Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Schematic overview of the workflow for the characterization of anti-nephrin autoantibodies . Panel A. Paired serum and urine samples from 20 healthy donors, 100 NS patients were analyzed using an ELISA assay to detect anti-nephrin autoantibodies. Panel B. The same paired samples were analyzed using a pull-down assay to verify whether the identified anti-nephrin positive samples could also immunoprecipitate the extracellular domain of human nephrin. Panel C. The fucose content of IgG from the same serum samples was measured using an ELISA assay using the fucose-binding lectins. INS, Idiopathic nephrotic syndrome; FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; MN, membranous nephropathy; LN, lupus nephritis; SDNS, steroid-dependent NS; MDNS, multidrug-dependent NS; MRNS, multidrug-resistant NS.

Article Snippet: Positive and negative controls included 20 ng of sheep anti-human nephrin IgG (R&D Systems Cat# AF4269) and rabbit anti-human albumin IgG (Merck Cat# A3293), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Pull Down Assay, Binding Assay

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Circulating antibodies against the extracellular domain of nephrin in idiopathic nephrotic syndrome (INS) patients . Panel A shows the levels of circulating anti-nephrin antibodies measured by ELISA in 10 healthy donors and 100 INS patients stratified based on biopsy. FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; MN, membranous nephropathy; LN, lupus nephritis. Additionally, in patients with FSGS and MCD, anti-nephrin levels were further stratified based on the type of INS. SDNS, steroid-dependent NS; MDNS, multidrug-dependent NS; MRNS, multidrug-resistant NS. Panel B shows the titer of immunoprecipitating anti-nephrin antibodies in the serum and urine of the same subjects as in panel A. Panel C shows the prevalence of circulating anti-nephrin autoantibodies in the investigated cohorts of children and young adults with INS, with red and light gray colors representing positive and negative percentages, respectively. Panel D shows the prevalence of circulating anti-nephrin autoantibodies in FSGS and MCD samples stratified based on their pharmacological response. The dotted line indicates the statistically significant threshold.

Article Snippet: Positive and negative controls included 20 ng of sheep anti-human nephrin IgG (R&D Systems Cat# AF4269) and rabbit anti-human albumin IgG (Merck Cat# A3293), respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Correlation between anti-nephrin antibody titers and urinary proteinuria in patients with anti-nephrin-associated MCD or FSGS . Panels A and B show the correlation between anti-nephrin antibody titers in serum, measured by ELISA (A) or immunoprecipitation (B), and 24-h proteinuria. Panels C and D show the corresponding correlation between urinary anti-nephrin antibody titers, measured by ELISA (C) or immunoprecipitation (D), and proteinuria. R = Spearman's correlation coefficient.

Article Snippet: Positive and negative controls included 20 ng of sheep anti-human nephrin IgG (R&D Systems Cat# AF4269) and rabbit anti-human albumin IgG (Merck Cat# A3293), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Evaluation of fucose in IgG by enzyme-linked immunosorbent assay (ELISA) with biotinylated lectins. Panels A and B show the levels of core-fucosylated IgG (α1,6-linked fucose) and antennary-fucosylated IgG (α1,2-linked fucose), respectively, measured using AAL and UEA-I lectins in the serum of healthy donors and NS patients, stratified by circulating anti-nephrin autoantibodies. Panels C and D display the inverse correlation between 24-h proteinuria and serum IgG fucosylation levels, as measured by AAL (Panel C) and UEA-I (Panel D), in patients with anti-nephrin–associated MCD or FSGS. Panels E and F show the corresponding levels of core and antennary fucosylation in IgG from urine samples, measured by AAL and UEA-I, and stratified by anti-nephrin antibody status. Red dots indicate patients positive for anti-nephrin autoantibodies, as confirmed by both ELISA and immunoprecipitation.

