1. Volume 64, Issue 6, Pages 2052-2063 (December 2003)
Cyclic AMP reverses radiocontrast media–induced apoptosis in LLC-PK1 cells by activating A kinase/PI3 kinase 
Takahisa Yano, Yoshinori Itoh, Toshiaki Sendo, Toshio Kubota, Ryozo Oishi 
Kidney International 
Volume 64, Issue 6, Pages (December 2003)
DOI: /j x
Copyright © 2003 International Society of Nephrology Terms and Conditions

2. Figure 1
Decrease in the viability of a porcine proximal cell line LLC-PK1 cells by various iodinated and magnetic resonance contrast media (A) and the lack of correlation between the reduction of cell viability and the osmolality of iodinated contrast media (B). Cells were exposed for 30 minutes at 37°C to eight different iodinated contrast media (each 150mg iodine/mL), including ioxaglate (IOX), sodium iotalamate (IOT), and amidotrizoate (AMD) for ionic agents and iohexol (IHX), iopamidol (IPD), ioversol (IVS), iomeprol (IMP), and iotrolan (ITL) for nonionic contrast media, and two different magnetic resonance contrast media (each ×2 dilution) such as gadopentetate (GPT) and gadoteridol (GTD) in serum-free medium, followed by further incubation for 24hours at 37°C in the absence of contrast media. Cell viability was assessed by the mitochondrial dehydrogenase activity using WST-8. In (B), the osmolality of 150mg iodine/mL of each iodinated contrast media solution was measured by the freezing point depression method using a calibrated osmometer. Data show the means ± SEM (N = 6 ∼ 8). *P < 0.05:; **P < 0.01 vs. control (Dunnett test)
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

3. Figure 2
Concentration-dependent (A) and time-dependent (B) decrease in cell viability after a transient exposure of LLC-PK1 cells to ioversol. In (A), cells were exposed to various concentrations of ioversol for 30 minutes at 37°C with or without adjustment of the osmolality (530 mOsm/kg) using mannitol. Then, the cells were further incubated for 24hours in the absence of ioversol or mannitol. In nontreated group, cells were incubated in serum-free medium for 24hours. In (B), cells were treated with 100mg iodine/mL of ioversol for 30 minutes at 37°C, then incubated for various periods indicated in the absence of ioversol. The cell viability was assessed by WST-8 assay. Data show the means ± SEM (N = 4 ∼ 6). **P < 0.01 vs. control group (Dunnett test). C is control; NT is not treated.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

4. Figure 3
Involvement of caspase-3 and caspase-9 but not caspase-8 in the ioversol-induced injury in LLC-PK1 cells as assessed by annexin V and propidium iodide (PI) stains (×100 magnification). (A) Cells not treated. (B) Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24hours at 37°C in the absence of ioversol. Caspase inhibitors, including (C) zVAD-fmk (pan caspase inhibitor, 30 μmol/L), (D) zDEVD-fmk (caspase 3 inhibitor, 50 μmol/L), (E) zIETD-fmk (caspase 8 inhibitor, 50 μmol/L), and (F) zLEHD-fmk (caspase 9 inhibitor, 50 μmol/L), were added 1hour before ioversol treatment and included throughout the experiment. Cells were visualized using a fluorescent microscopy under ×100 magnification.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

5. Figure 4
Reversal by inhibitors of caspase-3 and caspase-9 but not caspase-8 of ioversol-induced DNA ladder (A) and the activation of various classes of caspases after exposure of LLC-PK1 cells to ioversol (B). Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24hours at 37°C in the absence of ioversol. Caspase inhibitors, including zVAD-fmk, zDEVD-fmk, zIETD-fmk, and zLEHD-fmk, were added 1hour before ioversol treatment and included throughout the experiment. In (A), the extracted DNA was applied to agarose gel electrophoresis. Lane M, DNA molecular weight marker VIII (Roche Diagnostics Corporation, Indianapolis, IN, USA); lane 1, control; lane 2, ioversol (100mg iodine/mL); lane 3, ioversol + zVAD-fmk (30 μmol/L); lane 4, ioversol + zDEVDfmk (50 μmol/L); lane 5, ioversol + zIETD-fmk (50 μmol/L); and lane 6, ioversol + zLEHD-fmk (50 μmol/L). In (B), cells were dissolved in lysis buffer and incubated for 1hour at 37°C in the presence of fluorescent caspase substrates specific for caspase-3, caspase-9, or caspase-8. Caspase activity was determined in the absence or presence of specific caspase inhibitors (50 μmol/L), including zDEVD-fmk for caspase-3, zIETD-fmk for caspase-8 and zLEHD-fmk for caspase-9. Data show the means ± SEM (N = 4). *P < 0.05; **P < 0.01 vs. control (Dunnett test).
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

