Telomere shortening and cellular senescence induced by oxalate and nephrolithiasis urine in HK-2 cells

Kidney stone disease is a common urologic problem worldwide, especially in the tropics such as Thailand. It is known as a multifactorial condition, and aging increases the risk of stone development. The major type of stones is calcium oxalate (CaOx), and its formation is driven by increased urinary oxalate excretion and calcium oxalate monohydrate (COM) crystallization. Both oxalate and COM are known to induce reactive oxygen species (ROS) production and cause oxidative stress. Furthermore, patients with CaOx stone have increased extent of oxidative stress. In this study, we investigated the induction of cellular senescence and telomere shortening through oxidative stress by oxalate, COM and urine obtained from CaOx kidney stone (KS) patients (n=5) in HK-2 cells. Five urine samples from the age- and sex-matched non-stone (NS) subjects were used as urine control. HK-2 cells were treated with H2O2 (representative of ROS), oxalate, COM and urine samples (10% v/v) for 72 h. The result shown that the number of senescent (SA-βgal positive) cells were significantly higher in H2O2-, oxalate-, COM- and KS urine-treated conditions than that of the NS urine-treated and untreated conditions. Oxidative stress, indicated by increased protein carbinyl level and decreased total antioxidant capacity, was significantly increased in cells treated with H2O2, oxalate, COM and KS urine relative to the untreated control and NS urine. The expression of p16 protein was clearly increased in the H2O2-, oxalate-, COM- and KS urine-treated cells compared with the untreated control and NS urine-treated cells. In contrast, relative telomere length was significantly decreased in the H2O2-, oxalate-, COM- and KS urine-treated cells compared with the untreated control and NS urine-treated cells. Expression of TRF1, TRF2 and POT1 mRNAs was significantly lower in cells treated with H2O2, oxalate, COM and KS urine than that of the untreated control and NS urine. In conclusion, this is the first study showing that oxalate, COM and KS urine induce cellular senescence and telomere shortening in renal proximal tubular cells. This senescent induction is mediated through oxidative stress and associated with upregulation of p16 and downregulation of shelterin complex genes. Our findings suggest that oxidative stress-mediated senescence and telomere shortening in renal proximal tubular cells induced by lithogenic factors, particularly oxalate and COM, may contribute to the development of CaOx kidney stone disease.