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Hormones – Cytokines – Signaling

Kidney International (2001) 60, 553–567; doi:10.1046/j.1523-1755.2001.060002553.x

Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells

Yoshio Terada, Seiji Inoshita, Satoko Hanada, Haruko Shimamura, Michio Kuwahara, Wataru Ogawa, Masato Kasuga, Sei Sasaki and Fumiaki Marumo

Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, Tokyo, and the Second Department of Internal Medicine, Kobe University, School of Medicine, Kobe, Japan

Correspondence: Yoshio Terada, M.D., Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: yterada.kid@tmd.ac.jp

Received 27 June 2000; Revised 15 February 2001; Accepted 14 March 2001.

Abstract

Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells.

Background

The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in (1) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and (2) in the inner medulla of dehydrated rat in vivo.

Methods

The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis.

Results

The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H₂O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis.

Conclusion

The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.

Keywords:

serine-threonine kinase, cell signaling, phosphatidylinositol 3-kinase, MDCK cells, phosphorylation, dehydration, renal medulla collecting duct, osmotic stress

When a normal human is dehydrated and the urine becomes highly concentrated, the osmolality in the renal medulla increases to more than 1000 mOsm/kg H₂O¹. Renal medulla collecting duct cells can tolerate this hyperosmotic condition. The Madin-Darby canine kidney (MDCK) epithelial cell line is a useful model for cellular responses to hyperosmolality, since it is considered to have the characteristics of distal nephron segments and can tolerate extremes of osmolality². Hyperosmotic stress has been shown to induce cell damage and apoptosis in polymorphonuclear cells and mesothelial cells, suggesting that the biochemical pathways related to apoptosis are involved in a hyperosmotic condition^3,4. Raising the medium osmolality to 600 mOsm/kg H₂O final osmolality by the addition of NaCl transiently arrests the growth of murine inner medullary collecting duct cells (mIMCD), increases the expression of GADD45 (a growth-arrest and DNA damage-inducible protein), and possibly leads to cellular DNA damage^5,6. A recent report by Dmitrieva et al demonstrated the protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation⁷. Thus, anti-apoptotic/cell survival pathways may confer the balance between cell survival and apoptosis in MDCK cells in hyperosmotic conditions. Although the accumulation of osmolytes such as betaine, inositol, and glycerophosphorylcholine has been demonstrated under hyperosmolar conditions^8,9, much less is known about the signaling events leading to cell survival and anti-apoptosis in renal medullary cells¹⁰.

Growing evidence suggests that the serine-threonine kinase Akt/protein kinase B (PKB) is a critical enzyme in a cell survival pathway that protects cells from apoptosis^11,12,13,14. Upstream signaling pathways leading to Akt activation include phosphatidylinositol 3-kinase (PI3-K)^15,16. Cellular stresses such as heat shock and oxidative stress have been reported to stimulate the PI3-K and Akt pathways^17,18,19. Recently, Zhang et al reported that PI3-K signaling and activation of Akt were involved in inner medullary cell response to urea²⁰. However, the regulation and functional roles of the Akt pathway in hyperosmolality are poorly understood. Our study investigated the roles of the Akt pathway and apoptotic signals in MDCK cells in a hyperosmotic condition and in rat inner medulla in vivo.

METHODS

Materials

Wortmannin, LY294002 (a PI3-K inhibitor) and staurosporine (a protein kinase C inhibitor) were purchased from Sigma Chemical (St. Louis, MO, USA).

Recombinant adenoviruses

Replication-defective, recombinant adenoviruses encoding a dominant negative PI3-K (AxCAPIK85), dominant-active myristoylated Akt (AxCAmyrAkt), and dominant-negative Akt (AxCAAktAA) were prepared as described previously. In brief, the binding site for the catalytic subunit in regulatory subunit of PI3-K was deleted (AxCAPIK85)²¹. Rat Akt1 was replaced with the myristoylated form (myrAkt)²². Both Thr308 and Ser473 of rat Akt1 were replaced by alanine (AktAA)²¹. The recombinant adenovirus expressing the LacZ gene (AxCALacZ) was prepared as described previously²³. Each adenovirus preparation was titrated by plaque assay on 293 cells. Viral stocks [10¹¹ plaque-forming units (pfu)/mL] were stored at -80°C and were thawed on ice just before use.

Cell culture and preparation of extracts

Madin-Darby canine kidney cells and LLC-PK1 originally purchased from the American Type Culture Collection (Rockville, MD, USA) were grown in a defined medium: a 50:50 mixture of Dulbecco's modified Eagle's medium (DMEM) with 5 mmol/L glucose and Coon's modified Eagle's medium supplemented with 10 mmol/L HEPES, 5 pmol/L triiodothyronine, 50 mol/L hydrocortisone, 10 nmol/L Na₂SeO₃, 5 g/mL transferrin, 25 ng/mL prostaglandin E₁, 2 mmol/L L-glutamine, 50 IU/mL penicillin, and 50 g/mL streptomycin²⁴. The osmolality of the medium was adjusted to 300 mOsm/kg H₂O final osmolality by adding 30 mmol/L NaHCO₃. The medium was made hypertonic by adding NaCl or urea to 400, 500, and 600 mOsm/kg H₂O final osmolality. After varying lengths of incubation in hyperosmotic medium, the cells were scraped into a total volume of 0.5 mL of extraction buffer containing 50 mmol/L -glycerophosphate, pH 7.3, 1.5 mmol/L egtazic acid (EGTA), 0.1 mmol/L Na₃VO₄, 1 mmol/L dithiothreitol, 10 g/mL leupeptin, 10 g/mL aprotinin, 2 g/mL pepstatin A, and 1 mmol/L benzamidine. The cells in the extraction buffer were sonicated for 20 seconds (Sonicator, Central Co., Tokyo, Japan) and centrifuged at 100,000 g for 20 minutes at 4°C (TL-100 centrifuge; Beckman, Palo Alto, CA, USA). Supernatants were stored frozen at -80°C.

