Expression and Function of Ets-1 during Experimental Acute Renal Failure in Rats

Cell Culture and Exposure to Hypoxia

LLC-PK1 cells, originally purchased from American Type Culture Collection (Rockville, MD), were grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin, and 10% heat-inactivated FCS (Life Technologies). Cells were cultured at 37°C in 20% O₂ and 5% CO₂ (referred to as the normoxic condition). For the hypoxia experiments, cells were placed in a hypoxic chamber (Bellow Glass, Vineland, NJ) that contained 0% O₂ and 5% CO₂, which was maintained at 37°C.

Plasmid Constructs

The Ets-1 reporter construct used for the luciferase assays contained a mouse Ets-1 promoter (−2.1 kbp) cloned upstream of the luciferase gene (28). The cyclin D1 reporter construct used for the luciferase assays contained a human cyclin D1 promoter from residues −944 to 139 cloned upstream of the luciferase gene (a gift of Dr. M. Eilers Zentrum für Molekularbiologie Heidelberg, Heidelberg, Germany) (29). Expression vectors coding for HIF-1α and dominant-negative (dn) HIF-1α were prepared as described previously (30,31⇓) using the following primers: forward 5′-GGAAGACAACGCGGGCAC-3′ and reverse 5′-GGAGCTGTGAATGTGCTGTGATCTGGC-3′ and dnHIF-1α forward 5′-CCGCTCGAGACCATGCGAAGCAAAGAGTCTG-3′ and reverse 5′-GGGGTACCTCACTTATCA- AAAAGGCAGCT-3′. An open reading frame coding for the dnHIF-1α is a 1.1-kbp fragment whose product lacks both the DNA binding and transactivation domain. In both cases, RNA out of a C57BL mouse kidney was used as a template. The PCR fragment was subcloned into pcDNA3.1 (Invitrogen, San Diego, CA) and was processed to dideoxy-DNA sequencing.

Transient Transfection and Luciferase Assay

LLC-PK1 cells were transfected by the electroporation method with plasmid DNA (10 μg). Data are representative of at least four independent experiments performed in duplicate and are expressed as an n-fold increase in the luciferase activity calculated relative to the indicated level of Ets-1 promoter activity. Normalization was achieved by co-transfecting a β-galactosidase reporter construct, as described previously (12). Luciferase and β-galactosidase activities were measured according to the Promega (Madison, WI) protocol.

Recombinant Adenoviruses

Replication-defective, recombinant adenoviruses encoding human Ets-1 (Adets-1) and a control adenovirus (Adnull) were prepared as described previously (32). Replication-defective, recombinant adenoviruses encoding the rat dominant-negative Akt (AdAktDN) were also prepared as described previously (33). In this adenovirus, both Thr308 and Ser473 of the rat Akt1 were replaced by alanine (33).

Induction of ARF

Male Sprague-Dawley rats (Saitama Experimental Animal Supply, Saitama, Japan) that weighed 150 to 200 g were anesthetized intraperitoneally with sodium pentobarbital (30 mg/kg) at surgery. The left renal artery was occluded with Sugita aneurysm clips (Mizuho Ikakogyo, Tokyo, Japan) for 60 min. The clamps were removed after 60 min; the incisions were closed; and the rats were killed at 0, 6, 12, 24, 48, and 72 h (n = 5). The left kidney was removed quickly and processed for histologic evaluation, protein extraction, or RNA extraction. Age- and weight-matched Sprague-Dawley rats also received sham operations in a similar manner, except for clamping of the renal arteries at 6, 12, and 24 h (n = 5).

Isolation of Kidney Tissue and Histologic Examination

Rats were anesthetized with pentobarbital at indicated times after the ischemic event. Their kidneys were perfused with sterile PBS. The left kidney was excised quickly, frozen in liquid nitrogen, and homogenized in the SDS sample buffer described later.

For immunohistochemical studies, renal tissues were fixed in formalin overnight and then dehydrated and embedded in paraffin. Thin sections were examined with periodic acid-Schiff staining as described previously (34). Immunohistochemical staining was performed by a streptavidin and biotin technique using an anti–Ets-1–specific antibody (Santa Cruz Biochemical; N-276, cs-111), anti–proliferating cell nuclear antigen (PCNA)-specific antibody (Santa Cruz Biochemical; PC-10, cs-56), and anti–aquaporin-1–specific antibody (Santa Cruz Biochemical; B-11, sc-25287) as markers for proximal tubules, as described previously (35–37⇓⇓).

