Journal: Archives of Toxicology

Article Title: The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro

doi: 10.1007/s00204-022-03306-1

Figure Lengend Snippet: Effect of remdesivir on the viability of kidney cells. a , b NRK-52E cells ( n = 6–7) or c , d RPTEC/TERT1 ( n = 5) were treated with increasing concentrations of remdesivir or zalcitabine for 24 h. Viability was determined by measuring ATP levels. Data were normalized to the mean of the solvent control (dmso = 100%; dashed line). Data are presented with a non-linear curve-fit. Ordinary one-way ANOVA, Dunnett’s multiple comparisons test. e The relative cell number was determined by nuclei staining 24 h after remdesivir treatment in NRK-52E cells ( n = 8–9). f , g Proliferation of NRK-52E cells was measured by f 3 H-thymidine incorporation within 6 h after remdesivir treatment ( n = 5) and g by cell cycle analysis ( n = 7). h NRK-52E ( n = 5–9) or i RPTEC/TERT1 ( n = 6–9) cells were treated with increasing concentrations of remdesivir for indicated time periods. Lactate concentration was measured in supernatants. j NRK-52E or k RPTEC/TERT1 cells were treated with increasing concentrations of remdesivir for 24 h. 1 h after removal of remdesivir the oxygen consumption rate (OCR) was in the presence of the inhibitors of the respiratory chain oligomycin, FCCP or rotenone/antimycin A. Base OCR was derived from the difference of the OCR before oligomycin injection and after rotenone/antimycin A ( n = 5–6). * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Ordinary one-way ANOVA, Dunnett’s multiple comparisons test. e The relative cell number was determined by nuclei staining 24 h after remdesivir treatment in NRK-52E cells ( n = 8–9). f , g Proliferation of NRK-52E cells was measured by f 3 H-thymidine incorporation within 6 h after remdesivir treatment ( n = 5) and g by cell cycle analysis ( n = 7). h NRK-52E ( n = 5–9) or i RPTEC/TERT1 ( n = 6–9) cells were treated with increasing concentrations of remdesivir for indicated time periods.

Techniques: Staining, Cell Cycle Assay, Concentration Assay, Derivative Assay, Injection

Journal: Archives of Toxicology

Article Title: The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro

doi: 10.1007/s00204-022-03306-1

Figure Lengend Snippet: GO-ontology based analysis of differentially regulated proteins with DAVID

Article Snippet: Ordinary one-way ANOVA, Dunnett’s multiple comparisons test. e The relative cell number was determined by nuclei staining 24 h after remdesivir treatment in NRK-52E cells ( n = 8–9). f , g Proliferation of NRK-52E cells was measured by f 3 H-thymidine incorporation within 6 h after remdesivir treatment ( n = 5) and g by cell cycle analysis ( n = 7). h NRK-52E ( n = 5–9) or i RPTEC/TERT1 ( n = 6–9) cells were treated with increasing concentrations of remdesivir for indicated time periods.

Techniques: Protein Binding

Journal: International Journal of Molecular Sciences

Article Title: Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins

doi: 10.3390/ijms25010081

Figure Lengend Snippet: Differentiation protocol and physiological changes provoked by it. ( A ) Scheme of the treatment of the hiPSC F4- and b4 adapted from the protocol of Kandasamy et al. . Morphological appearance of the F4-hiPSC and cells on day 3 and day 9 ( B ) as well as of b4-hiPSC and cells on day 3 and day 9 ( C ) of the differentiation process compared to a ( D ) commercially available kidney biopsy-derived proximal tubular cell line (RPTEC/TERT1). Reduced proliferation after initiation of the differentiation process in F4- ( E ) and b4-hiPSC ( F ), quantified by the incorporation of fluorescent 5-ethynyl-2′-deoxyuridine (EdU) into S-phase cells. The mean % of proliferating cells of 3 independent experiments and representative pictures are shown. *** p < 0.001 vs. hiPSC, °° p < 0.01, °°° p < 0.001 vs. diffD3 (One-way ANOVA). The scale bars represent 50 µm. Yellow circles highlight dome-like and yellow arrows show tubule-like patterns in the cell layers of the differentiated cells. BMP = bone morphogenetic protein, diffD = differentiation day, hiPSC = human induced pluripotent stem cells, PTELC = proximal tubular epithelial-like cells, REGM = renal epithelial cell growth medium, ROCK = rho-associated, coiled-coil-containing protein kinase.

