Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct
2023; Elsevier BV; Volume: 300; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2023.105524
ISSN1083-351X
AutoresKyrylo Pyrshev, Anna Atamanchuk-Stavniichuk, Mariya Kordysh, Oleg Zaika, Viktor Tomilin, Oleh Pochynyuk,
Tópico(s)Blood properties and coagulation
ResumoThe renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid–base stimuli. The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid–base stimuli. Mechanosensitivity is an universal stress-responsive mechanism allowing effective adaption to external physical forces induced by physiological as well as pathological states/conditions (1Martino F. Perestrelo A.R. Vinarsky V. Pagliari S. Forte G. Cellular mechanotransduction: from tension to function.Front. Physiol. 2018; 9: 824Crossref PubMed Scopus (500) Google Scholar). Most common cellular responses to mechanical stimuli include cytoskeleton reorganization, activation of various plasma membrane delineated intracellular signaling cascades, and a direct flux of electrolytes via mechanoactivated channels (1Martino F. Perestrelo A.R. Vinarsky V. Pagliari S. Forte G. Cellular mechanotransduction: from tension to function.Front. Physiol. 2018; 9: 824Crossref PubMed Scopus (500) Google Scholar, 2Jin P. Jan L.Y. Jan Y.N. Mechanosensitive ion channels: structural features relevant to mechanotransduction mechanisms.Annu. Rev. Neurosci. 2020; 43: 207-229Crossref PubMed Scopus (114) Google Scholar). Piezo1 was identified as a long-anticipated molecular conduit of the mechanically activated cation currents observed in a variety of cells (3Coste B. Xiao B. Santos J.S. Syeda R. Grandl J. Spencer K.S. et al.Piezo proteins are pore-forming subunits of mechanically activated channels.Nature. 2012; 483: 176-181Crossref PubMed Scopus (712) Google Scholar, 4Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1770) Google Scholar). Functional Piezo1 channel is comprised of three subunits each having 14 transmembrane domains to form a single central cation-conducting pore (5Ge J. Li W. Zhao Q. Li N. Chen M. Zhi P. et al.Architecture of the mammalian mechanosensitive Piezo1 channel.Nature. 2015; 527: 64-69Crossref PubMed Scopus (314) Google Scholar). The overall structure resembles a propeller with three flexible extracellular blades, which are well-suited to transduce mechanical force to the gate of the central pore (5Ge J. Li W. Zhao Q. Li N. Chen M. Zhi P. et al.Architecture of the mammalian mechanosensitive Piezo1 channel.Nature. 2015; 527: 64-69Crossref PubMed Scopus (314) Google Scholar, 6Mulhall E.M. Gharpure A. Lee R.M. Dubin A.E. Aaron J.S. Marshall K.L. et al.Direct observation of the conformational states of PIEZO1.Nature. 2023; 620: 1117-1125Crossref PubMed Scopus (6) Google Scholar). Patch clamp analysis of Piezo1 in native cells and over-expression systems revealed that application of a negative pressure to the recording pipette (i.e. membrane stretch) induces a rapidly inactivating cation-selective current with single channel conductance around 20 pS (4Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1770) Google Scholar, 7Nosyreva E.D. Thompson D. Syeda R. Identification and functional characterization of the Piezo1 channel pore domain.J. Biol. Chem. 2021; 296100225Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Further development of channel-selective agonists/activators Yoda-1 (8Syeda R. Xu J. Dubin A.E. Coste B. Mathur J. Huynh T. et al.Chemical activation of the mechanotransduction channel Piezo1.eLife. 2015; 4: e07369Crossref PubMed Scopus (382) Google Scholar) and Jedi-2 (9Wang Y. Chi S. Guo H. Li G. Wang L. Zhao Q. et al.A lever-like transduction pathway for long-distance chemical- and mechano-gating of the mechanosensitive Piezo1 channel.Nat. Commun. 