1Department of Pediatrics, Mount Sinai School of Medicine, New York 10029-6574; and2Department of Mechanical Engineering and Center for Biomedical Engineering, The City College of New York, New York, New York 10031
Submitted 19 February 2003 ; accepted in final form 25 June 2003
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ABSTRACT |
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cilium; cytoskeletal deformation; fluid shear stress; mechanotransduction; fura 2; intracellular calcium concentration
We have recently shown that an acute increase in tubular fluid flow rate in the microperfused CCD, associated with an 20% increase in tubular diameter, leads to a rapid >150 nM increase in intracellular Ca2+ concentration ([Ca2+]i) in both principal and intercalated cells comprising this segment (44).1 Whether the increase in [Ca2+]i reflects increased Ca2+ influx from the external solutions, Ca2+ mobilization from internal stores, and/or decreased Ca2+ efflux from the cell remains uncertain. Also unknown is the identity of the stimulus for the flow and/or stretch-induced [Ca2+]i transient. Like endothelial cells lining the vasculature (6, 38), renal tubular epithelial cells likely experience at least three types of mechanical forces in response to variations in urinary flow rate: hydrostatic pressure, circumferential stretch, and fluid flow-induced shear or drag forces.
A likely candidate for the proximate flow sensor in the tubule is the apical primary cilium, a nonmotile structure projecting from the centriole of all renal tubular cells except intercalated cells of the collecting duct (18, 24, 34). Madin-Darby canine kidney (MDCK) principal cells express a primary cilium that is 8 µm in length (26). Praetorius and Spring (26) recently reported that bending of the cilium of MDCK cells either directly with a micropipette or by increasing the rate of flow superfusing the apical surface of monolayers resulted in an increase in [Ca2+]i, a response attributed to external Ca2+ entry through mechanosensitive channels followed by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive internal stores. Direct mechanical stimulation of the apical membrane of MDCK cells also led to a transient increase in [Ca2+]i that differed from the response induced by bending of the cilium in its larger amplitude and shorter time delay between stimulation and peak [Ca2+]i (
10 vs.
40 s for bending of the cilium). Furthermore, the response was not affected by removal of extracellular Ca2+, suggesting that direct mechanical manipulation of the apical membrane stimulates release from internal stores and does not require external Ca2+ entry. The physiological importance of structurally and functionally intact cilia in renal epithelial cells is underscored by the growing body of evidence that disruption of proteins localized to the cilia, such as polaris (45), cystin (15), and polycystin 1 (22) in orpk, cpk, and Pkd1del34/del34 mice, respectively, is associated with a renal cystic phenotype.
The flow channel used by Praetorius and Spring (26) in the studies described above had a cross section 3-mm wide and 3-mm deep and thus generated an average linear velocity of 110 µm/s at a flow rate of 1 µl/s. The authors attempted to simulate in their flow chamber the average velocity that would be encountered in an intact perfused tubule. An average velocity of 110 µm/s corresponds to a tubular fluid flow rate of 7 nl/min when the internal diameter of the tubule is assumed to be 37 µm, the value used by Praetorius and Spring (26). Model calculations performed in the present study, however, assume an internal tubular diameter of 25 µm, a dimension we (30, 44) and others (42) have previously reported for the rabbit CCD. Yet, as will be demonstrated in the present study, it is not the average velocity that one should attempt to reproduce in simulating mechanotransduction in a flow chamber, but rather a dynamic similarity of the fluid shear stress acting on the apical surface of the cells and, more importantly, the hydrodynamic forces and torques required to bend the cilia, at least in the case of principal cells.
The internal structure of the principal cell cilium is characterized by a 9 + 0 organization of microtubules (34, 40). The deformation of this type of cilium in a cultured kidney epithelial cell line was studied by Schwartz et al. (34) using nonlinear beam theory ("heavy elastica" model). These authors were primarily interested in determining the flexural rigidity of the cilium. Because a detailed hydrodynamic model for determining the drag forces on the cilia and their hydrodynamic interaction with one another was not available until recently (12), Schwartz et al. (34) used an empirical equation for the flow past a single cylinder to calculate the drag on the cilium. Using this approach, the authors were able to demonstrate good agreement between theoretical predictions and experimental observations of the bending response of the cilium subject to a physiologically appropriate range of flow rates. These authors also raised a question relevant to the studies performed by Praetorius and Spring (26) and that posed in the present study; specifically, does stretch-activation of channels (e.g., Ca2+ channels) reflect bending deformation of the cilium with resultant tension in its plasma membrane or deformation of the cortical cytoskeleton in the terminal web at the base of the cilium arising from the resisting moment of the linkages that attach the cilium to the cortical network of microtubules and actin filaments at its base? Both of these possibilities will be explored in the present study using a mathematical model.
Although the data summarized above provide compelling evidence that the cilium is a flow sensor in collecting duct epithelia, two important differences are evident on comparing the response of the native collecting duct (44) with that of the cultured cell line. First, in the perfused tubule, both principal and intercalated cells, the latter devoid of cilia, respond to an increase in flow with an increase in [Ca2+]i (44). In addition, there is no evidence of gap junctional communication in native tubules (44), whereas in MDCK cells the Ca2+ signal spreads laterally by the diffusion of IP3 through gap junctions (26).
The purpose of the present study was thus to identify the cellular mechanisms underlying the flow and/or stretch-induced [Ca2+]i transient in the native CCD. We specifically sought to determine whether 1) flow across the apical membrane of the CCD, comprised of principal cells with apical cilia and intercalated cells decorated with apical microvilli/microplicae, and/or circumferential stretch mediates the flow-induced increase in [Ca2+]i in this segment and 2) the [Ca2+]i transient induced by flow/stretch reflects external Ca2+ entry and/or mobilization of internal stores. To examine these questions, we used the Ca2+-sensitive fluorescent dye fura 2 to measure changes in principal and intercalated cell [Ca2+]i in isolated CCDs microperfused in vitro in their native geometry (and thus subject to both flow and stretch), split-open to expose the luminal surface to superfusate flow in the absence of stretch (simulating an epithelial monolayer preparation) or occluded to isolate the effect of circumferential stretch in response to transient fluid flow loading of increasing magnitude and duration. We also performed a rigorous hydrodynamic analysis of the flow in our perfusion chamber and then developed a detailed model to predict the fluid shear stress at the apical surface of the split-open tubule and the hydrodynamic forces and torques acting on the cilia. Finally, these predictions were quantitatively compared with the equivalent forces and torques in the intact perfused tubule by suitably modifying the theoretical model developed for brush-border microvilli by Guo et al. (12). We show that the local velocities, forces, and torques reproduced in existing flow chambers are significantly smaller than those encountered in vivo because the cilia lie within a hydrodynamic boundary layer at the base of the flow chamber where the local velocity at the cilia tips in current experiments is far less than encountered in vivo.
