A primary anti-ADM antibody was obtained from Abcam (Cambridge, MA, USA), and the anti-HIF-1α, anti-E-cadherin, anti-tight junction protein-1 (ZO-1), anti-vimentin and anti-α-smooth muscle actin (α-SMA) antibodies were purchased from Santa Cruz Biotechnology Inc., (Dallas, TX, USA). The antibodies against total extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), and β-actin were purchased from Cell Signaling Technology Inc., (Danvers, MA, USA). Human ADM and ADM 22–52 (an ADM receptor antagonist), and apigenin (an activator of ERK) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The HK-2 and HKC human proximal tubular epithelial cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 (Invitrogen Life Technologies, Carlsbad, CA, USA), supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 36 ng/ml hydrocortisone, 4 pg/ml tri-iodo-L-thyronine, 10 ng/ml epidermal growth factor, 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 mM glutamine (all Invitrogen Life Technologies, Carlsbad, CA, USA). The HK-2 and HKC cells from passages 6–16 were used in the following experiments. The cells were starved of serum overnight, prior to experimentation, to avoid the effects of serum-induced signaling. The cells were maintained at 37°C in a humidified atmosphere, under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 12, 24 or 48 h with 95% N₂ and 5% CO₂. In some experiments, the cells were exposed to normoxia or hypoxia in the absence or presence of the following compounds: 0.1 or 1.0 μM ADM; 5 nM ADM22-52; and 100 μM apigenin.

The HK-2 cells from the sixth passage were seeded in 6-well plates (1×10⁵ cells/well) and allowed to adhere for 24 h, prior to transfection. The commercial ADM shRNA plasmid was purchased from Santa Cruz (Santa Cruz Biotechnology, Dallas, Texas, USA). The plasmid is based on a pool of 3 target-specific lentiviral vector plasmids, each encoding 19–25 nt (plus hairpin) shRNAs designed to knockdown human ADM gene expression. Each plasmid contains a puromycin resistance gene and a copGFP gene for the selection of cells stably expressing shRNA. The shRNAs were transfected using specifically designed reagent for shRNA transfection, including Santa Cruz Biotechnology’s shRNA Plasmid Transfection Reagent (sc-108061, 0.2 ml) and shRNA Plasmid Transfection Medium (sc-108062, 20 ml) according to manufacturer’s protocol. Then the cells were incubated for 8 h at 37°C in a 95% air/5% CO₂ incubator. In addition, the cells were transfected with a non-targeting negative-control shRNA (NC-shRNA) (Santa Cruz Biotechnology, Dallas, Texas, USA) to monitor and optimize transfection efficiency. At 72 h post-transfection, puromycin (7.0 μg/ml) was added to the culture medium for selection and further characterization. The transfection efficiency of the HK-2 cells expressing ADM-shRNA (copGFP) was assessed using flow cytometry. In brief, transfected cell were fixed in cold 70% ethanol, washed twice and resuspended in cold phosphate-buffered saline buffer. The cell suspensions were analyzed on an FC500/MPL flow cytometer (Beckman Coulter, Brea, CA), using the FlowJo software (Tree Star, Ashland, OR, USA). GFP was detected on the FITC channel using a 488 nm laser, and the transfection efficiency was no less than 85%.. The cells were cultured for 48 h following transfection and then exposed to hypoxia for 12–48 h.

The treated HK-2 and HKC cells were harvested and lysed for 30 min on ice using lysis buffer (50 mM Tris–HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 μg/ml each aprotinin, leupetin, and pepstatin; 1 mM Na₃VO₄; 1 mM NaF). The samples were then centrifuged at 14,000 × g for 10 min at 4°C, and the protein concentrations of the cell lysates were determined using the Bradford method. The proteins (50 μg/lane) were loaded onto 10% SDS-containing polyacrylamide gels and were separated by SDS-PAGE, prior to a transfer to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk in Tris-buffered saline (TBS) with Tween^® 20 (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween^® 20) for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C with the primary antibodies, in blocking buffer containing 5% milk, at the dilutions specified in the manufacturer’s instructions. Following extensive washing in TBS, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, Dallas, Texas, USA), for 1 h at room temperature in 5% nonfat milk dissolved in TBS. The membranes were visualized using a chemiluminescent substrate and developed following exposure to Kodak X-OMAT film (Kodak, Rochester, NY, USA). The immunoreactive protein bands were quantified by densitometry using QuantityOne Image Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA) and were normalized to the β-actin values.

Total RNA was extracted from treated cells using the RNAiso Plus kit (Takara Biotechnology Co., Ltd., Dalian, China). A total of 1 μg RNA was used for first-strand cDNA synthesis using the PrimeScript reverse trasncription kit reagents (Takara Biotechnology Co., Ltd.). Quantification was performed using the iCycler iQ™ Sequence Detection system (Bio-Rad, USA), and the SYBR Green Premix kit (Takara Biotechnology Co., Ltd.) was used as the fluorophore. The following oligonucleotides were used as primers: ADM forward, 5′-ACTTGGCAGATCACTCTCTTAGCA-3′, and reverse, 5′-ATCAGGGCGACGGAAACC-3′; β-actin forward, 5′-ATCCTGCCAGTAGCATATGC-3′, and reverse, 5′-ACCGGGTTGGTTTTGATCTG-3′. For each primer pair, the annealing temperature was optimized using a gradient PCR reaction. The expression levels of each target mRNA, relative to the β-actin mRNA expression levels, were calculated from the cycle threshold (Ct) using the 2^−ΔΔCt formula. For each sample, the experiment was repeated in triplicate, using RNA from three separate culture wells.

