Effects of miR-200b-3p inhibition on the TRPC6 and BKCa channels of podocytes

Accepted Manuscript Effects of miR-200b-3p inhibition on the TRPC6 and BKCa channels of podocytes Hongqiang Yin, Xiaochen Zhang, Kai Li, Zhigui Li, Zhuo Yang PII:

S0003-9861(18)30191-7

DOI:

10.1016/j.abb.2018.06.013

Reference:

YABBI 7759

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 9 March 2018 Revised Date:

22 June 2018

Accepted Date: 25 June 2018

Please cite this article as: H. Yin, X. Zhang, K. Li, Z. Li, Z. Yang, Effects of miR-200b-3p inhibition on the TRPC6 and BKCa channels of podocytes, Archives of Biochemistry and Biophysics (2018), doi: 10.1016/j.abb.2018.06.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Effects of miR-200b-3p inhibition on the TRPC6 and BKCa channels of podocytes Hongqiang Yin1, Xiaochen Zhang1, Kai Li1, Zhigui Li1,2, Zhuo Yang1* 1

College of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of

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Bioactive Materials for Ministry of Education, Nankai University, Tianjin 300071, China Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of

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Medicine, New Haven, Connecticut 06510, USA

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*Correspondence for Proofs:

Professor Yang Zhuo, College of Medicine, Nankai University, Tianjin, 300071, China Tel: 86-22-23504364

Fax: 86-22-23502554

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E-mail: [email protected]

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Abstract Transient receptor potential canonical 6 (TRPC6) and large-conductance 2+ Ca -activated K+ channels (BKCa), two of the key ion channels for blood filtration function of podocytes, have been implicated in the pathogenesis of kidney diseases. Moreover, it has been reported that miR-200b plays an important role in regulating the biological processes of podocytes. In this study, we aimed to examine whether there was a relationship between miR-200b-3p and the two ion channels. It was suggested that miR-200b-3p down-regulation inhibited the currents of TRPC6 and BKCa channels. It also showed that miR-200b-3p inhibition reduced the levels of protein expression and mRNA transcription of TRPC6 and BKCa channels. Moreover, the down-regulation of miR-200b-3p resulted in the decrease of the intracellular Ca2+ concentration. It was also suggested that the decrease of BKCa currents resulting from miR-200b-3p inhibition could be regulated by TRPC6 channels. TRPC6 blockage also inhibited BKCa currents and reduced the level of BKCa expression. These results together suggested that miR-200b-3p inhibition reduced the currents of TRPC6, which led to the decrease of intracellular Ca2+ concentration. The decrease of Ca2+ source required for BKCa activation may result in the inhibition of BKCa currents. Key words: Podocytes; TRPC6; Ca2+; BKCa

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1 Introduction Podocytes, also called glomerular epithelial cells, are terminally differentiated cells that are attached to the glomerular basement membrane (GBM). Podocytes consists of three parts including the cell body, the primary processes and foot processes. Owing to the special structure, podocytes together with glomerular basement membrane constitute the glomerular blood filtration, which play an important role as a barrier to prevent protein loss. Accumulating results show that the development and process of many kidney diseases, including focal segmental glomerulosclerosis (FSGS) and proteinuria, are closely related to the damage and functional abnormalities of podocytes [1, 2]. microRNA (miRNA) is a class of endogenous single-stranded, non-coding RNA molecules including about 22 nucleotides in length. It is thought to modulate gene transcription and expression by translational inhibition and destabilization of messenger RNAs (mRNAs) by binding to its target gene to form a complete or incomplete complementary pairing [3, 4]. Some studies have showed that knocking out some special enzymes in podocytes such as Dicer or Drosha that plays an important role in the production of miRNAs could lead to proteinuria and glomerulosclerosis [5-7]. Moreover, in our previous study it was suggested that miR-200b family could promote podocyte differentiation by the repression of RSAD2 [8]. The function of TRPC6 and BKCa channels has a great effect on the physiological process of podocytes and also plays an important role in promoting the pathogenesis and development of different types of kidney diseases [9-11]. TRPC6 channels, a nonselective Ca2+-permeable cation channels in podocytes, interact with a

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number of different proteins, including scaffolding molecules, signaling proteins, cytoskeletal elements, and other types of ion channels [12]. It is reported that BKCa and TRPC6 channels are co-expression in podocytes, and TRPC6 could provide a Ca2+ source for BKCa activation [13]. Moreover, BKCa plays an important role in regulating the self-balance of Ca2+ in podocytes [14]. The aim of this study was to determine whether miR-200b-3p could regulate TRPC6 channels and BKCa channels in podocytes.

