Observations of the Effects of Angiotensin II Receptor Blocker on Angiotensin II-Induced Morphological and Mechanical Changes in Renal Tubular Epithelial Cells Using Atomic Force Microscopy

Objective Angiotensin II (Ang II) plays a profibrotic role in the kidneys. Although many pathways of Ang II have been discovered, the morphological and mechanical aspects have not been well investigated. We observed the changes in tubular epithelial cells (TECs) after Ang II treatment with or without Ang II receptor blockers (ARBs) using atomic force microscopy (AFM). Methods TECs were stimulated with Ang II with or without telmisartan, PD123319, and blebbistatin. AFM was performed to measure the cellular stiffness, cell volume, and cell surface roughness. Epithelial to mesenchymal transition markers were determined via immunocytochemistry. Results After Ang II stimulation, cells transformed to a flattened and elongated mesenchymal morphology. Cell surface roughness and volume significantly increased in Ang II treated TECs. Ang II also induced an increase in phospho-myosin light chain and F-actin and a decrease in E-cadherin. Ang II coincubation with either telmisartan or blebbistatin attenuated these Ang II-induced changes. Conclusion We report, for the first time, the use of AFM in directly observing the changes in TECs after Ang II treatment with or without ARBs. Simultaneously, we successfully measured the selective effect of PD123319 or blebbistatin. AFM could be a noninvasive evaluating strategy for cellular processes in TECs.


Introduction
Renal fibrosis, characterized by increased extracellular matrix (ECM) accumulation on the kidney parenchyma, is the final common manifestation of chronic kidney disease (CKD), regardless of the primary causes [1,2]. Previous studies reported that renal tubular epithelial cells (TECs) played an important role in the development of renal tubulointerstitial fibrosis [3]. TECs release chemokines and profibrogenic cytokines and undergo epithelial to mesenchymal transition (EMT) in pathological conditions [4][5][6]. Therefore, understanding the changes of TECs are important for the prevention and effective treatment of renal fibrosis.
Angiotensin II (Ang II), a major component of the renin angiotensin aldosterone system (RAAS), is known to be a crucial mediator of renal fibrosis [7,8]. Several studies have demonstrated the ability of Ang II to induce EMT of TECs by regulating the synthesis of ECM and production of profibrotic molecules such as transforming growth factor- [9]. Ang II binds to two specific receptors, angiotensin type 1 (AT 1 ) and angiotensin type 2 (AT 2 ) receptor [10]. AT 1 receptor is known to mediate most of the classical physiologic and pathologic effects of Ang II, while the role of AT 2 receptor is not completely established [11]. Many in vitro and in vivo studies have established that RAAS blockade using AT 1 receptor blockers has therapeutic effects 2 BioMed Research International on renal tubulointerstitial fibrosis. [12,13]. However, most of these studies demonstrated this mechanism in indirect ways, including gene and protein expression, associated with renal fibrosis and RAAS. Therefore, further studies with direct measurement of the morphological and mechanical changes of TECs during Ang II stimulation and treatment with Ang II receptor blockers (ARBs) are needed.
Atomic force microscopy (AFM), invented in 1986 by Binnig et al. [14], has become a useful noninvasive imaging tool in biological and medical research [15]. AFM shows the force-distance (FD) curve by measuring the force between its probe tip and the sample surface and can be used to evaluate a sample's physical properties. Hence, the stiffness and adhesive characteristics of cell membranes can be evaluated by AFM [16]. Recently, many studies suggested that the information obtained via AFM helps in understanding the biological and physical mechanism of renal injury [17,18]. Our group previously used AFM to monitor Ang IIinduced conformational changes in mesangial cells [19], and we also successfully observed that the changes in the Ang II-stimulated mesangial cells were effectively disrupted by treatment with telmisartan, a specific AT 1 receptor blocker [20]. However, only a few studies investigated the changes of TECs using AFM. In this study, we used AFM to observe the Ang II-induced morphological and mechanical changes in TECs. Moreover, the effects of various ARBs on Ang II treated TECs were also investigated [21].

Cell Culture and Treatment.
A well characterized, normal rat kidney cell line (NRK-52E; Sigma-Aldrich, MO, USA) was used in this study. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco-Invitrogen, CA, USA) containing 4.5 g/L of glucose with 10% fetal calf serum in a humidified 5% CO 2 incubator at 37 ∘ C and passaged twice a week. NRK-52E cells between the 28th and 30th passages were used. In preparation for AFM observation, the cells were seeded into a collagen type I-coated 60 mm cell culture dish. After the cells reached confluence, they were washed once with filtered phosphate buffered saline (PBS; pH 7.4), and new DMEM was added. Cells were then incubated for 24 hours with Ang II (Sigma-Aldrich, MO, USA) in the presence or absence of telmisartan (Sigma-Aldrich, MO, USA), an AT 1 receptor antagonist. We also used PD123319 (Sigma-Aldrich, MO, USA), an AT 2 receptor antagonist, as a negative control and blebbistatin (Sigma-Aldrich, MO, USA), a myosin II inhibitor, as a positive control for telmisartan at the same concentration (1 × 10 −6 M) for 24 hours.

