INTRODUCTION

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THE PROCESS OF CREATING a concentrated urine requires that renal medullary cells survive and function in high concentrations of NaCl and urea. Interestingly, renal epithelial cells exhibit distinctly different responses to NaCl and urea. Hyperosmolar NaCl is known to activate multiple families of mitogen-activated protein kinases (14, 20, 31, 39, 40), stimulate gene transcription via a tonicity/osmotic response element (11, 38), and enhance expression of molecular chaperones (8, 30, 33) and genes responsible for accumulating organic osmolytes (2, 15). In contrast, hyperosmolar urea activates protein kinase C, stimulates extracellular signal-regulated kinase (ERK) and, via the actions of serum response factor (SRF) and ERK-responsive transcription factor (Elk-1), enhances expression of two transcription factors, Egr-1 and c-fos (4, 5). These actions of hyperosmolar urea are urea specific and renal epithelial cell specific (6), suggesting that urea, acting through a urea sensor/receptor, activates a specific program of gene expression (7). Hyperosmolar urea does not increase expression of the same set of genes as NaCl, and, to date, the downstream physiological responses to urea remain largely unknown.

In the present study, we evaluated the effects of hyperosmolar NaCl and urea on the survival and function of murine inner medullary collecting duct (mIMCD3) cells (29). The results indicate that cells acutely exposed to increasing doses of NaCl or urea die of apoptosis. Unexpectedly, a combination of NaCl and urea was associated with enhanced survival, suggesting mIMCD3 cells are specifically programmed to respond to a combination of NaCl and urea.

	METHODS

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Cell culture and viability assays. mIMCD3 cells were grown to confluence in plastic dishes in Dulbecco's modified Eagle's medium/ Ham's F12 (1:1) supplemented with 10% fetal bovine serum (JRH Biosciences) and 2% penicillin/streptomycin (Life Technologies). For hyperosmolality experiments, cells were exposed to either isosmolar or hyperosmolar medium supplemented with NaCl, urea, or both. The crystal violet assay was used to assess viability, as has been done in many previous studies (17, 21, 23-25, 32, 36, 37). Cells were seeded at 10⁴ cells/well in 96-well flat-bottom plates, incubated in 5% CO₂ atmosphere at 37°C until they reached confluence, and treated for 4-24 h, under the appropriate osmotic conditions. After treatment, DNA of remained adherent cells was stained with 20 µl/well of 0.75% crystal violet in 30% acetic acid for 15 min, rinsed, and dried. Methanol was added to solubilize the stained cells, and the absorbance of each well was read at 630 nm with a Vmax-Kinetic Microplate Reader (Molecular Devices) (12). Percent viability of treated cells was defined as the absorbance relative to control cells. Independent analysis of viability using trypan blue exclusion confirmed the results of the crystal violet assay.

Assessment of mIMCD3 cells by light microscopy. mIMCD3 cells were seeded in 12-well plates until they reached confluence and treated with hyperosmolar medium (NaCl or urea). After 24 h, the supernatants and trypsinized cells were collected. Both collections were cytocentrifuged onto a slide, at 750 rpm, for 6 min, fixed, stained with Wright-Giemsa, and examined by light microscopy in a blinded-label fashion, and, whenever possible, at least 100 cells (adherent and nonadherent) were evaluated (19).

[³H]uridine and [³H]leucine incorporation. mIMCD3 cells were seeded in 96-well plates and grown to confluence in the presence of complete medium. The medium was replaced by isosmotic or hyperosmotic medium (NaCl, urea, or both). Concomitantly, cells received a pulse of labeled substrate (NEN) as follows: 1 µCi/ml [³H]uridine and 0.5 µCi/ml [³H]leucine. After 4 h, cells were trypsinized for 30 min and harvested, using a 1205 Betaplate system (Wallack, Finland). The results were obtained by scintillation counting in the presence of Betaplate Scint.