Western blot analysis

The cell lysates were mixed 1:4 with 5 Laemmli buffer and heated for five minutes at 95°C. Soluble lysates (20 g) were loaded in each lane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 5 and 20% acrylamide for stacking and resolving gels, respectively. Protein was transferred to nitrocellulose (pore size 0.45 m; Schleicher and Schuell, Keene, NH, USA) and probed with polyclonal antibodies against Serine 174-phospho-specific Akt, total-Akt, serine 15-phospho-p53, and total-p53 (New England Biolabs, Beverly, MA, USA). For detection of the phosphospecific-Akt, total-Akt, phosphospecific-p53 (Ser15), and total-p53, the primary antibodies (diluted 1/1000) and a second antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (diluted 1/2000), were used. The bands were visualized by the New England Biolabs detection system with 5 to 10 minutes of exposure after extensive washing of the membranes. The films were scanned using an Epson scanner (Nagano, Japan). Signals on Western blots were quantified by densitometry using an image analysis software application (NIH Image 1.47).

Caspase-3 assays

A caspase-3 fluorometric protease assay kit (MBL, Tokyo, Japan) was used for measurement of caspase-3 activities as previously described²⁵. In brief, cells were plated in six-well dishes and cultured in medium of different osmolalities. The cells were collected and lysed in lysis buffer at indicated times, and the protein concentration was normalized by the Bradford assay. The lysates were incubated with the same amounts of reaction buffer and 50 mol/L Asp-Glu-Val-Asp-p-nitroanilide (DEVD-pNA) substrate for two hours at 37°C. Fluorescence was monitored with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

Ladder assays

Adherent and floating cells were collected and lysed in the following (in mmol/L): Tris (pH 8.0) 10, NaCl 100, and EDTA 25, 0.5% SDS, and 1.0 mg/mL proteinase K at 37°C for four hours. DNA was extracted from the digested cells as previously described²⁶, and 30 g DNA were subjected to electrophoresis on 1.5% agarose gels.

Cell death enzyme-linked immunosorbent assay

Histone-associated DNA fragments were quantitated by enzyme-linked immunosorbent assay (ELISA; Boehringer Mannheim, Mannheim, Germany). All cells from each well were collected by trypsinization and pipetting and were pelleted (800 rpm, 5 min), lysed, and subjected to the capture ELISA according to the manufacturer's protocol. Cytosolic proteins were collected using cell lysis buffer according to the manufacturer's protocol. After 30 minutes of incubation of the cells with cell lysis buffer, samples were centrifuged for 10 minutes (15,000 rpm). The nucleus formed into a pellet, and the cytoplasmic fraction became supernatant. Supernatants were collected for the ELISA assay. Each experiment was carried out in triplicate and repeated in at least five independent experiments.

Water restriction experiments

The water restriction experiments were performed using male Sprague-Dawley rats weighing 100 to 150 g (N = 10 per group). The normal-rat group was given a standard diet and allowed free access to tap water. Rats in the dehydration-experiment group were deprived of water for 24 or 48 hours before death. Urine osmolality was measured with a Wescor vapor pressure osmometer.

Immunocytochemistry

The rat kidneys were fixed in 10% buffered-formaldehyde (Wako Biochemical, Tokyo, Japan) overnight and then dehydrated by 70% ethanol for one hour followed by 100% ethanol for four hours. Next, they were treated with xylane and then paraffin embedded. DNA fragmentation was detected in situ by the methods of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) using a commercial kit (Boehringer Mannheim) according to the manufacturer's instructions. Counterstaining was performed with hematoxylin and eosin staining.

Statistics

The results were given as means SEM. The differences were tested using two way-analysis of variance followed by the Scheffe's test for multiple comparisons. Two groups were compared by the unpaired t test. P < 0.05 was considered significant.

RESULTS

Dehydration caused Akt activation and apoptotic changes in rat renal medulla

The normal-rat group was given a standard diet and allowed free access to tap water, while rats in the dehydration-experiment group were deprived of water for 24 or 48 hours before death. The state of dehydration of the rats was evaluated by measuring urine osmolality. Urine osmolality was significantly elevated in both the 24- and 48-hour water-restricted animals (1420 165, 1825 176 mOsmol/kg H₂O, respectively) compared with control values (956 145 mOsmol/kg H₂O, P < 0.05). Phosphorylation of Akt was detected by immunoblots using an antibody specific to the phosphorylated form of Akt. As shown in Figure 1a, phosphorylation of Akt was significantly increased in the renal medulla of 24- and 48-hour water-restricted rats. Densitometric analysis showed that phosphorylation of Akt was significantly increased in the renal medulla of water-restricted animals compared with that of the control animal. In contrast, phosphorylation of Akt was not changed in the renal cortex. We also examined the abundance of the Akt protein using immunoblots with antibody to total Akt protein. There were no significant changes in the abundance of total Akt in dehydrated rat kidneys. Furthermore, no apoptotic cells were observed in the kidneys of either water-restricted rats or control rats examined by TUNEL staining Figure 1b. These in vivo observations suggest the presence of cell-protecting systems. To investigate the precise mechanisms of Akt activation and apoptotic change in hyperosmotic condition, we employed an in vitro culture system.

