Modulation of Hypertonicity-Induced Aquaporin-1 by Sodium Chloride, Urea, Betaine, and Heat Shock in Murine Renal Medullary Cells
Fuminori Umenishi*,,
Shigemi Yoshihara,
Takefumi Narikiyo* and
Robert W. Schrier*
* Department of Medicine and Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado; and Department of Pediatrics, Dokkyo University School of Medicine, Tochigi, Japan
Address correspondence to: Dr. Fuminori Umenishi, Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box C281, Denver, CO 80262. Phone: 303-315-6715; Fax: 303-315-4852; E-mail: fuminori.umenishi{at}uchsc.edu
Aquaporin-1 (AQP1) expression is induced by hypertonicity inrenal medullary cells. The purpose of the present study wasto elucidate the role of sodium chloride (NaCl), urea, betaine,and heat shock on hypertonicity-induced AQP1 expression in culturedmurine renal medullary-K2 (mIMCD-K2) cells. AQP1 expressionwas maximally induced under mild hypertonic medium supplementedwith 100 mM NaCl (N100), whereas severe hypertonic medium supplementedwith 150 mM NaCl (N150) caused little AQP1 induction. The reductionof AQP1 expression in N150 was associated with reduced cellviability. When cells were exposed continuously to N100, hypertonicity-inducedAQP1 expression was elevated, whereas the return to isotonicmedium reduced AQP1 expression in a time-dependent manner. Thehalf-life of AQP1 protein in isotonic conditions was approximately4 h, whereas hypertonicity markedly increased its half-life.These results indicate that hypertonicity plays an importantrole in AQP1 induction, stability, and degradation. On the contrary,urea inhibited hypertonicity-induced AQP1 expression in a dose-dependentmanner. The addition of organic osmolyte betaine in N150 enhancedhypertonicity-induced AQP1 expression, whereas it decreasedAQP1 expression in N100. This suggests that the excessive accumulationof betaine may counteract hypertonic stress and thus attenuatehypertonicity-induced AQP1 expression. Heat shock treatmentpromoted hypertonicity-induced AQP1 and heat shock protein 70(HSP70) expression in both N100 and N150, suggesting an effecton the stability of hypertonicity-induced AQP1 expression. Takentogether, NaCl, urea, betaine, and heat shock that regulatehypertonicity-induced AQP1 expression are potentially importantfactors in urinary concentration and contribute to the steady-statelevel of AQP1 expression.
The renal medullary cells, unlike most cells in mammals, areroutinely exposed to extremely high concentrations of sodiumchloride (NaCl) and urea. The adaptation of medullary cellsto hyperosmotic stress involves acute cellular efflux of water,cell shrinkage by NaCl, chronic accumulation of compatible organicosmolytes, and acute activation of immediate-early and heatshock genes (1–4). Moreover, hypertonicity with solutesthat are impermeable to the plasma membrane elevates the expressionof a number of genes, including the sodium/ myo-inositol cotransporter(5), sodium/chloride/betaine cotransporter (6), aldose reductase(7), and urea transporter (8). In addition, aquaporin-1 (AQP1),which provides a pathway of water movement across plasma membranesin renal cells, is a water channel protein that is induced byhypertonicity (9,10). Urea, which is permeant to the plasmamembrane, also upregulates several immediate-early genes, includingEgr-1 (11). Thus, NaCl, water, organic osmolytes, heat shockproteins (HSP), and urea may play an important role in the adaptationof renal medullary cells during urine concentration.
AQP1 plays an important role in constitutive water reabsorptionin the epithelial cells of the renal medulla. Specifically,AQP1 expression in the descending limb of Henle and vasa rectaeof the renal medulla is critical in generating and maintainingan axial osmotic gradient through the medulla (12). AQP1 osmoregulationin the epithelial cells of the renal medulla has been studiedusing cultured murine renal medullary-3 (mIMCD-3) cells. Inthese studies, AQP1 expression was demonstrated to be upregulatedby hypertonicity and its induction to be mediated by mitogen-activatedprotein kinase pathways (ERK, p38, and JNK) and a hypertonicityresponse element in the AQP1 gene (9,13). Furthermore, bothtranscriptional and posttranscriptional regulation was foundto be involved in hypertonicity-induced AQP1 expression (9),and a previous report suggested that protein stability was importantto AQP1 induction by hypertonicity in mouse BALB/c fibroblasts(14). Thus, hypertonicity is crucial on AQP1 expression andurinary concentration.
