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TRPV4 Dysfunction Promotes Renal Cystogenesis in Autosomal Recessive Polycystic Kidney Disease

Oleg Zaika, Mykola Mamenko, Jonathan Berrout, Nabila Boukelmoune, Roger G. O’Neil, and Oleh Pochynyuk

Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas

ABSTRACT The molecular mechanism of cyst formation and expansion in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood, but impaired mechanosensitivity to tubular flow and dysfunctional calcium signaling are important contributors. The activity of the mechanosensitive Ca2+-permeable TRPV4 channel underlies flow-dependent Ca2+ signaling in murine collecting duct (CD) cells, suggesting that this channel may contribute to cystogenesis in ARPKD. Here, we developed a method to isolate CD-derived cysts and studied TRPV4 function in these cysts laid open as monolayers and in nondilated split-open CDs in a rat model of ARPKD. In freshly isolated CD-derived cyst monolayers, we observed markedly impaired TRPV4 activity, abnormal subcellular localization of the channel, disrupted TRPV4 glycosylation, decreased 2+ 2+ basal [Ca ]i,andlossofflow-mediated [Ca ]i signaling. In contrast, nondilated CDs of these rats exhibi- ted functional TRPV4 with largely preserved mechanosensitive properties. Long-term systemic augmen- tation of TRPV4 activity with a selective TRPV4 activator significantly attenuated the renal manifestations of ARPKD in a time-dependent manner. At the cellular level, selective activation of TRPV4 restored mecha- nosensitive Ca2+ signaling as well as the function and subcellular distribution of TRPV4. In conclusion, the functional status of TRPV4, which underlies mechanosensitive Ca2+ signaling in CD cells, inversely corre- lates with renal cystogenesis in ARPKD. Augmenting TRPV4 activity may have therapeutic potential in ARPKD.

J Am Soc Nephrol 24: 604–616, 2013. doi: 10.1681/ASN.2012050442

Polycystickidneydisease(PKD)isacohortofmono- PC2, possibly participating in mechanotransduc- genic disorders that result in development and tion.10–12 1–5 2+ subsequent growth of renal cysts filled with fluid. It is accepted that the CD cells elevate [Ca ]i in Cyst enlargement compromises function of sur- response to mechanical stress arising from varia- rounding nephrons and progresses to ESRD.1,6 In tions in tubular flow or tubular composition.13–23 2+ the more common form of PKD, autosomal dom- Impaired mechanosensitive [Ca ]i responses, reported inant PKD (ADPKD), which is caused by muta- for both cultured ADPKD24 and ARPKD25,26 tions of (PC1) and (PC2), renal cysts are formed along the full length of the nephron with prevalence to the collecting Received May 2, 2012. Accepted December 19, 2012. duct (CD).1,7 In the rarer and more severe autosomal O.Z. and M.M. contributed equally to this work. recessive PKD (ARPKD), renal cyst formation is Published online ahead of print. Publication date available at 1,2,5,8 virtually restricted to the CD. of www.jasn.org. the PKHD1 encoding fibrocystin underlie Correspondence: Dr. Oleh Pochynyuk, Department of Inte- 6,8,9 the genetic basis of the disease. Although the grative Biology and Pharmacology, University of Texas Health exact function of the protein is unknown, fibro- Science Center at Houston, 6431 Fannin, Houston, TX 77030. cystin was shown to be expressed in primary cilia Email: [email protected] where it can interact and form complexes with Copyright © 2013 by the American Society of Nephrology

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2+ cells, point to a possible fundamental role of disrupted [Ca ]i also suggests pharmacologic potential of targeting TRPV4 ac- signaling in cystogenesis. The central cilia and cilia-associated tivity as a treatment strategy in retarding development of PC1 and PC2 were proposed to mediate flow-induced cellular ARPKD. responses.19,27 However, homomeric PC2 channels are not 2+ mechanosensitive and fail to increase [Ca ]i in response to flow and hypotonicity.28,29 Furthermore, intercalated cells, which RESULTS lack primary cilia, respond to flow changes with comparable 2+ 2+ increases in [Ca ]i as observed in principal cells, which have Accumulatingevidencepointstoimpairedflow-dependentCa primary cilia.16,30 Therefore, additional mechanisms confer- signalization in cultured ARPKD cells.25,26 To assess mechano- ring mechanosensitivity to the CD cells need to be considered. sensitive properties of native cyst cells, we developed and im- Transient receptor potential (TRP) channels are known to plemented mechanical isolation of CD-derived cyst monolayers participate in cellular responses to a variety of environmental from 2-month-old ARPKD PCK453 rats. This novel technique 2+ stimuli, including thermosensation, chemosensation, and enables real-time monitoring changes in [Ca ]i at the single mechanical forces (reviewed in Song and Yuan31). Several cell level in native tissue in response to mechanical and phar- TRP channels, including TRPC3, TRPC6, and TRPV4, can macologic inputs (Figure 1). CD origin of the isolated cyst frag- be detected in the native CD cells and CD-originated cultured ments was confirmed using with AQP2, as discussed in lines.19,32–34 Among these channels, TRPV4 has routinely the Concise Methods. We used freshly isolated split-opened been shown to be activated by mechanical stimuli.34–38 In- CDs from age-matched Sprague-Dawley (S/D) rats as controls. deed, we documented that endogenous TRPV4 in M-1 CD An abrupt 103 elevation in flow (producing shear stress of cells is stimulated by increases in flow, a response that is abol- approximately 3 dyn/cm2, as discussed in the Concise Methods) ished by TRPV4 small interfering RNA knockdown.34,38 on the apical surface caused a rapid and sustained increase (at 2+ 2+ We further demonstrated a lack of flow-mediated [Ca ]i el- least for 10 minutes) (Supplemental Figure 1) in [Ca ]i in evations in CD from TRPV42/2 mice.30 Consistently, flow- control CD cells, whereas it had a minimal effect on cyst cells 2+ + 2+ mediated Ca -dependent K secretion in the CD is disrupted (Figure 2A). Importantly, basal [Ca ]i levels are prominently in TRPV4 knockout animals.39 TRPV4 directly interacts with decreased in ARPKD cells. PC2 to form mechanosensitive heteromeric complexes.28,29 ThefactthatPC2interactswithbothPC140 and fibrocys- tin10–12 suggests that TRPV4 could be an essential part of this mechanotransducing sensory complex. Current PKD management is directed toward pharmaco- logic interference with abnormal signaling pathways causing exaggeratedcell proliferation,dedifferentiation, apoptosis,and cyst growth.41 Specifically, PKD is associated with elevated circulating vasopressin levels, increased basal cellular cAMP levels, and strong upregulation of cAMP-dependent fluid se- cretion and proliferation.42,43 V2 antagonism greatly dimin- ishes disease progression in rodent models of both ADPKD and ARPKD.42,44 Elevated cAMP levels might be directly re- 2+ lated to the reduced [Ca ]i, possibly due to impaired ability to sense changes in flow.42,44,45 This raises the possibility that manipulation with the mechanosensitivity in the CD along 2+ the TRPV4 axis modulates [Ca ]i signalization and, in turn, renal cystogenesis. In this study, we developed a new approach to isolate native CD-derived cyst monolayers and nondilated CDs from a rat model of ARPKD to thoroughly investigate how functional 2+ TRPV4 status determines the development and growth of renal Figure 1. Ca imaging in individual cells of normal CDs and CD- cysts. We found that the disease leads to disruption of derived cyst monolayers of ARPKD. The top row shows repre- mechanosensitive [Ca2+] signaling and impaired TRPV4 ac- sentative micrographs of a typical kidney slice (left) from a control i Sprague-Dawley (S/D) rat and respective split-opened CD after tivity specifically in CD cysts but not in nondilated CDs. Long- loading with Fura-2 taken with bright-field illumination (middle) term pharmacologic potentiation of TRPV4 activity gradually and 380 nm excitation (right). The bottom row shows represen- restores mechanosensitivity in cyst cells and greatly blunts re- tative micrographs of a kidney slice (left) from a PCK453 rat and nal ARPKD progression. From a global prospective, this study respective freshly isolated CD-derived cyst monolayer after establishes a temporal link between disruption of TRPV4- loading with Fura-2 taken with bright-field illumination (middle) based mechanosensitivity in the CD and cystogenesis. This and 380 nm excitation (right).

