PAR1-mediated Non-periodical Synchronized Calcium Oscillations in Human Mesangial Cells

Abstract Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases such as thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 G-protein coupled receptors-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and transient receptor potential canonical 3 (TRPC3) channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells, contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.


Introduction
Mesangial cells (MCs) are specialized contractile cells, which abut the glomerular vasculature and basement membrane esta b lishing the central stalk of the glomerulus. 1 , 2 MCs generate an extracellular matrix that joins together the basement membr ane , glomerular capillaries, and the contr actile mac hinery of MCs. 3 Aside from structurally supporting the glomerular capillaries, this intertwining network forms a contractile biomechanical unit, allowing for fine-tuning of the intraglomerular blood volume and filtration surface.Indeed, the mechanisms of acute regulation of glomerular blood flow, including the response to changes in the concentration of NaCl in the lumen of the cortically thick ascending limb of Henle or tubuloglomerular feedbac k mec hanism, str ongl y de pend on extra glomerular MC function and the corresponding constriction of intraglomerular afferent arterioles. 4 , 5Mor eov er, MCs r emov e molecular a ggr egates and debris from the basement membrane by phagocytosis, further contributing to maintenance of ultr afiltr ation.In healthy glomeruli, MCs secrete and respond to numerous signaling molecules, including v asoacti v e a gents, cytokines, and hormones, allowing them to perform a range of physiological functions. 3 , 6Cs have been assiduously studied in relation to the pathogenesis of chronic kidney diseases, such as diabetic nephropathy (DN).Hyperfiltration in the early stages of DN has been associated with functional abnormalities in glomerular mesangium, including decreased contractility and increased surface area. 7xcessi v e pr oliferation of MCs and de position of extracellular matrix in the mesangium is a consistent glomerular hallmark of advanced DN. 2 The resulting mesangial expansion decr eases b lood flow in glomerular capillaries, and matrix accum ulation r esults in basement membr ane thic kening and reduction of the glomerular filtr ation r ate . 8Ultimately, DN leads to glomerulosclerosis and tubulointerstitial fibrosis c har acterized by persistent fibrin deposition.Thrombin, a serine proteinase that cleaves fibrinogen into fibrin, is a potent profibrotic factor and a major pathogenetic determinant of DN. 9 , 10 In addition to its key role in blood coagulation, thrombin is a potent vasoconstrictor, mitogen, pr oinflammator y a gent, and a powerful acti v ator of several cell types in the glomerulus, including MCs. 11-13 Thrombin has been shown to stimulate MC proliferation and synthesis of pr osta glandins, nitric o xide, endothelin-1, e xtracellular matrix components, and chemokines. 14 , 15This wide array of functions is achie ved b y the acti v ation of a G-pr otein coupled r ece ptor (GPCR), known as pr otease-acti v ated r ece ptor 1 (PAR1), expressed on the surface of MCs. 16ARs are activated by proteolytic cleavage of their extracellular N-termin us. 17Thr ombin or other serine pr oteases cleav e the r ece ptor at a specific site that acts as a tethered ligand acti v ating the signal transduction thr ough G-pr oteins.TFLLR-NH 2 is a synthetic peptide mimicking the tethered ligand, specifically activating PAR1 without thrombin cleavage. 18Similarly, other short peptides could be used for activation and allow the study of specific signaling pathways and physiological responses mediated by PAR r ece ptors famil y.
Acti v ation of PAR1 signaling r e portedl y contributes to DN. PAR1 expression is upregulated in glomeruli isolated from diabetic db/db mice and str e ptozocin-induced dia betic rats. 19 , 20enetic knockout, knockdown, or pharmacological inhibition of PAR1 attenuates DN in streptozotocin-induced diabetic mice and rats. 21 , 22Our r ecentl y pub lished data indicate that elevation of serine proteases and PAR1 signaling directly mediate intracellular calcium dynamics in glomerular podocytes. 23or eov er, the pathological acti v ation of serine proteases in diabetes further promotes PAR1-TRPC6 (transient receptor potential canonical 6) channel acti v ation, leading to podocyte apoptosis and the development of albuminuria. 24It was also reported that PAR1 blockade ameliorates DN and reduces mesangial proliferation in type I diabetic Akita mice with reduced expression of endothelial nitric oxide synthase. 22Yet, the specific molecular determinants and the relation of PAR1 signaling to MC function, calcium influx, and corresponding ion channels acti v ation modulating MC contractility ar e poorl y understood.Here, for the first time, we r e port that the acti v ation of PAR1 mediates intracellular calcium oscillations in primary human renal mesangial cells (HRMCs).Confocal fluorescent microscopy and patch-clamp electrophysiology were combined with pharmacological approaches to dissect the contribution of specific membrane channels to PAR1-mediated calcium signals.