In the present study, urea, organic osmolytes, and HSP are proposedto be important factors in modulating AQP1 expression in renalmedulla under hypertonic conditions. Urea, in contrast to NaCl,is permeant to cell membrane. When a high concentration of ureais present, it denatures protein and provides harmful effectsin the medulla (2). However, the combination of NaCl and ureaenhances the survival rate of cells compared with NaCl or ureaalone at the same osmolality (15). Chronic adaptation by hypertonicityleads to accumulation of compatible organic osmolytes such asbetaine (16). The accumulation of such osmolytes protects thecells from hypertonic stress by relatively lowering cellularinorganic salts. Thus, the uptake and accumulation of organicosmolytes allow medullary cells to maintain intracellular ionconcentrations and to stabilize cellular proteins during hypertonicconditions. The HSP family, which is also called molecular chaperones,stabilizes macromolecules (17). It has been demonstrated thatincreased HSP70 expression or overexpression of HSP70 protectscells from apoptosis in hyperosmotic conditions (18,19). Todate, the effect of NaCl, urea, organic osmolytes, and HSP onhypertonicity-induced AQP1 expression in renal medullary cellshas not been studied. The objective of the present study thereforewas to examine (1) whether hypertonicity affects the inductionand stability of AQP1; (2) whether urea alters hypertonicity-inducedAQP1 expression; (3) whether betaine, which is one of the majororganic osmolytes, alters hypertonicity-induced AQP1 expression;and (4) whether heat shock, which induces HSP, is involved inhypertonicity-induced AQP1 expression.
Betaine, cycloheximide, protease inhibitor cocktail, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis,MO).
Cell Line and Culture Condition
Murine renal medullary mIMCD-K2 cells (20), derived from theinitial half of the inner medulla, were cultured at 37°Cand 5% CO2 in Opti-MEM medium (Life Technologies, Gaithersburg,MD) that contained 10% FBS. This cell line was chosen becausethe cell attachment on culture dishes in a severe hypertonicenvironment (isotonic medium + 150 mM NaCl [N150]) was similarto that in a mild hypertonic environment (isotonic medium +100 mM NaCl [N100]). A previous study showed that the cell viabilitywas not significantly reduced under N150 in mIMCD-K2 cells (21).On the contrary, in mIMCD-3 cell line, which was used in ourprevious study (9), most of the cells were detached from culturedishes under N150. Therefore, mIMCD-K2 cell line was used inthe present study.
Immunoblot Analysis
Cells grown on 6-cm dishes were washed with ice-cold PBS, suspendedwith extraction buffer (10 mM Tris-HCl, 200 mM sucrose, andprotease inhibitor cocktail [pH 7.5]), and homogenized by 10passages through a 27-G needle. The homogenate was centrifugedat 3000 x g for 10 min at 4°C. The supernatant was collected,and protein concentration was measured using the Bradford proteinassay method (Bio-Rad, Hercules, CA). Protein (10 µg)from the cell extract was treated with Laemmli sample buffer,resolved on a 12% SDS–polyacrylamide gel, and transferredto a PVDF membrane. The membrane was incubated with rabbit polyclonalanti-human AQP1 antibody (9). For the analysis of HSP70, themembrane was incubated with mouse monoclonal anti-HSP70 antibody(StressGen, Victoria, BC, Canada). After washing, the membranewas incubated with anti-IgG horseradish peroxidase secondaryantibody (Amersham, Piscataway, NJ). The immunoreactive bandswere visualized by enhanced chemiluminescence method (NEN LifeScience, Boston, MA). The bands on the film were scanned andanalyzed by using the NIH image software. To confirm that equalamounts of protein samples were loaded, we stained duplicategels with Coomassie Brilliant Blue.
