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Developmental changes in the expression and function of TRPC6 channels related the F-actin organization during differentiation in podocytes
Cell Calcium 58 (2015) 541–548
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Cell Calcium journal homepage: www.elsevier.com/locate/ceca
Developmental changes in the expression and function of TRPC6 channels related the F-actin organization during differentiation in podocytes Zhaowei Liu a,1 , Jiajia Yang b,c,1 , Xiaochen Zhang a , Pengjuan Xu a , Tao Zhang c , Zhuo Yang a,∗ a College of Medicine, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Nankai University, Tianjin 300071, China b Tianjin University, Tianjin 300072, China c College of Life Science, Nankai University, Tianjin 300071, China
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Article history: Received 23 March 2015 Received in revised form 2 August 2015 Accepted 1 September 2015 Available online 3 September 2015 Keywords: Podocyte TRPC6 channel Differentiation F-actin
a b s t r a c t The transient receptor potential canonical (TRPC) 6 channel is an important ion channel located in podocytes, which plays an essential role in regulating calcium homeostasis of the cell signaling. Podocytes are specialized, terminally differentiated cells surrounding glomerular capillaries, and are the subject of keen interest because of their key roles in kidney development and disease. Here we wonder whether TRPC6 channels undergo developmental changes in the expression and function during the podocyte differentiation, and whether they contribute to the maturation of podocytes. Using morphological, immunohistochemical and electrophysiological techniques, we investigated the development of distribution and expression of TRPC6 in conditionally immortalized mouse podocyte cell line. Our results showed that the distribution of TRPC6 channels changed with the maturity of podocyte differentiation. The fluorescent intensity of TRPC6 on cell surface increased, which was accompanied by a corresponding increase in the density of current flowing through the channels. TRPC6 inhibition by TRPC6 siRNA or SKF-96365, a blocker or TRP cation channels, resulted in F-actin cytoskeleton disruption only on the developmental stage of podocytes. These results strongly support the conclusion that TPRC6 is an essential component of the slit diaphragm and is required for development of glomerulus. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Transient receptor potential (TRP) superfamily member the transient receptor potential canonical (TRPC) 6 is confirmed to be the main cation-permeable channel in excitable and non-excitable cells, which enables the influx of Ca2+ and Na+ ions into cells [1–3]. This superfamily consists of a group of six transmembrane domain-containing ion channels and plays an essential role in regulating Ca2+ homeostasis, which consequently mediates a cascade of signaling events [4,5]. Roles for TRPC6 in kidney were revealed by studies showing that mutations in human TRPC6 gene caused inherited focal segmental glomerulosclerosis (FSGS), and overexpression of TRPC6 in glomeruli could induce several acquired human proteinuric kidney diseases [6–8].
∗ Corresponding author. E-mail address: [email protected] (Z. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ceca.2015.09.001 0143-4160/© 2015 Elsevier Ltd. All rights reserved.
Podocytes are specialized, terminally differentiated cells around glomerular capillaries, which provide physical support for capillary loops responding to changes in intraglomerular pressure [2]. Various studies suggest that TRPC6 is expressed in the podocyte cell body, major processes and foot processes near the slit diaphragm. The alterations of TRPC6 in podocytes have recently been attributed to proteinuric kidney diseases and foot process effacement [7–9]. The morphology of podocyte is defined by the underlying actomyosin cytoskeleton, and organization of the actin network is required for proper kidney function [10]. TRPC6 may affect the function of slit diaphragm where TRPC6 localizes by inducing the remodeling of actomyosin cytoskeleton [11]. The results of our previous studies revealed that TRPC6 was expressed in glomerulus and tubules but showed a decreased expression during the postnatal maturation of mouse renal cortex [12]. Nevertheless, we still wonder the development expression of TPRC6 in podocytes. The aim of this research was to clarify the developmental expression of TRPC6 ion channels on podocytes during the
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differentiation to the mature phenotype, and its role in F-actin cytoskeleton organization related to kidney function.
was excited at 488 nm laser line and the fluorescence signal was acquired at 522 nm. The fluorescence of Fluo-3 induced by 1-oleoyl2-acetyl-sn-glycerol (OAG, 100 M) was measured every 10 s.
2. Material and methods 2.5. Transient transfection in vitro 2.1. Podocyte culture and drug treatment Conditionally immortalized mouse podocyte cell line (a kind gift from Prof. Peter Mundel) was cultured at 33 ◦ C in RPMI1640 medium (Gibco, Gaithersburg, MD, USA), which contained 10% fetal bovine serum (Gibco), 10 U/ml of recombinant mouse ␥interferon and 100 U/ml of penicillin-streptomycin (Sigma, USA). Then podocytes were cultured at 37 ◦ C without ␥-interferon (growth restrictive conditions) to induce differentiation in two weeks. Experimental studies were conducted during the period of differentiation. All experiments were performed at least three times. In some experiments, the podocytes were treated with La3+ (100 M), OAG (100 M), which were purchased from Sigma (USA). 2.2. Laser-scanning confocal microscopy Cultured cells were washed in PBS, fixed by 15 min exposure to 4% paraformaldehyde in PBS, rinsed in PBS, blocked with 10% normal goat serum, and then permeabilized in PBS containing 0.3% Triton X-100. After that, cells were incubated with primary antibodies including rabbit anti-TRPC6 antibodies (1:1000, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C. Then cells were incubated with Alexa 488-conjugated anti-rabbit IgG (working dilution 1:1000, Invitrogen, San Diego, CA, USA) for 3 h at room temperature after being washed three times for 10 min with PBS at room temperature. The fluorescent signals were examined using a Leica TCS SP5 laser-scanning confocal microscope. The fluorescence quantitative of TRPC6 was semi quantitatively determined results, and Image-Pro Plus 7.0 software was used in the process. The fluorescence pictures were converted to 8-bit gray scale, and “invert contrast” and “apply contrast” buttons were pressed. Then, the optical density was corrected, and the “IOD” and “area” were selected to measure the pictures. Next the cell part in the field of view except for nucleus was drawn to measure, and the data could be read from SUM area and SUM view in the “view statistics” window. Density mean = (IOD SUM)/(area sum), and this was the average fluorescent intensity of the cells in our photograph. Enough cells were taken to make statistical analysis.
