Among the mono- and dichloroanilines, 3,5-Dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (~4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0 mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0 mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro.

The administration of intravenous iodinated radiocontrast media (RCM) to visualize internal structures during diagnostic procedures has increased exponentially since their first use in 1928. A serious side effect of RCM exposure is contrast-induced acute kidney injury (CI-AKI), which is defined as an abrupt and prolonged decline in renal function occurring 48-72 hours after injection. Multiple attempts have been made to decrease the toxicity of RCM by altering ionic strength and osmolarity, yet there is little evidence to substantiate that a specific RCM is superior in avoiding CI-AKI. RCM-associated kidney dysfunction is largely attributed to alterations in renal hemodynamics, specifically renal vasoconstriction; however, numerous studies indicate direct cytotoxicity as a source of epithelial damage. Exposure of in vitro renal proximal tubule cells to RCM has been shown to affect proximal tubule epithelium in the following manner: 1) changes to cellular morphology in the form of vacuolization; 2) increased production of reactive oxygen species, resulting in oxidative stress; 3) mitochondrial dysfunction, resulting in decreased efficiency of the electron transport chain and ATP production; 4) perturbation of the protein folding capacity of the endoplasmic reticulum (ER) (activating the unfolded protein response and inducing ER stress); and 5) decreased activity of cell survival kinases. The present review focuses on the direct cytotoxicity of RCM on proximal tubule cells in the absence of in vivo complications, such as alterations in renal hemodynamics or cytokine influence.

Chloroanilines are widely used in the manufacture of drugs, pesticides and industrial intermediates. Among the trichloroanilines, 3,4,5-trichloroaniline (TCA) is the most potent nephrotoxicant in vivo. The purpose of this study was to examine the nephrotoxic potential of TCA in vitro and to determine if renal biotransformation and/or free radicals contributed to TCA cytotoxicity using isolated renal cortical cells (IRCC) from male Fischer 344 rats as the animal model. IRCC (~4 million cells/mL; 3 mL) were incubated with TCA (0, 0.1, 0.25, 0.5 or 1.0 mM) for 60–120 min. In some experiments, IRCC were pretreated with an antioxidant or a cytochrome P450 (CYP), flavin monooxygenase (FMO), cyclooxygenase or peroxidase inhibitor prior to incubation with dimethyl sulfoxide (control) or TCA (0.5 mM) for 120 min. At 60 min, TCA did not induce cytotoxicity, but induced cytotoxicity as early as 90 min with 0.5 mM or higher TCA and at 120 min with 0.1 mM or higher TCA, as evidenced by increased lactate dehydrogenase (LDH) release. Pretreatment with the CYP inhibitor piperonyl butoxide, the cyclooxygenase inhibitor indomethacin or the peroxidase inhibitor mercaptosuccinate attenuated TCA cytotoxicity, while pretreatment with FMO inhibitors or the CYP inhibitor metyrapone had no effect on TCA nephrotoxicity. Pretreatment with an antioxidant (α-tocopherol, glutathione, ascorbate or N-acetyl-l-cysteine) also reduced or completely blocked TCA cytotoxicity. These results indicate that TCA is directly nephrotoxic to IRCC in a time and concentration dependent manner. Bioactivation of TCA to toxic metabolites by CYP, cyclooxygenase and/or peroxidase contributes to the mechanism of TCA nephrotoxicity. Lastly, free radicals play a role in TCA cytotoxicity, although the exact nature of the origin of these radicals remains to be determined.

Contrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital associated kidney damage. Potential mechanisms of CI-AKI may involve diminished renal hemodynamics, inflammatory responses, and direct cytotoxicity. The hypothesis for this study is that diatrizoic acid (DA) induces direct cytotoxicity to human proximal tubule (HK-2) cells via calcium dysregulation, mitochondrial dysfunction, and oxidative stress. HK-2 cells were exposed to 0–30 mg I/mL DA or vehicle for 2–24 h. Conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion indicated a decrease in mitochondrial and cell viability within 2 and 24 h, respectively. Mitochondrial dysfunction was apparent within 8 h post exposure to 15 mg I/mL DA as shown by Seahorse XF cell mito and Glycolysis Stress tests. Mitophagy was increased at 8 h by 15 mg I/mL DA as confirmed by elevated LC3BII/I expression ratio. HK-2 cells pretreated with calcium level modulators BAPTA-AM, EGTA, or 2-aminophenyl borinate abrogated DA-induced mitochondrial damage. DA increased oxidative stress biomarkers of protein carbonylation and 4-hydroxynonenol (4HNE) adduct formation. Caspase 3 and 12 activation was induced by DA compared to vehicle at 24 h. These studies indicate that clinically relevant concentrations of DA impair HK-2 cells by dysregulating calcium, inducing mitochondrial turnover and oxidative stress, and activating apoptosis.