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MATERIALS AND METHODS |
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Adult female New Zealand White rabbits were obtained from Covance (Denver, PA) and housed in the Mount Sinai School of Medicine animal care facility. The animals were fed standard rabbit chow and given free access to food and water. Animals were killed by intraperitoneal injection of a lethal dose of pentobarbital sodium (100 mg/kg). All experiments were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.
The kidneys were removed, and single tubules were dissected freehand in cold (4°C) dissection solution containing (in mM): 145 NaCl, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 sodium lactate, 1.0 sodium citrate, 6.0 L-alanine, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O (44). A single tubule was studied from each animal.
Isolated Tubules
Isolated tubules were either microperfused in vitro (native geometry) or split open to expose the luminal surfaces of all cells to superfusate flow, as previously described (44). Tubules were perfused and bathed at 37°C with Burg's solution, which resembled the dissection solution except that 25 mM NaCl was replaced by NaHCO3, and the solution was gassed with 95% O2-5% CO2 at room temperature to reach a pH of 7.4 (44). In some experiments, CCDs were perfused with Burg's solution prepared without Ca2+ (Ca2+-free perfusate).
To distinguish between the contribution of circumferential stretch and a flow-induced response in tubules perfused in their native geometry, the distal ends of some perfused CCDs were occluded manually, using a blunt pipette, after baseline [Ca2+]i was measured, and the pressure in the tubule was increased by raising the height of the perfusate reservoir. The dilation of the tubule creates a transient fluid flow whose magnitude and duration depends on the rate at which the reservoir is elevated, the final height of the reservoir, and the distance [length (L), µm] of the measurement location from the occlusion site. The instantaneous average velocity uav (in µm/s) at the measurement site is given by uav = (2L/R)dR/dt, where R(t) is the instantaneous tubule radius (in µm) and dR/dt is the rate at which it is changing in response to the time-dependent increase in pressure. When the pressure reservoir is raised slowly, dR/dt 0, leading to a purely elastic response; rapid increases in height generate a transient flow impulse in addition to the circumferential stretch response. Thus the rate at which the reservoir height is raised enables the elastic response to be distinguished from a combined response that involves both fluid flow and circumferential stretch.
Measurement of [Ca2+]i
After equilibration, tubules were loaded with 20 µM of the acetoxymethyl ester of fura 2 (Molecular Probes, Eugene, OR) added to the bath for 20 min. In several experiments, rhodamine-labeled peanut lectin (PNA; Vector Laboratories, Burlingame, CA) was added to the luminal perfusate for 5 min to identify intercalated cells; rabbit principal cells do not bind PNA (31). [Ca2+]i was measured in individually identified fura 2-loaded cells, as previously described (44). Two to four principal and intercalated cells residing in the lateral wall of each perfused CCD or localized to the center of each split-open segment were analyzed. For each cell in each tubule, three (for peak) to five (for baseline) measurements of [Ca2+]i were averaged to generate a mean value for baseline (obtained immediately before flow was increased) and peak (maximal) concentrations. The time to peak was defined as the time interval between the onset of high flow rate and the detection of the maximal [Ca2+]i value. The mean responses of principal and intercalated cells in each tubule were used for further cell-specific data analysis.
To assess the source of Ca2+ contributing the [Ca2+]i transient, CCDs were studied in the absence of luminal and/or basolateral Ca2+ or pretreated with basolateral 2-aminoethoxydiphenyl borate (2-APB; 10 µM), a cell-permeant inhibitor of the IP3 receptor (10, 19), or basolateral thapsigargin (100 nM), an irreversible inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase that prevents refilling of intracellular Ca2+ pools and leads to depletion of internal stores. Several additional studies were performed in microperfused CCDs bathed in Burg's solution containing apyrase (10 U/ml; Sigma-Aldrich), an enzyme that rapidly hydrolyzes 5'-nucleotide triphosphates to monophosphates, to examine whether autocrine/paracrine signaling by 5'-nucleotides mediates the response of the CCD to flow/stretch.
Effect of Flow on Gap Junctional Intercellular Communication
Randomly identified individual cells in split-open CCDs were injected with Lucifer Yellow for monitoring of intercellular coupling, as previously described (44), before and after initiation of high superfusate flow (25 µl/s).
Measurement of Cilia Length
Cilia length was measured by indirect immunofluorescence microscopy using a monoclonal antibody specific for acetylated -tubulin (6-11B-1; generous gift from G. Piperno; see Ref. 25) and a commercially available anti-bovine
-tubulin mouse monoclonal antibody (Molecular Probes). Split-open CCDs were fixed for 15 min in 2.5% paraformaldehyde at room temperature, permeabilized with Triton X-100 (0.1% for 30 min for 6-11B-1 and 0.3% for 15 min for anti-tubulin antibody) in PBS containing BSA (15 mg/ml), and incubated for 2 h at room temperature with 6-11B-1 or overnight at 4°C with
-tubulin antibody (1 µg/ml). After a thorough washing, the tubules were incubated for 2 h with a 1:1,000 dilution of the secondary antibody, a FITC-conjugated anti-mouse IgG (Sigma, St. Louis, MO), at room temperature. After being washed with PBS three times, an 8-µl droplet of Prolong anti-fade solution was deposited on the tubule, which was then covered with a glass coverslip applied with slight pressure to flatten and bend the apical cilia in a uniform direction. Confocal microscopic analysis of six tubules from three different rabbits (n = 2 cilia/tubule) revealed that the cilia length was 2.2 ± 0.4 µm with cilia possessing bulbous distal ends (Fig. 1).