Cell Culture
Primary HRMCs [male (lot #12445) and female (lot #17554)] w ere pur c hased from the ScienCell Resear c h Labor atories (San Diego , C A, USA).In our experiments, we used both male and female cell lines.We did not find significant sex differences in confocal microscopy experiments, and most of the statistical datasets were shown for the male cell line.Cells were cultured in RPMI-1640 medium (Gibco, #11875085), containing Insulin-Transferrin-Selenium (Gibco, #41400045), penicillin str e ptomycin (Cyti v a, #SV30010), and 10% fetal bovine serum (Corning, #35011CV), in a 5% CO 2 incubator at 37 • C. HRMCs in the passages between 4 and 10 were sub-cultured at 90% confluence and used for further experiments.Seeding density was 6 × 10 3 cells/cm 2 .

Confocal Microscopy
HRMCs wer e cultur ed on glass-bottom dishes (Mattek, #P35G-0-14-C, 35 mm dish, No. 0 coverslip, 14 mm glass diameter, uncoated) until reaching 90% confluence, before being utilized for confocal experiments.The cells were loaded with a 5 μm Fluo-8H, AM fluorescent dye (AAT Bioquest, #21090) and incubated at 37 • C for 1 h in a CO 2 incubator.For the RWJ 56110, GSK 2833503A, and tcY-NH 2 preincubation, the drug or corresponding v ehicle wer e added to the cell media 1 h before the confocal imaging experiments.Before performing the confocal ima ging, cells wer e w ashed to r emov e unincorpor ated dy e , and the media was replaced with a 2 m m (or zero) Ca 2 softw ar e and curr ent acti vity w as anal yzed using Clampfit 11.2 softw ar e (Molecular Devices, San Jose, CA, USA).Channels activity were determined during the 100 s recording period at baseline and after the acute application of TFLLR-NH 2 or corresponding pharmacology.The total number of events were used to measure the channels activity within a patch and total current through the clamped membr ane w ere calculated as an inte gr al for the 100 s interv als befor e and after drug application, as pr eviousl y r e ported. 35

Glomeruli Volume Dynamics Assay
Sixteen-w eek Wistar Ky oto male r ats w er e obtained fr om Charles Ri v er and wer e ke pt in a light-contr olled envir onment with a 12:12-h light/dark cycle and gi v en fr ee access to w ater (filter ed, R O w ater) and food (5V75-PicoLa b Verified 75 IF, La bDiet, USA).All animal pr ocedur es wer e appr ov ed by the Institutional Animal Care and Use Committee at the Medical Uni v ersity of South Carolina in accordance with the Guide for the Care and Use of La borator y Animals and followed the ARRIVE guidelines.
Experimental pr ocedur es wer e performed as pr eviousl y described. 36Briefly, male Wistar rat kidneys were harvested, decapsulated, and used to isolate glomeruli by differential sieving.Fr eshl y isolated glomeruli were collected and stored on ice in a 15-mL tube with a 5% BSA (Sigma-Aldrich, # A8327)/RPMI-1640 solution with non-permea b le 150 kDa TRITC-dextran (1 mg/mL, TdB Labs, #TD150, Uppsala, Sweden).Glomeruli volume c hanges w er e measur ed using fast confocal 3D ima ging before and after acute application of 60 μm of Angiotensin II (Bachem, #4006473) or 10 μm of TFLLR-NH 2 .Glomeruli were attached to pol y-l -l ysine cov er ed glass-bottom dishes and covered with the extracellular solution containing 150 kDa TRITC dextr an.Z-stac ks with 18 consecuti v e focal planes (3 μm each) were collected every 1 min, allowing glomeruli volume reconstruction.Output files were imported into Imaris Softw ar e (Oxford Bitplane, version 9.6.1),reconstructed in 3D, and processed to calculate glomerular volume.

