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Aldosterone induces epithelial-mesenchymal transition via ROS of mitochondrial origin

Division of Nephrology, University of Utah and Veterans Affairs Medical Center, Salt Lake City, Utah

Submitted 5 December 2006 ; accepted in final form 11 May 2007

	ABSTRACT

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It has been well appreciated that aldosterone (Aldo) plays a direct profibrotic role in the kidney but the underlying mechanism is unclear. We examined the role of Aldo in epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Exposure of human renal proximal tubular cells to Aldo for 48 h dose dependently induced EMT as evidenced by conversion to the spindle-like morphology, loss of E-cadherin, and de novo expression of -smooth muscle actin (SMA); the effect was noticeable at 50 nM and maximal at 100 nM. The EMT was completely blocked by the selective mineralocorticoid receptor (MR) antagonist eplerenone. Aldo time dependently increased intracellular reactive oxygen species (ROS) production that was detectable at 15 min and peaked (2.3-fold) at 60 min, as assessed by 2',7'-dichlorofluorescin diacetate fluorescence. Aldo-induced oxidative stress and EMT were both abolished by the mitochondrial respiratory chain complex I inhibitor rotenone, but not the NADPH oxidase inhibitor apocynin. Aldo induced phosphorylation of ERK1/2 that was completely blocked by rotenone. Male 129-C57/BL6 mice were treated with deoxycorticosterone acetate (DOCA) salt (subcutaneous implantation of 50 mg of DOCA pellet plus 1% NaCl as drinking fluid) for 3 wk and animals were treated with vehicle or rotenone (600 ppm in diet) for the last week. DOCA salt induced a 2.5-fold increase in -SMA and a 30% reduction of E-cadherin, as assessed by real-time RT-PCR, that were both restricted to renal epithelial cells, as determined by immunohistochemistry. In contrast, DOCA salt-induced changes in -SMA and E-cadherin were completely blocked by treatment with rotenone. These observations suggest that Aldo induces EMT via MR-mediated, mitochondrial-originated, ROS-dependent ERK1/2 activation in renal tubular epithelial cells.

reactive oxygen species; renal tubular epithelial cells

Epithelial-mesenchymal transition (EMT) defines a phenotypic conversion in epithelial cells, leading to the loss of epithelial cell-cell-basement membrane contacts and structural/functional polarity and the acquisition of a fibroblastic phenotype (6, 11, 31). Emerging evidence has established EMT as a major mechanism of tubulointerstitial fibrosis (4, 5, 21, 28). EMT typically occurs in response to a number of environmental stresses and associated cytokine/growth factor stimuli, such as mechanical stretch (27), cyclosporine treatment (13), exposure to advanced glycation end products (AGE consequence of hyperglycemia) (10), hypoxia (12), oxidative stress (22), activated monocyte supernatant (17), interleukin-1 (32), and oncostatin M treatment (17), as well as the culturing of cells on collagen I (36). The goal of the present study is to test whether Aldo induces EMT in renal epithelial cells and if so to determine the underlying mechanism.

	MATERIALS AND METHODS

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Reagents and antibodies. Aldo, apocynin, rotenone, N-acetyl-L-cysteine (NAC), and mouse monoclonal anti--smooth muscle actin (SMA) antibody were purchased from Sigma (St. Louis, MO). Extracellular signal-regulated kinases 1/2 (ERK 1/2) inhibitor, U0126, was purchased from Calbiochem (Cambridge, MA). Eplerenone, a specific MR blocker, was provided by Pfizer (New York, NY). Mouse monoclonal anti-E-cadherin antibody was provided from BD PharMingen (San Diego, CA). Rabbit monoclonal antiphospho-p44/42 ERK1/2 (Thr202/Tyr204) and anti-total p44/42 ERK1/2 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-MR polyclonal antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA).

Mice and experimental protocol. Eight-week-old male 129/Sv-C57BL/6J mice (2530 g body wt) were implanted subcutaneously with 21-day-release DOCA pellets containing 50 mg DOCA (Innovative Research of America, Sarasota, FL) by an incision of the right flank region under light 3% isoflurane anesthesia. After 14 days, DOCA-treated animals received vehicle or rotenone (600 ppm in diet) for 7 days. All animals (control, DOCA salt, and DOCA salt plus rotenone) received 1% NaCl in tap water starting on the first day of DOCA treatment. All mice were placed on standard pelleted rodent chow and housed in an air-conditioned room with a 12:12-h light-dark cycle. All animal procedures were approved by the University of Utah Institutional Animal Care and Use Committee.

