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Am J Physiol Renal Physiol 294: F909-F918, 2008. First published February 6, 2008; doi:10.1152/ajprenal.00606.2007
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Mediation of angiotensin II-induced Ca2+ signaling by polycystin 2 in glomerular mesangial cells

Juan Du,1,2 Min Ding,1 Sherry Sours-Brothers,1 Sarabeth Graham,1 and Rong Ma1

1Department of Integrative Physiology, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas; and 2Department of Physiology, Anhui Medical University, Hefei, Peoples Republic of China

Submitted 20 December 2007 ; accepted in final form 31 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
Ca+ influx across the plasma membrane is a major component of mesangial cell (MC) response to vasoconstrictors. Polycystin 2 (PC2), the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as a nonselective cation channel in a variety of cell types. The present study was performed to test the hypothesis that PC2 and its binding partners constitute a Ca2+-permeable channel and contribute to ANG II-induced Ca2+ signaling in MCs. Western blot and immunocytochemistry showed PC2 expression in cultured human MCs. The existence of PC2 in MCs was further confirmed by immunohistochemsitry in rat kidney sections. Coimmunoprecipitation displayed a selective interaction of PC2 with canonical transient receptor potential (TRPC) proteins TRPC1 and TRPC4. Cell-attached patch-clamp experiments revealed that ANG II-induced membrane currents were enhanced by overexpression of pkd2 but significantly inhibited by knock down of pkd2, 30 µM Gd3+ (a PC2 channel blocker), and dominant-negative pkd2 mutant (pkd2-D511V). Corresponding to the increase in channel currents, ANG II stimulation increased expression of PC2 on the cell surface of MCs and interaction with TRPC1 and TRPC4. Furthermore, ANG II-induced MC contraction was significantly reduced in pkd2-knocked down MCs. These data suggest that PC2 selectively assembles with TRPC1 and TRPC4 to form channel complexes mediating ANG II-induced Ca2+ responses in MCs.

calcium channel

GLOMERULAR MESANGIAL CELLS (MCs) are contractile cells that surround the glomerular capillaries of the kidney and regulate glomerular filtration rate by altering the surface area of the capillaries (24, 43). Ca2+ influx across the plasma membrane is critical for MC contraction in response to vasoactive peptides. A variety of ion channels contribute directly or indirectly to Ca2+ signaling in MCs and therefore regulate the contractile function of the cells. These include Ca2+-activated Cl– channels, volume-regulated Cl– channels, nonselective cation channels (NSCC), voltage-operated Ca2+ channels, receptor-operated Ca2+ channels, store-operated Ca2+ channels, and K+ channels (primarily Ca2+-activated K+ channel) (20). Despite intensive studies over past decades, the molecular entity of most Ca2+-conductive channels in MCs remains unclear. Our group (4, 42) recently found that selective members of the canonical transient receptor potential (TRPC) protein family were expressed in MCs and that some of these participated in agonist-stimulated Ca2+ entry, suggesting that specific TRPC proteins might be candidates for Ca2+-permeable channels in MCs. However, it is unknown whether there are any additional molecular candidates for these channels or whether the TRPC proteins require binding partners to form functional channels.

Polycystin 2 (PC2), a protein product of pkd2 gene mutation that causes autosomal dominant polycystic kidney disease (ADPKD), is an integral membrane protein sharing predicted membrane topology with TRPC channels (26, 27). Recent studies have demonstrated that PC2 functions as a Ca2+-permeable channel that mediates Ca2+ influxes in the plasma membrane (7, 10, 18, 48) and in the membrane of the endoplasmic reticulum (ER) (16) and primary cilia (28, 30, 41). We recently found that PC2 was a plasma membrane Ca2+ channel linked to receptor tyrosine kinase-phospholipase C signaling pathway in the epithelial cells of the renal proximal tubule (19). Studies in vascular smooth muscle cells from wild-type and pkd2+/– mice demonstrated that PC2-mediated Ca2+ signaling played an important role in proliferation and contraction of vascular smooth muscle cells (14, 36, 37). Because MCs are biophysically and phenotypically similar to vascular smooth muscle cells, we speculated that the PC2 channel might also participate in a Ca2+ response on agonist stimulation in glomerular MCs. In addition, previous studies revealed that PC2 required other membrane-associated proteins, such as PC1 or TRPC1, to form protein complexes for its channel function (10, 28, 30, 31, 46). In the present study, we further examined whether PC2 had any physical interactions with endogenous TRPC proteins in MCs in the present study. Our results indicate that PC2 protein was endogenously expressed in glomerular MCs and participated in agonist-stimulated Ca2+ entry and contraction of MCs. The PC2-involved channel might include two additional components, TRPC1 and TRPC4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
MC culture and transient transfection. Human MCs were purchased from Cambrex. MCs were subcultured to no more than 10 generations by standard methods (21). All plasmids were transiently transfected into MCs using Lipofectamine and Plus reagents (Invitrogen-BRL, Carlsbad, CA), following the protocols provided by the manufacturer.

Patch-clamp procedure. Conventional cell-attached voltage clamp was employed as described in our previous study (4). Single-channel analysis was made with a Warner PC-505B amplifier (Warner Instrument, Hamden, CT) and pCLAMP 9.2 (Axon Instruments, Foster City, CA). The extracellular solution contained (in mM) 135 NaCl, 5 KCl, 10 HEPES, 2 MgCl2, and 1 CaCl2. The pipette solution contained (in mM) 140 NaCl, 0.1 CaCl2, and 10 HEPES. At the time of the experiment, the pipette solution was supplemented with 100 µM niflumic acid and 10 µM MRS-1845 to block Ca2+-activated Cl– channels and store-operated Ca2+ channels, respectively. To exclude the influence of fluid flow on channel activity on ANG II infusion, the bathing solution continuously flowed throughout experiments. The flow rate was adjusted by gravity and controlled by a multiple channel perfusion system (ValveLink 8; Automate Scientific). Channel activity was indicated by single-channel open probability (NPO). Clampfit 9.2 software (Axon Instruments) was used to analyze single-channel currents.