Northern analyses. As described previously (22), at appropriate time points, cells were washed twice with phosphate-buffered saline (PBS), and total RNA was isolated using the RNAzol B method (Tel-Test). Total RNA (10 µg) was fractionated in a 1% agarose/0.7% formaldehyde denaturing gel and transferred overnight to a nylon membrane. cDNA probes for heat shock protein 70 (Hsp70, American Type Culture Collection) were labeled with [³²P]dCTP (10⁶ cpm/ml) and hybridized overnight at 42°C in 40% formamide, 10% dextran sulfate, 7 mM Tris (pH 7.6), 4× SSC (1× SSC contains 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), 0.8× Denhardt's solution (1× Denhardt's solution consisted of 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 20 mg/ml salmon sperm DNA, and 0.5% SDS. Blots were washed at room temperature (2× SSC and 0.1% SDS for 30 min), then at 50°C (0.2× SSC and 0.1% SDS for 20 min), and autoradiographed. The Hsp70 probe detects both the inducible (Hsp70) and constitutive (Hsc70) mRNAs.

Flow cytometry and cell cycle. Staining was performed according to the Dana Farber Core Flow Cytometry Center protocol. Cells were treated for 4 h with isotonic or hyperosmotic medium (NaCl, urea, or NaCl + urea), trypsinized from the plates, washed with ice-cold PBS (without divalent cations), and resuspended in ice-cold PBS to a concentration of 2 × 10⁶ cells/ml. One milliliter of iced cell suspension was vortexed while 1 ml of ice-cold 80% ethanol was added in a drop-wise fashion. For fixation, cells were incubated for 30 min on ice. Fixed cells were washed and raised in 1 ml of PBS containing propidium iodide (2.5 µg/ml) and RNase (50 µg/ml). Cells were incubated for 30 min at 37°C in the dark. Subsequently, the material was submitted to flow cytometric analysis (Scalibur Scan, Becton-Dickinson, and CellFit software).

Statistical analyses. Statistical analyses were performed using ANOVA and the Bonferroni multiple comparison procedure, with True Epistat Software (Epistat Services, Richardson, TX). Data are expressed as means ± SE, and significance was assigned to a P < 0.05.

	RESULTS

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To evaluate the ability of mIMCD3 cells to survive hyperosmotic stress, cells were exposed for 4, 8, or 24 h to media made hyperosmotic (300-1,260 mosmol/kgH₂O) with NaCl, urea, or both. Of note, the hyperosmolar media contained 10% serum, in contrast to previous works that excluded or at least reduced it. Exposure to hyperosmolar NaCl or urea (Fig. 1) caused dose- and time-dependent increases in cell death. At equivalent osmolalities, the responses to NaCl and urea were comparable. For example, after 24 h (Fig. 1C), NaCl and urea produced similar toxicity effects on mIMCD3 cells, killing more than half the cells at ~600 mosmol/kgH₂ and >90% of the cells at 800-900 mosmol/kgH₂O. Because renal medullary cells are exposed to both NaCl and urea in vivo, we evaluated the effect of a combination of these solutes on viability. Surprisingly, an equimolar combination of NaCl and urea greatly enhanced cell survival compared with either solute alone (Fig. 1). In fact, after 24 h, 40% of the cells survived 900 mosmol/kgH₂O. Not shown, these levels of viability were sustained for at least 48 h. Moreover, enhanced survival associated with the combination of NaCl and urea was also observed in Madin-Darby canine kidney (MDCK) cells but not human umbilical vein endothelial cells (HUVECs). Thus mIMCD3 and MDCK cells possess an inherent survival mechanism when exposed to a combination of NaCl and urea at concentrations known to exist in the renal medulla.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effects of NaCl, urea, and NaCl + urea hyperosmolar media on murine inner medullary collecting duct (mIMCD3) cell viability evaluated by crystal violet assay. Cells were exposed to increasing concentrations of hyperosmolar NaCl, urea, or NaCl + urea, and viability was assayed after 4 (A), 8 (B), and 24 h (C). When used in combination, NaCl and urea were added in equimolar amounts. Each point is the mean ± SE of 8-51 observations (C), and the majority of the points that comprise the NaCl + urea curve are statistically different from NaCl or urea alone (P < 0.05).