Human kidney-2 cells. HK-2 cells, the immortalized human proximal tubular cell line, were grown in keratinocyte serum-free media (KSFM) supplemented with bovine pituitary extract and epidermal growth factor (Invitrogen). The cells were grown at 37°C in a humidified 5% CO₂ incubator and subcultured at 50–80% confluence using 0.05% trypsin-0.02% EDTA (Invitrogen).

RT-PCR and real-time RT-PCR. Total RNA was isolated from HK-2 cells or whole kidney tissues using TRIzol Total RNA Isolation kit (Invitrogen) according to the manufacturer's protocol. The RNA was eluted with RNase-free water. Reverse transcription was performed using the Superscript III RT kit (Invitrogen) according to the manufacturer's protocols. Briefly, the reactions were incubated at 65°C for 5 min and then at 50°C for 60 min. Oligonucleotides (Table 1) were designed by Primer3 software (available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and synthesized at the University of Utah. Real-time PCR amplification was performed using the SYBR Green master mix (Applied Biosystems) and the Prism 7500 Real-time PCR Detection System (Applied Biosystems). Cycling conditions were 95°C for 10 min followed by 40 repeats of 95°C for 15 s and 60°C for 1 min. Relative amounts of mRNA were normalized by -actin or GAPDH and calculated using the delta-delta method from threshold cycle numbers (35).

Fluorescent immunohistochemistry. The kidneys were fixed in 4% paraformaldehyde and imbedded with paraffin. Paraffin sections of each specimen were cut at 5 µm (Cryostat 2800 Frigocut-E, Leica Instruments) and a standard protocol using xylene and graded ethanol was employed to deparaffinize and rehydrate. The sections were washed with K⁺-free PBS and treated with blocking buffer containing 50 mM NH₄Cl, 2% BSA, 0.05% saponin in K⁺-free PBS for 20 min at room temperature for permeabilization. The sections were then incubated overnight at 4°C with mouse monoclonal anti--SMA or anti-E-cadherin antibodies at 2–5 µg/ml. The sections were rinsed and incubated for 30 min with FITC-labeled goat anti-mouse IgG at 2 µg/ml. In control slides, equal amounts of mouse IgG were used instead of the primary antibodies. All stainings were visualized under confocal laser-scanning microscopy.

Western blotting. HK-2 cells were lysed and subsequently sonicated in PBS containing 1% Triton X-100, 250 µM phenylmethanesulfonyl fluoride (PMSF), 2 mM EDTA, and 5 mM dithiothrietol (DTT; pH 7.5). Protein concentrations were determined by Coomassie reagent. Thirty micrograms of protein from whole cell lysates were denatured in boiling water for 10 min, separated by SDS-PAGE gel, and transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for 1 h with mouse anti--SMA or E-cadherin or rabbit anti-total ERK1/2 antibodies at a dilution of 1:1,000. After being washed with TBS, blots were incubated with a goat anti-horseradish peroxidase-conjugated secondary antibody and visualized with ECL kits (Amersham).

Phosphorylation of MAP kinases. HK-2 cells grown in a six-well plate were lysed by sonication for 10 s in 200 µl of 1x Laemmli sample buffer containing 10 mM Tris, 1.4% SDS, and 40 mM DTT (pH 6.8). The protein samples were heated at 60°C for 15 min and electrophoresis was performed as described above. The blots were blocked in 5% nonfat dry milk for 1 h and incubated overnight at 4°C with the primary antibodies against phospho-ERK1/2 at a dilution of 1:1,000. The secondary antibody and ECL reaction were the same as described above.

DCFDA fluorescence measurement of reactive oxygen species. The fluorogenic substrate 2',7'-dichlorofluorescein diacetate (DCFDA) is a cell-permeable dye that is oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by H₂O₂ and can therefore be used to monitor intracellular generation of reactive oxygen species (ROS). For measurement of ROS, cells were grown on glass coverslides. When the cells reached confluence, they were washed twice with PBS and incubated for 30 min with 10 µM DCFDA and then treated with Aldo in the presence or absence of apocynin or rotenone at appropriate concentrations. At the end of the incubation period, the cells were again washed twice with PBS and imaged by fluorescence microscopy. To quantitate ROS levels, cells were seeded to a 96-well plate and treated as described above. Relative fluorescence was measured using a fluorescence plate reader (FLUOstar OPTIMA) at excitation and emission wavelengths of 485 and 528 nm, respectively, three times at 90-s intervals.