Article Snippet: The human primary renal proximal tubular epithelial cell line (RPTEC-TERT1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded as described by [ ].

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins

doi: 10.3390/ijms25010081

Figure Lengend Snippet: Changes in expression of differentiation markers in F4- and b4-hiPSC differentiated into proximal tubular epithelial cell-like cells (PTELC). ( A ) mRNA expression of stem cell, epithelial cell, and PTEC markers in differentiated F4- and b4hiPSC relative to expression in undifferentiated hiPSC analyzed by quantitative RT-PCR. ( B ) Visualization of selected proteins by immunocytochemical staining on F4-hiPSC and F4 on differentiation day 9. Antibodies against the different markers are visualized with FITC-coupled secondary antibodies, and nuclei are stained with DAPI. ( C ) Flow cytometry analysis of the PTEC typical protein aquaporin-1 in F4-hiPSC and F4 on differentiation day 9. Data from at least 3 independent experiments (qPCR also included 3 technical replicates) are shown as mean + SD. * p ≤ 0.05, ** p < 0.01 and *** p < 0.001 vs. hiPSC (Student’s t -test). Aqp-1 = aquaporin-1, Cad-16 = cadherin 16, CD13 = alanyl aminopeptidase, DAPI = 4′,6-diamidino-2-phenylindole, diffD = differentiation day, E-cad = E-cadherin, FITC = fluorescein isothiocyanate, Gapdh = glyceraldehyde 3-phosphate dehydrogenase, Glut-5 = fructose transporter 5, hiPSC = human induced pluripotent cells, Nanog = homeobox protein, N-cad = N-cadherin, Oct-3/4 = octamer-binding transcription factor 3/4, PTEC = proximal tubular epithelial cells, Sox-2 = sex determining region Y-box 2, Uro-10 = urothelial glycoprotein, Zo-1 = zonula occludens-1.

Article Snippet: The human primary renal proximal tubular epithelial cell line (RPTEC-TERT1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded as described by [ ].

Techniques: Expressing, Quantitative RT-PCR, Staining, Flow Cytometry, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins

doi: 10.3390/ijms25010081

Figure Lengend Snippet: Expression changes in functional proteins in hiPSC differentiated into proximal tubule epithelial cell (PTEC)-like cells. ( A ) mRNA expression of PTEC transporters and transporters for cisplatin relative to expression in F4- (left) and b4- (right) hiPSC analyzed by quantitative RT-PCR. ( B ) Expression of selected transporter proteins in F4 cells analyzed by Western blotting. Shown are representative blots as well as their quantification. ( C ) Visualization of selected proteins by immunocytochemical staining on F4-hiPSC and F4 on differentiation day 9. Antibodies against the different markers are visualized with FITC-coupled secondary antibodies, and nuclei are stained with DAPI. Data from at least 3 independent experiments (qPCR also included 3 technical replicates) are shown as mean ± SD. * p ≤ 0.05, ** p < 0.01, *** p < 0.001 vs. hiPSC (Student’s t -test). Ctr-1/2 = copper transpor½ 1/2, Cubn = cubilin, DAPI = 4′,6-diamidino-2-phenylindole, diffD9 = differentiation day 9, FITC = fluorescein isothiocyanate, hiPSC = human induced pluripotent cells, Mdr-1 = multidrug resistance protein 1, Meg = megalin, Oat-1/3 = organic anion transporter 1, Oct-2 = organic cation transporter 2, Octn-2 = organic cation/carnitine transporter 2, Pept-1/2 = peptide transpor½ 1/2, Sglt-2 = sodium/glucose cotransporter 2.

Article Snippet: The human primary renal proximal tubular epithelial cell line (RPTEC-TERT1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded as described by [ ].