Western Blot Analysis

Homogenized total renal tissue or LLC-PK1 cells were lysed in an SDS sample buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM PMSF, and 0.1 mM sodium orthovanadate) at 4°C (38). Protein was transferred to a nitrocellulose membrane and probed with the anti–Ets-1–specific antibody. The primary antibodies were detected using horseradish peroxidase–conjugated rabbit anti-mouse IgG and visualized by the Amersham ECL system (Amersham, Arlington Heights, IL).

Electrophoretic Mobility Shift Assay

Nuclear extracts from the renal cortex were prepared as described previously (39). The extracts (10 μg) underwent a reaction in a premixed incubation buffer (Gel Shift Assay Kit; Promega) with the γ-³²P-end-labeled ets-1 binding site of a cyclin D1 promoter-lesion (5′-GATCTCGAGCAGGAAGTTCGA-3′) for 30 min at 25°C. To establish the specificity of the reaction, we performed competition assays with 100-fold excess of unlabeled ets-1 binding oligonucleotides (heterologous competitor DNA). To perform supershift assay, we added 5 μg of anti–Ets-1 antibody (Santa Cruz; N-276, cs-111) to the nuclear extracts, incubated extracts for 1 h at 4°C, and performed gel shift assay. After the reaction, the samples were analyzed on a 6% nondenaturing polyacrylamide gel. The gel was dried, and the protein-DNA complexes were visualized by autoradiography.

Real-Time Quantitative PCR

We performed a reverse transcription–PCR (RT-PCR) reaction for Ets-1 from RNA extracted from the ischemia-reperfusion kidneys. Total RNA was harvested from renal tissue using TRI-REAGENT (Life Technologies) (40). One microgram of total RNA samples was used for the RT-PCR as follows. The real-time quantitative PCR method was used to detect accurately the changes in Ets-1 gene copies. Total RNA was harvested from renal tissue. Rat Ets-1 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA were amplified. The primers for rat Ets-1 were 5′-GCCCAGCTTCATCACAGAGT-3′ (upper) and 5′-TGTTGAAAGATGACTGGCTG-3′ (lower) (23). The predominant cDNA amplification product was predicted to be 296 bp in length. The RT-PCR of GAPDH served as positive controls. The primers for rat GAPDH were 5′-TCCCTCAAGATTGTCAGCAA-3′ (upper) and 5′-AGATCCACAACGGATACATT-3′ (lower) (41). The predominant cDNA amplification product was predicted to be 309 bp in length. PCR products were detected and quantified in real time using the LightCycler Real-Time PCR (Roche Molecular Biochemicals, Tokyo, Japan) as described previously (42,43⇓). A three-step PCR was performed for 35 cycles. Denaturation was performed at 94°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s. The PCR products of Ets-1 and GAPDH were subcloned to the TA cloning vector (Promega, Biotec, Madison, WI) as described previously (40). The plasmids that contained Ets-1 cDNA and GAPDH cDNA were used to make standard curves of quantitative PCR.

Cell Proliferation Analysis by [³H]Thymidine Incorporation

After transfection, the LLC-PK1 cells were plated in 24-well plates and incubated in a medium without FCS for 20 h. For the last 4 h, the cells were pulsed with 1 μCi [³H]thymidine (Amersham). After the incubation, the cells were redissolved in 0.5 M NaOH and counted in an Aquasol-2 scintillation cocktail (NEN Research Products, Boston, MA) (44).

Caspase 3 Assays

A Caspase 3 Fluorometric Protease Assay Kit (MBL, Tokyo, Japan) was used for the measurement of caspase 3 activities as described previously (33). In brief, the cells were plated in six-well dishes and infected with adenoviruses. The cell lysates then were incubated with the same amounts of reaction buffer and a 50-mM DEVD-AFC substrate for 2 h at 37°C. Fluorescence was monitored with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

Cell Death ELISA

Histone-associated DNA fragments were quantified by ELISA (Boehringer Mannheim). All cells from each well were collected by trypsinization and pipetting, then pelleted (800 rpm, 5 min), lysed, and subjected to ELISA capture according to the manufacturer’s protocol (33). Cytosolic proteins were collected using a cell lysis buffer and centrifuged according to the manufacturer’s protocol. The nucleus formed into a pellet, and the cytoplasmic fraction became the supernatant. These supernatants were collected for the ELISA assay. Each experiment was carried out in triplicate and repeated in at least five independent experiments.