MTT Assay
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells grown in 24-well plateswere incubated with 1 ml of isotonic or hypertonic medium. After16 h, 100 µl of MTT (5 mg/ml; Sigma) was added directlyto the medium and incubated at 37°C for 4 h. The resultingMTT reaction product was dissolved with 1 ml of isopropanolcontaining 0.1 N HCl, and the plates were agitated gently atroom temperature for 10 min. The absorbance at 570 and 690 nmwas measured, and the results were assessed by the subtractionthe absorbance at 690 nm (background) from the absorbance at570 nm.
Protein Stability
Cells grown on 6-cm dishes were incubated with isotonic or hypertonicmedium supplemented with 100 mM NaCl for 16 h followed by treatingwith 20 µg/ml cycloheximide (Sigma) for 1 h to block newlysynthesized proteins. The cells were incubated further in isotonicor hypertonic medium supplemented with 100 mM NaCl containing20 µg/ml cycloheximide for various time periods. At eachtime point, cells were homogenized in extraction buffer andprotein was immunoblotted with anti-AQP1 antibody as describedabove. For comparing the amount of -actin in each sample asa loading control, the membrane was stripped and incubated withanti–-actin antibody (Sigma).
Statistical Analyses
All values represent means ± SEM. Statistical analysiswas performed by unpaired t test. P < 0.05 was consideredsignificant.
AQP1 Induction by Hypertonicity
To investigate AQP1 expression in media with different osmolalities,murine renal medullary-K2 (mIMCD-K2) cells were treated by theaddition of NaCl (100 to 300 mM) or urea (200 to 600 mM) tothe isotonic medium. After 16 h, total cell extract was isolatedand immunoblot analysis was performed to detect AQP1 protein.As shown in Figure 1A, the addition of N100 to the isotonicmedium revealed the highest induction of AQP1. The additionof N150 or higher concentrations of NaCl showed little AQP1expression. AQP1 expression was dramatically decreased by 93± 3% for N150 as compared with N100. Urea had no effecton AQP1 induction. Moreover, the addition of 200 mM glyceroland DMSO did not induce AQP1 expression (data not shown). Theseresults suggest that a hypertonic gradient, not hyperosmolality,is necessary for AQP1 induction. To investigate whether AQP1expression by hyperosmolality was associated with changes incell viability, we performed MTT assay. The addition of NaClor urea to the medium caused significant cell death in a dose-dependentmanner (Figure 1B). Therefore, it was concluded that the reductionof AQP1 expression in N150 was associated with reduced cellviability. Next, to determine the maximum AQP1 induction byNaCl, cells were incubated with hypertonic medium supplementedwith 25, 50, 75, 100, 125, or 150 mM NaCl for 16 h. As shownin Figure 1C, N100 showed the highest AQP1 expression. To examineAQP1 induction by different impermeable osmolytes, cells wereincubated in medium supplemented with NaCl or impermeable osmolytes(raffinose, glucose, sorbitol, sucrose, or mannitol) for 16h. The expression level of AQP1 protein was significantly increasedby the addition of all impermeable osmolytes tested. The relativeexpression level was NaCl > mannitol > raffinose >sorbitol > glucose > sucrose (Figure 1D). The result indicatesthat impermeable osmolytes also induce AQP1 expression.