Article Snippet: Positive and negative controls included 20 ng of sheep anti-human nephrin IgG (R&D Systems Cat# AF4269) and rabbit anti-human albumin IgG (Merck Cat# A3293), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Journal of the American Society of Nephrology

Article Title: Podocyte-Specific Overexpression of the Antioxidant Metallothionein Reduces Diabetic Nephropathy

doi: 10.1681/asn.2007080967

Figure Lengend Snippet: Figure 4. Altered expression of slit diaphragm proteins in isolated glomeruli. (A) Nephrin, podocalyxin, and GAPDH protein in glomeruli; FVB (F), OVE26 (O), and OVE26Nmt7 (ON). (B and C) Expression of nephrin (B) and podocalyxin (C), normalized to GAPDH expression. OVE26 and OVE26Nmt7 are always less than FVB (*) and podocalyxin OVE26Nmt7 is greater than OVE26 (**) (P 0.05 by Kruskal Wallis ANOVA).

Article Snippet: Antibodies used were nephrin (1:1000, guinea pig; Fitzgerald, Concord, MA), podocalyxin (1:3000; Alpha Diagnos- tic Int.

Techniques: Expressing, Isolation

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice

doi: 10.2147/dddt.s170840

Figure Lengend Snippet: Figure 3 Effect of AS-IV on miR-21 overexpression-induced podocyte dedifferentiation and MC activation. (A–D) Real-time PCR and Western blot results showed that miR-21 overexpression decreased nephrin expression and increased α-SMA expression in podocyte. AS-IV treatment reversed this effect. (E and F) Real-time PCR and Western blot results showed that AS-IV treatment decreased the levels of α-SMA in miR-21-overexpression-stimulated MCs. Data are presented as mean ± SD. n=3. *Compared with NC, P,0.05; #compared with miR-21 mimics group, P,0.05. Abbreviations: α-SMA, α-smooth muscle actin; AS-IV, astragaloside IV; DAPI, 4′,6-diamidino-2-phenylindole; DKD, diabetic kidney disease; HG, high glucose; MC, mesangial cell; miR-21, microRNA-21; NC, normal control.

Article Snippet: Rabbit monoclonal anti-nephrin antibody was provided by Novus Biologicals (Littleton, CO, USA). miR-21 mimics and negative controls were synthesized by Ribobio (Guangzhou, China). cell culture As previously described, primary podocytes were obtained from male C57BL/6J mice (30–40 g).18 Under brief diethyl ether anesthesia, mice kidneys were excised.

Techniques: Over Expression, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice

doi: 10.2147/dddt.s170840

Figure Lengend Snippet: Figure 4 Effect of AS-IV on the expression of nephrin and α-SMA in vivo. Immunofluorescence images showed that AS-IV increased the expression of nephrin and decreased the expression of α-SMA in glomerulus (white arrows) compared with the untreated DKD mice. Abbreviations: α-SMA, α-smooth muscle actin; AS-IV, astragaloside IV; DKD, diabetic kidney disease.

Article Snippet: Rabbit monoclonal anti-nephrin antibody was provided by Novus Biologicals (Littleton, CO, USA). miR-21 mimics and negative controls were synthesized by Ribobio (Guangzhou, China). cell culture As previously described, primary podocytes were obtained from male C57BL/6J mice (30–40 g).18 Under brief diethyl ether anesthesia, mice kidneys were excised.

Techniques: Expressing, In Vivo, Immunofluorescence

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice

doi: 10.2147/dddt.s170840

Figure Lengend Snippet: Figure 7 Effect of the Wnt/β-catenin pathway and the TGF-β1/Smads pathway on podocyte dedifferentiation and MC activation. (A–C) Western blot results showed that both SB431542 and XAV-939 decreased nephrin expression in podocytes and increased the expression of α-SMA in podocytes and MCs co-treated with miR-21 and AS-IV. Data are presented as mean ± SD. n=3. *Compared with co-treatment with miR-21 mimics and AS-IV group, P,0.05. Abbreviations: α-SMA, α-smooth muscle actin; AS-IV, astragaloside IV; MC, mesangial cell; miR-21, microRNA-21; TGF, transforming growth factor.

Article Snippet: Rabbit monoclonal anti-nephrin antibody was provided by Novus Biologicals (Littleton, CO, USA). miR-21 mimics and negative controls were synthesized by Ribobio (Guangzhou, China). cell culture As previously described, primary podocytes were obtained from male C57BL/6J mice (30–40 g).18 Under brief diethyl ether anesthesia, mice kidneys were excised.