Sta tistics Anal ysis
Changes in Ca 2 + fluor escence wer e calculated for individual cells, with appr oximatel y 10 cells per dish analyzed.Each experiment was repeated a minimum of 4 times on the new unexposed cells.For r e pr esentati v e Figur es, the r esponses fr om one single experiment were summarized.The statistical graphs r e pr esent an indi vidual cell r esponses and mean ± SE values.For

Basal Activity and Pharmacological Modulation of Ion Channels in HRMCs
Ther e ar e sev er al types of ion c hannels expressed in MCs. 38wever, the knowledge about signaling pathways mediated by GPCR PAR and the acti v ation of corr esponding ion channels in MCs is limited.To build a strong foundation for our study about the PAR1-mediated calcium influx, we initiall y explor ed basal single-channel activity in HRMCs using single-channel electrophysiolo gy.The e xperiments pr esented in Figur e 1 demonstr ated that STIM1/Or ai1 complex and TRPC3 channels predominantly contributed to baseline activity, which represent multiple ev ents fr om differ ent ion channels on the membr ane .Inhibition of STIM1/Orai1 and TRPC3 channels with a high concentration of pyrazole deri v ati v e Pyr6 (see the "Materials and Methods" section for details) resulted in a significant decrease in basal current acti vity ( Figur e 1 A and B).In addition, the application of the specific TRPC6 blocker BI-749327 revealed that this channel plays a minor but detecta b le r ole in basal activity in HRMCs ( Figure 1 C).

PAR1-mediated Calcium Flux in Cultured HRMCs
To explore PAR1-mediated Ca 2 + flux, we used li v e cell confocal imaging.HRMCs displayed high sensitivity to a specific PAR1 a gonist pe ptide TFLLR-NH 2 ( Figur e 2 A).The EC 50 v alues wer e in the nanomolar range of 3.0 ± 0.8 and 6.3 ± 0.2 n m for male-and female-deri v ed cultur ed cells, r especti v el y.Shown in Figur e 2 B is the dose-r esponse curv e for male HRMCs.The observed sensitivity to a PAR1 agonist peptide was much higher (nanomolar versus tens of micromolar concentration range) in comparison with our r e ports in other glomerular cells, like podocytes, 24 or brain cells like astrocytes. 39In contrast, Ca 2 + response to ATP and the acti v ation of corr esponding purinergic r ece ptors wer e in the range of 100 μm (different reports show a 10-100 μm ATP EC 50 values for MCs 40 , 41 ), which is compara b le with pr eviousl y r e ported v alues for podocytes. 42

PAR1 Signaling Promotes Intracellular Ca 2 + Oscillations in HRMCs
We further decided to explore PAR-mediated calcium oscillations in MCs, gi v en the smooth muscle-like nature of these cells.It is known that smooth muscle cells' functional contractility is mediated by periodic pulses of cytosolic Ca 2 + trigger oscillations, which may be r esponsib le for contraction and membrane depolarization that couples the individual oscillators together, mediating the synchronization. 43First, the acute application of TFLLR-NH 2 in the nanomolar range resulted in a single calcium peak, which could be blocked by preincubation with a specific PAR1 signaling antagonist, RWJ 56110 ( Figure 3 A).However, when we increased the TFLLR-NH 2 concentration up to saturation levels of 1 μm and achieved maximum PAR1 signaling acti v ation (see Figure 2 B), the initial increase in cytosolic Ca 2 + lev els w as follow ed by sync hronized, damped Ca 2 + oscillations (r e peated calcium peaks appeared after the initial one) with a lag of 6.74 ± 0.84 minutes between first and second peaks ( Figure 3 B and Supplemental Video S1 ).Nota b l y, the oscillation pattern w as a bsent in extracellular zer o calcium solutions, suggesting a crucial role for ionotropic Ca 2 + entry and resident ion channel acti v ation ( Figur e 3 B and C).To make a mor e dir ect connection between the Ca 2 + oscillations and presumed cellular contr actions w e performed glomerular volume c hanges assay using fr eshl y isolated Wistar rat glomeruli.The acute application of Ang II result in strong contraction of the mesangium matrix and reduce glomerular volume up to 20.7% ( Figure 4 A).The application of PAR1 acti v ating pe ptide TFLLR-NH 2 r esulted in the v olume reduction up to 7.6% ( Figure 4 B).

Intr acellular Ca 2 + Oscilla tions in Response to T hrombin and Role of PAR1
Beyond the coagulation functions, thrombin is a crucial activator of PARs signaling, which primarily activates PAR1 and PAR4 on platelets and endothelial cells, initiating a cascade of intracellular signaling pathways.To test if thrombin promotes cytosolic Ca 2 + oscillations, we performed confocal imaging experiments using acute applications of thrombin peptide.As shown in Figure 5 A, the application of thrombin promotes synchronized Ca 2 + oscillations, similar to PAR1 agonist peptide TFLLR-NH 2 (see Supplemental Video S2 ).Inter estingl y, pr eincubation of cells with specific PAR1 antagonist RWJ 56110 significantly inhibits the first peak (store release, see Figure 3 B) and eliminates the second peak, r esponsib le for the extracellular influx and oscillations ( Figure 5 A and B, and Supplemental Video S3 ).We perform the following experiments to explore further if the PAR4 signaling cascade may be inv olv ed in MC signaling.HMRCs wer e pr eincubated in RWJ 56110 to block PAR1 response on the acute application of TFLLR-NH 2 peptide.The following application of PAR4 acti v ating pe ptide AY-NH 2 pr omotes r obust intracellular Ca 2 + release ( Figure 6 A), confirming the functional presence of PAR4 in MCs.In the prolonged recording, the same concentrations of PAR4 acti v ating pe ptide did not pr omote synchr onized Ca 2 + oscillations and can be efficiently blocked by the application of PAR4 antagonist tcY-NH 2 ( Figure 6 B).Moreover, the applications of thrombin in the presence of tcY-NH 2 and corresponding PAR4 blockade successfully produce Ca 2 + oscillations, as evidenced by statistical data showing the presence of a second peak in Figure 6 C.  In addition, we performed a series of experiments to test the possible involvement of PAR2 in the described Ca 2 + oscillation behavior in response to thrombin.As shown in Figure 6 D, thrombin-mediated oscillations in individual cells are not affected by the application of the PAR2 agonist AZ 3451.The statistical analysis ( Figure 6 E) indicates that preincubation with AZ 3451 also does not affect the first (store release) peak.

Pharmacological Inhibition of PAR1-mediated Oscillations
PARs are G protein-coupled receptors and may adopt distinct acti v e conformations and signal to di v erse effectors in many cells. 44According to the data a bov e, PAR1 signaling in HRMCs consists of initial intracellular store release and synchronized oscillations mediated by plasma membrane ion channels.To dissect the contribution of the store-operated (Orai1) and second messenger-operated (TRPC) plasma membr ane c hannels to the calcium oscillations, w e performed confocal imaging experiments in the presence of specific pharmacological blockers (see the "Materials and Methods" section for detailed information about drugs and vehicle).We measured the changes in the intracellular Ca 2 + amplitude of the first response (depo release) and the amplitude of the second peak (r e pr esenting ionotr opic influx) after applying 1 μm TFLLR-NH 2 in the presence of a vehicle or corresponding drugs ( Supplemental Figure S1 ).Pyr6, which inhibits the store-operated calcium (SOCs) channels at the low (5 μm ) concentration, significantly attenuated the oscillation amplitudes (r e pr esented as the maxim um amplitude of the second calcium peak; Figure 7 A).Similarly, the TRPC3/6 inhibitor (GSK 2833503A), reduced the oscillation amplitude up to 40% (two-way ANOVA, * P < 0.0001 compared to vehicle).The simultaneous application of both drugs resulted in an additi v e effect with a total of 65% blockade of the second peak amplitude (two-way ANOVA, * P < 0.0001 compared to vehicle) ( Figure 7 A).Further experiments shown in Figure 7 B include high Pyr6 concentration (over 15 μm ) with or without a TRPC3/6 inhibitor (GSK 2833503A) to sim ultaneousl y bloc k STIM1/Or ai1 + TRPC3 c hannels and bloc kade TRPC6 with a selecti v e inhibitor BI-749327.Note, that preincubation with high Pyr6 concentration ( Figure 7 B) also cause significant inhibition of first response, and then may reflect on overall deficit in intracellular Ca 2 + pool, which later may significantly inhibit oscillations amplitudes.These studies indicate that oscillation is pr esuma b l y mediated by the acti v ation of STIM1/Orai1 and TRPC3 channels and does not depend on TRPC6 activity.

Single Channel Activity in Response to PAR1 Signaling Activ a tion in HRMCs
We used patch clamp electrophysiology to confirm our findings to r ev eal ion channel acti v ation in response to acute PAR1 agonist application.The addition of saturated concentrations of TFLLR-NH 2 (1 μm ) into the bath solution promoted a rapid increase in a number of events and overall current density ( Figure 8 A and B), suggesting the presence of ion channelmediated calcium influx which was observed in the confocal microscopy experiments in Figure 3 B. Similarly, PAR1-mediated ion channels activity could be significantly reduced by application of a high concentration of Pyr6 (15 μm ) and blockade of STIM1/Or ai1 + TRPC3 c hannels ( F igure 8 C).