AFM Observations.
Contact mode AFM images were obtained using a NANO Station II (Surface Imaging Systems, Herzogenrath, Germany). The AFM was placed on an active vibration isolation table (TS-150; S.I.S., Herzogenrath, Germany) inside a passive vibration isolation table (Pucotech, Seoul, Korea) to eliminate external noise. Silicon cantilevers with the reflective side coated with gold were used for the measurements under liquid conditions. The properties of the probe used in contact mode were as follows: resonance frequency: 13 kHz (±4 kHz); force constant: 0.2 N/m (±0.14 N/m); cantilever length: 450 m (±10 m); cantilever width: 38 m (±5 m); cantilever thickness: 2 m (±1 m); tip radius: 5 nm (±1 nm); and tip height: of 17 m (±2 m). The AFM probe tips were stabilized with DMEM or PBS for at least 10 minutes prior to scanning.
For AFM imaging, the cells were washed twice with filtered PBS and fixed for 20 min in 2.5% glutaraldehyde in PBS at room temperature and 5 ml PBS was added to culture dishes containing fixed cells. TECs fixed with glutaraldehyde were scanned in PBS solution at a resolution of 512 × 512 pixels, at a scan speed of 0.5 line/s. We fixed the cells with glutaraldehyde to get the high resolution image.
The cell stiffness was obtained from the force-distance (FD) curve on live TECs after 24 hours of Ang II or various ARBs application. The live cells were first identified using the contact imaging mode to determine the appropriate site for the FD curve without defects or impurities, and force data were obtained at locations with similar height to prevent edge effects. Live TECs were scanned in DMEM solution at a resolution of 256 × 256 pixels and a scan rate of 2 lines/s. The loading force was adjusted to below 1-2 nN to minimize cell damage. We calculated cell , the cellular spring constant, by modeling the cell-tip interaction as two springs to quantify cell elasticity [16]. cell was defined as 1/ cell = 1/ eff − 1/ cantilever , where eff is the slope of the linear region of the FD curve for a cell and cantilever is determined from each cantilever using a clean culture dish containing DMEM. Data acquisition and image processing were performed with SPIP6 (Scanning Probe Image Processor Version 5.0.3, Image Metrology, Denmark). The fixation process could damage the cytoskeleton; we determine the FD curve in live cell without fixation. After FD curve measurements were completed, a second image was obtained to ensure that the cell had not shifted or was damaged.

Immunocytochemistry.
TECs were washed in PBS before fixing in 4% paraformaldehyde for 1 hour at room temperature (RT). Cells were permeabilized with 0.1% Triton X-100 for 15 min at RT. Nonspecific antibody binding was blocked by incubating in 1% BSA for 30 min at RT, followed by overnight incubation with anti-rabbit phospho-myosin light chain (pMLC; Cell Signaling, #3671; 1 : 200 dilution) or antirabbit E-cadherin (Cell signaling, #3195; 1 : 200 dilution) antibodies at 4 ∘ C. The cells were then incubated with secondary antibodies consisting of anti-rabbit IgG FITC (Sigma, F0382; 1 : 500 dilution) for 2 hours at RT. Rhodamine phalloidin (Invitrogen, R415) is a high-affinity probe for F-actin that is synthesized from a mushroom toxin conjugated with the orange-fluorescent dye, tetramethylrhodamine (TRITC). F-actin staining was carried out for 2 hours at RT with rhodamine phalloidin (0.2 U/mL dilution). Finally, the slides were mounted using the VECTASHIELD HardSet Antifade Mounting Medium with DAPI (H-1500, Vector labs) and detected using a fluorescence or confocal microscope.

Statistical Analysis.
The calculated spring constants of TECs are expressed as mean ± standard deviation (SD).  ANOVA was used to evaluate the significance of the differences between the groups. All statistical analyses were carried out using SPSS software version 19.0 (SPSS Inc., Chicago, IL, USA); < 0.05 was considered to be statistically significant.

Morphological Changes in TECs after Treatment with Ang
II and ARB Treatment. Figure 1 shows representative AFM topography (upper panels) and deflection images (lower panels) taken from TECs fixed with glutaraldehyde in liquid conditions. After 24 hours in culture, control cells exhibited a typical epithelial cuboidal shape with cobblestone-like appearance. Cell bodies were convex, and many microvilli were regularly spread over the cell surface (Figure 1(a)). However, TECs cultured in Ang II revealed profound morphological changes. As shown in Figure 1(b), the cells became flattened and elongated and changed to a spindle-like shape. There were small bumps around the nucleus on the cell surface, and microvilli presence decreased when compared to control cells. Simultaneous incubation with telmisartan or blebbistatin disrupted the Ang II-induced morphological changes in majority of the cells, while retaining a cobblestonelike appearance, with the absence of hypertrophy and elongated morphology. (Figures 1(c) and 1(e)). PD12319, which was used as a negative control, had no significant effect on the morphological change of the TECs treated with Ang II (Figure 1(d)).

