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Divisions of Nephrology and Molecular Medicine, Oregon Health Sciences University and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypertonic NaCl upregulated two sensitive and specific
biochemical indices of apoptosis, caspase-3 activation and annexin V
binding, in a time- and dose-dependent fashion in renal medullary mIMCD3 cells. Pretreatment with urea (200 mM for 30 min) protected from
the proapoptotic effect of hypertonic stress (200 mosmol/kgH2O) in this model. The protective effect of urea
was dose dependent and was effective even when applied a short time
(
1 h) following NaCl exposure; this protective effect was not
observed in the nonrenal 3T3 cell line. In both mIMCD3 and 3T3 cells,
urea failed to protect from the proapoptotic stressor, ultraviolet
(UV)-B irradiation. The ability of urea to protect from hypertonic
stress was approximately comparable to the protective effect of peptide mitogens epidermal growth factor and insulin-like growth factor (IGF), but it potentiated the IGF effect. Interestingly, the
tyrosine kinase inhibitor, genistein, potentiated the proapoptotic
effect of urea yet abrogated the proapoptotic effect of hypertonic
stress. In aggregate, these data indicate that urea protects from the proapoptotic effect of hypertonic stress in a potentially cell type-specific and stimulus-specific fashion.
apoptosis; hypertonicity; caspase; annexin; stress; osmotic
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ON AN OSMOLAR BASIS, urea and NaCl are the principal constituent solutes of the renal inner medullary interstitium. Each activates a unique subset of signaling intermediates and thereby regulates downstream expression of effector gene products in a relatively solute-specific fashion (1, 3). The interrelationship of these two solutes has come under increasing scrutiny in the context of medullary cell survival in this inhospitable milieu. Hypertonic NaCl appears to limit the proapoptotic effect of elevated concentrations of urea (19, 22). In the course of investigating signaling pathways conferring osmotic resistance, we uncovered the converse relationship: urea protects from the proapoptotic effect of hypertonic NaCl in the renal medullary mIMCD3 cell line, but not in nonrenal 3T3 cells.
Aberrant regulation of apoptosis has been implicated in developmental
anomalies, neoplasia, and response to toxic or ischemic injury. The
osmotic strength of the renal inner medulla is sufficient to cause
apoptosis in diverse cell types in vitro. Resident cells of the inner
medulla tolerate a wide range of ambient osmolarities, in part because
of regulated accumulation of organic osmolytes (reviewed in Ref. 12).
Hyperosmotic urea and NaCl induce distinct programs of osmolyte
accumulation (12). Urea signaling exhibits hallmarks of a
peptide mitogen-like signaling pathway in cells of the renal medulla,
including transcription (9) and expression (5) of immediate-early genes and activation of the
signaling intermediates and receptor tyrosine kinase effectors ERK and
Elk-1 (4); Shc, Grb2, SOS, and Ras (24,
27); phospholipase-C
(8); and PI3K, Akt,
and p70 S6 kinase (27). Because it has recently
been shown that the peptide mitogen insulin-like growth factor (IGF) is
protective of hypertonicity-inducible apoptosis in cells of neural
origin (17), the ability of urea to mediate an analogous
phenomenon in renal medullary cells was investigated.
Earlier investigations relied upon relatively nonspecific endpoints of cell viability. The caspases, members of a family of aspartate-directed cysteine proteases, are responsible for much of the execution of the apoptotic program (reviewed in Ref. 23). The tetrapeptide recognition sequence differs among members of the caspase family. Caspase-3 specifically recognizes and cleaves the ASP-GLU-VAL-ASP (DEVD) motif (23); a modified peptide substrate incorporating this motif serves as the basis for a sensitive fluorometric assay of caspase-3 protease activity. An early consequence of initiation of the apoptotic program is disorganization of membrane lipid polarity. Phosphatidylserine, normally restricted to the inner leaflet of the cell membrane, translocates to the cell surface (16) where it can be labeled and detected by a fluorescent conjugate of the phosphatidylserine binding protein, annexin V (reviewed in Ref. 21). Herein, we used these sensitive and specific endpoints to examine the ability of urea to influence the proapoptotic effect of hypertonic and ultraviolet stress.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture and treatment. Cells in culture (mIMCD3, Ref. 20; and 3T3) were maintained, propagated, and treated with solute as previously described (5, 24). Briefly, cells were treated with gentle dropwise addition of 1/20 volume stock of 2.1 M NaCl or 4.2 M urea. For experiments requiring the use of other solutes (e.g., mannitol; see Fig. 4), a complete medium change to premade medium supplemented with the relevant concentration of solute was made for all samples. For consistency with mIMCD3 signaling data, cells were serum-deprived overnight prior to solute exposure. For ultraviolet (UV)-B exposure, cells in confluent monolayers were irradiated from below with a UV transilluminator (IBI model TF-20M; peak emission 312 nm; 6 tubes at 15 W) for the indicated interval. Because the surface of the transilluminator rapidly became warm to the touch (>42°C by thermistor-based measurement), dishes to be treated were elevated above the surface by ~0.5 cm and cooled by a constant room-temperature air jet between the dishes and the transilluminator surface to eliminate the confounding variable of heat shock. In pilot experiments, this configuration permitted maintenance of dishes at 37°C (±0.4°C by thermistor-based measurement) for longer than the 10-min interval required for maximal UV administration (12,000 J/m2) in the depicted experiments. UV flux was monitored at 300 and 250 nm using a UVX digital radiometer and UVX-25 and UVX-31 sensors (UVP) in accordance with the manufacturer's directions as described by Iordanov et al. (13). The bottom of the tissue-culture dish attenuated the exposure at 300 nm by ~55%; appropriate dose adjustments were made. Absorbance of the tissue-culture dish across the UV spectrum was determined spectrophotometrically (13).