Discussion
The seminal studies from the 90's highlighted that thrombin is a potent regulator of MC function, influencing not only the contractile behavior of these cells but also their role in matrix production and overall renal physiology. 14 , 45Thrombin's actions ar e m ultifaceted, impacting cellular pr ocesses ranging fr om contr action to bioc hemical mediator synthesis, whic h are essential for the normal and pathological states of the kidney's filtering mechanism.Importantly, the high sensitivity of MCs to thrombin observed in our studies with specific PAR1 agonist peptide ( Figure 2 ) was supported by the similar thrombin applications, wher e significant Ca 2 + r esponse w as detected at concentrations around 0.1 U/mL (approximately 1 n m ). 14 , 46As mentioned above, podocytes express PARs but require much higher concentrations for the intr acellular Ca 2 + tr ansient.The other cells with known functional expression of PARs are platelets, endothelial, vascular smooth muscle, monocytes and macrophages, and neuronal cells could be divided into three groups for their sensitivity to thrombin: Some cell types, such as platelets, ar e highl y sensiti v e to thr ombin.Ev en low concentrations (0.1 to 1 U/mL) ar e sufficient to induce Ca 2 + mobilization in these cells, leading to platelet secretion and aggregation. 47oderate sensitivity.Concentrations around 0.5 to 2 U/mL are often required to activate endothelial and vascular smooth muscle cells. 48Endothelial cells utilize thrombin to regulate barrier function and inflammatory responses through Ca 2 + signaling. 49Vascular smooth muscle cells primarily use PAR signaling for contraction and proliferation, which is also an important property for mesangial cells.
Other cell types, such as monocytes and neuronal cells, demonstr ate a low er sensiti vity to thr ombin.These cells generall y r equir e higher thr ombin concentrations (1-5 U/mL) to activate intracellular Ca 2 + signaling.This activation plays a role in monocyte adhesion and migr ation, whic h is crucial for inflammator y r esponses. 50In the nerv ous system, thr ombin can influence neuron and glial function, as we have also described in our previous studies, 32 impacting cell survi v al and inflammator y processes. 51ur data suggest that MCs appear to have high sensitivity, similar to platelets, to promote initial Ca 2 + response but r equir e higher close to moderate range concentrations to r ev eal Ca 2 + oscillations.
Intracellular calcium is an essential second messenger regulating multiple aspects of MC function, modulating responses to v asoacti v e hormones, pol ype ptide gr owth factors, and cytokines. 2 , 38 , 52Importantly, changes in intracellular calcium concentration are the key requirement for cell contractility.Thus, calcium flux and corresponding MC contraction can dir ectl y modulate glomerular blood flow and renal hemodynamics. 7Thrombin and PARs are known to mediate intracellular calcium signaling and acti v ate a v ariety of intracellular cascades. 47 , 53An acti v ation of the PAR1 r ece ptor can stimulate G q protein and trigger subsequent IP 3 -dependent calcium release from the sarco/endoplasmic reticulum (SR/ER). 54alcium mobilization depletes the intracellular stores and triggers the opening of calcium channels on the plasma membr ane .The resulting calcium influx from the extracellular compartment allows r e plenishment of intracellular stores and is termed SOCE.Numerous findings support the presence of SOCE in MCs, 52 where it can be mediated by a highl y calcium-selecti v e ORAI1 channel or nonselecti v e transient r ece ptor potential canonical channels, such as TRPC1, 3, 4, and 6. 38 However, TRPC channels can be activ ated inde pendentl y as r ece ptor-oper ated calcium c hannels by various ligands, including via the PAR1 pathway. 24 , 55Our data suggest that PAR1 acti v ation in MCs triggers calcium r elease fr om the SR/ER at nanomolar concentrations.Moreover, saturated PAR1 agonist concentrations, in addition to SR/ER calcium r elease, acti v ate ionotr opic calcium influx from STIM1/Orai1 and TRPC3 channels and mediate intracellular calcium oscillations ( Figure 9 ).The observed spontaneous cellular oscillations are sync hronized betw een cells in the monolayer and may r e pr esent a typical smooth muscle contr actility phenotype . 43MC oscillations can be c har acterized by a quick spike in Ca 2 + followed by a more extended phase of oscillations, which takes minutes (our recordings usually take up to 20 min; see supplemental video for more information).It is important to note that MC may possess properties similar to v ascular smooth m uscle cells, and cytosolic Ca 2 + oscillations may lead to cell contraction or proliferation.Mor eov er, under pathophysiological conditions, this type of cells may develop either contractile or synthetic (pr oliferati v e) phenotype. 56The questions are essential for understanding the physiology and pathophysiology of mesangial cells since both mesangial matrix contr actility and prolifer ation are the basis of most glomerular diseases.
Our data suggest the selecti v e r ole of PAR1, and not PAR4 or PAR2, in triggering cytosolic Ca 2 + oscillations in MCs.While both r ece ptors enga ge distinct GPCR mechanisms, PAR1 mediates its effects through interactions with G αq, G αi, and G α12/13 proteins, facilitating rapid and versatile cellular responses.Conv ersel y, PAR4, which primarily associates with G αq and G α12/13, exhibits a slower acti v ation r esponse but maintains signaling over a longer period. 57This prolonged activation is crucial for sustained thrombin signaling in the context of chronic disease pr ogr ession and r ole of PAR4 r equir ed further detailed investigation.Our study contributes to the broader understanding of GPCR-mediated calcium signaling oscillations, illustrating its complexity and di v ersity acr oss differ ent cell types, including MCs. 58 Mounting evidence links SOCE in MCs to extracellular matrix protein synthesis and deposition.59-62 Changes in SOCE parallel mesangial expansion and the fibrotic glomerular phenotype in DN. 52 , 61 Mor eov er, PAR1 ov eracti v ation and high activity of serine proteases have recently been linked to glomerular pathologies like FSGS and DN. 24 , 55Inter estingl y, the a bov ementioned studies emphasize the key role of TRPC6 channels in PAR1-mediated podocyte and glomerular damage.In contrast, MCs mediate contractility through the STIM1/Orai1 and TRPC3 channels, where the expression of TRPC6 plays a minor role and does not significantly contribute to the observed phenomenon.Our data indicate high sensitivity of MCs to the PAR-1 acti v ating peptide , whic h may contribute to both physiological and pathophysiological functions.For instance, high serine proteases activity may induce frequent cytosolic oscillations and changes in membrane potential in MCs, which can lead to hyperfiltration and glomerular blood flow disturbance or mesangial matrix accumulation and consecutive aberrant mesangial cell proliferation leading to glomerulosclerosis.Our stud y pro vides direct evidence of the possible role of PAR1 receptors in glomerular and MC pathology and raises the question about the potential use of PAR1 as a therapeutical target in glomerular diseases.Ther efor e, further inv estigation of this pathw ay, including r esear c h using various animal models, is r equir ed.