Techniques: Expressing, Functional Assay, Quantitative RT-PCR, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins

doi: 10.3390/ijms25010081

Figure Lengend Snippet: Gain of functional albumin transport in hiPSC differentiated into proximal tubule epithelial cell (PTEC)-like cells. Analysis of albumin uptake into the undifferentiated and differentiated F4- ( A ) and b4- ( B ) cells, as well as in RPTEC- ( C ) and LLC-PK1 ( D ) cells with the help of FITC-labelled BSA. Shown are representative pictures. BSA = bovine serum albumin, DAPI = 4′,6-diamidino-2-phenylindole, diffD9 = differentiation day 9, FITC = fluorescein isothiocyanate, hiPSC = human induced pluripotent cells, RPTEC = renal proximal tubule epithelial cells.

Article Snippet: The human primary renal proximal tubular epithelial cell line (RPTEC-TERT1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded as described by [ ].

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins

doi: 10.3390/ijms25010081

Figure Lengend Snippet: Expression changes in selected genes in F4- and b4-hiPSC differentiated into proximal tubular epithelial cell (PTEC)-like cells compared to the expression in a commercial renal proximal tubule epithelial kidney cell line (RPTEC/TERT1). mRNA expression of a stem cell marker, an epithelial marker, several PTEC markers, and transporters relative to expression in RPTEC/TERT1 were analyzed by quantitative RT-PCR. Data from at least 3 independent experiments (qPCR also included 3 technical replicates) are shown as mean ± SD. * p ≤ 0.05, ** p < 0.01 and *** p < 0.001 vs. RPTEC/TERT1, °° p < 0.01, °°° p < 0.001 vs. F4 (ANOVA). BSA = bovine serum albumin, DAPI = 4′,6-diamidino-2-phenylindole, diffD9 = differentiation day 9, FITC = fluorescein isothiocyanate, hiPSC = human induced pluripotent cell. Aqp-1 = aquaporin-1, CD13 = alanyl aminopeptidase, Ctr-1 = copper transporter 1, Cubn = cubilin, diffD9 = differentiation day 9, E-cad = E-cadherin, Glut-5 = fructose transporter 5, hiPSC = human induced pluripotent cells, Meg = megalin, Oct-3/4 = octamer-binding transcription factor 3/4, RPTEC = renal proximal tubule epithelial cells.

Article Snippet: The human primary renal proximal tubular epithelial cell line (RPTEC-TERT1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded as described by [ ].

Techniques: Expressing, Marker, Quantitative RT-PCR, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Sensitivity of Human Induced Pluripotent Stem Cells and Thereof Differentiated Kidney Proximal Tubular Cells towards Selected Nephrotoxins

doi: 10.3390/ijms25010081

Figure Lengend Snippet: Effects of cisplatin and cyclosporin A on marker gene expression and transport function in F4-hiPSC differentiated into proximal tubular epithelial cell (PTEC)-like cells. mRNA expression of marker genes in diffD9 F4 cells after 24 h treatment with the IC 20 and IC 50 concentrations of cisplatin ( A ) and cyclosporin A ( D ) relative to their expression in F4-hiPSC analyzed by quantitative RT-PCR. Flow cytometry analysis of the PTEC typical protein aquaporin-1 in diffD9 F4 cells treated for 24 h with the IC 20 and IC 50 concentrations of cisplatin ( B ) and cyclosporin A ( E ). Flow cytometric analysis of albumin uptake into diffD9 F4 cells after 24 h treatment with the IC 20 and IC 50 concentrations of cisplatin ( C ) and cyclosporin A ( F ) with the help of FITC-labelled albumin. Data from at least 3 independent experiments (qPCR included 3 technical replicates) are shown as mean + SD. * p ≤ 0.05, ** p < 0.01 and *** p < 0.001 vs. treated F4-hiPSC, ° p ≤ 0.05, °° p < 0.01, °°° p < 0.001 vs. diffD9 cells treated with IC 20 (ANOVA). Aqp-1 = aquaporin-1, Cad-16 = cadherin 16, CD13 = alanyl aminopeptidase, Ctr-1/2 = copper transporter 1/2, Cubn = cubilin, diffD9 = differentiation day 9, E-cad = E-cadherin, Glut-5 = fructose transporter 5, hiPSC = human induced pluripotent cells, Meg = megalin, Pept-1 = peptide transporter 1, Zo-1 = zonula occludens-1.

Article Snippet: The human primary renal proximal tubular epithelial cell line (RPTEC-TERT1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded as described by [ ].

Techniques: Marker, Gene Expression, Expressing, Quantitative RT-PCR, Flow Cytometry