Journal: Frontiers in Medicine

Article Title: Selective Inhibition of Histone Deacetylase Class IIa With MC1568 Ameliorates Podocyte Injury

doi: 10.3389/fmed.2022.848938

Figure Lengend Snippet: Histone deacetylase IIa inhibition with MC1568 protects podocyte from injury in vitro . (A,B) Immunoblotting and quantification analysis of HDAC IIa members, desmin and α-SMA in ADR-stimulated podocytes with or without MC1568 treatment. (C) Phalloidin staining in ADR-stimulated podocytes with or without MC1568 treatment. Scale bar, 50 μm. Statistical analysis was performed with two-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Histone deacetylase 4 (Proteintech, 17449-1-AP, WB, 1:1,000, IHC, 1:100), HDAC5 (Proteintech, 16166-1-AP, WB, 1:1,000, IHC, 1:100), HDAC7 (Santa Cruz, sc-74563, WB, 1:1,000, IHC, 1:100), HDAC9 (Santa Cruz, sc-398003, WB, 1:1,000, IHC, 1:100), Desmin (Proteintech, 16520-1-AP, WB, 1:1,000, IHC), α-SMA (Proteintech, 14395-1-AP, WB, 1:1,000, IHC, 1:200), Active β-catenin (CST, 8814, WB, 1:3,000, IHC, 1:200), GAPDH (Proteintech, 10494-1-AP, WB, 1:10,000), β-tubulin (Proteintech, 10094-1-AP, WB, 1:10,000).

Techniques: Histone Deacetylase Assay, Inhibition, In Vitro, Western Blot, Staining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: MiR-29b promotes podocyte injury by targeting PGC-1α. (A) Bioinformatics analysis shows the predicted binding sites of miR-29b in the PGC-1α 3′-untranslated region (UTR) using the TargetScan software. (B) Sequence validation of the wild type or mutant PGC-1α 3′-UTR for the luciferase reporter construction. The wild-type miR-29b binding site in PGC-1α 3′-UTR (upper) and the mutated one (bottom) in the region corresponding to the miR-29b seed sequence are shown. (C) Luciferase reporter assay show that miR-29b decreased the luciferase activity in 293T cells co-transfected with wild-type PGC-1α 3′ UTR, but not with mutant PGC-1α 3′ UTR. *** P < 0.001 versus control group (n=4). (D) Mouse podocytes (MPC5) were transfected with miR-29b mimic or negative control (miR-Ctrl, NC) for 24h. qRT-PCR analysis shows the relative levels of miR-29b. ** P < 0.01 versus control group (n=3). (E) Immunostaining of ZO-1 were presented. Arrows indicate positive staining. Bar = 25μm. (F) Representative western blot showing expression of ZO-1, podocalyxin and Desmin in two groups. Numbers (1-3) indicate each individual culture in each given group. (G) Representative western blot showing expression of PGC-1α, TFAM, TOMM20 and COX1 in two groups. Numbers (1-3) indicate each individual culture in each given group. (H) Representative micrographs show mitotracker staining, immunostaining of TOMM20 and mitoSOX probe staining. Arrows indicate positive staining. Bar = 20μm or 50μm. (I-J) Graphical representations of basal OCR, maximal OCR, ATP-linked OCR, spare respiratory capacity and FAO-linked OCR in different groups. * P < 0.05, ** P < 0.01 versus control group (n=3). (K) MPC5 cells were transfected with miR-29b inhibitor (anti-miR-29b) or control (anti-miR-Ctrl, NC) for 24 h. qRT-PCR analysis shows the relative levels of miR-29b.*** P < 0.01 versus control group (n=3). (L) Representative western blot showing expression of PGC-1α, Zo-1 and podocalyxin in two groups. Numbers (1-3) indicate each individual culture in each given group. (M) MPC5 cells were transfected with pDel-β-catenin or co-transfected with miR-29b inhibitor for 24 h. Representative western blot showing expression of PGC-1α, Zo-1 and Desmin among three groups. Numbers (1-3) indicate each individual culture in each given group.