Figure 1. Hypertonicity-induced aquaporin-1 (AQP1) expression in murine renal medullary-K2 (mIMCD-K2) cells. (A) Effect of AQP1 induction by NaCl or urea with different hyperosmolalities. Cells were incubated in medium supplemented with NaCl (100, 150, 200, 250, or 300 mM) or urea (200, 300, 400, 500, or 600 mM) for 16 h. Cells were harvested, and total protein was analyzed by immunoblot with anti-AQP1 antibody. The data are representative of three independent experiments. (B) Cell viability. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to assess cell viability as described in the Materials and Methods section. Cell viability in isotonic medium was assigned as 100%. Values represent the mean ± SEM of four independent sets of experiments. * P < 0.005 versus isotonic cells. (C) AQP1 induction by NaCl with different hyperosmolality. Cells were incubated in medium supplemented with NaCl (25, 50, 75, 100, 125, or 150 mM) for 16 h. Cells were harvested, and total protein was analyzed by immunoblot. The data are representative of three independent experiments. (D) AQP1 induction by different organic osmolytes. Cells were incubated in isotonic or hypertonic medium supplemented with NaCl (100 mM), raffinose (100 mM), glucose (200 mM), sorbitol (200 mM), sucrose (200 mM), and mannitol (200 mM) for 16 h. Cells were harvested, and total protein was analyzed by immunoblot. The data are representative of three independent experiments.
Hypertonicity-Induced AQP1 Expression Is Decreased by Returning to Isotonic Medium
To investigate whether a continuous hypertonic stress is requiredfor hypertonicity-induced AQP1 expression, cells were treatedwith N100 for 16 h to induce AQP1, and then replaced the mediumwith isotonic or continuous N100. When the medium was returnedto isotonic, hypertonicity-induced AQP1 expression was reducedafter 8 h of incubation and its expression level decreased by50% after 20 h of incubation. On the contrary, continuous exposureof cells to N100 elevated hypertonicity-induced AQP1 expressionby 2.5-fold in a time-dependent manner (Figure 2). These datasuggest that a continuous hypertonic stress is necessary forthe steady-state level of AQP1 expression.
Figure 2. Comparison of hypertonicity-induced AQP1 expression in isotonic or hypertonic medium. Cells were incubated with hypertonic medium supplemented with 100 mM NaCl for 16 h to induce AQP1 expression, and then the medium was replaced by isotonic () or hypertonic medium supplemented with 100 nM NaCl (•). Samples were harvested at specific times (0, 2, 4, 8, 12, and 24 h) for immunoblot analysis. Each protein blot was analyzed by densitometry. AQP1 expression in hypertonic medium at 0 h was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments.
Comparison of AQP1 Protein Stability in Isotonic versus Hypertonic Conditions
To compare the stability of AQP1 in isotonic versus hypertonicconditions, cells were incubated with N100 for 16 h to induceAQP1 followed by treating with cycloheximide for 1 h to blocknewly synthesized proteins. Cycloheximide-treated cells wereincubated further in isotonic or N100 with cycloheximide forvarious time periods. As shown in Figure 3, the half-life ofAQP1 protein in isotonic conditions was approximately 4 h. Incontrast, AQP1 protein in N100 was still sustained at a levelof >70% of the protein after 8 h of incubation. Cycloheximidedid not affect the protein level of -actin in the experiment.This result indicates that hypertonicity enhances AQP1 proteinstability.
Figure 3. Stability of AQP1 protein in isotonic versus hypertonic conditions. Cells were stimulated with hypertonic medium supplemented with 100 mM NaCl for 16 h to induce AQP1 expression and then treated with cycloheximide (20 µg/ml) for 1 h to block newly synthesized proteins. Cycloheximide-treated cells were further incubated in isotonic () or hypertonic medium supplemented with 100 mM NaCl (•) in the presence of cycloheximide (20 µg/ml). Samples were harvested at specific times (0, 2, 4, 6, and 8 h), and total protein (10 µg) was analyzed by immunoblot. Each protein blot was analyzed by densitometry. AQP1 expression at 0 h was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments. The membrane was also incubated with anti–-actin antibody as a loading control.
Effect of Urea on Hypertonicity-Induced AQP1 Expression
Previous studies have shown that under severe hypertonic conditions(>600 mOsm/kg), apoptosis is induced and cell death occurs(22), whereas under mild hypertonic conditions (<600 mOsm/kg),apoptosis is blocked by activated p53, cell cycles cease, andthe DNA repair mechanism is activated (23–25). Nevertheless,it has been reported that a combination of NaCl and urea enhancescell survival compared with NaCl or urea alone at the same osmolality(15). Therefore, we investigated whether urea promoted or preventedhypertonicity-induced AQP1 expression. Cells were pretreatedwith N100 or N150 for 16 h, and then urea (100 to 300 mM) wasadded to the hypertonic medium and incubated for 24 h. As shownin Figure 4A, urea inhibited hypertonicity-induced AQP1 expressionin a dose-dependent manner. However, the addition of urea inboth N100 and N150 did not significantly decrease cell viabilitycompared with N100 or N150 alone (Figure 4B). Our data indicatethat urea counteracts AQP1 expression under both mild and severehypertonic conditions.