Podocytes were transiently transfected with TRPC6 siRNA (RiboBio, Guangzhou, GD, China) and its negative control using transfection reagent (RiboBio, Guangzhou, GD, China) according to the manufacturer’s instructions. These cells were used for the following experiments after transfection for 24 h. 2.6. Western blot analysis Proteins were run on a 10% SDS/PAGE gel and transferred onto PVDF membranes and blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% fat-free dry milk. The membranes were incubated with the primary antibodies, anti-TRPC6 (1:1000, Abcam, Cambridge, MA, USA), and then with horseradish peroxidaseconjugated secondary antibodies. The immunoreactive bands were detected using chemiluminescence methods and visualized on Xray films (Kodak, Rochester, NY, USA). The analysis of images was performed using Image J software (NIH, Bethesda, MD, USA). 2.7. Fluorescence staining of F-actin Podocytes were placed on poly-l-lysine-coated glass coverslips. After washing with PBS, the cells were fixed with 4% formaldehyde in PBS at room temperature for 30 min. The 0.1% Triton was added in PBS for 5 min to increase permeability. F-actin was detected using green fluorescent Phalloidin Conjugate working solution (Abcam, Cambridge, MA, USA) for 60 min at room temperature. Podocytes with distinct F-actin were counted as previous described [13]. 2.8. Data acquisition and statistical analysis Data were acquired using an EPC10 amplifier (HEKA, Germany) connected to a computer and stored on a hard disk using pulse 8.52 software (HEKA, Germany), analyzed off-line using the pCLAMP 9.0 and Origin 8.0. All quantitative data are presented as mean ± S.E.M. Data were analyzed by one-way ANOVA followed by post hoc analysis. Throughout, P < 0.05 is regarded as significant. 3. Results
2.3. Electrophysiology recording 3.1. Expression of TRPC6 in podocytes Conventional whole-cell patch-clamp recording was carried out at room temperature (22–24 ◦ C). Experiments were performed on podocytes attached to glass coverslips and transferred to a recording chamber on an Olympus inverted microscope stage immediately before recording. For recording TRPC6 currents, the pipette solution contained 130 mM CsOH, 130 mM l-aspartic acid, 2 mM MgCl2 , 0.3 mM CaCl2 , 10 mM HEPES, 10 mM EGTA and 3 mM ATP-Na at pH 7.3. And the external solution contained 130 mM NaCl, 4 mM KCl, 1 mM MgCl2 , 10 mM HEPES, 10 mM glucose and 2 mM CaCl2 at pH 7.3. The sample rate was 4 kHz (filtered at 2 kHz). The currents were measured at −100 and +100 mV and normalized by membrane capacitance. 2.4. Calcium imaging Cells were washed three times in Hanks’ solution and then loaded with Fluo 3-AM (5 M) (Sigma, USA) in 1 ml of Hanks’ solution at 37 ◦ C for 30 min. After three washings with D-Hanks’ solution, cells were incubated in 1 ml of Hanks’ solution and imaged at room temperature. The calcium-dependent fluorescence
All the experiments were performed in a conditionally immortalized mouse podocyte cell line in different differentiation status, including five groups of undifferentiated cultured podocytes (d0), 3, 6, 9 and 12 days after induction of differentiation (d3, d6, d9 and d12), and mature podocytes (14 or more days after induction of differentiation). Using confocal microscopy, we addressed the distribution of TRPC6 in podocytes of different differentiation status. First, the cell morphology was observed, which indicated the conversion of typical cobblestone appearance into arborized cells within 2 weeks (Fig. 1a–f). Groups d0 and d3 showed the classic “cobblestone” appearance. While at the late stage of differentiation (d9 and d12), cells showed a more differentiated morphology characterized by arborization, processes extending from the cell body. In the mature state, podocytes reduced their proliferative capacity (data not shown) and developed specialized junctions between the cell body and foot processes. Hence we observed a process of maturation in podocytes in vitro, which was analogous to the developmental maturation of podocytes in vivo.
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Fig. 1. The localization of TRPC6 in podocytes at different development stages. In different stages of differentiating podocytes, TRPC6 was gradually translocated to the membrane (d3-mature). Subcellular distribution of TRPC6 (g) and dapi (h), double immunolabeling (i) showing TRPC6 was mainly expressed in the nucleus in undifferentiated podocytes (d0).
Fig. 1a also showed that TRPC6 proteins were mainly expressed in the nuclear regions of podocytes in group d0, with hardly distinguishable signals toward the membrane of the cells. The results can be showed more clearly in Fig. 1g–i by double immunolabeling of TRPC6 and nucleus. And from group d3 to d12, the images exhibited gradually translocating of TRPC6 channels to cytoplasm. The image of mature podocyte indicated that TRPC6 was evenly
distributed inside the cell but almost empty in nuclear (mature), especially in cytoplasm and membrane surface. The expression of TRPC6 in nuclear regions seemed almost all translocated to other regions and increased in the cell membrane with the differentiation process. We also quantified the fluorescent intensity of TRPC6 on cell surface which was shown in Fig. 2. It showed a significant increase 9 days after induction of differentiation. Meanwhile, the
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Fig. 2. Fluorescent intensity of TRPC6 on podocyte membrane at different differentiation stages. Bar graphs showed the relative fluorescent intensity of TRPC6 on the cell surface of podocytes. Data were presented as the means ± S.E.M. *P < 0.05 vs. adjacent stages group.
TRPC6 protein levels increased in mature podocytes compared to undifferentiated ones (Fig. 3a). The summarized data are shown in Fig. 3b. In addition, we provided the developmental expression of a specific podocyte molecule, nephrin, which is one of markers for maturation of podocytes. The images in Fig. 1 indicated that the change of fluorescent intensity of TRPC6 was due to the differentiation of podocytes. 3.2. Whole-cell recordings of TRPC6 currents Morphological and biochemical data alone might not be enough to characterize the membrane expression change of TRPC6 in podocytes. Functional tests should be considered. Using more quantitative electrophysiological methods, TRPC6 current density was assayed by means of conventional whole-cell recordings in five groups. TRPC6 currents were activated using a voltage-stimulation mode, ramp protocol (1 mV/ms, from −100 mV to 100 mV, over 200 ms) every 5 s. Eight points were chosen on current curves to process statistical analysis and the currents at +75 mV for drawing histograms. The I–V curves in Fig. 4a showed that both the inward and the outward currents were substantially increased in different groups. And the average current density in these groups at +75 mV was shown in the bar graph (Fig. 4b), which is consistent with an increase in the number of active channels with the development of podocytes, since the TRPC6 protein increased according to Figs. 1–3. There were significant increases between d3 and d6, d6 and d9, d12 and mature cell groups. Therefore, we believe that the increased current density reflected an increased expression of TRPC6 channels in podocytes, which is consistent with the result of confocal microscopy.
Previous studies on TRPC6 channels in heterologous expression systems have shown that their activation gives rise to outwardly rectifying macroscopic cationic currents that are much larger in the presence of OAG, a membrane-permeable analog of diacylglycerol (DAG) analogs and the currents can be blocked by La3+ [14]. In d6 and mature podocytes, we observed an activation of macroscopic currents with these characteristics, which had distinctive inward and outward rectification properties as seen in representative examples (Fig. 5a, c). Furthermore, the cationic currents had an average reversal potential of 0 mV (0 ± 4 mV) under the recording conditions. These currents were substantially larger in cells exposed for 5 min to 100 M OAG (Fig. 5a, b), and they were also blocked by superfusion of 100 M La3+ (Fig. 5a, d), which blocked TRPC6 channels [15]. The pharmacological and electrophysiological characteristics of the nonselective currents in podocytes suggested that the membrane currents we recorded from podocytes were TRPC6 currents. In addition, the results suggested that the effects of OAG and La3+ on TRPC6 channel during development were similar to mature stage in podocytes.
3.3. Intracellular calcium concentration increase during the development in podocytes Commonly, TRPC channels associate with the cytoskeleton in order to link changes in membrane cationic flux with alteration in the actin-based cytoskeleton [16–18]. And intracellular calcium concentration was tested in the next step. To investigate the [Ca2+ ]i during the development of podocytes, cells were incubated with the calcium indicator Fluo 3-AM (5 M). The [Ca2+ ]i was significantly increased in d3 and d6, d6 and d9, d9 and d12 groups (Fig. 6).