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2 Materials and methods 2.1 Podocytes culture Conditionally immortalized mouse podocyte cell line (MPC5), obtained from Prof. Peter Mundel [15], was cultured at 33 in RPMI 1640 medium (Hyclone, USA) contained 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 100U/ml of penicillin-streptomycin (Sigma, St. Louis, MO, USA) and 10U/ml of recombinant mouse γ-interferon (INF-γ, Sigma, St. Louis, MO, USA) in a humidified atmosphere of 5% CO2. To induce differentiation of podocytes (DMPC5), the cells were cultured at 37℃ in the absence of INF-γ for at least two weeks. At least three times were repeated in all experiments. OAG (100 µM, sigma, USA) and La3+ (100 µΜ, sigma, USA) were used to treat podocytes in the experiments. Moreover, in order to examine the effect of TRPC6 blockage on BKCa currents, we used La3+ to block the TRPC6 channels. The currents of BKCa in the absence of 100 µM La3+ were recorded as control, and the currents were recorded after cells were incubated with La3+ for 24h, which was used as TRPC6 blockage group. 2.2 Electrophysiology recording The TRPC6 and BKCa currents were recorded in the conventional whole-cell patch-clamp configuration. All experiments were performed at room temperature (22-24℃). The differentiated podocytes were attached to glass coverslips and then immediately transferred to a recording chamber mounted on the stage of an Olympus invented microscope. For whole-cell recording of TRPC6 currents, the internal solution contained (in mM): 2 MgCl2, 0.3 CaCl2, 130 CsOH, 130 L-aspartic acid, 10 HEPES, 3 ATP-Na and adjusted to pH 7.3. The external solution contained (in mM): 1 MgCl2, 130 NaCl, 4 KCl, 10 HEPES, 10 Glucose, 2 CaCl2 and adjusted to pH 7.3. For whole-cell recording of BKCa currents, the external solution contained (in mM): 150 NaCl, 5.4 KCl, 0.8 MgCl2, 5.4 CaCl2, and 10 HEPES and adjusted to pH 7.4 with NaOH. The pipette solution contained (mM): 140 KCl, 1 MgCl2, 10 HEPES, 10 EGTA, 5.76 CaCl2 (free calcium concentration 200 nM), and pH was adjusted to pH 7.2 with KOH. The ionic conditions in pipette solution provided sufficient intracellular Ca2+ for activation of BKCa currents by command depolarized potential pulses. Whole-cell BKCa currents were evoked by a series of 800 ms depolarized duration under a holding potential of -60 mV. In every series, the cells were depolarized from -50 mV to +130 mV in increments of 10 mV to ensure the BKCa channels activity could be detected. The currents of TRPC6 and BKCa channels were amplified with an EPC10