Figure 1 .
Figure 1.Functional basal activity of STIM1/Orai1, TRPC3, and TRPC6.(A) Re pr esentati v e trace showing inhibition of store-operated calcium entry (SOCE) and TRPC3 baseline activity in human renal mesangial cells (HRMCs) by the acute application of pyrazole deri v ati v e Pyr6 (15 μm ) (holding potential is −60 mV).(B) The total number of events (top, single channel openings) and total current (bottom, calculated as an inte gr al for the 100 s intervals before and after STIM1/Orai1 + TRPC3 inhibition).Shown individual data points (left) and summary graphs (right).Data were analyzed using a one-way RM ANOVA ( * P < 0.05).(C) Representative trace showing partial inhibition of baseline activity by TRPC6 channel blocker BI-749327 (1 μm ) and strong inhibition of baseline activity by pyrazole deri v ati v e Pyr6 (15 μm ) in HRMCs (holding potential is −60 mV).Expanded fragments show baseline activity, inhibition of TRPC6 channel after application of BI-749327, and inhibition of STIM1/Orai1 + TRPC3 currents after application of Pyr6, respectively.normalized maximum [Ca 2 + ] i amplitudes shown in Figure 6 : corr esponding v ehicle r esponse w as taken for 100% and each cell response to drug was normalized to mean vehicle value.Corresponding non-normalized values and statistics for Figure 6 data are shown in Supplemental Figure S1 .Data were analyzed with ANOVA, and multiple-comparison adjustments (Tukey post hoc test) were conducted only if the ANOVA F value was significant.P -values of < 0.05 were considered significant.The doser esponse curv e with v aria b le Hill slope fit w as generated by a Nonlinear Curve Fit (DoseResp) module with the corresponding Levenber g-Marquar dt algorithm, as r ecentl y described. 37The generated model was adjusted to impr ov e the adj.R -Square ≥0.98.All statistical analyses were performed in Origin-Pr o 2021b softw ar e (Micr ocal Softw ar e, Northampton, MA, USA).

Figure 4 .
Figure 4.The contraction of the glomerular mesangial matrix in response to Ang II and PAR1 agonist acute applications.(A) Fast confocal 3D imaging shows changes in glomerular volume in response to acute application of Ang II (60 μm ).(B) Fast confocal 3D imaging shows changes in glomerular volume in response to acute application of PAR1 agonist peptide TFLLR-NH 2 (10 μm ).The volume c hanges w er e calculated using the Imaris Ima ge Anal ysis Softw ar e packa ge.