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Mathematical Models for Shear Stresses, Forces, and Torques in Perfused and Split-Open CCDs
As indicated above, the microperfused CCD subject to an increase in tubular fluid flow rate experiences a shear stress on the apical membranes of the cells comprising the segment, a drag and torque on their primary cilium, and circumferential stretch (44). To examine the isolated response to apical shear stress, we studied split-open tubule monolayers. To compare the shear forces acting on the apical surface of the tubular epithelial cells and the forces and torques acting on the cilia, mathematical models were developed to describe the fluid flow past the cilia in the experimental chamber used for measurement of [Ca2+]i in split-open CCDs. These predictions were then compared with those of a newly developed mathematical model for the flow past the cilia in a perfused tubule.
Flow in the channel. A sketch of the geometry (transverse and longitudinal sections) of the flow chamber is shown in Fig. 2. The channel has a free water surface that is maintained at a constant height of 1.5 mm by a vacuum siphon placed just at the top of the bevel in the diagram. The tip of the flow inlet (a bent 18-gauge needle, with sharp tip removed) was positioned just above the floor of the specimen chamber. The split-open tubule is positioned in the center of the bottom surface of the chamber with the tubule axis aligned parallel to the longitudinal section. An en face view of the specimen with representative cellular dimensions is shown in Fig. 3A. Because the dimensions of the cross section of the chamber are much greater than the width or height of the split-open tubule, the basic flow in the tubule can be calculated as if the tubule was absent. This flow can be closely approximated as a unidirectional viscous flow in a beveled trough that satisfies no-slip boundary conditions on its bottom and slanted side walls and a zero fluid shear stress at the free surface. Because of the complex cross-sectional geometry, the governing Navier-Stokes equation was solved numerically. The relationship between the shear stress on the center line at the bottom wall of the channel, w(0) in dyn/cm2, and the flow rate Q, is
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Velocity in cilia layer of split-open tubule. It is a difficult and subtle problem to accurately calculate the flow in the disturbed region at the apical surface of the split-open tubule because of the presence of the protruding cilia. Our idealized mathematical model is shown in Fig. 3A. The cells are arranged in a hexagonal array. Given that intercalated cells account for only 25% of the total number of cells comprising the rabbit CCD, this cell population is neglected in the model. At the center of each hexagonal cell is a vertical cilium that is approximated as a circular cylinder of 2.5 µm height and 0.2 µm diameter (1). Because the height of the cilia is small compared with the width of the open tubule sheet (78.5 µm), the flow in the central region of the sheet can be approximated as a two-dimensional flow in which edge effects from the lateral boundaries of the sheet can be neglected. The Reynold number based on the cilia tip velocity and height is of the order of 10-4 and, thus, in the Stokes slow flow regime. The viscous dissipation resulting from the protruding cilia can be described by a distributed body force, or Darcy resistance. An approximate governing equation for the flow in the disturbed layer is thus given by a modified Stokes equation with a Darcy term, as described by Bird et al. (3)
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The use of Eq. 2 to determine the average velocity field and the forces and torques on the cilia has been examined in a hydrodynamically equivalent problem, the Stokes flow past a periodic array of slender vertical fibers in a parallel walled channel (39). Comparison with exact Stokes solutions for the average flow past each fiber and the resulting drag show excellent agreement for fiber solid fractions up to 0.7.
The solution of Eq. 2 with the foregoing boundary conditions is given in APPENDIX 1. Representative solutions for the disturbed average velocity profile are shown in Fig. 4 for cilia of different lengths ranging in height from 2.5 µm in the adult rabbit principal cell to 8 µm in confluent monolayers of MDCK cells examined by Praetorius and Spring (26). The nearly straight line labeled "without cilia" in Fig. 4 is the velocity profile that would exist at the bottom of the channel in the absence of a split-open tubule. The distortion of the velocity profiles is significantly greater for the MDCK cilia because of their greater protrusion length.
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At a high superfusate flow rate of 25 µl/s, the maximum velocity in the channel is 6.56 mm/s, but the local velocity at a distance 2.5 µm (height of the cilia) off the bottom wall on the channel centerline is only 17.9 µm/s, a value 360 times smaller (Table 1). At a low flow rate of 3.2 µl/s, the velocities, which scale linearly, are 12.7% of those just cited. In contrast, we shall show that, for a tubule perfusion rate of 5 nl/min, the velocity at the tip of a 2.5-µm cilia (Fig. 1) in a typical tubule of 25-µm internal diameter (42) is 119 µm/s, whereas the peak velocity at the tubule center line is 350 µm/s (Table 1). It is clear from these theoretical predictions that the maximum or average channel velocity has little bearing when trying to achieve dynamic similitude with the forces and torques experienced by cilia in intact tubules.
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Forces and torques on cilia in split-open CCDs. With the use of the theoretical approach developed by Guo et al. (12), local solutions of the Stokes equations for creeping flow can be used to calculate the local drag on the cilia per unit length. This drag is proportional to the local velocity U(z) given by Eq. A5 in APPENDIX 1. The local drag can be integrated along the length of the cilia to provide the total drag on each cilium. If this local drag is multiplied by the local moment arm from the base of the cilia at z = 0, and this local moment is integrated along the length of the cilium, the total torque can be obtained. The expressions for the total drag and torque are given by Eqs. A8 and A9 in APPENDIX 1.
Velocity profile forces and torques on cilia in perfused tubules. Guo et al. (12) developed a theoretical model to describe the flow in the proximal tubule and the forces and torques acting on the brush-border microvilli. The flow in the brush-border layer is described by the axisymmetric equivalent of Eq. 2. The flow geometry for the proximal tubule is conceptually equivalent to our model for the CCD shown in Fig. 3B except that, in the case of the proximal tubule, the layer of microvilli at the tubule wall forms a relatively dense ordered hexagonal array in which there is virtually no axial flow except in the immediate vicinity of the microvilli tips. The same basic model can be applied to the flow in the intact CCD, although the flow now can easily pass through the border layer of much more widely separated cilia that line the wall of the CCD. The disturbed velocity profiles predicted by the model are shown in Fig. 5 where one observes that the presence of the cilia causes only a modest distortion of the parabolic profile for Poiseuille flow in a tube without cilia and a modest change in the wall shear stress. These calculations assume a flow rate of 5 nl/min, an inner tubule diameter of 25 µm, and a value for k based on the separation distances of the cilia tips (see Eq. A7 in APPENDIX 1). The forces and torques acting on the cilia are determined in the same manner as described above for the split-open tubule using the velocity profiles shown in Fig. 5.