Techniques: Activation Assay, Western Blot, Expressing

Journal: International Journal of Nephrology

Article Title: Urinary Nephrin: A Potential Biomarker of Early Glomerular Injury in a Cohort of Pregnant Women Attending Routine Antenatal Care Services

doi: 10.1155/2024/9089557

Figure Lengend Snippet: Association of maternal urinary and serum indices with albuminuria categories.

Article Snippet: The urinary nephrin was quantified using a human nephrin ELISA kit (Elabscience Biotech Co. Ltd., Wuhan, Hubei Province, China).

Techniques:

Journal: International Journal of Nephrology

Article Title: Urinary Nephrin: A Potential Biomarker of Early Glomerular Injury in a Cohort of Pregnant Women Attending Routine Antenatal Care Services

doi: 10.1155/2024/9089557

Figure Lengend Snippet: Sensitivity, specificity and reliability of urinary nephrin for determining glomerular injury in (A) all pregnant women, (B) normal control group and (C) those with comorbidities.

Article Snippet: The urinary nephrin was quantified using a human nephrin ELISA kit (Elabscience Biotech Co. Ltd., Wuhan, Hubei Province, China).

Techniques: Control

Journal: International Journal of Nephrology

Article Title: Urinary Nephrin: A Potential Biomarker of Early Glomerular Injury in a Cohort of Pregnant Women Attending Routine Antenatal Care Services

doi: 10.1155/2024/9089557

Figure Lengend Snippet: Predictive performance of urinary nephrin and ACR for PE (PE: N = 26 vs. non-PE: N = 247).

Article Snippet: The urinary nephrin was quantified using a human nephrin ELISA kit (Elabscience Biotech Co. Ltd., Wuhan, Hubei Province, China).

Techniques:

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: Evaluation of the effects of lack of APOL1 and Nephrin on the stability of adherens complex. ( A ) DPDS were transfected with either scrambled (SCR), APOL1- or nephrin-siRNAs (n = 10). Protein blots of control ( C ), DPD-transfected with SCR/SiRNA nephrin/siRNA APOL1 were probed for CD2AP, nephrin, APOL1, dendrin, and CTSL (n = 10). The same blots were reprobed for GAPDH. Gels from three different cellular lysates are displayed. ( B – F ) Cumulative densitometric data for each variable (protein/GAPDH) are shown as dot plots. ***p < 0.001 vs respective control and SCR; ### p < 0.001 vs. respective control and SCR. For clarity lanes were cropped from different gels. Full-length blots are presented in Fig. .

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Transfection, Control

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: Effect of Ectopic overexpression or silencing of APOL1 on the stability of adherens complexes. ( A ) PDs stably expressing vector (PDV) and ectopic APOL1G0 (PDG0) were differentiated for ten days and the transfected with either scrambled or APOL1 siRNA (n = 5). Protein blots were probed for APOL1, CD2AP, CTSL, and dynamin and reprobed for GAPDH. The Gels from two different lysates are displayed. ( B ) Protein blots from the above lysates were probed for nephrin and dendrin and reprobed for GAPDH. The gels from two different lysates are shown. ( C – H ) Densitometric data for each variable (protein/GAPDH) are shown as dot plots. ( C ) *P < 0.05 compared with V/SCR; ***P < 0.001 compared with V/SCR; ### P < 0.001 compared with G0/SCR. ( D ) **P < 0.01 compared with V/SCR; ### P < 0.001 compared with G0/SCR. ( E ) *P < 0.0.5 compared with V/SCR; **P < 0.01 compared with V/SCR; ### P < 0.001 compared with G0/SCR. ( F ) *P < 0.05 compared with V/SCR; **P < 0.01 compared with V/SCR; ### P < 0.001 compared with G0/SCR. ( G ) **P < 0.0a compared with V/SCR; ### P < 0.001 compared with G0/SCR. For clarity lanes were cropped from different gels. Full-length blots are presented in Fig. .