Statistical analysis. Values shown represent means ± SE. Statistical analysis was performed by one-way ANOVA and Bonferroni tests with a P value of <0.05 being considered statistically significant.

	RESULTS

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Aldo-induced EMT. To evaluate EMT, we used three independent parameters: cell morphology, the expression of E-cadherin, and -SMA. The morphological changes were assessed by phase contrast microscopy. Control HK-2 cells formed a confluent monolayer and exhibited cobblestone-like morphology (Fig. 1A); while following exposure to 100 nM Aldo for 48 h, the cells exhibited striking morphological changes, displaying an elongated and fibroblast-like morphology (Fig. 1B).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Aldosterone (Aldo)-induced epithelial-mesenchymal transition (EMT). A and B: morphological changes. The cells were grown in 6-well plates until 80% confluence and then treated with vehicle (A) and Aldo (100 nM; B) for 48 h. Photographs were taken using a Nikon microscope (phase contrast). C and D: real-time RT-PCR analysis for E-cadherin and -smooth muscle actin (SMA) expression. The cells were treated with Aldo (10–100 nM) for 12 h and E-cadherin (C) and -SMA (D) expression was detected by real-time RT-PCR. E and F: Western blotting analysis for E-cadherin and -SMA. The cells were treated with Aldo (10–100 nM) for 48 h and E-cadherin (E) and -SMA (F) expression was detected by immunoblotting. Top: representative immunoblots. Bottom: densitometric analysis; n = 3 for each group. *P < 0.01 vs. control (P < 0.01, 1-way ANOVA for C, D, E, and F).

View larger version (103K): [in this window] [in a new window]   Fig. 2. Fluorescent immunocytochemistry for E-cadherin and -SMA. The cells were grown on the coverslides until 80% confluence and then treated with vehicle (A, C) or Aldo (100 nM; B, D) for 48 h. Immunofluorescence was performed using mouse monoclonal anti-E-cadherin (A, B) or anti--SMA (C, D) and FITC-labeled goat anti-mouse antibodies. Cells were visualized and photographed by fluorescence microscopy at x200 magnification for E-cadherin and x400 magnification for -SMA.

Role of MR in Aldo-induced EMT. To address this issue, we first examined whether MR was expressed in cultured HK-2 cells. RT-PCR and immunoblotting revealed the presence of both MR mRNA and protein in these cells (Fig. 3, A and B). Immunofluorescence showed staining of MR antibody almost exclusively in the cytosol in basal state (Fig. 3C) but in the nucleus following 60 min of Aldo treatment, indicating nearly complete translocation of MR (Fig. 3D). To address the functional role of MR, we examined the effect of the MR antagonist eplerenone on Aldo-induced EMT. As shown in Fig. 4, in the presence of eplerenone, the EMT was almost completely prevented as assessed by changes in morphology, -SMA, and E-cadherin.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Expression of mineralocorticoid receptor (MR) in HK-2 cells. A: RT-PCR analysis of the MR was performed from RNA isolated from HK-2 cells. Ethidium bromide-stained agarose gels for the MR. B: immunoblotting of the MR from HK-2 cells. C: fluorescent immunocytochemistry of the MR in HK-2 cells. HK-2 cells were incubated in keratinocyte serum-free media (KSFM) in the absence (left) and presence (right) of Aldo for 60 min and then were fixed with acetone-methanol and stained with MR antibody. Magnification x400.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Aldo-induced EMT via MR. The cells were grown in 6-well plates until 80% confluence and then pretreated with eplerenone (10 µM) for 30 min followed by incubation with Aldo (100 nM) for further 48 h. A–C: morphological changes (A: vehicle; B: Aldo; C: Aldo plus eplerenone). Photographs were taken using a Nikon microscope (phase contrast). D and E: real-time RT-PCR analysis for -SMA (D) and E-cadherin (E) expression. F and G: Western blotting analysis for -SMA (F) and E-cadherin (G). Top: representative immunoblots. Bottom: densitometric analysis; n = 3 for each group. *P < 0.01 vs. the Aldo alone group (P < 0.01, 1-way ANOVA for D, E, F, and G).