Figure 4. Effect of urea on hypertonicity-induced AQP1 expression. (A) Cells were pretreated with hypertonic medium supplemented with 100 mM NaCl or 150 mM NaCl. After 16 h, cells were incubated further with 100, 150, 200, 250, or 300 mM urea for 24 h. Cells were harvested, and total protein was analyzed by immunoblot. Each protein blot was analyzed by densitometry. AQP1 expression in N100 was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments. (B) Cell viability. MTT assay was performed to assess cell viability as described in the Materials and Methods section. Cell viability in isotonic medium was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments.
Effect of Betaine on Hypertonicity-Induced AQP1 Expression
In hypertonic conditions, the accumulation of organic osmolytessuch as betaine protects cells from hypertonic stress (16).To investigate whether betaine influences hypertonicity-inducedAQP1 expression under mild and severe hypertonic conditions,cells were incubated with N100, or N150 that contained 0, 5,or 10 mM betaine for 16 h. The medium used in this study containedno betaine. There was no detectable AQP1 in the presence ofbetaine under isotonic conditions (data not shown). Hypertonicity-inducedAQP1 expression in N100 significantly decreased in the presenceof betaine in a dose-dependent manner. On the contrary, AQP1expression in N150 was strongly enhanced in the presence ofbetaine (Figure 5A). The addition of betaine in both N100 andN150 did not significantly increase cell viability (Figure 5B).These results suggest that the additional accumulation of betaineunder mild and severe hypertonic conditions inversely influenceshypertonicity-induced AQP1 expression.
Figure 5. Effect of betaine on hypertonicity-induced AQP1 expression. (A) AQP1 expression. Cells were incubated with N100 or N150 that contained 0, 5, or 10 mM betaine. After 16 h, cells were harvested and total protein was analyzed by immunoblot. Each protein blot was analyzed by densitometry. AQP1 expression in N100 was assigned as 100%. Values represent the mean ± SEM of three to five independent sets of experiments. (B) Cell viability. MTT assay was performed to assess cell viability as described in the Materials and Methods section. Cell viability in isotonic medium was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments.
Effect of Heat Shock on Hypertonicity-Induced AQP1 Expression
Some of the HSP, including HSP70, are strongly induced by bothheat shock and hypertonicity (3,26). A potential role of HSP70in response to hypertonicity to stabilize protein conformationhas been proposed. Also, increased HSP70 expression or overexpressionof HSP70 protects cells from apoptosis in hyperosmotic conditions(18,19). To investigate whether heat shock influences AQP1 expression,cells were pretreated at 42°C for 30 min and then incubatedthem with isotonic, N100, or N150 for 16 h at 37°C. Heatshock did not induce AQP1 expression under isotonic conditions.However, AQP1 expression was significantly enhanced by heatshock under both mild and severe hypertonic conditions (Figure 6A).HSP70 was also increased under both hypertonic conditionsas well as heat-shock treatment. Furthermore, its expressionlevel in heat shock followed by hypertonic conditions was comparableto that in hypertonic shock alone (Figure 6B). Nevertheless,heat shock significantly reduced cell viability in both isotonicand hypertonic conditions (Figure 6C). This result suggeststhat heat shock enhances AQP1 expression under both mild andsevere hypertonic conditions, although cell viability is notassociated with hypertonicity-induced AQP1 expression. HSP70may be associated with the stability of hypertonicity-inducedAQP1 expression.
