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

Journal: Frontiers in Physiology

Article Title: Spleen Tyrosine Kinase Inhibition Ameliorates Tubular Inflammation in IgA Nephropathy

doi: 10.3389/fphys.2021.650888

Figure Lengend Snippet: Inhibition of Syk by R406 suppresses inflammatory cytokine expression in proximal tubular epithelial cells (PTECs). (A) qPCR and (B) ELISA with quantitative analysis on IL-6, IL-8, and ICAM-1 expression in PTECs incubated with conditioned medium from patients with IgAN (IgAN-CM), conditioned medium from healthy control subjects (control-CM) and medium control. * P < 0.05; ** P < 0.01 and *** P < 0.001 between groups as indicated.

Article Snippet: Human primary renal proximal tubular epithelial cells (PTECs) were obtained from Lonza and cultured in renal epithelial cell growth basal medium (REBM) with growth supplements at 37°C in 5% CO 2 atmosphere.

Techniques: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Frontiers in Physiology

Article Title: Spleen Tyrosine Kinase Inhibition Ameliorates Tubular Inflammation in IgA Nephropathy

doi: 10.3389/fphys.2021.650888

Figure Lengend Snippet: Inhibition of Syk by R406 attenuates activation of NF-κB and MAPK signaling pathway in PTECs. Western blot analysis with quantitative analysis on expression of (A) p-p65 and total p65 of NF-κB and (B) p-p42/p-p44 and total p42/p44 of MAPK in PTECs incubated with conditioned medium from patients with IgAN (IgAN-CM) with and without R406, and conditioned medium from healthy control subjects (control-CM). β-actin was used as loading control. * p < 0.05 and ** p < 0.01 between groups as indicated.

Article Snippet: Human primary renal proximal tubular epithelial cells (PTECs) were obtained from Lonza and cultured in renal epithelial cell growth basal medium (REBM) with growth supplements at 37°C in 5% CO 2 atmosphere.

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Incubation, Control

Journal: Frontiers in Physiology

Article Title: Spleen Tyrosine Kinase Inhibition Ameliorates Tubular Inflammation in IgA Nephropathy

doi: 10.3389/fphys.2021.650888

Figure Lengend Snippet: Inhibition of Syk by R406 reduces TNF-α-induced inflammatory cytokine production in PTECs. ELISA with quantitative analysis on protein expression of (A) IL-6, (B) IL-8 and (C) ICAM-1 in culture supernatant from PTECs incubated with control medium, TNF-α and TNF-α pretreated with R406. * p < 0.05; ** p < 0.01 and *** p < 0.001 between groups as indicated.

Article Snippet: Human primary renal proximal tubular epithelial cells (PTECs) were obtained from Lonza and cultured in renal epithelial cell growth basal medium (REBM) with growth supplements at 37°C in 5% CO 2 atmosphere.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Control

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: High glucose (HG) induces cell senescence in proximal tubular epithelial cells (PTECs). ( A ) The effect of HG on morphological changes and senescence-associated β-galactosidase (SAβ-Gal) staining of human PTECs. PTECs were incubated under normal glucose (NG, 6.2 mM) and HG (30 mM) conditions for seven days and nine days. Cell senescence was assessed using SAβ-Gal staining. HG increased p53 ( B ) (D3: n = 6, D4: n = 4) and p27 ( C ) (D3: n = 3, D4: n = 3), and decreased cyclin E1 ( D ) (D3: n = 3, D4: n = 4) and cyclin E2 ( E ) (D3: n = 3, D4: n = 4) protein expression in human PTECs after three and four days of treatment. Protein levels were assessed by western blot. The expression of p53 in the proximal tubule of kidneys of mice ( F ) and humans ( G ). The kidney sections of C57BL/6 mice, non-diabetic db/m mice, and diabetic db/db mice, and human donors (upper tract urothelial carcinoma, UTUC with normal kidney function and normal glomerulus and proximal tubule) and patients with diabetic nephropathy (DN) were stained with p53 (brown). The images quantification was performed using the IHC Profiler Plugin of ImageJ Software. The bar graph represents the mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Staining, Incubation, Expressing, Western Blot, Software