Statistical Analyses

The results are given as means ± SEM. The differences were tested using a two-way ANOVA followed by the Scheffe test for multiple comparisons. Two groups were compared by the unpaired t test. P < 0.05 was considered significant.

Western Blot Analysis of the Protein Expression of Ets-1 and HIF-1α after Ischemic Renal Failure

The left renal artery was clamped for 60 min and the left kidney was excised at 0, 6, 12, 24, 48, and 72 h after reperfusion. Western blot analysis was used to detect the protein levels of Ets-1 and actin. The expression of Ets-1 was weak in the control kidney (0 h) and sham-operated kidneys (6 and 12 h; Figure 1). The expression of Ets-1 was dramatically increased at 6 to 24 h after ischemia-reperfusion (Figure 1A). The upregulation of Ets-1 protein expression was temporary, with the intensity of the Ets-1 band decreasing at 48 and 72 h after ischemia-reperfusion. The protein levels of Ets-1 exhibited no changes after 6 to 12 h in the sham-operated rats (the data of 24 h are not shown). Western blot analysis was used to detect the protein levels of HIF-1α and actin. The expression of HIF-1α was weak in the control kidney (0 h) and sham-operated kidneys (6 and 12 h; Figure 1). The expression of HIF-1α was dramatically increased as early as 6 h after ischemia-reperfusion (Figure 1A). The upregulation of HIF-1α protein expression was temporary, with the intensity of the Ets-1 band decreasing at 48 and 72 h after ischemia-reperfusion. We performed an immunoblot for actin as a loading marker, and there were no significant changes in actin during ischemia-reperfusion. The protein levels of HIF-1α exhibited no changes after 6 to 12 h in the sham-operated rats (the data of 24 h are not shown).

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Figure 1. Protein expressions of Ets-1 and hypoxia-inducible factor-1α (HIF-1α) in the kidneys of rats that were subjected to 60 min of renal ischemia. The left renal artery was clamped for 60 min, and the left kidney was excised at 0, 6, 12, 24, 48, and 72 h after reperfusion (A). A sham operation was performed, and the left kidney was excised at 6 and 12 h (B). Extracted protein (20 μg) from renal tissue was separated by SDS-PAGE gels. Ets-1, HIF-1α, and actin protein levels were detected by Western blot analysis.

Immunohistochemical Examination of Ets-1 in ARF

Next, we performed immunohistologic studies on Ets-1 in ARF (Figures 2 and 3⇓). In a low-power view examination, Ets-1 expression was observed in cortical renal tubules at 12 h after ischemia-reperfusion (Figure 2A). We used the anti–aquaporin-1 antibody as a marker of proximal tubules (35–37⇓⇓). As shown in Figure 2, A and B, the expression of Ets-1 is co-localized with aquaporin-1 in the low-power view examination in continuous sections. Conversely, only a slight Ets-1 expression is observed in the renal cortex form control rats (Figure 2E), whereas the expression of aquaporin-1 is clearly observed in the renal cortex form control rats (Figure 2F). From these results, Ets-1 was expressed mainly in the proximal tubules of the renal cortex 12 h after ischemia-reperfusion (Figure 2). In the renal medulla, Ets-1 could not be detected in either the ischemia-reperfusion kidney (Figure 2C) or the control kidney (data not shown). In a higher-power view, Ets-1 staining was observed in the nucleus of proximal tubular cells (Figure 3A). As shown in Figure 3B, the expression of Ets-1 is co-localized with aquaporin-1 in continuous sections. To examine the specificity of the antibody, we used a blocking peptide (sc-111p; Santa Cruz Biotechnology). The nuclear signal diminished in the presence of the blocking peptide in the cortex of ischemia-reperfusion kidney (Figures 2G and 3C⇓).

