International Journal of Molecular Sciences

Article Rearrangements Modulate TRPC6 Channel Activity in Podocytes

Alexey Shalygin 1,†, Leonid S. Shuyskiy 1,†, Ruslan Bohovyk 2,†, Oleg Palygin 2, Alexander Staruschenko 2,3,* and Elena Kaznacheyeva 1,*

1 Institute of Cytology, Russian Academy of Sciences, 194064 Saint-Petersburg, Russia; [email protected] (A.S.); [email protected] (L.S.S.) 2 Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA; [email protected] (R.B.); [email protected] (O.P.) 3 Clement J. Zablocki VA Medical Center, Milwaukee, WI 53295, USA * Correspondence: [email protected] (A.S.); [email protected] (E.K.) † These authors contributed equally to this work.

Abstract: The cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canon- ical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migra- tion properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D  increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the  channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported

Citation: Shalygin, A.; Shuyskiy, L.S.; that in ACTN4, which encodes α--4, causes focal segmental glomerulosclerosis and Bohovyk, R.; Palygin, O.; solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the Staruschenko, A.; Kaznacheyeva, E. activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in Cytoskeleton Rearrangements CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction Modulate TRPC6 Channel Activity in between the structure of the actin cytoskeleton and TRPC6 activity. Podocytes. Int. J. Mol. Sci. 2021, 22, 4396. https://doi.org/10.3390/ Keywords: podocyte; focal segmental glomerulosclerosis; FSGS; TRPC6; actin cytoskeleton; α- ijms22094396 actinin-4

Academic Editor: Malgorzata Kloc

Received: 5 April 2021 1. Introduction Accepted: 20 April 2021 Published: 22 April 2021 Podocytes are terminally differentiated epithelial cells whose foot processes wrap the capillaries and form a barrier, which filters the blood in the glomeruli to form primary

Publisher’s Note: MDPI stays neutral urine [1]. Podocyte function depends on the actin cytoskeleton and is regulated by multiple with regard to jurisdictional claims in and signaling pathways [2]. For many years, angiotensin-converting enzyme published maps and institutional affil- inhibitors and angiotensin receptor blockers have been used as the first line of standard iations. nephropathy treatment. A study of the mechanisms of their anti-proteinuric, podocyte- specific effects has demonstrated that calcium-mediated signaling connects angiotensin receptor type 1 with the rearrangement of the cytoskeleton in podocytes [3]. Angiotensin II (Ang II) also plays a critical role in TRPC6-mediated calcium signaling in podocytes [4,5]. TRPC6 (Transient Receptor Potential Canonical 6) calcium channels are essential compo- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. nents of the podocyte slit diaphragm, where they are integrated into a signaling complex α This article is an open access article that interacts with nephrin, podocin, -actinin-4, calcineurin, and other proteins [6,7]. distributed under the terms and Several independent laboratories have identified TRPC6 associated with the conditions of the Creative Commons autosomal dominant form of focal segmental glomerulosclerosis (FSGS) [8–10] and the Attribution (CC BY) license (https:// critical importance of TRPC6 in the maintenance of glomerular filtration. In addition to creativecommons.org/licenses/by/ gain- or loss-of-function mutations that cause FSGS, changes in channel expression may 4.0/). also contribute to the disease [11].

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Dysregulation of the actin cytoskeleton leads to foot process retraction, podocyte dam- age, and proteinuria [12]. TRPC6 activation induces cytoskeleton rearrangements through calcium-dependent signaling in podocytes [13]. Several TRPC6 mutations associated with FSGS result in decreased calmodulin binding and calcium-dependent inactivation of the channels. Increased TRPC6 channel activity changes actin organization from parallel stress fibers to disorganized F-actin structures [14]. TRPC6 channel activation by Ang II also induces cytoskeleton reassembly, decreases the number of actin stress fibers, and promotes a contractile phenotype [15]. Furthermore, it has been shown that TRPC6 inhibition induces cytoskeletal rearrangements of conditionally immortalized mouse podocytes during the differentiation stage [16]. The association between TRPC6 and podocyte actin cytoskeleton rearrangement in diabetic kidney disease has also recently been discussed in a review by Wang et al. [13]. For instance, it was reported that talin1, a focal adhesion molecule, and calpain 1 and 2 are important for maintaining podocyte cytoskeletal stability [17,18] and the TRPC6-mediated Ca2+-dependent calcineurin signal transduction pathway [19]. TRPC6 also binds to and activates the actin regulatory proteins caldesmon and calpain 1 and 2, proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin and [20]. α-actinin-4, encoded by the ACTN4 , cross-links filamentous actin into thick bun- dles with defined spacing and is also regulated by calcium signaling. The binding efficacy of α-actinin-4 to actin is modified by the calmodulin-like domain of α-actinin-4 [21]. More- over, α-actinin-4 interaction with the cytoskeleton depends on phosphorylation [22,23], which is diminished in cells with the FSGS-causing mutation ACTN4 K255E [24]. ACTN4 K255E is a gain-of-function mutation which increases actin binding and eliminates sen- sitivity to tension [25]. Binding of mutant α-actinin-4 enhances cytoskeleton stiffness, contractile force, and recovery time after stretch [26,27], and decreases cell spreading [28]. Many cases of FSGS are caused by disruption of TRPC6 calcium entry or the cytoskeleton’s rearrangements [29]. Since mutations in TRPC6 are known to cause cytoskeleton rear- rangement, we propose there is a relationship between cytoskeleton dynamics and TRPC6 channel activity. The goal of this study was to study how cytoskeleton reorganization influences TRPC6 channel activity.