Caspase-3 assay. Caspase-3 (cpp32) microfluorescence assay was performed according to a modification of the method of Enari et al. (11). Briefly, cells were washed twice with ice-cold PBS, scraped into 50 µl of extraction buffer (50 mM PIPES-NaOH, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, 20 µM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg/ml antipain, and 10 µg/ml chymopapain), and lysed with five freeze-thaw cycles. Following centrifugation at 10,000 g for 12 min at 4°C, supernatants were assayed for protein concentration as above. Cell lysate (25 µg) was incubated in a reaction volume of 50 µl with fluorogenic substrate [N-acetyl-DEVD-MCA; 10 µM (BioMol)], 100 mM HEPES-KOH, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM dithiothreitol, and 0.1 mg/ml ovalbumin for 60 min at 30°C in a 96-well microtiter plate (Falcon). Enzyme activity was detected by Cytofluor II (PerSeptive Biosystems, Framingham, MA) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Among the ~40+ independent caspase-3 assays performed, the basal level of caspase activity varied between 7 and 45 U/20 µg lysate. This variability appeared to correlate with the elapsed interval since prior medium change as well as unknown variables (data not shown). Although not uniformly observed, there was a tendency for activation in response to solute stress (expressed as fold induction) to be greater in experiments exhibiting low basal caspase-3 activity. In this and other assays, statistical significance was assigned to P < 0.05 with respect to the indicated control (e.g., vehicle treatment) of data from three or more independent experiments compared via t-test (Excel, Microsoft). The qualitative effects reported herein (and their ordinal relationships) were observed in virtually all experiments; magnitude of error bars is largely reflective of varying degrees of induction of caspase-3 and annexin V binding in response to applied stressors.
Annexin V binding assay. Annexin binding was determined using the ApoAlert annexin V apoptosis kit (Clontech) in accordance with the manufacturer's directions for adherent cells (full details available at http://www.clontech.com/techinfo/manuals/PDF/PT3050-1.pdf). Cells were stained with propidium iodide (PI) or 7-amino actinomycin D (7AAD) and annexin V-FITC as directed and sorted (FL1 for annexin V-FITC, FL2 for PI, or FL3 for 7AAD; see below) on a Becton-Dickinson Calibur instrument, following gating upon a representative population of cells established by forward- and side-angle light scatter. Apoptosis (early) was ascribed to cells exhibiting annexin staining >40 fluorescence units (FL1) and PI staining <300 fluorescence units (FL2). For PI staining, cells labeled with only PI were used to compensate the FL2 signal out of FITC (FL1), and cells labeled with only FITC-annexin V were used to compensate the FITC signal out of FL2. Because the PI concentration was kept low, there was no excess signal overlapping into FL1. For the 7AAD protocol, FL3 was used as the DNA channel, and there was no need to compensate. Similar to the caspase-3 assay described above, there was a tendency for activation in response to solute stress to be greater in experiments exhibiting low basal annexin V binding activity. Subsequent to these studies, a preliminary observation was made that trypsinization, which is recommended by the manufacturer of the ApoAlert kit and was performed in these studies, may influence phosphatidylserine localization in the cell membrane (25). ApoAlert product literature indicated that the annexin binding assay may appear to overestimate apoptosis relative to other indices (Clontech). Whether this is a consequence of trypsinization or other manipulations is unexplored. We have been unable to reliably demonstrate annexin V-FITC binding to confluent (untrypsinized) monolayers by fluorescence microscopy, even when markedly proapoptotic stimuli (e.g., NaCl 400 mosmol/kgH2O) were applied for 1-24 h (data not shown). We have, however, qualitatively observed an increase in the abundance of PI-stained nuclei and of condensed PI-stained nuclei in confluent monolayers subjected to NaCl 200 mosmol/kgH2O relative to control treatment that was consistent with PI (or 7AAD) positivity in the trypsinized and sorted cells (see Fig. 2; two top quadrants).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NaCl induces apoptosis in mIMCD3 cells.