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Over Expression, Stable Transfection, Expressing, Plasmid Preparation, Transfection

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: APOL1 risk alleles destabilize the adherens complex. ( A ) Protein blots of DPDs stably over-expressing Vector (V), G0, G1, and G2 were probed for APOL1, nephrin, CD2AP, dynamin, CTSL, and GAPDH (n = 5–7). Representative gels are displayed. ( B ) Protein blots of the above lysates were probed for dendrin and reprobed for GAPDH (n = 5). ( C – H ) Cumulative densitometric data on each variable (protein/GAPDH) are shown as dot plots. *<0.05 compared with respective V; **P < 0.01 compared with respective V; † P < 0.05 compared with respective G0; ‡ P < 0.01 compared with respective G1 and G2. ( I ) PDs expressing vector, G0, G1, and G2 grown on coverslips were differentiated and labeled for dendrin (n = 3). Nuclei were stained with DAPI. Cells were imaged under a confocal microscope. Representative fluoromicrographs are displayed. DPDs expressing vector and G0 showed predominantly cytosolic dendrin (indicated by green fluorescence). However, DPD expressing G0 and G1 showed predominantly nuclear localization of dendrin. Horizontal solid white bar represents the scale as 100 µM. ( J ) PDs expressing vector, G0, G1, and G2 grown on coverslips were differentiated (n = 3). Subsequently, cells were co-labeled for F-actin (by phalloidin) and dynamin. Nuclei were stained with DAPI. Cells were examined under a confocal microscope. Representative fluoromicrographs are shown. DPDs expressing vector and G0 displayed well- spread and intact actin filaments (indicated by red fluorescence). DPDs expressing G1 and G2 displayed predominantly cortical labeling for F-actin. DPDs expressing vector and G0 displayed moderate expression of dynamin (indicated by green fluorescence). DPDs expressing G1 and G2 displayed minimal expression of dynamin. Horizontal solid white bar represents the scale as 50 µm. ( K ) The cortical F-actin score (CFS) index was calculated under control and experimental conditions. DPDAPOLG1 (G1) and DPDAPOL1G2 (G2) displayed an increased CFS index compared with DPDV (Vector) and DPDAPOL1G0 (G0). ***P < 0.001 compared with V and G0; ### P < 0.001 compared to V and G0. For clarity lanes were cropped from different gels. Full-length blots are presented in Fig. .

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Labeling, Staining, Microscopy, Fluorescence, Control

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: APOL1 binds with one of the constituents of adherens complex. ( A ) Cellular lysates of DPDVs (DPDs expressing vector) and DPDG0s (DPDs expressing APOL1 G0) were probed for APOL1, CD2AP, dendrin, nephrin, and reprobed for actin (n = 3). Gels from three different lysates are displayed. ( B ) Cumulative densitometric data from protein blots displayed in 5A. **P < 0.01 compared with respective vector variables. ( C ) The cellular lysates of DPDVs and DPDG0s in the 5A were immunoprecipitated (IP) with an anti-APOL1 antibody (n = 3). IP fractions were probed for APOL1, CD2AP, dendrin, nephrin, and IgG. Gels from three different fractions are displayed. ( D ) Cumulative densitometric data from protein blots displayed in 5C. **P < 0.01 compared to respective Vec. ( E ) The cellular lysates of DPDVs, DPDG0s, DPDG1s, and DPDG2s were probed for APOL1, nephrin, CD2AP, CTSL, dynamin, and GAPDH (n = 3). Gels from three different lysates are displayed. ( F ) Cumulative densitometric data from protein blots displayed in 5E. *P < 0.05 compared to respective Vec, G1, and G2. ( G ) The cellular lysates in the 5E were immunoprecipitated with an anti-CD2AP antibody. IP fractions of three different lysates were probed for APOL1, CD2AP, dendrin, nephrin, and IgG. Gels from there different IP fractions have been displayed. ( H ) Cumulative densitometric data from protein blots displayed in 5G. *P < 0.05 compared to respective Vec, G1, and G2. ( I ) The cellular lysates of DPDVs, DPDG0s, DPDG1s, and DPDG2s in the 5E were immunoprecipitated (IP) with a non-specific mouse IgG (n = 3). IP fractions were probed for APOL1, CD2AP, dendrin, nephrin, and IgG. Gels from three different fractions are displayed. For clarity lanes were cropped from different gels. Full-length blots are presented in Fig. .