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Binding Assay, Software, Sequencing, Biomarker Discovery, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Transfection, Control, Negative Control, Quantitative RT-PCR, Immunostaining, Staining, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Inhibition of miR-29b mitigates high glucose-induced podocyte injury by increasing PGC-1α. (A) qRT-PCR analysis shows the relative levels of miR-29b after high glucose treatment for 48h. ** P < 0.01 versus control group (n=3), ††† P < 0.001 versus high glucose group (n=3). (B-E) Representative western blot and quantitative data showing expression of podocalyxin, WT1 and Desmin. Numbers (1-3) indicate each individual culture in each given group. * P < 0.05, ** P < 0.01 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3). (F) Representative micrographs showing immunostaining of F-actin. Arrows indicate actin skeleton rearrangement. Bar = 15μm. Representative TEM micrographs showing mitochondrial ultrastructure following different treatments. Arrows indicate injured mitochondria. Bar = 1μm. (G-K) Representative western blot and quantitative data showing expression of PGC-1α, TFAM, TOMM20 and COX1. Numbers (1-3) indicate each individual culture in each given group. * P < 0.05, ** P < 0.01 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3). (L) Graphical representation of mitochondrial membrane potential (MMP). MMP was detected by JC-1 staining and analyzed by flow cytometry. The data is shown as the ratio of the fluorescence intensity at absorbance of 590 nm (JC-1 aggregate) to 520 nm (JC-1 monomer). *** P < 0.001 versus control group; † P < 0.05 versus high glucose group (n=3). (M) Representative micrographs show immunostaining of TOMM20 staining. Arrows indicate positive staining. Bar = 25μm. (N) Representative micrographs show mitoSOX probe staining. Arrows indicate positive staining. Bar = 25μm. (O-P) Graphical representations of basal OCR, maximal OCR, ATP-linked OCR, spare respiratory and FAO-linked OCR in different groups. * P < 0.05, ** P < 0.01, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3). (Q) Graphical representations of the relative mRNA abundance of PPARα, CPT1a, CPT2 and ACOX1 in different groups. ** P < 0.01, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01 versus high glucose group (n=3).

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Inhibition, Quantitative RT-PCR, Control, Western Blot, Expressing, Immunostaining, Membrane, Staining, Flow Cytometry, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Ectopic expression of miR-29b aggravates podocyte injury in ADR nephropathy. (A) Schematic diagram shows the experimental procedure. (B) qPCR analyses show renal miR-29b level in two groups as indicated. *** P < 0.001 versus ADR mice group (n=5). (C) Ectopic expression of miR-29b augmented proteinuria in ADR mice. Urinary albumin was expressed as mg/mg urinary creatinine. * P < 0.05 versus ADR mice group (n=5). (D) Representative micrographs show PAS staining in different groups. Arrows indicate positive staining. Bar = 50μm. (E-J) Representative western blot and quantitative data showing expression of fibronectin, Podocalyxin, nephrin and Desmin in two groups. Numbers (1-5) indicate each individual animal in each given group. * P < 0.05, ** P < 0.01 versus ADR mice group (n=5). (K) Representative micrographs show immunostaining of fibronectin and nephrin. Arrows indicate positive staining. Bar = 50μm and 25μm. (L-M) Representative western blot and quantitative data showing expression of PGC-1α in two groups. Numbers (1-5) indicate each individual animal in each given group. *** P < 0.001 versus ADR mice group (n=5). (N) Representative micrographs show immunostaining of PGC-1α. Arrows indicate positive staining. Bar = 50μm. (O) Representative TEM micrographs showing podocytes foot process in two groups. Bar = 1μm.

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Expressing, Staining, Western Blot, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Inhibition to miR-29b mitigates mitochondrial dysfunction and podocyte injury in db/db mice. (A) Schematic diagram shows the experimental procedure. (B) qPCR analyses show renal miR-29b level in two groups as indicated. * P < 0.05 versus db/db mice group (n=5). (C) Inhibition of miR-29b by antagomiR mitigated proteinuria in db/db mice. Urinary albumin was expressed as mg/mg urinary creatinine. * P < 0.05 versus db/db mice group (n=5). (D, F-G) Representative western blot and quantitative data showing expression of fibronectin, Desmin, Podocalyxin and nephrin in two groups. Numbers (1-4) indicate each individual animal in each given group. * P < 0.05, ** P < 0.01, *** P < 0.001 versus db/db mice group (n=5). (E) Representative micrographs show immunostaining of fibronectin, podocalyxin and Zo-1. Arrows indicate positive staining. Bar = 25μm. (H, J-M) Representative western blot and quantitative data showing expression of PGC-1α, TFAM, TOMM20 and Cytb in two groups. Numbers (1-4) indicate each individual animal in each given group. * P < 0.05, *** P < 0.001 versus db/db mice group (n=5). (I) Co-staining of nephrin and TOMM20 in glomerular podocytes in different groups. Arrows indicate the co-localization of nephrin and TOMM20. Bar = 25μm. (N) Graph showing the mtDNA level in 2 groups. *** P < 0.001 versus db/db mice group (n=5). (O) Graphical representations of the relative mRNA abundance of PPARα, CPT1a, CPT2 and ACOX1 in different groups. * P < 0.05 versus db/db mice group (n=5). (P) Co-staining of PGC-1α (red) and WT1 (green), ADRP (red) and synaptopodin (green) in glomerular podocytes in different groups. Arrows indicate positive staining. Bar = 25μm.

