Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: High glucose– (6 or 24 hours, 30 mM) induced csGRP78 expression, assessed by biotinylation, was increased in ( A ) PTEC ( n = 6) and ( B ) renal fibroblasts ( n = 3). Production of α2M (24 and 48 hours) by PTEC ( C ) and renal fibroblasts ( D ) was increased by high glucose (30 mM, n = 5 and 8, respectively). Similar results were observed for α2M activation ( E and F , respectively) (high glucose 48 hours, 30 mM, n = 5 and 4, respectively). Inhibition of csGRP78 interaction with α2M* using the GRP78-targeting antibody C38 prevented high glucose– (30 mM, 48 hours) induced fibronectin and collagen IV production in both ( G ) PTEC ( n = 4–6) and ( H ) renal fibroblasts ( n = 3–5). Similarly, α2M* inhibition with the Fα2M antibody attenuated matrix protein production in high glucose (30 mM, 48 hours, n = 5–6 PTEC and 4 renal fibroblasts ( I = PTEC and J = renal fibroblasts). Peptide inhibition of the csGRP78/α2M* interaction also prevented matrix protein production in high glucose (30 mM, 48 hours) by ( K ) PTEC ( n = 4–9) and ( L ) renal fibroblasts ( n = 3–6) (* P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001).

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Expressing, Activation Assay, Inhibition

Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: TGF-β1 (5 ng/mL, 6 or 24 hours) increased localization of GRP78 to the surface of both PTEC and renal fibroblasts, assessed by biotinylation ( A and B , respectively) ( n = 4). Similarly, TGF-β1– (5 ng/mL) induced α2M production (24 and 48 hours) and activation (48 hours) were increased in PTEC ( n = 6 production and 10 activation) ( C and E ) and renal fibroblasts ( n = 8–9 production and 7 activation) ( D and F ). TGF-β1– (5 ng/mL, 48 hours induced fibronectin and collagen IV production were attenuated by csGRP78 inhibition ( G and H for PTEC and renal fibroblasts, respectively) ( n = 4 for both). Similarly, α2M* inhibition in PTEC and renal fibroblasts prevented TGF-β1-induced matrix protein production ( I and J ) (5 ng/mL, 48 hours, n = 4 and 6) (* P < 0.05, ** P < 0.01, *** P < 0.005; Kruskal-Wallis test used for α2M in D ).

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Inhibition

Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: High glucose– (30 mM, 48 hours) induced activation of Smad3 (measured as phosphorylation at Ser473/475) was prevented by csGRP78 inhibition in PTEC ( n = 3–4) ( A ) and renal fibroblasts ( n = 5) ( B ). Similarly, α2M* inhibition attenuated Smad3 activation by high glucose with either neutralizing antibody ( n = 6 PTEC and 4 renal fibroblasts) ( C = PTEC and D = renal fibroblasts) or inhibitory peptide ( n = 4–5 PTEC and 3–4 renal fibroblasts) ( E = PTEC and F = renal fibroblasts). In both PTEC and renal fibroblasts, csGRP78 (C38, 10 μg) did not prevent TGF-β1– (5 ng/mL, 48 hours) induced Smad3 activation ( n = 4 and 6) ( G and J , respectively). TGF-β1–induced Smad3 activation was also not prevented by α2M* inhibition (Fα2M, 10 μg) in PTEC or renal fibroblasts ( n = 6 for both) ( H and K , respectively). We confirmed these results using the Smad3-mediated reporter CAGA 12 -luciferase. TGF-β1-induced luciferase activation was not prevented by csGRP78 inhibition in either PTEC or renal fibroblasts ( n = 8 for both) ( I and L , respectively). Similarly, inhibition of α2M* did not prevent activation by TGF-β1 ( I and L , respectively) (0.05 ng/mL, 24 hours, n = 8 for both) (* P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001; Kruskal-Wallis test used for CAGA 12 -luciferase in K ).

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Activation Assay, Phospho-proteomics, Inhibition, Luciferase

Journal: JCI Insight

Article Title: Inhibition of cell surface GRP78 and activated α 2M interaction attenuates kidney fibrosis

doi: 10.1172/jci.insight.183998

Figure Lengend Snippet: Increased YAP and TAZ in response to TGF-β1 (5 ng/mL, 48 hours) were prevented by inhibition of csGRP78 ( n = 4–6 PTEC and 6 renal fibroblasts) ( A = PTEC and B = renal fibroblasts) and α2M* ( n = 4–6 PTEC and 6–8 renal fibroblasts) ( C = PTEC and D = renal fibroblasts). Using the TEAD-luciferase reporter construct, we confirmed that inhibition of both csGRP78 and α2M* in PTEC ( n = 8–10) ( E ) and renal fibroblasts ( n = 7–8) ( F ) prevented YAP/TAZ signaling in response to TGF-β1. High glucose– (30 mM, 48 hours) induced YAP and TAZ expression were also attenuated by csGRP78 ( n = 4 PTEC and 4–6 renal fibroblasts) ( G = PTEC and H = renal fibroblasts) and α2M* ( n = 4–6 PTEC and 4 renal fibroblasts) ( I = PTEC and J = renal fibroblasts) inhibition, as well as by the peptide inhibitor of csGRP78/α2M* interaction ( n = 3–4, PTEC, K ; and n = 4, renal fibroblasts, L ). (* P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.0001.)

Article Snippet: Primary rat renal fibroblasts (Cell Biologics, RN-6016) and immortalized human PTEC (HK2 cells, ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS).

Techniques: Inhibition, Luciferase, Construct, Expressing

Journal: Renal Failure

Article Title: FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner

doi: 10.1080/0886022X.2024.2322037

Figure Lengend Snippet: Overexpression of FTO in PTECs diminished TNF-α-induced viability decrease and apoptosis induction, while its silence did oppositely. (A–C) Validation on the transfection efficiency of FTO-specific overexpression plasmid and shRNA into human PTECs HK-2 via quantitative real-time PCR and western blot. β-actin was the housekeeping control. (D) CCK-8 assay results displaying the relative cell viability (%) of human PTECs HK-2 following the intervention of TNF-α and the transfection of FTO-specific overexpression plasmid and shRNA (48 hours). (E) Representative TUNEL staining results hinting the possible effects of TNF-α exposure and FTO-specific overexpression plasmid and shRNA intervention on the apoptosis of human PTECs HK-2 (indicated as green fluorescence). Magnification: 200 times. Scale bar = 50 µm. All experiments were performed in independent triplicates, and the data are expressed as mean ± standard deviation ( n = 3). *** p or ^^^ p or ### p or +++ p or ΔΔΔ p < 0.001. * vs. shNC; ^ vs. NC; # vs. Control; + vs. TNF-α+shNC; Δ vs. TNF-α+NC. Abbreviations: FTO, FTO alpha-ketoglutarate-dependent dioxygenase; TNF-α, tumor necrosis factor-α; PTECs, proximal tubular epithelial cells; shRNA, short-hairpin RNA; NC, negative control; CCK-8, cell counting kit-8; DAPI, 4’,6-Diamidino-2-phenylindole dihydrochloride; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: Human proximal tubular epithelial cells (PTECs) HK-2 (CL-0109, Procell, Wuhan, China) were maintained in non-essential amino acid-enriched minimal essential medium (PM150410, Procell, China) supplemented with 10% fetal bovine serum (164210, Procell, China) and 1% penicillin–streptomycin (PB180120, Procell, China), and incubated in a HeracellTM 240i CO 2 incubator (51032875, ThermoFisher, Waltham, MA, USA) at 37 °C with 5% CO 2 .

Techniques: Over Expression, Biomarker Discovery, Transfection, Plasmid Preparation, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Control, CCK-8 Assay, TUNEL Assay, Staining, Fluorescence, Standard Deviation, Negative Control, Cell Counting

Journal: Renal Failure

Article Title: FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner

doi: 10.1080/0886022X.2024.2322037

Figure Lengend Snippet: Overexpression of FTO reversed TNF-α-induced damage to apoptosis-related proteins, PTECs and the AQP3/β-Catenin axis, while downregulation of FTO did the opposite. (A–D) Western blot was used to measure the expression of Bax, Cleaved-caspase 3 and Bcl-2. (E–F) Flow cytometry (equipped with a DCFH-DA probe) was adopted to assess ROS generation in PTECs with various interventions. (G–H) Relevant quantification on SOD and MDA contents in TNF-α-induced PTECs with intervention of FTO-specific overexpression plasmid and shRNA. (I) Representative protein bands displaying AQP3 and β-Catenin protein expressions in TNF-α-induced PTECs with intervention of FTO-specific overexpression plasmid and shRNA based on western blotting. (J–K) Quantified AQP3 (J) and β-Catenin (K) protein expressions in TNF-α-induced PTECs with intervention of FTO-specific overexpression plasmid and shRNA based on western blotting. β-actin was the housekeeping control. All experiments were performed in independent triplicates, and the data are expressed as mean ± standard deviation ( n = 3). + p or Δ p < 0.05. ΔΔ p or ## p < 0.01. ### p or +++ p or ΔΔΔ p < 0.001. # vs. Control; + vs. TNF-α+shNC; Δ vs. TNF-α+NC. Abbreviations: ROS, reactive oxygen species; DCFH-DA, 2’,7’-Diochlorofluorescin Diacetate; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; SOD, superoxide dismutase; MDA, malonaldehyde; AQP3, Aquaporin 3.

Article Snippet: Human proximal tubular epithelial cells (PTECs) HK-2 (CL-0109, Procell, Wuhan, China) were maintained in non-essential amino acid-enriched minimal essential medium (PM150410, Procell, China) supplemented with 10% fetal bovine serum (164210, Procell, China) and 1% penicillin–streptomycin (PB180120, Procell, China), and incubated in a HeracellTM 240i CO 2 incubator (51032875, ThermoFisher, Waltham, MA, USA) at 37 °C with 5% CO 2 .

Techniques: Over Expression, Western Blot, Expressing, Flow Cytometry, Plasmid Preparation, shRNA, Control, Standard Deviation, Fluorescence

Journal: Renal Failure

Article Title: FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner

doi: 10.1080/0886022X.2024.2322037

Figure Lengend Snippet: FTO negatively mediated AQP3 level in RTECs in an m 6 A-depenent manner and detriminished AQP3 stability. (A–C) The possible interaction between FTO and AQP3 was investigated based on the results from quantitative real-time PCR and western blot. β-actin was the housekeeping control. (D–E) Agarose gel electrophoresis (D) and MeRIP (E) were implemented to reveal the presence of AQP3 m 6 A modification. (F) MeRIP was carried out again to reveal the impact of FTO silencing on AQP3 m 6 A modification. (G) The results from mRNA stability assay addressing the effects of FTO silencing on the stability of AQP3 in PTECs using Actinomycin D for 0, 3 and 6 hours. β-actin was the housekeeping control. (H–I) Validation on AQP3 overexpression plasmid transfection efficiency via quantitative real-time PCR And western blot. β-actin was the housekeeping control. All experiments were performed in independent triplicates, and the data are expressed as mean ± standard deviation ( n = 3). ^^ p <0.01, *** p or ^^^ p or ε ε ε p < 0.001. * vs. shNC, ε vs. IgG; ^ vs. NC. Abbreviations: m 6 A, N6-methyladenosine; MeRIP, Methylated RNA immunoprecipitation.