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Lack of flow-mediated Ca2+ responses 2+ and diminished basal [Ca ]i levels in ARPKD cyst cells indicate TRPV4 dysfunc- tion. Indeed, stimulation of TRPV4 with a highly selective activator, GSK1016790A 2+ (30 nM), caused [Ca ]i elevations in con- trol CDs that were four times greater than in cyst cells (Figure 2D). GSK1016790A had no effect when TRPV4 was inhibited with red (Supplemental Figure 2). We concluded that functional activity of TRPV4 is drastically diminished in ARPKD cyst cells contributing to the impaired flow-mediated Ca2+ signalization. TRPV4 dysfunction in cyst monolayers may result from decreased levels of the protein. Figure 3A shows a representative Western blot monitoring TRPV4 expression in whole kidney homogenates from S/D and PCK453 rats. As expected, TRPV4 antibod- ies recognized both nonglycosylated (lower) and glycosylated (upper) bands around 95 fl 2+ Figure 2. Cyst cells in ARPKD have disrupted ow sensitivity, decreased [Ca ]i,and kD in S/D rats. Renal TRPV4 levels were impaired TRPV4 activity. (A) The average time course of [Ca2+] changes in individual i modestly but significantly reduced in CD cells (black) and CD-derived cyst cells (gray) in response to an abrupt 103 ele- vation in flow (for 90 seconds) from apical side (shown with a bar on top). (B) The PCK453 rats. Strikingly, the glycosylated 2+ form of TRPV4 was virtually absent in these average time course of [Ca ]i changes in individual cells within split-opened area of CDs from Sprague-Dawley (S/D) rats in response to elevated flow (gray bars) in the animals (Figure 3). We concluded that control conditions and in the presence of TRPV4 inhibition with 4 mM of HC067047 post-translational modification of TRPV4 (black bar). All other conditions are the same as in A. (C) The left side shows a summary is compromised during ARPKD. graph of the magnitudes of flow-induced Ca2+ responses in CD cells from S/D rats We next probed subcellular TRPV4 (black bars) in the control, after pretreatment with 4 mM of HC067047 and 2 mMof distribution in control split-opened CDs (RuR), and in CD-derived cyst cells from PCK453 rats (gray bar). The from S/D rats and CD-derived cyst mono- 2+ right side shows the summary graph of the basal [Ca ]i in CD cells from S/D rats (black layers from PCK453 rats with immu- m bars) in the control and after pretreatment with 4 M of HC067047 and in CD-derived nofluorescence. TRPV4 is distributed in cyst cells from PCK453 rats (gray bar). *Significant decrease versus S/D rats. (D) The 2+ subapical cytosolic compartments and at average time course of [Ca ]i changes in individual CD cells from S/D rats (black) and cyst cells within CD-derived cyst monolayers (gray) in response to 3-minute application the apical plasma membrane, with stronger of TRPV4 activator GSK1016790A (30 nM) followed by RuR (2 mM). expression in AQP2-positive cells (Figure 4A; see Supplemental Figure 3 for a lower magnification). Surprisingly, we detected a Wedemonstrated that geneticdeletion of the Ca2+-permeable striking shift of the TRPV4-reporting fluorescent signal to- 2+ TRPV4 channel abolished flow-mediated [Ca ]i elevations in ward the apical membrane in CD-derived cyst monolayers CD cells of mice.30 To probe whether TRPV4 is also critical for (Figure 4B; see Supplemental Figure 3 for a lower magnifica- mechanosensitive properties in rat CD cells, we took advantage tion). We also observed AQP2 targeting to the apical side, of a potent and selective TRPV4 antagonist, HC067047. Acute which is consistent with reported upregulation of cAMP levels treatment with HC067047 (4 mM) nearly abolished elevations in ARPKD cells.42,46 For quantitative estimation, we utilized 2+ in [Ca ]i to the mechanical stimulation (Figure 2B). Pretreat- line-scan analysis (Figure 4C) of TRPV4-reporting fluorescent ment with a less selective TRPV4 antagonist, Ruthenium red signal distribution along the z-axis in cross-sections of three- (2 mM) yielded similar results (Figure 2C). Inhibition dimensional stacks similar to that shown in Figure 4, A and B. of TRPV4 with HC067047 also significantly decreased basal Cyst cells clearly exhibit a leftward narrowing of the bell-shape 2+ [Ca ]i levels (Figure 2B). These results suggest a key role of distribution of TRPV4 along the z-axis toward the apical TRPV4 in mediating flow-dependent Ca2+ responses and plasma membrane. As summarized in Figure 4D, the half- 2+ shaping basal [Ca ]i in rat CD cells. Importantly, pharma- width was significantly reduced from 2.6360.12 mminthe cologic inhibition of TRPV4 recapitulates the state of com- control to 1.8960.03 mm in CD and cyst cells, respectively. 2+ promised [Ca ]i signalization observed in cyst cells (Figure To exclude the possibility that isolation of CD-derived cysts 2, A and C). might potentially cause subcellular TRPV4 redistribution,