2. Results 2.1. Disruption of the Actin Cytoskeleton with Cytochalasin D Affects Filopodial Extension and To test the remodeling effects of the actin cytoskeleton on podocyte morphology, we employed a novel scanning ion-conductance microscopy (SICM) approach. SICM generates electron microscopy resolution images of live cells in physiologically relevant solutions over time. Experiments using SICM demonstrate that disrupting the actin cytoskeleton integrity by application of cytochalasin D prevents changes in podocyte foot process and migration area. Cytochalasin D is a cell-permeable fungal toxin that binds to the barbed end of actin filament, inhibiting both the association and dissociation of actin subunits. Actin dynamics allow cells to change shape in response to specific stimuli. The actin cytoskeleton form highly organized dynamic structures, such as filopodia and lamellipodia. Lamellipodia (red arrows, Figure1a) and filopodia (yellow arrows, Figure1a) are actin-based protrusions particularly relevant to cell motility. Figure1a shows a detailed 3D topographical image of a podocyte foot process scanned in real-time to observe the changes during the physiological migration process and under treatment with cytochalasin D. To estimate the activity of cell edge migration, we calculated the scanned area at different time points during normal physiological conditions and after the application of cytochalasin D (Figure1). A stable increase in the cell area was calculated in the scanned cell fragment (Figure1a,c (images 1 and 2); time frame from −70 to 0 min). The application of cytochalasin D resulted in the retraction of the podocyte migratory cell edge, thereby decreasing the calculated area of the cell edge (Figure1a,c (images 3 and 4); time frame from 0 to +120 min). A full cell scan (Figure1b) requires a lack of sample movement for its long duration (~40 min), therefore Int. J. Mol. Sci. 2021, 22, 4396 3 of 14

scanned cell fragment (Figure 1a,c (images 1 and 2); time frame from −70 to 0 min). The application of cytochalasin D resulted in the retraction of the podocyte migratory cell Int. J. Mol. Sci. 2021, 22, 4396 3 of 12 edge, thereby decreasing the calculated area of the cell edge (Figure 1a,c (images 3 and 4); time frame from 0 to +120 min). A full cell scan (Figure 1b) requires a lack of sample movement for its long duration (~40 min), therefore the full image was made after the disruptionthe full image of the was actin made cytoskeleton after the disruption and prev ofention the actin of cell cytoskeleton migration andby cytochalasin prevention ofD treatment.cell migration by cytochalasin D treatment.

Figure 1. Scanning Ion Conductance Microscopy (SICM) analysis analysis of of human human podocyte podocyte morphology morphology in response response to to cytoch cytochalasinalasin D D treatment. treatment. (A (A) Representative) Representative scans scans of ofpodocyte podocyte lamellipodia lamellipodia (red (red arrows) and filopodia filopodia (yellow (yellow arrows) arrows) at at different different time time points points (60 (60 and and 20 20 min min before before (1–2) (1–2) and and 20 20 and 120 min afterafter (3–4)(3–4) cytochalasincytochalasin D D (10 (10µ µm)m) application; application; scan scan area area 30 30µ MµM× ×30 30µ µM).M). (B (B) Expanded) Ex- panded topographical map showing the complete podocyte from a after the application of cyto- topographical map showing the complete podocyte from a after the application of cytochalasin D chalasin D (scan area 60 µM × 60 µM). (C) Timeline of changes to the podocyte foot process area (scan area 60 µM × 60 µM). (C) Timeline of changes to the podocyte foot process area during normal during normal migration and after cytochalasin D application. Numbers 1–4 represent time points A shownmigration in a and. Asafter seencytochalasin in this summary, D application. cytochalasin Numbers D completely 1–4 represent stopped time the points migration shown of in the ( po-). As docyteseen in filopodium. this summary, cytochalasin D completely stopped the migration of the podocyte filopodium. 2.2. Acute Disruption of Actin Microfilaments with Cytochalasin D Markedly Increases TRPC6 Activity2.2. Acute (NP Disruptiono) in CHO of Cells Actin and Rat Podocytes with Cytochalasin D Markedly Increases TRPC6 Activity (NPo) in CHO Cells and Rat Podocytes To study the regulation of TRPC6 by disruption of actin cytoskeleton dynamics, we testedTo study the effectsthe regulation of cytochalasin of TRPC6 D by on disrup TRPC6tion channel of actin activity. cytoskeleton Several dynamics, members we of testedthe TRPC the effects family, of including cytochalasin TRPC3, D on TRPC5, TRPC6 and channel TRPC6, activity. have Several been implicated members inof thethe pathogenesisTRPC family, ofincluding proteinuric TRPC3, kidney TRPC5, diseases and TRPC6, [30–32]. have Therefore, been implicated to test the in effect the patho- of the genesisactin cytoskeleton of proteinuric on TRPC6 kidney channels, diseases we[30–32]. heterologously Therefore, overexpressed to test the effect TRPC6 of the in CHOactin (Chinesecytoskeleton Hamster on TRPC6 Ovary) channels, cells. The we application heterologously of 10 ofoverexpressedµM cytochalasin TRPC6 D toina CHO bath (Chinesesolution ofHamster cell-attached Ovary) patchescells. The induced application activation of 10 of singleµM cytochalasin channels (Figure D to a2 batha) with solu- a tioncurrent-voltage of cell-attached relationship patches typicalinduced for activation TRPC6 (Figure of single2b). Applicationchannels (Figure of cytochalasin 2a) with a current-voltageD did not affect relationship conductance typical of the for recorded TRPC6 channels(Figure 2b). (18 Application± 1 pS (n = of 3) cytochalasin and 20 ± 1 pS D (didn = not 5) beforeaffect conductance and after cytochalasin of the recorded D application, channels (18 respectively). ± 1 pS (n = 3) The and positive 20 ± 1 pS reversal (n = 5) potentialbefore and of after the current-voltage cytochalasin D relationshipapplication, reflectsrespectively). the plasma The membranepositive reversal potential potential of the ofcell. the After current-voltage excision to inside-out relationship configuration, reflects the the plasma reversal membrane potential potential was shifted of tothe 0 cell. mV, Afterand the excision conductance to inside-out increased configuration, to 23 ± 1 pSthe (reversaln = 6). Control potential cells was transfected shifted to 0 with mV, GFP and thealone conductance lacked TRPC6 increased activity to before23 ± 1 andpS (n after = 6). cytochalasin Control cells D transfected application. with Basal GFP activity alone (NPlackedo) of TRPC6 overexpressed activity before TRPC6 and channels after cytochalasin was increased D afterapplication. the application Basal activity of cytochalasin (NPo) of overexpressedD (Figure2c). In TRPC6 experiments channels with was no basal increased activity, after 10 µtheM ofapplication cytochalasin of Dcytochalasin also activated D TRPC6(Figure channels2c). In experiments but with less with activity, no basal which activity, most 10 likely µM of represents cytochalasin a smaller D also number activated of TRPC6active channels. channels Onbut average, with less NP activity,o increased which from most 0.45 likely± 0.23 represents to 1.69 ± a 0.66smaller (Figure number2c). of activeIn channels. our next experiments,On average, NP weo tested increased the effect from of0.45 inhibiting ± 0.23 to actin 1.69 cytoskeleton± 0.66 (Figure dynamics 2c). on endogenous TRPC channels. We performed cell-attached patch-clamp experiments on podocytes from freshly isolated, decapsulated glomeruli. In cell-attached experiments, the addition of 10 µM of cytochalasin D to the bath solution induced activation of currents (Figure3a) with current-voltage relationships and conductance of 23 ± 1 pS, typical for TRPC6 channels (Figure3b). The increase in calcium entry is caused by the activation of silent TRPC6 channels and is not associated with changes in their conductivity or other biophysical properties (Figure3). The NP o of silent channels increased from 0.00 to 0.87 ± 0.44 (n = 8; Figure3c). In contrast to experiments with CHO cells, basal TRPC6 activity was not significantly increased after cytochalasin D application (1.46 ± 0.25 vs. 1.52 ± 0.34, n = 7, Figure3d).