View larger version (54K): [in this window] [in a new window]   Fig. 5. Aldo-induced reactive oxygen species (ROS) production of mitochondrial origin. A and B: confluent HK-2 cells in chamber slides were exposed to vehicle (A) or Aldo (100 nM; B) for 60 min in the presence of 2',7'-dichlorofluorescein diacetate (DCFDA). 2',7'-Dichlorofluorescein (DCF) fluorescence was visualized by fluorescence microscopy. C and D: quantitation of DCF fluorescence. Confluent HK-2 cells in 96-well plates were treated with 100 nM Aldo for the indicated periods of time (0–60 min; C) or pretreated with eplerenone (10 µM), apocynin (100 µM), or rotenone (10 µM), and then stimulated by 100 nM Aldo for further 60 min in the presence of DCF (D). Fluorescence was quantified using FLUOstar OPTIMA. E and F: real-time RT-PCR analysis for p47phox and gp91phox expression. The cells were grown in 6-well plates until 80% confluence and then incubated with Aldo at the indicated concentrations (0–100 nM) for 12 h. Values represent means ± SE, n = 4. *P < 0.05. P < 0.01 vs. control. #P < 0.01 vs. the Aldo alone group. NS, no significant difference vs. the Aldo alone group (P < 0.01, 1-way ANOVA for C and D).

Activation of ERK1/2 by mitochondrial-derived ROS. Given the potential role of ERK1/2 in mediating TGF-1-induced EMT (22, 33), we examined whether ERK1/2 served as a target of ROS in Aldo-induced signaling pathway in cultured HK-2 cells. Within minutes, Aldo treatment significantly increased phosphorylation of ERK1/2 without affecting the total abundance of the proteins (Fig. 6A). The activation of ERK1/2 was almost completely abolished by eplerenone and NAC contrasting with a less significant effect of apocynin (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Aldo-induced ERK1/2 activation and its inhibition by eplerenone, N-acetyl-L-cysteine (NAC), and rotenone. A: time course of Aldo-stimulated ERK1/2 activity in HK-2 cells. The cells were stimulated with 100 nM Aldo for the indicated periods of time (0–30 min). B: effect of eplerenone, NAC, rotenone, and apocynin on the Aldo-induced ERK1/2 activation. HK-2 cells were pretreated with eplerenone (10 µM), NAC (5 mM), rotenone (10 µM), and apocynin (100 µM) for 30 min and then stimulated with Aldo (100 nM) for further 30 min. Top: representative immunoblots. Bottom: densitometric analysis; n = 3 for each group. *P < 0.01 vs. control (A: graph) or vs. the Aldo alone group. NS, no significant difference vs. the Aldo alone group (P < 0.01, 1-way ANOVA for A and B).

View larger version (13K): [in this window] [in a new window]   Fig. 7. Aldo-stimulated Snail-1 mRNA expression. A: dose response of Aldo-induced snail-1 gene expression. HK-2 cells were stimulated with the indicated concentrations of Aldo (0–100 nM) for 6 h. B: effect of eplerenone, NAC, rotenone, apocynin, or U0126 on the Aldo-induced Snail-1 expression. HK-2 cells were pretreated with eplerenone (10 µM), NAC (5 mM), rotenone (10 µM), apocynin (100 µM), or U0126 (10 µM) for 30 min and then stimulated with Aldo (100 nM) for 6 h. Snail-1 mRNA expression was determined by real-time RT-PCR. *P < 0.01 vs. control. P < 0.01 vs. the Aldo alone group. NS, no significant difference vs. the Aldo alone group (P < 0.01, 1-way ANOVA for A and B).

View larger version (32K): [in this window] [in a new window]   Fig. 8. Effect of rotenone and U0126 on Aldo-induced EMT. The cells were grown in 6-well plates until 80% confluence and then pretreated with rotenone (10 µM) or U0126 (10 µM) for 30 min followed by incubation with Aldo (100 nM) for further 12 h (for real-time RT-PCR) or 48 h (for immunoblotting). A–D: morphologic changes (A: vehicle; B: Aldo alone; C: Aldo plus rotenone; D: Aldo plus U0126). Photographs were taken using a Nikon microscope (phase contrast). E and F: real-time RT-PCR analysis for -SMA (E) and E-cadherin (F) expression. G and H: Western blotting analysis for -SMA (G) and E-cadherin (H). Top: representative immunoblots. Bottom: densitometric analysis; n = 3 for each group. *P < 0.01 vs. the Aldo alone group (P < 0.01, 1-way ANOVA for all graphs).

View larger version (80K): [in this window] [in a new window]   Fig. 9. Rotenone inhibited DOCA salt-induced EMT in mice. DOCA salt-treated wild-type mice received vehicle or rotenone for 1 wk. -SMA and E-cadherin expression was determined by real-time RT-PCR (A: -SMA; B: E-cadherin) and immunofluorescence (C, D, and E: -SMA; F, G, and H: E-cadherin; C and F: control; D and G: DOCA salt/vehicle; E and H: DOCA salt/rotenone), respectively. V, vasculature. *P < 0.01 vs. control. P < 0.01 vs. the DOCA salt alone group (P < 0.01, 1-way ANOVA for A and B).