3.4. OAG induced intracellular calcium concentration increase at different differentiation stages To investigate the relationship between the TRPC6 currents and the increase of [Ca2+ ]i, OAG was used in the next experiment. OAG induced [Ca2+ ]i increase in d6 and mature podocytes, and the trends in mature cells were more significant compared to d6 cells, but no statistically significant difference (Fig. 7). The results suggested the increase of [Ca2+ ]i during the development in podocytes partly depend on Ca2+ influx through TRPC6 channel, since OAG could induce TRPC6 current increase.
3.5. TRPC6 siRNA transfection blocked the expression of TRPC6 protein Western blot analysis was used to confirm the expression of TRPC6 in podocytes using negative control siRNA as a control. The successful transfection was shown compared to the controls (Fig. 8).
Fig. 3. TRPC6 protein expression levels at undifferentiated and mature podocyte. (a) Relative expression levels of TRPC6 were normalized to the -actin. (b) Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
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Fig. 4. The development of TRPC6 currents in cultured podocytes at different development stages. The current densities–voltage relationships of TRPC6 currents of podocytes in different development stages (a). Quantification of current densities at the potential of +75 mV (b), numbers in parentheses indicated the number of cells tested in each group. Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
Fig. 5. The characteristic of TRPC6 currents in cultured d6 and mature podocytes. Examples of membrane current recorded from d6 and mature podocytes (a, c). The current was induced by a 200-ms voltage ramp protocol (1 mV/ms, from −100 mV to +100 mV, holding potential −60 mV) every 5 s. Currents were normalized by membrane capacitance. Note that the current was increased after OAG (100 M) perfusion (a), and inhibited by La3+ (100 M) (c). The summarized results were shown in bar graphs (b, d). Data were presented as the means ± S.E.M. *P < 0.05 vs. d6 control group and # P < 0.05 vs. mature control group.
Fig. 6. Fluorescent intensity of intracellular Ca2+ in cultured podocytes at different differentiation stages. Microscopic images of intracellular Ca2+ using Fluo 3-AM staining were represented (a). Bar graphs showed the relative fluorescent intensity of intracellular Ca2+ in podocytes (b). Data were presented as the means ± S.E.M. *P < 0.05 vs. adjacent stages group.
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Fig. 7. OAG-induced intracellular Ca2+ increase in cultured podocytes at different differentiation stages. After measuring the baseline fluorescence of Fluo 3-AM, OAG (100 M) was applied at d6 and mature podocytes at the time indicated by an arrow. The results were expressed as the percent changes in fluorescence intensity relative to the baseline. Each trace represented the mean valve that was derived from 10 to 20 cells in a single experiment. Data were presented as means ± S.E.M. *P < 0.05 vs. control group.
3.6. TRPC6 inhibition induced F-actin cytoskeleton rearrangement on the developmental stage of podocytes TRPC6 may affect function of the slit diaphragm by inducing the remodeling of the actomyosin cytoskeleton. TRPC6 localizes to the glomerular slit diaphragm, where it associates with core components such as podocin and nephrin as well as the cytoskeleton. To investigate the function of TRPC6 during the development of podocytes, TRPC6 siRNA, La3+ (100 M) and SKF-96365 (5 M), which is a blocker of TRP cation channels, were used to inhibit the activity of TRPC6 channels. We tested whether the decreased TRPC6 expression in cultured podocytes would affect the structure of the actin cytoskeleton. Cells on SKF96365, La3+ and TRPC6-si groups displayed loss of actin stress fibers, which normally were found in control cells on d6. In mature podocytes, the inhibition of TRPC6 had no effects on F-actin structure (Fig. 9). These studies suggest that normal expression of TRPC6 is functionally associated with the actin cytoskeleton during podocyte development. 4. Discussion Podocytes are typically fully differentiated in newborns, while undifferentiated podocytes could be found in fetuses in most species besides some fish and shark ones [19]. Immature, undifferentiated and proliferating podocytes switch to a mature, differentiated and quiescent phenotype during glomerulogenesis. The normal development of podocytes is essential to maintain the structural integrity of glomerulus and the prevention of serious
kidney disease [20]. Recent studies have indicated that podocytes in vitro could mimic the development of podocytes in vivo in appropriate culture condition, therefore the cells should be good for development study [21–23]. During development periods, expressions of different kinds of proteins vary greatly. For example, nestin is shown to be expressed at the early stage of development and becomes confined to podocytes in mature glomeruli [24]. Ion channel function is the final mark for cell maturation [25] and hence changes in ion channel are necessary to the study of podocyte development. The expression and distribution of TRPC6 may be significantly important in the podocyte function and development [20]. The results we observed from immunofluorescence staining demonstrated a distribution change of TRPC6 in podocytes and especially an increase in functional expression on the podocyte membrane with the cell maturity. Further, patch clamp electrophysiology offers a direct measurement of channel activity if and when TPRC6 proteins are present in the plasma membrane. In addition, TRPC6 protein expression increased in mature podocytes compared to d 0 cells, and the results suggest that the upregulation of TRPC6 currents is due to both an increased protein expression and increased channel translocation to the plasma membrane. TRPC6 in podocytes are six times more permeable for Ca2+ than Na+ with a single-channel conductance [26,27]. In our study, treatment with OAG induced the increase in TRPC6 currents meanwhile [Ca2+ ]i rise in different stages of podocytes. That suggest the [Ca2+ ]i increase with podocyte development shown in Fig. 7 partly depend on TRPC6 channels function. Ca2+ is an important mediator of cellular homeostasis owing to its ability to elicit a dynamic changes and regulate biochemical responses, and Ca2+ regulates diverse vital processes such as muscle contraction, cytoskeletal structure and vesicle secretion. So it is impossible that the function of TRPC6 in podocyte development partly dependents on the influx of Ca2+ . According to Ariano [28], TRPC channels were shown to be involved in Ca2+ -dependent proliferation, and a reduction in proliferation following treatment with SKF-96365 in gonadotropin-releasing hormone (GnRH)-secreting neurons. In our study, podocytes reduced their proliferative capacity in the mature state, but TRPC6 had an increase in functional expression. That suggested the decreased proliferative capacity in mature podocytes did not attribute to less Ca2+ influx through TRPC6 channel. Previous studies have reported that TRPC6 protein has to be transported from the cell body into the foot processes in order for the protein to reach the slit diaphragm [3], which is consistent with our results that may happen during the process of development. The discovery of ion channels, whose open probability is increased by membrane stretch, provides the first direct experimental evidence indicating that podocytes may be able to respond acutely to mechanical stress. It has been known that the gating of TRPC6 is stretch-sensitive [29]. Therefore, TRPC6 could contribute to the mechanosensitivity of Ca2+ regulatory systems in podocytes.