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FigureFigure 2. Effect 2. Effect of cytochalasin of cytochalasin D on TRPC6 D (Transient on TRPC6 Receptor (Transient Potential Receptor CanonicalPotential 6) channels Canonical 6) channels overexpressed in CHO (Chinese Hamster Ovary) cells. (A) Representative trace of TRPC6 channel activityoverexpressed before and after in CHO application (Chinese of 10 µM Hamster cytochalasin Ovary) D at cells.a holding (A potential) Representative of –40 mV. Ex- trace of TRPC6 channel pandedactivity fragments before are and shown after below. application Closed (c) and of open 10 µ (o)M states cytochalasin are indicated. D ( atB) Cur- a holding potential of –40 mV. rent-voltageExpanded relationship fragments of overexpressed are shown below.TRPC6 with Closed estimated (c) andconductance open (o)20 ± states 1 pS in are indicated. (B) Current- cell-attached mode (black squares) and subsequent excision to inside-out mode (grey squares). (C) Summaryvoltage graph relationship demonstrating of overexpressed the effect of the TRPC6application with of 10 estimated µM cytochalasin conductance D on TRPC6 20 ± 1 pS in cell-attached channelmode activity (black (NP squares)o). Wilcoxon and nonparametric subsequent test excision for paired to samples; inside-out * = p < mode0.05. (grey squares). (C) Summary graph demonstrating the effect of the application of 10 µM cytochalasin D on TRPC6 channel activity Int. J. Mol. Sci. 2021, 22, 4396 In our next experiments, we tested the effect of inhibiting actin cytoskeleton5 ofdy- 14

namics(NPo). on Wilcoxon endogenous nonparametric TRPC channels. test forWe pairedperformed samples; cell-attached * = p < 0.05.patch-clamp ex- periments on podocytes from freshly isolated, decapsulated glomeruli. In cell-attached experiments, the addition of 10 µM of cytochalasin D to the bath solution induced acti- vation of currents (Figure 3a) with current-voltage relationships and conductance of 23 ± 1 pS, typical for TRPC6 channels (Figure 3b). The increase in calcium entry is caused by the activation of silent TRPC6 channels and is not associated with changes in their con- ductivity or other biophysical properties (Figure 3). The NPo of silent channels increased from 0.00 to 0.87 ± 0.44 (n = 8; Figure 3c). In contrast to experiments with CHO cells, basal TRPC6 activity was not significantly increased after cytochalasin D application (1.46 ± 0.25 vs 1.52 ± 0.34, n = 7, Figure 3d).