Mechanical Changes in Live TECs Induced by Ang II and
ARBs. We calculated cell , the cellular spring constant, from the FD curve in Figure 2. In this study, FD measurements were obtained for 30 cells in each group.

Immunofluorescent Findings.
To confirm the transformation of TECs into a fibroblastic phenotype, the expression of pMLC, F-actin, and E-cadherin was investigated  via immunofluorescent staining. As shown in Figure 3, pMLC (upper panels) and F-actin (mid panels) expressions markedly increased in TECs with Ang II treatment when compared to the control cells (Figures 3(a) and 3(b)). Conversely, E-cadherin expression (lower panels) markedly decreased in TECs with Ang II treatment when compared to the control cells (Figure 3(c)). As shown in Figures 3(d)

Discussion
In the present study, we observed the Ang II-induced morphological and mechanical changes in TECs and investigated the effect of ARBs on Ang II-stimulated TECs. To our knowledge, this is the first study to visualize and characterize the changes in TECs induced by Ang II and ARBs using AFM. Our principle findings were as follows: (1) after treatment with Ang II, TECs exhibited notable morphological and mechanical changes; (2) Ang II caused the expression of EMT markers, including decreased expression of E-cadherin and increased expression of pMLC and F-actin; and (3) these changes and phenotypic conversion were disrupted by the addition of telmisartan.
Ang II has been reported to promote renal fibrosis by regulating ECM accumulation, inflammation, and cellular proliferation [9,13]. Activation of the RAAS is also widely known to play a crucial role in the EMT of TECs [22]. Many studies have demonstrated that the suppression of RAAS results in renal protective effects and prevents renal fibrosis [10]. Therefore, understanding the changes in TECs and RAAS activation during the renal fibrosis process is important in understanding the mechanisms underlying renal damage.
Although several studies investigated changes in TECs after Ang II treatment, studies that demonstrate morphological and mechanical aspects are limited. In this study, we effectively examined the cell response to Ang II and ARBs with AFM imaging and FD curve measurement. As shown in Figure 1, we performed AFM imaging to directly observe the morphological changes in TECs after treatment with Ang II with or without ARBs. Although the fixation process could lead to cell damage, we used TECs fixed with glutaraldehyde to get high resolution images. After treatment with Ang II, TECs exhibited marked hypertrophy, lost their cobblestonelike morphology, and became elongated in shape, which is typical of fibroblasts. These morphological changes were accompanied with phenotypic changes. Immunofluorescent staining showed that TECs treated with Ang II lost their epithelial marker and newly acquired mesenchymal markers ( Figure 3).
The structural and physical changes of TECs are difficult to visualize. Rabinovich et al. [23] found the existence of repulsive forces between the AFM tip and renal tubular epithelial cells. They reported that the oxalate treatment of renal TECs gave rise to increase elastic modulus of the cells. In the present study, we also used AFM to monitor and obtain mechanical properties and cell stiffness. Table 1 shows the mechanical changes in TECs during Ang II-induced EMT. By using the AFM spring constant, we showed that the contractile response of TECs can generate stiffness, which may deform the surrounding ECM or exchange in tissue containing a TEC layer. In addition to the methods used in this study to calculate cells' stiffness, more advanced methods have been suggested [24,25]. To evaluate cell's elasticity, they determined Young's modulus by AFM. In this study, we calculated the spring constant to quantify cell elasticity. In our future study, we will also consider apply these methods.
As mentioned above, we revealed that Ang II-induced morphological and mechanical changes that were attenuated via telmisartan treatment. It is now widely recognized that RAAS blockade by ARB exhibits a therapeutic effect in renal injury [10]. Our results suggest that telmisartan may disrupt Ang II-induced renal damage by reducing the morphological changes and contraction of TECs. Several studies reported that ARBs diminished renal fibrosis and the expression of profibrotic growth factors such as transforming growth factor-and connective tissue growth factor [7,9,13]. The reduction in molecular and mechanical changes of TECs in our study were presumed to be due to telmisartan-induced biochemical modification. The morphological and mechanical changes of cells have been reported to be associated with the changes of cytoskeletal structures. Ang II could affect the increase of cytoskeleton activity and lead to the changes of the elastic modulus of the cell [26]. These dynamics of cytoskeletal structure could be considered the cause of cell stiffness, but further studies are needed to confirm their contribution.

Conclusion
In summary, we observed morphological changes in TECs induced by Ang II treatment with the help of AFM imaging. Furthermore, the mechanical changes in TECs were evaluated using FD curve analysis. We also demonstrated that these morphological and mechanical changes were effectively prevented by telmisartan treatment. Although the mechanisms underlying these physical changes in TECs have not yet been fully elucidated, AFM could provide noninvasive measurements of the cellular processes in TECs.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.