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amplifier (HEKA, Germany), low-pass filtered at 2 kHz. The patch pipettes were pulled from borosilicate glass on a multistage micropipette puller (P-97, Sutter Instruments, USA) and had a resistance ranged from 3 to 7 MΩ. 2.3 Western blot analysis Podocytes were washed with ice-cold PBS and lysed by RIPA buffer on ice. The cell lysates were cleared by centrifugation at 12000 r/min for 15 min at 4 . Then protein concentrations were determined by BCA Protein Assay Kits according to the manufacturer’s instruction (Beyotime, China). Equal samples (20 µg/well) were subjected to electrophoresis by 10% SDS/PAGE gel and transferred onto PVDF membranes. After transfer, the PVDF membranes were blocked in Tris-buffered saline containing 5% non-fat milk and 0.05% Tween 20 (TBST) for 2h at room temperature. The PVDF membranes were incubated with appropriate primary antibodies (anti-TRPC6, 1:1000, Abcam; anti-BKCa-α, 1:1000, Santa Cruz; β-actin, 1:2000, Abcam) diluted in blocking buffer overnight at 4 ℃. After washing 310min with TBST next day, the membranes were incubated with HRP-conjugated secondary antibodies (anti-rabbit IgG, 1:4000, Promega, USA; anti-mouse IgG, 1:4000, Promega, USA). The immunoreactive bands were detected with HRP substrate (Millipore, USA) and analyzed by a Gel Image Analysis System (Tanon-3500R, Shanghai, China). The quantitation analysis was performed with NIH Image J pro. β-actin was used as the internal control. 2.4 Transient transfection in vitro The cells were transiently transfected with miR-200b-3p inhibitors (miR2150821043313, RiboBio, Guangdong, China) and the negative control (miR02204, RiboBio, Guangdong, China) respectively using ribo FECT™ CP Transfection Kit (RiboBio, Guangdong, China) according to the manufacture’s instruction. After the transfection for 24 h, total protein and RNAs were extracted for Western blot analysis and qRT-PCR. 2.5 Fluorescent Ca2+-imaging After transfection for 24h, podocytes attached coverslips were washed three times with Hanks’ solution and then loaded with Fluo 3-AM (5 µM, Sigma, USA) in Hanks’ solution for 30 min at room temperature in dark. Subsequently, the cells were washed with D-Hanks’ solution for three times and incubated in Hanks’ solution at 37 ℃ for 30min. The fluorescence was activated at 488 nm laser line and the signal was obtained at 522 nm. 2.6 Real-time quantitative PCR Total RNA was extracted from podocytes with Trizol (Takara, Japan) according to the manufacturer’s protocols. The harvested RNA was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara, Japan) and then the cDNA was used for real-time PCR to quantify mRNA expressions of TRPC6 and Slo1 using SYBR Green qPCR Master Mix (Takara, Japan) with GAPDH as an internal control. U6 was also used as an internal control when miR-200b-3p expression was detected. The stem-loop primer and forward and reverse primer pairs for miR-200b-3p and U6 were designed by RiboBio (Guangzhou, Guangdong, China). The levels of TRPC6, BKCa △△ and miR-200b-3p were presented as values of 2- Ct. The primers’ sequences are as

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follows: TRPC6 forward primer, 5’-TGGTGCGGAAGATGCTAGAA-3’ reverse primer, 5’-AAAGCATCCCCAACTCGAGA-3’; Slo1 forward primer, 5’-TGGTAACGTGGACACCCTTG-3’ reverse primer, 5’-GGGTCATGCCTCATCAGCTT-3’; GAPDH forward primer, 5’-ACCCAGAAGACTGTGGATGG-3’ reverse primer, 5’-CACATTGGGGGTAGGAACAC-3’; 2.7 Data acquired and analysis The data were acquired by a patch clamp EPC10 amplifier (HEKA, Germany) and analyzed by Clampfit 9.0, Origin 8.0 and SPSS 11.5. For the activation curve of BKCa channels, currents at each command potential were converted to conductance (G) by the formula G=I/(Vm-Vr), where Vr is the reverse potential. The conductance value at each command potential is normalized to GMax. Then the value of G/GMax plots against each command potential to produce a voltage-gated relationship, which are fitted Boltzmann functions G/GMax=1/{1+exp[(Vm–Vh)/k]}, where Vh is the membrane potential at half-activation and k is a slope factor. Differences between groups were analyzed using one-way ANONA accompanied with Fisher’s least significant difference (LSD) post-test. For comparison between two groups, the two-tailed Student’s t-test was used. p<0.05 was considered significant.