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PCK453 rats. Exogenous ATP (10 mM) 2+ induced a transient [Ca ]i peak followed by a sustained plateau (Figure 7). The mag- nitude of the initial peak was indeed mark- edly increased in cyst cells compared with the control CD cells from S/D rats (Figure 7). However, the plateau phase was nearly absent in ARPKD cells. In nondilated CDs from PCK453 rats, ATP produced Ca2+ response undistinguishable from that ob- served in control CDs (Figure 7A). We re- Figure 3. Renal TRPV4 levels are modestly decreased and TRPV4 glycosylation is cently identified a dominant role of TRPV4 2+ disrupted in ARPKD rats. (A) Representative Western blot from whole kidney lysates of in generating ATP-induced Ca plateau in Sprague-Dawley (S/D) and PCK453 rats simultaneously probed with anti-TRPV4 and murine CD cells.18 The absence of the pla- anti-actin antibodies. Samples are run in triplicate. (B) Summary graph comparing total teau phase in cyst cells supports the con- renal TRPV4 expression (left), glycosylated (middle), and nonglycosylated (right) forms ception of TRPV4 dysfunction in ARPKD. of TRPV4 in S/D and PCK453 rats from Western blots similar to that shown in A. Intensities These results suggest malfunctioning of of TRPV4-reporting bands were normalized to the intensities of the respective actin bands. purinergic signaling in CD-derived cysts fi *Signi cant decrease versus S/D rats. g, glycosylated form of TRPV4. but not in nondilated CDs. Our results point to a severe TRPV4 we monitored TRPV4 localization in kidney sections from S/D dysfunctioninCD-derivedcystsofARPKD.Physiologic and PCK453 rats (Figure 5). TRPV4 was preferentially local- stimuli, including flow-mediated shear stress and activation ized to the AQP2-positive distal nephrons and cysts (respec- of purinergic cascade, fail to effectively stimulate TRPV4 to tive top rows in Figure 5). Higher-resolution images of the elicit proper mechanosensitive response. Moreover, impaired same respective kidney sections revealed that TRPV4- and TRPV4 functional status contributes to the decreased resting 2+ AQP2-reporting fluorescent signals are restricted to the api- [Ca ]i levels (Figures 2 and 7). However, TRPV4 can still be cal/subapical domain of cyst cells (Figure 5B, middle row). activated to some extent by strong pharmacologic inputs, such This is consistent with the results obtained in freshly isolated as GSK1016790A (Figure 2D). We hypothesized that augmen- cyst fragments (Figure 4B). In contrast, we detected diffuse tation of the mechanosensitivity on the TRPV4 axis will subcellular distribution of TRPV4 and AQP2 in nondilated interfere with ARPKD progression. Indeed, a recent report CDs (Figure 5B, bottom row). These expression patterns are provided initial evidence that prolonged injection of clearly different from observed in cyst cells and are reminis- GSK1016790A to PCK453 rats decreases renal cystic area.49 cent of those observed in control CD cells from S/D rats (Fig- Therefore, we next tested if GSK1016790A treatment inter- ure 5A). feres with ARPKD progression by restoring mechanosensitive Different subcellular TRPV4 distribution in nondilated CD properties of the CD-derived cyst cells. cells and cyst cells might indicate altered TRPV4 functionality. Systemic administration of GSK1016790A (3 mgzkg body Thus, we assessed mechanosensitive Ca2+ signalization and weight per day) for 1 and 2 months (Figure 8, A and B) to 1- functional TRPV4 status in nondilated CDs from PCK453 month-old PCK453 rats diminished renal cyst development 2+ rats. We detected prominent [Ca ]i response to elevations and growth compared with the control vehicle-treated group. 2+ in luminal flow and unchanged basal [Ca ]i values (Figure Specifically, we detected a significant reduction of relative cyst 6, A and B). Activation of TRPV4 with GSK1016790A also area (Figure 8C; also see low-powered section slices in Sup- 2+ produced a similar rise in [Ca ]i as in control CD cells plemental Figure 5) at both time points. We still observed the from Sprague-Dawley rats (Figure 6C and Supplemental Fig- disease progression in GSK1016790A-treated animals be- ure 4). Consistently, subcellular TRPV4 and AQP2 distribu- tween 1 and 2 months of treatment, although at a slower tion in freshly isolated nondilated CDs was not different from rate compared with that in vehicle-treated animals. The kid- that in control CDs as visualized with immunofluorescence ney/total body index was also reduced by GSK106790A treat- (Figure 6D). Therefore, the results in Figure 6 strongly argue ment (Figure 8D). In contrast, GSK1016790A (1 month) had for unchanged mechanosensitivity in nondilated CDs. no significant effect on kidney/total body index in control S/D Substantial experimental evidence supports a critical role of rats (0.8960.08 versus 0.8660.04 for treated and untreated 2+ paracrine ATP release in facilitating [Ca ]i responses in the animals, respectively). CD.21 Development of ARPKD results in a virtually closed We next examined the ramifications of systemic 2+ environment inside renal cysts, creating favorable conditions GSK1016790A administration on [Ca ]i signaling in cells for amplification of the purinergic signal.14,47,48 Thus, we next from residual CD-derived cysts. We detected gradual restora- 2+ characterized the functional status of purinergic cascade in tion of the flow-mediated [Ca ]i responses and return of 2+ CD-derived cyst monolayers and nondilated CDs from resting [Ca ]i levels to control values after 1 and 2 months

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We next subjected CD-derived cyst monolayers of GSK1016790A-treated PCK453 rats to immunofluorescence. Sys- temic GSK1016790Atreatment for 1month greatly reversed the apical TRPV4 trans- location observed in cysts from untreated animals of the same age (Figure 10; see Fig- ure 4B for comparison). Specifically, we detected a shift of the TRPV4-reporting fluorescent signal from apical membrane to cytosol. We also detected redistribution of AQP2 from the apical membrane, indicating a possible decrease in cellular cAMP levels. Treatment with GSK1016790A for 2 months yielded similar results (Supple- mental Figure 6). However, GSK1016790A fails to restore TRPV4 glycosylation in the kidney of PCK453 rats (Supplemental Fig- ure 7). Total TRPV4 levels were also not al- tered in GSK1016790A-treated PCK453 rats (87%63% versus 100%62% for trea- ted and untreated rats, respectively). We concluded that TRPV4 distribution in cyst cells of GSK1016790A-treated animals mainly recapitulates TRPV4 localization in CD cells of control Sprague-Dawley rats (Figure 4A). This occurs independently of significant changes in renal TRPV4 levels and TRPV4 glycosylation status.

