Voltage Dependence and pH Regulation of Human Polycystin-2-mediated Cation Channel Activity*

Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca 2 (cid:1) -permeable non-selective cation channel. Polycystin-2 is functionally expressed in the apical membrane of the human syncytiotrophoblast, where it may play a role in the control of fetal electrolyte homeostasis. Little is known, however, about the mechanisms that regulate polycys-tin-2 channel function. In this study, the role of pH in the regulation of polycystin-2 was assessed by ion channel reconstitution of both apical membranes of human syncytiotrophoblast and the purified FLAG-tagged protein from in vitro transcribed/translated material. A kinetic analysis of single channel currents, including dwell time histograms, confirmed two open and two close states for concentration of 150 K (cid:1) and 135 Cl (cid:3) , respectively. In Vitro Transcription/Translation of FLAG-tagged Polycystin- vitro FLAG-tagged polycystin-2 a 3.2-Kb I- Pst I fragment encoding the protein pVL1393-PKD2 was transferred to the Xba I-NsiI site of pGEM-7zf ( (cid:1) to generate the plasmid pGEM-PKD2. This cDNA was transcribed and translated in the TnT-T7-coupled reticulo-cyte lysate (Promega)

Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca 2؉  Autosomal dominant polycystic kidney disease (ADPKD) 1 is a prevalent human genetic disorder affecting 1:400 to 1:1000 individuals worldwide. Mutations in at least two genes, PKD1 and PKD2, are responsible for more than 90% of all cases of the disease (1). Despite little knowledge concerning the role(s) of the gene product of PKD1, polycystin-1, recent studies deter-mined that polycystin-2, the gene product of PKD-2, is a Ca 2ϩpermeable, non-selective cation channel (2,3). It is assumed that membrane-associated polycystin-1-polycystin-2 complexes may be part of a regulatory pathway involved in the control of membrane transport in target epithelia, including those of the kidney and the liver. Coiled-coil interactions between polycystin-1 and -2 are postulated to occur in vivo (4), and the two gene products, but neither one alone, were reported to increase the whole cell conductance when heterologously overexpressed in mammalian cells (5). Dysfunctional control of this transportassociated regulatory pathway has been implicated in cyst formation and cell proliferation. This may be partly explained by the cell location and developmental characteristics of the target tissue(s) in which polycystin-2 is expressed (6). Recent findings indicate, however, that polycystin-2 engages in ion channel activity in the absence of any other associated proteins (2). As wild type (2,3) as well as ADPKD-causing mutated polycystin-2 (7) both behave as functional ion channels in plasma membranes, it seems likely that as yet largely unknown regulatory mechanisms may contribute to the activation/regulation of polycystin-2 channel function in vivo.
In this report, we investigated the regulatory role of pH in the control of polycystin-2 channel function. Human polycystin-2 from two different sources, including hST apical membranes and the in vitro transcribed/transduced human gene product, was functionally reconstituted in a lipid bilayer system where the effect(s) of pH on its cation channel activity was determined. The data indicate that polycystin-2 is strongly voltage-dependent and contains a regulatory site that is highly sensitive to H ϩ ions, enabling its regulation as an ion channel as a function of intracellular pH.

EXPERIMENTAL PROCEDURES
Human Placenta Membrane Preparation-Syncytiotrophoblast membrane vesicles were obtained from term human placentae as described (8) with minor modifications (2). Apical membrane enrichment was ϳ26-fold from the initial homogenate, and the final pellet was resuspended in a buffer solution containing HEPES-KOH (10 mM), pH 7.14, sucrose (250 mM), and KCl (20 mM).
Solutions-Both sides of the lipid bilayer were bathed with solution containing 10 -15 M Ca 2ϩ , 10 mM MOPS-KOH, and 10 mM MES-KOH, pH 7.14. The final K ϩ concentration in the solution was ϳ15 mM. KCl was added to the cis side of the chamber to a final concentration of 150 mM. Whenever indicated, KCl was added to the trans compartment to a final concentration of 150 K ϩ and 135 Cl Ϫ , respectively.