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Inhibition, Western Blot, Expressing, Immunostaining, Staining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Activation of PGC-1α inhibits miR-29b-induced mitochondrial dysfunction and cell injury in podocytes. (A) Schematic diagram shows the experimental procedure. (B) qPCR analyses show renal miR-29b level in different groups as indicated. *** P < 0.001 versus control group (n=5). (C) Representative micrographs show PAS staining in different groups. Arrows indicate positive staining. Bar = 20μm. (D-E) Representative micrographs confirming the specific expression of miR-29b in podocytes through immunofluorescence staining with anti-Flag and nephrin antibodies. Arrows indicate positive staining. Bar = 250μm or 25μm. (F-I) Representative western blot and quantitative data showing expression of podocalyxin, nephrin and Desmin in different groups. Numbers (1-3) indicate each individual animal in each given group. * P < 0.05, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01, ††† P < 0.001 versus miR-29b plasmid group (n=5). (J) Representative micrographs show immunostaining of fibronectin, nephrin and PGC-1α. Arrows indicate positive staining. Bar = 20μm or 50 μm. (K-O) Representative western blot and quantitative data showing expression of PGC-1α, TFAM, TOMM20 and COX1 in different groups. ** P < 0.01, *** P < 0.001 versus control group; † P < 0.05, †† P < 0.01, ††† P < 0.001 versus miR-29b plasmid group (n=5).

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Activation Assay, Control, Staining, Expressing, Immunofluorescence, Western Blot, Plasmid Preparation, Immunostaining

Journal: International Journal of Biological Sciences

Article Title: MicroRNA-29b Plays a Vital Role in Podocyte Injury and Glomerular Diseases through Inducing Mitochondrial Dysfunction

doi: 10.7150/ijbs.93506

Figure Lengend Snippet: Specific ablation of miR-29b in podocytes mitigates high glucose-induced podocyte injury and mitochondrial dysfunction in glomerular mini-organ culture. (A) Representative image showing the isolated miR-29b flox/flox mice glomeruli under microscopy. (B) Graph showing the miR-29b level in Adv-NC group, Adv-NC+HG group and Adv-NPHS2-Cre+HG group. *** P <0.001 versus negative control group (Adv-NC); ††† P <0.001 versus high glucose group (Adv-NC+HG) (n=3). (C-D) Representative western blot and quantitative data showing expression of PGC-1α. Numbers (1-3) indicate each individual culture in each given group. ** P < 0.01 versus negative control group (Adv-NC); † P < 0.05 versus high glucose group (Adv-NC+HG) (n=3). (E) Graphical representations of the relative mRNA abundance of TFAM, COX1, Cytb and TOMM20 in different groups. * P < 0.05 versus negative control group (Adv-NC); † P < 0.05, †† P < 0.01 versus high glucose group (Adv-NC+HG) (n=3). (F-J) Representative western blot and quantitative data showing expression of podocalyxin, nephrin, WT1 and Desmin. Numbers (1-3) indicate each individual culture in each given group. * P < 0.05, ** P < 0.01, *** P < 0.001 versus negative control group (Adv-NC); † P < 0.05, †† P < 0.01 versus high glucose group (Adv-NC+HG) (n=3). (K-N) Graphical representations of the relative mRNA abundance of PPARα, CPT1a, CPT2 and ACOX1 in different groups. * P < 0.05, ** P < 0.01 versus negative control group (Adv-NC); † P < 0.05, †† P < 0.01 versus high glucose group (Adv-NC+HG) (n=3).

Article Snippet: Primary antibodies used in experiments were as follows: ZO-1 (QF215185; Life Technologies, Carlsbad, CA), podocalyxin (AF1556; R&D Systems), Desmin (PB0095; Boster, Wuhan, China), nephrin (ab58968; Abcam, Cambridge, UK), WT1 (sc-393498; Santa Cruz Biotechnology, Dallas, TX), PGC-1α (66369; proteintech), TFAM (GTX112760; Genetex), COX1 (SAB1301619; Sigma-Aldrich), TOMM20 (ab186735; Abcam, Cambridge, UK), Cytb (SAB1304939; Sigma-Aldrich), fibronectin (F3648; Sigma-Aldrich), anti-GAPDH (RM2001; Ray Antibody Biotech) and α-tubulin (BM3885; Boster, Wuhan, China).

Techniques: Organ Culture, Isolation, Microscopy, Negative Control, Western Blot, Expressing