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3 Results 3.1 miR-200b-3p inhibition downregulates expression and mRNA levels of TRPC6 and BKCa channels in podocytes To confirm the regulation of miR-200b-3p on TRPC6 in podocytes, we examined the changes in mRNA and protein levels of TRPC6 and BKCa after miR-200b-3p inhibition. The repression of miR-200b-3p expression reached about 43.36% compared with that of NC group (Fig. 1A). Moreover, we also excluded the off-target effects of miR-200b-3p inhibitors (Fig. S1), and it was suggested that miR-200b-3p inhibitors could specifically decrease miR-200b-3p in podocytes. The results indicated that the mRNA and protein levels of TRPC6 (Fig. 1B, 1D and 1E) and BKCa (Fig. 1C, 1D and 1F) were markedly decreased in the group of miR-200b-3p inhibition compared with those of the NC group. 3.2 miR-200b-3p inhibition reduces the currents of TRPC6 in podocytes Conventional whole-cell patch clamp recording was used to detect the effects of miR-200b-3p inhibition on TRPC6 currents. In these experiments, TRPC6 currents were evoked by a ramp voltage commands (-100 mV to +100 mV over 800ms) from a holding potential of -60 mV. Moreover, the transfection efficiency was examined to identify that the patched cells were all transfected with miR-200b-3p inhibitors (Fig. S2). The I-V curves of TRPC6 indicated that both inward and outward currents were significantly decreased in miR-200b-3p inhibition group compared with those of the

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NC group (Fig. 2A and 2B), which was consistent with the decrease in the number of active channels with the miR-200b-3p inhibition in podocytes, since the TRPC6 protein decreased (Fig. 1D and 1E). It has been showed that 1-oleoyl-2-acetyl-sn-glyceroldiacylglycerol (OAG), a membrane-permeable analog of diacylglycerol (DAG), can activate TRPC6 channels, and their activation gives rise to much larger outwardly rectifying macroscopic cationic currents. Moreover, TRPC6 currents could also be blocked by La3+ [16]. In miR-200b-3p inhibition group, we observed distinctive inward and outward rectification properties of TRPC6 currents in the presence of OAG as seen in representative graph (Fig. 2C). The cationic currents were significantly larger in podocytes exposed to 100 µM OAG (Fig. 2D). They were also blocked by 100 µM La3+ (Fig. 2E and 2F). Moreover, the macroscopic cationic currents showed an average reverse potential of 0~-4 mV under the recording condition used in these experiments. Thus, these evidences suggested that the currents we recorded from podocytes were TRPC6 currents. 3.3 miR-200b-3p inhibition influences the currents and the activation kinetics of BKCa channels in podocytes Furthermore, conventional whole-cell patch clamp recording was used to explore the effect of miR-200b-3p inhibition on BKCa channel currents. It’s known that macroscopic BKCa currents in podocytes have the property of slow activation kinetics. The previous study has showed that the outward currents evoked using these protocols can isolate macroscopic BKCa currents [15]. Moreover, we examined the sensitivity of the evoked currents to 100 nM paxilline, a special inhibitor of BKCa channels [17]. It was found that the currents were significantly inhibited in the presence of paxilline (Fig. 3A), which indicated that the evoked currents were BKCa currents. The I-V curve showed that BKCa currents were evoked in a voltage-dependent manner and at the same command potential, the mean currents significantly decreased in miR-200b-3p inhibition group compared to those of the NC group (Fig. 3B), which was consistent with the decrease in the number of BKCa channels with the miR-200b-3p inhibition of podocytes, since the BKCa protein decreased (Fig. 1D and 1F). Furthermore, we tested the kinetics of BKCa channels (Fig. 3C). The result showed the activation curve shifted to positive potential in miR-200b-3p inhibition compared to the NC group, which suggested that the opening number of BKCa channels was also decreased following miR-200b-3p inhibition. 3.4 miR-200b-3p inhibition decreases the intracellular calcium concentration TRPC6 and BKCa channels play crucial role in the Ca2+ homeostasis of podocytes [12], and according to the decrease of TRPC6 currents, we examined the change of [Ca]i in podocytes. The results showed that the [Ca]i was significantly decreased after miR-200b-3p inhibition compared with the NC group (Fig. 4A and 4B). It is known that TRPC6 can serve as a source of Ca2+ for the activation of BKCa channels [13]. 3.5 miR-200b-3p inhibition decreases BKCa currents via TRPC6 To investigate the decline of BKCa currents induced by miR-200b-3p inhibition via TRPC6, BKCa currents were recorded in podocytes exposed to La3+ or OAG

