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: Experimental physiology

Article Title: A PAI-1 mutant retards diabetic nephropathy in db/db mice through protecting podocytes

doi: 10.1113/expphysiol.2013.077610

Figure Lengend Snippet: Primers used for real time reverse transcriptase-PCR

Article Snippet: The rabbit polyclonal anti-nephrin antibody (TA306037) and rabbit polyclonal anti-zonula occludin-1 (ZO-1) antibody (61-7300) were obtained from OriGene Technologies and Invitrogen respectively.

Techniques: Sequencing

Journal: Experimental physiology

Article Title: A PAI-1 mutant retards diabetic nephropathy in db/db mice through protecting podocytes

doi: 10.1113/expphysiol.2013.077610

Figure Lengend Snippet: Representative photomicrographs (original magnification × 400) of the glomeruli obtained at 22 weeks from a normal control mouse (db/m), a diabetic db/db mouse treated with PBS (db/db) and a diabetic db/db mouse treated with PAI-1R (db/db-PAI-1R). Graphic representation of the mean nephrin and podocin staining scores of each group is shown on the right. *P<0.05, vs. db/m. #P<0.05, vs. db/db.

Article Snippet: The rabbit polyclonal anti-nephrin antibody (TA306037) and rabbit polyclonal anti-zonula occludin-1 (ZO-1) antibody (61-7300) were obtained from OriGene Technologies and Invitrogen respectively.

Techniques: Staining

Journal: Experimental physiology

Article Title: A PAI-1 mutant retards diabetic nephropathy in db/db mice through protecting podocytes

doi: 10.1113/expphysiol.2013.077610

Figure Lengend Snippet: Expression of mRNA in renal cortical tissues obtained from various mouse groups at 22 weeks was determined by real-time RT/PCR and corrected for β-actin for each samples. Db/m: non-diabetic normal control; db/db: diabetic db/db mouse treated with PBS; and db/db-PAI-R: diabetic db/db mouse treated with PAI-1R. A. Expression of nephrin mRNA, B. Expression of ZO-1 mRNA, C. Expression of desmin mRNA, D. Expression of B7-1 mRNA, *P<0.05, vs. db/m. #P<0.05, vs. db/db.

Article Snippet: The rabbit polyclonal anti-nephrin antibody (TA306037) and rabbit polyclonal anti-zonula occludin-1 (ZO-1) antibody (61-7300) were obtained from OriGene Technologies and Invitrogen respectively.

Techniques: Expressing, Quantitative RT-PCR

Journal: Experimental physiology

Article Title: A PAI-1 mutant retards diabetic nephropathy in db/db mice through protecting podocytes

doi: 10.1113/expphysiol.2013.077610

Figure Lengend Snippet: Quiescent mouse podocytes were incubated in the absence (normal control, Con) or the presence of TGFβ1 (TGFβ1, 2 ng/ml) or PAI-1R (150 nmol) for 24h, and western blot analysis was performed in cell lysates. A. Representative western blots showing nephrin, ZO-1 and B7-1 protein expression. β-actin was included in the blot as a protein loading control. The graphs (B-D) summarize the results of the respective band density measurements, normalized against the band intensity of β-actin. *P <0.05 vs. normal control.

Article Snippet: The rabbit polyclonal anti-nephrin antibody (TA306037) and rabbit polyclonal anti-zonula occludin-1 (ZO-1) antibody (61-7300) were obtained from OriGene Technologies and Invitrogen respectively.