Cell viability was quantified by staining DNA of cells that remained adherent to 96-well plates after treatment. Consequently, we performed morphological studies of adherent and nonadherent cells to confirm that detached cells were indeed nonviable. As shown in Table 1, the percentage of viable (adherent) cells decreased with increasing osmolality. Moreover, at lower osmolalities, adherent cells were largely viable and few apoptotic cells were observed, whereas, at higher osmolalities, viability was greatly reduced and more apoptotic cells were typically observed. In comparison, analysis of the nonadherent cells showed that, under hyperosmolar conditions, detached cells were nonviable and displayed characteristic signs of apoptosis, like chromatin condensation and fragmentation with plasma membrane integrity (16, 19). It is known that hyperosmolarity induces programmed cell death in SHEP human neuroblastoma cells (35) and PEER human lymphoid tumor cells (13). Not shown, hyperosmotic stress also induced characteristic DNA laddering. Thus nonadherent cells are nonviable, and cell detachment appears be one step in the process of hyperosmolality-induced cell death.

Hyperosmotic stress adversely affects many cellular processes, including RNA and protein synthesis (5, 6), which could potentially explain the loss of viability by the cells. To examine this issue, RNA and protein synthesis rates were measured in cells during the first 4 h of exposure to hyperosmolality (Fig. 2). Hyperosmolar NaCl greatly inhibited synthesis of RNA and protein, and the dose dependence was similar to that observed for viability at 24 h. In comparison, urea had no significant adverse effects on biosynthesis even at osmolalities higher than 900 mosmol/kgH₂O. Of particular note, 750 mosmol/kgH₂O failed to alter biosynthesis at 4 h but resulted in nearly complete cell death at 24 h. The combination of NaCl and urea gave an intermediate response that appeared to correlate with the enhanced survival observed at 24 h (Fig. 1C).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A comparison of effects of hyperosmolar NaCl and/or urea on rates of RNA (A) and protein (B) synthesis. mIMCD3 cells were exposed to increasing concentrations of hyperosmolar NaCl and/or urea for 4 h. To measure RNA and protein synthesis, cells were pulsed with [3H]uridine and [3H]leucine throughout the 4-h time period. When used in combination, NaCl and urea were added in equimolar amounts. Each point is the mean ± SE of 7-18 observations corrected by the number of cells. Majority of points that comprise the NaCl + urea curve are statistically different from NaCl or urea alone (P < 0.05).

Previous work found that hyperosmolar NaCl can increase Hsp70 expression (8, 30, 33). Moreover, enhanced Hsp70 expression can prevent apoptosis in cells exposed to heat shock, ethanol, osmotic shock, H₂O₂, and ultraviolet irradiation (13, 26). To investigate whether expression of molecular chaperones correlated with cell survival, we measured expression of Hsp70 4 h after exposure to hyperosmolality (Fig. 3). As shown previously in MDCK cells (8, 33), hyperosmolar NaCl but not urea increased inducible Hsp70 mRNA expression in mIMCD3 cells. Notable in the response to NaCl was the fact that Hsp70 expression was robust at lower osmolalities (500-600 mosmol/kgH₂O) when survival was greatest and was absent at the highest osmolalities when cell viability was lower. The combination of NaCl and urea shifted the peak of Hsp70 response to higher osmolalities (750 mosmol/kgH₂O), which correlated with the enhanced survival. The constitutive Hsc70 was unchanged with hyperosmolality.

