High NaCl Promotes Cellular Senescence

[Cell Cycle 6:24, 3108-3113; 15 December 2007]; ©2007 Landes Bioscience

Report

High NaCl Promotes Cellular Senescence Natalia I. Dmitrieva* Maurice B. Burg Laboratory of Kidney and Electrolyte Metabolism; National Heart, Lung and Blood Institute; National Institutes of Health; Department of Health and Human Services; Bethesda, Maryland USA *Correspondence to: Natalia I. Dmitrieva; Laboratory of Kidney and Electrolyte Metabolism; National Heart, Lung and Blood Institute; National Institutes of Health; 9000 Rockville Pike, Building 10, Rm. 6N260; Bethesda, Maryland 20892 USA; Email: [email protected] Original manuscript submitted: 09/14/07 Manuscript accepted: 09/19/07 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5084

Key words NaCl, senescence, aging, DNA damage, osmotic stress, C. elegans, kidney

Abstract High extracellular NaCl was previously shown to increase the number of DNA breaks in mammalian cells in tissue culture, renal medullary cells in vivo, C. elegans and marine invertebrates. It was also shown to increase reactive oxygen species in renal cells, resulting in oxidation of proteins and DNA. Cellular senescence is a common response to such damage. Therefore, in the present studies we looked for signs of senescence in cells exposed to high NaCl. We find that (1) the rate of proliferation of HeLa cells exposed to high NaCl decreases gradually to the point of arrest, and the cells display signs of senescence, including hypertrophy and increased auto fluorescence. (2) High NaCl accelerates the appearance of senescence in primary mouse embryonic fibroblasts, as measured by senescence-associated β-galactosidase activity (SA-β-gal). (3) High NaCl retards growth and markedly decreases the life span of C. elegans, accompanied by features of accelerated aging, such as decreased locomotion and increased number of SA-β-gal positive cells. (4) Mouse renal medullary cells, which are normally continuously exposed to high NaCl, express increased p16INK4 (another indicator of senescence) much earlier than do cells in the renal cortex, which has the same level of NaCl as peripheral blood. We conclude that high NaCl accelerates cellular senescence and aging, most likely secondary to the DNA breaks and oxidative damage that it causes.

Acknowledgements We thank C. Combs and D. Malide at NHLBI Light Microscopy Core Facility for help with microscopy and J. Handler for discussions and suggestions on the manu‑ script. This research was supported by the Intramural Research Programs of the NIH, NHLBI.

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Introduction Primary cells become senescent after serial passaging in vitro, a process histori‑ cally termed ‘replicative senescence’. The seminal work of Hayflick and Moorhead1 demonstrated that the number of doublings of most normal cells is limited (reviewed in ref. 2). For example, human fibroblasts in culture stop replicating after 50 to 80 population doublings.3 The initial explanation for replicative senescence emerged from studies on human fibroblasts. Their telomeres—the nucleoprotein structures that cap the chromosomes ends—act as a “cell‑division counters”. With each round of mitosis 50–100 bp of DNA typically are lost from the chromosome ends. Shortening of one or more telomeres to a critical length is believed to trigger senescence and impose growth arrest.4‑6 This model is strongly supported by the finding that expression of exogenous telomerase confers cellular immortality.7,8 More recently, additional causes of senescence have been recognized. Stress of various types can cause cells to become senescent well before they reach their normal replicative limit.9‑11 Foremost among these stresses is DNA damage, including DNA breaks and oxidative lesions caused by environmental insults or genetic defects.12‑15 In addition, overexpression of certain oncogenes, such as Ras and its downstream effectors, can induce senescence,16,17 and cells can senesce in response to epigenetic changes in chromatin organization, for example, those caused by pharma‑ cological agents or altered expression of proteins that modify DNA or histones.18,19 The activation of senescence in these situations occurs relatively rapidly, in a manner of days. Because these signals act before the ordinary replicative limit is reached, the process has been termed ‘stress‑induced premature senescence’. The attributes of senescence are inde‑ pendent of its cause. Cells entering senescence undergo dramatic changes in function and morphology. They become unresponsive to mitogenic stimuli, their chromatin structure and gene expression change, and they enlarge and flatten. Adhesion to the extracellular matrix strengthens and cell to cell contacts weaken. Senescent cells can remain in a viable, nondividing state for months.9‑11,15 Accumulation of senescent cells contributes to aging and age‑related pathology.20 Cell Cycle