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A central question that must be addressed in understanding mechanotransduction in the CCD is the relative magnitude of the drag force on cilia compared with the total drag force resulting from fluid shear on the apical membrane of an individual cell. The latter is the product of the wall shear stress w and the area of a hexagonal unit cell in Fig. 3A.
Statistical Analysis
Results are expressed as means ± SE; n equals the number of animals. Significant differences were determined by paired or unpaired t-tests, as appropriate, using the software program SigmaStat (SPSS, Chicago, IL). Significance was asserted at P < 0.05.
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RESULTS |
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In perfused tubules, a rapid increase in tubular fluid flow rate, sufficient to increase diameter by 14.4 ± 1.7% (n = 7) within 1 s, led to a prompt increase in [Ca2+]i in both principal (106.3 ± 15.7 to 338.9 ± 67.2 nM) and intercalated (124.1 ± 20.9 to 346.3 ± 48.4 nM; Figs. 6A and 7) cells within 10.6 ± 1.9 s in both cell types (Fig. 8), followed by a gradual decay to a plateau value. [Ca2+]i remained significantly elevated above baseline for at least 20 min during a period of sustained high flow (Figs. 6A and 7).
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Effect of High Superfusate Flow on [Ca2+]i in Split-Open CCDs
In split-open CCDs, an increase in superfusate flow from 0 to 3.2 µl/s failed to generate a [Ca2+]i response. However, an increase in superfusate flow from 3.2 to 25 µl/s led to an increase in [Ca2+]i in both principal (103.9 ± 9.2 to 213.9 ± 38.8 nM; P < 0.05) and intercalated (101.1 ± 15.8 to 200.9 ± 9.4 nM; P < 0.05) cells in four CCDs (Figs. 6B and 7). The time to peak [Ca2+]i averaged 36.3 ± 4.7 s in these preparations (P < 0.005 compared with perfused CCDs; Fig. 8). At 20 min of sustained high flow, [Ca2+]i was not significantly different [P = not significant (NS)] from the peak value attained and tended to be higher than baseline (P = 0.08). A reduction in superfusate flow from 25 to 3.2 µl/s led to a fall in principal (239.2 ± 41.5 to 149.2 ± 37.2 nM; P < 0.03) and intercalated (199.8 ± 13.2 to 127.4 ± 29.5 nM; P = 0.05) cell [Ca2+]i from peak values in three out of the four tubules studied; of note was that the flow-induced increase in principal [Ca2+]i in the fourth CCD was modest (35 nM) compared with the increase of 135.2 ± 35.8 nM detected in the other three CCDs. The recovery [Ca2+]i indicated above was not significantly different from those measured during the initial superfusion period at 3.2 µl/s.
Effect of Circumferential Stretch and Flow Rise Time on [Ca2+]i in Occluded CCDs
In the occluded CCD, a rapid increase in luminal volume, sufficient to increase tubular diameter by 19.5 ± 2.4% (n = 5) within 0.6 s, led to an increase in [Ca2+]i in both principal (83.9 ± 9.5 to 364.9 ± 16.9 nM; P < 0.05) and intercalated (84.2 ± 18.8 to 370.7 ± 79.1 nM; P < 0.05) cells (Figs. 6C and 7). Peak [Ca2+]i was reached within 7.6 ± 0.8 s in both cell types (Fig. 8). The magnitude of the change in [Ca2+]i in principal (281.0 ± 19.0 nM) and intercalated (286.6 ± 66.9 nM) cells in occluded CCDs exceeded that measured in split-open tubules (110.0 ± 35.7 and 99.8 ± 12.1 nM, respectively; P < 0.05). Similarly, a rapid increase in luminal volume, sufficient to increase tubular diameter by 5.4 ± 0.9% (n = 3), led to a significant increase in [Ca2+]i in both principal (133.3 ± 5.3 to 233.0 ± 37.5 nM; P < 0.05) and intercalated (155.0 ± 8.8 to 264.4 ± 17.3 nM; P < 0.05) cells. However, a slow increase (over 3-5 min) in tubular volume, to expand tubular diameter by 20%, failed to elicit a [Ca2+]i transient (data not shown; n = 3).
Source of Ca2+ Leading to Flow-Induced Rise in [Ca2+]i
We considered it likely that the flow-induced increase in [Ca2+]i in the perfused CCD was the result of release of internal Ca2+ stores and/or external Ca2+ influx. These possibilities were tested using an array of inhibitors. Pretreatment of CCDs with thapsigargin, in the presence of external Ca2+, led to a modest increase in [Ca2+]i as internal stores were emptied, followed by a slow decay (Fig. 9A). A subsequent increase in tubular fluid flow/stretch failed to elicit an increase in [Ca2+]i in principal (183.7 ± 26.2 to 207.5 ± 21.2 nM; P = NS) and intercalated (196.0 ± 36.4 to 208.8 ± 30.7; P = NS) cells in seven CCDs. Similarly, exposure of tubules (n = 4) to 2-APB, an IP3 receptor antagonist that had no effect on baseline [Ca2+]i (97.1 ± 22.5 nM in principal cells and 99.1 ± 18.4 nM in intercalated cells), completely abolished the [Ca2+]i response to an increase in flow rate/stretch (Fig. 9B). These data indicate that the flow-induced increase in [Ca2+]i requires release of IP3-sensitive internal Ca2+ stores.