Figure 1.

Phosphorylation of Akt and apoptotic changes in rat renal medulla by dehydration. (A) Phosphorylation of Akt in dehydrated renal medulla and cortex was detected by immunoblots using an antibody specific to the phosphorylated form of Akt (upper gel). The abundance of Akt protein was demonstrated by immunoblots with an antibody to total Akt (lower gel). Densitometric analysis of the phosphorylation of Akt [N = 5, mean SEM, *P < 0.05 vs. control (0 hour dehydration); middle panel]. (B) TUNEL staining of inner medulla of control and water-restricted (24 and 48 hours) rat kidney.

Hyperosmolality stimulated the phosphorylation of Akt in MDCK cells

To examine whether hyperosmolality stimulates the phosphorylation of Akt in MDCK cells, the cells were exposed to hyperosmotic media (600 mOsm/kg H₂O final osmolality) prepared by adding NaCl, urea, or raffinose for 1, 3, 6, 12, 24, and 48 hours. Densitometric analysis showed that exposure to hyperosmolar media caused two peaks of phosphorylation of Akt. The first and most prominent peak occurred at 1 to 3 hours, and the second peak occurred at 24 hours Figure 2a. There were no significant changes in the abundance of total Akt stimulated by NaCl, urea, and raffinose compared with the control values (data are not shown). To examine the osmolality dependence of phosphorylation of Akt, MDCK cells were incubated with media containing NaCl, urea, or raffinose at 300, 400, 500, or 600 mOsm/kg H₂O final osmolality for one hour. Densitometric analysis demonstrated that hyperosmotic media containing NaCl, urea, and raffinose induced the concentration-dependent phosphorylation of Akt at osmolalities from 300 to 600 mOsm/kg H₂O final osmolality Figure 2b. There was no significant change in the abundance of Akt (data not shown).

Figure 2.

Hyperosmolality stimulates the phosphorylation of Akt in MDCK cells. (A) Anti-phospho-Akt immunoblot of detergent lysates were prepared from MDCK cells treated with NaCl, urea, or raffinose at the indicated times (left figures). Densitometric analysis of the phosphorylation of Akt [*P < 0.05 vs. control (0 hour); right panels]. (B) Anti-phospho-Akt immunoblot of detergent lysates prepared from MDCK cells treated with an indicated osmolality of NaCl, urea, or raffinose for one hour (left panels). Densitometric analysis of the phosphorylation of Akt (*P < 0.05 vs. 300 mOsm/kg H₂O final osmolality; right panels). Data are N = 5, mean SEM.

Phosphatidylinositol 3-kinase inhibitors and the dominant-negative mutant of PI3-K (p85) inhibited phosphorylation of Akt in hyperosmolality-treated MDCK cells. To examine the possible contribution of PI3-K to hyperosmolality-induced Akt phosphorylation, MDCK cells were preincubated with PI3-K inhibitors [100 nmol/L Wortmannin, 10 mol/L LY294002, or 0.1% dimethyl sulfoxide (DMSO) as a solvent] and exposed to hyperosmolality or normo-osmolality (NaCl or urea at 500 or 300 mOsm/kg H₂O final osmolality) for one hour. The dominant-negative mutant of the PI3-K adenovirus (AxCAPI3Kp85) also was employed. MDCK cells were transfected with AxCAPI3Kp85 or AxCALacZ(10¹⁰ pfu/mL) for 48 hours and then exposed to hyperosmolality or control osmolality (NaCl or urea at 500 or 300 mOsm/kg H₂O final osmolality) for one hour. Wortmannin, LY294002, and AxPI3Kp85 inhibited hyperosmolality-induced Akt phosphorylation, as shown in Figure 3a. DMSO (solvent for Wortmannin and LY294002) and AxCALacZ did not attenuate the hyperosmolality-induced Akt phosphorylation. These results demonstrate that hyperosmolality-induced Akt phosphorylation is totally dependent on PI3-K activation. The basal level of Akt phosphorylation was very low in the 300 mOsm/kg H₂O final osmolality medium, and these inhibitors did not significantly change the Akt phosphorylation in that osmolality Figure 3b. Similar results were obtained in experiments with urea-induced hyperosmolality (data not shown). Thus, we speculated that the Akt pathway was not highly activated in the control-treated MDCK cells and that the DMSO (solvent) and adenovirus infection did not cause nonspecific toxic effects in the MDCK cells.

Figure 3.