Figure 6. Effect of heat shock on hypertonicity-induced AQP1 expression. After pretreatment at 42°C for 30 min, cells were incubated with isotonic, N100, or N150 for 16 h at 37°C. Cells were harvested, and total protein was analyzed by immunoblot. Each protein blot was analyzed by densitometry. (A) AQP1 expression. AQP1 expression in N100 was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments. (B) Heat shock protein 70 (HSP70) expression. The membrane was also incubated with anti-HSP70 antibody. The abundance in isotonic medium was assigned as onefold. Values represent the mean ± SEM of three independent sets of experiments. (C) Cell viability. MTT assay was performed to assess the cell viability as described in the Materials and Methods section. Cell viability in isotonic medium was assigned as 100%. Values represent the mean ± SEM of four independent sets of experiments.
The role of arginine vasopressin (AVP) in urinary concentrationin mammals is well known. However, maximal urinary concentration,as achieved during hyperosmolality associated with fluid deprivation,cannot be achieved by AVP alone, thus suggesting the involvementof other factors. The role of AVP in upregulating AQP2 waterchannel and enhancing AQP2 trafficking to the apical membraneof the collecting duct has been well defined (27). However,an important role of AQP1, which is located in the descendinglimb of Henle’s loop and vasa rectae in the renal medullaryregion (28), in urinary concentration in mice and men has beenshown to be independent of AVP (29,30). Although AQP1 is notregulated by AVP, recent results from our laboratory and othershave shown that AQP1 mRNA and protein are upregulated by hypertonicity(9,10). Thus, AQP1 has emerged as a potentially independentAVP action in urinary concentration.
The renal medullary cells are exposed to a hyperosmotic environmentduring urinary concentration in which other cells would causeinjury and death. Several adaptive mechanisms protect renalmedullary cells against the deleterious effects of hyperosmolality.Therefore, the present study was undertaken to examine whetherNaCl, urea, organic osmolyte betaine, and heat shock influencehypertonicity-induced AQP1 expression in renal medullary mIMCD-K2cells. This cell line showed the maximal induction of AQP1 undera mild hypertonic condition supplemented with 100 mM NaCl, whereasa severe hypertonic condition supplemented with 150 mM NaClcaused little AQP1 induction. The reduction of AQP1 expressionin N150 was associated with reduced cell viability. However,stepwise adaptation of cells to N150 by adding 50 mM NaCl, whichimproved cell viability, did not increase AQP1 expression aswell as N150 (unpublished observation, 2004). Thus, the improvementof cell viability in severe hypertonic conditions is not sufficientto rescue AQP1 expression.
When cells were incubated with hypertonic medium and then themedium was returned to isotonic condition, AQP1 expression leveldecreased by 50% after 20 h, whereas continued exposure by hypertonicmedium elevated its expression in a time-dependent manner. Thisindicates that hypertonicity may control the induction and degradationof AQP1. Hypertonicity-induced AQP5 expression in mouse lungepithelial cells also declined when returned to isotonic medium(31). These data suggest that hypertonicity may be associatedwith the stability of AQP1. Therefore, AQP1 stability was measuredby cycloheximide treatment, which inhibits protein synthesis.We demonstrated that hypertonicity significantly enhanced AQP1protein stability compared with the short half-life of 4 h inisotonic conditions. The similar observation was also reportedby Leitch et al. (14). We conclude that hypertonicity enhancesthe induction and stability of AQP1.
Urea, in contrast to NaCl, is permeant to cell membrane. Whena high concentration of urea is present, it denatures proteinand causes harmful effects to the medulla (2). However, thecombination of NaCl and urea enhances the survival rate of cellsexposed to hyperosmolality (15), suggesting that the coexistencewith NaCl and urea protects medullary cells from the proapoptoticeffect of NaCl (32). In the present study, an inhibitory effectof urea on hypertonicity-induced AQP1 expression was demonstrated.Urea in itself caused the adverse effect on AQP1 expressionby NaCl. It also has been reported that urea inhibits hypertonicity-inducedtonicity-responsive enhancer binding protein and aldose reductase(33). Thus, the upregulation of AQPs and osmotically responsiveproteins by hypertonicity is likely to be suppressed in thepresence of urea. Taken together, urea counteracts the actionto increase AQP1 expression by NaCl.