Figure 5 .
Figure 5.The acti v ation of pr otease-acti v r ece ptor 1 (PAR1) is r equir ed for thr ombin-mediated intracellular Ca 2 + [Ca 2 + ] i oscillations in human r enal mesangial cells.(A) Confocal imaging experiment (Fluo-8H, AM fluorescence) shows [Ca 2 + ] i oscillations in response to application of thrombin receptor agonist peptide (5 μm ) (black line).The preincubation of cells with the specific PAR1 inhibitor (RWJ 56110, 10 μm ) significantly inhibit 1 st peak (stor e r elease, white bar) and eliminate 2 nd peak (extracellular influx, gray bar) of [Ca 2 + ] i response to thrombin in HRMCs (red line).(B) Summary for confocal experiments shown mean (bars) and individual cell (data points) of maximum [Ca 2 +] i amplitudes for first (store release, 1 st peak, white bar) and second (extracellular influx, 2 nd peak, gray bar) peaks in response to thr ombin r ece ptor a gonist pe ptide (5 μm ) with or without the pr esence of PAR1 anta gonist RWJ 56110.One w ay ANOVA, * * p < 0.001 between control and RWJ 56110 tr eated gr oups.

Figure 6 .
Figure 6.The acti v ation of pr otease-acti v ated r ece ptor 4 or 2 (P AR4 or P AR2) is not r equir ed for thr ombin-mediated intracellular Ca 2 + [Ca 2 + ] i oscillations in human renal mesangial cells.(A) Confocal imaging experiment (Fluo-8H, AM fluorescence) shows the presence of PAR4-mediated [Ca 2 + ] i in HMRCs.Cells preincubated with the specific PAR1 inhibitor (RWJ 56110, 10 μm ) were not responsive to PAR1 agonist peptide TFLLR-NH 2 (10 n m ), but produced robust transient in response to PAR4 a gonist pe ptide AY-NH 2 (200 μm ).(B) Confocal imaging experiment (Fluo-8H, AM fluorescence) shows the a bsence of synchr onized [Ca 2 + ] i oscillations in response to PAR4 agonist peptide AY-NH 2 (200 μm ) (black line).The preincubation of cells with the specific PAR4 inhibitor (tcY-NH 2 , 50 μm ) inhibits the r esponse entir el y.Note the pr esence of non-synchr onized spontaneous [Ca 2 + ] i sparks in individual cells in both r ecords.(C) Summar y for confocal experiments shown mean (bars) and individual cell (data points) of maximum [Ca 2 + ] i amplitudes in response to thrombin receptor agonist peptide (5 μm ) with the presence of the specific PAR4 inhibitor (tcY-NH2, 50 μm ).(D) The example of synchronized non-periodical oscillations in individual cells after acute application of thrombin receptor agonist peptide in the presence of vehicle (DMSO, black line) or PAR2 agonist (AZ 3451, 100 n m , red line).(E) Summary for confocal experiments shown in D. Mean (bars) and individual cell (data points) of maximum [Ca 2 + ] i amplitudes in response to thrombin receptor agonist peptide (5 μm ) with the presence of the specific PAR2 inhibitor (AZ 3451, 100 n m ).

Figure 8 .
Figure 8. G-protein-coupled receptors PAR1 signaling activate TRPC3 STIM1/Orai1 channels in human renal mesangial cells (HRMCs).(A) Representative electr ophysiological r ecording of channels acti vity in HRMCs befor e and after application of PAR1 a gonist pe ptide TFLLR-NH 2 (1 μm ).The top left corner shows a photomicrograph of the electrophysiolo gical e xperiment.The single-channel trace insets show expanded r ecording interv als.(B) The total number of events (top, single channel openings) and total current (bottom, calculated as an inte gr al for the 100 s intervals before and after PAR1 activation) changes in response to TFLLR-NH 2 (1 μm ) application.Shown individual data points (left) and summary graphs (right).One-way RM-ANOVA, * P < 0.05.(C) Representative trace showing the activation of PAR1 by the specific agonist peptide TFLLR-NH 2 (1 μm ) and inhibition of PAR1-mediated STIM1/Or ai1 + TRPC3 c hannels activity by pyrazole deri v ati v e Pyr6 (15 μm ).Expanded fragments show baseline activity, increased current activity after application of TFLLR-NH 2 , and inhibition of currents after application of Pyr6, r especti v el y.All traces were recorded at −60 mV holding potential.