Mitotherapy inhibits against tenofovir induced nephrotoxicity on rat renal proximal tubular cells

Abstract

Tenofovir, as nucleotide reverse transcriptase inhibitors (NRTIs), is used to prevent and cure HIV/AIDS. Ample evidence confirmed that the nephrotoxicity of tenofovir has been linked to mitochondrial dysfunction. It seems that transplantation with healthy mitochondria instead of damaged mitochondria may be a beneficial approach to therapy. Therefore, it decided to investigate the impact of mitotherapy on tenofovir against renal proximal tubular cells (RPTCs) toxicity by measurement of oxidative stress and cytotoxicity biomarkers and restoring of mitochondrial function on isolated mitochondria. EC₅₀ of tenofovir was achieved at 40 μM following 2 h incubation in Earle's solution (pH = 7.4; 37 °C). Freshly isolated mitochondria (80 μg/ml) were added to damage RPTCs affected by tenofovir in treated groups. One Way ANOVA analysis showed that healthy mitochondrial transplantation decreased oxidative stress biomarkers following tenofovir toxicity in RPTCs. Our data revealed that mitotherapy makes cell survival possible in RPTCs affected by tenofovir. In addition, it supposed that a novel and ideal strategy for the treatment of chemicals-induced nephrotoxicity.

Keywords: Mitochondrial transplantation; Nephrotoxicity; Oxidative stress; Renal proximal tubular cells (RPTCs); Tenofovir.

Fig. 1

Effect of time on functionality of isolated mitochondria. Values are expressed as the mean of three separate experiments (±SD). NS. No significant difference compared to zero time as the control group (p > 0.05).

Fig. 2

Effect of Tenofovir concentrations on RPTCs viability. Values are expressed as the mean of three separate experiments (±SD). *p < 0.05 and ****p < 0.0001 compared with control groups.

Fig. 3

Protective effect of transplantation of isolated mitochondria (5–160 μg/ml) against tenofovir (40 μM) induced cytotoxicity on RPTCs. Values are expressed as the mean of three separate experiments (±SD). *p < 0.05 and ****p < 0.0001 compared with control groups.

Fig. 4

Effect of mitochondrial transplantation on RPTCs ROS level. It was measured by DCFH-DA (1.6 μM) following incubation of RPTCs. The fluorescence intensity was measured using a Shimadzu RF5000U fluorescence spectrophotometer with excitation and emission wavelengths 500 and 520 nm, respectively. The results were expressed as fluorescent intensity per 10⁶ cells. Values are expressed as the mean of three separate experiments (±SD). *p < 0.05 and ****p < 0.0001 compared with control groups.

Fig. 5

Effect of mitochondrial transplantation on RPTCs mitochondrial membrane potential. RPTCs (10⁶ cells/ml) were incubated for 2 h following the addition of Tenofovir (40 μM) and incubation with freshly isolated mitochondria. Mitochondrial membrane potential was determined as the difference in mitochondrial uptake of the rhodamine 123 between the control, treated cells, and mitotherapy (Freshly isolated mitochondria addition) group. Values are expressed as means of three separate experiments (±SD). *** Significant difference in comparison with control (p < 0.001). * Significant difference in comparison with the tenofovir group (p < 0.05).

Fig. 6

Effect of mitochondrial transplantation on RPTCs (A) GSH and (B) GSSG levels. Values are expressed as mean ± SD of separate experiments (n = 3). ** Significant difference in comparison with control group (P < 0.01). ** Significant difference in comparison with Tenofovir (P < 0.01).

Fig. 7

Effect of mitochondrial transplantation on RPTCs ATP contents. Values are expressed as mean ± SD of separate experiments (n = 3). ** Significant difference in comparison with control group (P < 0.01). ** Significant difference in comparison with tenofovir (P < 0.01).