To discriminate potentially subtle differences in proapoptotic events,
a highly quantitative approach was adopted with respect to apoptosis
detection. The renal medullary mIMCD3 cell line exhibits a
dose-dependent increase in activity of the pivotal apoptotic protease,
caspase-3, (as determined by cleavage of a fluorogenic substrate) in
response to hypertonic NaCl at 100 mosmol/kgH2O (50 mM) and
above (Fig. 1), consistent with the
proapoptotic effect observed by others in this model (22).
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Unlike impermeant solutes, urea fails to exert a proapoptotic
effect in mIMCD3 cells.
The effect of each of a panel of membrane-permeant and -impermeant
solutes, applied in an equiosmolar fashion, was examined with respect
to caspase-3 activity. The effect of the functionally impermeant solute
mannitol was indistinguishable from that of NaCl (Fig.
4). Urea (200 mM) and the
nonphysiological permeant solute glycerol (200 mM) failed to increase
caspase-3 activity. The effect of glucose (200 mM) was similar to that
of mannitol (n = 2 experiments; data not shown).
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Urea pretreatment protects from NaCl-associated apoptosis in mIMCD3
cells.
Others have suggested that NaCl may protect from the adverse effects of
elevated urea concentration. In light of the ability of peptide
mitogens to confer protection from hypertonic stress in cells of neural
origin (17), the ability of urea to protect from
hypertonicity was investigated in this cell line. Treatment of mIMCD3
monolayers with NaCl (200 mosmol/kgH2O for 4 h)
resulted in a 2.5-fold increase in caspase-3 activity (Fig.
5A). Pretreatment of control
cells treated with urea (200 mM for 30 min prior to sham treatment;
4.5 h total urea exposure) decreased caspase-3 activity by 23%, a
degree that, although reproducible, did not achieve statistical
significance. Pretreatment of mIMCD3 monolayers with urea (200 mM for
30 min) prior to NaCl treatment, however, inhibited caspase-3
activation by 37% (Fig. 5A). This represented a 61%
inhibition of the increment in caspase-3 activity inducible by NaCl
treatment. Urea also protected from the proapoptotic effect of nonionic
solutes such as mannitol (data not shown). The protective effect of
urea was evident out to 48 h of treatment (the last time point
examined), suggesting that urea was not operating through a delay of
apoptosis (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Using two independent biochemical indices of apoptosis, we show that pretreatment with urea protects renal medullary cells from the proapoptotic effect of hypertonic stress. The caspase-3 and annexin V assays were used rather than less quantitative approaches to maximize the likelihood of detecting a potentially subtle protective effect of urea. This protective effect is not complete; however, it does block greater than 60% of the increment in apoptosis inducible by modest degrees of hypertonic stress (e.g., 200-400 mosmol/kgH2O) and is a function of the applied urea concentration. Whether the ability of 200 mM urea to protect most effectively from 200 mosmol/kgH2O NaCl is a consequence of the equiosmolar solute ratio rather than the absolute concentration of urea remains unclear. A ratio-dependent protective effect has been described in the context of the counteracting osmolytes and urea stress (12). Detailed investigation of this possibility was precluded by the relative inability of very low doses of NaCl to exert a demonstrable apoptotic effect (Fig. 1) and by the proapoptotic effect associated with concentrations of urea markedly in excess of 200 mM.
Urea, NaCl, and cell death. Others have described a relationship among urea and NaCl concentration and cell viability that is converse to the present one (19, 22). Our studies, in contrast, utilized as outcome measures biochemical indices unique to apoptosis. Santos et al. (22) recently observed in the mIMCD3 model that high concentrations of either urea or NaCl decreased cell viability and that combining the two solutes enhanced cell survival of an osmotic shock. Neuhofer et al. (19) showed that pretreatment of renal epithelial MDCK cells with hypertonic stress (a phenomenon known to induce heat shock protein expression; Ref. 10) was associated with subsequent enhanced survival of urea stress. This group later implicated NaCl-inducible hsp72 expression in this preconditioning effect (18). In our studies, medullary cells were much less sensitive to urea than they were to NaCl (on a mosmol/kgH2O basis) and subapoptotic doses of urea protected from proapoptotic doses of NaCl. This protective effect of the "activating" solute urea paralleled in many respects the ability of peptide growth factors to protect medullary cells from hypertonic stress (Fig. 9).