Fig. 8. TRPC6 protein expression inhibited by TRPC6-si. Podocytes were transfected with TRPC6 siRNA for 24 h. Western blot analysis was used to determine the transfection efficiency (a). Relative expression levels of TRPC6 were normalized to the control (b). Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
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Fig. 9. TRPC6 inhibition inducing F-actin cytoskeletal rearrangement in the development stage of podocytes. The exposure of SKF96365, TRPC6-si and La3+ to podocytes for 24 h decreased the density of stress fibers and caused a redistribution of actin to the cell periphery on d6 but had no effects on the F-actin after the podocytes were mature (a). The summarized results showed the ration of podocytes that retain distinct longitudinal stress fibers (b). Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
For example, greater Ca2+ influx through TRPC6 channel when the foot process is subjected to mechanical stress, if pressure in glomerular arteries is elevated [30]. One recent study reported that TRPC6 could interact with the slit diaphragm protein nephrin and its total expression is increased in nephrin knockout mice [6]. TRPC6 also interacts with podocin, which may regulate its gating mechanism [31]. Besides, BKCa channels [32], which may play a role to maintain a driving force for Ca2+ influx in the process of TRPC6 activation [3,33]. What’s more, we have found a similar development change of BKCa in podocytes [34]. Consequently, the trafficking of TRPC6 channels may be regulated by these proteins during the development. Interestingly, we have found that the expression of TRPC6 was decreased during the postnatal development (P1–P49) in kidney cortex, although it could be detected in all stages during the glomeruli development. This novel finding indicated that TRPC6 might play an important function at the beginning of the kidney development [12]. Our results reported here, however, focused on its expression change in the podocyte differentiation, especially on membrane, which also indicated that TRPC6 was necessary during the podocyte differentiation. Actin dynamics plays a major role in defining changes in the podocyte morphology during development, when podocytes undergo remodeling to produce effacement, including alteration of podocyte intercellular junction with loss of the slit diaphragm. Thus, proteins regulating the plasticity of the podocyte actin cytoskeleton are critical for sustained function of the glomerulus [10,35]. According to Möller [8] and Jiang [36], over-expression TRPC6 induced the loss of actin stress fibers and reduced cell processes remarkably in cultured differentiated podocytes, and TRPC6 inhibition could recover the albumin induced F-actin cytoskeletal rearrangement [37], which suggest TRPC6 channels are associated with the actin cytoskeleton. However, recent work had also implicated TRPC5, in addition to TRPC6, as an important mediator of cytoskeletal changes induced by angiotensin II in podocytes [15]. The controversial results may be due to the different responses of podocytes to changed stimuli. This is the first study to investigate the relationship between TRPC6 and cytoskeleton in the development of podocytes. According to our results, TRPC6 inhibition leads to F-actin rearrangement only on d6, which suggest TRPC6 is necessary for normal podocyte development. 5. Conclusion In summary, there is an increase in the surface expression of TRPC6 channels, and the distribution of TRPC6 is different with the maturity of differentiation in cultured podocytes. This
development of ion channels may play an important role in maintain the stability of glomerular filtration during stimuli, which are normally associated with changes in renal hemodynamics. In addition, TRPC6 inhibition leads to F-actin rearrangement only on the developmental stage in podocytes. These results strongly support the conclusion that TPRC6 is an essential component of the slit diaphragm and is required for development of glomerulus. Conflicts of interest All the authors declared no conflicts of interest. Acknowledgements This work was supported by the grant of National Basic Research Program of China (2011CB944003) and the National Natural Science Foundation of China (31271074, 81571804). References [1] K. Kuwahara, Y. Wang, J. McAnally, J.A. Richardson, R. Bassel-Duby, J.A. Hill, E.N. Olson, TRPC6 fulfills a calcineurin signaling circuit during pathologic cardiac remodeling, J. Clin. Investig. 116 (2006) 3114–3126. [2] H. Pavenstadt, W. Kriz, M. Kretzler, Cell biology of the glomerular podocyte, Physiol. Rev. 83 (2003) 253–307. [3] M. Estacion, W.G. Sinkins, S.W. Jones, M.A. Applegate, W.P. Schilling, Human TRPC6 expressed in HEK 293 cells forms non-selective cation channels with limited Ca2+ permeability, J. Physiol. 572 (2006) 359–377. [4] M.A. Saleem, J. Zavadil, M. Bailly, K. McGee, I.R. Witherden, H. Pavenstadt, H. Hsu, J. Sanday, S.C. Satchell, R. Lennon, L. Ni, E.P. Bottinger, P. Mundel, P.W. Mathieson, The molecular and functional phenotype of glomerular podocytes reveals key features of contractile smooth muscle cells, Am. J. Physiol. Renal Physiol. 295 (2008) F959–F970. [5] J. Peti-Peterdi, Calcium wave of tubuloglomerular feedback, Am. J. Physiol. Renal Physiol. 291 (2006) F473–F480. [6] J. Reiser, K.R. Polu, C.C. Moller, P. Kenlan, M.M. Altintas, C. Wei, C. Faul, S. Herbert, I. Villegas, C. Avila-Casado, M. McGee, H. Sugimoto, D. Brown, R. Kalluri, P. Mundel, P.L. Smith, D.E. Clapham, M.R. Pollak, TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function, Nat. Genet. 37 (2005) 739–744. [7] M.P. Winn, P.J. Conlon, K.L. Lynn, M.K. Farrington, T. Creazzo, A.F. Hawkins, N. Daskalakis, S.Y. Kwan, S. Ebersviller, J.L. Burchette, M.A. Pericak-Vance, D.N. Howell, J.M. Vance, P.B. Rosenberg, A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis, Science 308 (2005) 1801–1804. [8] C.C. Moller, C. Wei, M.M. Altintas, J. Li, A. Greka, T. Ohse, J.W. Pippin, M.P. Rastaldi, S. Wawersik, S. Schiavi, A. Henger, M. Kretzler, S.J. Shankland, J. Reiser, Induction of TRPC6 channel in acquired forms of proteinuric kidney disease, J. Am. Soc. Nephrol. 18 (2007) 29–36. [9] X. Sun, Z. Fang, Z. Zhu, X. Yang, F. He, C. Zhang, Effect of down-regulation of TRPC6 on the puromycin aminonucleoside-induced apoptosis of mouse podocytes, J. Huazhong Univ. Sci. Technol. Med. Sci. 29 (2009) 417–422. [10] C. Faul, K. Asanuma, E. Yanagida-Asanuma, K. Kim, P. Mundel, Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton, Trends Cell Biol. 17 (2007) 428–437. [11] J. Reiser, K.R. Polu, C.C. Moller, P. Kenlan, M.M. Altintas, C.L. Wei, C. Faul, S. Herbert, I. Villegas, C. Avila-Casado, M. McGee, H. Sugimoto, D. Brown, R. Kalluri, P. Mundel, P.L. Smith, D.E. Clapham, M.R. Pollak, TRPC6 is a glomerular
