nephrin  (Bioss)

Journal: Frontiers in Pharmacology

Article Title: Hyperoside ameliorates lupus nephritis by suppressing AKT1-mediated PANoptosis in podocytes: integrating network pharmacology and experimental validation

doi: 10.3389/fphar.2025.1726254

Figure Lengend Snippet: PANoptosis contributes to renal pathology in lupus nephritis. (A) Comparison of 24-h urinary protein levels between MRL/MpJ and MRL/lpr mice (n = 6). (B) Representative renal histology evaluated by H&E staining (n = 6). (C) TUNEL staining showing cell death in glomeruli (n = 6). (D) IHC analysis of podocyte markers (Podocalyxin, Nephrin, Synaptopodin, Podocin) in renal tissues. (E–G) Immunofluorescence staining of PANoptosis-related markers (NLRP3, RIPK3, Caspase-9) in kidney sections. (H) WB analysis of PANoptosis executor proteins (caspase-3, caspase-1, p-MLKL) in renal tissues, with bar graphs showing quantitative results relative to GAPDH (n = 3). (I) IF triple-staining of caspase-3, caspase-1, and p-MLKL in mouse kidney tissues. (J) Viability of MPC-5 cells treated with ICs at different concentrations and time points, measured by CCK-8 assay. (K) Flow cytometric analysis of cell death in MPC5 cells pretreated with VX-765 (10 μM), Nec-1 (20 μM), or Z-VAD (5 μM), followed by IC stimulation for 24 h. Bar graph shows the percentage of dead cells (n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: Sequential sections (4 μm) were used for the following staining and analyses: Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes in renal tissues; immunohistochemistry (IHC) was conducted to examine the expression of synaptopodin (21064-1-AP, Proteintech, USA), nephrin (bs-10233R, Bioss, China), podocalyxin (bs-1345R, Bioss, China), and podocin (bs-6597R, Bioss, China); immunofluorescence (IF) staining was used to detect NLRP3 (DF7438, Affinity, UK), RIPK3 (bs-3551R, Bioss, China), and Caspase-9 (bs-0049R, Bioss, China) expression; and a TUNEL assay kit (E-CK-A320, Elabscience, China) was employed to assess cell death.

Techniques: Comparison, Staining, TUNEL Assay, Immunofluorescence, CCK-8 Assay

Journal: Frontiers in Pharmacology

Article Title: Hyperoside ameliorates lupus nephritis by suppressing AKT1-mediated PANoptosis in podocytes: integrating network pharmacology and experimental validation

doi: 10.3389/fphar.2025.1726254

Figure Lengend Snippet: AKT1 mediates podocyte PANoptosis and LN progression. (A–E) MRL/lpr mice were injected via tail vein with KD-NC or KD-AKT1 AAV and maintained for 6 weeks (n = 6). (A,B) Western blot analysis of AKT1 and PANoptosis markers (p-MLKL, Caspase-1, Caspase-3) in renal tissues (n = 3). (C) Renal pathology assessed by H&E staining. (D) Immunohistochemical staining of podocyte-associated proteins (Podocalyxin, Nephrin, Synaptopodin, Podocin). (E) Comparison of 24 h urinary protein levels between the two groups. (F–P) MPC-5 cells were transfected with Si-NC, Si-AKT1, OE-NC, or OE-AKT1, followed by IC treatment for 24 h. (F) Percentage of dead cells measured by flow cytometry (n = 6). (G) Representative flow cytometry plots showing apoptosis. (H–J) mRNA expression levels of PANoptosis-related genes (NLRP3, RIPK3, Caspase-9) detected by qPCR (n = 6). (K–P) Western blot analysis of PANoptosis executor proteins (p-MLKL, Caspase-1, Caspase-3, GSDMD-N, Caspase-8) in MPC5 cells (n = 3). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: Sequential sections (4 μm) were used for the following staining and analyses: Hematoxylin and eosin (H&E) staining was performed to evaluate pathological changes in renal tissues; immunohistochemistry (IHC) was conducted to examine the expression of synaptopodin (21064-1-AP, Proteintech, USA), nephrin (bs-10233R, Bioss, China), podocalyxin (bs-1345R, Bioss, China), and podocin (bs-6597R, Bioss, China); immunofluorescence (IF) staining was used to detect NLRP3 (DF7438, Affinity, UK), RIPK3 (bs-3551R, Bioss, China), and Caspase-9 (bs-0049R, Bioss, China) expression; and a TUNEL assay kit (E-CK-A320, Elabscience, China) was employed to assess cell death.

Techniques: Injection, Western Blot, Staining, Immunohistochemical staining, Comparison, Transfection, Flow Cytometry, Expressing

Journal: Renal Failure

Article Title: Aucubin ameliorates diabetic kidney disease by restoring hGENCs autophagy through promoting phosphorylation of ATG4B protein

doi: 10.1080/0886022X.2025.2605756

Figure Lengend Snippet: Protective effect of aucubin on DKD mice by promoting ATG4B protein phosphorylation and increasing autophagy. The expression levels of p-ATG4B proteins, ATG4B proteins, autophagy protein (LC3, p62, ATG5, and ATG7), apoptosis-related proteins (Bax, cleaved caspase3, and Bcl-2), EndMT marker proteins (CD31 and α-SMA), and nephrin protein in each group were detected by WB and the statistical analysis result of the proteins expression levels. *p < .05; ** p < .01. DKD: diabetic kidney disease; AU: aucubin.

