Proliferation and osmotic tolerance of renal inner medullary epithelial cells in vivo and in cell culture

Zheng Zhang1,*, Qi Cai1,*, Luis Michea1,2, Natalia I. Dmitrieva1, Peter Andrews3, and Maurice B. Burg1

1 Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung and Blood Institute, Bethesda, Maryland 20892; 2  Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, Universidad de los Andes, San Carlos Apoquindo 2200, Santiago, Chile; and 3 Department of Cell Biology, Georgetown University Medical Center, Washington, District of Columbia 20002


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Renal inner medullary (IM) cells survive interstitial osmolality that ranges from 600 to 1,700 mosmol/kgH2O or more. In contrast, much smaller acute changes killed the cells previously studied in tissue culture, such as mouse IM collecting duct 3 (mIMCD3) cells, that are immortalized with SV40 and proliferate rapidly. Proliferation and DNA replication sensitize mIMCD3 cells to hypertonicity. In the present studies, we observed that proliferating cells were scarce in rat IM. Then, we prepared passage 2 mouse IM epithelial (p2mIME) cells. They have a much lower incidence of DNA replication than do mIMCD3 cells. p2mIME cells survive much greater acute increases in NaCl than do mIMCD3 cells and also tolerate significantly greater acute increases of urea and of NaCl plus urea, but still not to levels as high as occur in vivo. We conclude that immortalization and continued DNA replication account for part of the previously observed difference in osmotic tolerance between IM cells in vivo and in cell culture but that other factors must also be involved.

sodium cloride; urea; renal papilla


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RENAL INNER MEDULLARY (IM) interstitial NaCl and urea concentrations normally vary widely, depending on urine concentration (6), yet the cells evidently survive and function. During water diuresis, interstitial osmolality is ~600 mosmol/kgH2O and is elevated above the level in peripheral blood mainly by increased NaCl (1). During antidiuresis, additional NaCl and urea raise osmolality up to 1,500 mosmol/kgH2O or more, depending on the species and conditions. The osmotic tolerance of IM cells has been studied extensively in tissue culture, generally starting from a baseline level of 300 mosmol/kgH2O. In contrast to the results in vivo, acutely increasing osmolality of immortalized cells, such as mouse IM collecting duct 3 (mIMCD3) cells (10), above 600 mosmol/kgH2O by the addition of NaCl or urea, or increasing it above 1,000 mosmol/kgH2O by the addition of a mixture of NaCl and urea, rapidly kills most of the cells by apoptosis (7). The purpose of the present studies was to examine possible reasons for the difference in osmotic tolerance between IM cells in culture and in vivo to better understand the factors that determine osmotic tolerance.

The mIMCD3 cell line originated from an IMCD dissected from an SV40 transgenic mouse (10). Constitutive expression of the SV40 oncogene immortalizes these cells and drives their rapid growth. In contrast to such immortalized cells, it seemed unlikely to us that renal IM cells proliferate very rapidly in vivo, although we were unaware of any studies that directly measured this. This point might be important because, in addition to growing more rapidly than normal cells, immortalized cells could also be more susceptible to osmotic stress than normal cells. This was suggested to us by the observation that continued proliferation and DNA replication increase the susceptibility of mIMCD3 cells to hypertonicity (3). Therefore, their immortalization and rapid proliferation might explain, in part, why mIMCD3 cells are more susceptible to osmotic stress than are IMCD cells in vivo (4).