2018; 9: 1300Crossref PubMed Scopus (134) Google Scholar) allowed quantification of the Piezo1-dependent Ca2+ influx in different cells without constraints associated with its rapid inactivation. Importantly, it was recently shown that chemical (Yoda-1) and mechanical (hypotonicity) stimuli induce similar actions on Piezo1 blade expansion, which correlate with channel activity (6Mulhall E.M. Gharpure A. Lee R.M. Dubin A.E. Aaron J.S. Marshall K.L. et al.Direct observation of the conformational states of PIEZO1.Nature. 2023; 620: 1117-1125Crossref PubMed Scopus (6) Google Scholar) indicative of the common underlying mechanism of Piezo1 stimulation. At the systemic level, Piezo1 was implicated in a variety of essential physiological processes, such as development of vasculature (10Li J. Hou B. Tumova S. Muraki K. Bruns A. Ludlow M.J. et al.Piezo1 integration of vascular architecture with physiological force.Nature. 2014; 515: 279-282Crossref PubMed Scopus (693) Google Scholar, 11Ranade S.S. Qiu Z. Woo S.H. Hur S.S. Murthy S.E. Cahalan S.M. et al.Piezo1, a mechanically activated ion channel, is required for vascular development in mice.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10347-10352Crossref PubMed Scopus (552) Google Scholar), blood pressure control (12Yang X. Zeng H. Wang L. Luo S. Zhou Y. Activation of Piezo1 downregulates renin in juxtaglomerular cells and contributes to blood pressure homeostasis.Cell Biosci. 2022; 12: 197Crossref PubMed Scopus (6) Google Scholar), and voiding function (13Ihara T. Mitsui T. Shimura H. Tsuchiya S. Kanda M. Kira S. et al.Different effects of GsMTx4 on nocturia associated with the circadian clock and Piezo1 expression in mice.Life Sci. 2021; 278119555Crossref PubMed Scopus (9) Google Scholar, 14Dalghi M.G. Ruiz W.G. Clayton D.R. Montalbetti N. Daugherty S.L. Beckel J.M. et al.Functional roles for PIEZO1 and PIEZO2 in urothelial mechanotransduction and lower urinary tract interoception.JCI Insight. 2021; 6: e152984Crossref PubMed Scopus (33) Google Scholar) to name a few. Piezo1 deletion caused embryonically lethal phenotype in mice due to profound deficiencies in the flow-dependent vascular remodeling during the development (10Li J. Hou B. Tumova S. Muraki K. Bruns A. Ludlow M.J. et al.Piezo1 integration of vascular architecture with physiological force.Nature. 2014; 515: 279-282Crossref PubMed Scopus (693) Google Scholar, 11Ranade S.S. Qiu Z. Woo S.H. Hur S.S. Murthy S.E. Cahalan S.M. et al.Piezo1, a mechanically activated ion channel, is required for vascular development in mice.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10347-10352Crossref PubMed Scopus (552) Google Scholar). On the other hand, gain-of-function Piezo1 mutations have been linked to several clinically relevant syndromes, such as hereditary xerocytosis with fetal hydrops (15Albuisson J. Murthy S.E. Bandell M. Coste B. Louis-Dit-Picard H. Mathur J. et al.Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.Nat. Commun. 2013; 4: 1884Crossref PubMed Scopus (249) Google Scholar, 16Beneteau C. Thierry G. Blesson S. Le Vaillant C. Picard V. Bene M.C. et al.Recurrent mutation in the PIEZO1 gene in two families of hereditary xerocytosis with fetal hydrops.Clin. Genet. 