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To discern whether external Ca2+ entry participates in the response, CCDs were perfused with a Ca2+-free perfusate (with or without 1 mM EGTA), and the response to flow/stretch was monitored. As shown in Fig. 9C, in the absence of luminal Ca2+ and presumably Ca2+ influx across the apical membrane, an increase in flow/stretch led to a rapid rise in [Ca2+]i in principal (113.9 ± 10.2 to 398.7 ± 53.6 nM; P < 0.005) and intercalated (138.3 ± 8.7 to 408.3 ± 63.9 nM; P = 0.005) cells in six CCDs, a response that could reflect either release of Ca2+ from internal stores or Ca2+ influx across the basolateral membrane. Unlike our observation of sustained plateau elevations of [Ca2+]i for at least 20 min in CCDs perfused at high flow rates with standard Ca2+-containing perfusate (Figs. 6 and 7), [Ca2+]i in CCDs perfused in the nominal absence of luminal Ca2+ fell to baseline levels (108.6 ± 18.0 and 113.3 ± 17.2 nM in principal and intercalated cells, respectively; P = NS for each value compared with paired baseline [Ca2+]i) by 10 min in the continued presence of fast flow. These data suggest that the flow-induced plateau elevation in [Ca2+]i in perfused CCDs is mediated by luminal Ca2+ entry. In the nominal absence of luminal (x10 min) and basolateral (x3 min) Ca2+, no [Ca2+]i increase was detected in CCDs (n = 4) subject to an increase in luminal flow rate (Fig. 9D). The latter observation provides compelling evidence for coupling between extracellular Ca2+ entry at the basolateral membrane and internal Ca2+ release. Of note are reports by others that interruption of store-operated channels (SOCs), either by genetic disruption (21) or by pharmacological inhibition, significantly attenuates IP3-mediated ER Ca2+ release. Apyrase added selectively to the bathing solution (n = 4) failed to inhibit the rapid high-amplitude flow and/or stretch-stimulated increase in [Ca2+]i in either principal ([Ca2+]i = 190 ± 65.9 nM from a baseline of 127.7 ± 16.7 nM within 7.8 ± 1.2 s) or intercalated (
[Ca2+]i = 193.5 ± 51.9 nM from a baseline of 115.3 ± 11.3 nM within 8.8 ± 1.3 s) cells in CCDs perfused in the nominal absence of Ca2+ (Fig. 9E); in these tubules, [Ca2+]i returned to baseline within 10 min (i.e., no sustained plateau elevation of [Ca2+]i was detected). These results suggest that 5'-nucleotides, acting as autocrine/paracrine mediators, potentially released at the basolateral membrane in response to epithelial stretch, do not mediate the flow-induced high-amplitude [Ca2+]i response.
Effect of Flow on Gap Junctional Intercellular Communication
Intercalated cells do not possess apical cilia, yet they respond to an increase in flow/stretch with an increase in [Ca2+]i. This could be explained if principal and intercalated cells are directly coupled and/or gap junctions are opened in response to flow. To examine these possibilities, individual cells in split-open CCDs were microinjected with the cell-impermeable fluorescent dye Lucifer Yellow. Superfusion for 5 min did not lead to intercellular spread or significant leakage of the dye (data not shown). Exposure of split-open CCDs to 0.4% trypan blue at the conclusion of the experiment (n = 3) revealed occasional trypan blue-positive cells along the edges of the split tubule; microinjected cells, however, excluded the viability marker.
Forces and Torques on Cilia in Split-Open and Perfused CCDs
Table 1 summarizes the major predictions of our theoretical models for the split-open and perfused CCDs and presents results that would have been obtained for a confluent monolayer of MDCK cells if they were exposed to shear in our fluid flow chamber. Although the results cannot be compared quantitatively with the experiments reported by Praetorius and Spring (26), since the geometry of their flow chamber differed substantially from our own, instructive qualitative comparisons can be made. The primary difference from a hydrodynamic viewpoint between the cells analyzed in this study and cultured MDCK cells is the large difference in length of the cilia (2.5 vs. 8 µm, respectively).
In split-open CCDs, an increase in superfusate flow from 0 to 3.2 µl/s failed to generate a [Ca2+]i response, whereas a further increase in flow rate to 25 µl/s led to a moderate response (Fig. 6B).2 The average velocity in our flow chamber at the high flow rate is about four times the maximum average velocity in the experiments reported by Praetorius and Spring (26), but, as indicated earlier, it is the local velocity at the tips of the cilia and not the average velocity of the flow in the chamber that determines the dynamic behavior of the cilia in response to flow.
The first important observation in Table 1 (also see Figs. 4 and 5) is that the tip velocity at the high flow rate for a cilium of 2.5-µm height and 0.2-µm diameter is 17.9 µm/s, approximately one-sixth of that anticipated in an intact tubule of 25-µm internal diameter (42) perfused at a rate of 5 nl/min. Both the wall shear stress on the apical membrane and the total drag on the cilia at the high flow rate are nearly one order of magnitude smaller than predicted for the perfused tubule, as is also true for the total torque (Table 1). The predictions for the 8-µm cilium provide new insight into the experiments with MDCK cells reported by Praetorius and Spring (26). Cilium tip velocities do not scale linearly with the length of the cilia because of the increasing hydrodynamic interactions between cilia as their length increases (Figs. 4 and 5). Thus the tip velocity on an 8-µm cilium is only about two times that on 2.5-µm cilium (Table 1). The total drag force, in that it is proportional to the product of tip velocity and cilia length, would scale as the square of the cilium length were it not for this cilium-cilium interaction. For the split-open tubule, the drag on the 8-µm cilium is less than six times greater than that on the 2.5-µm cilium at the flow rate of 25 µl/s (Table 1). Similarly, the torque, as a product of drag and moment arm, would scale as the cube of the tip length without the ciliumcilium interaction. Instead, the torque experienced by the 8-µm cilia is 22-fold greater than that predicted for the 2.5-µm cilium (Table 1). The torque on an 8-µm cilium at the low flow rate of 3.2 µl/s (0.37 pN · µm; results not shown in Table 1) significantly exceeds that on the 2.5-µm cilium at the high flow rate of 25 µl/s (0.13 pN · µm).
