Journal: Biochimica et biophysica acta

Article Title: Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury.

doi: 10.1016/j.bbamcr.2014.12.033

Figure Lengend Snippet: Fig. 1. Tubular (NRK-52E) and glomerular (RGE) cells differ in their sensitivity to platinum compounds. A, B: Logarithmically growing normal rat tubular epithelial cells (NRK-52E) (A) and glomerular endothelial cells (RGE) (B) were incubated with different concentrations of the platinum compounds for 72 h. Cell viability was measured using the MTT assay as described in methods. Results are expressed as viable cells in percent of the untreated control, which was set to 100%. Data shown are the mean ± SD from 3 to 5 independent experiments each performed in quadruplicate. *: significance of CisPt vs. OxaliPt; #: significance of CisPt vs. CarboPt; +: significance of OxaliPt vs. CarboPt. ***, ###, +++p ≤0.001; ++p ≤0.01. C: NRK-52E and RGE cells were treated with the corresponding IC80 of the different platinum compounds. Cell growth was measured by recording alterations in electrical impedance in real-time as described in methods. Shown are data from one representative experiment. Con, untreated control. D: Logarithmically growing rat tubular epithelial (NRK-52E) and glomerular endothelial (RGE) cells were incubated with the IC50 of the platinum compounds for 72 h. The number of viable cells was determined by Trypan Blue exclusion assay as described in methods. The number of viable (i.e. Trypan Blue negative) cells at the time of drug addition (Con2) was set to 100%. Data shown are the mean ± SD from one representative experiment performed in triplicate. Con1, untreated control after incubation period of 72 h; Con2, untreated control at the time of platinum treatment; CisPt, OxaliPt, CarboPt, percent viable cells 72 h after platinum treatment.

Article Snippet: Rat renal proximal tubular epithelial (NRK-52E) and rat glomerular endothelial (RGE) cells originate from the German Collection of Microorgansisms and Cell Culture (DSMZ) (Braunschweig, Germany).

Techniques: Incubation, MTT Assay, Control, Trypan Blue Exclusion Assay

Journal: British Journal of Pharmacology

Article Title: Identification of endogenous 1‐aminopyrene as a novel mediator of progressive chronic kidney disease via aryl hydrocarbon receptor activation

doi: 10.1111/bph.15062

Figure Lengend Snippet: Aryl hydrocarbon receptor (AhR) signalling was activated by 1‐aminopyrene (AP). (a) Cell viability after treatment with increasing concentrations of AP (0–40 μM) in NRK‐52E cells for 24 h and cell viability after treatment with 10‐nM AP at different time points. (b) Protein expression of AhR in the nuclei and cytoplasm of NRK‐52E cells induced by AP at the different concentrations. (c) Quantitative analysis of the AhR expression of NRK‐52E cells induced by AP at different concentrations. (d) Protein expression of AhR in the nuclei and cytoplasm of NRK‐52E cells induced by AP at different time points. (e) Quantitative analysis of the AhR expression of NRK‐52E cells induced by AP at different time points. (f) The mRNA expression levels of AhR in NRK‐52E cells induced by AP at different concentrations. (g) Luciferase assays of AhR activation in NRK‐52E cells induced by AP for 24 h at different concentrations. (h) The mRNA expression levels of AhR and its target genes including CYP1A1, CYP1A2 and CYP1B1 in mice induced by AP at Weeks 3, 6 and 9. (i) Protein expression of AhR in the nuclei and cytoplasm of mice induced by AP at Weeks 3, 6 and 9. (j) Quantitative analysis of the AhR expression of mice induced by AP at Weeks 3, 6 and 9. (k) Immunohistochemical findings with anti‐AhR in mice induced by AP at Week 9. (l) Quantitative analysis of immunohistochemistry with anti‐AhR in mice induced by AP. (m) mRNA expression of AhR studied by comparative qRT‐PCR in NRK‐52E transfected with scramble shRNA or AhR shRNA. (n) Representative MSI of AP in kidney tissues in mice induced by AP. (o) After transfection with AhR‐specific shRNA or scrambled shRNA, the mRNA expression levels of AhR target genes including CYP1A1, CYP1A2 and CYP1B1 in AP‐induced NRK‐52E cells at 24 h. (p) AhR mRNA expression levels in AhR shRNA‐treated mice at Week 9. (q) mRNA expression levels of AhR target genes including CYP1A1, CYP1A2 and CYP1B1 in AhR shRNA‐treated mice induced by AP at Week 9. (r) The levels of serum creatinine and urea in control and AP‐induced mice at Week 9. (s) Images for HE staining in control and AP‐induced mice at Week 9. (t) Images for Masson's trichrome staining from control and AP‐induced mice at Week 9. *P < 0.05 versus CTL group (n = 6). # P < 0.05 versus scramble shRNA or AhR shRNA group (n = 6)

Article Snippet: Normal rat kidney proximal tubular epithelial cells NRK‐52E (JCRB Cat# IFO50480, RRID:CVCL_0468) were transfected with 3 μl of 10‐μM shRNA aryl hydrocarbon receptor or scramble per well, as per by using Lipofectamine RNAiMAX (Invitrogen, New York, USA).