2014; 85: 293-295Crossref PubMed Scopus (26) Google Scholar). Piezo1 is broadly expressed in both excitable and non-excitable (most commonly epithelial) tissues, including the kidneys (4Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1770) Google Scholar). However, the physiological significance of Piezo1 in the renal tubule is just beginning to emerge. It was previously shown that conditional Piezo1 deletion does not affect urinary concentrating ability but delays urinary dilution upon rehydration (17Martins J.R. Penton D. Peyronnet R. Arhatte M. Moro C. Picard N. et al.Piezo1-dependent regulation of urinary osmolarity.Pflugers Arch. 2016; 468: 1197-1206Crossref PubMed Google Scholar). On the other hand, pathological upregulation of Piezo1 levels has been implicated in the progression of renal fibrosis (18Zhao X. Kong Y. Liang B. Xu J. Lin Y. Zhou N. et al.Mechanosensitive Piezo1 channels mediate renal fibrosis.JCI Insight. 2022; 7: e152330Crossref PubMed Scopus (34) Google Scholar). In the renal tubule, Piezo1 expression has been reported predominantly in the late segments from the cortical to inner medullary collecting ducts (17Martins J.R. Penton D. Peyronnet R. Arhatte M. Moro C. Picard N. et al.Piezo1-dependent regulation of urinary osmolarity.Pflugers Arch. 2016; 468: 1197-1206Crossref PubMed Google Scholar, 19Dalghi M.G. Clayton D.R. Ruiz W.G. Al-Bataineh M.M. Satlin L.M. Kleyman T.R. et al.Expression and distribution of PIEZO1 in the mouse urinary tract.Am. J. Physiol. Ren. Physiol. 2019; 317: F303-F321Crossref PubMed Scopus (64) Google Scholar), although a weaker diffuse staining was also detected in glomeruli (19Dalghi M.G. Clayton D.R. Ruiz W.G. Al-Bataineh M.M. Satlin L.M. Kleyman T.R. et al.Expression and distribution of PIEZO1 in the mouse urinary tract.Am. J. Physiol. Ren. Physiol. 2019; 317: F303-F321Crossref PubMed Scopus (64) Google Scholar) and proximal tubule (18Zhao X. Kong Y. Liang B. Xu J. Lin Y. Zhou N. et al.Mechanosensitive Piezo1 channels mediate renal fibrosis.JCI Insight. 2022; 7: e152330Crossref PubMed Scopus (34) Google Scholar). The collecting duct is comprised of two cell types: principal and intercalated (PCs and ICs, respectively) exhibiting distinct morphology and physiological roles (20Pearce D. Soundararajan R. Trimpert C. Kashlan O.B. Deen P.M. Kohan D.E. Collecting duct principal cell transport processes and their regulation.Clin. J. Am. Soc. Nephrol. 2015; 10: 135-146Crossref PubMed Scopus (207) Google Scholar, 21Roy A. Al-bataineh M.M. Pastor-Soler N.M. Collecting duct intercalated cell function and regulation.Clin. J. Am. Soc. Nephrol. 2015; 10: 305-324Crossref PubMed Scopus (155) Google Scholar). In particular, more abundant PCs are primarily involved in Na+-water reabsorption and K+ secretion, while ICs are critical for enacting acid–base transport (20Pearce D. Soundararajan R. Trimpert C. Kashlan O.B. Deen P.M. Kohan D.E. Collecting duct principal cell transport processes and their regulation.Clin. J. Am. Soc. Nephrol. 2015; 10: 135-146Crossref PubMed Scopus (207) Google Scholar, 21Roy A. Al-bataineh M.M. Pastor-Soler N.M. Collecting duct intercalated cell function and regulation.Clin. J. Am. Soc. Nephrol. 2015; 10: 305-324Crossref PubMed Scopus (155) Google Scholar). It is not currently known whether Piezo1 is functional in both cells types and whether it is regulated by the same or cell-type specific mechanisms. It is well appreciated that the collecting duct is subjected to the highest variations/changes in fluid flow rates and osmotic gradients depending on dietary electrolyte intake and water consumption (22Berrout J. Jin M. Mamenko M. Zaika O. Pochynyuk O. O'Neil R.G. Function of TRPV4 as a mechanical transducer in flow-sensitive segments of the renal collecting duct system.J. Biol. Chem. 2012; 287: 8782-8791Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Weinbaum S. Duan Y. Satlin L.M. Wang T. Weinstein A.M. Mechanotransduction in the renal tubule.Am. J. Physiol. Ren. Physiol. 2010; 299: F1220-1236Crossref PubMed Scopus (118) Google Scholar). These mechanical forces are known to induce comparable elevations in [Ca2+]i in both PCs and ICs (24Woda C.B. Leite Jr., M. Rohatgi R. Satlin L.M. Effects of luminal flow and nucleotides on [Ca(2+)](i) in rabbit cortical collecting duct.Am. J. Physiol. Ren. Physiol. 2002; 283: F437-446Crossref PubMed Google Scholar, 25Liu W. Xu S. Woda C. Kim P. Weinbaum S. Satlin L.M. Effect of flow and stretch on the [Ca2+]i response of principal and intercalated cells in cortical collecting duct.Am. J. Physiol. Ren. Physiol. 2003; 285: F998-F1012Crossref PubMed Google Scholar), which are instrumental in modulating water-electrolyte transport, for instance, by promoting the flow-induced K+ secretion (26Liu W. Morimoto T. Woda C. Kleyman T.R. Satlin L.M. Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct.Am. J. Physiol. Ren. Physiol. 2007; 293: F227-235Crossref PubMed Scopus (84) Google Scholar). However, published evidence suggests that Piezo1-reporting signal is largely accumulated on the basolateral side (17Martins J.R. Penton D. Peyronnet R. Arhatte M. Moro C. Picard N. et al.Piezo1-dependent regulation of urinary osmolarity.Pflugers Arch. 2016; 468: 1197-1206Crossref PubMed Google Scholar, 19Dalghi M.G. Clayton D.R. Ruiz W.G. Al-Bataineh M.M. Satlin L.M. Kleyman T.R. et al.Expression and distribution of PIEZO1 in the mouse urinary tract.Am. J. Physiol. Ren. Physiol. 2019; 317: F303-F321Crossref PubMed Scopus (64) Google Scholar), which does not align with its role as a flow-sensor. Indeed, we and others have previously shown that the activity of another mechanosensitive [Ca2+]i permeable channel, transitransient receptor potential vanilloid channel type 4 (TRPV4) is imperative for flow-dependent [Ca2+]i signaling (22Berrout J. Jin M. Mamenko M. Zaika O. Pochynyuk O. O'Neil R.G. Function of TRPV4 as a mechanical transducer in flow-sensitive segments of the renal collecting duct system.J. Biol. Chem. 2012; 287: 8782-8791Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and subsequently flow-induced K+ secretion in the collecting duct (27Mamenko M.V. Boukelmoune N. Tomilin V.N. Zaika O.L. Jensen V.B. O'Neil R.G. et al.The renal TRPV4 channel is essential for adaptation to increased dietary potassium.Kidney Int. 2017; 91: 1398-1409Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 28Stavniichuk A. Pyrshev K. Zaika O. Tomilin V.N. Kordysh M. Lakk M. et al.TRPV4 expression in the renal tubule is necessary for maintaining whole body K(+) homeostasis.Am. J. Physiol. Ren. Physiol. 2023; 324: F603-F616Crossref PubMed Scopus (3) Google Scholar, 29Pyrshev K. Stavniichuk A. Tomilin V.N. Zaika O. Pochynyuk O. Evolving concepts of TRPV4 in controlling flow-sensitivity of the renal nephron.Curr. Top. Membr. 2022; 89: 75-94Crossref PubMed Scopus (3) Google Scholar). Interestingly, a functional interplay was shown in different cell types, including acinar cells and chondrocytes, where an initial transient Ca2+ influx via Piezo1 could lead to sustained TRPV4 activation (30Swain S.M. Romac J.M. Shahid R.A. Pandol S.J. Liedtke W. Vigna S.R. et al.TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation.J. Clin. Invest. 2020; 130: 2527-2541Crossref PubMed Scopus (103) Google Scholar, 31Servin-Vences M.R. Moroni M. Lewin G.R. Poole K. Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes.eLife. 2017; 6: e21074Crossref PubMed Scopus (179) Google Scholar). It is not known whether a similar coupling exists between Piezo1 and TRPV4 in the collecting duct cells. The current study was undertaken to examine the significance of Piezo1 activation on [Ca2+]i signaling in native collecting duct cells. We focused on addressing the following research questions: (1) if Piezo1 can be found on both basolateral and apical membrane? (2) is there a functional interaction between mechanosensitive Piezo1 and TRPV4 in the collecting duct? (3) whether Piezo1 activity is comparable in PCs and ICs? and (4) what systemic stimuli modulate Piezo1 activity in these cell types? Previous studies using transgenic reporter mice showed the most abundant Piezo1 expression in the collecting duct (17Martins J.R. Penton D. Peyronnet R. Arhatte M. Moro C. Picard N. et al.Piezo1-dependent regulation of urinary osmolarity.Pflugers Arch. 2016; 468: 1197-1206Crossref PubMed Google Scholar, 19Dalghi M.G. Clayton D.R. Ruiz W.G. Al-Bataineh M.M. Satlin L.M. Kleyman T.R. et al.Expression and distribution of PIEZO1 in the mouse urinary tract.Am. J. Physiol. Ren. Physiol. 2019; 317: F303-F321Crossref PubMed Scopus (64) Google Scholar). In agreement, our immunofluorescence confocal microscopy found the strongest Piezo1 signal in AQP2-positive tubular segments in both cortical and medullary renal sections (Fig. 1). Here, Piezo1 signal was present on both apical and basolateral sides, whereas AQP2-negative tubular segments exhibited much weaker basolateral staining. We next probed whether activation of Piezo1 contributes to intracellular [Ca2+]i signaling in the collecting duct cells. Application of a selective channel agonist, Yoda-1 (20 μM for 5 min), induced a robust increase in [Ca2+]i in all cells within split-opened area of a freshly-isolated collecting duct (Fig. 2A). Interestingly, we detected two distinct patterns of cellular responses to the treatment with Yoda-1 with the majority of cells responding with a rapid large increase followed by a slow decline in [Ca2+]i, while the remaining population exhibited a slow progressive rise in [Ca2+]i. Postexperimental staining of the collecting duct with AQP2 revealed that the observed two populations corresponded to PCs and ICs, respectively (Fig. 2, A and B). The summary graph in Figure 2C shows significantly higher Yoda-1-induced [Ca2+]i elevations from the baseline (time-point 1) at the beginning (time-point 2) and at the end of application (time-point 3) in PCs than in ICs. As shown in Figure 3, application of another structurally nonrelated selective Piezo1 agonist, Jedi-2 (500 μM for 5 min) also elicited distinct responses in PCs (larger) and ICs (smaller). This directly implies that the observed differences in [Ca2+]i dynamics to Piezo1 stimulation are not attributable to a particular channel agonist.Figure 3Stimulation of Piezo1 with Jedi-2 induces greater [Ca2+]i responses in principal (PCs) than in intercalated (ICs) cells of the collecting duct. A, representative pseudocolor images (blue-low and red-high) of an isolated split-opened collecting duct loaded with Ca2+-sensitive dye fura-2 at the baseline (1), after 30 s (2), and 5 min (3) of 500 μM Jedi-2 application. Shown on the right is a confocal micrograph of the same split-opened collecting duct probed with anti-AQP2 (pseudocolor red) antibodies to discriminate PCs (highlighted with yellow arrows) and ICs (highlighted with white arrows). B, the averaged time courses of [Ca2+]i changes in PCs (black) and ICs (gray) upon application of Jedi-2 (shown with a bar on top). The time points shown in (A) are marked as 1 to 3. Number of individual cells is shown. Six different collecting ducts from three different mice were used for analysis. C, the summary graph comparing the magnitudes of Jedi-2-mediated [Ca2+]i elevations calculated as the difference in [Ca2+]i values before (time point 1), at the beginning (time point 2), and at the end (time point 3) of Jedi-2 application in individual PCs and ICs from the conditions in A. Bars and whiskers represent