Article Snippet: Human proximal tubular epithelial cells (PTECs) HK-2 (CL-0109, Procell, Wuhan, China) were maintained in non-essential amino acid-enriched minimal essential medium (PM150410, Procell, China) supplemented with 10% fetal bovine serum (164210, Procell, China) and 1% penicillin–streptomycin (PB180120, Procell, China), and incubated in a HeracellTM 240i CO 2 incubator (51032875, ThermoFisher, Waltham, MA, USA) at 37 °C with 5% CO 2 .

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control, Agarose Gel Electrophoresis, Modification, Stability Assay, Biomarker Discovery, Over Expression, Plasmid Preparation, Transfection, Standard Deviation, Methylation, RNA Immunoprecipitation

Journal: Renal Failure

Article Title: FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner

doi: 10.1080/0886022X.2024.2322037

Figure Lengend Snippet: AQP3 overexpression neutralized the effects of FTO overexpression on the TNF-α-induced PTECs viability and apoptosis. (A–B) The results from CCK-8 (A) and TUNEL staining assays (B) were summarized to reveal the interplay between AQP3 and FTO in TNF-α-induced PTECs. Magnification: 200 times. Scale bar = 50 µm. (C–F) The expression of Bax, Cleaved-caspase 3 and Bcl-2 was detected by western blot. All experiments were performed in independent triplicates, and the data are expressed as mean ± standard deviation ( n = 3). ^^ p or ω ω p < .01. ^^^ p or θ θ θ p or ω ω ω p < .001. ^ vs. NC; θ vs. AQP3; ω vs. FTO.

Article Snippet: Human proximal tubular epithelial cells (PTECs) HK-2 (CL-0109, Procell, Wuhan, China) were maintained in non-essential amino acid-enriched minimal essential medium (PM150410, Procell, China) supplemented with 10% fetal bovine serum (164210, Procell, China) and 1% penicillin–streptomycin (PB180120, Procell, China), and incubated in a HeracellTM 240i CO 2 incubator (51032875, ThermoFisher, Waltham, MA, USA) at 37 °C with 5% CO 2 .

Techniques: Over Expression, CCK-8 Assay, TUNEL Assay, Staining, Expressing, Western Blot, Standard Deviation

Journal: Renal Failure

Article Title: FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner

doi: 10.1080/0886022X.2024.2322037

Figure Lengend Snippet: AQP3 overexpression cancelled the effects of FTO overexpression in TNF-α-induced PTECs on PTECs damage and β-Catenin protein expression. (A–B) Flow cytometry (equipped with a DCFH-DA probe) was adopted to assess ROS generation in TNF-α-induced PTECs with FTO and/or AQP3 overexpression. (C–D) Relevant quantification on SOD and MDA contents in TNF-α-induced PTECs with intervention of FTO and/or AQP3 overexpression. (E) Representative protein bands displaying β-Catenin protein expressions in TNF-α-induced PTECs with intervention of FTO and/or AQP3 overexpression based on western blotting. (F) Quantified AQP3 protein expression in TNF-α-induced PTECs with FTO and/or AQP3 overexpression based on western blotting. β-actin was the housekeeping control. All experiments were performed in independent triplicates, and the data are expressed as mean ± standard deviation ( n = 3). ^^ p or θ θ p or ω ω p < 0.01. ^^^ p or θ θ θ p or ω ω ω p < 0.001. ^ vs. NC; θ vs. AQP3; ω vs. FTO.

Article Snippet: Human proximal tubular epithelial cells (PTECs) HK-2 (CL-0109, Procell, Wuhan, China) were maintained in non-essential amino acid-enriched minimal essential medium (PM150410, Procell, China) supplemented with 10% fetal bovine serum (164210, Procell, China) and 1% penicillin–streptomycin (PB180120, Procell, China), and incubated in a HeracellTM 240i CO 2 incubator (51032875, ThermoFisher, Waltham, MA, USA) at 37 °C with 5% CO 2 .

Techniques: Over Expression, Expressing, Flow Cytometry, Western Blot, Control, Standard Deviation