Premature Senescence and Telomere Shortening Induced by Oxidative Stress From Oxalate, Calcium Oxalate Monohydrate, and Urine From Patients With Calcium Oxalate Nephrolithiasis

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.

Keywords: calcium oxalate; cellular senescence; kidney calculi; nephrolithiasis; oxalates; oxidative stress; urine.

Figure 1

Induction of premature senescence by H₂O₂, sodium oxalate (NaOx), calcium oxalate monohydrate (COM), or urine from patients with kidney stones (KS). (A) SA-βgal staining in HK-2 cells treated for 72 h with H₂O₂ (25 µM), NaOx (900 µM), COM (25 µg/cm²), urine from patients with KS (10%, v/v) and those without KS (NS urine) (10%, v/v). More senescent cells (blue arrowheads) were observed in cultures of HK-2 cells treated with H₂O₂, NaOx, COM, and urine from patients with KS (KS urine) than in cultures treated with urine from those without KS or in untreated control cultures. The red arrowhead indicates CaOx formed after addition of NaOx. (B) The proportion of SA-βgal positive cells in cultures treated with H₂O₂, NaOx, COM, or urine from patients with KS were significantly higher than in untreated cultures or those treated with urine from donors without KS. Inset: proportion (%) of SA-βgal positive cells after treatment with individual samples of urine from patients with KS (KS samples) compared with treatment with individual samples from donors without KS (NS samples). Dashed line and number indicate mean. Error bars indicate SEM of at least triplicate treatments. (C) Cell viability assay showing treatment with H₂O₂, NaOx, COM, and urine at the concentrations used did not notably change the cell survival. Nuclei are stained by 4′,6-diamidino-2-phenylindole (DAPI). *P < 0.05 vs. Control, ^$ P < 0.05 vs. NS urine. Magnification 400×.

Figure 2

Oxidative stress induced by treatment of HK-2 cells with H₂O₂, sodium oxalate (NaOx), calcium oxalate monohydrate (COM), or urine from people with kidney stones (KS). (A) Levels of protein carbonylation after treatment with H₂O₂, NaOx, COM, or urine from patients with KS (KS urine) were significantly higher than those in untreated controls and after treatment with urine from patients without KS (NS urine). Inset: protein carbonyl level after treatment with individual samples of urine from patients with KS (KS samples) compared with treatment with individual samples from donors without KS (NS samples). (B) Total oxidant capacity (TAC) levels in HK-2 cells treated with H₂O₂, NaOx, COM, or urine from patients with KS were significantly lower than in untreated cells. TAC levels after treatment with NaOx, COM, and KS urine treatments were significantly lower than after treatment with NS urine. Inset: TAC level after treatment with individual samples of urine from patients with KS (KS samples) compared with treatment with individual samples from donors without KS (NS samples). Dashed line and number indicate mean. Error bars indicate SEM of at least triplicate treatments. *P < 0.05 vs. Control, ^$ P < 0.05 vs. NS urine.

Figure 3

Upregulation of p16 in senescent HK-2 cells induced by their treatment with sodium oxalate (NaOx), calcium oxalate monohydrate (COM), and urine from patients with KS (KS urine). (A) Dual staining from SA-βgal and p16 immunofluorescence. p16 immunoreactivity is intensely positive in SA-βgal positive cells. (B) Western blot showing increased p16 expression in HK-2 treated with H₂O₂, oxalate, and urine from patients with KS. Numbers indicates intensity ratios of p16-to-GAPDH normalized by untreated control. (C) Relative p16 expression, indicated by intensity ratios of p16-to-GAPDH, compared between conditions. KS urine induced p16 expression significantly higher than NS urine and in untreated control cells. Error bars indicate SEM. Scale bar indicates 50 µm. *P < 0.05 vs. Control, ^$ P < 0.05 vs. NS urine.

Figure 4

Shortening of telomeric length in HK-2 cells treated with sodium oxalate (NaOx), calcium oxalate monohydrate (COM), and urine from patients with KS (KS urine). The relative telomere length (RTL) was significantly reduced in HK-2 cells treated with NaOx, COM, or KS urine compared with the length in untreated control cells or those treated with urine from donors without KS (NS urine). In all instances KS urine reduced the RTL to almost half of that of the controls, and the extent of shortening was greater than after treatment with NS urine (inset). Dashed line and number indicate mean. Error bars indicate SEM of at least triplicate treatments. *P < 0.05 vs. Control, ^$P < 0.05 vs. NS urine.

Figure 5

Transcript expression of TRF1, TRF2, and POT1 in HK-2 cells exposed to lithogenic factors. (A) TRF1 mRNA expression was significantly lower in cells after treatment with H₂O₂ or urine from patients with KS (KS urine), but significantly higher after treatment with calcium oxalate monohydrate (COM), compared with that in untreated controls or after treatment with urine from donors without KS (NS urine). (B) Treatment of HK-2 cells with H₂O₂ or NaOx significantly decreased TRF2 mRNA expression compared with that in untreated control cells, but KS urine treatment significantly decreased TRF2 mRNA expression compared with that in both untreated control cells and those treated with NS urine. (C) POT1 mRNA expression was significantly decreased in HK-2 cells treated with H₂O₂, NaOx, COM, and KS urine compared with that in untreated control cells or those treated with NS urine. Insets show expression of TRF1, TRF2, and POT1 mRNA after treatment with individual samples of urine from patients with KS (KS samples) compared with treatment with inidividual samples from donors without KS (NS samples). Dashed line and number indicate mean. Error bars indicate SEM of at least triplicate treatments. *P < 0.05 vs. Control, ^$P < 0.05 vs. NS urine.