In Vitro Transcription/Translation of FLAG-tagged Polycystin-2-In vitro translated FLAG-tagged polycystin-2 was obtained as recently described (2). Briefly, a 3.2-Kb XbaI-PstI fragment encoding the protein from pVL1393-PKD2 was transferred to the XbaI-NsiI site of pGEM-7zf (ϩ) to generate the plasmid pGEM-PKD2. This cDNA was transcribed and translated in vitro with the TnT-T7-coupled reticulocyte lysate system (Promega) in the presence or absence of microsomal * This work was supported in part by an American Society of Nephrology grant-in-aid (to H. F. C.) and by Program Project Grant DK54711 (to K. K., M. E., and M. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
*  Ion Channel Reconstitution-Lipid bilayers were formed with a mixture of synthetic phospholipids (Avanti Polar Lipids, Birmingham, AL) in N-decane following methods reported previously (2,8). All phospholipids used were 1-palmitoyl-2-oleoyl-based, including 1-palmitoyl-2oleoyl phosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylethanolamine. The lipid solution (ϳ20 -25 mg/ml) was spread over the diameter aperture (250 m) of a polystyrene cuvette (CP13-150) of a bilayer chamber (model BCH-13, Warner Instruments Corp.) with a thin glass rod. The cuvette was inserted into a polyvinyl chloride holder, thus defining two aqueous compartments of volumes 800 and 1,600 l, respectively, and separated by a planar lipid film as originally described (9). Both sides of the lipid bilayer were bathed with a 10 -15 M Ca 2ϩ solution, which was buffered at pH 7.14 with 10 mM MOPS-KOH and 10 mM MES-KOH. The final K ϩ concentration in the solution was ϳ15 mM. Unless otherwise stated, experiments were initiated by bathing the trans side of the bilayer with this solution. 135 mM KCl was added to the cis side of the chamber to generate a trans bilayer osmotic gradient that promoted the vesicle-planar bilayer membrane fusion (10).
Changes in pH-Changes in pH in the cis and trans hemi-chambers of the lipid reconstitution chamber were conducted by addition of small volumes (1-10 l) of concentrated solutions of either HCl or KOH, ranging between 0.5 and 2.0 N. The final pH was calibrated with a pH mini-electrode, either by means of titration curves of comparable volumes and saline concentrations or directly from the final cis or trans solutions.
Electrical Recordings-Holding potentials (V h ) were applied from the trans chamber with either a DC voltage source or a wave function generator having the opposite, cis side defined as virtual ground. Unless otherwise stated, a cis minus trans voltage convention was utilized throughout the study. Bilayer formation was monitored by applying a 2.5-mV peak-to-peak 20-Hz triangular wave with a typical membrane capacitance of 100 -200 pF. All the experiments were performed at room temperature (20 -25°C). Electrical signals were recorded using a current-to-voltage converter with a 10-gigaohm feedback resistor. Output (voltage) signals were low pass filtered at 700 Hz (Ϫ3 db) with an eight-pole Bessel type filter (Frequency Devices, Haverhill, MA). Signals were displayed on an oscilloscope, and channel recordings were simultaneously digitized with a pulse code modulator (Sony PCM-501 ES) and stored in videotapes with a VCR (Toshiba HQ). Data were later transferred to a personal computer for subsequent analysis at 4 kHz (unless otherwise stated). Whenever indicated, single channel current tracings were further filtered (see "Results") for display purposes only.