DISCUSSION Figure 4. Subcellular TRPV4 distribution is shifted toward the apical membrane in cyst cells. Representative confocal plane micrographs (axes are shown) and corresponding This study provides two major advances in cross-sections (pointed by arrows) showing three-dimensional stacks of TRPV4 locali- our understanding of the relation between zation (anti-TRPV4, pseudocolor green), AQP2 localization (anti-AQP2, pseudocolor mechanosensitivity and cystogenesis in the red), and the combined image for a split-opened CD from a wild-type Sprague-Dawley CD. First, we demonstrate that the defect in (S/D) rat (A) and a CD-derived cyst monolayer from a PCK453 rat (B). Nuclear DAPI the fibrocystin gene inPCK453ratsdoesnot staining is shown by pseudocolor blue. (C) The distribution of averaged relative fl fluorescent signals representing TRPV4 localization along a line on z-axis in CD and cyst lead to immediate disruption of ow sen- cells. For each individual cell, the fluorescent signals are normalized to their corre- sitivity andcompromised TRPV4activity in sponding maximal value. The position of the apical and basolateral sides is shown with epithelial cells of nondilated CDs. Thus, the 2+ arrows at the top. (D) The summary graph of half-width means for distributions of intact mechanosensitive [Ca ]i signaliza- fluorescent signals shown in C. #Significant decrease versus control CDs. a, apical side; tion might have a permissive role for nor- b, basolateral side; DAPI, 4’,6-diamidino-2-phenylindole. mal tubular function and its disruption specifically occurs before, or as an early event in, cyst development. Second, we provide of treatment, respectively (Figure 9A). We also found progres- evidence that restoration of mechanosensitivity and TRPV4 2+ sive augmentation of [Ca ]i response to an acute application function in cyst cells drastically diminishes ARPKD progression. 2+ of GSK1016790A (Figure 9B). This suggests enhancement of This strongly supports the concept that flow-dependent [Ca ]i functional TRPV4 status in GSK1016790A-treated animals, signaling plays an important modulatory role in renal cyst de- likely due to TRPV4 sensitization. Consistently, we detected velopment. However, it does not seem that the disruption of appearance of the ATP-induced Ca2+ plateau (Figures 9C). mechanosensitivity per se initiates cystogenesis because Overall, we concluded that prolonged systemic administration TRPV42/2 mice50 and zebrafish29 do not have renal cysts. 2+ of GSK1016790A recovers the bulk of mechanosensitive We consistently observed diminished basal levels of [Ca ]i 2+ [Ca ]i signalinginCD-derivedcystcells. in native CD-derived cyst cells of ARPKD rats (Figures 2 and

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2+ due to augmentation of basal [Ca ]i (Figures 4 and 10). This explains, at least in part, the anticystogenic effect of GSK1016790A (Figure 8 and Supplemental Figure 5). The ob- servation that systemic GSK1016790A treatment enhances TRPV4 activity requires a comment. Unlike many other Ca2+- permeable channels, TRPV4 can be activated by modest eleva- 2+ tions in [Ca ]i via mechanisms involving direct binding of to the channel.52 Initial small activation of TRPV4 by GSK1016790A provides a source for such increases 2+ in [Ca ]i leading to sustained potentiation of TRPV4 activity. 2+ On a longer timescale, elevations of [Ca ]i will also promote phosphorylation of TRPV4 by Ca2+-dependent protein kinase C isoforms further activating the channel.53 In contrast, an exces- 2+ sive increase in [Ca ]i will lead to inhibition/desensitization of TRPV4 via a negative feedback mechanism52 protecting cells 2+ from the cytotoxic effects of elevated [Ca ]i. We demonstrate that TRPV4 functionality determines the 2+ status of flow-mediated [Ca ]i signaling in the CD. Diminished 2+ TRPV4 activity is associated with the lack of flow-sensitive [Ca ]i elevations in freshly isolated CD-derived cyst monolayers, whereas TRPV4 sensitization after systemic GSK1016790A 2+ treatment rejuvenates [Ca ]i responses to elevated flow in cyst cells. This enables us to correlate changes in mechanosen- sitivity at the cellular level with systemic progression of ARPKD.WefoundthatTRPV4dysfunctionincystcells is not likely associated with decreased TRPV4 protein levels Figure 5. Subcellular TRPV4 localization is different in cysts and nondilated CDs in PKC453 rats. (A) Immunofluorescence of (Figure 3) but rather with impaired ability of the channel to a kidney section from Sprague-Dawley (S/D) rat. A representative be activated (Figure 2D). Our results point to dramatically re- low-magnification 10-mm-thick kidney section (top row) shows duced glycosylation of TRPV4 in PCK453 rats (Figure 3). staining patterns for TRPV4 (pseudocolor green), AQP2 (pseu- Interestingly, a recent study demonstrated that mutations in docolor red), and the combined image. The area defined by TRPV4 associated with diminished glycosylation abolished re- a rectangle is shown below with a higher magnification. (B) Im- sponses to environmental stimuli (such as hypotonicity) and munofluorescence of kidney section from a PCK453 rat. A rep- decreased responses to GSK1016790A.54 Such impaired post- resentative low-magnification 10-mm-thick kidney section (top translational TRPV4 processing has been implicated in the de- row) shows staining patterns for TRPV4 (pseudocolor green), velopment of familial digital arthropathy-brachydactyly.54 fi AQP2 (pseudocolor red), and the combined image. Areas de ned Interestingly, the beneficial effects of TRPV4 activation by rectangles and containing a cyst (middle) and a nondilated CD during ARPKD might not be limited to the kidney. A recent (bottom) are shown below with a higher magnification. study identifies an antiproliferative role for TRPV4 in chol- angiocytes from ARPKD rats and TRPV4 activation has a tendency to decrease liver cysts.49 This study also provides 2+ 7). Similar decreases in resting [Ca ]i values were reported for initial observation that injection of GSK1016790A diminishes cultured human ADPKD and ARPKD cells.45 We further pro- renal cyst area. In our study, we manage, for the first time, vide strong experimental support to the conception that isolation of monolayers of CD-derived cysts to correlate 2+ TRPV4 activity is a critical determinant of basal [Ca ]i.In- TRPV4 function, mechanosensitivity of native CD cells, and 2+ hibition of TRPV4 with HC067047 decreases basal [Ca ]i renal cystogenesis at the timescale of ARPKD progression us- recapitulating the values observed in cyst cells that have ing per os administration of the TRPV4 activator, marked TRPV4 dysfunction (Figure 2, B–D). Restoration of GSK1016790A. However, it should be noted that excessive functional TRPV4 status in GSK1016790A-treated animals activation of TRPV4 by an acute administration of high doses 2+ brings [Ca ]i back to the levels detected in normal CD cells. of GSK1016790A increases permeability of the pulmonary 2+ 55 Decreased [Ca ]i values during PKD may contribute to the microvascular barrier leading to circulatory collapse. In con- abnormally elevated cAMP signaling.42,44,45 This is known to trast, gradual ingestion of small doses of the drug for up to 2 2 stimulate Cl secretion inside the cyst lumen and to promote months, as used in this study, is well tolerated and does not proliferation of cyst cells.51 By monitoring changes in AQP2 produce any detrimental side effects. subcellular distribution patterns, we provide evidence that This investigation also addresses the purinergic aspect of pharmacologic activation of TRPV4 blunts cAMP levels likely mechanosensitivity and its relation to TRPV4 function. ATP is