6. Figure 5
Reverse transcription-polymerase chain reaction (RT-PCR) analysis for bcl-2 and bax mRNA in LLC-PK1 cells after a transient exposure to ioversol. Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24hours at 37°C in the absence of ioversol. Then, RNA was isolated and bcl-2 and bax mRNA were determined by RT-PCR. The intensities of the PCR products were measured by densitometric analysis. Histograms show the relative concentration of mRNA for bcl-2 (A) and bax (B) to β-actin mRNA, and the ratio of bax mRNA to bcl-2 mRNA (C). Data show the means ± SEM (N = 4). *P < 0.05; **P < 0.01 vs. control (Student t test).
Kidney International  , DOI: ( /j x)
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7. Figure 6
Protective effect of dibutyl cyclic adenosine monophosphate (DBcAMP) against ioversol-induced decrease in the viability of LLC-PK1 cells. Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24h oursin the absence of ioversol. DBcAMP was added 1hour before ioversol treatment and included throughout the experiment. The cell viability was assessed by WST-8 assay. Data show the means ± SEM (N = 6). *P < 0.05 vs. control (Dunnett test).
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

8. Figure 7
Protective effect of dibutyl cyclic adenosine monophosphate (DBcAMP) against ioversol-induced injury in LLC-PK1 cells as determined by annexin V and propidium iodide (PI) stains. (A) Cells not treated. (B) Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24hours at 37°C in the absence of ioversol. (C) DBcAMP (1mmol/L) were added 1hour before ioversol treatment and included throughout the experiment. Cells were visualized using a fluorescent microscopy under ×100 magnification.
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

9. Figure 8
Involvement of protein kinase A (PKA) and phosphatidylinositol 3 (PI3) kinase in the reversal by dibutyl cyclic adenosine monophosphate (DBcAMP) of ioversol-induced DNA fragmentation in LLC-PK1 cells. Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24hours at 37°C in the absence of ioversol. H89 (30 μmol/L) or wortmannin (10nmol/L) was added 11/2 hours before, while DBcAMP (1mmol/L) was included 1hour before ioversol treatment. DNA was isolated and subjected to agarose gel electrophoresis. Lane M, DNA molecular weight marker VIII; lane 1, nontreatment; lane 2, ioversol (100mg iodine/mL); lane 3, ioversol + DBcAMP (1mmol/L); lane 4, ioversol + DBcAMP (1mmol/L) + H89 (30 μmol/L); lane 5, ioversol + DBcAMP (1mmol/L) + wortmannin (10nmol/L); lane 6, H89 (30 μmol/L); and lane 7, wortmannin (10nmol/L).
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions

10. Figure 9
Involvement of protein kinase A (PKA) and phosphatidylinositol 3 (PI3) kinase in the reversal by dibutyl cyclic adenosine monophosphate (DBcAMP) of ioversol-induced changes in the mRNA expression for bcl-2 (A), bax (B) and the ratio of bax mRNA/bcl-2 mRNA (C) in LLC-PK1 cells. Cells were exposed to ioversol (100mg iodine/mL) for 30 minutes at 37°C, followed by further incubation for 24hours at 37°C in the absence of ioversol. H89 (30 μmol/L) or wortmannin (10nmol/L) was added 11/2 hours before, while DBcAMP (1mmol/L) was added, and 1hour before ioversol treatment. The mRNA was isolated and mRNAs for bcl-2 and bax were measured by reverse transcription-polymerase chain reaction (RT-PCR). The intensities of PCR products were determined by densitometric analysis. Histograms show the relative concentration of mRNAs for bcl-2 (A) and bax (B) to β-actin mRNA. Data represent the ± SEM of four to six experiments. **P < 0.01 vs. ioversol + DBcAMP (Dunnett test).
Kidney International  , DOI: ( /j x)
Copyright © 2003 International Society of Nephrology Terms and Conditions