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respectively under the condition of miR-200b-3p inhibition. After cells were transfected with miR-200b-3p inhibitors for 24h, BKCa currents were recorded, and then the currents were recorded once again after application of OAG or La3+. As seen in representative graph (Fig. 5A), it showed that BKCa currents were visibly increased after application of OAG to podocytes compared with those of the miR-200b-3p inhibition group, and they were also significantly decreased in the presence of La3+. The I-V curves showed that the mean currents of BKCa were increased after application of OAG (Fig. 5B). The I-V curves of BKCa currents showed a decrease after application of La3+ (Fig. 5C). Moreover, the NC group also showed similar trends after application of OAG or La3+ (Fig. 5D and 5E). The activation curves of BKCa currents were also analyzed after application of OAG or La3+. The activation curves of BKCa currents shifted to negative potential after application of OAG, and it was suggested the opening number of BKCa channels was increased in podocytes exposed to OAG (Fig. 6A). The activation curves shifted positive potential after application of La3+ (Fig. 6B). Additionally, the activation curves of BKCa currents showed similar trends in NC group exposed to OAG or La3+ (Fig. 6C and 6D). 3.6 TRPC6 blockage reduces BKCa currents in podocytes Next, we examined the interactions between TRPC6 and BKCa channels in podocytes. La3+ was used to block the TRPC6 channels. The results showed that TRPC6 blockage reduced the currents of BKCa (Fig. 7C) and the activation curve of BKCa currents shifted to positive potential (Fig. 7D), which indicated that the mean current of BKCa significantly decreased compared to that of La3+ group at the same command potential. Moreover, the protein of BKCa channels was also examined after podocytes were incubated with La3+. The protein levels of TRPC6 could be inhibited by La3+ (Fig. 7A). The protein levels of BKCa were also decreased in La3+ group comparing with those of the control group (Fig. 7B). Moreover, we also investigated the contribution of RSAD2 in the miR-200b-3p/TRPC6/BKCa signal in Fig. S3, and it was suggested that RSAD2 may not play an important role in the signal of miR-200b-3p /TRPC6 /BKCa. 4 Discussion In our study, we showed the relationship between miR-200b-3p and the two ion channels of TRPC6 and BKCa. Our studies suggested that the repression of miR-200b-3p by RNA interference inhibited the currents of TRPC6 and BKCa channels. The levels of mRNA and proteins of TRPC6 and BKCa channels were also reduced. Moreover, the concentration of [Ca2+]i was significantly reduced in miR-200b-3p inhibition group. Under the condition of miR-200b-3p inhibition, the decrease of BKCa currents could be regulated by TRPC6 channels. This study showed that miR-200b-3p/TRPC6/BKCa signal may be a potential mechanism to regulate the physiological processes of podocytes. It is widely reported that miRNAs play crucial roles in biological and pathological processes of kidney and podocytes [18]. Moreover, there are important

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enzymes for the production of mature miRNAs [19]. The podocyte specific Dicer knockout mice as well as the inducible podocyte specific Drosha knockout mice resulted in foot process effacement of podocyte as well as loss of some podocyte specific marker proteins [6, 7]. In human hypertensive nephrosclerosis biopsies, the global miRNA profiling revealed a number of differentially expression of miR-200 family, including miR-200a, miR-200b, miR-141, miR-429, and miR-192 [20]. Our recent studies also suggested that miRNA-mRNA regulatory networks during podocyte differentiation [21], and miR-200 family could promote differentiation of podocytes via targeting RSAD2 [8]. TRPC6, a Ca2+-permeable channel, is located on podocyte membrane, where it is integrated into a signaling complex that interacts with nephrin, podocin [22], and some other proteins that are critical for podocyte function [23, 24]. It is well known that a mutation in TRPC6 is associated with the onset of the familial forms of focal segmental glomerulosclerosis (FSGS) [25, 26]. In the present study, we found that miR-200b-3p inhibition reduced the currents of TRPC6 recorded by whole-cell patch-clamp technique (Fig. 2A and 2B). Moreover, the levels of protein and mRNA of TRPC6 were also declined in the miR-200b-3p inhibition group (Fig. 1B and 1D). The role of intracellular calcium is critical for the physiological function of podocytes [27, 28]. Excessive calcium influx in podocytes may result in the effacement of foot processes, apoptosis, and subsequent glomeruli damage [29-31]. TRPC6 channels, acted as a Ca2+-permeable ion channels, could mediate enhanced calcium influx in podocytes [32]. Angiotensin II (Ang II) could induce a calcium-dependent pathway of programmed podocyte death[33]. Moreover, TRPC6 channels are associated with Ca2+-induced calcium influx in many renal cell types [34, 35]. In this study, the results showed that the concentration of intracellular calcium was decreased in miR-200b-3p inhibition group (Fig. 4A). The decrease of [Ca2+]i may result from the inhibition of TRPC6 currents caused by the downregulation of miR-200b-3p. Therefore, miR-200b-3p may be a promising biomarker for the TRPC6/Ca2+-dependent podocyte injury. BKCa channels, encoded by the Slo1 gene, play an important role in regulating the physiological membrane potential [36]. Moreover, many evidences suggested that some hormones, such as Ang II and insulin, as well as pathological environment could regulate the expression and function of BKCa and play important roles in podocyte injury [37, 38]. It is reported that Ang II could induce the increase of oxidative stress and podocyte death and inhibit the currents of BKCa in podocytes with facilitating the BKCa activation [38]. The exposure of podocytes to hypoxia environment caused a significant reduction in BKCa currents and shifted the activation curves toward more depolarized potential and slowed the activation kinetics [39]. In our present study, the results showed that the inhibition of miR-200b-3p could induce the reduction of BKCa currents in podocytes (Fig. 3B). Moreover, the activation curves of BKCa also shifted toward depolarized potential (Fig. 3C). From a number of researches, we can see that the expression and function of BKCa play a vital role in physiological processes of podocytes. From our study, it is suggested that miR-200b-3p may be a promising target to regulate the expression and function of BKCa in podocytes.