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Figure 2. Immunohistological examination of Ets-1 in ischemic-reperfusion kidneys. Immunohistochemical analyses at a low-power view (×100) of the renal cortex were performed with antibodies against Ets-1 (A) and aquaporin-1 (B) at 12 h after ischemic injury. Immunohistochemical analyses at a low-power view (×100) of the renal medulla were performed with antibodies against Ets-1 (C) and aquaporin-1 (D) at 12 h after ischemic injury. Immunohistochemical analyses at a low-power view (×100) of the renal cortex of control kidneys were performed with antibodies against Ets-1 (E) and aquaporin-1 (F). Immunohistochemical analyses at a low-power view (×100) of the renal cortex of ischemic kidneys were performed with antibodies against Ets-1 in the presence of a blocking peptide (G). Histologic examinations A and B, C and D, and E and F are continuous sections, respectively.

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Figure 3. Immunohistologic examination of Ets-1 in proximal tubules of ischemic-reperfusion kidneys. Immunohistochemical analyses at a high-power view (×400) of the renal cortex were performed with antibodies against Ets-1 (A) and aquaporin-1 (B) at 12 h after ischemic injury. Histologic examinations A and B are continuous sections. The arrows indicate Ets-1– and aquaporin-1–positive cells. When a blocking peptide was added to the anti–Ets-1 antibody solution, no positive signal was observed in ischemia-reperfusion kidney (12 h; C).

To co-localize Ets-1 with dividing cells, we examined Ets-1 staining at 24 h after ischemia-reperfusion (Figure 4). Ets-1 expression was observed at the proximal tubules of the cortex (Figure 4, A and C). PCNA staining was also observed at the proximal tubules of the cortex (Figure 4, B and D). The higher power view of Ets-1 staining also revealed co-localization of Ets-1 and PCNA in the same tubules at 24 h after ischemia-reperfusion in continuous sections (Figure 4, C and D). Conversely, only a slight Ets-1 expression is observed in the renal cortex from control rats (Figure 4E). No PCNA-positive tubules were observed from control rats (Figure 4F). To examine the specificity of the antibody, we used a blocking peptide (sc-9857p; Santa Cruz Biotechnology). The nuclear signal diminished in the presence of the blocking peptide in the cortex of ischemia-reperfusion kidney (Figure 4G).

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Figure 4. Immunohistologic co-localization of Ets-1 and proliferating cell nuclear antigen (PCNA) in the proximal tubules of ischemic-reperfusion kidneys. Immunohistochemical analyses at a low-power view (×100) of the renal cortex were performed with antibodies against Ets-1 (A) and PCNA (B) at 24 h after ischemic injury. Histologic examinations A and B are continuous sections. Immunohistochemical analyses at a high-power view (×400) of the renal cortex of ischemic kidneys were performed with antibodies against Ets-1 (C) and PCNA (D) at 24 h after ischemic injury. The arrows indicate Ets-1– and PCNA-positive cells. Immunohistochemical analyses at a low-power view (×100) of the renal cortex were performed with antibodies against Ets-1 (E) and PCNA (F) of control kidney. Histologic examinations E and F are continuous sections. When a blocking peptide was added to the anti-PCNA antibody solution, no positive signal was observed in ischemia-reperfusion kidney (12 h; G).

Real-Time PCR

Quantification of the Ets-1 mRNA transcript using the real-time quantitative PCR method revealed 8.0-fold (6 h), 3.8-fold (12 h), and 2.2-fold (24 h) increases in Ets-1 mRNA levels compared with the 0-h value (Figure 5). The linear curve between the cDNA amount and PCR product was observed in the same range utilized by using the Ets-1 cDNA plasmid. The signals of Ets-1 were not significantly changed in the sham-operated rat kidneys. The GAPDH signal was not significantly changed by ischemia-reperfusion (Figure 5A).

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Figure 5. Quantitative analysis of Ets-1 expression in ischemic-reperfusion kidneys using real-time PCR. The left renal arteries were clamped for 60 min, and kidneys were excised at 0, 6, 12, 24, 48, and 72 h after reperfusion. Sham-operated rats at 6 h were also examined. Extracted total RNA were subjected to quantitative PCR using the LightCycler Real-Time PCR for the estimation of relative Ets-1 mRNA levels and the Ets-1 to glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA ratio, as described in the Materials and Methods section. (A) The representative agarose gels for Ets-1 and GAPDH are shown. (B) Each column with a bar shows the mean ± SEM (n = 5). *P < 0.05 versus control rats.