In epithelial cells of renal medulla, five compatible organicosmolytes are mainly accumulated by prolonged exposure to hypertonicity:Betaine, myo-inositol, sorbitol, taurine, and glycerophosphorylcholine.The accumulation of such osmolytes protects cells from hypertonicstress by relatively lowering cellular inorganic salts. Whencells are exposed to hypertonicity or to the higher concentrationof betaine (>5 mM) in the medium, they accumulate a largeamount of organic osmolytes. A previous report demonstratedthat the addition of betaine to N100 strongly inhibited hypertonicity-inducedbetaine transporter expression in MDCK cells (34). In the presentstudy, we showed that the additional accumulation of betaineunder N100 decreased hypertonicity-induced AQP1 expression,whereas it was strongly improved by the addition of betaineunder N150. Although the uptake and accumulation of betaineprotects cells from severe hypertonicity, the addition of betaineunder mild hypertonic conditions counteracts hypertonicity-inducedAQP1 expression. Thus, the addition of betaine under severehypertonic conditions restores its expression because betaineprevents hypertonicity-induced cell damage and apoptosis (35).Although the protective effect of betaine on hypertonicity-inducedcell death after 48 h of incubation has been reported previously(36), our data showed that the adverse expression of AQP1 bythe addition of betaine in N100 and N150 was not due to changesin cell viability. This was because there was no significanteffect of betaine on cell viability after 16 h of incubation.This finding suggests that the excessive accumulation of betainemay simply counteract hypertonic stress and thus attenuate hypertonicity-inducedAQP1 expression.
The HSP family stabilizes macromolecules (17). It was demonstratedpreviously that HSP70 was strongly induced by not only heatshock but also hypertonicity (3,26). In the present study, weshowed that heat-shock treatment enhanced hypertonicity-inducedAQP1 expression in both mild and severe hypertonic conditions.It has been demonstrated that increased HSP70 expression oroverexpression of HSP70 protects cells from apoptosis in hypertonicconditions (18,19). In such severe conditions, HSP may be neededto assist in folding and degradation of proteins. In vivo, dehydrationelevates the expression of HSP70 in the inner medulla, whichis able to adapt to very high concentrations of NaCl (37). Amongseven members of the HSP70 family, HSP70.1 is induced by hypertonicity.When HSP70.1-deficient mice were exposed to osmotic stress,the renal medulla of these mice exhibited increased apoptosis,whereas wild-type mice did not (38). Thus, the HSP70 familymay play a critical role in the tolerance to osmotic stressin the kidney. In addition, Osp94 and HSP110, which are theHSP110/SSE subfamily induced by heat and osmotic stress, arehighest in the inner medulla (39). Our data suggest that HSP70and other HSP family members such as Osp94 and HSP110, whichare induced by hypertonicity, may be associated with the stabilityof hypertonicity-induced AQP1 expression.
In summary, hypertonicity with impermeant solutes such as NaClbut not the permeant solute urea induces and stabilizes AQP1protein, whereas urea inhibits hypertonicity-induced AQP1 expression.Organic osmolyte betaine and heat shock treatment promote hypertonicity-inducedAQP1 expression under severe hypertonic conditions. All of theseimportant factors that regulate hypertonicity-induced AQP1 expressionmay contribute to the steady-state level of AQP1 during urinaryconcentration.
Acknowledgments
This work was supported by the National Institutes of HealthGrant DK19928.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication March 26, 2004.
Accepted for publication November 24, 2004.
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Top
Abstract
Introduction
-actin in each sample as a loading control, the membrane was stripped and incubated with anti–
-B-crystallin expression.
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) or hypertonic medium supplemented with 100 nM NaCl (•). Samples were harvested at specific times (0, 2, 4, 8, 12, and 24 h) for immunoblot analysis. Each protein blot was analyzed by densitometry. AQP1 expression in hypertonic medium at 0 h was assigned as 100%. Values represent the mean ± SEM of three independent sets of experiments.