Data Acquisition and Analysis-Unless otherwise stated, pCLAMP Version 5.5.1 (Axon Instruments, Foster City, CA) was used for data analysis, and Sigmaplot Version 2.0 (Jandel Scientific, Corte Madera, CA) was used for statistical analysis and graphics. Single channel conductances (␥) under asymmetrical conditions were calculated by the best fitting of current-to-voltage experimental data to either a straight line or the Goldman-Hodgkin-Katz equation, such that ␥ ϭ I/(V h Ϫ E r ) was obtained from Eq. 1, where i represents the cation species (either K ϩ or Na ϩ ) in the trans compartment, and j represents the cation species in the cis compartment. V h is the holding electrical potential in mV; z i and z j , are the charge for species i and j, respectively. C cis and C trans are the cis and trans concentrations of j and i, respectively, and ␣ ϭ RTV h /z i F, and ␤ ϭ RTV h /z j F. P i and P j represent the permeability coefficient for either species i or j, respectively. F is the Faraday constant, 96,500 coulombs/ equivalent; R is the ideal gas constant, 0.082053 liter⅐atm/(mol K) or FIG. 3. Effect of low pH cis on hST polycystin-2 channel currents. A, representative single channel currents in either symmetrical pH (top tracing, cis and trans compartments, pH 7.14) or after reduction of pH cis to pH 6.40 (lower tracing). In B, single channel current amplitude (all-point) histograms were obtained by fitting experimental values (circles) with two-Gaussian distributions (solid lines). Single channel amplitude did not change after reduction of pH cis . The holding potential was 20 mV. Data are representative of four experiments. C, closed and open state dwell histograms (left and right, respectively) for tracings in panel A. The number of kinetic states (two) remained unchanged, but the time constants obtained by best fitting of the closed dwell histogram data with two exponentials were 0.79 and 44.4 ms for the closed states, and 1.78 and 16.7 ms for the open states, respectively. The data indicate that the second rate constant for the closed state increased after reduction in pH cis . D, current-to-voltage relationships for single channel currents under either control (pH cis and pH trans ϭ 7.14, filled circles) or low pH cis (6.40, filled squares). Data are representative of five experiments. In E, voltage dependence of the open probability of the channel in the presence of pH cis ϭ 6.40 (filled circles, n ϭ 3) was fitted to the Boltzmann equation (solid line), showed a shift to the left, indicative of a change in voltage sensitivity as compared with normal pH cis (dashed line, see "Experimental Procedures" and "Results" for fitted values).
1.9872 cal/(mol K); and T is the absolute temperature, 293 K in our calculations.
The single channel open probability (P o ) was obtained from dwell time histograms by fitting experimental residence times with PClamp software. The voltage dependence of the P o was assessed by fitting P o versus V h , the holding voltage potential, to the Boltzmann equation, shown in Eq. 2, where P max is the maximal value for P o , kF/RT represents the slope, k is equal to the z times ␦ product, z and ␦ are the charge of, and the fractional voltage drop sensed by, the gating particle, respectively. V m is the holding potential at which P o ϭ 0.5. F, R, and T have their usual meaning. Whenever indicated, statistical significance was obtained by paired t test comparison of sample groups of similar size (11). Average data values were expressed as the mean Ϯ S.E. under each condition, and n represents the number of experiments analyzed. Statistical significance was accepted at p Ͻ 0.05. Patlak's mean versus variance analysis to enhance detection of brief subconductance states was conducted as originally described (12). Tracings of 12.5 s in duration (50,000 points) were used to perform this analysis. Five-point segments or "sliding windows" were considered where each window started one point later than the previous one. The mean versus variance of the windows was plotted by connecting lines following the temporal sequence in the original traces. Histograms were constructed for those mean current values whose variance was either less or equal to that corresponding to the closed state variance. Thus, mean current values corresponding to "in-between" substate transitions (large variance) were discarded (12 where H cis ϩ , H trans ϩ , and H site ϩ represent the H ϩ ions in the cis and trans compartments and the protonation site, respectively. [H ϩ ] cis and [H ϩ ] trans represent the H ϩ concentrations in either compartment. Further, b 1 , b Ϫ1 , b 2 , and b Ϫ2 are the voltage-independent (V h ϭ 0 mV) components of the velocity constants for the reaction. Any departure from zero mV would entail a correction for each velocity constant, which is affected by an exponential factor taking into consideration the holding potential (13).
Following Woodhull's interpretation of this model for the pH inhibition of macroscopic Na ϩ currents in nerve cells (13), several assumptions were made. The H ϩ association and dissociation reactions with the channel site change exponentially with respect to the V h , as in Eyring rate constant theory (14). Whenever H ϩ occupies the blocking site, polycystin-2-permeable ions, in this case K ϩ , do not permeate the channel. Thus, ions other than H ϩ do not interfere with the regulatory (protonation) site, and in this sense, K ϩ and H ϩ ions do not compete with each other. This model is not concerned with polycystin-2 opening and closing mechanisms but rather with the fractional probability of open channel blockage by H ϩ occupation of the regulatory site. Another important assumption of the Woodhull's model is that the current driven by H ϩ ions as they enter the channel is negligible as compared with that observed by K ϩ ions. Thus, H ϩ movement does not make a contribution to the open channel current. This is further supported by the fact that there is no change in the single channel conductance at different pHs. Finally, the H ϩ ion-H ϩ site system is in steady state, namely, the time constants for the open channel state are long as compared with the time taken by the H ϩ ion to reach a steady state concentration inside the channel.