Phosphatidylinositol 3-kinase (PI3-K) inhibitors and the dominant-negative mutant of PI3-K (p85) inhibit phosphorylation of Akt in hyperosmolality-treated MDCK cells. (A) Anti-phospho-Akt immunoblot of detergent lysates prepared from MDCK cells treated with PI3-K inhibitors (wortmannin or LY294002) for one hour and exposed to hyperosmolality (NaCl at 500 mOsm/kg H₂O final osmolality) for one hour. Anti-phospho-Akt immunoblot of detergent lysates prepared from MDCK cells transfected with AxCAPI3Kp85 or AxCALacZ (10¹⁰ pfu/mL) for 48 hours and exposed to hyperosmolality (NaCl at 500 mOsm/kg H₂O final osmolality) for one hour (upper gel). The abundance of Akt protein was demonstrated by immunoblots with an antibody to total Akt (lower gel). Band intensities of phospho-Akt were obtained by densitometry of immunoblots, and expression is presented as a fraction of the expression in control (500 mOsm/kg H₂O final osmolality; lower panel). (B) Anti-phospho-Akt immunoblot of detergent lysates prepared from MDCK cells treated with PI3-K inhibitors (wortmannin or LY294002) for one hour and exposed to control osmolality (NaCl at 300 mOsm/kg H₂O final osmolality) for one hour. Anti-phospho-Akt immunoblot of detergent lysates prepared from MDCK cells transfected with AxCAPI3Kp85 or AxCALacZ (10¹⁰ pfu/mL) for 48 hours and exposed to normo-osmolality (NaCl at 300 mOsm/kg H₂O final osmolality) for 1 hour (upper gel). The abundance of Akt protein was demonstrated by immunoblots with an antibody to total Akt (lower gel). Band intensities of phospho-Akt were obtained by densitometry of immunoblots, and expression is presented as a fraction of the expression in control (300 mOsm/kg H₂O final osmolality; lower panel). Results are means SEM of four independent experiments. *P < 0.05 vs. control. Similar results were obtained in experiments with urea-induced hyperosmolality (data not shown).

Hyperosmolality stimulated caspase cascades and induced apoptosis in MDCK cells

We next investigated the effects of hyperosmolality on apoptosis in MDCK cells. To examine whether hyperosmolality leads to apoptotic change in MDCK cells, a caspase-3 assay, cell death ELISA assay, and agarose gel electrophoresis of fragmented DNA after hyperosmolar stimuli were performed. To investigate the time course of hyperosmolality-induced caspase-3 activity in MDCK cells, cells were exposed to hyperosmotic medium (600 mOsm/kg H₂O final osmolality with NaCl or urea) for 1, 3, 6, 12, and 24 hours. The activity of caspase-3 increased from three hours after exposure of MDCK cells to hyperosmolar NaCl or urea, and reached its peak at 12 hours Figure 4a. The concentration dependence of caspase-3 activation was investigated next at 12 hours of incubation with 300, 400, 500, and 600 mOsm/kg H₂O final osmolality medium with NaCl or urea. Compared with the 300 mOsm/kg H₂O final osmolality medium, the caspase-3 activity was significantly increased in the 500 and 600 mOsm/kg H₂O final osmolality mediums, but was not significantly changed in the 400 mOsm/kg H₂O final osmolality medium Figure 4b. Furthermore, the osmolality dependence of cell death was examined using the ELISA assay and DNA laddering at osmolalities of 300, 400, 500, and 600 mOsm/kg H₂O final osmolality with NaCl or urea. MDCK cells incubated in 300 and 400 mOsm/kg H₂O final osmolality medium (NaCl or urea) demonstrated no significant change in the cell death ELISA assay and no nucleosomal laddering of DNA on agarose gel electrophoresis (Figure 4 C, D). In contrast, the cells incubated in the 500 and 600 mOsm/kg H₂O final osmolality mediums demonstrated an increased activity of cell death with the ELISA assay and nucleosomal laddering of DNA, and both of these apoptotic signals were most evident in the 600 mOsm/kg H₂O final osmolality medium (Figure 4 C, D). These results demonstrate that MDCK cells induce apoptosis when incubated with 500 and 600 mOsm/kg H₂O final osmolality mediums, but not with 300 and 400 mOsm/kg H₂O final osmolality mediums. Therefore, our subsequent experiment examined the Akt pathway and apoptosis in the 500 and 600 mOsm/kg H₂O final osmolality medium conditions. Results of a statistical analysis between NaCl and urea are shown in Figure 4 A–C. The only significant difference in caspase-3 activity was observed at 12 hours. In cell death ELISA assay, no difference was observed between NaCl and urea. It is very difficult to conclude from these data that there were significant differences between the urea- and NaCl-induced apoptotic changes in our experimental conditions.

Figure 4.

Hyperosmolality stimulates caspase-3 activity, increases the activity of cell death ELISA assay, and induces nucleosomal laddering of DNA in MDCK cells. (A) MDCK cells were exposed to hyperosmotic medium (600 mOsm/kg H₂O final osmolality with NaCl () or urea () for the indicated times. Cell lysate was used for caspase-3 assay (*P < 0.05 vs. control, #P < 0.05 NaCl vs. urea). (B) MDCK cells were exposed to normo-osmotic or hyperosmotic medium (300, 400, 500, 600 mOsm/kg H₂O final osmolality) for 12 hours with NaCl () or urea (). Cell lysate was used for caspase-3 assay (N = 5, mean SEM, *P < 0.05 vs. 300 mOsm/kg H₂O final osmolality, #P < 0.05 NaCl vs. urea). (C) MDCK cells were exposed to normo-osmotic or hyperosmotic medium (300, 400, 500, 600 mOsm/kg H₂O final osmolality) with NaCl () or urea () for 12 hours. Cell lysate from adherent and floating cells was subjected to cell death ELISA assay (*P < 0.05 vs. 300 mOsm/kg H₂O final osmolality). (D) MDCK cells were exposed to normo-osmotic or hyperosmotic medium (300, 400, 500, 600 mOsm/kg H₂O final osmolality) with NaCl or urea for 12 hours. Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose gels. Data are N = 5, mean SEM.