FigureFigure 3. Application 3. Application of cytochalasin of cytochalasin D to podocytes D to podocytesfrom freshly fromisolated freshly glomeruli isolated induces glomeruli induces TRPC6 TRPC6 channel activation. (A) Representative example of basal channel activity before and after 10 µMchannel cytochalasin activation. D application (A) Representativeat –40 mV holding potential. example Expanded of basal fragments channel are activity shown below. before and after 10 µM Opencytochalasin and closed D channel application states are at indi –40cated mV with holding (o) and potential. (c), respectively. Expanded (B) Current-voltage fragments are shown below. Open relationship of recorded channels. (C) Summary graph of the endogenous silent TRPC6 activity inducedand closed by 10 channelµM cytochalasin states D are in podocytes. indicated (D with) Summary (o) and graph (c), demonstrating respectively. the (B effect) Current-voltage of the relationship applicationof recorded of 10 channels. µM cytochalasin (C) D Summary on constituti graphve endogenous of the endogenousTRPC6-like channel silent activity TRPC6 (NPo) activity induced by in10 freshlyµM cytochalasin isolated glomeruli. D in Wilcoxon podocytes. nonparametric (D) Summary test for paired graph samples; demonstrating * = p < 0.05. the effect of the application of 10Interestingly,µM cytochalasin in addition D on to constitutive TRPC6-like channels endogenous (n = 15), TRPC6-like we observed channel activity activity of (NPo) in freshly anotherisolated endogenous glomeruli. channel Wilcoxon (Figure nonparametric 4a) with 7.0 ± test 0.1 pS for conductance paired samples; (n = 6, *Figure = p < 4b); 0.05. in 3 experiments, both types of channels were observed, while in 8 attempts there was no channel activity. Cytochalasin D increased the activity of 7 pS channels with NPo aug- mented from 0.61 ± 0.35 to 1.33 ± 0.66 (Figure 4c). Additional studies are required to de- termine the identity of this channel.

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Interestingly, in addition to TRPC6-like channels (n = 15), we observed activity of another endogenous channel (Figure4a) with 7.0 ± 0.1 pS conductance (n = 6, Figure4b ); in 3 experiments, both types of channels were observed, while in 8 attempts there was no channel activity. Cytochalasin D increased the activity of 7 pS channels with NPo Int. J. Mol. Sci. 2021, 22, 4396 augmented from 0.61 ± 0.35 to 1.33 ± 0.66 (Figure4c). Additional studies are6 of required 14 to

determine the identity of this channel.

Figure 4. 4. ApplicationApplication of ofcytochalasin cytochalasin D to D podocytes to podocytes induces induces activation activation of a native of a channel native channel with 7 with 7 pS pSconductance. conductance. ( A(A)) Representative Representative trace trace of of basal basal channel channel activity activity before before and after and after10 µM 10 cyto-µM cytochalasin chalasin D at a holding potential of –40 mV. Expanded fragments are shown below. Closed (c) and D at a holding potential of –40 mV. Expanded fragments are shown below. Closed (c) and open open (oi) states are indicated. (B) Current-voltage relationship of channels with estimated con- ductance(oi) states 7.0 are ± 0.2 indicated. pS. (C) Summary (B) Current-voltage graph of the effect relationship of 10 µM cytochalasin of channels D with on 7 estimatedpS channel conductance activity7.0 ± 0.2 (NP pSo)..( WilcoxonC) Summary nonparametric graph of thetest effectfor paired of 10 samples;µM cytochalasin * = p < 0.05 Dlevel. on 7 pS channel activity (NPo). Wilcoxon nonparametric test for paired samples; * = p < 0.05 level. 2.3. ACTN4 K255E Reduces TRPC6 Activity (NPo) and Conductance 2.3. ACTN4Since the K255E actin-binding Reduces TRPC6 α-actinin-4 Activity(NP playso) andan essential Conductance role in podocyte func- tion andSince mutations the actin-binding in the ACTN4 protein gene,α which-actinin-4 encodes plays this an protein, essential are role associated in podocyte with function FSGS,and mutations we interrogated in the whetherACTN4 gene,α-actinin-4 which can encodes modulate this TRPC6 protein, channel are associated activity. The with FSGS, FSGS-causingwe interrogated ACTN4 whether mutationα-actinin-4 K255E canincreases modulate podocyte TRPC6 cytoskeleton channel activity. stiffness The FSGS- [26,27,33];causing ACTN4 thus, we mutation hypothesized K255E that increases it could podocyte affect TRPC6 cytoskeleton activity. stiffnessTo test this, [26 ,27we, 33]; thus, comparedwe hypothesized the activity that of itTRPC6 could channels affect TRPC6 in CHO activity. cells co-transfected To test this, with we wild-type compared (wt) the activity ACTN4 or the K255E mutant. Moderate basal activity of TRPC6 channels was observed of TRPC6 channels in CHO cells co-transfected with wild-type (wt) ACTN4 or the K255E using cell-attached experiments in CHO cells co-transfected with TRPC6 and wild type mutant. Moderate basal activity of TRPC6 channels was observed using cell-attached exper- ACTN4 (TRPC6/ACTN4), whereas no basal activity was observed in CHO cells with TRPC6iments and in CHO ACTN4 cells K255E co-transfected (TRPC6/ACTN4 with K255E) TRPC6 (Figure and wild 5). To type further ACTN4 explore (TRPC6/ACTN4), the ef- fectswhereas of α-actinin-4 no basal on activity TRPC6 activity, was observed we applied in CHOOAG, cellsa membrane-permeant with TRPC6 and analog ACTN4 of K255E diacylglycerol,(TRPC6/ACTN4 which K255E) is an (Figureagonist 5of). TRPC To further channels. explore In cell-attached the effects ofexperiments,α-actinin-4 the on TRPC6 additionactivity, weof 100 applied µM of OAG, OAG ainto membrane-permeant the bath solution induced analog TRPC6 of diacylglycerol, channel activity which in is an ago- bothnist ofTRPC6/ACTN4 TRPC channels. and In TRPC6/ACTN4 cell-attached experiments,K255E cells (Figure the addition 5). Current-voltage of 100 µM of OAGrela- into the tionshipsbath solution demonstrated induced TRPC6that the channelconductance activity of TRPC6 in both channels TRPC6/ACTN4 in TRPC6/ACTN4 and TRPC6/ACTN4 cells wasK255E 19 cells± 1 pS, (Figure similar5). to Current-voltage the properties of relationships TRPC6 channels demonstrated in podocytes that or theCHO conductance cells of transfectedTRPC6 channels with inTRPC6 TRPC6/ACTN4 only (Figure cells was5b). 19Interestingly,± 1 pS, similar TRPC6 to the channels properties in of TRPC6 TRPC6/ACTN4channels in podocytes K255E cells or CHOexhibited cells a transfectedsignificantly withlower TRPC6 conductance only (Figure of around5b). 12 Interestingly, ± 2 pS [34]. Estimation of OAG-induced TRPC6 activity in TRPC6/ACTN4 cells showed that TRPC6 channels in TRPC6/ACTN4 K255E cells exhibited a significantly lower conductance NPo increased from 0.09 ± 0.05 to 1.24 ± 0.23 (n = 7), comparable with OAG-induced of around 12 ± 2 pS [34]. Estimation of OAG-induced TRPC6 activity in TRPC6/ACTN4 TRPC6 activity CHO cells transfected with TRPC6 alone (from 0.05 ± 0.04 to 1.40 ± 0.33 (n ± ± =cells 8)). showedIn TRPC6/ACTN4 that NPo K255Eincreased cells, from channel 0.09 activity0.05 upon to 1.24 OAG0.23 stimulation (n = 7), increased comparable with fromOAG-induced 0.00 to 0.65 TRPC6 ± 0.12 activity (n = 7). CHOTherefore, cells transfectedthe ACTN4 withK255E TRPC6 mutation alone reduces (from 0.05both ± 0.04 to TRPC6 channel conductance and current activity.