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As we known, the activation of BKCa channels at depolarized membrane potentials usually requires micromolar concentrations of free Ca2+ or even higher [15]. Therefore, the functional coupling of BKCa channels with Ca2+-permeable channels usually requires very close colocalization [40, 41]. There is literature on excitable cells suggesting that BKCa channels bind directly to voltage-gated Ca2+ channels, which ensures the necessary co-localization for functional coupling of their gating. However, podocytes are not excitable cells and do not express voltage-gated Ca2+ channels. Instead, it was reported that the subunits of BKCa channels could bind to TRPC6 channels in podocytes [41]. These interactions may allow Ca2+ influx through TRPC6 channels to cause a coordinate activation of BKCa channels. The co-expression of TRPC6 with certain Slo1 splice variants increased the surface expression and currents of BKCa channels, whereas siRNA knockdown of TRPC6 reduced the surface expression of endogenous BKCa channels in podocytes. In our study, it was suggested that the decrease of BKCa currents induced by miR-200b-3p inhibition could be regulated by TRPC6 channels (Fig. 6B and 6C). Moreover, the inhibition of TRPC6 by La3+ also results in the reduction of BKCa currents (Fig. 7C and 7D). The protein levels of BKCa were also reduced in podocytes incubated in La3+ for 24h (Fig. 7B). In our present study, it was suggested that the inhibition of miR-200b-3p reduced the currents of TRPC6 channels and the inhibition of BKCa currents possibly resulted from the decline of Ca2+ influx through TRPC6 channels in podocytes. From our study, we can see miR-200b-3p/TRPC6/BKCa signaling may provide a promising molecular mechanism in the biological and pathological processes of podocytes. However, it’s a pity that the gene, which miR-200b-3p targets to regulate the expression and function of TRPC6 and BKCa channels, is not determined in the study.

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5 Conclusion In summary, the study showed that miR-200b-3p inhibition could reduce the currents of TRPC6 and BKCa channels of podocytes. Moreover, the mRNA and protein levels of the two channels were also downregulated under the condition of miR-200b-3p inhibition. The [Ca2+]i was also declined in the miR-200b-3p inhibition group. Therefore, the inhibition of BKCa channels may result from the decrease of [Ca2+]i influx through TRPC6 channels. miR-200b-3p/TRPC6/BKCa signaling may be a promising molecular mechanism to regulate the biological and pathological processes of podocytes. 6 Conflicts of interest All the authors declared no conflicts of interest. 7 Acknowledgements This study was supported by the National Natural Science Foundation of China (81771979, 81571804). 8 References