Techniques: Incubation, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: MRL.lpr mice were injected i.p. with free KN93 (10 μg/wk), anti-podocin or anti-nephrin antibody–coated KN93-loaded nlg (10 μg of KN93/week), or empty nlg at from 8 to 16 weeks of age (n = 5–7 mice in each group). (A) Urine albumin/creatinine (Alb/Cre) ratio. Urine samples were obtained biweekly, and albumin and creatinine levels were determined by ELISA. Error bars represent mean ± SEM. *P < 0.05, 2-way ANOVA with Bonferroni’s post test. (B and C) Representative images of glomeruli from 16-week- old MRL.lpr mice treated with anti-nephrin antibody–coated empty nlg or KN93-loaded nlg. PAS (B) and Masson’s trichrome staining (C) are shown. The arrows point to the crescent. Scale bars: 50 μm. (D) Mean histological scores in the kidneys of mice from the indicated treatment groups (n = 5 in each group). *P < 0.05; **P < 0.01, Student’s t test. (E and F) Electron microscopic images of a glomerulus from a mouse treated with anti-nephrin empty nlg (left) showing diffuse podocyte foot process (FP) effacement with slit diaphragm occlusion (arrows) and subepithelial dense deposits (asterisks) and a mouse treated with anti-nephrin KN93-loaded nlg (right), which shows podocytes with normal foot processes and slit diaphragms and no deposits in the basement membrane. Original magnification, ×8,000 (E); ×30,000 (F). Three glomeruli were evaluated in each of 3 mice in each experimental condition. (G) C3 and IgG deposition was significantly suppressed in the glomeruli of MRL.lpr mice treated with KN93 targeted to podocytes, as indicated. Kidney sections from MRL/MpJ (16 weeks of age) mice are shown as controls (n = 5 mice in each group). (H) Nephrin (green) and synaptopodin (red) expression detected by immunofluorescence. Scale bars: 50 μm. (I) The fluorescence intensity of nephrin and synaptopodin quantified in glomeruli from mice subjected to the indicated treatments. ****P < 0.0001, Student’s t test. (J) Nphs1, Nphs2, and Synpo expression in glomeruli from mice treated with anti-nephrin–coated empty or KN93-loaded nlg. Results were normalized to the expression of GAPDH (n = 5 in each group). ****P < 0.0001, Student’s t test.

Article Snippet: Isolated cells were stained for flow cytometry with antibodies against nephrin (bs-10233R-A488, Bioss Antibodies) and podocin (bs-6597R-A647, Bioss Antibodies).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Immunofluorescence, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: Mice treated with KN93-loaded nlg targeted to podocytes and CaMK4-deficient mice develop proteinuria after exposure to LPS. Each B6 or B6 Camk4–/– mouse was injected i.p. with LPS on day 0. (A) Urine albumin/creatinine ratio of mice treated with anti-nephrin antibody–coated nlg either empty or loaded with KN93. Nlg were injected i.p. on day –1 (n = 10 mice in each group). (B) Urine albumin/creatinine ratio of B6 or B6 Camk4–/– mice (n = 6 in each group). Error bars represent mean ± SEM. *P < 0.05; **P< 0.01, 2-way ANOVA with Bonferroni’s post test. (C) Representative immunofluorescent images of nephrin (upper panels) and synaptopodin (lower panels) in the kidneys of mice injected with LPS or PBS. Scale bar: 50 μm. (D) Nphs1 expression in human podocytes after stimulation with LPS with or without KN93. Cells were treated with KN93 1 hour before stimulation. Results were normalized by the expression of GAPDH. Four independent experiments were performed. *P < 0.05, 1-way ANOVA with Tukey’s post test. (E and F) Mean percentage of nephrin-positive human podocytes evaluated by flow cytometry after stimulation with LPS for 72 hours with KN93 (E) or CAMK4 siRNA (F). Four independent experiments were performed. *P < 0.05, 1-way ANOVA with Tukey’s post test.

Article Snippet: Isolated cells were stained for flow cytometry with antibodies against nephrin (bs-10233R-A488, Bioss Antibodies) and podocin (bs-6597R-A647, Bioss Antibodies).

Techniques: Injection, Expressing, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