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Z. Liu et al. / Cell Calcium 58 (2015) 541–548 slit diaphragm-associated channel required for normal renal function, Nat. Genet. 37 (2005) 739–744. P. Xu, J. Xu, Z. Li, Z. Yang, Expression of TRPC6 in renal cortex and hippocampus of mouse during postnatal development, PLoS ONE 7 (2012) e38503. L. Yang, S.R. Zheng, P.N. Epstein, Metallothionein over-expression in podocytes reduces adriamycin nephrotoxicity, Free Radic. Res. 43 (2009) 174–182. M. Estacion, S. Li, W.G. Sinkins, M. Gosling, P. Bahra, C. Poll, J. Westwick, W.P. Schilling, Activation of human TRPC6 channels by receptor stimulation, J. Biol. Chem. 279 (2004) 22047–22056. D. Tian, S.M. Jacobo, D. Billing, A. Rozkalne, S.D. Gage, T. Anagnostou, H. Pavenstadt, H.H. Hsu, J. Schlondorff, A. Ramos, A. Greka, Antagonistic regulation of actin dynamics and cell motility by TRPC5 and TRPC6 channels, Sci. Signal. 3 (145) (2010) ra77. D. Becker, J. Bereiter-Hahn, M. Jendrach, Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation, Eur. J. Cell Biol. 88 (2009) 141–152. V.J. Bezzerides, I.S. Ramsey, S. Kotecha, A. Greka, D.E. Clapham, Rapid vesicular translocation and insertion of TRP channels, Nat. Cell Biol. 6 (2004) 709–720. K. Clark, J. Middelbeek, F.N. van Leeuwen, Interplay between TRP channels and the cytoskeleton in health and disease, Eur. J. Cell Biol. 87 (2008) 631–640. H. Haller, K. de Groot, F. Bahlmann, M. Elger, D. Fliser, Stem cells and progenitor cells in renal disease, Kidney Int. 68 (2005) 1932–1936. S.E. Quaggin, J.A. Kreidberg, Development of the renal glomerulus: good neighbors and good fences, Development 135 (2008) 609–620. A.C. Leapley, C.C. Lee, C.A. Batchelder, M.C. Yoder, D.G. Matsell, A.F. Tarantal, Characterization and culture of fetal rhesus monkey renal cortical cells, Pediatr. Res. 66 (2009) 448–454. P. Mundel, J. Reiser, W. Kriz, Induction of differentiation in cultured rat and human podocytes, J. Am. Soc. Nephrol. 8 (1997) 697–705. M.R. Vaughan, J.W. Pippin, S.V. Griffin, R. Krofft, M. Fleet, L. Haseley, S.J. Shankland, ATRA induces podocyte differentiation and alters nephrin and podocin expression in vitro and in vivo, Kidney Int. 68 (2005) 133–144. M. Ishizaki, T. Ishiwata, A. Adachi, N. Tamura, M. Ghazizadeh, H. Kitamura, Y. Sugisaki, N. Yamanaka, Z. Naito, Y. Fukuda, Expression of nestin in rat and human glomerular podocytes, J. Submicrosc. Cytol. Pathol. 38 (2006) 193–200. C. Bellardita, F. Bolzoni, M. Sorosina, G. Marfia, S. Carelli, A. Gorio, A. Formenti, Voltage-dependent ionic channels in differentiating neural precursor cells collected from adult mouse brains six hours post-mortem, J. Neurosci. Res. 90 (2012) 751–758.
[26] T. Hofmann, A.G. Obukhov, M. Schaefer, C. Harteneck, T. Gudermann, G. Schultz, Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol, Nature 397 (1999) 259–263. [27] I.S. Ramsey, M. Delling, D.E. Clapham, An introduction to TRP channels, Annu. Rev. Physiol. 68 (2006) 619–647. [28] P. Ariano, S. Dalmazzo, G. Owsianik, B. Nilius, D. Lovisolo, TRPC channels are involved in calcium-dependent migration and proliferation in immortalized GnRH neurons, Cell Calcium (2011) 387–394. [29] M.A. Spassova, T. Hewavitharana, W. Xu, J. Soboloff, D.L. Gill, A common mechanism underlies stretch activation and receptor activation of TRPC6 channels, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 16586–16591. [30] W. Kriz, E. Hackenthal, R. Nobiling, T. Sakai, M. Elger, B. Hahnel, A role for podocytes to counteract capillary wall distension, Kidney Int. 45 (1994) 369–376. [31] T.B. Huber, B. Schermer, R.U. Muller, M. Hohne, M. Bartram, A. Calixto, H. Hagmann, C. Reinhardt, F. Koos, K. Kunzelmann, E. Shirokova, D. Krautwurst, C. Harteneck, M. Simons, H. Pavenstadt, D. Kerjaschki, C. Thiele, G. Walz, M. Chalfie, T. Benzing, Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 17079–17086. [32] E.Y. Kim, C.P. Alvarez-Baron, S.E. Dryer, Canonical transient receptor potential channel (TRPC)3 and TRPC6 associate with large-conductance Ca2+ -activated K+ (BKCa) channels: role in BKCa trafficking to the surface of cultured podocytes, Mol. Pharmacol. 75 (2009) 466–477. [33] S.E. Dryer, J. Reiser, TRPC6 channels and their binding partners in podocytes: role in glomerular filtration and pathophysiology, Am. J. Physiol. Renal Physiol. 299 (2010) F689–F701. [34] J. Yang, P. Xu, Y. Xie, Z. Li, J. Xu, T. Zhang, Z. Yang, Developmental changes of BK Ca channels depend on differentiation status in cultured podocytes, In Vitro Cell. Dev. Biol.: Anim. (2013) 205–211. [35] S. Somlo, P. Mundel, Getting a foothold in nephrotic syndrome, Nat. Genet. 24 (2000) 333–335. [36] L.N. Jiang, J.E. Ding, H.J. Tsai, L. Li, Q.C. Feng, J. Miao, Q.F. Fan, Over-expressing transient receptor potential cation channel 6 in podocytes induces cytoskeleton rearrangement through increases of intracellular Ca2+ and RhoA activation, Exp. Biol. Med. 236 (2011) 184–193. [37] S. Chen, F.F. He, H. Wang, Z. Fang, N. Shao, X.J. Tian, J.S. Liu, Z.H. Zhu, Y.M. Wang, S. Wang, K. Huang, C. Zhang, Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes, Cell Calcium 50 (2011) 523–529.
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Developmental changes in the expression and function of TRPC6 channels related the F-actin organization during differentiation in podocytes Zhaowei Liu a,1 , Jiajia Yang b,c,1 , Xiaochen Zhang a , Pengjuan Xu a , Tao Zhang c , Zhuo Yang a,∗ a College of Medicine, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Nankai University, Tianjin 300071, China b Tianjin University, Tianjin 300072, China c College of Life Science, Nankai University, Tianjin 300071, China
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Article history: Received 23 March 2015 Received in revised form 2 August 2015 Accepted 1 September 2015 Available online 3 September 2015 Keywords: Podocyte TRPC6 channel Differentiation F-actin
a b s t r a c t The transient receptor potential canonical (TRPC) 6 channel is an important ion channel located in podocytes, which plays an essential role in regulating calcium homeostasis of the cell signaling. Podocytes are specialized, terminally differentiated cells surrounding glomerular capillaries, and are the subject of keen interest because of their key roles in kidney development and disease. Here we wonder whether TRPC6 channels undergo developmental changes in the expression and function during the podocyte differentiation, and whether they contribute to the maturation of podocytes. Using morphological, immunohistochemical and electrophysiological techniques, we investigated the development of distribution and expression of TRPC6 in conditionally immortalized mouse podocyte cell line. Our results showed that the distribution of TRPC6 channels changed with the maturity of podocyte differentiation. The fluorescent intensity of TRPC6 on cell surface increased, which was accompanied by a corresponding increase in the density of current flowing through the channels. TRPC6 inhibition by TRPC6 siRNA or SKF-96365, a blocker or TRP cation channels, resulted in F-actin cytoskeleton disruption only on the developmental stage of podocytes. These results strongly support the conclusion that TPRC6 is an essential component of the slit diaphragm and is required for development of glomerulus. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Transient receptor potential (TRP) superfamily member the transient receptor potential canonical (TRPC) 6 is confirmed to be the main cation-permeable channel in excitable and non-excitable cells, which enables the influx of Ca2+ and Na+ ions into cells [1–3]. This superfamily consists of a group of six transmembrane domain-containing ion channels and plays an essential role in regulating Ca2+ homeostasis, which consequently mediates a cascade of signaling events [4,5]. Roles for TRPC6 in kidney were revealed by studies showing that mutations in human TRPC6 gene caused inherited focal segmental glomerulosclerosis (FSGS), and overexpression of TRPC6 in glomeruli could induce several acquired human proteinuric kidney diseases [6–8].