Another major result is the prediction of the relative magnitude of the total shear force on the apical membrane of the principal cells compared with the drag force on the primary cilium. The total drag resulting from fluid shear on the plasma membrane in our high-flow experiment, assuming hexagonal cells whose surface area is 111 µm2, is 0.62 pN. In contrast, the drag on a 2.5-µm cilium itself at a flow of 25 µl/s is only 0.078 pN, or nearly one order of magnitude smaller than the drag resulting from fluid shear. The predicted tip deflection (Table 1) resulting from this drag, while small for the 2.5-µm cilium (only 1.9 nm or 0.08% of cilium length), increases to 227 nm for the 8-µm cilium at the same flow rate. The predicted shapes of the deformed cilia for the split-open tubule for a flow of 25 µl/s are shown in Fig. 10. The flexural rigidity of the cilia used in these calculations is 1.4 x 10-23 N · m2 (34). Unfortunately, the calculation for the tip deflection could not be performed for an 8-µm cilium in a perfused tubule since the predicted tip deflection exceeded the limit of validity of the small deflection theory applied herein. For these large deflections, an "elastica" model would need to be employed similar to that used by Schwartz et al. (34). For comparison, the tip deflection of a 2.5-µm cilium in a perfused tubule subject to a flow of 5 nl/min is 13 nm or 0.52% of the cilium length. As a rough guide, the tip deflection increases as the fourth power of the cilia length and the deflection of an 8-µm cilium is 100 fold greater than the 2.5-µm cilium just described.
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DISCUSSION |
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Microperfused CCDs
Increases in tubular fluid flow rate in the CCD, isolated from its surrounding interstitium and microperfused in vitro, were associated with circumferential stretch and increases of up to 20% in tubular diameter. Note that, although circumferential stretch of this magnitude would not likely occur in the kidney with an intact capsule under conditions of health, comparable increases in collecting duct diameter have been reported after ureteric obstruction (16, 43). Both principal and intercalated cells within the CCD responded to increases in flow/circumferential stretch (
5% increase in tubular diameter) with an increase in [Ca2+]i (Figs. 6A and 7).
The source of the Ca2+ giving rise to the flow and/or stretch-induced rise of [Ca2+]i includes release from internal stores and influx from the extracellular space. The complete inhibition of the response by thapsigargin and 2-APB (Fig. 9) points to a requirement of IP3-mediated release of ER Ca2+ stores in the response. Typically, depletion of Ca2+ from internal stores triggers the capacitative influx of extracellular Ca2+ across the plasma membrane through SOC or Ca2+ release-activated Ca2+ currents (2, 23). Praetorius and Spring (26) proposed that the Ca2+ signal generated by bending of the cilium in MDCK cells was the result of Ca2+ influx through mechanosensitive channels residing in the cilium or its base. We thus considered it likely that the flow and/or stretch-induced Ca2+ influx in the CCD studied in its native geometry was also localized to the apical membrane. Indeed, our finding that removal of Ca2+ from the luminal perfusate led to a fall in [Ca2+]i to baseline levels within 10 min in the continued presence of high flow rate, an observation that contrasted with the sustained (>20 min; Figs. 6 and 7) elevation in [Ca2+]i in CCDs perfused with our standard Ca2+-containing perfusate, suggests that the flow-induced plateau elevation in [Ca2+]i in perfused CCDs is mediated by luminal Ca2+ entry in the epithelial cells. The complete inhibition of the flow and/or stretch-induced increase in [Ca2+]i in tubules perfused and briefly bathed in the absence of Ca2+ suggests that basolateral Ca2+ entry pathways exist in the CCD and may be coupled to internal Ca2+ release.
Compatible with a basolateral Ca2+ entry pathway linked to release of internal Ca2+ stores are recent reports of cell-specific and polarized expression of IP3 receptor isoforms in the kidney in patterns that suggest compartmentalization of distinct IP3-sensitive Ca2+ pools (20). Molecular cloning studies have demonstrated three types of IP3 receptor (types 1-3) derived from different genes (42). Type 2 is expressed exclusively in collecting duct intercalated cells in a diffuse cytoplasmic distribution, whereas principal cells express the type 3 receptor mainly in the basolateral portion of the cytoplasm. Of note are recent reports that the IP3 receptor in the ER and SOCs in the plasma membrane may directly interact (2, 19).
Split-Open CCD
In split-open CCDs, an increase in superfusate flow from 0 to 3.2 µl/s failed to elicit a response. However, an increase in flow rate to 25 µl/s led to a modest sustained increase in [Ca2+]i with an 30-s time delay between stimulus and response (Figs. 6B and 7). Note that, even at the high flow rate, the forces and torques experienced by the cilium are approximately sixfold lower than those predicted in the microperfused CCD (Table 1). Thus the flow-induced approximately twofold increase in [Ca2+]i in principal and intercalated cells in split-open CCDs may be below the potential maximal response, which, for the MDCK cells used by Praetorius and Spring (26), was also a twofold increase. Although the highest average velocity in the flow chamber in the latter study was less than in the present experiments, Table 1 shows that the torque on an 8-µm cilium in MDCK cells is 19 times that on a 2.5-µm cilium for the same flow conditions. This observation suggests that, if the torque is the triggering mechanism for the response, a saturation behavior could have been achieved for the 8-µm cilium studied by Praetorius and Spring (26), even for a lower tip velocity. A different type of flow chamber is needed for future studies where local velocities, drag forces, and torques can be increased by an order of magnitude to quantify the maximum response. To test for the maximum cilium-generated response in split-open CCDs, the bending response of micropipette suction should be compared with that resulting from fluid flow.
Our theoretical model allowed us to predict the relative magnitude of the total shear force on the apical membrane of the principal cells compared with the drag force on the primary cilium. The response to fluid shear of endothelial cells has been studied extensively. A [Ca2+]i response has been observed for fluid shear stresses as low as 1 dyn/cm2 (6). An important question that arises from the data in the CCD is whether it is the high total drag resulting from fluid shear on the plasma membrane or the lower drag on the cilium itself that is the primary mechanism involved in the flow-induced [Ca2+]i response for the split-open CCD. The observation that the fluid shear stress on the apical membrane at the high flow rate is only 0.056 dyn/cm2, a value substantially below the shear stress threshold for a [Ca2+]i response in endothelial cells, and the surface area of an endothelial cell is at least three times larger than a CCD principal cell suggests that it is not the drag force or shear stress per se that is the activating mechanical stimulus but either the torque on the anchoring microtubules and actin filaments at the base of the cilium where it attaches to the cytoskeleton or, alternatively, the bending deformation of the cilium and the resulting opening of stretch-activated Ca2+ channels.