Fluorescence measurement of intracellular Ca2+ concentration. Measurements of intracellular Ca2+ concentration ([Ca2+]i) in MCs were performed using fura 2 dual-excitation wavelength fluorescence microscopy. MCs, grown on coverslips (22 x 22 mm), were loaded with fura 2 by incubation for ~50 min at room temperature in the dark in physiological saline solution containing 2 µM acetoxymethyl ester of fura 2 (fura 2-AM), 0.09 g/dl DMSO, and 0.018 g/dl Pluronic F-127 (Molecular Probes, Eugene, OR) followed by washing three times with physiological saline solution. The cells were then incubated with fura 2-free physiological saline solution for an additional ~20 min. The coverslip was then placed in a perfusion chamber (model RC-2OH; Warner) and mounted on the stage of a Nikon Diaphot inverted microscope. Fura 2 fluorescence was monitored by a ratio technique (excitation at 340 and 380 nm, emission at 510 nm) using Metafluor software (Universal Imaging, West Chester, PA).

Immunoprecipitation and immunoblotting. Cell monolayers were collected with PBS and then lysed in 1% Triton X-100 buffer containing (in mM) 150 NaCl, 10 Tris·HCl (pH 7.5), 1 EGTA, 0.2 sodium orthovanadate, 0.2 phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, aprotinin (1 µg/ml), pepstatin (1 µg/ml), and proteinase inhibitor cocktail (Roche Applied Science, Indianapolis, IN) followed by centrifugation at 6,000 g for 15 min at 4°C. For coimmunoprecipitation (co-IP) experiments, the cell lysates were incubated for 2 h with the primary antibody indicated, followed by immunoprecipitation (IP) for 1 h with 30-µl slurry of protein G or A (Amersham Biosciences, Piscataway, NJ) in 50 mM Tris·HCl. Immunocomplexes were then washed five times in lysis buffer. The cell lysates (without precipitation, for inputs) and immunoprecipitates (for co-IP) were analyzed by Western blot.

For immunoblotting experiments, all samples were fractionated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with the indicated primary antibodies. Bound antibodies were visualized with Super Signal West Femto luminol/enhancer solution (Pierce Biotechnology, Rockford, IL) using AlphaEase FC imaging system (Alpha Innotechnology).

Biotinylation assays. When cell monolayers were 80% confluent, the cells were washed twice with PBS and then scraped in 1 ml of PBS (per 10-cm plate). The cell suspension was centrifuged at 6,000 g for 15 min at 4°C, and the pellet was resuspended with 1 ml of PBS and biotinylated with 0.05 mg/ml of sulfo-NHS-SS-biotin (Pierce) for 30 min at room temperature. After centrifugation at 6,000 g for 15 min at 4°C, the pellet was washed three times with PBS and then lysed in 1 ml of lysis buffer. The cell lysates were then centrifuged at 21,000 g for 15 min at 4°C. Lysates (300 µl) were saved for immunoblotting. Supernatants (700 µl) were mixed with 300 µl of lysis buffer (total volume: 1 ml) and then incubated with 30 µl of slurry of immobilized streptavidin beads (Pierce) for 1 h with shaking. The beads were spun down and washed two times with lysis buffer. The biotinylated samples were then analyzed by Western blot.

Fluorescent immunocytochemistry. MCs were plated on 22 x 22 x1-mm coverslips, fixed with ice-cold acetone/methanol, and permeabilized with 0.2% Triton X-100. The cells were then incubated with blocking buffer (2% heat-inactivated goat serum and 0.2% Triton X-100 in PBS) overnight at 4°C, followed by incubation with PC2 antibody or control rabbit IgG for 1 h at room temperature. The cells were then incubated with secondary antibodies (goat anti-rabbit conjugated with Alexa Fluor 488; Molecular Probes) for 1 h at room temperature. Cells were washed three times with PBS and processed for confocal microscopy using a confocal laser scanning microscope (Zeiss LSM410).

Fluorescent immunohistochemistry. Fluorescent immunohistochemistry was performed as described in our previous publication (42). All animals were treated according to guidelines approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center. In brief, adult male Sprague-Dawley rats (weighing 200–250 g) were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Kidneys were perfused with physiological saline solution via a catheter inserted into the abdominal aorta followed by 4% paraformaldehyde and then excised from the animals. The kidneys were fixed in 2% paraformaldehyde in K+-free PBS over 2 h at 4°C, immersed in 30% sucrose overnight at 4°C, and cryosectioned at 5-µm thickness (Cryostat 2800 Frigocut-E; Leica Instruments). The sections were washed with K+-free PBS and treated with blocking buffer containing 50 mM NH4Cl, 2% BSA, and 0.05% saponin in K+-free PBS for 20 min at room temperature for permeabilization. The sections were then incubated overnight at 4°C in blocking buffer containing rabbit polyclonal anti-PC2 antibody. To label glomerular MCs, we also incubated the sections with mouse monoclonal anti-Thy1.1 (OX-7) antibody overnight at 4°C. The concentrations for both primary antibodies were 5 µg/ml. The sections were rinsed and incubated for 30 min at room temperature with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes). The concentrations of the secondary antibodies were 2 µg/ml. In control slides, equal amounts of rabbit IgG or mouse IgG without immunoreactivity were used instead of the primary antibodies. All stainings were visualized under confocal laser scanning microscopy (Zeiss LSM410).

MC contractility assay. ANG II-induced MC contraction was measured by changes in planar surface area as described in our previous publication (4). In brief, MCs were visualized under an inverted fluorescent microscope (Nikon TE-2000S, upgraded). The perfusates were heated through an inline heater, and the temperature was controlled by an automatic temperature controller (TC-324B; World Precision Instruments, Sarasota, FL). Changes in the planar surface area of MCs in response to ANG II were monitored at 37°C with 95% O2-5% CO2 gassing. With the use of a digital camera, images of the same cells were captured serially every 5 min for 30 min. The perimeters of individual cells with clearly defined borders were outlined, and the planar surface areas were calculated with ImageJ software (National Institutes of Health).