For holding potentials between Ϫ30 and 10 mV, for which P o (V h ) is almost constant as a function of voltage, the equilibrium constant of the

FIG. 4. Effect of low pH cis on hST polycystin-2 channel current substates.
Left, representative single channel currents in either symmetrical pH (cis and trans compartments pH 7.55) after reduction of pH cis to pH 6.60 and upon return of pH cis to 7.55. Several single channel substates are more frequent after lowering pH cis . Clearly distinguishable single channel conductance levels at control pH were 51, 109, and 144 pS, respectively. Similar values with different probabilities were observed at low pH. The holding potential was 60 mV for all tracings. Data are representative of four experiments. Right, the presence of channel substates was made more evident by Patlak's mean versus variance analysis (12), where variance deflections ( 2 , pA 2 ) were plotted versus mean values for sliding windows (abscissa, see "Experimental Procedures"). Various single channel substates were observed that started at ϳ5 pA under control conditions (top deflections histogram), which shifted to transitions starting at zero level after lowering pH cis (middle deflections histogram). Channel resumed maximal conductance upon return to normal pH (bottom deflections histogram). Data are representative of four experiments.
In our study, b 2 and b Ϫ2 are negligible as compared with b 1 and b Ϫ1 such that K(V h ) can be reduced to Eq. 6, where z is the charge of the membrane potential sensor, ␦ is the electrical distance sensed from the cis (cytoplasmic) side of the channel, and F, R, and T have their usual meaning. Fitting the values to this equation, the z␦ product was obtained, as well as the value K for V h ϭ 0 mV, representing the dissociation constant K (0) ϭ (b Ϫ1 /b 1 ).

Kinetic Analysis of Single Channel Currents of hST Polycystin-2-Human
syncytiotrophoblast apical membranes were reconstituted in the presence of asymmetrical KCl (150 mM) in the cis side and 15 mM K ϩ in the trans side. Polycystin-2 single channel currents were obtained at different holding potentials (Fig. 1A), and current amplitudes and open probabilities were calculated from all-point histograms (Fig. 1B). The mean open probability (area under open state) largely depended on the holding potential. The main single channel conductance was 157 Ϯ 4.90 pS (n ϭ 7), as recently reported (2). Kinetic analysis of single channel currents (Fig. 2, A and B) indicated that the channel had two open and closed states, as indicated by the closed and open dwell histograms (Fig. 2C, left and right, respectively), which were best fitted with two exponentials for each state. The open probability of the channel (P o ) was strongly dependent on the holding potential (Fig. 2D). The voltage dependence of the P o was further assessed by fitting experimental data to the Boltzmann equation (see "Experimental Procedures"). The fitted parameters indicated a P max of 0.72 Ϯ 0.05 (n ϭ 5) with k ϭ Ϫ2.3 Ϯ 0.7 (n ϭ 5), and a V m ϭ 29.4 Ϯ 3.9 mV (n ϭ 5).