To investigate these possibilities, we first tested for proliferation of renal IM cells in vivo by determining the incidence of cells that express proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation (2). On finding that very few IM cells were proliferating, as indicated by scant PCNA expression, we prepared and characterized passage 2 mouse IM epithelial (p2mIME) cells in the anticipation that fewer of these cells than of mIMCD3 cells would replicate their DNA. On finding this to be the case, we compared the acute osmotic tolerance of the p2mIME cells to that of mIMCD3 cells. Also, we compared the osmotic tolerance of proliferating (subconfluent) and more quiescent (confluent) p2mIME cells (4).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Determination of IM cell proliferation and apoptosis in vivo. Pathogen-free male Sprague-Dawley rats (200-250 g body wt) were separated into a control group (n = 2) with free access to food and water and an antidiurectic group (n = 2) in which each member was deprived of water for 18 h and then received a single-dose injection of aqueous arginine-vasopressin (1 nmol/100 g body wt im). Spontaneously voided urine was collected 3 h later for determination of osmolality (Advanced Osmometer, Norwood, MA), and kidneys were immediately harvested for microscopic examination. Mean osmolality of urine from the control rats was 768 mosmol/kgH2O and that from the antidiuretic rats was 2,014 mosmol/kgH2O.

The kidneys were fixed by vascular perfusion. The rats were anesthetized with Inactin (thiobutalbarbital, 100 mg/kg body wt ip). The abdominal aorta was cannulated below the renal arteries, the vena cava was severed, and a physiological saline solution (37°C) was perfused retrograde through the kidneys at a pressure of 140 mmHg. When the kidneys were clear of blood, a phosphate-buffered 10% formalin solution (pH 7.2) was perfused for ~3 min without loss of pressure. Then, the kidneys were excised and immersed overnight in buffered 10% formalin. After fixation, the kidneys were embedded in paraffin, sectioned (3-5 µm), and mounted on glass slides.

PCNA was detected by using the PCNA immunohistochemical detection kit (Zymed) according to the manufacturer's instructions. Briefly, kidney sections were deparaffinized in two changes of xylene for 5 min each, then rehydrated in a series of baths that contained decreasing ethanol concentrations. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min, followed by rinsing three times with PBS. The sections were blocked and stained with biotinylated anti-PCNA antibody (ready-to-use solution) and developed by using streptavidin-peroxidase and diaminobenzidine. Finally, the sections were counterstained with hematoxylin and mounted with Histomount. Intestine was used as a positive control. Additional imunodetection experiments in the kidney sections were performed by using the mouse monoclonal anti-PCNA antibody (1:100 and 1:50 dilutions; clone PC-10, Sigma, St. Louis, MO). Antibody attachment was detected with Alexa 488-conjugated anti-mouse IgG (1:200 dilution, Molecular Probes, Eugene, OR). Propidium iodide (PI; 10 µg/ml; Sigma) was used as a nuclear counterstain.

Apoptosis was determined with an in situ apoptosis detection kit (ApopTag, Intergen, Purchase, NY) according to the manufacturer's instructions [terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method]. We also used 4',6-diamidine-2'-phenylindole, dihydrochloride (0.1% solution) in SlowFade antifade solution (Molecular Probes) as another fluorescent stain to detect apoptotic nuclei. The kidney sections were analyzed by using an epifluorescence microscope (Olympus, Melville, NY).

Cultures of kidney IM cells. Pathogen-free male mice (4-8 wk old; Taconics, New York, NY) were killed by cervical dislocation, and their kidneys were quickly removed and processed under aseptic conditions. Kidney medullas were dissected under ×10 magnification and transferred to hyperosmotic (120 mM NaCl and 80 mM urea added, which yielded a total osmolality of 640 mosmol/kgH2O) enzyme solution that contained 12 ml DMEM-Ham's F-12 medium without phenol red (GIBCO BRL), plus 24 mg collagenase B (Roche, Indianapolis, IN) and 8.5 mg hyaluronidase (Worthington Biochemical, Lakewood, NJ). The solutions used in all subsequent steps were also made hyperosmotic to 640 mosmol/ kgH2O by the addition of 120 mM NaCl and 80 mM urea. The inner medullas from 2 to 4 mice were minced (1-2 mm), then digested in the enzyme solution for 90 min at 37°C under continuous agitation (300 revolutions/min) in a humidified incubator (5% CO2-95% O2). The resulting cell suspension was centrifuged 160 g for 1 min, and then the cellular pellet was washed three times in prewarmed, enzyme-free hyperosmotic DMEM-Ham's F-12 medium. Finally the cells were resuspended in hyperosmotic medium that contained 50% low-glucose DMEM (Irvine Scientific, Santa Ana, CA), 50% Coon's Improved Ham's F-12 medium (Cellgro, Mediatech, Herndon, VA), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin sulfate, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo-L-thyronine, 1 nM sodium selenate, 5 mg/l transferrin, and 10% fetal bovine serum (vol/vol). The cell suspension obtained from two kidneys was plated in a 6-cm plastic petri dish (Costar). Cells were fed every 24 h and reached confluence after 48 h. The confluent cultures were split sixfold with hyperosmotic Ca2+/Mg2+-free Dulbecco's PBS (DPBS) that contained 4% trypsin. After 72 h, these first passage cells reached confluence, and they were split again and seeded (25,000 cells/chamber) onto eight-chamber plastic slides (Permanox, Nalge Nunc International, Naperville, IL). The cells from this second passage reached confluence after 48 h, then were switched to the same medium without serum and with (640 mosmol/kgH2O) or without (300 mosmol/kgH2O) added NaCl and urea for 48 h before the experiments began.