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: APOL1 risk alleles induce disruption in APOL1-miR193a axis. ( A ) RNAs were extracted from DPDs expressing vector, G0, G1, and G2 (n = 3). RNAs were assayed for miR193a. Cumulative data are shown in bar graphs. *P < 0.05 compared with V; **P < 0.01 compared with all other variables. ( B ) Protein blots of DPDs expressing vector, G0, G1, and G2 were probed for nephrin and reprobed for actin (n = 3). Gels from two different lysates are displayed in the upper panel. Cumulative densitometric data are shown as bar graphs in the lower panel. *P < 0.05 compared with V; **P < 0.01 compared with all other variables. ( C ) DPDs were transfected with either empty vector (25 nM, EV) or mir193a plasmid (25 nM). Protein blots of control and experimental DPDs (transfected with either EV or miR193a plasmid) were probed for APOL1, WT1, nephrin, CD2AP and reprobed for GAPDH. Gels from three different lysates are shown. ( D ) The lysates in 6C were probed for CTSL and dynamin and reprobed for GAPDH. Gels from three different lysates are shown. ( E ) Cumulative densitometric data (from 6C) are shown in bar graphs. **P < 0.01 compared with respective control and EV; ***P < 0.001 compared with respective control and EV. ( F ) Cumulative densitometric data (from D) are shown in bar graphs. **P < 0.01 compared with respective control and EV; ***P < 0.001 compared with respective control and EV. For clarity lanes were cropped from different gels. Full-length blots are presented in Fig. .

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Disruption, Expressing, Plasmid Preparation, Transfection, Control

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: VDA down regulates PAN-induced DPD expression of miR193a. ( A ) DPDs were transfected with either empty vector (EV) or miR193a inhibitor (plasmid, 25 nM), or treated with VDA (EB1089, 10 nM) in the presence or absence of PAN (30 ng/ml) for 48 hours (n = 3). RNAs were extracted and assayed for miR193a. Cumulative data are shown in bar graphs. *P < 0.05 compared with respective EV. **P < 0.01 compared with untreated EV; # P < 0.01 compared with PAN-treated EV. ( B ) DPDs were incubated in media containing either buffer (Control, C), vehicle (Veh, DMSO), or PAN (30 ng/ml) with or without VDA (EB1089, 10 nM) for 48 hours (n = 3). Protein blots were probed for CD2AP, nephrin, APOL1, dynamin, and CTSL. The same blots were reprobed for GAPDH. Gels from three different lysates are shown. ( C ) The lysates in 8B were probed for dendrin (n = 3). Gels from three different lysates are displayed. ( D ) Cumulative densitometric data from the blots of 8B are shown in bar graphs. *P < 0.05 compared to respective C and Veh; **P < 0.05 compared to respective C and Veh; # P < 0.05 vs. PAN alone; ## P < 0.01 vs. PAN alone; ### P < 0.001 vs. PAN alone. ( E ) Cumulative densitometric data from the blots of 8C are shown in bar graphs. For clarity lanes were cropped from different gels. Full-length blots are presented in Fig. .

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Control

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: Nuclear import of dendrin in podocytes in APOL1G1 transgenic mice. ( A ) Renal cortical sections of FVB/N, APOL1G0 (G0), and APOL1G1 (G1) transgenic mice (n = 4) were labeled for nephrin, APOL1, and dendrin. Representative microphotographs are displayed. Podocytes of an FVB/N mouse showed cytosolic labeling for nephrin (purple fluorescence) and dendrin (green fluorescence). Tubular cells also showed labeling for dendrin. Podocytes in an APOL1G0 transgenic mouse showed cytosolic labeling for APOL1 (red fluorescence), nephrin (purple fluorescence), and dendrin (green fluorescence). Tubular cells also showed labeling for APOL1 (red fluorescence). On the other hand, an APOLG1 transgenic mouse showed predominantly nuclear import of dendrin (green fluorescence, indicated by white arrows). Original Mag.X400. ( B ) A pipeline of modules of Broad Institute’s CellProfiler suite was used to analyze dendrin expression in podocytes. Representative original images of FVB/N, APOL1G0 and APOL1G1 glomeruli showing expression of nephrin (purple) dendrin (green) and DAPI (blue) were captured using Slidebook 6.0 software. Images were then processed using Cell Profiler pipeline to analyze expression of dendrin in cytosol and nucleus. Dendrin expressing pixels co-labeled with either nephrin or DAPI were measured and the total area occupied was calculated. Processed images are showing randomly denoted colors of the area occupied by pixels expressing dendrin and nephrin or dendrin and DAPI. Mag.X400. ( C ) Randomly selected glomeruli from FVB/N, APOL1G0 and APOL1G1 mice (Fig. 9B) were analyzed and data of the area occupied by pixels expressing dendrin and DAPI was collected. A bar graph is showing the number of pixels co-expressing dendrin and DAPI. ( D ) Randomly selected glomeruli from FVB/N, APOL1G0 and APOL1G1 mice (Fig. 9B) were analyzed and data of the area occupied by pixels expressing dendrin and nephrin was collected. A bar is showing the number of pixels co-expressing dendrin and nephrin.