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2+ Figure 6. Mechanosensitivity and TRPV4 function are intact in nondilated CDs of PCK453 rats. (A) The average time course of [Ca ]i changes in individual cells from nondilated CDs (red) in response to an abrupt 103 elevation in flow (for 90 seconds) from the apical 2+ side (shown with a bar on top). The average time course of [Ca ]i changes in CD cells from Sprague-Dawley (S/D) rats (black) in re- sponse to elevated flow is reproduced from Figure 2A. (B) Summary graph of relative responses to elevated flow in nondilated CDs and CD-derived cysts. The mean amplitude of flow-mediated Ca2+ response in control CD cells from S/D rats was used for normalization. (C) Summary graph of relative responses to application of the selective TRPV4 activator GSK1016790A (30 nM) in nondilated CDs and CD-derived cysts. The responses were normalized to the mean value for CD cells from S/D rats. $Significant decrease versus CD PCK453. (D) Representative confocal plane micrographs and corresponding cross-sections (shown by arrows) of three-dimensional stacks of TRPV4 localization (pseudocolor green), AQP2 localization (pseudocolor red), and the combined image for a split-opened nondilated CD from PCK453 rat. a, apical side; b, basolateral side. constitutively released from CD cells and this process is pharmacologic target to manage PKD.58 Despite the fact that strongly potentiated by mechanical stimuli.21,56 It becomes paracrine ATP release might be an important component of increasingly appreciated that paracrine purinergic signaling mechanosensitivity in normal CDs,18 this paradoxical upreg- is an important facilitator of mechanosensitivity in the CD. ulation of purinergic signaling in PKD pathology does not Indeed, we demonstrated that genetic deletion of the major improve mechanosensitive properties of cyst cells. We found 2+ purinergic receptor, P2Y2, markedly blunts cellular responses that the initial transient ATP-induced [Ca ]i peak was ele- to elevated flow and hypotonicity.18 Remodeling kidney tissue vated in CD-derived cyst cells. In contrast, the sustained pla- during development of PKD creates a virtually closed environ- teau is nearly absent in cyst cells. This suggests that the chronic ment inside cysts that can trap ATP.14 Several reports suggest a potentiation of purinergic cascade in ARPKD will have a min- 2+ marked augmentation of purinergic cascade and an increased imal effect on [Ca ]i levels. We identified a dominant role of 47,48 2+ ATP concentration inside the cyst lumen in PKD. This TRPV4 in the generation of the ATP-induced [Ca ]i plateau potentiation was shown to have detrimental renal effects by and found that genetic deletion of TRPV4 nearly abolishes the 2 47,51,57 2+ 18 promoting Cl secretion and cyst growth. Pharmaco- sustained elevation of [Ca ]i in response to ATP. The ab- logic inhibition of purinergic signaling could be a putative sence of the plateau in cyst cells is consistent with TRPV4

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2+ 2+ Figure 7. Lack of ATP-induced Ca plateau in CD-derived cyst cells. (A) The average time course of [Ca ]i changes in response to 10- minute application of ATP (10 mM, shown with a bar) for CD cells from Sprague-Dawley (S/D) rats (black), cells of nondilated CD from 2+ PCK453 rats (red), and CD-derived cyst cells from PCK453 rats (gray). (B) Summary graph of the basal [Ca ]i values (basal), the magnitudes of ATP-induced Ca2+ peak (peak), and the absolute values of the sustained plateau phase (plateau) for cells described in Figure 6A. $Significant changes versus cyst PCK453. dysfunction and impaired mechanical perception and vice maintained on standard rodent regimen (Purina #5001) and had free versa, restoration of TRPV4 functionality in GSK1016790A- access to tap water. For some experiments, 1-month-old PCK453 rats treated animals causes appearance of ATP-induced Ca2+ pla- received either tap water or tap water containing GSK1016790A (3 teau (Figure 9C). Therefore, our results allow consolidating mgzkg body weight per day) for either 1 or 2 months. This concen- the upregulation of purinergic signaling inside cyst lumen and tration is 100 times lower than the reported lethal dose for an acute the loss of mechanosensitivity in ARPKD. intravenous injection of the substance.55 We did not observe any In summary, this study directly demonstrates the relation noticeable morbidity in the GSK1016790A-treated groups during between TRPV4 activity, mechanosensitivity in CD cells, and thetimecourseofthedrugadministration. After the end of the fi renal cyst development in ARPKD. By identifying TRPV4 treatment, rats were sacri ced by CO2 administration followed by dysfunction as an underlying cause for disrupted flow percep- cervical dislocation and the kidneys were immediately removed and tion by the cyst cells, we provide evidence that augmentation of weighted. TRPV4 status limits renal ARPKD manifestations. It is also temptingto speculatethatpharmacologic targeting of signaling Tissue Isolation pathways that possess stimulatory actions on TRPV4 would The procedure for isolation of the CDs from Sprague-Dawley rats and yield similar results. Finally, although our investigation focuses nondilated CDs from PCK453 rats suitable for Ca2+ imaging and exclusively on ARPKD, we anticipate that systemic enhance- immunofluorescence closely follows our previously reported rat ment of TRPV4 function will also be beneficial for treatment of and mouse protocols.18,59–61 Kidneys were cut into thin slices (,1 ADPKD because both pathologies are associated with dis- mm), with slices placed into ice-cold physiologic saline solution buff- 2+ rupted mechanosensitive [Ca ]i signalization. ered with HEPES (pH 7.4). The CD was visually identified by its morphologic features (pale color, coarse surface, and, in some cases, bifurcations) and was mechanically isolated from kidney slices by CONCISE METHODS microdissection using watchmaker forceps under a stereomicro- scope. Isolated CDs were attached to 535 mm coverglass coated Materials and Animals with poly-L-lysine. A coverglass containing CD was placed in a per- All chemicals and materials were from Sigma (St. Louis, MO), VWR fusion chamber mounted on an inverted Nikon Eclipse Ti micro- (Radnor, PA), and Tocris (Ellisville, MO) unless noted otherwise and scope and perfused with room temperature HEPES-buffered (pH were of reagent grade. Animal use and welfare adhered to the National 7.4) saline solution. CDs were split-opened with two sharpened mi- Institutes of Health Guide for the Care and Use of Laboratory Animals cropipettes, controlled with different micromanipulators, to gain ac- following a protocol reviewed and approved by the Institutional cess to the apical membrane. The nephrons were used within 1–2 Laboratory Animal Care and Use Committee of the Universityof Texas hours of isolation. Health Science Center at Houston. For experiments, 1- to 3-month- Using a similar approach, CD-derived cyst monolayers were gently old control Sprague-Dawley (S/D) and ARPKD PCK453 rats (Charles teased from open cyst cavities in kidney slices from PCK453 rats River Laboratories, Wilmington, MA) were used. Animals were using watchmaker forceps under a stereomicroscope. ARPKD cyst