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1.40 ± 0.33 (n = 8)). In TRPC6/ACTN4 K255E cells, channel activity upon OAG stimulation Int. J. Mol. Sci. 2021, 22, 4396 increased from 0.00 to 0.65 ± 0.12 (n = 7). Therefore, the ACTN4 K255E mutation7 of 14 reduces both TRPC6 channel conductance and current activity.

FigureFigure 5. 5. ActivitiesActivities of ofTRPC6 TRPC6 channels channels in response in response to OAG to in OAG CHO in cells CHO co-transfected cells co-transfected with wild with wild type ACTN4 (α-actinin-4) or ACTN4 K255E. (A) Typical trace of OAG-induced TRPC6 activity in type ACTN4 (α-actinin-4) or ACTN4 K255E. (A) Typical trace of OAG-induced TRPC6 activity in CHO cells co-transfected with ACTN4; (c) and (oi) denote closed and open states of the channel, CHOrespectively; cells co-transfected a full recording with(upper ACTN4; row) and (c) fragme and (ontsi) of denote a recording closed at a and larger open scale states are shown. of the channel, respectively;The recording awas full obtained recording at holding (upper potential row) and –60 fragments mV. (B) Summarized of a recording current-voltage at a larger rela- scale are shown. Thetionship recording curve of was TRPC6 obtained currents at holding of CHOpotential cells co-transfected –60 mV. ( Bwith) Summarized ACTN4 wild-type current-voltage (triangle) relationship with estimated conductance 19±1 pS and ACTN4 K255E mutant (square) with estimated conduct- curve of TRPC6 currents of CHO cells co-transfected with ACTN4 wild-type (triangle) with estimated ance 12 ± 2 pS. (C) Summary graphs of the NPo of the TRPC6 channels recorded in CHO cells conductanceco-transfected 19with± TRPC61 pS and and ACTN4 ACTN4 K255Eor ACTN mutant4 K255E (square) before and with after estimated OAG (100 conductance µM) stimula- 12 ± 2 pS. (tion;C) Summary * denotes statistical graphs of significance the NPo of (pthe < 0.05). TRPC6 channels recorded in CHO cells co-transfected with TRPC6 and ACTN4 or ACTN4 K255E before and after OAG (100 µM) stimulation; * denotes statistical significanceTo confirm (p < 0.05).that actin cytoskeleton rearrangements by ACTN4 K255E overexpression did in fact lead to the observed reduction of TRPC6 single-channel activity, we analyzed actin Tocytoskeleton confirm that arrangement actin cytoskeleton using confoc rearrangementsal scanning microsco by ACTN4py. K255ERepresentative overexpression didimages infact of actin lead cytoskeleton to the observed staining reduction by rhodamine-phalloidine of TRPC6 single-channel in CHO cells activity, transfected we analyzed actinwith ACTN4 cytoskeleton wt and arrangement ACTN4 K255E using mutant confocal are pr scanningesented in microscopy.Figure 6a,b. The Representative structure of images the actin cytoskeleton in cells transfected with ACTN4 K255E mutant was altered com- of actin cytoskeleton staining by rhodamine-phalloidine in CHO cells transfected with pared to controls transfected with wild-type ACTN4. ACTN4 K255E expression resulted ACTN4 wt and ACTN4 K255E mutant are presented in Figure6a,b. The structure of the in enhanced actin bundle thickness (Figure 6b panels a, d, e) and abnormal actin cyto- actinskeleton cytoskeleton distribution in (Figure cells transfected6b panels a an withd b). ACTN4 These results K255E are mutant consistent was with altered previ- compared toously controls published transfected data showing with that wild-type the K255E ACTN4. mutation ACTN4 in ACTN4 K255E alters expression the actin cyto- resulted in enhancedskeleton structure actin bundle and impacts thickness different (Figure cell6 processes,b panels a, including d, e) and migration, abnormal contractility, actin cytoskeleton distributionand tensile response (Figure [24,27,33].6b panels a and b). These results are consistent with previously published data showing that the K255E mutation in ACTN4 alters the actin cytoskeleton structure and impacts different cell processes, including migration, contractility, and tensile response [24,27,33].