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Salt, Senescence and Aging

Previous studies showed that high NaCl increases the number of DNA breaks in mammalian cells in tissue culture,21,22 in mouse renal inner medullary cells in vivo,23 in cells of the soil nematode Caenorhabditis elegans (C. elegans)24 and in Marine invertebrates.25 The breaks occur at levels of NaCl lower than those that cause apoptosis, and the cells remain viable even in the continued pres‑ ence of the breaks.23,26 When the high NaCl is reduced, the breaks are rapidly repaired. Acute elevation of osmolality from 300 to 500–600 mosmol/kg by adding NaCl to medium bathing mIMCD3 cells not only increases the number of DNA breaks,21,22 but arrests proliferation in all phases of the cell cycle.27,28 The cell cycle arrest is transient. After several hours, the cells adapt and begin proliferating again, despite the continued presence of high NaCl.29,30 Even after the cells adapt to high NaCl and reenter the cell cycle, however, numerous DNA breaks persist.23 In addition to the DNA breaks, high NaCl also increases reactive oxygen species, resulting in oxida‑ tion of proteins both in cell culture and in cells of renal inner medulla in vivo.31 Given that high NaCl causes DNA damage and oxidative stress and that these conditions can induce senescence, the purpose of the present studies was to test for senescence in cells exposed to high NaCl.

previously described in ref. 33. In brief, cells were fixed with 0.2% glutaraldehyde and 2% formaldehyde in PBS for 10 minutes at room temperature, washed twice in PBS and incubated overnight at 37˚C in staining solution (1 mg of 5‑bromo‑4‑chloro‑3‑indolyl P3‑D‑galactoside (X‑Gal) per ml; stock = 20 mg of X‑Gal per 1 ml of dimethylformamide); 40 mM citric acid/sodium phosphate, pH 6.0; 5 mM potassium ferrocyanide; 150 mM NaCl; 2 mM MgCl2). Senescent cells develop a blue color. HeLa cell size. A Cellometer Automatic Cell Counter (Nexcelom Bioscience LLC, Lawrence, MA) was used. Adherent HeLa cells were suspended by treatment with 0.05% trypsin for 15 minutes. 20 ml of the suspension was injected into the Cellometer counting chamber and mean cell diameter was determined, using the Cellometer Automatic Cell Counter image analysis software. C. elegans. Strains and culture. Wild type N2 var. Bristol C. elegans were provided by Caenorhabditis Genetic Center (CGC, Minneapolis, MN). They were grown on Nematode Growth Medium agar plates spread with E. coli bacterial strain OP50 (obtained from CGC). Cultures were maintained at room temperature (about 20˚C). Control Nematode Growth Medium contains 51 mM NaCl, 1 mM MgSO4, 1 mM CaCl2, 25 mM KPO4, 5 mg/ml cholesterol, 2.5 g/l peptone, and 17 g/l agar.34 300 mM NaCl was added to produce high NaCl medium. Methods Estimation of life span. L2–L3 larvae were transferred to control Cell culture. HeLa cells were purchased from ATCC (#CCL‑2). or high NaCl agar plates. Every other day the original worms were Primary mouse embryonic fibroblasts (Mefs) were donated by Andre transferred to new plates to separate them from their progeny. The Nuzzensweig. The Mefs were isolated from embryos 13.5 days after number surviving was determined every day. Worms were consid‑ plug formation as previously described in ref. 32 and frozen in liquid ered to be dead if they did not respond to repeated prodding with a nitrogen at passage 1. Both HeLa cells and Mefs were grown in platinum wire. Estimation of body size. Worms were photographed through DMEM containing 10% fetal bovine serum (HyClone, Logan, UT). a Leica MZ FL III Stereomicroscope. Their length (L) and width Osmolality of control (“isotonic”) medium was 300 mosmol/kg. Hypertonic medium was prepared by adding NaCl to the total osmo‑ (W) within a relatively constant portion of the body near the midpoint were measured on the image using Scion Image software lality indicated on figures and in the text. Rate of proliferation of HeLa cells. Cells were cultured at 300, (Scion Corporation, Frederick, MD) and volume (V) was calculated 400, 450 or 500 mosmol/kg, splitting them to new dishes before assuming right cylindrical form (V = pLW2/4). Staining for senescence associated b‑Galactosidase (SA‑b‑gal) they became confluent to ensure logarithmic growth. The number of cells at every passage was measured with a Cellometer Automatic cell activity. A Senescence b‑Galactosidase Staining Kit (Cell Signaling, Beverly, MA) was used with modifications of the protocol described counter (Nexcelom Bioscience LLC, Lawrence, MA). Autofluorescence. To detect autofluorescence, HeLa cells were above for cultured cells. After a brief wash in PBS, worms were trans‑ plated on eight chamber slides. The cells were fixed for 10 minutes ferred in fixative, frozen immediately on dry ice to crack their cuticle, in 4% formaldehyde (#18814, Polysciences, Inc, Warrington, PA) thawed, and then left in fixative for 10 min at room temperature. at room temperature, washed twice with PBS and mounted with After fixation, the worms were washed twice with PBS and incubated antifade reagent (#P36934, Invitrogen, Carlsbad, CA). Images overnight at 37˚C in staining solution. The worms were mounted were photographed through a Leica SP1 Laser Scanning Confocal on slides with 70% glycerol, then observed microscopically for the Microscope. Excitation was at 488 nm, and emission autofluores‑ development of blue color in senescent cells. Immunohistological detection of p16Ink4 and Hsp70 in kidney cence was recognized in the range of 510–570 nm. F‑actin immunostaining. Cells grown on eight chamber slides sections. Mouse kidneys were fixed overnight in 4% paraformal‑ were fixed for 10 minutes in 4% formaldehyde (#18814, Polysciences, dehyde at 4˚C and embedded in paraffin. Sections were cut, then Inc., Warrington, PA) at room temperature, washed with PBS, mounted on silanized slides (American Histolabs, Inc., Gaithersburg, permeabilized with 0.1% Triton, blocked with 3% bovine serum MD). Sections were deparaffinized with xylene and rehydrated in a albumin for 1 h at room temperature, then incubated for 20 minutes graded series of ethanol concentrations. Endogenous peroxidase was with the F‑actin probe, phalloidin, conjugated with Alexa Fluor 594 quenched by placing the slides in 3% hydrogen peroxide in PBS for nm (Invitrogen, Carlsbad, CA). After two washes with PBS, cells 10 min. Heat‑induced epitope retrieval was performed by boiling were stained with DAPI (DNA stain) (Invitrogen, Carlsbad, CA) the slides for 6 min in citrate buffer solution, pH 6.0 (Invitrogen, and mounted with antifade reagent (#P36934, Invitrogen, Carlsbad, Carlsbad, CA). Slides were washed with PBS, then stained with CA). Images were photographed through the Leica SP1 Laser anti‑p16 (sc‑1207: Santa Cruz, Santa Cruz, CA) or anti‑Hsp70 (#386035, Calbiochem) antibody, using the Histostain‑Plus Kit Scanning Confocal Microscope. Staining of mefs for SA‑b‑gal activity. The Senescence b‑Galac‑ (Invitrogen, Carlsbad, CA), according to the manufacturer’s instruc‑ tosidase Staining Kit (Cell Signaling, Beverly, MA) was used, as tions. In brief, after exposure to serum blocking solution, the sections www.landesbioscience.com

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Figure 2. High NaCl promotes senescence of Mefs. Early passage Mefs were cultured at 300 or 500 mosmol/kg (NaCl added) for 10 days at passage 2. Cells were stained for senescence associated b‑galactosidase (SA‑b‑gal) activity to identify senescence or with DAPI (blue) and the F‑actin probe, phalloidin, (red) to facilitate counting them. Left panel: SA‑b‑galactosidase activity. Middle panel: DAPI and phalloidin staining. Right panel: Percent SA‑b‑gal positive cells.

cells is limited because the cells carry human papilomavirus (HPV), resulting in expression of the E6 and E7 transforming proteins. Figure 1. High NaCl promotes senescence of HeLa cells. (A) Effect of high NaCl on proliferation of HeLa cells. The cells were cultured for 18 days E6 stimulates telomerase activity, which prevents ordinary replica‑ at 300 mosmol/kg or in media made hyperosmolal to 400, 450 or 500 tive senescence.37 E7 neutralizes the retinoblastoma pathway,38 mosmol/kg by addition of NaCl. At each passage the cells were transferred inhibiting both replicative and stress‑induced senescence.10 Despite to new dishes before they became confluent to ensure logarithmic growth. such immortalization, agents that damage DNA can still induce (B and C) High NaCl induces cellular hypertrophy. (B) Cells stained with DAPI (blue) and F‑actin probe phalloidin (red) to show cell shape. (C) Cell senescence in some cancer cells, including HeLa, provided they have volume (see methods for details); (D) Autofluorescence of cells. Analyses in active p53 and Rb.39 Nevertheless, because HeLa cells express foreign (B–D) were performed after 18 days of exposure to high NaCl. proteins that affect senescence, the findings with high salt might not apply directly to normal cells. Therefore, we next tested the effect of high NaCl on normal early passage fibroblasts. Normal mammalian were incubated successively with primary antibody, biotinylated fibroblasts have a limited proliferative lifespan, after which they enter secondary antibody and streptavadin‑HRP conjugate. Peroxidase a state of senescence (i.e., permanent growth arrest). The number of activity was analyzed by addition of 3,3'‑Diaminobenzidine tetra‑ divisions at which a particular cell stops dividing is stochastic. Thus, hydrochloride (DAB) substrate, which produces a brown colored populations of early passage fibroblasts include both proliferating deposit upon reaction with peroxidase. A Nikon E800 Widefield and senescent cells. As more and more cells reach the end of their Microscope was used for photography. lifespan, the fraction of cells that are proliferating decreases steadily until all the cells are senescent and proliferation stops completely.40 The number of population doublings (PD) a culture will undergo Results depends on species. Normal human fibroblasts can sustain about High NaCl induces senescence of HeLa cells. Following an 60 PD,3 whereas mouse embryonic fibroblasts (MEFs) senesce in increase of extracellular NaCl that raises total osmolality from 300 to culture after 8‑10 population doublings.41 We cultured MEFs at 400–450 mosmol/kg, HeLa (human cervical epithelial carcinoma) passage 2 (3–4 PD) in high NaCl media, and identified senescent cells continue to proliferate, albeit at a slower rate than at 300 cells by their expression of b‑galactosidase (Fig. 2).33 After 10 days at mosmol/kg (Fig. 1A). At 500 mosmol/kg proliferation is even slower 500 mosmol/kg, the fraction of cells that are senescent (i.e., express for 15 days, then stops entirely (Fig. 1A). Further, at 500 mosmol/kg senescence associated b‑galactosidase) is much greater at 500 than there is gradual appearance of cells that have a senescent morphology, at 300 mosmol/kg (Fig. 2). Since high NaCl retards rather than i.e., are flat and fibroblast‑like, in sharp contrast to cells kept at 300 increases proliferation, the senescence evidently occurred after fewer mosmol/kg (Fig. 1B). By 18 days all of the cells at 500 mosmol/kg population doublings. We conclude that high NaCl accelerates senes‑ appear senescent. The measured volume of the cells increases at cence of normal cells in culture. 500 mosmol/kg so that by 18 days it is more than twice that at High NaCl accelerates senescence and aging of C. elegans. The 300 mosmol/kg (Fig. 1C). Senescent cells accumulate lipofuscin, studies presented above demonstrate that high NaCl causes senescence which causes them to autoflouresce.35,36 The HeLa cells exposed of transformed and normal cells in tissue culture. We next sought to for 18 days to high NaCl exhibit much more autoflourescence than test whether this also occurs in vivo. Accumulation of the somatic those kept at 300 mosmol/kg (Fig. 1D), which provides additional damage associated with senescence is a major cause of aging in species evidence that they are senescent. We conclude that high NaCl varying from nematodes and insects to mice and humans.12,20,42 Soil induces senescence of HeLa cells. dwellers like C. elegans can be exposed naturally to high NaCl during High NaCl accelerates senescence of normal mouse embryo drought. In our next experiments we tested the effect of high NaCl fibroblasts. Generalization from appearance of senescence in HeLa on the nematode, C. elegans. C. elegans can survive and reproduce 3110

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Figure 3. High NaCl accelerates aging of C. elegans. (A) Effect of high NaCl on longevity of C. elegans. C. elegans at L2/L3 larva stage were placed on plates containing 50 mM or 350 mM of NaCl. Every two days worms were transferred to new plates to separate them from their progeny. Left panel: % of animals surviving. Right panel: duration of life (mean ± SEM). (B) Size at different ages (mean ± SEM). (C) Staining for senescence associated‑b‑galactosidase activity (SA‑b‑gal).

while exposed to high NaCl.24,43 Nevertheless, we now find that, when exposed to high NaCl, their lifespan decreases (Fig. 3A), their rate of growth and maximal size diminishes (Fig. 3B), the number of senescent cells that they contain increases faster (elevated SA‑b‑gal activity, Fig. 3C), and the retardation of locomotion that is associated with their aging44 is accelerated (data not shown). We conclude that high NaCl accelerates senescence and aging of C. elegans in vivo. Aging and senescence are accelerated in renal medullary cells in vivo, in their normal high NaCl environment. Given this evidence that high NaCl causes cellular senescence and aging, we hypoth‑ esized that those processes might be accelerated in renal medullary cells in vivo because they are normally exposed to extremely high salt concentrations.45 Senescent cells accumulate with age in many human and rodent tissues,46 including kidney,47‑51 as indicated by elevation of p16INK4 and accumulation of cells that stain for SA‑b‑gal. In order to test whether aging and senescence are acceler‑ ated in renal medullary cells, we compared the number of senescent cells at various ages in the renal medulla, where salt concentration normally is always high, to the number in the renal cortex where the salt concentration is similar to that in peripheral blood. We used the abundance of the cell cycle regulator p16INK4 to identify senescent www.landesbioscience.com

Figure 4. Number of senescent cells is greater and they appear earlier in mouse renal inner medulla compared to cortex. Immunocytochemical analysis of p16INK4 and Hsp70 levels in mouse kidney. Positive staining is brown. (A) p16INK4 level is higher in renal medulla than in cortex and increases more with age. (B) Immunostaining of adjacent sections with anti‑ p16INK4 and anti‑Hsp70. p16INK4 positive cells (arrows) have lower Hsp‑70, consistent with senescence.

cells since P16INK4 is upregulated in them making it a biomarker of senescence and aging.10,50,52 Increase of renal p16INK4 expression with age correlates with decline of renal function and is associ‑ ated with cellular senescence.47,51 Immunostaining for p16INK4 in kidneys from 3 and 12 months old mice shows a much more rapid increase in the renal medulla that in the cortex (Fig. 4A). Hsp70 is normally elevated in renal inner medullary cells because of the high osmolality.53 However, the p16INK4 positive cells in inner medullas of older mice have a reduced level of Hsp70 (Fig. 4B), which is consistent with their being senescent since senescence reduces expres‑ sion of Hsp70.54,55 We conclude that aging and senescence are accelerated in renal medullary cells, probably because they are always exposed to high NaCl.

Discussion Effects of high NaCl that in retrospect are consistent with senes‑ cence have been noted in tissue cultures for more than 40 years. For example, the rate of proliferation is maximal at about 100–130 mM of NaCl and, as NaCl increases, proliferation decreases gradually to the point at which it entirely ceases,56 which is characteristics of senescence.10 High NaCl changes the appearance of epithelial cells to a fibroblast‑like morphology57 with increase in cell volume and protein content.56 Those changes are characteristic of presenescent and senescent cells.10,58 High NaCl damages DNA and causes

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chromosomal deletions and exchanges.59,60 Cells with large amounts of unrepaired DNA and damaged chromosomes become senescent.61 Thus, previously recognized effects of high NaCl on cells in culture are consistent with senescence. Nevertheless, despite these similari‑ ties, there was no previous direct evidence tying high NaCl to cellular senescence. Also, many of the previous cell culture studies involved acute application of high NaCl for only a few hours59,60,62 or used such high levels of NaCl that most cells died, leaving a small popula‑ tion that resumed proliferation with stable changes in karyotype.63 In our present studies we tested whether levels of NaCl to which cells can adapt cause senescence. The reason for suspecting that this might be the case is that we already knew that cells adapted to elevated NaCl have increased DNA breaks23,25,26 and oxidative stress,31 which in other settings lead to senescence. Recent progress in senes‑ cence research made it possible to test this conjecture directly. Thus, markers had been discovered to identify senescent cells directly, such as elevated autofluorescence,35,36 SA‑b‑Gal activity33 and expression of p16INK4.50,61 Using SA‑b‑Gal activity we found that exposure to high NaCl accelerates senescence of early passage Mefs (Fig. 2) and of C. elegans in vivo (Fig. 3). Using p16INK4 expression, we found that in mouse renal medullary cells, which are normally exposed to high NaCl in vivo, there are more senescent cells than in the renal cortex in which NaCl is not high, and also that age‑related accumulation of senescent cells is accelerated in the renal medulla (Fig. 4). Thus, prolonged exposure to high NaCl promotes senescence of normal cells both in culture and in vivo. References 1. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961; 25:585‑621. 2. Hayflick L. How and why we age. Experimental Gerontology 1998; 33:639‑53. 3. Hayflick L. The limited in vitro lifetime of human diploid cell strains. Experimental Cell Research 1965; 37:614‑36. 4. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibro‑ blasts. Nature 1990; 345:458‑60. 5. McEachern MJ, Blackburn EH. Cap‑prevented recombination between terminal telomeric repeat arrays (telomere CPR) maintains telomeres in Kluyveromyces lactis lacking telomer‑ ase. Genes Dev 1996; 10:1822‑34. 6. van Steensel B, Smogorzewska A, de Lange T. TRF2 protects human telomeres from end‑to‑end fusions. Cell 1998; 92:401‑13. 7. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE. Extension of life‑span by introduction of telomerase into nor‑ mal human cells. Science 1998; 279:349‑52. 8. de Lange T. Cell Biology. Telomeres and senescence: Ending the debate. Science 1998; 279:334‑5. 9. Serrano M, Blasco MA. Putting the stress on senescence. Curr Opin Cell Biol 2001; 13:748‑53. 10. Ben‑Porath I, Weinberg RA. The signals and pathways activating cellular senescence. Int J Biochem Cell Biol 2005; 37:961‑76. 11. Lloyd AC. Limits to lifespan. Nat Cell Biol 2002; 4:E25‑7. 12. Hasty P, Campisi J, Hoeijmakers J, van SH, Vijg J. Aging and genome maintenance: Lessons from the mouse? Science 2003; 299:1355‑9. 13. Di Leonardo A, Linke SP, Clarkin K, Wahl GM. DNA damage triggers a prolonged p53‑dependent G1 arrest and long‑term induction of Cip1 in normal human fibroblasts. Genes Dev 1994; 8:2540‑51. 14. Samper E, Nicholls DG, Melov S. Mitochondrial oxidative stress causes chromosomal instability of mouse embryonic fibroblasts. Aging Cell 2003; 2:277‑85. 15. Toussaint O, Medrano EE, von Zglinicki T. Cellular and molecular mechanisms of stress‑induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Experimental Gerontology 2000; 35:927‑45. 16. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes pre‑ mature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997; 88:593‑602. 17. Bringold F, Serrano M. Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol 2000; 35:317‑29. 18. Bandyopadhyay D, Medrano EE. The emerging role of epigenetics in cellular and organis‑ mal aging. Exp Gerontol 2003; 38:1299‑307. 19. Narita M, Lowe SW. Executing cell senescence. Cell Cycle 2004; 3:244‑6.

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Cell Cycle

2007; Vol. 6 Issue 24

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