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the microscope stage of an InCa Imaging Work- station (Intracellular Imaging Inc, Cincinnati, OH). Cells were imaged with a 203 Nikon Su- per Fluor objective and regions of interest drawn for individual cells. The Fura-2 fluorescence in- tensity ratio was determined by excitation (an average for approximately 300 ms) at 340 nm and 380 nm and calculating the ratio of the emission intensities at 511 nm in the usual man- ner every 5 seconds. We observed no significant Fura-2 bleaching and minimal Fura-2 leakage at both wavelengths during experiments. The changes in the ratio are converted to intracellu- lar Ca2+ concentrations using the calibration methods as previously described.18,59,62 At least three individual CDs or cysts from three rats were used for each experimental set.

Western Blot Analyses Immediately after dissection, kidneys were placed on ice, decapsulated, and homogenized in 3 volumes of ice-cold hypotonic lysis buffer containing 50 mM Tris, 1% Triton X-100, 5 mM EDTA (pH 7.4) supplemented with 1 mM PMSF and 2 mg/ml protease inhibitor cocktail (Com- plete Mini; Roche Diagnostics, Indianapolis, IN). Protein concentration was determined with a Bradford assay using IgG as a standard. The samples were diluted with hypotonic lysis buffer, denatured, and reduced in Laemmli buffer supplemented with 10 mM of dithio- threitol at +75°C for 10 minutes to obtain the Figure 8. Systemic treatment with the TRPV4 activator GSK1016790A attenuates renal final protein concentration of 4 mg/ml. The ARPKD manifestations. Representative kidney sections from PCK453 rats treated with samples (10 mg/lane) were separated on 9% vehicle and GSK1016790A for 1 month (A) and 2 months (B), respectively. (C) Summary graphs comparing relative visible cyst areas for kidney slices similar to that shown in polyacrylamide gels at 150 V for 1.75 hours A and B. (D) Summary graph of kidneys to total body weight ratios for PCK453 rats treated and transferred to nitrocellulose membrane with vehicle and GSK1016790A for 1 and 2 months. *Significant decrease versus vehicle. for 1.5 hours at 100 V. Subsequently, the nitro- cellulose membrane was incubated with anti- TRPV4 (1:500; Alomone Labs, Jerusalem, monolayers were further mechanically separated from surrounding Israel) and anti-actin (1:1000; Abcam, Cambridge, UK) primary anti- tissue/nephrons and attached with the basal side to 535mm bodies for 2 hours at room temperature. We previously tested spec- coverglass coated with poly-L-lysine. ificity of the TRPV4 antibodies in expression systems,38,63 cultured M1 cells,34 and TRPV42/2 mice.30 Upon washout (three times for 2+ 10 minutes in TBS-Tween), the membrane was incubated with [Ca ]i Measurements Unless otherwise noted, 2-month-old Sprague-Dawley and PCK453 peroxidase-conjugated goat anti-rabbit secondary antibodies rats were used for experiments. Intracellular calcium levels were (1:30,000; Bio-Rad, Hercules, CA) for 1 hour at room temperature. measured in individual cells of the split-opened CDs and CD-derived Blots were quantified using ImageJ 1.47 software (National Institutes cyst monolayers using Fura-2 fluorescence radiometric imaging as of Health, Bethesda, MD). The intensities for TRPV4 protein bands described previously.18,34,38 Split-opened CDs and cysts fragments were normalized to the intensities of the corresponding actin bands, were loaded with Fura-2 by incubation with 2 mM Fura-2/AM in a used as a loading control. All experiments were repeated three times. bath solution for 45 minutes at room temperature. Subsequently, the tissue samples were washed and incubated for an additional 10–15 Immunofluorescence and Renal minutes before experimentation. The CDs and cyst monolayers were Kidneys from PCK453 rats were briefly fixed in situ via intracardiac then placed in an open-top imaging study chamber (Warner RC-10) perfusion and subjected to standard immunohistochemical proce- with a bottom coverslip viewing window and the chamber attached to duresasdonebefore.30 Briefly, animals were anesthetized by

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2+ Figure 9. Mechanosensitive [Ca ]i signaling is restored in CD-derived cyst cells of GSK1016790A-treated PCK453 rats. The average 2+ time course of [Ca ]i changes in response to an abrupt 103 elevation in flow (A), application of GSK1016790A (30 nM) followed by 2 mM Ruthenium red (B), and application of 10 mM ATP (C) for individual cells within freshly isolated CD-derived cyst monolayers from PCK453 rats treated with GSK1016790A for 1 (green) and 2 months (blue), respectively. The time courses of control CD cells from Sprague-Dawley (S/D) rats (black) and CD-derived cyst monolayers from untreated PCK453 rats (gray) are reproduced from Figure 2A. Numbers of all individual recordings for each condition are shown in the respective color. Durations of treatments are shown on the top with bars.

washed in PBS. Sections were then blocked with 1% donkey serum, and incubated over- night at 4°C with primary antibody: anti- TRPV4 (1:500; Alomone Labs) and anti-AQP2 tagged with ATTO 550 (1:200; Alomone). Sec- tions were subsequently washed and incubated at room temperature for 3 hours with secondary antibody Cy2 anti-rabbit (1:500; Jackson Im- munoResearch, West Grove, PA). The tissue was then mounted in ProLong Gold antifade reagent with 4’,6-diamidino-2-phenylindole (Invitrogen, Carlsbad, CA). Figure 10. Systemic GSK1016790A treatment normalizes subcellular distribution of For histologic assessment of renal ARPKD TRPV4 and AQP2 in freshly isolated CD-derived cyst monolayers. Representative manifestation, kidney sections (30 mM) from confocal plane micrographs (axes are shown) and corresponding cross-sections (arrows) untreated and GSK106790A-treated PCK453 of three-dimensional stacks of TRPV4 localization (pseudocolor green), AQP2 locali- rats were obtained as described above. Sections zation (pseudocolor red), and the combined image for a CD-derived cyst monolayer were stained with hematoxylin and eosin with from PCK453 rat treated with GSK1016790A for 1 month. Nuclear staining with DAPI is standard procedures and used to quantify cystic ’ shown by pseudocolor blue. a, apical side; b, basolateral side; DAPI, 4 ,6-diamidino-2- area. Image analysis was performed using NIS phenylindole. elements 4.00 software (Nikon, Nelville, NY). At least four different sections from three different isoflurane inhalation and kidneys prepared by intracardiac perfusion animals were used for the assessment. by a fixation solution (20 ml, 4% paraformaldehyde and 0.1 M caco- Freshly isolated CD-derived cyst monolayers and split-opened dylate buffer in ice-cold PBS, pH 7.4). Kidneys were removed and CDs were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 placed in 4% paraformaldehyde overnight, and then mounted in minutes at room temperature (RT). After fixation the samples were tissue freezing medium (Tissue Tek; Sakura Finetek Inc., Torrance, permeabilized by addition of 0.1% Triton in PBS for 5 minutes and CA) and frozen at 230°C. Sagittal sections (10 mmthick)wereob- washed in PBS three times for 5 minutes. Nonspecificstainingwas tained, using an OTF 5000 cryostat (Bright Instrument, Huntingdon, blocked with 10% normal goat serum (NGS; Jackson ImmunoRe- UK). Sections were allowed to warm to room temperature and search) in PBS for 30 minutes at RT. After washing with PBS (three

