Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Activation of TLR4/MyD88/PKCδ/SHP-1 signaling cascade was involved in hyperglycemic podocyte damage in vivo and in vitro . (A) The protein levels of TLR4 were determined in renal tissues from mice at 12 and 16 weeks by western blot analysis. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05 ( n = 8). (B) Representative western blot of the expression of MyD88, p-PKCδ (Y311), PKCδ and SHP-1 in renal cortex from mice at 12 and 16 weeks. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 8). (C) Representative double IF staining of glomerular SHP-1 and synaptopodin expression in mice at 16 weeks. Scale bar, 20 μm ( n = 8). (D) The expression of TLR4, MyD88, p-PKCδ, PKCδ and SHP-1 in cultured mouse podocytes from treatments of LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (E) IF staining of podocyte proteins p-cadherin and synaptopodin after LG or HG stimulation for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Magnification 400 × . ( n = 3 independent experiments). (F) Determination of SD protein podocin and injury marker desmin in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Activation Assay, In Vivo, In Vitro, Western Blot, Expressing, Staining, Cell Culture, Transfection, Marker

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Hyperglycemia triggered PKCδ activation and subsequent binding to downstream SHP-1 following TLR4/MyD88 signaling to induce podocyte damage. (A) Determination of expression of p-PKCδ, PKCδ and SHP-1 in cultured podocytes from treatments of HG for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; *** P < 0.01; **** P < 0.001; ## P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (B) The expression of podocin and desmin in podocytes treated with HG for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; ## P < 0.01 ( n = 3 independent experiments). (C) The representative western blot to show the expression of TLR4 and MyD88 in podocytes after HG stimulation for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Data are presented as mean ± SD. P values were determined by one-way ANOVA and data are presented as mean ± SD; P > 0.05 ( n = 3 independent experiments). (DF) Surface diagram of the docking model and their interfacing residues between MyD88 and PKCδ (D) (MyD88, purple; PKCδ, yellow; hydrogen bonds, dotted line), and between PKCδ and SHP-1 (F) (PKCδ, yellow; SHP-1, blue; hydrogen bonds, dotted line). (EG) CO-IP analysis of the interaction between MyD88 and PKCδ (E), and between PKCδ and SHP-1 (G) in podocytes under HG condition in vitro . ( n = 3 independent experiments).

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Activation Assay, Binding Assay, Expressing, Cell Culture, Transfection, Western Blot, Co-Immunoprecipitation Assay, In Vitro

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: TLR4/MyD88/PKCδ/SHP-1 signaling cascade induced podocyte damage through regulating ER stress in DKD and podocyte damage. (A) The protein levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 were determined in renal tissues from mice at 12 and 16 weeks by western blot. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 8). (B) Representative double IF staining of the expression ATF4 and podocyte marker synaptopodin in mice at 16 weeks. Scale bar, 20 μm ( n = 8). (C) Representative western blot to assess the levels of ER stress markers BIP, sXBP-1, ATF4 and ATF6 in cultured mouse podocytes from treatments of LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ### P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (D) ER morphological alterations and ER stress illustrated by ER-tracker staining in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or MCP-1 shRNAs, or pretreatment of 4-PBA. Scale bar, 10 μm. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; # P < 0.05 ( n = 3 independent experiments). (E) Representative western blot to detect the expression of ER stress proteins BIP, sXBP-1, ATF4 and ATF6 in cultured podocytes after HG stimulation for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; *** P < 0.01; **** P < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Western Blot, Staining, Expressing, Marker, Cell Culture, Transfection

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Further validation of PKCδ activation being indispensable for ER stress and MCP-1 production through SHP-1 in diabetic podocyte injury. (A) Schematic representation of PKCδ plasmids to express PKCδ proteins in DN (tyrosine mutation at amino acid 311), CA or WT form. (B) Representative western blot to validate the expression of DN-PKCδ, CA-PKCδ and WT-PKCδ plasmids in HEK-293 T cells by using antibodies specific for HA-tag, with GAPDH detection as a loading control. ( n = 3 independent experiments). (CDE) Determination of protein levels of SHP-1 (C), ER stress proteins BIP, sXBP-1, ATF4 and ATF6 (D), and MCP-1 (E) in cultured mouse podocytes transfected with DN-, CA- and WT-PKCδ plasmids, or control plasmids under LG or HG conditions. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.01; **** P < 0.01; ## P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments).