Article Snippet: Proteins were transferred to polyvinylidene difluoride membranes (PVDF) and then incubated with specific primary antibodies against ATG4B, p-ATG4B(Ser383), ATG5, ATG7 (1:1,000, CST); Bcl-2, Bax (1:500, Immunoway); p62, cleaved caspase3 (1:500, Immunoway); LC3 (1:1,000, Abcam); CD31, Vimentin (1:2,000, Proteintech); nephrin (1:1,000, Affinity) and α-SMA, GAPDH(1:1,000, Proteintech), and secondary antibodies conjugated to HRP (Proteintech).

Techniques: Phospho-proteomics, Expressing, Marker

Journal: RNA Biology

Article Title: EIF4A3-induced circFAT1 promotes high glucose-induced podocyte damage via miR-30e-5p/SOX4 axis

doi: 10.1080/15476286.2025.2563865

Figure Lengend Snippet: Elevated expression of circFAT1 in HG-treated HPCs. (A) cell morphology (left) and viability measured by CCK-8 assay (right) after 48 h treatments. (B) immunofluorescence staining of Nephrin and FSP1. (C) CircFAT1 expression by qRT-PCR. (D) schematic diagram of the composition of circFAT1. (E) PCR-agarose gel electrophoresis was used to verify the back splicing sequence and RT-PCR product of circFAT1. (F) the circFAT1 and FAT1 mRNA abundance after RNase R treatment was determined via qRT-PCR. (G) RNA stability after actinomycin D (5 μg/mL) treatment. (H) nuclear and cytoplasmic fractionation showed the distribution of circFAT1 in HPCs. *** p < 0.001.

Article Snippet: The following antibodies were utilized: WT-1 antibody (#12609–1-AP, 1: 1000), Nephrin antibody (#66970–1-Ig, 1:1000), FSP1 antibody (#20886–1-AP, 1:5000) and Desmin antibody (#67793–1-Ig, 1:5000) and GAPDH antibody (#60004–1-Ig 1:10000) were obtained from Proteintech (Wuhan, China).

Techniques: Expressing, CCK-8 Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Agarose Gel Electrophoresis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Fractionation

Journal: RNA Biology

Article Title: EIF4A3-induced circFAT1 promotes high glucose-induced podocyte damage via miR-30e-5p/SOX4 axis

doi: 10.1080/15476286.2025.2563865

Figure Lengend Snippet: Depletion of circFAT1 attenuates HG-induced podocyte migration and EMT. (A) levels of circFAT1 and FAT1 in HPCs transfected with si-circFAT1. (B) the apoptosis of HPCs was determined using flow cytometry. (C) the motility of HPCs was determined using Transwell assays. (D) Western blotting demonstrated the expression of WT-1, Nephrin, FSP1, and Desmin under different conditions. (E) immunofluorescence staining of Nephrin and FSP1. Bar = 50 μm, * p < 0.05, *** p < 0.001.

Article Snippet: The following antibodies were utilized: WT-1 antibody (#12609–1-AP, 1: 1000), Nephrin antibody (#66970–1-Ig, 1:1000), FSP1 antibody (#20886–1-AP, 1:5000) and Desmin antibody (#67793–1-Ig, 1:5000) and GAPDH antibody (#60004–1-Ig 1:10000) were obtained from Proteintech (Wuhan, China).

Techniques: Migration, Transfection, Flow Cytometry, Western Blot, Expressing, Immunofluorescence, Staining

Journal: RNA Biology

Article Title: EIF4A3-induced circFAT1 promotes high glucose-induced podocyte damage via miR-30e-5p/SOX4 axis

doi: 10.1080/15476286.2025.2563865

Figure Lengend Snippet: SOX4 overexpression attenuated the protective effect of circFAT1 knockdown against HG-induced podocyte damage. (A) SOX4 mRNA expression in HPCs transfected with SOX4-expressing plasmids. *** p < 0.001. (B) SOX4 protein expression in HPCs transfected with SOX4-expressing plasmids. *** p < 0.001 (C) SOX4 protein expression in HPCs transfected with NC, si-circFAT1, si-circFAT1 + Vector or si-circFAT1 + SOX4_oe, under HG condition. (D) apoptosis by annexin V/PI staining. (E) migration by Transwell assay. (F) protein levels of podocyte (WT-1, Nephrin) and mesenchymal (FSP1, Desmin) markers. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following antibodies were utilized: WT-1 antibody (#12609–1-AP, 1: 1000), Nephrin antibody (#66970–1-Ig, 1:1000), FSP1 antibody (#20886–1-AP, 1:5000) and Desmin antibody (#67793–1-Ig, 1:5000) and GAPDH antibody (#60004–1-Ig 1:10000) were obtained from Proteintech (Wuhan, China).

Techniques: Over Expression, Knockdown, Expressing, Transfection, Plasmid Preparation, Staining, Migration, Transwell Assay