Dominant-active form of Akt prevents mild hyperosmolality-induced apoptosis in MDCK cells

To examine the functional roles of Akt, adenovirus-mediated gene transfer of the dominant-active mutant of Akt (AxCAmyrAkt) to MDCK cells was employed. The effects of AxCAmyrAkt and AxCALacZ on hyperosmolality-induced apoptotic phenomenona and on control-treated (300 mOsm/kg H₂O final osmolality) cells were examined. Transfection of AxCAmyrAkt reduced the caspase-3 activity to 42 12% and 81 17% in 12-hour incubations with 500 and 600 mOsm/kg H₂O final osmolality mediums with NaCl, respectively Figure 5a. Transfection of AxCAmyrAkt reduced the caspase-3 activity to 40 15% and 77 18% in 12-hour incubations with 500 and 600 mOsm/kg H₂O final osmolality mediums with urea, respectively Figure 5a. Cell death ELISA examination of MDCK cells exposed to the mild hyperosmotic medium (500 mOsm/kg H₂O final osmolality) for 12 hours showed that the apoptotic signals were significantly inhibited by transfection of AxCAmyrAkt Figure 5b. In contrast, cell death ELISA examination of MDCK cells exposed to hyperosmotic medium (600 mOsm/kg H₂O final osmolality) for 12 hours showed that the apoptotic signals were not significantly inhibited by transfection of AxCAmyrAkt in either NaCl or urea Figure 5b. However, transfection with AxCAmyrAkt significantly reduced nucleosomal laddering of MDCK cells induced by a 12-hour incubation with the 500 mOsm/kg H₂O final osmolality medium with NaCl or urea Figure 5c. As shown in Figure 5 A and B, caspase-3 activity and cell death ELISA were not significantly increased by transfection of control adenovirus (AxCALacZ) in 300, 500, or 600 mOsm/kg H₂O final osmolality medium. Transfection of these adenoviruses did not induce significant changes of caspase-3 activity, cell death ELISA assay, and DNA fragmentation in control-treated (300 mOsm/kg H₂O final osmolality) MDCK cells Figure 5.

Figure 5.

Dominant-active form of Akt prevents hyperosmolality-induced apoptosis in MDCK cells. (A) MDCK cells were transfected with AxCAmyrAkt (), AxCALacZ (), and control (; no-adenovirus-infection) for 48 hours and were exposed to normo-osmotic or hyperosmotic medium (300, 500, or 600 mOsm/kg H₂O final osmolality with NaCl or urea for 12 hours. Cell lysate was used for caspase-3 assay. (B) MDCK cells were transfected with AxCAmyrAkt (), AxCALacZ (), and control (; no-adenovirus-infection) for 48 hours and were exposed to normo-osmotic or hyperosmotic medium (300, 500, or 600 mOsm/kg H₂O final osmolality with NaCl or urea for 12 hours. Cell lysate from adherent and floating cells was subjected to cell death ELISA assay Data for panels A and B are N = 5, mean SEM, *P < 0.05.

(C) MDCK cells were transfected with AxCAmyrAkt and AxCALacZ for 48 hours and exposed to hyperosmotic medium (500 mOsm/kg H₂O final osmolality) or normo-osmotic medium (300 mOsm/kg H₂O final osmolality) with NaCl or urea for 12 hours. Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose gels.

These results demonstrate that activated-Akt substantially inhibited the apoptotic pathway, including case-3, in a mildly hyperosmotic condition (500 mOsm/kg H₂O final osmolality), whereas it only partially inhibited the apoptotic pathway induced by the 600 mOsm/kg H₂O final osmolar condition.

Phosphatidylinositol 3-kinase inhibitors, the dominant-negative mutant of PI3-K (p85), and the dominant-negative mutant of Akt promote apoptosis in hyperosmolality-treated MDCK cells. To investigate the functional roles of the hyperosmolality-induced Akt pathway further, we examined the effects of PI3-K inhibitors (100 nmol/L wortmannin or 10 mol/L LY294002), 0.1% DMSO (solvent of wortmannin and LY294002), dominant-negative mutant of PI3-K (AxCAPI3Kp85), dominant-negative mutant of Akt (AxCAAkt-AA), and AxCALacZ on the apoptotic pathway in MDCK cells. Wortmannin, Ly294002, and transfection of AxCAPI3Kp85 and AxCAAkt-AA increased caspase-3 activity in MDCK cells treated with medium of 400 mOsm/kg H₂O final osmolality with NaCl or urea Figure 6a. DMSO and AxCALacZ did not significantly change the caspase-3 activity. Cell death ELISA assay of MDCK cells treated with medium of 400 mOsm/kg H₂O final osmolality revealed that the inhibition of the PI3-K–Akt pathway induced a marked increase of apoptosis Figure 6c. DMSO and AxCALacZ did not significantly change the activity of cell death ELISA assay. DNA fragmentation of the cells treated with the 400 mOsm/kg H₂O final osmolality (NaCl or urea) revealed that the inhibitions of the PI3-K–Akt pathway by the PI3-K inhibitors, AxCAPI3Kp85, and AxCAAkt-AA caused marked increases of DNA fragmentation (Figure 6 E, F). DMSO and AxCALacZ did not cause DNA fragmentation in the 400 mOsm/kg H₂O final osmolality (NaCl or urea). These results suggested that inhibition of PI3-K and the Akt pathway promotes apoptotic changes in MDCK cells in mild hyperosmolality (400 mOsm/kg H₂O final osmolality).

Figure 6.