Of note are recent studies that show that polycystin-1 (PC1) and -2 (PC2), two genes implicated in autosomal dominant polycystic kidney disease, participate in fluid-flow sensation by the primary cilium in renal epithelial cells. Cells isolated from embryonic transgenic mice lacking functional PC1, and grown as monolayers on coverslips, formed cilia but did not exhibit a superfusate flow-induced Ca2+ influx as did control cells (22). Blocking antibodies directed against PC2, a Ca2+-permeable cation channel (9) that interacts with PC1 (13), also abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor (22). Based on these data, Nauli et al. (22) proposed that conformational changes of ciliary PC1 transduce a mechanical signal in a chemical response by activating associated PC2 Ca2+ channels; the local Ca2+ influx in the cilium subsequently triggers internal Ca2+ release. This paradigm is similar to that proposed by Praetorius and Spring (26), i.e., an initial Ca2+ influx in the primary cilium is important for the flow-induced Ca2+ response. In contrast, the data in the present study suggest that the flow-induced Ca2+ increase is generated by the cilium transmitting a mechanical force to the cytoskeleton, which is associated, at least in part, with basolateral Ca2+ entry that appears to be associated with Ca2+-induced Ca2+ release from the intracellular IP3-dependent Ca2+ stores. This hypothesis is based on a model for cilium torque. The discrepancy between the findings in perfused tubules and cell monolayers (22, 26) raises the possibility of two distinct mechanisms underlying the Ca2+ response observed in the CCD studied in its native geometry: flow and stretch activation.
The differences (temporal delay, amplitude of response) in [Ca2+]i response noted between the split-open CCD and the microperfused tubules also suggest that different Ca2+ signaling pathways mediate the two responses. Praetorius and Spring (26) proposed that the Ca2+ signal generated by bending of the cilium in MDCK cells was the result of Ca2+ influx through mechanosensitive channels residing in the cilium or its base. They demonstrated that the [Ca2+]i response resulting from the bending of the cilium by either flow or micropipette suction was abolished in confluent MDCK monolayers when extracellular Ca2+ was removed. However, under the latter conditions, the response to direct mechanical stimulation of the apical membrane was retained.
Because our split-open tubule preparation does not allow for the selective manipulation of apical and basolateral superfusate composition, we could not examine the effects of apical vs. basolateral Ca2+-free solution or Ca2+ channel inhibitors to discern whether the flow response in the native CCD, like that reported in MDCK cell monolayers (26), is absolutely dependent on external Ca2+ through stretch-activated Ca2+ channels in the cilium. However, the very small tip deflections for a 2.5-µm cilium (1.9 nm) elicited by a flow rate sufficient to generate a Ca2+ response, as predicted by our model in Table 1, would not be expected to be a sufficient stimulus to open mechanosensitive Ca2+ channels in the plasmalemma of the cilium. This suggests that the primary site of activation may be at the base of the cilium because of the transmission of the torque on the cilium to the actin cytoskeleton in the terminal web.
Weinbaum et al. (41) have reported that there is a large amplification of the force on the axial structural elements for brush-border microvilli, because of the resisting moment at the base. In the case of the microvilli, this resisting moment led to a 38-fold increase in the force on the axial actin filaments, since the moment arm at the base is small compared with the length of the microvilli. The strong wind blowing across a tall tree will seldom split its trunk but instead topple the tree at its roots. The 9 + 0 axial structure can be simplified to consider eight symmetrical microtubule pairs at the base of the axoneme part of the cilium where it enters the transition zone at the top of the basal body (Fig. 11). If the bending axis is in the center plane of the cross section passing through two of the microtubule pairs 1 and 5, the other six must provide the resisting moment applied by the torque on the protruding cilium. Assuming that the microtubule pairs lie on a circle of 200-nm diameter, it is relatively straightforward to compute the tensile and compressive forces on microtubule pairs 6, 7, and 8 and 2, 3, and 4, respectively. The symmetry of the arrangement predicts that the tensile and compressive forces are equal and opposite. The compressive forces on individual microtubule pairs are listed in Table 1. For a 2.5-µm cilium, the compressive force on the outermost microtubule pair 3 (0.86 pN) is 11-fold greater than the drag force on the cilium. For an 8-µm cilium, this compressive force increases to 16 pN at the same flow rate, whereas, for a 2.5-µm cilium in a perfused tubule, this force is 6 pN for a flow rate of 5 nl/min. Thus the forces exerted on the supporting structures in the basal body greatly exceed the typical 0.1-pN forces measured for the deformation of the cortical actin filament network beneath the plasma membrane (25) when membrane proteins are dragged in the plane of the membrane by optical traps and their cytoplasmic tails collide with microdomain boundaries. This suggests that the basal body must be anchored by more rigid supporting structures such as the microtubule complex shown in Fig. 11.
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Occluded Tubules
Analysis of the results generated in occluded tubules provides valuable insight into the nature of the mechanical signal that triggers the flow-induced [Ca2+]i response. Measurements of [Ca2+]i were obtained at a distance approximating two tubular diameters proximal to the occlusion site. Thus the average velocity for the flow transient in the tubule elicited during the "filling" period, uav = (2L/R)dR/dt, can be approximated by 8 dR/dt. For the rapid increase in luminal volume leading to a 20% increase in tubular diameter, dt was at most 600 ms (i.e., the increase in diameter was achieved between two image acquisition frames using our digital camera), dR was 7 µm, and thus uav was at least 93 µm/s. This value is fivefold greater than the 17.9-µm/s tip velocity predicted for the split-open tubule at the high flow rate (Table 1). If the [Ca2+]i response in the occluded tubule is triggered by a shortduration deformation of the cytoskeleton in the terminal web in which the cilium is anchored, then it is no surprise that this increase in [Ca2+]i actually exceeds the response elicited by the high flow rate in the split-open tubule. The notion that it is not the magnitude of epithelial stretch but the initial impulse loading resulting from the flow transient is supported by the important observation that, when a 20% stretch is achieved slowly over 3 min, where uav is <1 µm/s, no [Ca2+]i response was observed. Finally, a rapid small 5% circumferential stretch produced a peak flow velocity of 23 µm/s (
25% of the 93 µm/s response for the 20% increase in diameter), a rate comparable to the 17.9-µm/s tip velocity in the split-open tubule subject to high flow (Table 1). Of note is that the magnitude of the increase in [Ca2+]i in the latter series of tubules was similar to that detected in the split-open tubule superfused at high flow. In summary, these observations tentatively suggest that circumferential stretch may not be the triggering mechanism for the [Ca2+]i response but that the magnitude of the tip velocity and hence the torque on the cilium is the important mechanical signal. To confirm this conjecture, one needs to rule out rate-dependent stretch, not just stretch magnitude, and establish a threshold for fluid shear.