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Transgenic Assay, Labeling, Fluorescence, Expressing, Software

Journal: Scientific Reports

Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

doi: 10.1038/s41598-019-39376-y

Figure Lengend Snippet: Proposed hypothetical schemes. ( A ) APOL1G0 preserves actin cytoskeleton through down-regulation of miR193a and stimulation of nephrin expression, one of the constituents of the adherens complex. ( B ) APOL1G1 and APOL1G2 disrupt APOL1-miR193a axis and induce upregulation of miR193a resulting down-regulation of WT1 and associated attenuated expression of nephrin, one of the constituents of the complex.

Article Snippet: DPDs were transfected with scrambled siRNA (control), APOL1 siRNA (20 nM; Santa Cruz), or nephrin siRNA (25 nM; Santa Cruz) with Lipofectamine RNAiMAX transfection reagent according to the manufacturer’s protocol (Thermo Fisher).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Glomerular Damage in Trichloroethylene-Sensitized Mice: Targeting Cathepsin L-Induced Hyperactive mTOR Signaling

doi: 10.3389/fphar.2021.639878

Figure Lengend Snippet: Loss of podocytes occurred in OMDT patients and TCE-sensitized mice. (A) Expression of urine nephrin before and after clinical treatment in OMDT patients ( n = 6). (B–D) Representative results of immunofluorescence test in mice ( n = 5 per group). (B1–B4) Expression of nephrin in blank control group (B1) , vehicle control group (B2) , TCEpos group (B3) , and TCEneg group (B4) . (C1–C4) Expression of integrin β1 in blank control group (C1) , vehicle control group (C2) , TCEpos group (C3) , and TCEneg group (C4) . (D1–D4) Expression of α-actinin-4 in blank control group (D1) , vehicle control group (D2) , TCEpos group (D3) , and TCEneg group (D4) . (E-G) The fluorescence intensity of nephrin, integrin β1, and α-actinin-4 calculated in Image J software. Data are presented as mean ± SD and determined by paired t -test or one-way ANOVA, * p < 0.05, ns P >0.05; a P < 0.05, vs. blank control group; b P <0.05, vs. vehicle control group; c P <0.05, vs. TCEneg group.

Article Snippet: The human nephrin DuoSet ELISA kit was obtained from R&D System (Minneapolis, United States).

Techniques: Expressing, Immunofluorescence, Control, Fluorescence, Software

Journal: Drug Design, Development and Therapy

Article Title:

Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling

doi: 10.2147/dddt.s234262

Figure Lengend Snippet: Figure 5 TRPC6 activates RhoA/ROCK1 signaling. (A) Analysis ofof TRPC6, RhoA and ROCK1 mRNA by RT-qPCR analysis. (B) Analysis of TRPC6, RhoA-GTP/RhoA and ROCK1, nephrin and synaptopodin protein levels by Western blot. Differences were analyzed using one-way ANOVA. Significant differences with p < 0.05 are indicated by different letters. NC: containing blank lentivirus vector. blank:normal MPC5 podocyte.TRPC6: TRPC6-overexpressing.

Article Snippet: PVDF membranes were blocked with skim milk powder, and incubated with primary antibodies. against TRPC6 (1: 1000, Santa Cruz), Synaptopodin (1: 100; Santa Cruz), Nephrin (1: 100; Proteintech), ROCK1 (1: 1000; Proteintech), anti-RhoA antibody (1: 1000; Proteintech, Wuhan, China), and β-actin (1: 1000; Santa Cruz) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Plasmid Preparation