Phosphatidylinositol 3-kinase (PI3-K) inhibitors, dominant-negative mutant of PI3-kinase (p85), and dominant-negative mutant of Akt promote apoptosis in hyperosmolality-treated MDCK cells. Symbols are: () NaCl; () urea. (A) MDCK cells were initially transfected with either AxCAPI3Kp85, AxCAAkt-AA, or AxCALacZ for 48 hours or were exposed to PI3-K inhibitors (wortmannin, Ly294002) or 0.1% DMSO for 1 hour, and next, they were exposed to mild-hyperosmotic medium (400 mOsm/kg H₂O final osmolality with NaCl or urea for 12 hours. Cell lysate was used for caspase-3 assay (N = 5, *P < 0.05 vs. NaCl control). (B) MDCK cells were initially either transfected with AxCAPI3Kp85, AxCAAkt-AA, or AxCALacZ for 48 hours or were exposed to PI3-K inhibitors (wortmannin, Ly294002) or 0.1% DMSO for 1 hour, and next, they were exposed to normo-osmolality (300 mOsm/kg H₂O final osmolality with NaCl or urea for 12 hours. Cell lysate was used for caspase-3 assay (N = 5, *P < 0.05 vs. NaCl control). (C) MDCK cells were initially transfected with either AxCAPI3Kp85, AxCAAkt-AA, or AxCALacZ for 48 hours or exposed to PI3-K inhibitors (wortmannin, Ly294002) or 0.1% DMSO for 1 hour, and next, they were exposed to a mildly hyperosmotic medium (400 /kg H₂O final osmolality with NaCl) for 12 hours. Cell lysate from adherent and floating cells was subjected to cell death ELISA assay (N = 5, *P < 0.05 vs. control). (D) MDCK cells were initially either transfected with AxCAPI3Kp85, AxCAAkt-AA, or AxCALacZ for 48 hours or were exposed to PI3-K inhibitors (wortmannin, Ly294002) or 0.1% DMSO for 1 hour, and next, they were exposed to normo-osmolality (300 mOsm/kg H₂O final osmolality with NaCl for 12 hours. Cell lysate from adherent and floating cells was subjected to cell death ELISA assay (N = 5).

(E) MDCK cells were initially either transfected with AxCAPI3Kp85, AxCAAkt-AA, or AxCALacZ for 48 hours or were exposed to PI3-K inhibitors (wortmannin, Ly294002) or 0.1% DMSO for 1 hour, and next, they were exposed to the mild-hyperosmotic medium (400 mOsm/kg H₂O final osmolality with NaCl) or normo-osmolality (300 mOsm/kg H₂O final osmolality with NaCl for 12 hours. Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose gels. (F) MDCK cells were initially either transfected with AxCAPI3Kp85, AxCAAkt-AA, or AxCALacZ for 48 hours or exposed to PI3-K inhibitors (wortmannin, Ly294002) or 0.1% DMSO for 1 hour, and next they were exposed to a mildly hyperosmotic medium (400 mOsm/kg H₂O final osmolality with urea) or normo-osmolality (300 mOsm/kg H₂O final osmolality with urea) for 12 hours. Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose cells. All data are mean SEM.

The effects of wortmanin, Ly294002, DMSO (solvent of wortmanin, Ly294002), and AxCAPI3Kp85, AxCALacZ, and AxCAAkt-AA on caspase-3 activity were examined using cell death ELISA and DNA fragmentation in control-treated (300 mOsm/kg H₂O final osmolality) MDCK cells to determine whether the apoptotic effects of PI3-K inhibitors, AxCAPI3Kp85, and AxCAAkt-AA were specific in the mildly hyperosmolar condition (400 mOsm/kg H₂O final osmolality). Although the basal level of caspase-3 and the absorbance of cell death ELISA assay were very low in the 300 mOsm/kg H₂O final osmolality medium, these inhibitors (AxCAPI3Kdp85, and AxCAAkt-AA) did not significantly change caspase-3 activity with regards to cell death as measured by ELISA in the 300 mOsm/kg H₂O final osmolality, nor was any DNA fragmentation observed (Figure 6 B, D, E, F).

Specificity of hyperosomolality in Akt phosphorylation and cell-type specificity of Akt phosphorylation induced by hyperosmolality

To investigate whether the phosphorylation of Akt protein is specific to hyperosmolality induced-apoptosis, we examined Akt phosphorylation in association with apoptotic changes induced by staurosporine (1 g) by looking at increments of caspase-3 activity, cell death ELISA assay, and DNA fragmentation. As shown in Figure 7 A–C, staurosporine caused apoptotic changes in MDCK cells. However, phosphorylation of Akt was not changed in staurosporine-treated MDCK cells Figure 7d, suggesting that the phosphorylation of the Akt protein is specific to hyperosmolality-induced apoptosis, at least in our experimental conditions. Next, the cell type specificity for hyperosmolality-induced Akt phosphorylation was examined. In LLC-PK1 cells, phosphorylation of Akt was observed from one to three hours in the 600 mOsm/kg H₂O final osmolality medium with NaCl Figure 7e. The LLC-PK1 cells were completely detached from the culture dishes six hours after the addition of the 600 mOsm/kg H₂O final osmolality medium. Thus, immunoblots could not performed later than 12 hours. From these data, phosphorylation of Akt was observed not only in MDCK cells, but also in LLC-PK1 cells after at least one to three hours of exposure to hyperosmolality.

Figure 7.