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Identification of potential genes associated with cell senescence in PTECs in DN. ( A ) Flowchart of identification of potential genes associated with cell senescence in PTECs. ( B ) Display of differential expression patterns of normal and diabetic PTECs from deep RNA sequencing by volcano plot. ( C ) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in DAVID database. The 612 differentially expressed genes in diabetic PTECs were uploaded into DAVID database for enrichment analysis. The top seven KEGG pathway analysis results of these dysregulated genes in diabetic PTECs are displayed in a pie chart. The pie chart indicates the-Log10 (false discovery rate, FDR) of each KEGG term, and the numbers that are shown at the outside of each pie segment indicates the number of genes involved in each term. ( D ) The protein-protein interaction network analysis of 14 genes associated cell cycle of KEGG pathway using STRING database. S-phase kinase protein 2 (Skp2) correlated with cell cycle markers, such as cyclin D2 (CCND2), cyclin B1 (CCNB1), cyclin-dependent kinase inhibitor 1C (CDKN1C), and CDKN2C. ( E ) The potential network of Skp2 mediating cell cycle in Core analysis of Ingenuity Pathway Analysis (IPA) software. Skp2 correlated with cyclins.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Quantitative Proteomics, RNA Sequencing, Software

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: The networks associated with genes differentially expressed in diabetic PTECs in IPA database.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Histone Deacetylase Assay, Cell Function Assay

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Decreased Skp2 expression is associated with cell senescence of PTECs in DN. ( A ) Decreased Skp2 mRNA expression was found in PTECs from a type 2 diabetes mellitus (DM) patient ( n = 3). ( B ) HG decreased Skp2 mRNA expression in human PTECs after three days of treatment ( n = 4). Skp2 mRNA levels were assessed by quantitative real-time polymerase chain reaction (PCR). ( C ) HG suppressed Skp2 protein expression in human PTECs after three and four days of treatment (D3: n = 3, D4: n = 3). Skp2 protein levels were assessed by western blot. The expression of Skp2 in the proximal tubule of kidneys of mice ( D ) and humans ( E ). The kidney sections of C57BL/6 mice, non-diabetic db/m mice, and diabetic db/db mice, and human donors (UTUC with normal kidney function and normal glomerulus and proximal tubule) and patients with DN were stained with Skp2 (brown). The images quantification was performed using the IHC Profiler Plugin of ImageJ Software. The bar graph represents the mean ± S.E.M. * p < 0.05, *** p < 0.001 by Student’s t test.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Software

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Identification of miR-378i-Skp2 interaction in cell senescence in diabetic PTECs. ( A ) The heat map revealed differentially expressed miRNAs from normal and diabetic PTECs with Z-score values. ( B ) The regulation of miR-378i on cell cycle, as predicted by IPA. ( C ) Increased miR-378i levels were found in PTECs from a type 2 DM patient ( n = 3). ( D ) HG increased miR-378i levels in human PTECs after three days of treatment ( n = 3). miR-378i levels were assessed by quantitative real-time PCR. ( E ) A schematic representation of sequence alignment of miR-378i and Skp2 mRNA 3’UTR. ( F ) miR-378i mimic suppressed Skp2 expression in HEK 293 cells. Cells were transfected with either control mimic or miR-378i mimic (100 nM) using DharmaFECT No. 1 Transfection Reagent. After 72 h transfection, western blot was utilized to measure Skp2 protein expression. The bar graph represents the mean ± S.E.M. ** p < 0.01 by Student’s t test.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Expressing, Transfection, Control, Western Blot

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Potential microRNA–mRNA interactions identified in diabetic PTECs.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Illustration of the interaction between miR-378i and Skp2 inducing cell senescence in PTECs in DN.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: