(X-ray sensitive Chinese Hamster Ovary, no.CRL-2348, American Type Culture
Collection, Manassas, VA) is a mutant cell line which was derived from CHO-K1
cells (no.CCL-61, American Type
Culture Collection) by treating the cells with ethyl methanesulphonate.
These cells belong to X-ray complementation group 5 and are mutant in the p86
subunit of the Ku autoantigen [7
]. We grew the cells in DMEM plus 10% fetal bovine serum (HyClone, Logan, UT). Osmolality of
control ("isotonic") medium was 300 mosmol/kg. High
NaCl medium was prepared by adding NaCl to the total osmolality indicated.
Staining of cells for SA-β-gal
The Senescence β-Galactosidase
Staining Kit (Cell Signaling, Beverly, MA) was used, as previously described
]. Senescent cells are indicated by blue color.
strains and culture
Bristol N2 (Wild type) and cku80 (ok861)
elegans were provided by Caenorhabditis
Genetic Center (CGC, Minneapolis, MN). The cku80(ok861)
strain contains a homozygous 1,646-bp deletion,
including a large section of coding sequence, in the cku80
deletion was confirmed by PCR in our previous publication [6
]. The worms were
grown on Nematode Growth Medium agar plates spread with E. coli
(obtained from CGC). Cultures were maintained at room temperature (about 20
Control Nematode Growth Medium contains 51mM NaCl, 1mM MgSO4
, 25mM KPO4
, 5μg/ml cholesterol, 2.5g/l
peptone, and 17g/l agar [31
]. We increased NaCl by adding 300 mM, as indicated.
To measure longevity we
transferred L2-L3 larvae or adult C. elegans
control or high NaCl agar plates. Every other day the original worms were
transferred to new plates to separate them from their progeny. The number
surviving was counted every day. Worms were considered dead if they did not
respond to repeated prodding with a platinum wire.
detection of p16Ink4 in kidney sections.
Mouse kidneys were fixed
overnight in 4% paraformaldehyde at 4°C, and then embedded in paraffin.
Sections were cut and mounted on silanized slides by American Histolabs (Gaithersburg, MD). Sections were stained with anti-p16 (sc-1207: Santa Cruz, Santa Cruz, CA) as previously described [4
]. A Nikon E800 Widefield Microscope was used
of water balance.
The Ku86-/- mice used in this study were previously
]. Wild type mice were purchased at age of 2-3 months from Taconic
(129S6, Model no.129SVE, Taconic Farms, Inc, Hudson, NY) and housed in the
NHLBI animal facility. All mouse studies were done under approved National
Heart, Lung, and Blood Institute and National Cancer Institute animal study
protocols and mice were housed in an Association for Assessment and
Accreditation of Laboratory Animal Care-accredited facilities. Mice were
maintained in mouse metabolic cages (Hatteras Instruments, Cary, NC) during the study under controlled temperature and light conditions (12-h light and dark
design is shown on Figure . Initially, all mice received gelled food
containing 43% of water. The gelled food contained 3 ml of deionized water, 4 g
of balanced purified rodent diet (AIN-76A, Research Diets, New Brunswick, NJ),
and 70 mg of agar per 7 g of the food. Food in the metabolic cages wasprovided in excess so the mice could eat what they
wanted. Drinking water was provided ad libitum during this period. After 2 days
of adaptation, mice were subjected to 3 consecutive periods of differing water
availability (Figure ). During period I mice had free access to water and the
gel food containing 43% water. Then, supplemental drinking water was removed so
the only water was that contained in the gel food (period II). During period
III, the amount of water in gel food was decreased to 30% (1.7ml of water, 4g
of the rodent diet powder and 57 mg of agar). Body weight, urine volume, food
consumption, urine osmolality and urine Arginine Vasopressin (AVP)
concentration were measured every 24h. Urine was collected under mineral oil in
pre-weighed collection vials. Urine volume was measured gravimetrically, by
assuming a density of one. Gel food was supplied in preweighed plastic cups to
facilitate measurement of consumed food. Urine osmolality was measured using
Fiske Model 210 Freezing-Point Micro-Osmometer (Fiske Associates, Norwood, MA). AVP concentration in urine was measured using Vasopressin Enzyme Immunoassay
Kit (no. 900-017, Assay Designs, Ann Arbor, MI).
Average values during each period were normalized to
period (I). Data were evaluated by t-test, paired t-test comparison to period
(I), unpaired t-test for comparison between groups. A p-value less than 0.05
was considered significant.