J Am Soc Nephrol 24: 604–616, 2013 A Role of TRPV4 in ARPKD 613 BASIC RESEARCH www.jasn.org times for 5 minutes) the samples were incubated for 3 hours at RT in 3. Torres VE, Harris PC: Polycystic kidney disease in 2011: Connecting the dark with the mix of anti-TRPV4 (1:500; Alomone Labs), anti-AQP2 dots toward a polycystic kidney disease therapy. Nat Rev Nephrol 8: – tagged with ATTO 550 (1:100; Alomone Labs) in 1% serum + 0.1% 66 68, 2012 4. Torres VE, Harris PC: Mechanisms of disease: Autosomal dominant and Triton in PBS. Subsequently, samples were washed three times with recessivepolycystickidneydiseases.Nat Clin Pract Nephrol 2: 40–55, PBS and incubated for 1.5 hours at RT in dark with goat anti-rabbit quiz 55, 2006 IgG labeled with Alexa Fluor 488 (1:400 dilution; Invitrogen) in 1% 5. Wilson PD: Polycystic kidney disease. NEnglJMed350: 151–164, 2004 NGS + 0.1% Triton in PBS. After washing with PBS (three times for 5 6. Sweeney WE Jr, Avner ED: Molecular and cellular pathophysiology of min) the samples were stained with 4’,6-diamidino-2-phenylindole autosomal recessive polycystic kidney disease (ARPKD). Cell Tissue Res 326: 671–685, 2006 (300 nM concentration; Calbiochem, San Diego, CA) to visualize 7. Torres VE, Harris PC, Pirson Y: Autosomal dominant polycystic kidney nuclei. Subsequently the samples were dehydrated, and mounted disease. Lancet 369: 1287–1301, 2007 with permanent mounting media (Thermo Scientific, Pittsburg, 8. Bergmann C, Senderek J, Küpper F, Schneider F, Dornia C, Windelen E, PA). Labeled tissue samples were examined with an inverted Nikon Eggermann T, Rudnik-Schöneborn S, Kirfel J, Furu L, Onuchic LF, Eclipse Ti fluorescent microscope using a 403 Plan-Fluoroil-immersion Rossetti S, Harris PC, Somlo S, Guay-Woodford L, Germino GG, Moser (1.3 NA) objective. Samples were excited with 405, 488, and 561 nm M, Büttner R, Zerres K: PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD). Hum Mutat 23: 453–463, 2004 2 laser diodes and emission captured with a 16-bit Cool SNAP HQ 9. Wallace DP: Cyclic AMP-mediated cyst expansion. Biochim Biophys camera (Photometrics, Tucson, AZ) interfaced to a PC running NIS Acta 1812: 1291–1300, 2011 4.00 elements software. 10. Kim I, Fu Y, Hui K, Moeckel G, Mai W, Li C, Liang D, Zhao P, Ma J, Chen XZ, George AL Jr, Coffey RJ, Feng ZP, Wu G: Fibrocystin/polyductin Solutions modulates renal tubular formation by regulating polycystin-2 expres- Typical bath solution was as follows: 150 mM NaCl, 5 mM KCl, 1 mM sion and function. JAmSocNephrol19: 455–468, 2008 11. Kim I, Li C, Liang D, Chen XZ, Coffy RJ, Ma J, Zhao P, Wu G: Polycystin-2 CaCl2, 2 mM MgCl2, 5 mM , and 10 mM HEPES (pH 7.4). All expression is regulated by a PC2-binding domain in the intracellular reagents were applied by perfusing the experimental chamber at 1.5 portion of fibrocystin. JBiolChem283: 31559–31566, 2008 fl 2+ ml/min. To test the effect of elevated ow on [Ca ]i,therateof 12. Wang S, Zhang J, Nauli SM, Li X, Starremans PG, Luo Y, Roberts KA, perfusion was instantly increased from 1.5 ml/min (approximately Zhou J: Fibrocystin/polyductin, found in the same protein complex with

15 mm H2O) to 15 ml/min (approximately 80 mm H2O). Using a polycystin-2, regulates calcium responses in kidney epithelia. Mol Cell – parallel plate chamber, we recently estimated that this maneuver pro- Biol 27: 3241 3252, 2007 2 30 fi 13. Geyti CS, Odgaard E, Overgaard MT, Jensen ME, Leipziger J, duces shear stress of approximately 3 dyn/cm . This value ts well 2+ Praetorius HA: Slow spontaneous [Ca ] i oscillations reflect nucleotide within the physiologic range of shear stress present in the rat and release from renal epithelia. Pflugers Arch 455: 1105–1117, 2008 mouse CD as assessed previously.34,64 14. Hovater MB, Olteanu D, Welty EA, Schwiebert EM: Purinergic signaling in the lumen of a normal nephron and in remodeled PKD encapsulated Statistical Analyses cysts. Purinergic Signal 4: 109–124, 2008 All summarized data are reported as mean 6 SEM. Data were com- 15. Leipziger J: Control of epithelial transport via luminal P2 receptors. Am – pared using the t test or an one-way ANOVA as appropriate. P#0.05 J Physiol Renal Physiol 284: F419 F432, 2003 16. Liu W, Xu S, Woda C, Kim P, Weinbaum S, Satlin LM: Effect of flow and was considered significant. 2+ stretch on the [Ca ]i response of principal and intercalated cells in cortical collecting duct. Am J Physiol Renal Physiol 285: F998–F1012, 2003 17. Liu W, Morimoto T, Woda C, Kleyman TR, Satlin LM: Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting ACKNOWLEDGMENTS duct. Am J Physiol Renal Physiol 293: F227–F235, 2007 18. Mamenko M, Zaika O, Jin M, O’Neil RG, Pochynyuk O: Purinergic ac- This research was supported by grants from the American Heart tivation of Ca2+-permeable TRPV4 channels is essential for mechano- Association (SDG2230391 to O.P.) and the National Institute of Di- sensitivity in the aldosterone-sensitive distal nephron. PLoS ONE 6: abetes and Digestive and Kidney Diseases of the National Institutes of e22824, 2011 Health (DK095029 to O.P.), as well as a Carl W. Gottschalk research 19. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J: Polycystins 1 and 2 mediate scholar grant from the American Society of Nephrology (to O.P.). mechanosensation in the primary cilium of kidney cells. Nat Genet 33: 129–137, 2003 20. Praetorius HA, Leipziger J: Released nucleotides amplify the cilium- 2+ DISCLOSURES dependent, flow-induced [Ca ]i response in MDCK cells. Acta Physiol None. (Oxf) 197: 241–251, 2009 21. Praetorius HA, Leipziger J: Intrarenal purinergic signaling in the control of renal tubular transport. Annu Rev Physiol 72: 377–393, 2010 22. Satlin LM, Carattino MD, Liu W, Kleyman TR: Regulation of cation REFERENCES transport in the distal nephron by mechanical forces. Am J Physiol Renal Physiol 291: F923–F931, 2006 1. Harris PC, Torres VE: Polycystic kidney disease. Annu Rev Med 60: 321– 23. Woda CB, Leite M Jr, Rohatgi R, Satlin LM: Effects of luminal flow and 2+ 337, 2009 nucleotides on [Ca( )](i) in rabbit cortical collecting duct. Am J Physiol 2. Siroky BJ, Guay-Woodford LM: Renal cystic disease: The role of the Renal Physiol 283: F437–F446, 2002 primary cilium/centrosome complex in pathogenesis. Adv Chronic 24. Xu C, Shmukler BE, Nishimura K, Kaczmarek E, Rossetti S, Harris PC, Kidney Dis 13: 131–137, 2006 Wandinger-Ness A, Bacallao RL, Alper SL: Attenuated, flow-induced