SE and SD, respectively. Mean and median values are denoted with a dot and a line, respectively. ∗ - significant decreases (p < 0.05, one-way ANOVA with post hoc Tukey test) between experimental groups shown with lines on the top. AQP2, aquaporine 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Yoda-1-induced [Ca2+]i elevations might involve both direct Ca2+ entry via Piezo1 as well as accompanying Ca2+ release from the endoplasmic reticulum (ER) intracellular stores. To further test this, removal of extracellular Ca2+ (buffered with 5 mM EGTA) abolished responses to Piezo1 stimulation in both PCs and ICs (Fig. 4A) suggesting that Yoda-1 triggered a direct Ca2+ influx across the plasma membrane. We next quantified responses to Yoda-1 upon inhibition of the endoplasmic Ca2+ ATPase with thapsigargin (4 μM) to deplete ER storages. As expected, thapsigargin led to a mild increase in basal [Ca2+]i levels, which was more pronounced in PCs than in ICs (Fig. 4B). However, this maneuver had no significant effect on the magnitude and different kinetics of Yoda-1-induced [Ca2+]i elevations in both PCs and ICs (Fig. 4C). This strongly argues against a notable role of the intracellular ER stores in the Ca2+ responses to Piezo1 stimulation with Yoda-1. Overall, our results in Figure 2, Figure 3, Figure 4 show that functional Piezo1 is present in all collecting duct cells. However, the Piezo1-dependent Ca2+ influx is markedly greater in PCs than in ICs. We next monitored subcellular localization of Piezo1 in split-opened collecting ducts with confocal immunofluorescence microscopy (Fig. 5) to gain insights on the distinct kinetic profiles of [Ca2+]i responses to Yoda-1 and Jedi-2 in PCs and ICs. As shown on the representative images of a 3-D scan, Piezo1-reporting signal is distributed on the apical and basolateral sites in all cells (Fig. 5A). Importantly, the intensity of the signal was significantly higher in AQP2-positive PCs than in AQP2-negative ICs within the same collecting duct, as summarized in Figure 5B. Thus, we show that the lower channel expression is in agreement with lesser [Ca2+]i responses to Piezo1 agonists in ICs (Figs. 2 and 4). Since fluorescence Piezo1-reporting signal is apparent at the apical and basolateral sides in the collecting ducts (Fig. 5), we next used patch clamp electrophysiology to inquire whether the channel is indeed functional at both membranes. For this, pipettes were backfilled with Yoda-1 (20 μM) allowing diffusion of the agonist to a patch delineated membrane surface. We observed a gradual activation of a silent or a near silent channel(s) in approximately 30% of cell-attached experiments (10 out of 30) performed on split-opened collecting ducts. A typical experiment is shown in Figure 6A. Further analysis revealed that this is a nonselective cation channel with fast kinetics and 18.6 ± 0.7 pS conductance (Fig. 6B). These biophysical properties are reminiscent of those reported for Piezo1 in native cells and over-expression systems (4Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. et al.Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1770) Google Scholar, 7Nosyreva E.D. Thompson D. Syeda R. Identification and functional characterization of the Piezo1 channel pore domain.J. Biol. Chem. 2021; 296100225Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). A Yoda-1-stimulated channel with identical properties was also recorded in cell-attached experiments on the basolateral membrane in freshly isolated collecting ducts (Fig. 6C). A summary graph in Figure 6D quantifies activation of Piezo1 by Yoda-1 in cell-attached experiments on the apical and basolateral sides. As is clear, the presence of Yoda-1 in a recording pipette exhibited a comparable activation of Piezo1 on both membranes, though the overall activity (NPo) was moderately higher on the basolateral side. We also attempted to distinguish PCs and ICs by morphology (polygonal and round shape, respectively (insets in Fig. 6, A and C), as described previously (32Madsen K.M. Tisher C.C. Structural-functional relationship along the distal nephron.Am. J. Physiol. 