Techniques: Expressing, Luciferase, Activation Assay, Immunohistochemical staining, Immunohistochemistry, Quantitative RT-PCR, Transfection, shRNA, Staining

Journal: British Journal of Pharmacology

Article Title: Identification of endogenous 1‐aminopyrene as a novel mediator of progressive chronic kidney disease via aryl hydrocarbon receptor activation

doi: 10.1111/bph.15062

Figure Lengend Snippet: Flavonoids mitigate epithelial‐to‐mesenchymal transition (EMT) through blocking aryl hydrocarbon receptor (AhR) in 1‐aminopyrene (AP)‐induced NRK‐52E cells. (a) Chemical structure of three flavonoids including 5,7,3′,4′,5′‐pentahydroxy flavanone (PHF), barleriside A (BSA) and rhoifolin (RHO). (b) The interaction of PHF, BSA and RHO with the amino acids of rat AhR. (c) Representative immunofluorescent staining indicating AP‐induced AhR expression in AP‐induced NRK‐52E cells upon treatment with flavonoids or CH223191. (d) Protein expression of AhR in nuclei and cytoplasm in the different groups. (e) Quantitative analysis of AhR in nuclei and cytoplasm from the different groups, as indicated. (f) The mRNA expression levels of AhR target genes including CYP1A1, CYP1A2 and CYP1B1 from the different groups, as indicated. (g) Luciferase assays of AhR activation from the different groups, as indicated. (h) Protein expression of collagen I, α‐smooth muscle actin(α‐SMA), fibronectin and E‐cadherin in the different groups, as indicated. (i) Quantitative analysis of collagen I, α‐SMA, fibronectin and E‐cadherin in the different groups, as indicated. The dot represents a single‐data point in the bar graph. * P < 0.05 versus CTL group (n = 6). # P < 0.05 versus AP‐induced group (n = 6)

Article Snippet: Normal rat kidney proximal tubular epithelial cells NRK‐52E (JCRB Cat# IFO50480, RRID:CVCL_0468) were transfected with 3 μl of 10‐μM shRNA aryl hydrocarbon receptor or scramble per well, as per by using Lipofectamine RNAiMAX (Invitrogen, New York, USA).

Techniques: Blocking Assay, Staining, Expressing, Luciferase, Activation Assay

Journal: British Journal of Pharmacology

Article Title: Identification of endogenous 1‐aminopyrene as a novel mediator of progressive chronic kidney disease via aryl hydrocarbon receptor activation

doi: 10.1111/bph.15062

Figure Lengend Snippet: Barleriside A (BSA) mitigated epithelial‐to‐mesenchymal transition (EMT) via partly targeting the antagonism of aryl hydrocarbon receptor (AhR) activation. (a) The protein expression of AhR in NRK‐52E cells induced by AhR shRNA. (b) Quantitative analysis of AhR expression in NRK‐52E cells induced by AhR shRNA. (c) mRNA expression levels of AhR target genes including CYP1A1, CYP1A2 and CYP1B1 in 1‐aminopyrene (AP)‐induced NRK‐52E cells treated with BSA after AhR overexpression. (d) Protein expression of collagen I, α‐SMA, fibronectin and E‐cadherin in AP‐induced NRK‐52E cells treated with BSA after transfection with AhR‐specific shRNA or scrambled shRNA. (e) Quantitative analyses of protein expression of collagen I, α‐SMA, fibronectin and E‐cadherin in AP‐induced NRK‐52E cells treated with BSA. *P < 0.05 versus CTL group (n = 6); # P < 0.05 versus AP group (n = 6)

Article Snippet: Normal rat kidney proximal tubular epithelial cells NRK‐52E (JCRB Cat# IFO50480, RRID:CVCL_0468) were transfected with 3 μl of 10‐μM shRNA aryl hydrocarbon receptor or scramble per well, as per by using Lipofectamine RNAiMAX (Invitrogen, New York, USA).

Techniques: Activation Assay, Expressing, shRNA, Over Expression, Transfection