mIMCD3 cell culture. mIMCD3 cells (12) were grown on 100-mm Falcon plastic dishes and used between passages 16 and 20. Cells were fed with 1:1 Irvine DMEM-Ham's F-12 medium that contained 2 mM L-glutamine and 10% fetal bovine serum at 37°C in 5% CO2. The osmolality of the basal medium was 300 mosmol/kgH2O (Advanced Osmometer). The cells were harvested by trypsinization, as above, in Ca2+/Mg2+-free DPBS and seeded (12,000 cells/chamber) in eight-chamber plastic slides (Nalge Nunc International). After 48 h, they reached confluence and were switched for 48 h before the experiments began to a serum-free medium identical to that used at 300 mosmol/kgH2O for the p2mIME cells.

Cell fixation and PI staining. To count them, analyze their cell cycle, and detect apoptosis, cells were fixed in 100% methanol at -20°C for 20 min. After fixation, the cells were permeabilized with 0.1% Triton X-100, incubated with 1 mg/ml RNase (Sigma) for 15 min, stained with 10 µg/ml PI for 5 min, and then mounted with 200 µl of SlowFade antifade solution according to the manufacturer's instructions.

Immunofluorescence. To detect cytokeratin (Cyt) and alpha -smooth muscle actin (SMA), cells were fixed with 500 µl of methanol (-20°C) for 2 h, washed three times (5 min each) with 500 µl of 50 mM Tris · HCl, pH = 7.4, 150 mM NaCl, and 0.1% Triton X-100 (TBST) at room temperature, incubated in 5% bovine serum albumin (fraction V, Sigma) in TBST for 1 h, then incubated with mouse monoclonal anti-alpha -SMA conjugated to Cy3 (1:100 dilution; clone 1A4, Sigma) and/or mouse monoclonal anti-pan Cyt (1:100 dilution; clone C11, Sigma). To detect epithelial-specific antigen (ESA), the cells were incubated with mouse IgG1 monoclonal antibody (1:200 dilution; clone VU-1D9, Sigma) in 3% bovine serum albumin in TBST. After cells were washed with TBST (3 times, 5 min each), they were incubated for 1 h with 1:200 dilution of goat anti-mouse IgG antibody linked to Alexa 488, counterstained with PI (10 µg/ml) in DPBS that contained ribonuclease A (0.5 mg/ml; Sigma), mounted with SlowFade, and examined with the epifluorescence microscope of a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA). Digital images were acquired with a Kodak Digital Science DC 120 Zoom digital camera.