1986; 250: F1-F15Crossref Google Scholar, 33Rice W.L. Van Hoek A.N. Paunescu T.G. Huynh C. Goetze B. Singh B. et al.High resolution helium ion scanning microscopy of the rat kidney.PLoS One. 2013; 8e57051Crossref Scopus (65) Google Scholar). As highlighted in gray (Fig. 6D), Yoda-1 was less potent in ICs than in PCs, which is consistent with lower [Ca2+]i responses (Fig. 2) and expression (Fig. 5) in the former. Piezo1-dependent Ca2+ influx was shown to stimulate TRPV4 in different cell types (30Swain S.M. Romac J.M. Shahid R.A. Pandol S.J. Liedtke W. Vigna S.R. et al.TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation.J. Clin. Invest. 2020; 130: 2527-2541Crossref PubMed Scopus (103) Google Scholar, 31Servin-Vences M.R. Moroni M. Lewin G.R. Poole K. Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes.eLife. 2017; 6: e21074Crossref PubMed Scopus (179) Google Scholar). TRPV4 is a mechanosensitive Ca2+ permeable channel abundantly expressed in the collecting duct (22Berrout J. Jin M. Mamenko M. Zaika O. Pochynyuk O. O'Neil R.G. Function of TRPV4 as a mechanical transducer in flow-sensitive segments of the renal collecting duct system.J. Biol. Chem. 2012; 287: 8782-8791Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Pyrshev K. Stavniichuk A. Tomilin V.N. Zaika O. Pochynyuk O. Evolving concepts of TRPV4 in controlling flow-sensitivity of the renal nephron.Curr. Top. Membr. 2022; 89: 75-94Crossref PubMed Scopus (3) Google Scholar). Thus, we next probed potential functional coupling between Piezo1 and TRPV4 in split-opened collecting ducts using pharmacological and genetic tools. In the first set of experiments (Fig. 7A), we monitored Yoda-1 stimulation of Piezo1 in PCs and ICs, when TRPV4 activity was blocked by pretreatment with a mixture of selective antagonists HC-067047 (4 μM) and GSK2798745 (40 nM). To exclude an incomplete inhibition and/or putative adverse effects of the blockers, we also documented Piezo1 activation with Yoda-1 in split-opened collecting ducts isolated from TRPV4−/− mice (Fig. 7B). In both cases, we observed similar [Ca2+]i elevations in PCs (Fig. 7C) and ICs (Fig. 7D) at the peak (time-point 2) and at the end (time-point 3) of Yoda-1 application. It should be noted that there was a slight decrease in the basal [Ca2+]i levels due to the acute TRPV4 inhibition, as we reported previously (34Zaika O. Mamenko M. Berrout J. Boukelmoune N. O'Neil R.G. Pochynyuk O. TRPV4 dysfunction promotes renal cystogenesis in autosomal recessive polycystic kidney disease.J. Am. Soc. Nephrol. 2013; 24: 604-616Crossref PubMed Scopus (53) Google Scholar, 35Mamenko M. Zaika O.L. Boukelmoune N. Berrout J. O'Neil R.G. Pochynyuk O. Discrete control of TRPV4 channel function in the distal nephron by protein kinases A and C.J. Biol. Chem. 2013; 288: 20306-20314Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 36Tomilin V. Reif G.A. Zaika O. Wallace D.P. Pochynyuk O. Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca(2+)]i homeostasis in human autosomal-dominant polycystic kidney disease cells.FASEB J. 2018; 32: 4612-4623Crossref PubMed Scopus (18) Google Scholar). Thus, the amplitude of Yoda-1-induced [Ca2+]i elevations were assessed from this new baseline. Interest
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