INTRODUCTION

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IT IS NOW WELL ESTABLISHED that organic osmolytes play a major role in the maintenance of cell volume and functional integrity in the renal inner medulla (for review, see Refs. 2, 9, 10, 23, 24, 26). The presence of organic osmolytes is related to the unique composition of the extracellular fluid to which these cells are exposed during urinary concentration. The epithelium of the urinary bladder is also exposed to high and changing osmolalities. Recently, Kwon et al. (16) and Mähler et al. (18) observed that urinary bladder epithelial cells also contain organic osmolytes, whose concentration was shown to depend on the hydration state of the animals. Therefore, the question arose as to what mechanisms regulate organic osmolyte content in this tissue.

An in vitro system that is readily available could be a useful tool to study the role of organic osmolytes in the bladder epithelium, since in contrast to animal models, which were used by other authors (e.g., Ref. 16), this system can be manipulated under controlled conditions. As the availability of human specimens for a culture of human bladder epithelium is very restricted, a model system obtained from bladders of pigs derived from animals killed for food has been developed. This primary culture is well characterized (7, 13, 14) and was successfully used for in vitro toxicology studies (6, 8). It was also shown that the differentiation of the cells (morphological features, enzymatic functions) is preserved for a certain period in culture.

In the current study we used this cell culture model of porcine urinary bladder epithelial cells (PUBEC) to investigate sorbitol as a representative for an organic osmolyte, since the polyalcohol showed the most marked increase when diuretic and thirsted rabbits were compared (16). In the medulla, the regulation of cellular sorbitol content is rapid, extensive, and due to alterations both in metabolism and membrane permeability (1, 12, 22). Whether these mechanisms are also involved in regulating the sorbitol content in the bladder epithelium is investigated in this study.

	MATERIALS AND METHODS

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Materials. Porcine urinary bladders were obtained immediately after slaughter from a local slaughterhouse. Cell culture medium, antibiotics, and antimycotics were purchased from GIBCO (Eggenstein, Germany) or Biochrom (Berlin, Germany). All other medium supplements were from Sigma (Deisenhofen, Germany), with the exception of insulin (Serva, Darmstadt, Germany).

All other chemicals of analytical grade were obtained from Merck (Darmstadt, Germany) or Serva. Plastic materials were obtained from Greiner (Solingen, Germany).

Isolation of cells. Urinary bladders were obtained from slaughtered pigs immediately after opening of the abdominal wall and transferred aseptically to the laboratory in ice-cold 0.9% sodium chloride solution supplemented with 100 µg/ml streptomycin and 100 U/ml penicillin. Under sterile conditions, the bladders were opened and cut into stripes of 1-15 cm width and 6-8 cm length. With a sterile glass slide, the epithelial mucosa of the bladder was carefully scraped from the underlying muscle layer and transferred into culture medium. Isolated cells were harvested by centrifugation at 50 g for 5 min. The pellet was resuspended and washed twice with culture medium (50 g for 5 min). The cell number was determined, and viability was judged by trypan blue exclusion (50 µl 0.4% trypan blue + 100 µl cell suspension + 850 µl culture medium).

Routinely, 2-3 × 10⁷ cells per bladder were isolated, and viability of the cells ranged between 70 and 90%. Cells from preparations with a viability >75% were accepted. Moreover, adverse effects resulting from digestion with proteolytic enzymes could be excluded, reflected by a plating efficiency of 60-80%. Preparations with a plating efficiency >65% were accepted for further experiments.

Cell culture. A quantity of 5 × 10⁵ cells was seeded in 25-cm² culture flasks (coated with 50 µl collagen type I) and incubated at 37°C in 5% CO₂ in a humidified incubator. The next day the culture medium was changed to remove nonattached cells. To raise extracellular osmolality, NaCl or urea was added stepwise (150 mosmol/l per day) to cultured cells close to confluence. Cells were kept at the final osmolality for 24 h and then harvested.

Culture medium. Ham's F-12 medium (with 146 mg/l glutamine) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1.25 µg/ml amphotericin, 5 µg/ml human transferrin, 10 µg/ml bovine insulin, 0.1 mM nonessential amino acids, 2.7 mg/ml glucose, 1 µg/ml hydrocortisone, and 20 ng/ml epidermal growth factor (mouse) was used as culture medium.

Measurement of osmolality. Osmolalities of urine and culture medium were determined with a Semi Micro Osmometer (Knauer, Berlin, Germany). Basic osmolality of the culture medium was 300 mosmol/kg.

Determination of urea. For measurements of intracellular urea in freshly isolated cells, the urine of the bladders was removed by a syringe, and the bladder was opened and cut into strips. Remaining urine was washed off with ice-cold phosphate-buffered saline (PBS). To avoid loss of intracellular osmolytes, epithelial cells were scraped off immediately and directly suspended in 0.1% Triton X-100.

Cultured cells were washed twice with ice-cold PBS and then scraped with a rubber policeman in 0.1% Triton X-100.

Urea was measured according to the method described by Crocker (4) with an assay kit from Sigma Diagnostics, based on the reaction of urea with diacetylmonoxime to a pink chromogen and hydroxylamine. Urea concentration is directly proportional to the intensity of the color measured spectrophotometrically between 515 and 540 nm.

Determination of sorbitol. For measurements of intracellular sorbitol in freshly isolated cells, the cells were treated as described for the determination of urea (see above).

Cultured cells were rinsed once with ice-cold PBS, exposed to 0.03% (CHAPS) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in PBS at 4°C for 10 min, and then removed from the flasks by the aid of a rubber policeman. All cell preparations were frozen at 20°C, thawed immediately before the measurement, and centrifuged (50 g, 5 min), and the supernatant was used for determination of intracellular sorbitol. Sorbitol was assayed using a test kit from Boehringer (Mannheim, Germany). D-Sorbitol is oxidized by NAD to D-fructose in the presence of the enzyme sorbitol dehydrogenase with the formation of reduced NADH. Under the assay conditions, the equilibrium of the reaction lies far on the side of NAD and D-sorbitol. However, it is favorably displaced, as the formed NADH is removed by a subsequent reaction in which NADH reduces iodonitrotetrazolium chloride to a formazan in the presence of diaphorase. The absorbance of the formazan is measured at 492 nm.

Sorbitol release. After adaptation of the cells to 900 mosmol/kg, medium was removed, replaced by 1 ml medium of different osmolalities (reduction of NaCl), and further incubated at 37°C. At the time points indicated, aliquots of the supernatant were removed, heated to 100°C for 2 min, and centrifuged at 4°C (5 min at 16,000 g). Thereafter, sorbitol content was determined as described above.

Lactate dehydrogenase release. At the indicated time points, an aliquot of the culture medium was withdrawn and supplemented with an equal volume of 0.1% Triton X-100. For determination of the intracellular lactate dehydrogenase (LDH) activity, the culture medium was withdrawn completely at the end of the experiment, and the cells were harvested and suspended in 1 ml 0.1% Triton X-100.

Both medium samples and cell samples were centrifuged for 5 min at 5,000 g, and the supernatant was used for the assay. LDH was determined using a commercially available kit (Boehringer) in which the reduction of pyruvate by NADH to lactate and NAD is monitored by the rate of decrease in absorbance at 340 nm. Absorbance changes were recorded at 37°C every minute over a total period of 6 min in a Beckman model DU 6 spectrophotometer (12). The activity found in the supernatant is expressed in percent of total LDH activity (sum of the activity in the supernatant and in the cells).

Determination of aldose reductase and protein content. Cultured cells were treated as described above for determination of sorbitol. Frozen samples were thawed and disrupted with a Polytron homogenizer (model PT 1200, Kinematica). Homogenization was carried out on ice at the maximum speed four times for 20 s with an interval of 1 min. The resulting suspension was centrifuged for 10 min at 16,000 g and 4°C. The supernatant was concentrated in a Centricon 10 microconcentrator (Amicon, Beverly, MA).

Aldose reductase was determined spectrophotometrically according to the method of Das and Skrivastava (5) by monitoring the decrease in NADPH absorption at 340 nm at 37°C using DL-glyceraldehyde as substrate. Unspecific NADPH dehydrogenase activity was recorded for 5 min; then DL-glyceraldehyde was added, and the incubation was continued for another 5 min. Values of aldose reductase activity given in this study represent the difference between the rate of NADPH oxidation in the presence and absence of substrate.

Protein content was determined after precipitation in ice-cold trichloroacetic acid according to Lowry et al. (17), using bovine serum albumin as standard.

Determination of electrolyte content in porcine urine. Magnesium was determined photometrically by the formation of a red complex with xylidyl blue I, which is proportional to the magnesium concentration. Extinction was measured at 492 nm with an Eppendorf Photometer 1101 M. Chloride was measured with diphenylcarbazone as an indicator by titration with mercury (II) nitrate at pH 3.0 up to the appearance of a violet color. Sodium, potassium, and calcium were detected by flame photometry according to the method of Pirke and Stamm (20).

Statistical analysis. Statistical significance was evaluated using Student's t-test. P < 0.05 was considered as significant.

	RESULTS

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Correlation between sorbitol tissue content and urine osmolality. In a first series of investigations, we examined whether there was a correlation between the sorbitol content of freshly isolated urinary bladder cells and total urine osmolality. As shown in Fig. 1A, the sorbitol content of the surface cells changed in parallel with the osmolality to which the cells had been exposed before and during death of the animals. This strong correlation suggested a role of sorbitol as an osmolyte in the osmoregulation of the urinary bladder cells. As shown in Fig. 1B, changes of osmolality in the porcine urine were due to changes both in urea content and electrolyte content. Na, Cl, and K are the most important electrolytes determining osmolality in porcine urine, as amounts of Mg and Ca are very low (see Fig. 1B, electrolytes without Ca/Mg). The effects of urea and electrolytes (here NaCl) were subsequently analyzed separately in the cell culture system.

View larger version (15K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Fig. 1.   A: dependence of intracellular sorbitol content of freshly isolated urinary bladder cells on osmolality of urine from corresponding bladders (n = 14). B: content of urea () and electrolytes with Ca/Mg () and without Ca/Mg () in porcine urine in relation to urinary osmolality. Urea was measured as stated in MATERIALS AND METHODS. For electrolytes, the sum of Na, Cl, Mg, K, and Ca for electrolytes with Ca/Mg () and the sum of Na, Cl, and K for electrolytes without Ca/Mg () are given.

Effect of osmolality on cell growth. When the osmolality of the culture medium was increased by addition of NaCl or urea, it was observed that the growth rate of PUBEC was altered.

In cultures kept permanently at 300 mosmol/kg (control), protein content doubled from day 5 after seeding up to day 10 (end of the experiment, measurements of sorbitol, aldose reductase). When osmolality of the culture medium was stepwise increased by addition of NaCl or urea from day 7 (150 mosmol/kg per day) to day 9 to reach 600 mosmol/kg, protein content was about 60% (NaCl) or 70% (urea) at the end of the experiment (day 10) compared with the control cultures. Addition of NaCl or urea to reach 900 mosmol/kg (start of addition at day 5) resulted in a protein content of 62% or 52% at day 10, respectively, compared with the control. These results show that an increased external osmolality inhibited cell proliferation under these culture conditions as growth rate was reduced compared with control cultures, irrespective of the osmolyte used.

Effect of NaCl and urea on the sorbitol content in cultured urinary bladder cells. Primary cultures of urinary bladder cells were exposed to three different osmolalities either by raising the NaCl content or the urea content of the medium. As depicted in Fig. 2, the sorbitol content of the cells increased when the NaCl concentration was augmented, from 0.84 ± 0.02 nmol/mg protein at 300 mosmol/kg to 21.7 ± 0.95 nmol/mg protein at 600 mosmol/kg and to 59.5 ± 2.8 nmol/mg protein at 900 mosmol/kg; thus an increase of ~70-fold was observed when the highest and lowest osmolalities were compared. Negligible changes in intracellular sorbitol content were found, however, when the osmolality of the culture medium was increased by the addition of urea instead of NaCl. Here, values at 300 mosmol/kg of 3.8 nmol/mg protein, at 600 mosmol/kg of 4.92 nmol/mg protein, and at 900 mosmol/kg of 4.97 nmol/mg protein were measured, respectively. In inner medullary collecting duct cells and in papillary epithelial cells, similar changes have been observed, and both an increased intracellular synthesis of sorbitol and a decreased sorbitol efflux have been involved in this response (1, 9, 10).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Intracellular sorbitol content in cultured urinary bladder cells at different external osmolalities adjusted with NaCl () or urea (). Sorbitol was determined in adapted cells 24 h after adjustment of the stated osmolalities. Values are means of at least 4 cultures determined in quadruplicate; error lines are ±SD.

Effect of NaCl and urea on aldose reductase activity in cultured urinary bladder cells. The activity of aldose reductase, the main enzyme involved in sorbitol synthesis, at different osmolalities of the cell culture medium is shown in Table 1. At 300 mosmol/kg the lowest activity was observed, and the activity increased almost linearly with the NaCl content of the medium. At 600 mosmol/kg the stimulation was ~6.8-fold, and at 900 mosmol/kg the activity was 11.6-fold higher than at 300 mosmol/kg. As also shown in Table 1, an increase in urea did not affect the activity of the aldose reductase.

Effect of osmolality on the rate of sorbitol efflux in cultured urinary bladder cells. When cells grown at 900 mosmol/kg were exposed to different osmolalities (adjusted with NaCl) and the amount of sorbitol released into the medium was determined, the highest release was observed into the 300 mosmol/kg medium. The release into the 600 mosmol/kg medium was much lower, and release into the 900 mosmol/kg medium was the lowest. For example, efflux after 30 min at 900 mosmol/kg was 63 ± 16 nmol, at 600 mosmol/kg the efflux was 162 ± 20 nmol, and at 300 mosmol/kg the efflux was 389 ± 52 nmol (Fig. 3A). During this experiment, the integrity of the cells was controlled by a simultaneous measurement of the LDH leakage into the culture medium. The results demonstrated that the viability was not altered, as leakage was very low, between 4 and 7% under all experimental conditions (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Fig. 3.   A: efflux of sorbitol from cultured urinary bladder cells into solutions of different osmolalities. Cells were adapted to 900 mosmol/kg (adjusted by addition of NaCl) and exposed to medium of 900 (), 600 (), or 300 () mosmol/kg (removal of NaCl). Sorbitol release was determined by measuring sorbitol in the culture medium at the stated time points. Values are means of 4 cultures determined in duplicates; error lines are ±SD. B: lactate dehydrogenase (LDH) leakage into the culture medium in cell cultures of porcine urinary bladder epithelial cells exposed to medium of 900 (), 600 (), or 300 () mosmol/kg after preceding adaptation to 900 mosmol/kg (adjusted by addition of NaCl). For LDH determination, aliquots of culture medium were withdrawn simultaneously with aliquots for sorbitol measurement (see A). Values are means of 3 experiments measured in quadruplicate; SD lines fell within the symbols.

Effect of extracellular urea concentration on intracellular urea content in cultured PUBEC. The results presented above suggest that NaCl but not urea acts as a volume-perturbing osmolyte for the PUBEC in culture. If this were the case, then one would expect that urea equilibrates readily across the cell membranes. As shown in Fig. 4, indeed, intracellular urea content changed in parallel with the extracellular urea concentration.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Intracellular urea content in PUBEC cultured in medium without urea or in media whose osmolalities were raised by addition of urea (280 or 560 mM, which creates osmolalities of 600 and 900 mosmol/kg, respectively). Values are in triplicate; error lines are ±SD.

	DISCUSSION

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The current studies indicate that increased NaCl and urea inhibit cellular growth in primary cultures of porcine urinary cells and that intracellular sorbitol concentration changes with extracellular NaCl concentration but not urea concentration. The former effect is similar to the observations of Yancey and Burg (25), who showed that in MDCK cells both high concentrations of urea and NaCl reduce the colony-forming efficiency. With respect to urea, this might be due to the widespread ability of urea to inhibit isolated macromolecular physiological functions (25, 26). The lack of response of aldose reductase to an increase in urea concentrations of the medium in our studies can, however, not be explained by such adverse effects alone, since on the one hand the osmotic activity of urea in these cells is very limited (see below) and on the other hand the unspecific NADPH dehydrogenase activity measured in conjunction with the aldose reductase is not significantly altered by the urea content (no urea, 0.438 ± 0.029; 600 mosmol urea, 0.477 ± 0.002 µmol · h¹ · mg protein¹; n = 4). The effect of NaCl might be explained by the lack of reaching complete osmotic balance due to a limited synthesis of organic osmolytes.

The response of sorbitol content, aldose reductase activity, and sorbitol efflux to varying NaCl concentrations in PUBEC is qualitatively very similar to that observed in the intact renal papilla (2), in isolated inner medullary collecting duct cells (15), and papillary epithelium cells in culture (1). Such a behavior might be expected when the common embryological origin of the urinary bladder and the terminal parts of the nephron during ontogenesis of the urinary tract is considered.

Quantitatively, however, the sorbitol content of the urinary bladder cells of ~20 mmol/l at 900 mosmol/kg (calculated assuming a cellular volume of 3 µl/mg protein; see Ref. 11) is only about one-tenth or less of that observed in the papillary collecting duct of the kidney (22). These findings are in good agreement with the measurements of Kwon et al. (16), who found comparable low contents of sorbitol and other organic osmolytes in rat bladder epithelium. The authors also observed an increase in organic osmolytes during antidiuresis. In contrast to our findings in PUBEC, no sorbitol was found in rat urothelium in diuretic animals. This may be attributed to a species difference between rats and pigs. As it is known that rats do not store their urine for longer periods in contrast to pigs, a constitutive level of sorbitol in porcine urothelium is imaginable.

The current study demonstrates that the increase at higher osmolalities is probably due to both an increase in synthesis of sorbitol and a decrease in membrane permeability. The relatively low concentration of intracellular organic osmolytes might be explained by the fact that, in contrast to the papillary collecting duct cells, in the urinary bladder only one cell side (the luminal one) is exposed to changes in extracellular osmolality, whereas the other is exposed to isotonic interstitial fluid; this arrangement does not require (or even forbids) high intracellular concentrations of organic osmolytes. In addition, it has been shown that the apical membrane of dissected rabbit bladder epithelium, for example, has an extremely low permeability to water (3, 19, 21), thus attenuating any effect of high urine osmolality. These studies have also demonstrated a low permeability to sodium and chloride.

To what extent in culture the changes in extracellular osmolarity lead to alterations in intracellular osmolality remains to be elucidated, since the relative permeabilities for Na, Cl, and water in cultured urinary cells are not known. However, the observed increases in sorbitol and aldose reductase could suggest that transient alterations of cell volume occur that in other cells trigger the response of the sorbitol system.

It is also of note that in cell culture intracellular urea closely follows the extracellular content. This result is apparently in contrast to the findings in intact rabbit bladder, where the apical membrane was found to have a very low permeability for urea (3, 19). For the cell culture, the discrepancy might be due to the fact that the apical permeability barrier has not been functionally established, although morphologically cell-to-cell contacts and a differentiation of the apical membrane have been observed (13). Thus the basolateral cell sides might be the major route for urea entry. Another explanation might be a species difference in that porcine urinary bladder epithelium in general has a higher urea permeability than rabbit urinary bladder epithelium.