Growth factor protection from hypertonic stress. Matthews et al. (17) showed that IGF-I confers protection from hypertonic stress in a human neuroblastoma cell line. In Rat-1 fibroblasts, IGF-I protects from the proapoptotic effect of UV irradiation, and this phenomenon is Akt/PI3K dependent (14). We have previously implicated a PI3K-dependent signaling pathway in acquisition of resistance to both urea and hypertonic stress (27). Interestingly, in the Rat-1 model, overexpression of the relevant cell surface IGF receptor resulted in PI3K independence, implicating involvement of a second signaling pathway (14). We were unable to examine the role of these pathways in the present model, because pharmacological inhibition of PI3K-dependent signaling sharply sensitized medullary cells to osmotic stressors (27). Specifically, whereas PI3K inhibition exerted only a modest proapoptotic effect upon control-treated mIMCD3 cells in culture, it markedly potentiated the proapoptotic effect of high-dose urea (>400 mM) and both moderate (200 mosmol/kgH2O) and severe (400 mosmol/kgH2O) hypertonic stress (27). It was previously concluded on this basis that a PI3K-dependent pathway was potentially instrumental in protecting medullary cells from the adverse effects of hypertonic and urea stress. We sought to establish parallels between peptide mitogen-associated protection and that of urea in the renal medullary model. The tyrosine kinase inhibitor genistein inhibits elements of urea-mediated signaling in renal medullary cells (8); therefore, the effects of this compound were examined with respect to the protective effect of urea. Although the general tyrosine kinase inhibitor genistein failed to influence the protective effect of urea upon hypertonic stress, these data were confounded by the striking ability of genistein to prevent hypertonicity-inducible caspase-3 activation and by the ability of this inhibitor to potentiate the adverse effect of urea exposure upon this index of apoptosis. These data serve to further underscore the dissimilar physiological responses engendered by equiosmolar concentrations of urea and NaCl.
Mechanism of urea protection. We originally hypothesized that the effect of urea upon NaCl-inducible apoptosis would represent a signaling-dependent and not a physicochemical effect. Data presented here both support and refute this notion. The parallel abilities of urea, which exhibits selected biochemical hallmarks of a renal epithelial cell growth factor, and of the peptide mitogens, IGF and EGF, to protect from hypertonicity-inducible apoptosis support a signaling-dependent mechanism of protection. Efforts to ascertain the contribution of specific urea signaling effector pathways to the protective effect were confounded by the proapoptotic effect of the inhibitors themselves as described above (Ref. 27 and data not shown). Inhibition of the urea-activatable (and peptide mitogen-activatable) Ras effector arm through inducible high-level expression of a dominant negative-acting N17Ras mutant failed to influence mIMCD3 cell resistance to either urea or hypertonic stress (24). It could be argued, in contrast, that the ability of urea to add with a receptor-saturating dose of peptide mitogen does not support a common mechanism. By extension, however, the additivity of IGF- and EGF-inducible protection (n = 2; data not shown) from hypertonicity-inducible apoptosis would similarly implicate two distinct mechanisms, a less likely possibility. It would therefore appear plausible that a submaximal protective response is activated by either urea or peptide mitogen and that additivity need not equate to independence of the responsible signaling events.