doi: 10.1172/JCI99507

Figure Lengend Snippet: Each mouse was injected i.v. with adriamycin (ADM) on day 0. (A–C) Anti-nephrin antibody–coated empty or KN93-loaded nlg were injected i.p. into BALB/c mice on day –1 and day 3 (n = 7 in each group). (A) Mean urine albumin/creatinine ratio from mice subjected to the indicated treatment. Error bars represent mean ± SEM. ****P < 0.0001, 2-way ANOVA with Bonferroni’s post test (B) Representative images showing PAS staining of kidney from BALB/c mice treated with anti-nephrin antibody–coated empty nlg or KN93-loaded nlg. Scale bar: 50 μm. (C) Representative immunofluorescence images of nephrin and synaptopodin expression in glomeruli. Scale bar: 100 μm. (D) Free KN93 (10 μg/wk), anti-nephrin antibody–coated empty, or KN93-loaded nlg (10 μg of KN93/wk) were injected i.p. into BALB/c mice on day 7 (n = 5 in each group). **P < 0.01; ***P < 0.001, 2-way ANOVA with Bonferroni’s post test. (E) Mean urine albumin/creatinine ratio of B6 or B6 Camk4–/– mice treated with adriamycin. (n = 5 in each group; 2 independent experiments were performed). ****P < 0.0001, 2-way ANOVA with Bonferroni’s post-test. (F) Representative electron microscopy images of glomeruli from BALB/c mice at 7 days after exposure to adriamycin, treated with anti-nephrin antibody–coated empty nlg (left) or KN93-loaded nlg (right). Original magnification, ×8,000.

Article Snippet: Isolated cells were stained for flow cytometry with antibodies against nephrin (bs-10233R-A488, Bioss Antibodies) and podocin (bs-6597R-A647, Bioss Antibodies).

Techniques: Injection, Staining, Immunofluorescence, Expressing, Electron Microscopy

Journal: Experimental Animals

Article Title: Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease

doi: 10.1538/expanim.21-0054

Figure Lengend Snippet: Expression of nephrin and podocin by immunofluorescence. (a)–(c): anti-nephrin stain. Control mice showed linear pattern along the capillary walls. On the other hand, the staining pattern of both proteins became granular and weak in mito-miceΔ with more than 90% ΔmtDNA accumulation. (d)–(f): anti-podocin stain. Control mice showed linear pattern along the capillary walls. On the other hand, the staining pattern of both proteins became granular and weak in mito-miceΔ with more than 90% ΔmtDNA accumulation. (a,d) C57/B6 control. (b, e) Sample No. 6 (ΔmtDNA 79.3%). (c, f) Sample No. 17 (ΔmtDNA 92.9%).

Article Snippet: As primary antibodies, rabbit polyclonal antibodies recognizing C terminal sequence of both nephrin and podocin (nephrin: CEDPRGIYDQVAADMD (neph-C: 1212–1226), podocin: CVEPLNPKKKDSPML (pod-C: 372–385)) were generated (QIAGEN, Peptide synthesis service, Tokyo, Japan).

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: PLoS ONE

Article Title: The Dose-Dependent Organ-Specific Effects of a Dipeptidyl Peptidase-4 Inhibitor on Cardiovascular Complications in a Model of Type 2 Diabetes

doi: 10.1371/journal.pone.0150745

Figure Lengend Snippet: Representative images of nephrin and WT-1 (A and C) and quantitative analyses of both nephrin and WT-1 levels (B and D) in db/m, db/db, 0.04% (w/w) gemigliptin-treated db/db, and 0.4% (w/w) gemigliptin-treated db/db mice. TUNEL staining detecting apoptotic injury to the glomeruli (E). Quantitative analysis of the numbers of TUNEL-positive cells (F) in the four experimental groups. Analysis of the levels of the pro-apoptotic Bax and anti-apoptotic Bcl2 proteins via Western blotting (G). TUNEL staining detecting injury to the kidney (H). Quantitative analyses of the numbers of TUNEL-positive cells and the Bax/Bcl2 ratios, derived using NIH ImageJ software, are shown for the four experimental groups (I and J). The results are expressed as means ± SEMs. **p < 0.01, ***p < 0.001 vs. db/m; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. db/db. Original magnification ×400.

Article Snippet: Sections were incubated for 15 min or overnight at ambient temperature with primary polyclonal antibodies against nephrin (1:100, ENZO Life Sciences, NY, USA), WT-1 (1:100, Santa Cruz Biochemicals, Santa Cruz, CA, USA), CD31 (1:100, Abcam, MA, USA), and α-SMA (1:100, Abcam, MA, USA) followed by a biotin-free polymeric horseradish peroxidase-linked antibody conjugate system in a Bond-maX automatic slide stainer (Vision BioSystems).

Techniques: TUNEL Assay, Staining, Western Blot, Derivative Assay, Software