Laser scanning cytometry. The number of normal and apoptotic cells and their position in the cell cycle were determined with an LSC that measured the integral of PI fluorescence and its peak intensity in each nucleus (3). The integral of PI fluorescence correlated with DNA content that corresponded to position in the cell cycle. Histograms of PI integral fluorescence were utilized to display cell cycle distribution and the relative numbers of cells in G1, S, and G2/M phases were calculated with WinCyte software (CompuCyte). Nuclear chromatin condenses during apoptosis, increasing the peak intensity (highest pixel value) of PI fluorescence. This was displayed in bivariate cytograms that plotted peak PI fluorescence vs. total PI fluorescence. The normal upper limit of normal PI peak fluorescence was determined by eye. It was the same for cells at 300 and 640 mosmol/kgH2O. The validity of this limit was confirmed by microscopic examination of the cells. Cells from particular regions of the cytograms were located on the slides, using spatial coordinates recorded by the LSC, and were visualized with the charge-coupled device camera of the LSC in the epifluorescence mode. Nuclei of cells with above-normal peak PI fluorescence were visibly shrunken and bright, consistent with the hypercondensation of chromatin that occurs relatively early in apoptosis. The limit was applied to cytograms of cells in different experimental conditions. In all cases, above-normal peak fluorescence corresponded to nuclei that were bright and shrunken, consistent with apoptosis. Finally, the number of normal cells was calculated as the number of nuclei with normal peak fluorescence and DNA content between 2N and 4N.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cellular proliferation and apoptosis in inner medullas from control and acutely antidiuretic rats. Cellular proliferation was characterized by PCNA expression (2). Adult rats were allowed free access to food and water (control group) or deprived of water for 18 h followed by injection of vasopressin (antidiuresis group). Three hours later, the animals were killed and kidney sections were prepared. Kidneys from both control rats (not shown) and antidiuretic rats (Fig. 1A) contain only 1-2 PCNA positive nuclei/medullary section, which contrasts to the much larger number in intestine (Fig. 1B). Identical results were obtained with two other anti-PCNA antibodies (data not shown).


View larger version (106K):
[in this window]
[in a new window]
 
Fig. 1.   Cell proliferation and apoptosis in vivo. Cell proliferation in rat inner medulla kidney sections (×20) was analyzed by proliferating cell nuclear antigen staining (brown, arrows); the counterstain is hematoxilin (blue). During antidiuresis, only occasional nuclei are stained in the kidney medulla (A) compared with the high percentage of labeling observed in the intestinal crypts (positive control; B). There are also very few apoptotic cells detected by the terminal deoxynucleotidyl transferase dUTP neck end labeling assay [green with red propidium iodide (PI) counterstain for nuclear visualization, ×40] in controls (C) and during antidiuresis (D). Identical results were observed in two independent experiments.

Apoptosis was analyzed by TUNEL assay, which detects DNA fragmentation (5). Using this method, no apoptotic cells were detected in inner medullas of rats on ad libitum fluid intake or after 24 or 48 h of thirsting (12). In confirmation, we find only occasional TUNEL-positive nuclei in kidney sections from control and antidiuretic rats (Fig. 1, C and D). The same result was obtained by using 4',6-diamidine-2'-phenylindole, dihydrochloride nuclear staining to detect hypercondensed chromatin in apoptotic nuclei (not shown).

We conclude that turnover of cells in the rat kidney inner medulla is very slow and is not markedly affected by fluctuations in osmolality, which contrasts to mIMCD3 cells that proliferate rapidly and undergo apoptosis after changes in osmolality much smaller than those that normally occur in renal medullas in vivo.

Characterization of the IM cells in culture. To characterize the cell type, confluent cells were stained with antibodies specific for SMA, which is a marker of myo-fibroblasts, and for Cyt or ESA, which are markers of epithelial cells. Specific staining was evaluated by immunofluorescence. Most of the cells express Cyt and ESA but not SMA (Fig. 2), indicating that they are epithelial cells. However, aquaporin-2, which is characteristic of collecting duct principal cells, is expressed by only ~7% of the cells (data not shown). Thus the cells are epithelial, but it is not clear whether the majority are collecting duct cells that fail to express aquaporin-2 under these conditions or some other type of epithelial cells. Therefore, we will refer to them as passage 2 mouse IM epithelial (p2mIME) cells.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 2.   Passage 2 mouse IM epithelial (p2mIME) cells express epithelial-specific antigen and cytokeratin, which are markers of epithelial cells. Confluent cultures were stained with PI (red, nucleus). Most cells are labeled by an antibody to epithelial-specific antigen (green; A) or Cy3 anti-cytokeratin antibody (green; B). C: few cells are labeled by Cy3 anti-smooth muscle actin antibody (myofibroblastic marker, orange).

Cell cycle distribution of mIMCD3 and p2mIME cells. The interstitial osmolality near the tip of the renal papilla can exceed 1,500 mosmol/kgH2O, depending on the species and conditions, yet the cells survive and function. In contrast, mIMCD3 cells die when NaCl and urea are added above 1,000 mosmol/kgH2O (7). As just described, the rate of proliferation of IM cells is very low in vivo. Because the mIMCD3 cells are immortalized by expression of SV40 (10), they might have a high incidence of DNA replication, even when confluent, which could make them more susceptible to osmotic stress. Therefore, we determined the cell cycle status of growing and confluent mIMCD3 cells (Table 1). Twenty-seven percent of subconfluent mIMCD3 cells were in the S phase of the cell cycle, which meant that they were replicating DNA. Even when they become confluent, 20% are in the S phase. In contrast, although 15% of subconfluent p2mIME cells are in the S phase, only 5% are in the S phase when they become confluent. Thus confluent p2mIME cells have a much lower incidence of DNA replication than do mIMCD3 cells and, in this respect, more closely resemble IM cells in vivo. In the remaining experiments, we compared the osmotic tolerance of confluent mIMCD3 and p2mIME cells.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Percentage of proliferating and confluent mIMCD3 and p2mIME cells in the S phase of the cell cycle

Tolerance for high NaCl. The confluent p2mIME cells were exposed to various levels of NaCl and urea to determine their osmotic tolerance. The number of cells, their distribution in the cell cycle, and the incidence of apoptosis were analyzed by laser scanning cytometry. Figure 3A shows representative bivariate cytograms of p2mIME cells grown at 640 mosmol/kgH2O, switched to 300 mosmol/kgH2O serum-free medium for 48 h after they reached confluence, and then exposed to serum-free media that contained various levels of NaCl, which resulted in osmolalities that ranged from 300 to 1,300 mosmol/kgH2O. After 16 h, the cells were fixed, stained with PI, and analyzed for integral PI fluorescence and maximal PI brightness in each nucleus. Confluent p2mIME cell cultures are not noticeably affected by NaCl concentration between 300 and 900 mosmol/kgH2O. At 1,100 mosmol/kgH2O and above, the number of viable (nonapoptotic) cells decreases (Fig. 3B) and the percentage of apoptotic ones increases (Fig. 3C). Almost no cells are viable at 1,300 mosmol/kgH2O.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Confluent p2mIME cells tolerate higher levels of NaCl than do confluent mIMCD3 cells. p2mIME cells were grown at 640 mosmol/kgH2O, then switched to serum-free medium at 300 mosmol/kgH2O for 48 h, followed by addition of NaCl, which elevated osmolality to various levels up to 1,300 mosmol/kgH2O for 16 h. Mouse inner medullary collecting duct 3 (mIMCD3) cells were grown in 300 mosmol/kgH2O medium, then switched to serum-free medium for 48 h before the addition of NaCl. Nuclei of all cells were stained with PI. A: representative cytograms of PI fluorescence peak (red max pixel values) vs. PI fluorescence integral (DNA content). High red max pixel values identify apoptotic cells, in which nuclei are hypercondensed (above the dashed red lines). Viable cells are below the lines. B: number of viable cells 16 h after NaCl is increased. C: percentage of cells that are apoptotic. Values are means ± SE, n = 3-5. * P < 0.05.

Confluent mIMCD3 cells were subjected to the same levels of NaCl. Consistent with the previous results with mIMCD3 cells (7), increasing osmolality to as little as 700 mosmol/kgH2O by the addition of NaCl markedly decreases the number of viable mIMCD3 cells (Fig. 3B), which is significantly different from p2mIME cells. At 900 mosmol/kgH2O, essentially no cells remain viable (Fig. 3B), which is also a significantly greater effect than with p2mIME cells. Thus p2mIME cells tolerate acute exposure to much higher levels of NaCl than do mIMCD3 cells.

Tolerance for high urea. p2mIME cells also have significantly greater tolerance for added urea than do mIMCD3 cells when both start at 300 mosmol/kgH2O (Fig. 4), but the difference is not as great as for added NaCl. The p2mIME cells were grown to confluence at 640 mosmol/kgH2O, then switched to 300 mosmol/kgH2O serum-free medium for 48 h before the addition of urea for 16 h. The mIMCD3 cells were grown to confluence at 300 mosmol/kgH2O, then also switched to 300 mosmol/kgH2O serum-free medium for 48 h before the addition of urea for 16 h. Significantly more p2mIME cells than mIMCD3 cells survive (Fig. 4A) and fewer are apoptotic (Fig. 4B) when 400 mosmol/kgH2O urea is added (P < 0.01), but not with other amounts of added urea.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Confluent p2mIME cells tolerate higher levels of urea than do confluent mIMCD3 cells. A and B: p2mIME cells were grown at 640 mosmol/kgH2O, then switched to serum-free medium at 300 mosmol/kgH2O for 48 h, followed by addition of between 400 and 1,000 mM urea for 16 h. mIMCD3 cells were grown in 300 mosmol/kgH2O medium, then switched to serum-free medium for 48 h before the addition of urea. Nuclei of all cells were stained with PI. A: number of viable cells 16 h after urea is increased. B: percentage of cells that are apoptotic. C and D: p2mIME cells were grown at 640 mosmol/kgH2O, then switched to serum-free medium at either 640 or 300 mosmol/kgH2O for 48 h, followed by addition of between 400 and 1,000 mM urea for 16 h. C: number of viable cells 16 h after urea is increased. D: percentage of cells that are apoptotic. Values are means ± SE, n = 3. * P < 0.05.

p2mIME cells have a greater tolerance for added urea when they start at 640 mosmol/kgH2O than when they start at 300 mosmol/kgH2O. Confluent cultures of p2mIME cells at 640 mosmol/kgH2O were switched to serum-free medium at 600 or 300 mosmol/kgH2O for 48 h before urea was added for 16 h. When 400-1,000 mM urea is added, more of the cells starting at 600 mosmol/kgH2O than of those starting at 300 mosmol/kgH2O remain viable (Fig. 4C) and fewer are apoptotic (Fig. 4D).

Tolerance for high NaCl plus urea. In vivo, interstitial NaCl and urea both increase during antidiuresis. Similar to the results of the addition of NaCl or urea separately, when both NaCl and urea are added together, survival of p2mIME cells exceeds that of mIMCD3 cells. The p2mIME cells were switched to serum-free 300 mosmol/kgH2O medium for 48 h, followed by an increase in osmolality by the addition of equiosmolal NaCl and urea to the medium (Fig. 5). Survival of mIMCD3 cells is significantly lessened (Fig. 5A) and apoptosis is significantly greater (Fig. 5B) than is the case for p2mIME cells when osmolality is raised to 1,100 mosmol/kgH2O or above (P < 0.05). As previously observed (7, 11), both cell types tolerate a higher total osmolality when both NaCl and urea are added together than when either is used alone. Thus the effects of NaCl and urea are not additive.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Confluent p2mIME cells tolerate higher levels of NaCl plus urea than do confluent mIMCD3 cells. p2mIME cells were grown at 640 mosmol/kgH2O, then switched to serum-free medium at 300 mosmol/kgH2O for 48 h, followed by addition of an equiosmolal mixture of NaCl and urea, which elevated osmolality to various levels up to 1,500 mosmol/kgH2O for 16 h. mIMCD3 cells were grown in 300 mosmol/kgH2O medium, then switched to serum-free medium for 48 h before the addition of NaCl and urea. Nuclei of all cells were stained with PI. A: number of viable cells 16 h after NaCl and urea were increased. B: percentage of cells that are apoptotic. Values are means ± SE, n = 3. * P < 0.05.

Effect of confluence on osmotic tolerance of p2mIME cells. To test whether proliferation is a factor in osmotic tolerance, we compared the effect of high NaCl on subconfluent and confluent p2mIME cells (Fig. 6). The subconfluent cells proliferated in 640 mosmol/kgH2O medium that contained 10% serum. The medium used with the confluent cells was serum free beginning 48 h before the osmolality was increased. The number of viable cells is significantly greater in confluent than in subconfluent cultures at 1,040 mosmol/kgH2O (P < 0.05), but not at the other osmolalities. The results are consistent with the hypothesis that proliferation reduces tolerance for high NaCl, but the effect is small.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Confluent p2mIME cells tolerate higher levels of NaCl than did proliferating subconfluent p2mIME cells. p2mIME cells were grown to confluence, then switched to serum-free medium at 640 mosmol/kgH2O for 48 h. Subconfluent p2mIME cells were also grown at 640 mosmol/kgH2O and maintained in serum throughout the experiment. Variable amounts of NaCl were added to all cultures, which elevated osmolality to levels up to 1,440 mosmol/kgH2O for 16 h. Nuclei of all cells were stained with PI. Values are means ± SE, n = 3-4. * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although renal IM cells survive the large changes in interstitial NaCl and urea concentrations that occur when urine concentration changes in vivo (6), much smaller changes kill IM cells in tissue culture (7, 11). One difference is that the cells in tissue culture generally have been immortalized to facilitate their use. For example, mIMCD3 cells, which have been widely studied, are immortalized by constitutive expression of the oncogene SV40 (10), which resulted in continued rapid growth. DNA replication increases the susceptibility of mIMCD3 cells to hypertonicity (4). In vivo, there were few renal IM cells that proliferated (Fig. 1). We hypothesized that immortalization and rapid DNA replication might explain, in part, why the mIMCD3 cells in tissue culture are more susceptible to osmotic stress than are IMCD cells in vivo. To test this possibility, we compared mIMCD3 cells to p2mIME cells, which we expected to be more nearly normal in this regard.

As a measure of DNA replication, we determined the percentage of p2mIME and mIMCD3 cells in the S phase of their cell cycle when they were both subconfluent and confluent (Table 1). Even when they are confluent, 20% of mIMCD3 cells are in the S phase, whereas only 5% of p2mIME cells are. Therefore, far fewer of the confluent p2mIME cells actively replicated DNA, which is closer to the situation in vivo.

We cultured the p2mIME cells at 640 mosmol/kgH2O, which approximates the lowest normal renal IM osmolality in vivo. The mIMCD3 cells were cultured at 300 mosmol/kgH2O, which is routine for this cell line. To directly compare their osmotic tolerance, we switched confluent p2mIME cells to 300 mosmol/kgH2O for 48 h before the experiment to equalize the starting conditions. The principal finding was that the osmotic tolerance of p2mIME cells is greater than that of mIMCD3 cells. Thus confluent p2mIME cell cultures are not noticeably affected by the addition of NaCl to increase osmolality from 300 to 900 mosmol/kgH2O, whereas increasing osmolality to as little as 700 mosmol/kgH2O by the addition of NaCl markedly decreased the number of viable mIMCD3 cells (Fig. 3). p2mIME cells also tolerate high urea significantly better than do mIMCD3 cells (Fig. 4), but the difference is much less than for added NaCl. Previously, continued proliferation and DNA replication were shown to increase the susceptibility of mIMCD3 cells to hypertonicity (4). Thus the higher prevalence of DNA replication in mIMCD3 cells than in p2mIME cells (Table 1) may contribute to the greater osmotic tolerance of the p2mIME cells and, by inference, to the greater osmotic tolerance of IM cells in vivo. Also consistent with this view is the finding that confluent p2mIME cells, fewer of which replicated DNA (Table 1), tolerate higher levels of NaCl than do subconfluent cells (Fig. 6).

Interestingly, p2mIME cells tolerate a much greater increase in urea if they start at 640 mosmol/kgH2O than if they start at 300 mosmol/kgH2O (Fig. 4). The 640 mosmol/kgH2O starting medium contained 300 mosmol/kgH2O more NaCl than the 300 mosmol/kgH2O medium. The effect is consistent with previous results with Madin-Darby canine kidney cells (9) in which preequilibration with high NaCl led to a greater tolerance for high urea. This was attributed to a protective effect of heat shock protein 70, which is elevated by high NaCl but not by high urea (8).

Although p2mIME cells tolerate higher osmolality than do mIMCD3 cells, they are still killed by an acute increase in NaCl plus urea above 1,500 mosmol/kgH2O (Fig. 5), which is lower than the level that is tolerated in vivo. Therefore, there may be additional factors, which remain to be discovered, that account for the higher osmotic tolerance of IM cells in vivo than in cell culture.


    FOOTNOTES

* Z. Zhang and Q. Cai contributed equally to this work.

Address for reprint requests and other correspondence: Z. Zhang, Rm. 6N315, Bldg. 10, National Institutes of Health, Bethesda, MD 20892-1603 (E-mail: Zhangz{at}nhlbi.nih.gov).

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. Section 1734 solely to indicate this fact.

March 5, 2002;10.1152/ajprenal.00038.2002

Received 29 January 2002; accepted in final form 25 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bankir, L. The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 1996.

2.   Connolly, KM, and Bogdanffy MS. Evaluation of proliferating cell nuclear antigen (PCNA) as an endogenous marker of cell proliferation in rat liver: a dual-stain comparison with 5-bromo-2'-deoxyuridine. J Histochem Cytochem 41: 1-6, 1993[Abstract/Free Full Text].

3.   Dmitrieva, N, Kultz D, Michea L, Ferraris J, and Burg M. Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation. J Biol Chem 275: 18243-18247, 2000[Abstract/Free Full Text].

4.   Dmitrieva, N, Michea L, and Burg MB. p53 tumor suppressor protein protects renal inner medullary cells from hypertonic stress by restricting DNA replication. Am J Physiol Renal Physiol 281: F522-F530, 2001[Abstract/Free Full Text].

5.   Kim, J, Cha JH, Tisher CC, and Madsen KM. Role of apoptotic and nonapoptotic cell death in removal of intercalated cells from developing rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 270: F575-F592, 1996[Abstract/Free Full Text].

6.   Masilamani, S, Knepper MA, and Burg MB. Urine concentration and dilution. In: The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 2000, p. 595-636.

7.   Michea, L, Ferguson DR, Peters EM, Andrews PM, Kirby MR, and Burg MB. Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells. Am J Physiol Renal Physiol 278: F209-F218, 2000[Abstract/Free Full Text].

8.   Neuhofer, W, Lugmayr K, Fraek ML, and Beck FX. 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 12: 2565-2571, 2001[Abstract/Free Full Text].

9.   Neuhofer, W, Muller E, Burger-Kentischer A, Fraek ML, Thurau K, and Beck F. Pretreatment with hypertonic NaCl protects MDCK cells against high urea concentrations. Pflügers Arch 435: 407-414, 1998[ISI][Medline].

10.   Rauchman, MI, Nigam SK, Delpire E, and Gullans SR. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse. Am J Physiol Renal Fluid Electrolyte Physiol 265: F416-F424, 1993[Abstract/Free Full Text].

11.   Santos, BC, Chevaile A, Hebert MJ, Zagajeski J, and Gullans SR. A combination of NaCl and urea enhances survival of IMCD cells to hyperosmolality. Am J Physiol Renal Physiol 274: F1167-F1173, 1998[Abstract/Free Full Text].

12.   Terada, Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, and Marumo F. Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells. Kidney Int 60: 553-567, 2001[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 283(2):F302-F308