Materials. Inactivating constructs directing the expression of short hairpin RNAs specific for human pkd2 (designated as shRNA-pkd2) were generated as described previously (19). The pkd2 expression plasmids (human hemagglutinin-pkd2), mouse monoclonal 1F1 TRPC1 antibody, and rabbit polyclonal PC2 antibody were obtained from Dr. Leonidas Tsiokas (University of Oklahoma Health Sciences Center, Oklahoma City, OK). The other primary antibodies and all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Statistical analysis. In all immunoblot experiments, at least three independent experiments were performed for each group. Data are reported as means ± SE. One-way ANOVA and Student-Newman-Keuls test, Student's unpaired t-test, or Student's paired t-test were used to analyze the differences among multiple groups, between two groups, and before and after treatment in the same group, respectively. P < 0.05 was considered statistically significant. Statistical analysis was performed with SigmaStat (Jandel Scientific, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 GRANTS
 REFERENCES
 
Expression of endogenous PC2 protein in MCs. Western blot and immunofluorescence staining were performed to detect the presence of PC2 protein in glomeruli and glomerular MCs using a well-characterized PC2 antibody (19, 40). Figure 1A shows immunoblots in rat glomerular extracts and lysates of cultured human MCs. These bands were ~110 kDa and the same size as the PC2 band indicated in pkd2-transfected human embryonic kidney (HEK)-293T cells. The specificity of the PC2 band in transfected HEK-293T cells was verified by inhibition of the protein expression by cotransfecting RNAi plasmids (shRNA-pkd2) that produced shRNAs specifically targeting human and porcine pkd2 (Fig. 1A, right). The amounts of native PC2 in glomeruli or glomerular MCs appear to be compatible with those in inner medullary collecting duct cells in which PC2 is highly expressed (18). This observation was further confirmed by fluorescence immunocytochemistry, which showed a punctuate pattern of staining in PC2 antibody-incubated MCs but not in control rabbit IgG-incubated MCs (Fig. 1B). It appears that the intracellular PC2 immunofluorescence aligned in tracks radiating out from the perinuclear region of MCs reminiscent of cargo transport via the tubulovesicular pathway.


Figure 1
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  		Fig. 1. Expression of polycystin 2 (PC2) protein in mesangial cells (MCs). A: Western blot. In left, samples included lysates of human embryonic kidney (HEK)-293T cells transfected with human pkd2 expression plasmids (serving as a positive control), lysates of cultured human MCs, extracts of rat glomeruli, and lysates of inner medullary collecting duct (IMCD) cells. In right, expression levels of PC2 in HEK-293T cells with transient transfection of pUB empty vector (mock), pkd2 expression plasmids (pkd2), and cotransfection of pkd2 with short hairpin RNA (shRNA)-pkd2 are shown. Actin was used as loading control. Primary antibody was rabbit {alpha}-PC2. B: immunofluorescence staining for PC2 in human MCs. Primary antibody was rabbit {alpha}-PC2 (1:200), and secondary antibody was goat Alexa Fluor 488-conjugated anti-rabbit (1:2,000). For a negative control, non-immunoreactive rabbit IgG instead of {alpha}-PC2 primary antibody was used.

 
Immunohistochemistry was performed to further detect PC2 expression in glomerular MCs in vivo. In rat kidney sections, glomeruli were easily distinguished by their characteristic circular morphological aspect bordered by peripheral lumen. As shown in Fig. 2A, glomerular MCs were identified by red signals (left) and probed with an antibody against Thy1.1 (OX-7), a MC marker. PC2 protein was labeled with green signals (Fig. 2A, middle). Yellow signals in the merged image (Fig. 2A, right) indicate localization of PC2 protein in MCs. The PC2 stainings were also found in the regions that were not stained by OX-7 antibody, suggesting that PC2 protein might also reside in other types of glomerular cells. However, these signals were not seen in control experiments (Fig. 2B), suggesting a specificity of the stainings.


Figure 2
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  		Fig. 2. Immunohistochemistry, showing expression of PC2 protein in rat kidney section. A: glomerular MCs were labeled with OX-7 mouse monoclonal antibodies (1:200) followed by Alexa Fluor 568-conjugated secondary antibody (1:2,000; red fluorescence). PC2 was stained with {alpha}-PC2 antibody (1:100) followed by an Alexa Fluor 488-conjugated secondary antibody (1:2,000; green fluorescence). Localization of PC2 in MCs was indicated by yellow signals in the overlap image. B: non-immunoreactive rabbit and mouse IgG instead of the primary antibodies was used as negative control (left). Right presents bright-field image of left, showing morphology of glomerulus.

 
PC2 mediated ANG II-stimulated membrane currents in MCs. Because PC2 protein was highly expressed in MCs, we then wanted to know its function in this type of glomerular cell. PC2 has been demonstrated to be a Ca2+-permeable channel in renal tubular epithelial cells and vascular smooth muscle cells (7, 10, 16, 18, 19, 28, 30, 41, 48). We speculated that PC2 might also function as a plasma membrane channel mediating agonist-stimulated ionic responses in MC. To test this, we performed cell-attached patch-clamp experiments in cultured human MCs to examine the effects of PC2 expression levels on ANG II-induced single-channel currents. PC2 was downregulated by transient transfection with RNAi plasmids (shRNA-pkd2) that produced shRNAs specifically targeting human and porcine pkd2. Upregulation of PC2 was achieved by transient transfection with human hemagglutinin-pkd2. Consistent with our previous report (19), both constructs successfully changed the expression levels of PC2 (Fig. 1A, right). Green fluorescent protein expression plasmids were cotransfected with these constructs or pUB vectors (for mock control) at 10-fold less for identification of successfully transfected cells. As shown in Fig. 3, A and B, ANG II (1 µM) significantly increased channel activity in mock-transfected cells. NPO of the observed channels was raised from 0.047 ± 0.017 at resting state to 0.135 ± 0.048 ~5 min after ANG II treatment, representing an increase of ~187 ± 42.8% (P < 0.05). This ANG II-evoked channel activation was significantly enhanced by overexpression of PC2, indicated by a rise of NPO from 0.114 ± 0.015 to 0.688 ± 0.095, representing an increase of ~542 ± 60.1% (P < 0.05, compared with mock). However, in pkd2-knocked down cells, ANG II was not able to produce significant channel activation. The NPO slightly increased from 0.022 ± 0.007 to 0.038 ± 0.012 in response to ANG II (P > 0.05), representing an increase of ~70.8 ± 10.3% (P < 0.05, compared with mock). Moreover, the basal channel activity was also significantly elevated in pkd2-overexpressing cells and inhibited in pkd2-knocked down cells, suggesting that PC2 not only participated in agonist-stimulated responses but also contributed to tonic channel activity in MCs.


Figure 3
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  		Fig. 3. Cell-attached patch-clamp recordings, showing the effect of PC2 expression levels on ANG II-induced membrane currents. A: representative traces, displaying single-channel activity before (pre-ANG II) and after (post-ANG II) ANG II stimulation in MCs transfected with pUB empty vector (mock), pkd2 expression plasmids (pkd2), and shRNA-pkd2 RNAi constructs (shRNA-pkd2). Arrows indicate closed state of channels. Downward deflections indicate inward currents. Holding potential was 80 mV (pipette). B: open probability (NPO) of single channels before and after application of ANG II (1 µM) in mock, pkd2, and shRNA-pkd2 cells; n = no. of cells analyzed. Holding potential was 80 mV (pipette). *P < 0.05, pre-ANG II vs. post-ANG II in the same group of cells. #P < 0.05, compared with pre-ANG II in mock group. C: current-voltage (I-V) relationship curves, showing development of ANG II-induced single-channel currents with changes in membrane potentials in mock-transfected MCs. Membrane potentials were expressed as negative pipette potentials (–Vp). The calculated single-channel conductance was 28.3 pS. The estimated reversal potential was 32 mV (–Vp).

