In vitro cytotoxicity and mitochondrial toxicity of tenofovir alone and in combination with other antiretrovirals in human renal proximal tubule cells

Abstract

We assessed the in vitro toxicity of tenofovir (TFV) and compared it with those of zidovudine (AZT), didanosine (ddI), ritonavir (RTV), and lopinavir (LPV) alone and in combination in human renal proximal tubule epithelial cells (RPTECs). The cells were treated with various concentrations and combinations of the tested antiretrovirals for up to 22 days, and cytotoxicity was determined. In addition, we assessed the levels of mitochondrial DNA (mtDNA) and cytochrome oxidase II (COII) mRNA in RPTECs treated with reverse transcriptase inhibitors. TFV alone was not associated with significant cytotoxicity. ddI showed pronounced cytotoxicity that was greater than those of AZT (P = 0.002) and TFV (P = 0.0001). The combination of 10 muM RTV and 40 muM LPV significantly reduced RPTEC viability (P < 0.0001), and TFV tended to partially reduce this effect. TFV alone affected neither mtDNA nor COII mRNA levels, whereas ddI caused a profound depletion of mtDNA and a parallel reduction in COII mRNA expression. The effects of ddI, but not those of AZT, on mtDNA and COII mRNA were further enhanced in the presence of TFV, a finding consistent with the inhibition of ddI clearance by TFV. The addition of TFV to ddI or AZT appeared to slightly increase the COII mRNA/mtDNA ratio relative to that in cells treated with ddI or AZT alone. Together, these in vitro results indicate that combination with other antiretrovirals does not significantly increase the toxic potential of TFV in RPTECs.

FIG. 1.

Renal cell viability (expressed in percent in relation to a control) of RPTECs cultured with ddI, AZT, and TFV alone and in combination at the three concentration for all drugs. Cultures were maintained for 15 and 22 days (represented in red and black, respectively). Exposure to TFV only during the last 5 days is represented in blue. Descriptive values for each group at day 22 are detailed as means [95% CIs]. Groups marked with an asterisk are statistically significantly different (Bonferroni adjusted) from the control at day 22.

FIG. 2.

Renal cell viability (expressed in percent in relation to a control) of RPTECs cultured with LPV, RTV, and TFV alone and in combination. Cultures were maintained for 12 days. Values are detailed as means [95% CIs]. Groups marked with an asterisk are statistically significantly different from the control group (Bonferroni adjusted).

FIG. 3.

mtDNA contents in RPTECs treated with different doses of TFV, ddI, and AZT alone or in combination for 22 days. Data are expressed as percentages relative to the mean control values. The line within the box marks the median, the upper boundary of the box indicates the 75th percentile, and the lower boundary of the box indicates the 25th percentile. Error bars above and below the box indicate the 100th and 0 percentiles. Groups marked with an asterisk are statistically significantly different from the control group (Bonferroni adjusted). Effects of TFV alone compared to the untreated control were not statistically significant (3 μM, P = 0.15; 30 μM, P = 0.20; 300 μM P = 0.1 [all Bonferroni adjusted]). Groups marked with # are statistically different from the corresponding non-TFV treated group (unadjusted pairwise comparisons).

FIG. 4.

Expression of the mtDNA-encoded mRNA for COII mRNA in RPTECs treated with different doses of TFV, ddI, and AZT alone or in combination for 22 days. Data are expressed as percentages relative to the mean control values. The line within the box marks the median, the upper boundary of the box indicates the 75th percentile, and the lower boundary of the box indicates the 25th percentile. Error bars above and below the box indicate the 100th and 0 percentiles from four independent experiments. Groups marked with an asterisk are statistically significantly different from the control group (Bonferroni adjusted). Effects of TFV alone compared to the untreated control were not statistically significant (3 μM, P = 1; 30 μM, P = 1; 300 μM P = 1 [all Bonferroni adjusted]). Groups marked with # are statistically different from the corresponding non-TFV treated group (unadjusted pairwise comparisons).