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: 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

Journal: Scientific reports

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

doi: 10.1038/srep43934

Figure Lengend Snippet: Figure 4. 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.

Article Snippet: After blocking with casein blocking buffer for two hours (Rockland Inc, Cat: MB-070), blots were incubated with specific primary antibodies against nephrin (Novus Biologicals, Cat: NBP1-30130), WT-1 (Novus Biologicals, Cat: NB120-15249), podocin (Sigma-Aldrich, Cat: P0372), CD2AP (Cell Signaling, Cat: 2135), synaptopodin (Progen, Cat: 55294), phospho-Y416-Src (Cell Signaling, Cat: 6943), phospho-T423-PAK (Cell Signaling, Cat: 2601), GAPDH (Sigma-Aldrich, Cat: G8795), and α -tubulin (Sigma-Aldrich, Cat: T5158) at 4 °C overnight.

Techniques: Functional Assay, Western Blot, Control

Journal: Scientific reports

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

doi: 10.1038/srep43934

Figure Lengend Snippet: Figure 5. Extent of mechanotransduction is extracellular matrix independent ECM coating with (A) collagen I of reduced density from 5 μg/cm2 to 1 μg/cm2: 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/cm2): 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/cm2): 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/cm2): 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).

Article Snippet: After blocking with casein blocking buffer for two hours (Rockland Inc, Cat: MB-070), blots were incubated with specific primary antibodies against nephrin (Novus Biologicals, Cat: NBP1-30130), WT-1 (Novus Biologicals, Cat: NB120-15249), podocin (Sigma-Aldrich, Cat: P0372), CD2AP (Cell Signaling, Cat: 2135), synaptopodin (Progen, Cat: 55294), phospho-Y416-Src (Cell Signaling, Cat: 6943), phospho-T423-PAK (Cell Signaling, Cat: 2601), GAPDH (Sigma-Aldrich, Cat: G8795), and α -tubulin (Sigma-Aldrich, Cat: T5158) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Two Tailed Test

Journal: Endocrinology

Article Title: CXCR4 promotes renal tubular cell survival in male diabetic rats: implications for ligand inactivation in the human kidney.

doi: 10.1210/en.2014-1650

Figure Lengend Snippet: Figure 3. CXCR4 blockade with AMD3100 exerts minimal effects on podocytes or glomerular and peritubular capillaries in rats with diabetes for 4 weeks (n 12/group except transmission EM n 4/group). A–D, Transmission electron micrographs of podocytes from (A) control PBS, (B) control AMD3100, (C) diabetes PBS, and (D) diabetes AMD3100. The images in C and D show podocytes with electron dense cytoplasm (arrows) in diabetic rats treated with either PBS or AMD3100 with no difference between the two groups. E, Podocyte foot process width. F, Percentage of podocytes displaying morphological abnormalities. G–J, Immunostaining for WT1 in kidney sections from (G) control PBS, (H) control AMD3100, (I) diabetes PBS, and (J) diabetes AMD3100. Original magnification, 400. K, WT1-positive nuclei per glomerular profile. L–O, Immunostaining for nephrin in kidney sections from (L) control PBS, (M) control AMD3100, (N) diabetes PBS, and (O) diabetes AMD3100. Original magnification, 400. P, Quantitation of glomerular nephrin immunostaining. Q–T, Immunostaining for glomerular endothelial cells with the monoclonal antibody JG-12 in kidney sections from (Q) control PBS, (R) control AMD3100, (S) diabetes PBS, and (T) diabetes AMD3100. Original magnification, 400. U, Quantitation of glomerular JG-12 immunostaining. V–Y, Immunostaining for peritubular endothelial cells with the monoclonal antibody JG-12 in kidney sections from (V) control PBS, (W) control AMD3100, (X) diabetes PBS, and (Y) diabetes AMD3100. Original magnification, 160. Z, Quantitation of peritubular JG-12 immunostaining.

Article Snippet: Immunohistochemistry Immunohistochemistry was performed on formalin-fixed paraffin-embedded kidney tissue as previously described (11, 12, 16) with antibodies in the following concentrations: CXCR4 ab7199 1:150 (Abcam, Cambridge, MA) (rat kidney sections), CXCR4 ab2074 1:50 (Abcam) (human kidney sections), SDF-1 1:25 (R&D Systems, Minneapolis, MN), WT1 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA), nephrin 1:400 (R&D Systems), CD2-associated protein (CD2AP) 1:400 (ThermoFisher Scientific Inc., Rockford, IL), podocalyxin 1:200 (Novus Biologicals, Littleton, CO), JG-12 1:1000 (Bender Medsystems GbdH, Vienna, Austria), phosphorylated serine residue 339 (S339) CXCR4 1:50 (Abcam), and matrix metalloproteinase 9 (MMP-9) 1:200 (Abcam).

Techniques: Transmission Assay, Control, Immunostaining, Quantitation Assay