Hypoxia Stimulates Ets-1 Expression and Promoter Activity via HIF-1α in LLC-PK1 Cells

To examine whether hypoxia-reperfusion stimulates Ets-1 expression and promoter activity in renal tubular cells, we used a hypoxic culture system with LLC-PK1 cells. We exposed LLC-PK1 cells to hypoxia for 3, 6, and 6 h + reoxygenation 6 h and examined the promoter activity of Ets-1 and the expression of Ets-1 protein by Western blot analysis. In the promoter assay experiments, the Ets-1 promoter-luciferase plasmid was transfected in LLC-PK1 cells 48 h before hypoxia. As shown in Figure 6B, Ets-1 promoter activity was increased 7.1-fold at 3 h, 9.1-fold at 6 h, and 12.1-fold at 6 h + reoxygenation 6 h. Hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells (Figure 6A). There were no significant changes in actin during hypoxia/reoxygenation.

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Figure 6. Hypoxia stimulates Ets-1 expression and promoter activity via HIF-1α in LLC-PK1 cells. To examine whether hypoxia/reoxygenation stimulates Ets-1 expression and promoter activity in renal tubular cells, we used a hypoxic culture system with LLC-PK1 cells. We exposed LLC-PK1 cells to hypoxia for 3, 6, and 6 h + reoxygenation 6 h and examined the promoter activity of Ets-1 and the expression of Ets-1 protein by Western blot analysis. (A) Hypoxia induced changes in the Ets-1 protein level in LLC-PK1 cells by Western blot analysis. (B) Ets-1 promoter activity was increased 7.1-fold at 3 h, 9.1-fold at 6 h, and 12.2-fold at 6 h + reoxygenation. (C and D) To test whether the upregulation of Ets-1 is dependent on HIF-1α, we examined Ets-1 protein expression and promoter activity in HIF-1α–transfected LLC-PK1 cells and dnHIF-1α–transfected LLC-PK1 cells; n = 5, mean ± SEM; *P < 0.05 versus control.

To test whether the upregulation of Ets-1 is dependent of HIF-1α, we examined Ets-1 protein expression and promoter activity in HIF-1α–transfected LLC-PK1 cells and dnHIF-1α–transfected LLC-PK1 cells. The hypoxic inducibility of the Ets-1 promoter assay in HIF-1α–overexpressing and dnHIF-1α–overexpressing cells was 17.8- and 1.5-fold, respectively, as compared with 9.1-fold in control LLC-PK1 cells at 6 h (Figure 6D). The Western blot analysis demonstrated that transfection of dnHIF-1α reduced the increment of Ets-1 protein by hypoxia (Figure 6C). These results indicated that the upregulation of Ets-1 by hypoxia is dependent on HIF-1α.

Electrophoretic Mobility Shift Assay

To examine the induction of cyclin D1 expression via the ets-1 binding site, we performed an electrophoretic mobility shift assay using nuclear extracts from a rat renal cortex. Figure 7 shows that the nuclear extract from the ischemia-reperfusion rat renal cortex gave rise to a protein-DNA complex (Figure 7, lane 2). However, we did not detect protein-DNA complex band in control rat renal cortex (Figure 7, lane 3). The band was completed using 100-fold excess of the unlabeled oligonucleotides (heterologous competitor DNA; Figure 7, lane 4). When nuclear extracts from the ischemia-reperfusion rat renal cortex were preincubated with anti–Ets-1 antibody, the supershift of the band was observed (Figure 7, lane 6). These results confirm that Ets-1 binds to the ets-1 binding site of the cyclin D1 promoter in the ischemia-reperfusion condition.