 
We also plotted current-voltage relationships within the range from –80 to –20 mV of command potentials (currents at the command potentials more positive than –20 mV were indiscernible) in mock control MCs to estimate the biophysical features of the ANG II-sensitive channels (Fig. 3C). Because the resting membrane potential of the MCs was unknown, the membrane potentials were expressed as negative pipette potentials (–Vp). The calculated single-channel conductance was ~28.3 pS. The extrapolated linear current-voltage relationships revealed that the currents were reversed at –Vp of 32 mV.

The ANG II-induced channel activity in MCs was also significantly inhibited by 30 µM Gd3+ (Fig. 4A), a known inhibitor of NSCC (PC2 is a NSCC) (7).


Figure 4
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  		Fig. 4. Inhibitory effect of 30 µM Gd3+ (A) and dominant-negative pkd2 mutant pkd2-D511V (B) on ANG II-induced channel activity in MCs. Channel activity was expressed as percent change in NPO after ANG II (1 µM) treatment; n = no. of cells analyzed in each group. *Significant difference (P < 0.05) compared with control in A and mock in B. C. Western blot showing expression of PC2 in MCs with transfection of pSHAG vector (mock) and pkd2-D511V constructs.

 
To provide additional evidence that PC2 mediated ANG II-induced currents and whether channel activity rather than a scaffolding role for PC2 was essential for ANG II-induced conductance, we measured the ANG II response in MCs that were transiently transfected with pkd2-D511V (Fig. 4C). It has been known that pkd2-D511V is a pathogenic mutant (39) and maintains the homodimerization domain of PC2 (47). We reasoned that it should function as a dominant-negative allele if endogenous PC2 mediated ANG II-induced currents. Consistent with our prediction, ANG II failed to induce a significant response in these transfected cells (Fig. 4B). These data suggested that PC2 channel activity was required for ANG II-evoked ionic responses in MCs.

PC2 participated in ANG II-stimulated Ca2+ response in MCs. We next determined whether PC2-mediated membrane conductance contributed to ANG II-induced Ca2+ responses in MCs. Fura 2 fluorescence-indicated [Ca2+]i was measured in cultured MCs with and without knockdown or overexpression of pkd2. YFP expression plasmids were cotransfected at 10-fold less for identification of positively transfected cells. Consistent with our previous studies (4), ANG II evoked a rapid and striking cytosolic Ca2+ transient followed by a steady state of lower elevation of [Ca2+]i in the presence of 1 mM Ca2+ extracellular solution. Removal of extracellular Ca2+ immediately reduced the [Ca2+]i to a level lower than baseline. Readmission of Ca2+ resulted in an increase in [Ca2+]i that is attributed to Ca2+ influx (21) (Fig. 5, A–C). Overexpression or knockdown of pkd2 did not alter the profile of the ANG II-induced Ca2+ responses. However, the ANG II-stimulated Ca2+ entry in response to Ca2+ readdition was significantly inhibited by knockdown of pkd2 (Fig. 5, C and D) and enhanced by overexpression of pkd2 (Fig. 5, B and D). The changes in Ca2+ entry were not due to changes in ANG II-induced Ca2+ release from ER because the initial Ca2+ transients were comparable among mock control and other treated cells (Fig. 5, A–C). Thus the Ca2+ imaging data suggested that the PC2 channel was involved in an ANG II Ca2+ signaling pathway in MCs.


Figure 5
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  		Fig. 5. Fura 2 fluorescence ratiometry measurement of intracellular Ca2+ concentration ([Ca2+]i). A–C: representative Ca2+ imaging experiments showing ANG II-stimulated Ca2+ responses in mock-transfected (A), PC2-upregulated (B), and PC2-downregulated (C) MCs. Ca2+ influx in response to Ca2+ readmission is indicated by arrows. [Ca2+]B, Ca2+ concentration in the bathing solution; HA, human hemagglutinin. Application of ANG II (1 µM) is indicated by the top horizontal bars. D: summary data showing the ANG II-induced Ca2+ influxes in response to Ca2+ readmission in untransfected (UT), mock, shRNA-pkd2, and HA-pkd2 MCs. Data are expressed as difference in [Ca2+]i ({Delta}[Ca2+]i) before and after Ca2+ readmission; n = no. of cells analyzed. *Significant difference (P < 0.05) between the indicated groups.

 
ANG II stimulation promoted the interaction between PC2 and TRPC proteins. Previous studies revealed that PC2 required other membrane-associated proteins, such as PC1 or TRPC1, to form protein complexes for its channel function (3, 10, 28, 30, 31, 46). We reasoned that, in native MCs, endogenous PC2 might require binding partners to form functional channels. Our previous study has demonstrated that MCs selectively express TRPC1, -3, -4, and -6 (42), all of which have structural similarity to PC2. Further studies revealed that some isoforms of the TRPC proteins also participated in ANG II-induced Ca2+ signaling (4, 8). Thus we examined whether these TRPC proteins were components of PC2 channel complexes and whether ANG II stimulation influenced the formation of the PC2-TRPC heteromultimers. co-IP was performed to detect the interaction between PC2 and each endogenous TRPC isoform in MCs. As shown in Fig. 6, A and C, IP of PC2 pulled down TRPC1 and TRPC4 (Fig. 6A) but not TRPC3 and TRPC6 (Fig. 6C). It appeared that PC2 had a stronger interaction with TRPC4 than with TRPC1 at the resting state (Fig. 6, A and B). Interestingly, the PC2-TRPC1 and PC2-TRPC4 interactions were significantly promoted by ANG II stimulation. After a 4-min treatment with ANG II, PC2-TRPC1 and PC2-TRPC4 interactions were increased by approximately six- and threefold, respectively (Fig. 6B). This biochemical evidence strongly indicated that agonist stimulation enhanced the interaction between PC2 and both TRPC1 and TRPC4 in MCs.


Figure 6
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  		Fig. 6. Coimmunoprecipitation (IP), showing a selective interaction of PC2 with canonical transient receptor potential (TRPC) protein TRPC1 (Aa) and TRPC4 (Ab) but not with TRPC3 and TRPC6 (C) and the influence of ANG II (AII) on the PC2-TRPC1 and PC2-TRPC4 interactions (A and B) in MCs. All inputs shown here were 1/20 of the proteins used for IP. B: summary of increases in the amount of TRPC1 (Ba) and TRPC4 (Bb) precipitated by PC2 after 4-min treatment with ANG II. Data are expressed as percentages of optical density of TRPC1 or TRPC4 immunoblots in IP samples to the corresponding inputs; n = no. of independent experiments. The optical density of immunoblots was measured with software provided by AlphaEaseFC Imaging System (Alpha Innotech). *Significant difference (P < 0.05) compared with pre-ANG II.

 
ANG II stimulation triggered plasma membrane translocation of PC2. It has been demonstrated that PC2 predominantly resides in intracellular compartments (16). Our immunocytochemistry data also displayed a major intracellular distribution of PC2. The intracellular PC2 staining showed a pattern similar to that of cargo transport via the tubulovesicular pathway (Fig. 1B). Thus we hypothesized that ANG II might trigger translocation of PC2 to the plasma membrane and the newly recruited PC2 bound to existing TRPC1 and TRPC4 to form additional functional channels. To test this hypothesis, we examined the membrane translocation of PC2 responding to ANG II treatment using a nonmembrane permeant agent (sulfo-NHS-SS-biotin) that biotinylates surface proteins and precipitated the proteins with avidin-linked streptavidin beads. As shown in Fig. 7, the expression of biotinylated PC2 protein was significantly increased in the cells treated with ANG II for 4 min (Fig. 7, top). The enrichment of cell membrane proteins in the biotinylated fractions and equal amounts of loaded proteins in different samples were confirmed by reprobing the blots with anti-integrin β1-antibody, a membrane marker (Fig. 7, middle). The biotinylated membrane fraction was not contaminated by nonplasma membrane proteins because calnexin, an ER protein, was not detected in the biotinylated fractions but was clearly detected in corresponding cell lysates (without biotinylation; Fig. 7, bottom). These data suggested that ANG II stimulated migration of intracellular PC2 to the cell surface.


Figure 7
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  		Fig. 7. Biotinylation of PC2 protein in MCs with and without ANG II stimulation. An increase in surface PC2 was observed in cells treated with 1 µM ANG II for 4 min (top). Con, control. PC2 in lysates indicates unbiotinylated PC2. The loading amount of lysates was 1/20 used for IP biotin. A cell membrane marker (integrin-β1) was used as a positive and loading control (middle). Calnexin, an endoplasmic reticulum marker, was used as a negative control for biotinylation (bottom). Immunoblots of integrin and calnexin were obtained by reprobing the same immunoblotting membrane as that for PC2 with corresponding antibodies.

 
PC2 contributed to contractile function of MCs. To determine the physiological relevance of the PC2 channel in MCs, we evaluated the role of PC2 in agonist-stimulated contraction of MCs by measuring the changes in planar surface area of MCs in response to ANG II. This contractility assay was carried out in MCs cotransfected with green fluorescent protein and pUB vector (mock control) or shRNA-pkd2 (knockdown of pkd2). Transfected cells were identified with green fluorescence. Changes in the planar surface area of green cells in response to 1 µM ANG II were monitored at 37°C and 95% O2-5% CO2. As shown in Fig. 8, ANG II treatment for 15 min decreased the surface area of control MCs by 32.2 ± 4.1%. However, this contractile response was reduced in MCs with knock down of pkd2 (10.8 ± 3.1%; P < 0.05, compared with Mock). These results suggested that PC2 contributed, at least partially, to the agonist-induced contraction of MCs.


Figure 8
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  		Fig. 8. Changes in the planar surface area of MCs with ANG II stimulation. MCs transfected with green fluorescent protein (GFP) (mock) or GFP + shRNA-pkd2 constructs were incubated with 1 µM ANG II for 15 min, and changes in the planar surface area of green cells were measured at 37°C with 95% O2-5% CO2 gassing, expressed as percent changes from the cell surface areas before ANG II stimulation. *Significant difference (P < 0.05) compared with Mock.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
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In addition to well-known L-type voltage-gated Ca2+ channels, several other types of Ca2+-permeable channels also contribute to Ca2+ signaling in MCs. These channels include the NSCC, receptor-operated Ca2+ channel, and store-operated Ca2+ channel (20). Physiologically, activation of these channels is linked to activation of the phospholipase C pathway by G-protein-coupled receptors or receptor tyrosine kinases. Despite intensive studies over the past decades, the molecular entity of these channels still remains undefined. The present study provided evidence that PC2 protein might be a functional subunit of one of these channels. The calculated single-channel conductance of the PC2-associated channel (Fig. 3C) was consistent with characteristics of NSCC described in rat MCs (17, 23). Also, the PC2-mediated currents were reversed at 32 mV of –Vp (Fig. 3C). With the consideration that intracellular K+ concentration in MCs is ~120 mM and that the resting membrane potential of MCs is estimated at about –40 mV (12, 17, 23), the reversal membrane potential for the PC2-dependent channel was estimated to be near zero, consistent with the property of NSCC (17, 23).

Like TRPC channel proteins, PC2 requires other membrane-associated proteins for its channel function. The known binding partners of PC2 include PC1 and TRPC1 (10, 28, 30, 31, 46). In the present study, we found that PC2 had physical interactions with TRPC1 and TRPC4 (Fig. 6, A and B), both of which are endogenous proteins in MCs (4, 42). Interestingly, we previously demonstrated that TRPC1 participated in ANG II-stimulated Ca2+ influxes in human MCs (4). Wang et al. (50) recently reported that TRPC4 might be a component of store-operated Ca2+ channels, which are physiologically activated by the phospholipase C pathway in mouse MCs. Thus it is possible that, in native MCs, PC2 heteromultimerizes with TRPC1 and TRPC4, and this PC2-TRPC1-TRPC4 complex mediates agonist (such as ANG II)-induced ionic responses. We inferred that PC2 might be a critical component or even a pore-forming subunit in this complex because ANG II-induced channel activation was nearly abolished not only by knock down of pkd2 (Fig. 3, A and B) but also by dominant-negative pkd2 mutant (Fig. 4B). It has been reported that pkd2-D511V is a pathogenic mutant (39) that loses its channel function but maintains the interaction domains of PC2 (47).

González-Perrett et al. (7) reported that the PC2 channel displays multiple single-channel subconductance states in human syncytiotrophoblasts and PC2-overexpressed Sf9 insect cells. However, this property was not seen in the present study. This discrepancy might be simply explained by distinct binding partners for PC2 in different cell types. In MCs, the PC2-dependent channel is composed of PC2, TRPC1, and TRPC4. However, the PC2 channel in other types of cells might have other binding partners that confer differential channel properties.

Another interesting finding from the present study was that ANG II stimulation triggered plasma membrane translocation of PC2 (Fig. 7) and promoted the interaction of PC2 with TRPC1 and TRPC4 (Fig. 6, A and B). These results suggested an underlying mechanism for ANG II-stimulated membrane currents, i.e., that ANG II triggered migration of intracellular PC2 to the cell surface and that the newly recruited PC2 incorporated with existing TRPC1 and TRPC4 in the plasma membrane to form PC2-TRPC1-TRPC4 channel complexes that mediated ion flow. PC2 trafficking between the intracellular compartments and the cell surface in MCs was also supported by the PC2 distribution, which showed a pattern reminiscent of cargo transport via the tubulovesicular pathway (Fig. 1B). The mechanism for ANG II-triggered PC2 trafficking is unknown. However, Köttgen et al. (15) recently proposed that PC2 trafficking is regulated by two phosphofurin acidic cluster sorting proteins (PACS-1 and PACS-2). Binding to the two proteins is dependent on the phosphorylation of PC2 by the protein kinase casein kinase 2. Thus ANG II could promote PC2 trafficking by regulation of PC2 phosphorylation. Our group (19) recently found that receptor tyrosine kinase activation can activate membrane PC2 channel by releasing PC2 from a tonic inhibition of membrane-delimited phosphatidylinositol-4,5-bisphosphate. The findings in the present study provide an alternative mechanism for agonist-induced PC2 currents, which is the increase in the number of functional channels through insertion of new PC2 protein into the plasma membrane on activation of G-protein-coupled receptors.

In several previous studies, PC2 was undetectable in glomeruli by immunohistochemistry (1, 5, 22). However, PC2 was clearly identified in MCs either in primary culture or rat kidney sections in the present study (Figs. 1 and 2). The reason for the discrepancy is presently unknown. However, one possibility could be the difference in PC2 antibodies used between the previous studies and ours. In agreement with our findings, glomerular cysts have been reported in animal models of polycystic kidney disease, including PC2-associated ADPKD (32, 44, 45). In addition, in severe early-onset cases and on the rare occasions that fetal ADPKD kidneys have been examined, glomerular cysts are the major manifestation of disease (25, 35, 38, 49). Also, glomerular hypertrophy and enlargement appear to be present in human ADPKD kidneys (56). These clinical and experimental findings support the existence of PC2 protein in glomeruli.

Glomerular MCs are contractile cells that surround the glomerular capillaries of the kidney and regulate glomerular filtration rate by regulating the surface area of the capillaries (24, 43). Like vascular smooth muscle cells, the contractile function of MCs is controlled by cytosolic Ca2+ concentration. Because the PC2 channel participates in Ca2+ signaling of MCs, this channel might play an important role in the contractile function of MCs that is supported by the present study (Fig. 8). Our findings may provide a new clue for the pathogenesis of ADPKD. The widely accepted model for cystogenesis in ADPKD is that dysfunction or defects in primary cilia of kidney epithelial cells contribute to the development of renal cysts (51, 55). The primary cilia physiologically function as a mechanosensor and regulate intracellular Ca2+ signaling of tubular epithelial cells by detecting the rate of urine flow (28, 30, 34, 53). Published studies have described a link of structural and functional defects of the primary cilia to the progression of ADPKD (29, 33, 55). However, a change in urinary flow, which will change the stimulus applied to the primary cilium in tubular epithelial cells, might also contribute to the formation of tubular cysts. Indeed, one early symptom of ADPKD is polyuria (6, 52). High fluid intake increases progression in patients with polycystic kidney disease and promotes renal cyst growth in rats, presumably through an increase in urine volume (9, 11, 13). Moreover, glomerular hyperfiltration has been suggested to be an early manifestation of ADPKD (2, 54). Because glomerular filtration is a determinant of urine flow rate and MC tone is an important physiological regulator of glomerular filtration, we can postulate that, at early stages of ADPKD, the contractile function of MCs is impaired due to a defect or dysfunction of PC2-associated Ca2+ signaling. The resulting glomerular hyperfiltration leads to an increase in urine flow rate that applies stronger stimulation to the primary cilia of tubular epithelial cells and consequently promotes tubular cyst formation and growth.


   NOTE ADDED IN PROOF
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In the original posting of this article on the APS Articles in PresS Web site (on February 6, 2008), Fig. 3A (inadvertently) contained elements (the top and bottom traces) from a previously published article (Ma et al., Ref. 19, supplemental material). The authors apologize for the error. The problem has been corrected here, in the final-published version of the manuscript, with a new version of Fig. 3.


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This study was supported by a scientist development grant from the American Heart Association (R. Ma) and grant-in-aid from the PKD Foundation (R. Ma).


   ACKNOWLEDGMENTS
 
We thank Dr. Leonidas Tsiokas (University of Oklahoma Health Sciences Center, Oklahoma City, OK) for providing rabbit {alpha}-PC2 antibody and mouse monoclonal 1F1 TRPC1 antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Ma, RES-302G, 3500 Camp Bowie Blvd., Dept. of Integrative Physiology, Univ. of North Texas Health Science Center, Fort Worth, TX 76107 (e-mail: rma{at}hsc.unt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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TOP
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 RESULTS
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  1. Chang MY, Parker E, Ibrahim S, Shortland JR, Nahas ME, Haylor JL, Ong AC. Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models. Nephrol Dial Transplant 21: 2078–2084, 2006.[Abstract/Free Full Text]
  2. Chapman A, Johnson A, Koehny W. Glomerular hyperfiltration: an early manifestation of autosomal dominant polycystic kidney disease. Kidney Int 35: 203A, 1989.
  3. Delmas P, Nauli SM, Li XG, Coste B, Osorio N, Crest M, Brown DA, Zhou J. Gating of the polycystin ion channel signaling complex in neurons and kidney cells. FASEB J 18: 740–742, 2004.[Abstract/Free Full Text]
  4. Du J, Sours-Brothers S, Coleman R, Ding M, Graham S, Kong D, Ma R. TRPC1 channel is involved in contractile function of glomerular mesangial cells. J Am Soc Nephrol 18: 1437–1445, 2007.[Abstract/Free Full Text]
  5. Foggensteiner L, Bevan AP, Thomas R, Coleman N, Boulter C, Bradley J, Ibraghimov-Beskrovnaya O, Klinger K, Sandford R. Cellular and subcellular distribution of polycystin-2, the protein product of the PKD2 gene. J Am Soc Nephrol 11: 814–827, 2000.[Abstract/Free Full Text]
  6. Gabow PA. Autosomal dominant polycystic kidney disease. Am J Kidney Dis 22: 511–512, 1993.[Web of Science][Medline]
  7. González-Perrett S, Kim K, Ibarra C, Damiano AE, Zotta E, Batelli M, Harris PC, Reisin IL, Arnaout MA, Cantiello HF. Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc Natl Acad Sci USA 98: 1182–1187, 2001.[Abstract/Free Full Text]
  8. Graham S, Ding M, Sours-Brothers S, Yorio T, Ma JX, Ma R. Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+ signaling in glomerular mesangial cells. Am J Physiol Renal Physiol 293: F1381–F1390, 2007.[Abstract/Free Full Text]
  9. Grantham JJ. The etiology, pathogenesis, and treatment of autosomal dominant polycystic kidney disease: recent advances. Am J Kidney Dis 28: 788–803, 1996.[Web of Science][Medline]
  10. Hanaoka K, Qian F, Boletta A, Bhunia AK, Piontek K, Tsiokas L, Sukhatme VP, Guggino WB, Germino GG. Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature 408: 990–994, 2000.[CrossRef][Medline]
  11. Hebert LA, Greene T, Levey A, Falkenhain ME, Klahr S. High urine volume and low urine osmolarity are risk factors for faster progression of renal disease. Am J Kidney Dis 41: 962–971, 2003.[CrossRef][Web of Science][Medline]
  12. Kasner SE, Ganz MB. Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye PBFI. Am J Physiol Renal Fluid Electrolyte Physiol 262: F462–F467, 1992.[Abstract/Free Full Text]
  13. Keith DS, Torres VE, Johnson CM, Holley KE. Effect of sodium chloride, enalapril, and losartan on the development of polycystic kidney disease in Han:SPRD rats. Am J Kidney Dis 24: 491–498, 1994.[Web of Science][Medline]
  14. Kip SN, Hunter LW, Ren Q, Harris PC, Somlo S, Torres VE, Sieck GC, Qian Q. [Ca2+]i reduction increases cellular proliferation and apoptosis in vascular smooth muscle cells. Circ Res 96: 873–880, 2005.[Abstract/Free Full Text]
  15. Köttgen M, Benzing T, Simmen T, Tauber R, Buchholz B, Feliciangeli S, Huber TB, Schermer B, Kramer-Zucker A, Höpker K, Simmen KC, Tschucke CC, Sandford R, Kim E, Thomas G, Walz G. Trafficking of TRPP2 by PACS proteins represents a novel mechanism of ion channel regulation. EMBO J 24: 705–716, 2005.[CrossRef][Web of Science][Medline]
  16. Koulen P, Cai YQ, Geng L, Maeda Y, Nishimura S, Witzgall R, Ehrlich BE, Somlo S. Polycystin-2 is an intracellular calcium release channel. Nat Cell Biol 4: 191–197, 2002.[CrossRef][Web of Science][Medline]
  17. Ling BN, Seal EE, Eaton DC. Regulation of mesangial cell ion channel by insulin and angiotensin II-possible role in diabetic glomerular hyperfiltration. J Clin Invest 92: 2141–2151, 1993.[Web of Science][Medline]
  18. Luo Y, Vassilev PM, Li X, Kawanabe Y, Zhou J. Native polycystin 2 functions as a plasma membrane Ca2+-permeable cation channel in renal epithelia. Mol Cell Biol 23: 2600–2607, 2003.[Abstract/Free Full Text]
  19. Ma R, Li WP, Rundle D, Kong J, Akbarali HI, Tsiokas L. PKD2 functions as an epidermal growth factor-activated plasma membrane channel. Mol Cell Biol 25: 8285–8298, 2005.[Abstract/Free Full Text]
  20. Ma R, Pluznick JL, Sansom SC. Ion channels in mesangial cells: function, malfunction, or fiction. Physiology 20: 102–111, 2005.[Abstract/Free Full Text]
  21. Ma R, Smith S, Child A, Carmines PK, Sansom SC. Store-operated Ca2+ channels in human glomerular mesangial cells. Am J Physiol Renal Physiol 278: F954–F961, 2000.[Abstract/Free Full Text]
  22. Markowitz GS, Cai Y, Li L, Wu G, Ward LC, Somlo S, D'Agati V. Polycystin-2 expression is developmentally regulated. Am J Physiol Renal Physiol 277: F17–F25, 1999.[Abstract/Free Full Text]
  23. Matsunaga H, Yamashita N, Miyajima Y, Okuda T, Chang H, Ogata E, Kurokawa K. Ion channel activities of cultured rat mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F808–F814, 1991.[Abstract/Free Full Text]
  24. Menè P, Simonson MS, Dunn MJ. Physiology of mesangial cell. Physiol Rev 69: 1347–1424, 1989.[Free Full Text]
  25. Michaud J, Russo P, Grignon A, Dallaire L, Bichet D, Rosenblat E, Lamothe E, Lambert M. Autosomal dominant polycystic kidney diseases in the fetus. Am J Med Genet 51: 240–246, 1994.[CrossRef][Web of Science][Medline]
  26. Mochizuki T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJ, Somlo S. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272: 1339–1342, 1996.[Abstract]