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60. Staruschenko A, Pochynyuk O, Vandewalle A, Bugaj V, Stockand JD: 63. Jin M, Berrout J, Chen L, O’Neil RG: Hypotonicity-induced TRPV4 Acute regulation of the epithelial Na+ channel by phosphatidylinositide function in renal collecting duct cells: Modulation by progressive cross- 3-OH kinase signaling in native collecting duct principal cells. JAmSoc talk with Ca2+-activated K+ channels. Cell Calcium 51: 131–139, 2012 Nephrol 18: 1652–1661, 2007 64. Cai Z, Xin J, Pollock DM, Pollock JS: Shear stress-mediated NO pro- 61. Zaika O, Mamenko M, O’Neil RG, Pochynyuk O: acutely duction in inner medullary collecting duct cells. Am J Physiol Renal inhibits activity of the epithelial Na+ channel in mammalian aldosterone- Physiol 279: F270–F274, 2000 sensitive distal nephron. Am J Physiol Renal Physiol 300: F1105–F1115, 2011 62. Jin M, Wu Z, Chen L, Jaimes J, Collins D, Walters ET, O’Neil RG: De- terminants of TRPV4 activity following selective activation by small This article contains supplemental material online at http://jasn.asnjournals. molecule agonist GSK1016790A. PLoS ONE 6: e16713, 2011 org/lookup/suppl/doi:10.1681/ASN.2012050442/-/DCSupplemental.

616 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 604–616, 2013 SUPPLEMENTARY FIGURES

2+ Figure S1. The average time course of [Ca ]i changes in individual CD cells in response to an abrupt 10x elevation in flow (for 10 min) from apical side (shown with a bar on top).

2+ Figure S2. The average time course of [Ca ]i changes in individual cells of split-opened CD from S/D rats (A) and cyst cells within a CD-derived cyst monolayer (B) upon application of RuR (2 μM, shown with a black bar) and GSK1016790A (30 nM, grey bar).

Figure S3. Representative confocal plane micrographs (axes are shown) and corresponding cross-sections (pointed by arrows) showing 3D-stacks of TRPV4 localization (anti-TRPV4, pseudocolor green), AQP2 localization (anti-AQP2, pseudocolor red), and the combined image for a split-opened CD from WT (S/D) rat (A) and a CD-derived cyst monolayer from PCK453 rat (B). Nuclear DAPI staining is shown by pseudocolor blue. “a” and “b” indicate apical and basolateral sides, respectively.

2+ Figure S4. The average time course of [Ca ]i changes in individual cells of split-opened non- dilated CD from PCK453 rats in response to application of GSK1016790A (30 nM, grey bar) followed by RuR (2 μM, black bar).

Figure S5. Representative micrographs of four tissue slices from the same kidney for age matched PCK453 rats treated with vehicle (top row) and GSK1016790A (bottom row) for 1 month (A) and 2 months (B), respectively.

Figure S6. Representative confocal plane micrographs (axes are shown) and corresponding cross-sections (pointed by arrows) of 3D-stacks of TRPV4 (pseudocolor green), AQP2 (pseudocolor red), and the combined image for a CD-derived cyst monolayer from PCK453 rat treated with GSK1016790A for 2 months. Nuclear staining with DAPI is shown by pseudocolor blue. a and b indicate apical and basolateral sides, respectively.

Figure S7. Representative Western blot from whole kidney lysates of S/D and PCK453 rats in the control and after 1 month treatment with GSK1016790A (experimental groups are shown with respective lines on the top) simultaneously probed with anti-TRPV4 (upper panel) and anti-actin (bottom panel) antibodies. Samples were run as triples. “g” indicates glycosylated form of TRPV4.

Figure S1

160 flow 150

140 130 , nM i ]

2+ 120 [Ca

110 100 n=39 90 0 200 400 600 800 Time, sec Figure S2

S/D rats PCK453 rats A. B.

220 220 200 200 GSK1016790A GSK1016790A RuR 180 RuR 180 160 160 140 140 120 120 n=60 n=56 100 100 80 80 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Time, sec Time, sec XY YZ A. Figure S3 50 µM

S/D rats

a b XZ b

TRPV4 AQP2 merged B.

50 µM

PCK453 rats Figure S4

Non-dilated CDs from PCK453 rats

600 GSK1016790A RuR 500

, nM 400 i ] 2+ 300 [Ca

200 n=111 100

0 100 200 300 400 500 600 700 Time, sec Figure S5 A. 2 month PCK453 rats Control

3 mm

GSK1016790A (1 month treatment) Figure S5 B. 3 month PCK453 rats Control

3 mm GSK1016790A (2 month treatment) Figure S6

PCK453 rats treated with GSK1016790A for 2 months

XY YZ

50 µM 50 µM 8 µm

a b XZ b TRPV4 AQP2 merged Figure S7

S/D PCK453 S/D PCK453 +GSK1016790A +GSK1016790A kDa g TRPV4 95

actin 44