Abstract
Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-βgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-βgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.
Highlights
Calculi or stones in the urinary tract form mostly in the kidneys, and the most common type is composed of calcium oxalate (CaOx) [1]
Representative micrographs showing viability of human kidney (HK)-2 cells treated with various concentrations (2.5%–40%, v/v) of urine are shown in Supplementary Figure 2
telomeric repeat binding factor 1 (TRF1) mRNA expression in HK-2 was significantly lower after treatment with H2O2 or urine from patients with kidney stones (KS), but higher after treatment with calcium oxalate monohydrate (COM), than it was after treatment with urine from donors without KS or in untreated control cell (Figure 5A)
Summary
Calculi or stones in the urinary tract form mostly in the kidneys, and the most common type is composed of calcium oxalate (CaOx) [1]. A proteomic analysis showed that inflammatory and fibrotic proteins are abundant in the urine and stone matrices of patients with nephrolithiasis [17]. This evidence highlights that oxidative stress and inflammation are critically associated with the development CaOx calculi, and that CaOx nephrolithiasis could be considered as an inflammatory disease mediated by oxidative stress. Because age is associated with increased risk of CaOx stone formation and with cellular senescence [18], ROS may mediate the onset of cellular senescence [19], and when senescent cells accumulate, inflammation occurs [20,21,22]. No study has previously investigated whether cell senescence contributes to the development of kidney stone disease
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