∗ Corresponding author. E-mail address: [email protected] (Z. Yang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ceca.2015.09.001 0143-4160/© 2015 Elsevier Ltd. All rights reserved.
Podocytes are specialized, terminally differentiated cells around glomerular capillaries, which provide physical support for capillary loops responding to changes in intraglomerular pressure [2]. Various studies suggest that TRPC6 is expressed in the podocyte cell body, major processes and foot processes near the slit diaphragm. The alterations of TRPC6 in podocytes have recently been attributed to proteinuric kidney diseases and foot process effacement [7–9]. The morphology of podocyte is defined by the underlying actomyosin cytoskeleton, and organization of the actin network is required for proper kidney function [10]. TRPC6 may affect the function of slit diaphragm where TRPC6 localizes by inducing the remodeling of actomyosin cytoskeleton [11]. The results of our previous studies revealed that TRPC6 was expressed in glomerulus and tubules but showed a decreased expression during the postnatal maturation of mouse renal cortex [12]. Nevertheless, we still wonder the development expression of TPRC6 in podocytes. The aim of this research was to clarify the developmental expression of TRPC6 ion channels on podocytes during the
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differentiation to the mature phenotype, and its role in F-actin cytoskeleton organization related to kidney function.
was excited at 488 nm laser line and the fluorescence signal was acquired at 522 nm. The fluorescence of Fluo-3 induced by 1-oleoyl2-acetyl-sn-glycerol (OAG, 100 M) was measured every 10 s.
2. Material and methods 2.5. Transient transfection in vitro 2.1. Podocyte culture and drug treatment Conditionally immortalized mouse podocyte cell line (a kind gift from Prof. Peter Mundel) was cultured at 33 ◦ C in RPMI1640 medium (Gibco, Gaithersburg, MD, USA), which contained 10% fetal bovine serum (Gibco), 10 U/ml of recombinant mouse ␥interferon and 100 U/ml of penicillin-streptomycin (Sigma, USA). Then podocytes were cultured at 37 ◦ C without ␥-interferon (growth restrictive conditions) to induce differentiation in two weeks. Experimental studies were conducted during the period of differentiation. All experiments were performed at least three times. In some experiments, the podocytes were treated with La3+ (100 M), OAG (100 M), which were purchased from Sigma (USA). 2.2. Laser-scanning confocal microscopy Cultured cells were washed in PBS, fixed by 15 min exposure to 4% paraformaldehyde in PBS, rinsed in PBS, blocked with 10% normal goat serum, and then permeabilized in PBS containing 0.3% Triton X-100. After that, cells were incubated with primary antibodies including rabbit anti-TRPC6 antibodies (1:1000, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C. Then cells were incubated with Alexa 488-conjugated anti-rabbit IgG (working dilution 1:1000, Invitrogen, San Diego, CA, USA) for 3 h at room temperature after being washed three times for 10 min with PBS at room temperature. The fluorescent signals were examined using a Leica TCS SP5 laser-scanning confocal microscope. The fluorescence quantitative of TRPC6 was semi quantitatively determined results, and Image-Pro Plus 7.0 software was used in the process. The fluorescence pictures were converted to 8-bit gray scale, and “invert contrast” and “apply contrast” buttons were pressed. Then, the optical density was corrected, and the “IOD” and “area” were selected to measure the pictures. Next the cell part in the field of view except for nucleus was drawn to measure, and the data could be read from SUM area and SUM view in the “view statistics” window. Density mean = (IOD SUM)/(area sum), and this was the average fluorescent intensity of the cells in our photograph. Enough cells were taken to make statistical analysis.
Podocytes were transiently transfected with TRPC6 siRNA (RiboBio, Guangzhou, GD, China) and its negative control using transfection reagent (RiboBio, Guangzhou, GD, China) according to the manufacturer’s instructions. These cells were used for the following experiments after transfection for 24 h. 2.6. Western blot analysis Proteins were run on a 10% SDS/PAGE gel and transferred onto PVDF membranes and blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% fat-free dry milk. The membranes were incubated with the primary antibodies, anti-TRPC6 (1:1000, Abcam, Cambridge, MA, USA), and then with horseradish peroxidaseconjugated secondary antibodies. The immunoreactive bands were detected using chemiluminescence methods and visualized on Xray films (Kodak, Rochester, NY, USA). The analysis of images was performed using Image J software (NIH, Bethesda, MD, USA). 2.7. Fluorescence staining of F-actin Podocytes were placed on poly-l-lysine-coated glass coverslips. After washing with PBS, the cells were fixed with 4% formaldehyde in PBS at room temperature for 30 min. The 0.1% Triton was added in PBS for 5 min to increase permeability. F-actin was detected using green fluorescent Phalloidin Conjugate working solution (Abcam, Cambridge, MA, USA) for 60 min at room temperature. Podocytes with distinct F-actin were counted as previous described [13]. 2.8. Data acquisition and statistical analysis Data were acquired using an EPC10 amplifier (HEKA, Germany) connected to a computer and stored on a hard disk using pulse 8.52 software (HEKA, Germany), analyzed off-line using the pCLAMP 9.0 and Origin 8.0. All quantitative data are presented as mean ± S.E.M. Data were analyzed by one-way ANOVA followed by post hoc analysis. Throughout, P < 0.05 is regarded as significant. 3. Results
2.3. Electrophysiology recording 3.1. Expression of TRPC6 in podocytes Conventional whole-cell patch-clamp recording was carried out at room temperature (22–24 ◦ C). Experiments were performed on podocytes attached to glass coverslips and transferred to a recording chamber on an Olympus inverted microscope stage immediately before recording. For recording TRPC6 currents, the pipette solution contained 130 mM CsOH, 130 mM l-aspartic acid, 2 mM MgCl2 , 0.3 mM CaCl2 , 10 mM HEPES, 10 mM EGTA and 3 mM ATP-Na at pH 7.3. And the external solution contained 130 mM NaCl, 4 mM KCl, 1 mM MgCl2 , 10 mM HEPES, 10 mM glucose and 2 mM CaCl2 at pH 7.3. The sample rate was 4 kHz (filtered at 2 kHz). The currents were measured at −100 and +100 mV and normalized by membrane capacitance. 2.4. Calcium imaging Cells were washed three times in Hanks’ solution and then loaded with Fluo 3-AM (5 M) (Sigma, USA) in 1 ml of Hanks’ solution at 37 ◦ C for 30 min. After three washings with D-Hanks’ solution, cells were incubated in 1 ml of Hanks’ solution and imaged at room temperature. The calcium-dependent fluorescence
All the experiments were performed in a conditionally immortalized mouse podocyte cell line in different differentiation status, including five groups of undifferentiated cultured podocytes (d0), 3, 6, 9 and 12 days after induction of differentiation (d3, d6, d9 and d12), and mature podocytes (14 or more days after induction of differentiation). Using confocal microscopy, we addressed the distribution of TRPC6 in podocytes of different differentiation status. First, the cell morphology was observed, which indicated the conversion of typical cobblestone appearance into arborized cells within 2 weeks (Fig. 1a–f). Groups d0 and d3 showed the classic “cobblestone” appearance. While at the late stage of differentiation (d9 and d12), cells showed a more differentiated morphology characterized by arborization, processes extending from the cell body. In the mature state, podocytes reduced their proliferative capacity (data not shown) and developed specialized junctions between the cell body and foot processes. Hence we observed a process of maturation in podocytes in vitro, which was analogous to the developmental maturation of podocytes in vivo.