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Figure 6. Effect of ACTN4 K255E mutant on actin cytoskeleton arrangement. CHO cells were Figuretransiently 6. Effect transfected of ACTN4 K255E with plasmids mutant on encoding actin cytoskeleton wild type ( Aarrangement.) or mutant K255ECHO cells ACTN4 were ( B). Scale transiently transfected with plasmids encoding wild type (A) or mutant K255E ACTN4 (B). Scale bar is 50 µm. a—rhodamine-phalloidine emission (red); b—histogram of relative fluorescence across bar is 50 µm. a—rhodamine-phalloidine emission (red); b—histogram of relative fluorescence a region of interest (yellow line); c—merged image of rhodamine-phalloidine (red), GFP-labeled across a region of interest (yellow line); с—merged image of rhodamine-phalloidine (red), GFP-labeledα-actinin-4 α-actinin-4 (green) and (green) Hoechst-33342 and Hoechst-33342 (nuclei, blue) (nuclei, emissions. blue) emissions. Panels (d) Panels and (e) (d) are and zoomed (e) are areas zoomedfrom areas panel from (c) (marked panel (c) by (marked white square), by white for square), rhodamine-phalloidine for rhodamine-phalloidine and GFP-labeled and α-actinin-4, GFP-labeledrespectively. α-actinin-4, respectively. 3. Discussion 3. Discussion Podocytes are exposed to repetitive stretch and shear stress, and their filtration func- Podocytes are exposed to repetitive stretch and shear stress, and their filtration tion requires the actin cytoskeleton [35]. It was previously shown that actin cytoskeleton function requires the actin cytoskeleton [35]. It was previously shown that actin cyto- rearrangements are induced by activation of small GTPases RhoA and Rac1, which modu- skeleton rearrangements are induced by activation of small GTPases RhoA and Rac1, late calcium signaling after TRPC channel activation [36]. It is also well established that which modulate calcium signaling after TRPC channel activation [36]. It is also well es- FSGS is associated with mutations in TRPC6 channels and actin cytoskeleton associated tablished that FSGS is associated with mutations in TRPC6 channels and actin cytoskel- proteins [29]. However, the ability of the actin cytoskeleton to influence TRPC6 activity has eton associated proteins [29]. However, the ability of the actin cytoskeleton to influence not previously been investigated. Our study demonstrates that TRPC6 channel activity is TRPC6 activity has not previously been investigated. Our study demonstrates that impacted by inhibition of the actin cytoskeleton in podocytes. Our results are in agreement TRPC6 channel activity is impacted by inhibition of the actin cytoskeleton in podocytes. with the observation that cytochalasin D increases stretch-evoked whole-cell currents in an Our results are in agreement with the observation that cytochalasin D increases immortalized human podocyte cell line [37]. TRPC6 channels are not inherently sensitive stretch-evokedto membrane whole-cell stretch, unlike currents the classicin an stretchimmortalized sensitive human channel podocyte Piezo1 [cell38,39 line]; however, [37]. TRPC6their channels mechanosensitivity are not inherently properties sensitiv may bee linkedto membrane to intracellular stretch, tethers. unlike Stretch the classic evoked stretchactivation sensitive of TRPC6 channel is modulatedPiezo1 [38,39]; by podocin however, [37 ]their or cytoskeleton mechanosensitivity components properties [31]. Our mayexperiments be linked to disrupting intracellular the tethers. actin cytoskeleton Stretch evoked suggest activation that TRPC6 of TRPC6 association is modulated with actin by networkspodocin [37] may or also cytoskeleton influence channelcomponents activity. [31]. Our experiments disrupting the actin cytoskeletonIn podocytes suggest from that freshlyTRPC6 isolated associatio glomeruli,n with weactin observed networks two may modes also of TRPC6influence chan- channelnel activity; activity. active channels that were not further modified by cytochalasin D application andIn silentpodocytes channels from that freshly were isolated activated glomeruli, by cytochalasin we observed D. Our two results modes demonstrate of TRPC6 that channelapplication activity; of cytochalasinactive channels D tothat podocytes were not induces further the modified activation by ofcytochalasin silent channels D ap- only plicationand is and not ablesilent to channels change the that properties were activated of highly by active cytochalasin channels. D. Furthermore,Our results demon- it appears stratethat that the numberapplication of active of cytochalasin channels does D to not podocytes change, at induces least during the activation acute treatment of silent with channelscytochalasin only and D. is not able to change the properties of highly active channels. Fur- thermore,In it addition appears to that TRPC6 the number channels, of active we observed channels additional does not change, channels at withleast during a smaller acuteconductance, treatment with that couldcytochalasin also be D. activated by the application of cytochalasin D. Channels withIn addition similar properties to TRPC6 havechannels, previously we observed been described additional in podocyteschannels with [34]. a Potentially,smaller conductance,they could that be composed could also of be TRPC activated heteromeres by the application with lower of permeability cytochalasin [40 D.–42 Channels]; however,