  27. Montell C, Birnbaumer L, Flockerzi V, Bindels R, Bruford EA, Caterina MJ, Clapham DE, Harteneck C, Heller S, Julius D, Kojima I, Mori Y, Penner R, Prawitt D, Scharenberg AM, Schultz G, Shimizu N, Zhu MX. A unified nomenclature for the superfamily of TRP cation channels. Mol Cell 9: 229–231, 2002.[CrossRef][Web of Science][Medline]
  28. Nauli SM, Alengha FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AEH, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33: 129–137, 2003.[CrossRef][Web of Science][Medline]
  29. Nauli SM, Rossetti S, Kolb RJ, Alenghat FJ, Consugar MB, Harris PC, Ingber DE, Loghman-Adham M, Zhou J. Loss of polycystin-1 in human cyst-lining epithelia leads to ciliary dysfunction. J Am Soc Nephrol 17: 1015–1025, 2005.[CrossRef][Web of Science]
  30. Nauli SM, Zhou J. Polycystins and mechanosensation in renal and nodal cilia. Bioessays 26: 844–856, 2004.[CrossRef][Web of Science][Medline]
  31. Newby LJ, Streets AJ, Zhao Y, Harris PC, Ward CJ, Ong ACM. Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex. J Biol Chem 277: 20763–20773, 2002.[Abstract/Free Full Text]
  32. Omori S, Hida M, Fujita H, Takahashi H, Tanimura S, Kohno M, Awazu M. Extracellular signal-regulated kinase inhibition slows disease progression in mice with polycystic kidney disease. J Am Soc Nephrol 17: 1604–1614, 2006.[Abstract/Free Full Text]
  33. Ong ACM, Harris PC. Molecular pathogenesis of ADPKD: the polycystin complex gets complex. Kidney Int 67: 1234–1247, 2005.[CrossRef][Web of Science][Medline]
  34. Praetorius HA, Spring KR. Bending the MDCK cell primary cilium increases intracellular calcium. J Membr Biol 184: 71–79, 2001.[CrossRef][Web of Science][Medline]
  35. Pritchard L, Sloane-Stanley JA, Sharpe JA, Aspinwall R, Lu W, Buckle V, Strmecki L, Walker D, Ward CJ, Alpers CE, Zhou J, Wood WG, Harris PC. A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype. Hum Mol Genet 9: 2617–2627, 2000.[Abstract/Free Full Text]
  36. Qian Q, Hunter LW, Li M, Marin-Padilla M, Prakash YS, Somlo S, Harris PC, Torres VE, Sieck GC. Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells. Hum Mol Genet 12: 1875–1880, 2003.[Abstract/Free Full Text]
  37. Qian Q, Li M, Cai Y, Ward CJ, Somlo S, Harris PC, Torres VE. Analysis of the polycystins in aortic vascular smooth muscle cells. J Am Soc Nephrol 14: 2280–2287, 2003.[Abstract/Free Full Text]
  38. Reeders ST, Zerres K, Gal A, Hogenkamp T, Propping P, Schidt W, Waldherr R, Dolata MM, Davies KE, Weatheral DJ. Prenatal diagnosis of autosomal dominant polycystic kidney disease with a DNA probe. Lancet 2: 6–8, 1986.[Web of Science][Medline]
  39. Reynolds DM. Aberrant splicing in the PKD2 gene as a cause of polycystic kidney disease. J Am Soc Nephrol 10: 2342–2351, 1999.[Abstract/Free Full Text]
  40. Rundle DR, Gorbsky G, Tsiokas L. PKD2 interacts and co-localizes with mDial1 to mitotic spindles of dividing cells. J Biol Chem 279: 29728–29739, 2004.[Abstract/Free Full Text]
  41. Siroky BJ, Ferguson WB, Fuson AL, Xie Y, Fintha A, Komlosi P, Yoder BK, Schwiebert EM, Guay-Woodford LM, Bell PD. Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells. Am J Physiol Renal Physiol 290: F1320–F1328, 2006.[Abstract/Free Full Text]
  42. Sours S, Du J, Chu S, Ding M, Zhou JX, Ma R. Expression of canonical transient receptor potential (TRPC) proteins in human glomerular mesangial cells. Am J Physiol Renal Physiol 290: F1507–F1515, 2006.[Abstract/Free Full Text]
  43. Stockand JD, Sansom SC. Glomerular mesangial cells: electrophysiology and regulation of contraction. Physiol Rev 78: 723–744, 1998.[Abstract/Free Full Text]
  44. Streets AJ, Moon DJ, Kane ME, Obara T, Ong ACM. Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localization of polycystin-2 in vivo and in vitro. Hum Mol Genet 15: 1465–1473, 2006.[Abstract/Free Full Text]
  45. Tanner GA, Tielker MA, Connors BA, Phillips CL, Tanner JA, Evan AP. Atubular glomeruli in a rat model of polycystic kidney disease. Kidney Int 62: 1947–1957, 2002.[CrossRef][Web of Science][Medline]
  46. Tsiokas L, Arnould T, Zhu C, Kim E, Walz G, Sukhatme VP. Specific association of the gene product of PKD2 with the TRPC1 channel. Proc Natl Acad Sci USA 96: 3934–3939, 1999.[Abstract/Free Full Text]
  47. Tsiokas L, Kim E, Arnould T, Sukhatme VP, Walz G. Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2. Proc Natl Acad Sci USA 94: 6965–6970, 1997.[Abstract/Free Full Text]
  48. Vassilev PM, Guo L, Chen XZ, Segal Y, Peng JB, Basora N, Babakhanlou H, Cruger G, Kanazirska M, Ye CP, Brown EM, Hediger MA, Zhou J. Polycystin-2 is a novel cation channel implicated in defective intracellular Ca2+ homeostasis in polycystic kidney disease. Biochem Biophys Res Commun 282: 341–350, 2001.[CrossRef][Web of Science][Medline]
  49. Waldherr R, Zerres K, Gall A, Enders H. Polycystic kidney disease in the fetus. Lancet 2: 274–275, 1989.[CrossRef][Web of Science][Medline]
  50. Wang X, Pluznick JL, Wei P, Padanilam BJ, Sansom SC. TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells. Am J Physiol Cell Physiol 287: C357–C364, 2004.[Abstract/Free Full Text]
  51. Weimbs T. Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystin-1. Am J Physiol Renal Physiol 293: F1423–F1432, 2007.[Abstract/Free Full Text]
  52. Wilson PD. Polycystic kidney disease: new understanding in the pathogenesis. Int J Biochem Cell Biol 36: 1868–1873, 2004.[CrossRef][Web of Science][Medline]
  53. Woda CB, Leite M, Rohatgi R, Satlin LM. Effects of luminal flow and nucleotides on [Ca2+]i in rabbit cortical collecting duct. Am J Physiol Renal Physiol 283: F437–F446, 2002.[Abstract/Free Full Text]
  54. Wongt H, Vivian L, Weiler G, Filler G. Patients with autosomal dominant polycystic kidney disease hyperfiltrate early in their disease. Am J Kidney Dis 43: 624–628, 2004.[CrossRef][Web of Science][Medline]
  55. Yoder BK, Mulroy S, Eustace H, Boucher C, Sandford R. Molecular pathogenesis of autosomal dominant polycystic kidney disease. Expert Rev Mol Med 8: 1–22, 2006.[Medline]
  56. Zeier M, Fehrenbach P, Geberth S. Renal histology in polycystic kidney disease with incipient and advanced renal failure. Kidney Int 42: 1259–1265, 1992.[Web of Science][Medline]



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