The effect of repeated passaging on the susceptibility of human proximal tubular HK-2 cells to toxic compounds

Abstract

The human proximal tubular HK-2 cell line is an immortalized cell line commonly used for studying proximal tubular toxicity. Even as their use is presently increasing, there unfortunately are no studies focused on functional changes in HK-2 cells associated with passaging. The aim of the present study, therefore, was to evaluate the functional stability of HK-2 cells during 13 weeks of continuous passaging after 6 and 24 h of treatment with model nephrotoxic compounds (i.e., acetaminophen, cisplatin, CdCl(2)). Short tandem repeat profile, the doubling time, cell diameter, glutathione concentration, and intracellular dehydrogenase activity were measured in HK-2 cells at each tested passage. The results showed that HK-2 cells exhibit stable morphology, cell size, and cell renewal during passaging. Mean doubling time was determined to be 54 h. On the other hand, we observed a significant effect of passaging on the susceptibility of HK-2 cells to toxic compounds. The largest difference in results was found in both cadmium and cisplatin treated cells across passages. We conclude that the outcomes of scientific studies on HK-2 cells can be affected by the number of passages even after medium-term cultivation and passaging for 13 weeks.

Fig. 1

Estimation of cell impairment in HK-2 cells after 6 h of treatment during repeated passaging Acetaminophen (APAP, 10 mM), cisplatin (CisPt, 100 μM), tert-butylhydroperoxide (tBHP, 50 μM), and CdCl₂ (Cd, 100 μM) A) Intracellular dehydrogenase activity in HK-2 cells in passages 3–15 was determined using the WST-1 test B) Intracellular GSH levels of HK-2 cells in each of passages 3–15 were determined using monochlorobimane assay Results are expressed as means ± SD (control = 100 %, n = 8–12, 3 independent experiments) One-way ANOVA with post-hoc test were used for comparison of means with control cells at appropriate number of passages (*, p < 0.05, **, p < 0.01, ***, p < 0.001).

Fig. 2

Estimation of cell impairment in HK-2 cells after 24 h of treatment during repeated passaging Acetami-nophen (APAP, 10 mM), cisplatin (CisPt, 100 μM), tert-butylhydroperoxide (tBHP, 50 μM), and CdCl₂ (Cd, 100 μM) A) Intracellular dehydrogenase activity in HK-2 cells in each of passages 3–15 was determined using the WST-1 test B) Intracellular GSH levels of HK-2 cells in passages 3–15 were determined using mono-chlorobimane assay Results are expressed as means ± SD (control = 100 %, n = 8–12, 3 independent experiments) One-way ANOVA with post-hoc test were used for comparison of means with control cells at appropriate number of passa-ges (**, p < 0.01, ***, p < 0.001).