Int. J. Mol. Sci. 2021, 22, 4396 8 of 12

it is also possible that other types of endogenous channels are activated in response to inhibition of actin cytoskeleton dynamics. Additional studies are required to reveal the identity of these channels. Both the application of cytochalasin D and the expression of the α-actinin-4 K255E mutant resulted in changes to cell morphology (Figures1 and6); however, their effects on TRPC6 activity were different. While in both cases we demonstrated that TRPC6 activity is under the control of the cytoskeleton, the different mechanisms of cytoskeletal perturbation cause disparate effects on channel activity. Thus, further experiments are needed to more fully understand the underlying dynamics by which the actin cytoskeleton influences TRPC6 behavior. It was reported that TRPC6 mechanosensitivity is linked to cytoskeleton rearrangement [37], while TRPC6 regulation by DAG after receptor activation is controlled by other pathways [43]. There are likely multiple factors at play that need additional investigation in order to understand these phenomena. α-actinin-4, encoded by ACTN4, is an essential protein controlling podocyte structure and function. Upregulation of α-actinin-4 is necessary for the differentiation of stem cells to podocytes [44,45]. Expression of α-actinin-4 is decreased in a model of diabetic nephropa- thy as well as in FSGS caused by the ACTN4 K255E mutation [46,47]. The ACTN4 K255E mutation also reduces foot process-like peripheral projections in a conditionally immor- talized podocyte cell line [33], similar to the disturbances in dendritic spine dynamics of neurons lacking α-actinin-4 [48]. In addition to podocytes, α-actinin-4 has been established as a critical mechanoresponsive protein in several other tissues [49,50]. Mutant ACTN4 K255E has increased binding affinity for actin [21] and slower recovery from tension [25,35]. α-actinin-4 binding to actin is regulated and reduced by calcium or phosphorylation at tyrosines 4 and 31 [28,51] or increased by phosphorylation at position S159 [23]. Mutant ACTN4 K255E is insensitive to calcium, while phosphorylation at tyrosines 4 and 31 de- creases its actin-binding [24]. There is limited information about the relationship between α-actinin-4 and ion channels. Our results demonstrate a novel regulatory effect of mutant α-actinin-4 on TRPC6 channel function. In cells expressing ACTN4 K255E, we observed reduced TRPC6 channel activity (both basal and OAG-activated), while the number of channels was unaltered. α-actinin-4 was also reported to be associated with acid-sensing ion channel ASIC1a. Similarly, it was shown that co-expression of α-actinin-4 with ASIC1a did not affect cell surface expression. In contrast, α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization [52]. These results suggest an additional potential mechanistic role for ACTN4 K255E to influence podocyte calcium entry through TRPC6 in the pathogenesis of FSGS. In summary, our data demonstrate that the dynamic rearrangements of the cytoskele- ton can affect calcium entry through TRPC6 channels, which in turn can influence the filtering properties of podocytes.

4. Materials and Methods 4.1. Cells CHO cells were cultured in a DMEM/F12 medium supplemented with 10% heat- inactivated FBS and 80 µg/mL of gentamicin. For experiments measuring TRPC6 activity, CHO cells were co-transfected using a PEI transfection reagent (Polysciences Inc., War- rington, FL, USA) with 0.5 µg TRPC6 and 0.25 µg GFP, 24–48 h before electrophysiological analysis. For control experiments, CHO cells were transfected with 0.7 µg GFP. For exper- iments with α-actinin-4, the cells were co-transfected with TRPC6 tagged with GFP and wild-type or mutant ACTN4 K255E tagged with GFP [27]. For control experiments with α-actinin-4, CHO cells were transfected with TRPC6 tagged with GFP. The weight ratio of plasmid DNAs was: TRPC6—0.7 µg; ACTN4—0.7 µg. For cytoskeleton analysis, CHO cells were transiently transfected by ACTN4 wt, or ACTN4 K255E mutant (0.7 µg of each DNA, respectively), 24 h before experiments. The immortalized human podocyte cell line AB 8/13 was kindly provided by M. Saleem and has been described previously [53,54]. Cells were cultured in an RPMI-1640 Int. J. Mol. Sci. 2021, 22, 4396 9 of 12

medium supplemented with 10% heat-inactivated FBS and insulin-transferrin-selenium supplement. Podocytes proliferate abundantly at 33 ◦C, and after thermo-switching to 37 ◦C (for 10 days), cells become mature podocytes and express key structural proteins such as nephrin and podocin.

4.2. Animals Male Wistar 9–14-week-old rats were used for experiments. The glomerular isolation protocol has been described previously [34]. Briefly, in anesthetized rats, the abdominal cavity was cut, and the kidneys were excised and placed in a cold saline solution. All actions were carried out in accordance with the rules of the local ethical committee. After isolation of the kidneys, the renal capsule was removed, and the kidney cortex was isolated with a thickness of 1–2 mm. The cortex of the kidney was crushed into small chunks and passed through a special mesh grid filters with numbers of 80, 120, and 200 (pore sizes of 150, 106, and 73.7 µm, respectively). After glomerular isolation, the podocytes are visible under a light microscope (40×). They have a round or oval shape and are located on the surface of the glomerular capillary loops.