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Figure 7. Electrophoretic mobility shift assay detected a protein binding to the promoter sequence of cyclin D1 in the nuclear extract of an ischemia-reperfusion rat kidney. A nuclear extract from renal cortex was prepared form the cortex of an ischemia-reperfusion rat kidney and a control rat kidney. The nuclear extract (10 μg) underwent a reaction in a premixed incubation buffer (gel shift assay kit) with a γ-³²P-end-labeled Ets-1 binding site of the cyclin D1 promoter lesion (5′-GATCTCGAGCAGGAAGTTCGA-3′) for 30 min at 25°C. To establish the specificity of the reaction, we performed competition assays with 100-fold excess of unlabeled ets-1 biding oligonucleotides (heterologous competitor DNA). To perform supershift assay, we added 5 μg of anti–Ets-1 antibody (N-276, cs-111) to the nuclear extracts, incubated the extracts for 1 h at 4°C, and performed gel shift assay. Negative control without nuclear extract (lane 1), nuclear extract of ischemia-reperfusion kidney (lane 2), nuclear extract of control kidney (lane 3), and nuclear extract of ischemia-reperfusion kidney with 100-fold excess of unlabeled oligonucleotide (heterologous competitor DNA; lane 4) are shown. Nuclear extract of ischemia-reperfusion kidney incubated without anti–Ets-1 antibody (lane 5) and nuclear extract of ischemia-reperfusion kidney incubated with anti–Ets-1 (lane 6, supershift) are shown.

Cell Proliferation and Cyclin D1 Expression by the Overexpression of Ets-1 in LLC-PK1 Cells

We first examined the effects of the overexpression of Ets-1, using an adenovirus, on the cell proliferation of LLC-PK1 cells by [³H]thymidine uptake. Figure 8A shows the effects of Ets-1 on [³H]thymidine uptake. Overexpression of Ets-1 stimulated the [³H]thymidine uptake to 225% dose-dependently. Overexpression of Adnull (control adenovirus) did not significantly change the [³H]thymidine uptake in LLC-PK1 cells. We next examined the role of Ets-1 in the regulation of cyclin D1 promoter activity and protein expression. We performed a transient transfection with the cyclin D1-luciferase reporter gene and the β-galactosidase expression vector and then infected it with Adets-1 or Adnull. When Ets-1 was overexpressed, cyclin D1 promoter activity increased significantly, by 3.6-fold, in LLC-PK1 cells. In the case of transfection of Adnull, there were no significant changes in cyclin D1 promoter activity or protein expression (Figure 8B). When Adets-1 was transfected, a higher level of cyclin D1 protein expression could be detected when compared with Adnull (Figure 8B).

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Figure 8. Cell proliferation and cyclin D1 expression by the overexpression of Ets-1 in LLC-PK1 cells. (A) We examined the effects of the overexpression of Ets-1 using an adenovirus on the cell proliferation of LLC-PK1 cells by [³H]thymidine uptake. A shows the effects of Ets-1 on [³H]thymidine uptake. (B) We next examined the role of Ets-1 in the regulation of cyclin D1 promoter activity and protein expression. We performed a transient transfection with the cyclin D1-luciferase reporter gene and the β-galactosidase expression vector and then infected it with either Adets-1 or Adnull (10⁹ pfu/ml). When Ets-1 was overexpressed, cyclin D1 promoter activity increased significantly, by 3.6-fold, in LLC-PK1 cells; n = 5, mean ± SEM; *P < 0.05 versus control.

Apoptotic Changes by the Overexpression of Ets-1 in LLC-PK1 Cells

In vascular endothelial cells, overexpression of Ets-1 causes apoptotic changes (30). Thus, we examined the effects of the overexpression of Ets-1 using an adenovirus on apoptotic changes of LLC-PK1 cells by caspase 3 activity and a cell death ELISA kit. We used AdAktDN as a positive control. Overexpression of AdAktDN caused apoptotic changes in renal tubular cells (33). Figure 9 shows the effects of Adets-1, AdAktDN, and Adnull on caspase 3 activity and the cell death ELISA. Overexpression of AdAktDN stimulated caspase 3 activity and the value of cell death ELISA. Overexpression of Adets-1 did not significantly change stimulated caspase 3 activity or the value of cell death ELISA in LLC-PK1 cells. These data demonstrated that Ets-1 does not cause apoptotic changes in LLC-PK1 cells.

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Figure 9. Apoptotic changes were not observed by the overexpression of Ets-1 in LLC-PK1 cells. We examined the effects of the overexpression of Ets-1 using an adenovirus on apoptotic changes of LLC-PK1 cells by caspase 3 activity and a cell death ELISA kit. AdAktDN was used as a positive control. Overexpression of AdAktDN stimulated caspase 3 activity (A) and the value of cell death ELISA (B). Overexpression of Adets-1 did not significantly change stimulated caspase 3 activity (A) or the value of cell death ELISA (B) in LLC-PK1 cells; n = 5, mean ± SEM; *P < 0.001 versus control.