All experimental data are expressed as the means ± standard error of the mean. The significant differences were evaluated using a one- or two-way analysis of variance, followed by multiple comparisons tests. Statistical analyses were performed using the commercially available software package SPSS version 13 (SPSS, Inc., Chicago, IL, USA). A P<0.05 was considered to indicate a statistically significant difference.

The HK-2 and HKC cells, cultured under chronic hypoxia for 48 h, exhibited a downregulation in the expression levels of the epithelial markers, E-cadherin and ZO-1, and an upregulation in the expression levels of the mesenchymal markers, vimentin and α-SMA, as compared with the cells cultured under normoxia (Fig. 1). The expression levels of the hypoxia-regulated molecule HIF-1α were also determined to confirm the hypoxic response. These results suggest that the epithelial cells underwent EMT, with a more fibroblast-like cell type, and indicate that hypoxia may promote EMT, one of the major mechanisms that mediates renal fibrosis. Furthermore, ADM expression levels were shown to be increased in HKC and HK-2 cells under hypoxic conditions, and was negatively correlated to the expression levels of E-cadherin and ZO-1.

ADM mRNA expression was evaluated in the HK-2 and HKC cells, by qPCR. The mRNA expression of β-actin was used as an internal control, and the ratio of ADM/β-actin was used to determine the relative mRNA expression levels of ADM. As shown in Fig. 2, the relative expression levels of ADM mRNA, in HKC and HK-2 cells, were significantly affected by both the time the cells were exposed to hypoxia (F_{time (3, 24)} = 52.43, P<0.0001) and the type of cell line (F_{cell lines (1, 24)} = 30.90, P<0.0001); these two factors also showed significant interactions (F_{time × cell lines (3, 24)} = 8.57, P<0.0005). A post hoc analysis showed that the mRNA expression levels of ADM significantly increased in both HKC and HK-2 cells, as a function of the level of hypoxia. Between the two cell lines, the HK-2 cells appeared more sensitive to hypoxic stress, as compared with the HKC cells (P<0.0001, at 48 h of hypoxia). These results suggest that expression of ADM was significantly enhanced in a time-dependent manner in human tubular cell lines under hypoxic conditions.

ADM was shown to be upregulated in hypoxia-stimulated HK-2 and HKC cells, therefore it was assessed as to whether exogenous ADM was capable of ameliorating hypoxia-induced EMT. HK-2 cells were exposed to hypoxia for 48 h in the presence of 0, 0.1 or 1.0 μM ADM. As shown in Fig. 3, the expression levels of E-cadherin were significantly increased in response to 1.0 μM ADM, as compared with the control cells not treated with ADM (P<0.0001); however, this upregulation did not occur when the ADM peptide inhibitor ADM 22–52 was co-administered with 1.0 μM ADM. Furthermore, the protein expression levels of vimentin were significantly decreased by ADM, in a dose-dependent manner (P<0.05 and P<0.0001, respectively); however, this downregulation did not occur when ADM was co-administered with ADM 22–52. These results suggest that exogenous ADM administration ameliorated hypoxia-induced EMT.

To further elucidate the effects of ADM on hypoxia-induced EMT in human tubular epithelial cells, a stable cell line was constructed, in which ADM expression was successfully knocked down. Because HK-2 cells appeared to have increased sensitivity to hypoxic stress, lentiviral ADM-shRNA was transfected into the HK-2 cells. Following the transfection, western blot analysis revealed that ADM protein expression levels were markedly decreased in the HK-2 cells, as compared with the parental cells (Fig. 4A, P<0.01). The expression levels of ADM were not different between the vector-control and untransfected parental HK-2 cells. Following 48 h exposure to hypoxia, the HK-2 cells that stably expressed ADM-shRNA exhibited significantly lower expression levels of E-cadherin, as compared with the untransfected cells (Fig. 4B, P<0.05). There was no difference in the vimentin protein expression levels between the parental and vector-control cells, under hypoxic conditions; however, vimentin expression levels were markedly upregulated in the hypoxic shRNA-ADM-transfected cells (P<0.05). These results indicate that ADM knockdown may accelerate EMT in tubular epithelial cells, and that this effect may be partly mediated by hypoxia-induced EMT processes.

It was investigated whether the upregulated expression of ADM ameliorated hypoxia-induced EMT, through inhibiting the activation of ERK. The HK-2 cells were exposed to hypoxia for 48 h in the presence of 1.0 μM ADM, with or without apigenin (100 μM). As shown in Fig. 5, the phosphorylation of ERK was significantly decreased in the ADM-treated cells, as compared with the control cells (P<0.01). However, when the HK-2 cells were treated with exogenous ADM in combination with apigenin, the levels of phospho-ERK were significantly elevated (P<0.05). Conversely, total ERK expression levels were not affected by either ADM nor apigenin treatment (F _{(2, 15)} = 0.1833, P=0.83). E-cadherin expression levels were significantly increased by 1.0 μM ADM (P<0.0001), as compared with the control cells not treated with ADM; however, 100 μM apigenin blocked the ADM-induced upregulation of E-cadherin. The expression levels of vimentin were significantly decreased by ADM (P<0.01); however, this downregulation was completely prevented by treatment with ADM in combination with apigenin (P<0.0001). These results suggest that the effects of hypoxia-induced ADM on the amelioration of EMT may be regulated by inhibition of the activation of ERK, and this effect can be effectively prevented by the administration of activators of ERK.