquired using a Zeiss 100x objective. ( G ) Elevated expression of ZEB2 and TRPC6 in human podocytes treated with FG-4592.

Techniques: Expressing, Phospho-proteomics, Permeability

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Immunoblot detection of APOL1 in podocytes treated in the absence (-INFγ, rightmost lane) or presence of INFγ (Ctrl, leftmost lane). APOL1 is not induced by IFNγ in three clonal populations derived from Celprogen human primary podocytes (C1KO, C2KO, C3KO) in which the APOL1 gene was subjected to CRISPR-mediated inactivation. β-actin served as loading control. B. Preservation of differentiated phenotype in Celprogen podocytes. Upper panels: immunoblot of nephrin in lysates of HEK-293 cells transiently overexpressing nephrin (lane 1), in SV40LgT-immortalized human podocytes (lane 2), in Celprogen primary human podocytes (lane 3) and in podocyte outgrowths from human glomeruli. Lower panels: immunoblots of WT1 and podocin in HEK-293 cells transiently overexpressing WT1 or podocin (lane 1), in SV40LgT-immortalized human podocytes (lane 2) and in Celprogen primary human podocytes (lane 3). β-actin served as loading control for both upper and lower panels. C. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right)) recorded from a representative human primary podocyte incubated 18 h in the absence (lower trace) or presence of IFNγ (10 ng/mL, upper trace). D. Whole cell IV curve (linear ramp from +100 (at left) to −100 mV (at right), recorded first in the absence (red trace) and then after 2 min in the presence of anti-APOL1 aa 1–238 antibody (black trace +Ab), and finally after 3 min of antibody washout (black trace “washout”). Seal resistances at the end of each period were 9.7 GΩ (-Ab), 8.3 GΩ (+Ab) and 4.0 GΩ (washout). E. Capacitance-normalized whole cell currents measured at −60 mV in normal human primary podocytes incubated 24 h in the absence (n=11, left three datasets) or presence of 10 ng/ml IFNγ (n=19, right three datasets); *, p<0.016 vs. minus IFNγ. Each cell was recorded first in the absence and then in the subsequent presence of anti-APOL1 N-terminal domain antibody for 2 min (2 μg/ml, +Ab; n=6 −IFNγ and n=11 +IFNγ), followed by 3 min antibody washout (w/o, n=3 −IFNγ and n 5 +IFNγ). Among the 19 IFNγ-pretreated cells, open circles represent cells for which patch seals did not survive the antibody exposure, black circles connected by black lines depict cells for which patch seals did not survive antibody washout, and black circles connected by red lines show cells for which patch seals survived all three recording conditions. #, p= 0.135, vs. −IFNγ before antibody; ##, p=0.008 vs. +IFNγ before antibody; one-way ANOVA on ranks with Dunnett’s post-test.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Western Blot, Derivative Assay, CRISPR, Preserving, Incubation

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. Representative on-cell current traces measured at −Vp = +50 mV in podocytes previously treated 24 h with vehicle (upper trace) or with IFNγ (10 ng/mL, lower trace). B. I-V curve of the predominant unitary conductance class (28 pS) of on-cell patch currents in a representative IFNγ-treated Celprogen human podocyte. C. NPo of predominant unitary current class in native Celprogen podocytes (WT, middle pair of bars) and in two Celprogen knockout cell lines (KO #1 and KO #2), each treated 24 h in the absence (left unfilled bars of each pair) and presence of IFNγ (right bars of each pair; data points overlap in all bars). Values are means ± s.e.m. for indicated (n). *, p<0.05 for WT+IFNγ vs −IFNγ, and for WT+IFNγ vs each KO+IFNγ; unpaired two-way t-tests.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Knock-Out

Journal: Pflugers Archiv : European journal of physiology

Article Title: Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes

doi: 10.1007/s00424-022-02767-8

Figure Lengend Snippet: A. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes treated without (n=14, white bar, data points overlap) or with 2 μg/ml anti-APOL1 N-terminal domain antibody (n=7, black bar, data points overlap) or nonspecific rabbit IgG (n=6, gray bar) in the pipette solution. Steady-state values at 2–3 min after achievement of seal. *, p=0.003 vs “none”; unpaired two-tailed t-test. B. NPo of unitary currents measured at −Vp = +50 mV in IFNγ-pretreated Celprogen podocytes (n=6) exposed to 3 μM recombinant N-terminal fragment of Serum Response-Associated (SRA) of Trypanosoma brucei rhodesiense in the pipette solution, and recorded immediately after establishment of a gigohm seal (gray bar) and ~110 s after seal establishment (black bar; data points overlap). *, p<0.041 vs ~0 sec; unpaired two-tailed t-test. Gigohm resistances were maintained throughout the recording periods.

Article Snippet: Human primary podocytes (Celprogen, Torrance, CA) were grown per supplier’s recommendations in Celprogen human podocyte primary cell culture complete growth medium and subcultured every 48 h on Celprogen podocyte cell culture extracellular matrix.

Techniques: Transferring, Two Tailed Test, Recombinant