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Biomarker Discovery, Activation Assay, Mutagenesis, Western Blot, Expressing, Control, Cell Culture, Transfection

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: TLR4/MyD88/PKCδ/SHP-1 signaling-dependent ER stress stimulated MCP-1 production via enhanced transcription activity by ATF4 binding to its promoter in hyperglycemic podocytes. (A) The altered expression of MCP-1 in renal tissue from mice at 12 and 16 weeks by western blot. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01 ( n = 8 samples). (B) Representative western blot to assess the levels of MCP-1 in cultured mouse podocytes from treatments of LG or HG for 48 h with or without transfection of TLR4, MyD88 or scrambled shRNAs. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (C) Representative western blot to show the expression of MCP-1 in podocytes after HG stimulation for 48 h with or without transfection of PKCδ, SHP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. ** P < 0.01; *** P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (DE) The expression of MCP-1 by western blot (D), and ER stress markers BIP, sXBP-1, ATF4 and ATF6 (E) after HG stimulation for 48 h with or without MCP-1 or scrambled shRNA transfection. P values were determined by one-way ANOVA and data are presented as mean ± SD. ## P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (F) Schematic description of primers spanning the distal, medial, and proximal regions of the MCP-1 promoter, encompassing predicted binding sites in the present study. (GH) ChIP analysis using anti-ATF4 or anti-IgG antibodies were performed either in cultured mouse podocytes after LG or HG treatment for 48 h (G), or HG stimulation with transfection of TLR4, PKCδ, MCP-1 or scrambled shRNA, or pretreatment of 4-PBA (H). Real-time PCR was used for the detection of the ChIP signals. P values were determined by Student’s t -test, or by one-way ANOVA, and data are presented as mean ± SD. * P < 0.05; ** P < 0.01; **** P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (I) Determination of ATF4 nuclear accumulation in podocytes under LG or HG conditions by western blot. P values were determined by Student’s t -test and data are presented as mean ± SD. ** P < 0.01; ## P < 0.01 ( n = 3 independent experiments). (J) Assessment of nuclear ATF4 protein levels in cultured podocytes transfected with TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreated with 4-PBA under HG conditions for 48 h. P values were determined by one-way ANOVA and data are presented as mean ± SD. * P < 0.05; * P < 0.01 ( n = 3 independent experiments).

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Activity Assay, Binding Assay, Expressing, Western Blot, Cell Culture, Transfection, shRNA, Real-time Polymerase Chain Reaction

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Blockade of TLR4/MyD88/PKCδ/SHP-1 signaling attenuated podocyte cytoskeletal remodeling and inflammation, and macrophage recruitment and infiltration through decreasing ER stress and MCP-1 production under hyperglycemic conditions. (A) Assessment of podocyte migration by transwell assay in cultured podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; ### P < 0.01; #### P < 0.01 ( n = 3 independent experiments). (B) Representative IF images by phalloidin staining to illustrate F-actin distribution and cytoskeletal remodeling in cultured podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Magnification 400 × . ( n = 3 independent experiments). (C) Assessment of the secretion of inflammatory factors IL-6 and IL-1β in podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, or scrambled shRNAs, or pretreatment of 4-PBA. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test and data are presented as mean ± SD. **** P < 0.01; # P < 0.05; ## P < 0.01; ### P < 0.01 ( n = 3 independent experiments). (D) IHC staining using anti-f4/80 antibodies to illustrate macrophage infiltration in the glomeruli from mice at 16 weeks (Red arrow). Scale bar, 20 μm. ( n = 8) (E) The model diaphragm of podocyte-macrophage co-culture. (F) Representative images and assessment of macrophage migration by transwell assay after co-cultured with podocytes treated with LG or HG for 48 h with or without transfection of TLR4, PKCδ, MCP-1 or scrambled shRNAs, or pretreatment of 4-PBA. Magnification 400 × . P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons test and data are presented as mean ± SD. **** P < 0.01; ## P < 0.01; ### P < 0.01 ( n = 3 independent experiments).

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Migration, Transwell Assay, Cell Culture, Transfection, Staining, Immunohistochemistry, Co-Culture Assay

Journal: Journal of Advanced Research

Article Title: Podocyte TLR4 deletion alleviates diabetic kidney disease through prohibiting PKCδ/SHP-1-dependent ER stress and relieving podocyte damage and inflammation

doi: 10.1016/j.jare.2025.07.013

Figure Lengend Snippet: Schematic diaphragm of TLR4/MyD88/PKCδ/SHP-1 signaling in podocytes in exposure of hyperglycemia. Upon the stimuli of hyperglycemia, TLR4/MyD88 signaling was activated in glomerular podocyte; and PKCδ was then phosphorylated and bound to SHP-1 to provoke downstream ER stress, leading to nuclear translocation of ATF4 and enhanced transcriptional activity of MCP-1, which exacerbated damage to SD proteins and disruption of the cytoskeletal structure, increased cell motility, promoted inflammation in podocytes, and finally induced local macrophage migration and infiltration.

Article Snippet: Primary antibodies used were as follows: PKCδ (14188–1-AP) were from Proteintech (IL, USA); SHP-1 (ab227503), MyD88 (ab219413) were from Abcam (MA, USA).

Techniques: Translocation Assay, Activity Assay, Disruption, Migration