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Fig. 1. The localization of TRPC6 in podocytes at different development stages. In different stages of differentiating podocytes, TRPC6 was gradually translocated to the membrane (d3-mature). Subcellular distribution of TRPC6 (g) and dapi (h), double immunolabeling (i) showing TRPC6 was mainly expressed in the nucleus in undifferentiated podocytes (d0).
Fig. 1a also showed that TRPC6 proteins were mainly expressed in the nuclear regions of podocytes in group d0, with hardly distinguishable signals toward the membrane of the cells. The results can be showed more clearly in Fig. 1g–i by double immunolabeling of TRPC6 and nucleus. And from group d3 to d12, the images exhibited gradually translocating of TRPC6 channels to cytoplasm. The image of mature podocyte indicated that TRPC6 was evenly
distributed inside the cell but almost empty in nuclear (mature), especially in cytoplasm and membrane surface. The expression of TRPC6 in nuclear regions seemed almost all translocated to other regions and increased in the cell membrane with the differentiation process. We also quantified the fluorescent intensity of TRPC6 on cell surface which was shown in Fig. 2. It showed a significant increase 9 days after induction of differentiation. Meanwhile, the
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Fig. 2. Fluorescent intensity of TRPC6 on podocyte membrane at different differentiation stages. Bar graphs showed the relative fluorescent intensity of TRPC6 on the cell surface of podocytes. Data were presented as the means ± S.E.M. *P < 0.05 vs. adjacent stages group.
TRPC6 protein levels increased in mature podocytes compared to undifferentiated ones (Fig. 3a). The summarized data are shown in Fig. 3b. In addition, we provided the developmental expression of a specific podocyte molecule, nephrin, which is one of markers for maturation of podocytes. The images in Fig. 1 indicated that the change of fluorescent intensity of TRPC6 was due to the differentiation of podocytes. 3.2. Whole-cell recordings of TRPC6 currents Morphological and biochemical data alone might not be enough to characterize the membrane expression change of TRPC6 in podocytes. Functional tests should be considered. Using more quantitative electrophysiological methods, TRPC6 current density was assayed by means of conventional whole-cell recordings in five groups. TRPC6 currents were activated using a voltage-stimulation mode, ramp protocol (1 mV/ms, from −100 mV to 100 mV, over 200 ms) every 5 s. Eight points were chosen on current curves to process statistical analysis and the currents at +75 mV for drawing histograms. The I–V curves in Fig. 4a showed that both the inward and the outward currents were substantially increased in different groups. And the average current density in these groups at +75 mV was shown in the bar graph (Fig. 4b), which is consistent with an increase in the number of active channels with the development of podocytes, since the TRPC6 protein increased according to Figs. 1–3. There were significant increases between d3 and d6, d6 and d9, d12 and mature cell groups. Therefore, we believe that the increased current density reflected an increased expression of TRPC6 channels in podocytes, which is consistent with the result of confocal microscopy.
Previous studies on TRPC6 channels in heterologous expression systems have shown that their activation gives rise to outwardly rectifying macroscopic cationic currents that are much larger in the presence of OAG, a membrane-permeable analog of diacylglycerol (DAG) analogs and the currents can be blocked by La3+ [14]. In d6 and mature podocytes, we observed an activation of macroscopic currents with these characteristics, which had distinctive inward and outward rectification properties as seen in representative examples (Fig. 5a, c). Furthermore, the cationic currents had an average reversal potential of 0 mV (0 ± 4 mV) under the recording conditions. These currents were substantially larger in cells exposed for 5 min to 100 M OAG (Fig. 5a, b), and they were also blocked by superfusion of 100 M La3+ (Fig. 5a, d), which blocked TRPC6 channels [15]. The pharmacological and electrophysiological characteristics of the nonselective currents in podocytes suggested that the membrane currents we recorded from podocytes were TRPC6 currents. In addition, the results suggested that the effects of OAG and La3+ on TRPC6 channel during development were similar to mature stage in podocytes.
3.3. Intracellular calcium concentration increase during the development in podocytes Commonly, TRPC channels associate with the cytoskeleton in order to link changes in membrane cationic flux with alteration in the actin-based cytoskeleton [16–18]. And intracellular calcium concentration was tested in the next step. To investigate the [Ca2+ ]i during the development of podocytes, cells were incubated with the calcium indicator Fluo 3-AM (5 M). The [Ca2+ ]i was significantly increased in d3 and d6, d6 and d9, d9 and d12 groups (Fig. 6).
3.4. OAG induced intracellular calcium concentration increase at different differentiation stages To investigate the relationship between the TRPC6 currents and the increase of [Ca2+ ]i, OAG was used in the next experiment. OAG induced [Ca2+ ]i increase in d6 and mature podocytes, and the trends in mature cells were more significant compared to d6 cells, but no statistically significant difference (Fig. 7). The results suggested the increase of [Ca2+ ]i during the development in podocytes partly depend on Ca2+ influx through TRPC6 channel, since OAG could induce TRPC6 current increase.
3.5. TRPC6 siRNA transfection blocked the expression of TRPC6 protein Western blot analysis was used to confirm the expression of TRPC6 in podocytes using negative control siRNA as a control. The successful transfection was shown compared to the controls (Fig. 8).
Fig. 3. TRPC6 protein expression levels at undifferentiated and mature podocyte. (a) Relative expression levels of TRPC6 were normalized to the -actin. (b) Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
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Fig. 4. The development of TRPC6 currents in cultured podocytes at different development stages. The current densities–voltage relationships of TRPC6 currents of podocytes in different development stages (a). Quantification of current densities at the potential of +75 mV (b), numbers in parentheses indicated the number of cells tested in each group. Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
Fig. 5. The characteristic of TRPC6 currents in cultured d6 and mature podocytes. Examples of membrane current recorded from d6 and mature podocytes (a, c). The current was induced by a 200-ms voltage ramp protocol (1 mV/ms, from −100 mV to +100 mV, holding potential −60 mV) every 5 s. Currents were normalized by membrane capacitance. Note that the current was increased after OAG (100 M) perfusion (a), and inhibited by La3+ (100 M) (c). The summarized results were shown in bar graphs (b, d). Data were presented as the means ± S.E.M. *P < 0.05 vs. d6 control group and # P < 0.05 vs. mature control group.
Fig. 6. Fluorescent intensity of intracellular Ca2+ in cultured podocytes at different differentiation stages. Microscopic images of intracellular Ca2+ using Fluo 3-AM staining were represented (a). Bar graphs showed the relative fluorescent intensity of intracellular Ca2+ in podocytes (b). Data were presented as the means ± S.E.M. *P < 0.05 vs. adjacent stages group.