4.3. Electrophysiological Analysis For cell-attached patch-clamp analysis, solutions were (in mM): bath—NaCl 130, CaCl2 1, HEPES 10, MgCl2 2, Glucose 10, pH 7.4 (in experiments with ACTN4—130 mM NaCl was replaced by 140 mM KCl, 5 mM NaCl); pipette—NaCl 126, CaCl2 1.5, HEPES 10, Glucose 10, TEACL 10. A number of blockers were added to the pipette solution, specifically 10 nM of iberiotoxin, 0.1 mM of DIDS, and 10 µM of nicardipine. The inhibitors prevented activation of non-TRPC cationic channels from interfering with patch-clamp recordings. Voltage is amplifier command potential. Positive current flows from the pipette into the cell. Additional filtering at 300 Hz was applied before analysis. Cytochalsin D and OAG was from Sigma-Aldrich (St. Louis, MO, USA). Single-channel unitary current (i) was determined from the best-fit Gaussian distribu- tion of amplitude histograms. The activity was analyzed as NPo = I/i, where I is the mean total current in a patch and i is unitary current at this voltage.

4.4. Imaging of the Cytoskeleton Fixation and staining of the transfected CHO cells were performed according to a standard protocol [55]. Cells were passaged onto coverslips (12 mm × 12 mm), washed with PBS the next day after transient transfection (by wild type ACTN4 or ACTN4 K255E mutant), and then fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Then, the cells were perforated with 0.1% Triton X-100 (5 min, room temperature) in PBS and incubated with 2 µM rhodamine-phalloidin solution (Sigma-Aldrich) for 15 min at 37 ◦C. Nuclei were stained with a Hoechst-33342 dye (5 µg/mL, 5 min incubation, room temperature) and mounted onto a slide using Vectashield medium (Vector Laboratories, Inc., Burlingame, CA, USA). The addition of each reagent (pre-dissolved in PBS) was followed by washing with PBS. Imaging was carried out using Olympus FV3000, × 63 lens, digital zoom. Lasers with excitation wavelengths of 405 nm (Hoechst-33342, emission maximum at 461 nm), 488 nm (GFP, emission maximum at 509 nm), and 561 nm (rhodamine- phalloidin, emission maximum at 565 nm) were used. Image analysis and processing were performed using open-source software ImageJ v1.53c (National Institutes of Health, USA, http://imagej.nih.gov/ij/).

4.5. Scanning Ion Conductance Microscopy (SICM) Analysis To perform SICM imaging, human podocytes were attached to poly-L-lysine glass surface and placed into the cell chamber filled with PSS solution. Samples were manually positioned in the x–y direction under the inverted optical microscope, Nikon TE2000-U (Nikon Instruments, Tokyo, Japan). For the hopping probe, SICM imaging borosilicate glass nanopipettes with a resistance of approximately 100 MΩ, which corresponds to Int. J. Mol. Sci. 2021, 22, 4396 10 of 12

an estimated tip diameter of around 120 nm, were used as described previously [56,57]. The nanopipettes were filled with the same PSS solution used for the bath and were positioned in the z-direction with a piezoelectric actuator. The ion current flowing through the nanopipettes was measured with an Axopatch 700B patch-clamp amplifier (Molecular Devices, San Jose, CA, USA) in voltage-clamp mode and monitored by the custom-modified universal controller (ICAPPIC Ltd., London, UK), which simultaneously controlled sample and pipette positioning [58].

4.6. Statistical Analysis Results are presented as a mean ± standard error of the mean. Unpaired Student t-test results were calculated using Origin software (Microcal Software, Northampton, MA, USA). Differences with p < 0.05 were considered statistically significant.

Author Contributions: Conceptualization, A.S. (Alexander Staruschenko) and E.K.; methodology, R.B.; formal analysis, A.S. (Alexey Shalygin), L.S.S., R.B., O.P.; investigation, A.S. (Alexey Shaly- gin), L.S.S., R.B., O.P.; writing—original draft preparation, A.S. (Alexey Shalygin), A.S. (Alexander Staruschenko), and E.K.; writing—review and editing, A.S. (Alexey Shalygin), L.S.S., R.B., O.P., A.S. (Alexander Staruschenko), and E.K.; supervision, A.S. (Alexander Staruschenko) and E.K.; project administration, A.S. (Alexander Staruschenko) and E.K.; funding acquisition, A.S. (Alexander Staruschenko) and E.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Heart, Lung, and Blood Institute grant R35 HL135749 (to A.S. (Alexander Staruschenko)), Department of Veteran Affairs grant I01 BX004024 (to A.S. (Alexander Staruschenko)), and Russian Science Foundation project No. 19-14-00114 (to E.K.). Institutional Review Board Statement: Animal use and welfare adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, following protocols reviewed and approved by either the Medical College of Wisconsin Institutional Animal Care and Use Committee (AUA1061) or the guidelines for the welfare of animals of the ethical committee of the Institute of Cytology, Russian Academy of Sciences (No. F18-00380, approved on 12 October 2017). Data Availability Statement: The data presented in this study are available upon reasonable request from the corresponding author. Acknowledgments: Plasmids encoding wild type ACTN4 and the K255E mutant were provided by Di Feng and Martin R. Pollak (Beth Israel Deaconess Medical Center; Harvard Medical School). Moin A. Saleem (University of Bristol, UK) is greatly appreciated for providing the human podocytes cell line. The authors also thank Christine A. Klemens for critical proof-reading of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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