	DISCUSSION

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MR has been suggested to mediate the pathogenic role of Aldo. We demonstrated the existence of MR in cultured HK-2 cells at both mRNA and protein levels. Furthermore, in response to Aldo treatment, MR was translocated from the cytosol to the nucleus in HK-2 cells, suggesting activation of MR. Blockade of MR with eplerenone remarkably inhibited Aldo-induced EMT. Taken together, the evidence obtained from the cell culture model suggests that MR is likely responsible for the profibrotic actions of Aldo in renal epithelial cells.

In line with the pivotal role of ROS in Aldo-induced renal injury, we observed a direct stimulatory effect of Aldo on ROS production in a human renal proximal tubular cell line. Subsequently, we attempted to determine the source of Aldo-induced ROS, e.g., the mitochondria electron-transfer chain vs. the NADPH oxidase using the mitochondrial respiratory chain complex I inhibitor rotenone and the NADPH oxidase inhibitor apocynin. Rotenone was highly effective not only in inhibiting Aldo-induced ROS production in cultured HK-2 cells but also in attenuating EMT in DOCA salt-treated mice. In contrast, apocynin was without an obvious effect on Aldo-induced ROS production. Also, Aldo treatment failed to elevate gene expression of gp91^phox and p47^phox in HK-2 cells. Taken together, these observations suggest that Aldo-induced ROS in HK-2 cells may be derived from mitochondria rather than NADPH oxidase. This notion is in contrast to a number of previous studies indicating NADPH oxidase as a source of Aldo-induced ROS production in rat mesangial cells (15) and rat ventricular cardiomyocytes (26). A possibility exists that Aldo utilizes different sources to generate ROS depending on individual cell types.

Given the important role of mitochondria in energy production, rotenone is suspected to induce some degree of toxicities. Therefore, we performed histological analysis of several internal organs as well as TUNEL assay on the kidney. These assays did not reveal obvious tissue injuries associated with rotenone treatment (data not shown). A study from National Toxicology Program reported that rotenone treatment for 14 days at 1,200 ppm or higher doses induced decreases in body weight gain in rats but not in mice and this dose range was even well tolerated for up to 2 yr. (1) It is unexpected that the relatively low dose and short duration of rotenone treatment in the present study will produce significant toxicities.

What would be the molecular targets of Aldo-induced ROS in renal epithelial cells? One of these targets appeared to be ERK1/2. We showed that Aldo treatment induced ERK1/2 activation that was significantly blocked by MR antagonist eplerenone, and by NAC and rotenone but not apocynin. We further demonstrated that the blockade of ERK1/2 with U0126 was able to prevent Aldo-induced EMT in HK-2 cells. It is likely that ERK1/2 acts downstream of mitochondrial-derived ROS in Aldo-induced signaling pathway in renal epithelial cells.

The Snail is a zinc finger transcription factor responsible for the loss of E-cadherin during EMT (3, 14, 34). We examined that possibility that the Snail may serve as a component in Aldo-induced signaling cascades in cultured renal epithelia cells. Indeed, Aldo treatment induced Snail-1 mRNA expression as early as 6 h preceding the occurrence of EMT. The stimulation was significantly blocked by eplerenone, NAC, and rotenone but not apocynin, similar to the pattern of changes in ERK1/2, suggesting that ERK1/2 and the Snail may both act downstream of mitochondrial-derived ROS. The effectiveness of U0126 in the inhibition of the Snail suggests that the transcription factor may serve as a downstream target of ERK1/2 in Aldo-induced cascades in HK-2 cells.

In summary, this study, for the first time, presents evidence that Aldo induces EMT in renal epithelial cells both in vitro and in vivo through MR-mediated, mitochondrial ROS-dependent activation of ERK1/2 and the Snail. These findings provide new insights into the renal deleterious effects of Aldo. The new information might be useful for the development of novel therapeutic strategies for renal fibrosis.

	GRANTS

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This work was supported by National Institutes of Health Grants RO-1 HL-079453, RO-1 DK-066592, and VA Merit Review (to T. Yang).

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Yang, Univ. of Utah, 30 N 1900 E, Rm 4R312, Salt Lake City, UT 84132 (e-mail: tianxin.yang{at}hsc.utah.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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