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Fig. 7. OAG-induced intracellular Ca2+ increase in cultured podocytes at different differentiation stages. After measuring the baseline fluorescence of Fluo 3-AM, OAG (100 M) was applied at d6 and mature podocytes at the time indicated by an arrow. The results were expressed as the percent changes in fluorescence intensity relative to the baseline. Each trace represented the mean valve that was derived from 10 to 20 cells in a single experiment. Data were presented as means ± S.E.M. *P < 0.05 vs. control group.
3.6. TRPC6 inhibition induced F-actin cytoskeleton rearrangement on the developmental stage of podocytes TRPC6 may affect function of the slit diaphragm by inducing the remodeling of the actomyosin cytoskeleton. TRPC6 localizes to the glomerular slit diaphragm, where it associates with core components such as podocin and nephrin as well as the cytoskeleton. To investigate the function of TRPC6 during the development of podocytes, TRPC6 siRNA, La3+ (100 M) and SKF-96365 (5 M), which is a blocker of TRP cation channels, were used to inhibit the activity of TRPC6 channels. We tested whether the decreased TRPC6 expression in cultured podocytes would affect the structure of the actin cytoskeleton. Cells on SKF96365, La3+ and TRPC6-si groups displayed loss of actin stress fibers, which normally were found in control cells on d6. In mature podocytes, the inhibition of TRPC6 had no effects on F-actin structure (Fig. 9). These studies suggest that normal expression of TRPC6 is functionally associated with the actin cytoskeleton during podocyte development. 4. Discussion Podocytes are typically fully differentiated in newborns, while undifferentiated podocytes could be found in fetuses in most species besides some fish and shark ones [19]. Immature, undifferentiated and proliferating podocytes switch to a mature, differentiated and quiescent phenotype during glomerulogenesis. The normal development of podocytes is essential to maintain the structural integrity of glomerulus and the prevention of serious
kidney disease [20]. Recent studies have indicated that podocytes in vitro could mimic the development of podocytes in vivo in appropriate culture condition, therefore the cells should be good for development study [21–23]. During development periods, expressions of different kinds of proteins vary greatly. For example, nestin is shown to be expressed at the early stage of development and becomes confined to podocytes in mature glomeruli [24]. Ion channel function is the final mark for cell maturation [25] and hence changes in ion channel are necessary to the study of podocyte development. The expression and distribution of TRPC6 may be significantly important in the podocyte function and development [20]. The results we observed from immunofluorescence staining demonstrated a distribution change of TRPC6 in podocytes and especially an increase in functional expression on the podocyte membrane with the cell maturity. Further, patch clamp electrophysiology offers a direct measurement of channel activity if and when TPRC6 proteins are present in the plasma membrane. In addition, TRPC6 protein expression increased in mature podocytes compared to d 0 cells, and the results suggest that the upregulation of TRPC6 currents is due to both an increased protein expression and increased channel translocation to the plasma membrane. TRPC6 in podocytes are six times more permeable for Ca2+ than Na+ with a single-channel conductance [26,27]. In our study, treatment with OAG induced the increase in TRPC6 currents meanwhile [Ca2+ ]i rise in different stages of podocytes. That suggest the [Ca2+ ]i increase with podocyte development shown in Fig. 7 partly depend on TRPC6 channels function. Ca2+ is an important mediator of cellular homeostasis owing to its ability to elicit a dynamic changes and regulate biochemical responses, and Ca2+ regulates diverse vital processes such as muscle contraction, cytoskeletal structure and vesicle secretion. So it is impossible that the function of TRPC6 in podocyte development partly dependents on the influx of Ca2+ . According to Ariano [28], TRPC channels were shown to be involved in Ca2+ -dependent proliferation, and a reduction in proliferation following treatment with SKF-96365 in gonadotropin-releasing hormone (GnRH)-secreting neurons. In our study, podocytes reduced their proliferative capacity in the mature state, but TRPC6 had an increase in functional expression. That suggested the decreased proliferative capacity in mature podocytes did not attribute to less Ca2+ influx through TRPC6 channel. Previous studies have reported that TRPC6 protein has to be transported from the cell body into the foot processes in order for the protein to reach the slit diaphragm [3], which is consistent with our results that may happen during the process of development. The discovery of ion channels, whose open probability is increased by membrane stretch, provides the first direct experimental evidence indicating that podocytes may be able to respond acutely to mechanical stress. It has been known that the gating of TRPC6 is stretch-sensitive [29]. Therefore, TRPC6 could contribute to the mechanosensitivity of Ca2+ regulatory systems in podocytes.
Fig. 8. TRPC6 protein expression inhibited by TRPC6-si. Podocytes were transfected with TRPC6 siRNA for 24 h. Western blot analysis was used to determine the transfection efficiency (a). Relative expression levels of TRPC6 were normalized to the control (b). Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
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Fig. 9. TRPC6 inhibition inducing F-actin cytoskeletal rearrangement in the development stage of podocytes. The exposure of SKF96365, TRPC6-si and La3+ to podocytes for 24 h decreased the density of stress fibers and caused a redistribution of actin to the cell periphery on d6 but had no effects on the F-actin after the podocytes were mature (a). The summarized results showed the ration of podocytes that retain distinct longitudinal stress fibers (b). Data were presented as the means ± S.E.M. *P < 0.05 vs. control group.
For example, greater Ca2+ influx through TRPC6 channel when the foot process is subjected to mechanical stress, if pressure in glomerular arteries is elevated [30]. One recent study reported that TRPC6 could interact with the slit diaphragm protein nephrin and its total expression is increased in nephrin knockout mice [6]. TRPC6 also interacts with podocin, which may regulate its gating mechanism [31]. Besides, BKCa channels [32], which may play a role to maintain a driving force for Ca2+ influx in the process of TRPC6 activation [3,33]. What’s more, we have found a similar development change of BKCa in podocytes [34]. Consequently, the trafficking of TRPC6 channels may be regulated by these proteins during the development. Interestingly, we have found that the expression of TRPC6 was decreased during the postnatal development (P1–P49) in kidney cortex, although it could be detected in all stages during the glomeruli development. This novel finding indicated that TRPC6 might play an important function at the beginning of the kidney development [12]. Our results reported here, however, focused on its expression change in the podocyte differentiation, especially on membrane, which also indicated that TRPC6 was necessary during the podocyte differentiation. Actin dynamics plays a major role in defining changes in the podocyte morphology during development, when podocytes undergo remodeling to produce effacement, including alteration of podocyte intercellular junction with loss of the slit diaphragm. Thus, proteins regulating the plasticity of the podocyte actin cytoskeleton are critical for sustained function of the glomerulus [10,35]. According to Möller [8] and Jiang [36], over-expression TRPC6 induced the loss of actin stress fibers and reduced cell processes remarkably in cultured differentiated podocytes, and TRPC6 inhibition could recover the albumin induced F-actin cytoskeletal rearrangement [37], which suggest TRPC6 channels are associated with the actin cytoskeleton. However, recent work had also implicated TRPC5, in addition to TRPC6, as an important mediator of cytoskeletal changes induced by angiotensin II in podocytes [15]. The controversial results may be due to the different responses of podocytes to changed stimuli. This is the first study to investigate the relationship between TRPC6 and cytoskeleton in the development of podocytes. According to our results, TRPC6 inhibition leads to F-actin rearrangement only on d6, which suggest TRPC6 is necessary for normal podocyte development. 5. Conclusion In summary, there is an increase in the surface expression of TRPC6 channels, and the distribution of TRPC6 is different with the maturity of differentiation in cultured podocytes. This
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