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ACCEPTED MANUSCRIPT Figure Captions Figure 1 miR-200b-3p inhibition downregulated expression and mRNA levels of TRPC6 and BKCa channels in podocytes. (A) The expression of miR-200b-3p was examined after transfection of miR-200b-3p inhibitors for 24h. (B) The relative mRNA expression of TRPC6 was examined after transfection of miR-200b-3p inhibitors for 24h. (C) The relative mRNA expression of BKCa was examined after transfection of miR-200b-3p inhibitors for 24h. (D) The representative immunoreactive bands of BK-α, TRPC6, β-actin of Con, miR-200b-3p inhibition and NC

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groups. (E) Quantitative analysis of TRPC6 was examined after transfection of miR-200b-3p inhibitors for 24h (n=3). (F) Quantitative analysis of BK-α was examined after transfection of miR-200b-3p inhibitors for 24h(n=3). The data are presented as the mean±S.E.M., *p<0.05, **p<0.01.

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Figure 2 miR-200b-3p inhibition reduces the currents of TRPC6 channels in podocytes. (A) The representative traces of TRPC6 currents were evoked by a ramp voltage commands (-100 mV to +100 mV over 800ms) at a holding potential of -60 mV. (B) The I-V curves of TRPC6 currents in different groups. (C-D) The representative traces of TRPC6 currents were induced (-100 mV to +100 mV over 800ms) at a holding potential of -60 mV and

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the amplitudes of TRPC6 in miR-200b-3p inhibition group and miR-200b-3p inhibition +OAG group. (E-F) The representative traces of TRPC6 currents were induced (-100 mV to +100 mV over 800ms) at a holding potential of -60 mV and the amplitudes of TRPC6 in miR-200b-3p inhibition group and miR-200b-3p inhibition +La3+ group. (n=15 cells per group). The data are presented as the mean±S.E.M., **p<0.01.

Figure 3 miR-200b-3p inhibition inhibited the currents of BKCa channels in podocytes. (A) The examples of BKCa

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currents were evoked at a holding potential of -60 mV and the inhibition of evoked currents by application of the BKCa inhibitor paxilline. (B) The I-V curves of BKCa currents in different groups. (C) The activation curves of BKCa currents in different groups. (n=15 cells per group) The data are presented as the mean±S.E.M., *p<0.05,

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**p<0.01.

Figure 4 miR-200b-3p inhibition inhibited the intracellular concentration of Ca2+ in podocytes. (A) The images of intracellular Ca2+ using Fluo 3-AM staining were represented in different groups. (B) The relative fluorescent

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intensity of intracellular Ca2+ in podocytes (n=6 cells per group). The data are presented as the mean±S.E.M., **p<0.01.

Figure 5 The decrease of BKCa currents in podocytes with miR-200b-3p inhibition was regulated by TRPC6 channels. (A) The representative graph of BKCa currents after application of OAG and La3+ in podocytes with miR-200b-3p inhibition and NC. (B-E) The I-V curves of BKCa currents after application of OAG and La3+ respectively in podocytes with miR-200b-3p inhibition and NC. (n=15 cells per group). The data are presented as the mean±S.E.M., *p<0.05, **p<0.01.

Figure 6 The kinetics of BKCa channels after application of OAG and La3+ in podocytes with miR-200b-3p inhibition and NC. (A-D) The activation curves of BKCa currents after application of OAG and La3+ in

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Figure 7 The blockage of TRPC6 channels inhibited BKCa channels in podocytes. (A) The relative protein expression of TRPC6 channels was examined after application of La3+ for 24h (n=3). (B) The relative protein expression of BKCa was examined after treatment with La3+ for 24h (n=3). (C) The I-V curves of BKCa currents in control group and La3+ group (n=15 cells per group). (D) The activation curves of BKCa currents in control group

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and La3+ group (n=15 cells per group). The data are presented as the mean±S.E.M., *p<0.05, **p<0.01.

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Highlights 1. The miR-200b-3p inhibition reduced the currents of TRPC6 and BKCa channels. 2. miR-200b-3p inhibition downregulated mRNA and protein levels of TRPC6 and BKCa. 3. The miR-200b-3p inhibition declined the concentration of intracellular Ca2+. 4. miR-200b-3p inhibition decreased BKCa currents via TRPC6 channels in podocytes. 5. TRPC6 blockage inhibited the currents and expression levels of BKCa channels.