Specificity of hyperosmolality in Akt phosphorylation and cell-type specificity of hyperosmolality-induced Akt phosphorylation. (A) MDCK cells were exposed to staurosporine (1 mol/L) for 12 hours. Cell lysate was used for caspase-3 assay. (B) MDCK cells were exposed to staurosporine (1 mol/L) for 12 hours. Cell lysate from adherent and floating cells was subjected to cell death ELISA assay. (C) MDCK cells were exposed to staurosporine (1 mol/L) for 12 hours. Extracted DNA from adherent and floating cells was subjected to electrophoresis on 1.5% agarose gels. (D) Anti-phospho-Akt immunoblot of detergent lysates prepared from MDCK cells treated with staurosporine (1 mol/L) for 12 hours (upper panel). The abundance of Akt protein was demonstrated by immunoblots with an antibody to total Akt (lower panel). (E) Anti-Akt immunoblot of detergent lysates prepared from LLC-PK1 cells treated with NaCl at indicated times (upper panel). The abundance of Akt protein was demonstrated by immunoblots with an antibody to total Akt (middle panel). Densitometric analysis of phosphorylation of Akt. (0 hour; bottom panel). All data are mean SEM, N = 5, *P < 0.05 vs. control.

Mild hyperosmolality stimulated the phosphorylation of p53 in MDCK cells

To examine whether hyperosmolality stimulates the protein abundance and phosphorylation of p53 in MDCK cells, the cells were incubated with media containing NaCl or urea at 300, 400, 500, or 600 mOsm/kg H₂O final osmolality for one hour. Exposure to hyperosmolar media with NaCl (400, and 500 mOsm/kg H₂O final osmolality) increased the protein abundance of p53, as shown in Figure 8a. Densitometric analysis showed that phosphorylation of p53 was increased in the 400 and 500 mOsm/kg H₂O final osmolality mediums. In the higher osmolality medium of 600 mOsm/kg H₂O final osmolality with NaCl, phosphorylation of p53 was decreased as shown in Figure 8a. On the other hand, exposure to hyperosmolar media with urea (400, 500, or 600 mOsm/kg H₂O final osmolality) did not significantly change the protein abundance of p53, as shown in Figure 8b. Phosphorylation of p53 was slightly increased in the 400 and 500 mOsm/kg H₂O final osmolality mediums with urea, and was decreased in the 600 mOsm/kg H₂O final osmolality medium Figure 8b.

Figure 8.

Mild hyperosmolality stimulated the phosphorylation of p53 in MDCK cells. To examine whether hyperosmolality stimulates the protein abundance and phosphorylation of p53 in MDCK cells, the cells were incubated with media containing NaCl or urea at 300, 400, 500, or 600 mOsm/kg H₂O final osmolality for one hour. (A) The abundance of phospho-p53 (Ser15) protein was demonstrated by immunoblots with an antibody to phospho-p53 (upper panel). Anti-p53 immunoblot of detergent lysates prepared from MDCK cells treated with NaCl at the indicated times (middle panel). Densitometric analysis of the phospho-p53 (Ser15) protein level (N = 5, mean SEM, *P < 0.05 vs. 300 mOsm/kg H₂O final osmolality; lower panel). (B) The abundance of Phospho-p53 (Ser15) protein was demonstrated by immunoblots with an antibody to phospho-p53 (upper panel). Anti-p53 immunoblot of detergent lysates prepared from MDCK cells treated with urea at indicated times (middle panel). Densitometric analysis of phospho-p53 (Ser15; N = 5, mean SEM, *P < 0.05 vs. 300 mOsm/kg H₂O final osmolality; lower panel).

DISCUSSION

The major findings of the present study are, first, that the Akt pathway is strongly activated by hyperosmolality in vitro and in vivo, and second, that the activated Akt prevents apoptotic changes induced by mild hyperosmolality (500 mOsm/kg H₂O final osmolality) in renal tubular cells. We demonstrated that the PI3-K/Akt pathways help to maintain the balance between cell survival and apoptosis in hyperosmolality-treated MDCK cells.

To our knowledge, this is the first study demonstrating the activation of the Akt pathway in the inner medulla of dehydrated rats. In a previous report, Zhang et al demonstrated the activation of PI3-K and Akt signaling by urea in cultured mIMCD3 cells²⁰. However, they did not examine the physiological stimulation of the PI3-K and Akt pathways in in vivo experiments²⁰. We and other investigators observed that hyperosmolality induces apoptotic change in cultured renal tubular cells. Nevertheless, to our surprise, no apoptotic changes were observed in dehydrated renal medulla at osmolalities raised to as high as 1850 mOsm/kg H₂O. We hypothesized that this phenomenon was at least partially mediated by the activated Akt pathway, which prevents an apoptotic change of the inner medulla of dehydrated rats that are in a hyperosmotic condition. This hypothesis was supported by the results of our in vitro experiments, which showed that the Akt pathway suppresses apoptotic changes of MDCK in a mild hyperosmotic condition.

Furthermore, we evaluated apoptotic signals and activation of Akt in MDCK cells in a hyperosmotic condition. While studying the effects of hyperosmolality, we were surprised to find that at the final osmolality of 600 mOsm/kg H₂O, apoptotic changes were clearly observed in MDCK cells in spite of a strong activation of Akt. Furthermore, transfection of dominant-active Akt only partially suppressed the apoptotic changes detected by caspase-3 activity, DNA fragmentation, and cell death ELISA assay in a severely hypertonic condition. In contrast, transfection of dominant-active Akt suppressed caspase-3 activity and played anti-apoptotic roles in mildly hyperosmotic (500 mOsm/kg H₂O final osmolality) conditions. It is likely that apoptotic pathway mechanisms exist that cannot be completely inhibited by Akt in severely hyperosmotic conditions in vitro. Many reports have suggested the co-presence of several apoptotic pathways such as the caspase family, JNK pathway, p38K pathway, and p53 pathway^27,28,29. Activated Akt inhibited capsase-9–induced apoptosis and activation of the cytochrome c-pathway^30,31. However, it is not known whether activated Akt could inhibit JNK- and p53-mediated apoptosis.