Intercalated Cell Response
Our detection of comparable flow-stimulated increases in [Ca2+]i in principal and intercalated cells, the latter devoid of apical cilia, indicates that the cilium is not the sole mechanotransducer of the flow response in the CCD. Given that principal and intercalated cells appear not to be coupled in the native epithelium (44), we speculate that flow over the microvilli and microplicae that decorate the apical surfaces of intercalated cells (8) generates a bending moment equivalent to that created by flow-induced bending of the apical cilium of principal cells. The flow-induced deformations of individual microvilli in the brush border of the proximal tubule (12) are predicted to be substantially smaller than the flow-induced bending response of the primary cilium (26, 34). Although the bending moment on each microvillus may be very small (0.01 pN · µm at a flow rate of 30 nl/min), these microvilli are quite numerous (4,000/cell) and collectively generate a combined torque (4 pN · µm; see Ref. 12) that exceeds that predicted for a central cilium (0.91 pN · µm) in a perfused CCD. A similar model must be developed for the microvilli/microplicae at the surface of the intercalated cell to provide a theoretical basis for understanding their response to flow.
Alternatively, the response to flow/stretch in principal and intercalated cells may be mediated by paracrine/autocrine signaling secondary to stretch-induced release of an extracellular factor. It is well documented that extracellular nucleotides, including ATP and UTP, can induce a variety of cell responses, including increases in [Ca2+]i. Burnstock (4) proposed that deformation of cells in distended epithelial "tubes" leads to release of ATP by the epithelium. In fact, mechanical stress has been shown to lead to release of ATP and UTP across both apical and basolateral membranes in polarized airway epithelia (14). ATP functions as an extracellular signaling molecule through activation of members of the P2X and P2Y receptor families. P2X receptors are Ca2+-permeable, nonselective cation channels identified, at the mRNA level, in both principal and intercalated cells (36). Binding of ATP to G protein-coupled P2 purinergic receptors activates phospholipase C, leading to hydrolysis of phosphatidylinositol 4,5-bisphosphate to IP3 and release of internal Ca2+ stores (7, 27). We have recently identified functional P2Y2 but not P2X receptors on the apical surfaces of both principal and intercalated cells of the CCD (44). Furthermore, we reported that [Ca2+]i transients induced by an acute increase in tubular fluid flow in the CCD were not mediated by apical P2 purinergic receptor signaling (44). Our present observation that basolateral apyrase fails to prevent the flow and/or stretch-induced high-amplitude increase in [Ca2+]i suggests that 5'-purine and -pyrimidine nucleotides, possibly released at the basolateral membrane in response to epithelial stretch, do not mediate this response.
Physiological Significance
Nearly all epithelial and endothelial cells that are subjected to mechanical strains that exceed a few percent will exhibit [Ca2+]i transients. Thus the flow and/or stretch-induced increase in [Ca2+]i observed in the microperfused or occluded tubule is not unexpected. The central question is whether the force experienced by the isolated CCD (flow/circumferential stretch) or split-open tubule (no circumferential stretch) is more representative of the physiological condition. As stated above, it is unlikely that the diameter of the CCD increases by >15% under physiological conditions, since this would cause a substantial change in volume of the kidney and produce large compressive stresses on the tissue opposing expansion. We believe the more reasonable hypothesis to be that individual tubules in vivo experience only minimal circumferential stretch in response to increases in urinary flow rate and that the primary cilium in principal cells and microvilli/microplicae in intercalated cells serve as flow sensors. If this is indeed the case, the response observed in the split-open tubule is the more relevant, and the models developed herein for the forces and torques on these structures and the consequent deformation of the cytoskeleton provide the key to understanding the mechanical behavior of the afferent sensor.
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APPENDIX I |
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![]() | (A1a) |
![]() | (A1b) |
![]() | (A1c) |
The general solution of Eq. 2 is the sum of particular and homogeneous solutions
![]() | (A2) |
The solution for U(z) that satisfies Eqs. A1a and A1b is
![]() | (A3) |
The local drag force (drag force/unit fiber length) on each cilium, F(z), is related to the local velocity distribution U(z) and Darcy permeability coefficient k by
![]() | (A4) |
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Sangani and Acrivos (28) have obtained a numerical solution for the Stokes flow past a periodic fiber array shown in Fig. 12. These authors showed that the dimensionless drag, F/µU, can be approximated by
![]() | (A5) |
![]() | (A6) |
![]() | (A7) |
![]() | (A8) |
![]() | (A9) |
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Abstracts of this work were presented at the 2002 Annual Meetings of the Academic Pediatric Societies (Baltimore, MD; May 4-6, 2002) and American Society of Nephrology (Philadelphia, PA; December 1-3, 2002).
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FOOTNOTES |
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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.
1 The time interval between onset of flow and maximal [Ca2+]i response in perfused CCDs in the present study was 10 s, an interval calculated based on recordings of images obtained, at least during the initial 2-3 min after an increase in flow, every 600 ms. In the study in which we first described the flow dependence of [Ca2+]i (44), we did not rigorously measure the time interval between onset of flow and maximal response, nor were these values reported.
2 Although we did not attempt to precisely define the threshold value for flow that, when exceeded, elicits a [Ca2+]i response, we did note that an increase in superfusate rate to 15 µl/s failed to elicit an increase in [Ca2+]i in principal ([Ca2+]i = -1.9 nM) and intercalated (
[Ca2+]i = -6.9 nM) cells in a single experiment in a split-open CCD. An increase in superfusate flow rate to 20 µl/s led to increases in
[Ca2+]i of 13.3 and 30.9 nM in principal and 9.7 and 54.9 nM in intercalated cells in 2 CCDs. These data suggest that the threshold in our flow apparatus lies between flow rates of 15 and 20 µl/s, which correspond to tip velocities between 11 and 14 µm/s.
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REFERENCES |
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