View larger version (76K): [in this window] [in a new window]   Fig. 3.   Hsp70 mRNA expression in mIMCD3 cells exposed to hyperosmolar media. Cells were exposed to increasing concentrations of hyperosmolar NaCl and/or urea for 4 h. When used in combination, NaCl and urea were added in equimolar amounts. Hsc70 represents the constitutive member of the Hsp70 family, and its expression shows that lanes were evenly loaded. Each figure is representative of at least 3 different experiments.

Previous work in MDCK cells demonstrated that hyperosmolar NaCl slows cell cycle progression at the G₂/M phase (6), a stage known for its sensitivity to external stresses (34). To evaluate whether a combination of NaCl and urea could restore normal cell cycle progression, we performed flow cytometric analysis of cellular DNA content on mIMCD3 cells after 4 h of exposure to hyperosmolality, a time point that preceded significant cell death. As shown in Fig. 4, mIMCD3 cells exposed to hyperosmolar NaCl or urea (500 mosmol/kgH₂O) exhibited a decrease in the proportion of cells in the G₀/G₁ stage and a corresponding increase in the proportion of cells in G₂/M. Thus NaCl and urea have similar effects on cell cycle progression and appear to slow progression at the G₂/M stage. Exposure to a combination of NaCl and urea (600 mosmol/kgH₂O) produced a similar change in the cell cycle profile, with cells shifting from G₀/G₁ to G₂/M. Identical results were also observed with 450 mosmol/kgH₂O NaCl + urea. Thus exposure to a combination of NaCl and urea slowed cell cycle progression. Cell cycle analysis also revealed that, under diverse hyperosmolar conditions (500-750 mosmol/kgH₂O), there was no increase in the sub-G₀/G₁ population of cells, confirming the absence of significant apoptosis at this early time point.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   mIMCD3 cells and cell cycle in presence of hyperosmolar medium. Hyperosmolality caused a shift from the G0/G1 to the G2/M phase of the cell cycle. Cell cycle distribution of mIMCD3 cells was measured by flow cytometry after 4 h of treatment. With either isotonic medium (control) or hyperosmolar medium (500 mosmol/kgH2O, NaCl or urea; 600 mosmol/kgH2O, NaCl and urea). When used in combination, NaCl and urea were added in equimolar amounts. Each bar is mean ± SE of 3 observations, and G2/M phase of NaCl, urea, and NaCl + urea is statistically different from control (P < 0.05).

	DISCUSSION

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Under normal physiological circumstances, cells of the renal medulla survive and function, despite being exposed to extremely high concentrations of NaCl and urea. In the present study, we found that mIMCD3 cells possess an inherent mechanism that confers enhanced survival during exposure to a combination of NaCl and urea. In particular, mIMCD3 cells exposed to hyperosmolar NaCl + urea could survive osmolalities 200-300 mosmol/kgH₂O greater than cells exposed to either solute alone. This enhanced osmotolerance was also observed in MDCK cells but not in HUVECs, suggesting it is a characteristic of renal cells. The survival response required the presence of serum, suggesting a role for other unknown prosurvival cofactors. Morphological analysis and DNA laddering revealed that cell death was an apoptotic event. In this regard, cell death was time dependent, with reduced viability apparent within 4 h and reaching maximal levels after 8-24 h of hyperosmolar stress.

Individually NaCl and urea had quantitatively similar effects on cell survival. In both cases, ~50% of the cells were killed after 24 h of exposure to 600 mosmol/kgH₂O. However, these treatments had entirely different effects on the synthesis of RNA and protein. Hyperosmolar NaCl suppressed biosynthetic rates starting at 500 mosmol/kgH₂O, whereas hyperosmolar urea had no adverse effects until osmolality exceeded 900 mosmol/kgH₂O. Thus, at 700-750 mosmol/kgH₂O, an osmolality that killed 90% of the cells under both conditions, only NaCl suppressed cellular biosynthesis. These observations confirm and extend those made previously in MDCK cells at 500 mosmol/kgH₂O, wherein NaCl but not urea or glycerol inhibited DNA and protein synthesis (8). Notably, a combination of NaCl and urea, which enhanced cell survival, had intermediate effects on biosynthetic rates. Together, these data indicate that apoptosis induced by hyperosmolality is not directly related to disruption of biosynthetic processes.

Previous studies showed that expression of Hsp70 enhances survival during exposure to adverse stresses such as heat, heavy metals, and ischemia (1, 10, 28). In the present study, Hsp70 expression in mIMCD3 cells was increased within 4 h of exposure to either NaCl or NaCl + urea but not urea alone. When induced, Hsp70 expression was generally predictive of cell survival. In other words, at higher osmolalities (>600 mosmol/kgH₂O for NaCl, >750 mosmol/kgH₂O for NaCl + urea), the cells failed to mount an Hsp70 response and also died. In comparison, hyperosmolar urea failed to induce an Hsp70 response at any concentration, indicating that the cell signaling process responsible for Hsp70 induction was not activated. As protein unfolding is considered a potent stimulus for Hsp70 induction (18), this observation may suggest there is no urea-related protein unfolding at osmolalities of 300-600 mosmol/kgH₂O. Overall, these observations confirm and extends previous studies showing that Hsp70 mRNA and protein expression are increased with hyperosmolar NaCl or mannitol but not glycerol or urea (8, 30). Moreover, in vivo Hsp70 expression is highest in the renal medulla where osmolality is highest (27).

The role of Hsp70 in hyperosmolality has not been explicitly delineated. Of interest, Hsp70 is known to modulate apoptosis (13, 26). In U-937 and PEER human lymphoid cells, overexpression of Hsp70 prevented apoptosis induced by heat shock, osmotic shock, ethanol, or ultraviolet irradiation (13). Thus it is reasonable to speculate that Hsp70 may serve a similar role in renal medullary cell adaptation to hyperosmolality, such that moderate stress, which a cell can tolerate, will generate an Hsp70 response, thereby preventing cell suicide. With more severe stress, the Hsp70 response is either not activated or is unable to be activated, resulting in programmed cell death.

Flow cytometric analysis of DNA content revealed that hyperosmolar stress caused a shift in the cell cycle profile of mIMCD3 cells. Four hours of hyperosmolar NaCl or urea caused a decrease in the proportion of cells in G₁/G₀ and a corresponding increase in G₂/M cells. This is consistent with a slowing of the G₂/M phase, which has been observed previously with a variety of stress conditions (9, 34). A combination of NaCl and urea, while enhancing survival, did not alter this cell cycle profile, suggesting that slowing of the cell cycle is a normal physiological response and is not necessarily a sign of cell death.

The synergistic actions of NaCl and urea in promoting cell survival complement previous observations that NaCl and urea activate independent signal transduction systems. In renal epithelial cells and a variety of other cell types, hyperosmolar NaCl activates three families of signaling mitogen-activated protein kinases, including jun kinase (JNK) (14, 31), p38 (40), and ERK (20). This appears to be a response to tonicity and can lead to enhanced transcription via a specific enhancer element within a variety of genes (11, 38). In contrast, urea activates ERK family in a MEK-dependent fashion (3) and subsequently activates gene transcription via an SRF/Elk-1-dependent mechanism. The urea response is specific for renal epithelial cells (6). The present study indicates that the combined actions of NaCl and urea, presumably via these independent signaling pathways, enhance cell survival. This dual program of activation and survival suggests that epithelial cells of the renal medulla are endowed with a complex proactive program that confers enhanced survival and likely other physiological functions that characterize IMCD cells. Finally, it is reasonable to speculate that many of the signs of stress that are observed with moderate hyperosmolality, such as altered biosynthesis, slowing of the cell cycling, and stress protein induction, are not a response to toxic injury but, rather, represent normal adaptive responses of the cells. Moreover, exceeding the limits of adaptation activates a program of cell death, whereas moderate hyperosmolality promotes survival and other phenotypic changes that are characteristic of the IMCD.