Our findings demonstrate that activated Akt prevented apoptotic signaling in mildly hyperosmotic conditions but not in severely hyperosmotic conditions. A recent report by Dmitrieva et al described protective effects of p53 activation in hyperosmolality-induced apoptosis in mouse renal inner medullary collecting duct cells (mIMCD3)⁷, prompting us to examine activation of p53 in hyperosmolality-treated MDCK cells. Figure 7 shows that phosphorylation of p53 is increased in the 400 and 500 mOsm/kg H₂O final osmolality but is decreased in 600 mOsm/kg H₂O final osmolality. One possible explanation for the lack of Akt-induced suppression of apoptosis at the 600 mOsm/kg H₂O may be the decrease of p53 phosphorylation at 600 mOsm/kg H₂O. Not only Akt activation, but also phosphorylation of p53 might be necessary to prevent severe hyperosmolality (600 mOsm/kg H₂O final osmolality)-induced apoptosis. Further investigations are needed to explain this complex phenomenon.

Our findings are not completely in accord with the in vivo experiments, which did not show apoptosis in the inner medulla of dehydrated rats. This discrepancy may stem from the difference between in vivo and in vitro systems and/or other hormonal effects that are influenced by dehydration such as vasopression and the action of the renin-angiotensin system. There were discrepancies in osmolality between the in vivo and in vitro experiments. Although the usual osmolality in the inner medulla is not 300 mOsm/kg H₂O, the MDCK cells do not share identical characteristics with the inner medullary collecting duct cells in vivo. Many previous studies on the osmotic changes of MDCK cells or inner medullary epithelial cells relied on experiments in ranges from 300 to 600 or 300 to 800 mOsm/kg H₂O^{5,6,7,10,20,24}. Thus, our same experimental condition has been used quite frequently in these earlier experiments on hyperosmolality.

Another interesting in vitro finding was that phosphorylation of Akt in the presence of hyperosmotic media resulted in dual peaks, as shown in Figure 2. We have no clear explanation about the dual peaks of Akt phosphorylation. One possible explanation is that hyperosmolality stimulates not only Akt, but also many cytokines, enzymes such as cyclooxygenase-2, and many kinases such as ERK and p38 kinase^8,9,32. In turn, these enzymes and cytokines could stimulate the Akt phosphorylation. Another possible explanation is related to cell cycle arrest. Michea et al reported a cell cycle delay by hyperosmolality⁶, and many kinases related to cell cycle regulation may contribute to the late phase of Akt phosphorylation. Further experiments are necessary in future studies to resolve the precise mechanism(s).

Regarding the possible mechanism of the hyperosmolality-stimulated Akt signaling pathway Figure 9, our results raise questions as to how hyperosmolality stimulates the Akt pathway in MDCK cells and renal inner medulla. We and others have reported previously that hyperosmolality increased Ins 1,4,5-P₃ levels and mitogen-activated protein kinase activity in renal tubular cells^24,33. These findings suggest that cellular responses to hyperosmotic changes are mediated at least partially via phospholipase C- (PLC-). Recently, Zhang et al reported that urea treatment results in the activation of the SH2 domain-containing phospholipase, PLC-²⁰. They speculated that a urea-activatable (tyrosine-phosphorylated) upstream receptor or nonreceptor tyrosine kinase recruited and activated PLC-, and additionally may activate another tyrosine kinase effector, PI3-K²⁰. PI3-K phosphorylates membrane phosphoinositides at the D-3 position. These phospholipids act as second messengers that mediate the activation of Akt¹⁵. Phosphoinositide-dependent kinase I (PDKI) and PDKII phosphorylate Akt at residues Thr308 and Ser473 and activate Akt¹². Activated Akt inhibits activation of capsase 9- and cytochrome c-induced apoptosis^30,31. Thus, our most favored hypothesis is that the alteration of ionic and solute concentrations in the cell activates PLC- and PI3-K, and then the activated PI3-K phosphorylates Akt and inhibits apoptosis in MDCK cells. This process is summarized in Figure 9.

Figure 9.

Schema of the role of Akt in hyperosmolality-induced apoptosis in MDCK cells. MDCK cellular responses to hyperosmotic changes are mediated at least partially via phospholipase C- (PLC-). Osmolality-activatable (tyrosine-phosphorylated) upstream receptor (osmosensor) or nonreceptor tyrosine kinase recruited and activated PLC- and may activate another tyrosine kinase effector, PI3-K²⁰. PI3-K phosphorylates membrane phosphoinositides at the D-3 position. These phospholipids act as second messengers that mediate the activation of Akt¹⁴. Phosphoinositide-dependent kinase I (PDKI) and PDKII phosphorylate Akt at residues Thr308 and Ser473 and activate Akt¹¹. Activated Akt inhibited the activation of capsase 9- and cytochrome c-induced apoptosis^30,31.

In conclusion, we demonstrate that anti-apoptotic/cell survival pathways help to confer the balance between cell survival and apoptosis in hyperosmolality-treated MDCK cells and in the inner medulla of the dehydrated rat. These results provide insight into the highly organized signaling mechanisms coordinated by the Akt pathway and apoptotic signals in renal tubular cells in vitro and in vivo.

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