Limitations of the antiapoptotic effect of urea. The ability of urea to protect from apoptosis is subject to at least three constraints. First, based upon the limited sampling presented here, it may exhibit cell-type specificity. Such specificity could represent a consequence of the unique ability of renal epithelial cells to respond to exogenous urea through the activation of receptor tyrosine kinase effector pathways. Activation of such effector pathways, in a fashion similar to the cell response to a peptide mitogen, could permit urea treatment to mimic the known protective effect of these mitogens in other contexts. We have been unable to confirm a promitotic effect of urea in the mIMCD3 cell line, although enhanced DNA synthesis and total DNA content have been observed in other renal epithelial cell lines in the absence of an increase in cell number (6, 7, 15). With respect to cell-type specificity, it is of interest that concentrations of NaCl and urea in the renal medulla are generally (but not universally) regulated in parallel. It is conceivable that urea protects from the proapoptotic influence of hypertonic NaCl in this milieu in vivo. The inability of urea to protect from NaCl stress in the nonrenal 3T3 cell culture model supports this teleological explanation. Second, the protective effect of urea is steeply dose dependent; urea concentrations in excess of 50 mM are required for demonstrable protection from hypertonicity. Interestingly, this protection is not confined to pretreatment; urea inhibited hypertonicity-inducible caspase-3 activation even when applied up to 60 min after initial exposure to hypertonic stress. Based upon this latter observation, it appears likely that a substantial interval must be traversed before biochemical commitment to irreversible caspase-3 activation (and ultimately apoptosis) occurs. Third, the protective effect of urea applies to only a subset of proapoptotic stimuli. Specifically, urea protected from hypertonicity (both NaCl and mannitol induced) yet failed to protect from the proapoptotic effect of UV-B irradiation. As both urea (26) and UV irradiation (2) are potential oxidative stressors, this failure of protection may represent a consequence of additive pro-oxidant stress.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52494 and by the National Kidney Foundation and American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. M. Cohen, Mail Code PP262, Oregon Health Sciences Univ., 3314 S.W. US Veterans Hospital Rd., Portland, OR 97201 (E-mail: cohend{at}ohsu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 4 October 1999; accepted in final form 5 April 2000.
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Q. Cai, J. D. Ferraris, and M. B. Burg Greater tolerance of renal medullary cells for a slow increase in osmolality is associated with enhanced expression of HSP70 and other osmoprotective genes Am J Physiol Renal Physiol, January 1, 2004; 286(1): F58 - F67. [Abstract] [Full Text] [PDF]
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B. C. Santos, J. M. Pullman, A. Chevaile, W. J. Welch, and S. R. Gullans Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury Am J Physiol Renal Physiol, March 1, 2003; 284(3): F564 - F574. [Abstract] [Full Text] [PDF]
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Q. Cai, L. Michea, P. Andrews, Z. Zhang, G. Rocha, N. Dmitrieva, and M. B. Burg Rate of increase of osmolality determines osmotic tolerance of mouse inner medullary epithelial cells Am J Physiol Renal Physiol, October 1, 2002; 283(4): F792 - F798. [Abstract] [Full Text] [PDF]
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W. Tian and D. M. Cohen Urea stress is more akin to EGF exposure than to hypertonic stress in renal medullary cells Am J Physiol Renal Physiol, September 1, 2002; 283(3): F388 - F398. [Abstract] [Full Text] [PDF]
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A. Soupart, S. Silver, B. Schrooeder, R. Sterns, and G. Decaux Rapid (24-Hour) Reaccumulation of Brain Organic Osmolytes (Particularly myo-Inositol) in Azotemic Rats after Correction of Chronic Hyponatremia J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1433 - 1441. [Abstract] [Full Text] [PDF]
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W. Neuhofer, K. Lugmayr, M.-L. Fraek, and F.-X Beck Regulated Overexpression of Heat Shock Protein 72 Protects Madin-Darby Canine Kidney Cells from the Detrimental Effects of High Urea Concentrations J. Am. Soc. Nephrol., December 1, 2001; 12(12): 2565 - 2571. [Abstract] [Full Text] [PDF]
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W. Tian, H. L. Bonkovsky, S. Shibahara, and D. M. Cohen Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells Am J Physiol Renal Physiol, November 1, 2001; 281(5): F983 - F991. [Abstract] [Full Text] [PDF]
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W. Tian and D. M. Cohen Urea inhibits hypertonicity-inducible TonEBP expression and action Am J Physiol Renal Physiol, May 1, 2001; 280(5): F904 - F912. [Abstract] [Full Text] [PDF]
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C. Colmont, S. Michelet, D. Guivarc'h, and G. Rousselet Urea sensitizes mIMCD3 cells to heat shock-induced apoptosis: protection by NaCl Am J Physiol Cell Physiol, March 1, 2001; 280(3): C614 - C620. [Abstract] [Full Text] [PDF]
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P < 0.05 with respect to control (absence
of supplemental NaCl).
Urea) or
urea pretreatment (200 mM urea for 30 min and thereafter; +Urea) prior
to exposure to hypertonic stress (200 mosmol/kgH2O for
4 h). Cells were harvested for determination of caspase-3 activity
(units of cpp32 activity/20 µg protein expressed relative to control
activity; A) or annexin V binding (percentage of cells
exhibiting PI impermeance and FITC fluorescence by FACS analysis,
expressed relative to control; B). Depicted data are
means ± SE for 4-12 separate experiments.
