Comparative analysis of various senescence inducers in proximal renal tubular cells

Abstract

Senescence in renal cells has attracted wide attention as the critical factor promoting renal fibrosis and chronic kidney disease. Establishing a reliable cellular model is essential to study mechanisms underlying renal cell senescence. Herein, we compared various inducers to define the most suitable senescence inducer for HK-2 proximal tubular cells. These inducers included hydrogen peroxide (H2O2), high-temperature (HT), glucose, mannitol and hydroxyurea (HU). To screen for optimal concentration/level, the highest concentration/level of each inducer that did not increase cell death (to avoid severe toxicity) was selected for senescence induction and comparative analysis using the two most appropriate markers for HK-2 cell senescence as recently established. The data revealed that 0.4 mM, 43 °C, 80 mM, 80 mM and 100 μM were the optimal concentrations/levels of H2O2, HT, glucose, mannitol and HU, respectively. Comparative analysis using optimal concentration/level of each marker revealed that 0.4 mM H2O2, HT at 43 °C, 80 mM glucose and 80 mM mannitol were the weak senescence inducers. The most effective inducer for HK-2 senescence was 100 μM HU, which provided the greatest fold-changes of cell area and granularity when compared with other stimuli in a time-dependent manner. Based on these data comparing H2O2, HT, glucose, mannitol and HU at their optimal concentrations/levels, 100 μM HU seems to be most effective for senescence induction in HK-2 cells for in vitro study of proximal renal tubular cells.