Effect of pH cis on hST Polycystin-2 Channel Function-To assess the regulatory effect(s) of pH on polycystin-2 channel activity, the pH of the cis chamber was reduced by addition of a small volume of HCl (2.0 N). Single channel currents were reduced after lowering pH cis (Fig. 3, A and B). The single channel current amplitude of hST polycystin-2 in symmetrical KCl (150 mM) did not change by reducing pH cis from 7.14 to 6.4 (Fig. 3B). The kinetic behavior of the channel did not change because the dwell histograms were still best fitted by two exponentials (Fig. 3C). However, a change in pH reduced the open probability of the channel, which decreased from 0.74 Ϯ 0.11 (n ϭ 5) at pH 7.14 to 0.24 Ϯ 0.08 (n ϭ 3) at pH 6.4 for a holding potential of 20 mV. This was a reflection of the increase in the dwell zero time (second rate constant), which increased as a function of lowered pH. The single channel conductance for these tracings was also similar between control and low pH cis , as measured by K ϩ currents in the presence of the KCl chemical gradient (Fig. 3D). The non-selective cation channel conductance of hST polycystin-2 in symmetrical KCl (150 mM) was 135 Ϯ 11.2 pS (n ϭ 5), and it did not change by reducing pH cis from 7.14 to 6.4 (138 Ϯ 21 pS, n ϭ 3, p Ͻ 0.3). Fitting of the P o values as a function of the various holding potentials showed a displacement of the Boltzmann distribution determined after pH cis reduction (Fig. 3E). The P o versus voltage function shifted to the left, from V m ϭ 29.4 Ϯ 3.9 mV (n ϭ 5) at pH cis 7.14 to V m ϭ Ϫ14.7 Ϯ 2.6 mV (n ϭ 3) at pH cis 6.4, with a decrease in the slope k from Ϫ2.30 Ϯ 0.70 (n ϭ 5) at pH cis 7.14 to k ϭ Ϫ1.06 Ϯ 0.5 (n ϭ 3) at pH cis 6.4. Further, the reduction in pH cis also increased the number of single channel substates (Fig. 4), which were further manifested by the transitions observed in the mean versus variance plots (Fig. 4, right) (12). Thus, an increase in H ϩ concentration induced more fluctuations between the channel substates, but the current amplitudes themselves did not change. Conversely, an increase in pH cis increased the mean current by 123% (n ϭ 3, p Ͻ 0.045, Fig. 5) by increasing the open probability of the high conductance state of the channel but not the single channel conductance of hST polycystin-2 (Fig. 5B, histograms). Elimination of the pH gradient (pH cis ϭ pH trans ϭ 6.4) at low pH, however, was without effect in reversing the inhibitory effect of pH cis reduction alone (n ϭ 7, data not shown).
Effect of Changes in pH cis on the Purified Polycystin-2-To confirm that a regulatory site for H ϩ ions indeed exists in the polycystin-2 ion channel, the effect of changes in pH cis was

Voltage Dependence and pH Regulation of Polycystin-2 24964
further assessed on the ion channel activity of purified FLAGtagged human polycystin-2, obtained from the in vitro transcribed/translated material (Fig. 6). A reduction of pH cis decreased the single channel activity, including an increase in the subconductance state level of the channel (Fig. 6). A completely inhibited channel reactivated upon reinstating a higher pH in the cis side of the chamber (Fig. 6B). The data are in agreement with the effect of an H ϩ ion titration site, which is likely intrinsic to the channel protein by comparison of the similar effect in the hST and in vitro preparations.
Effect of pH cis on Polycystin-2 Open Probability-To further assess the nature of the regulation by pH on hST polycystin-2 single channel tracings, the mean open probability (P o ) was assessed as a function of the pH in the cis compartment (Fig. 7). Experimental values were fitted to Eq. 7, P o (pH)ϭ 10 pH 10 pH ϩ10 pKa (Eq. 7) which is based on the assumption that polycystin-2 presents a single H ϩ titration site accessible from the cytoplasmic domain. Orientation of the channel protein was confirmed in our original studies (2). The experimental data were best fitted with this equation, indicating that a regulatory site indeed exists for the regulation by pH cis of polycystin-2 ion channel activity, which has an equilibrium constant pK a of ϳ6.4. The equilibrium constant for the H ϩ regulatory site was validated by assessing the effect of pH on the single channel currents following a modification of the model described by Woodhull to assess the blocking effect of low pH on nerve Na ϩ currents (13). Theoretical current-to-voltage (I/V) relationships (Fig. 7) were obtained for single channel currents at two different pHs, namely, 7.14 and 6.40, for which single channel currents and open probabilities were fitted to the Goldman-Hodgking-Katz and Boltzmann equations, respectively (Figs. 2D and 3, D and E). The I/V plots, where the mean single channel currents are shown as I ϫ P o versus holding potentials in the range of Ϫ20 to 10 mV showed a strong voltage dependence of the pH effect on the mean channel currents (Fig. 7). By applying the Woodhull's model (13) to the pH 7.14 versus 6.40 substracted currents (Fig. 7), the product z␦ was calculated as 0.67, and the pK was calculated at ϳ6.5, where pK ϭ Ϫlog K for the protonation reaction.

DISCUSSION
The present study demonstrates that polycystin-2 cation channel function is voltage-dependent and highly sensitive to changes in pH. The data are most consistent with the assumption that polycystin-2 contains a highly sensitive pH site, which regulates its cation-selective channel activity by controlling the voltage dependence of the single channel kinetics. Dwell time histograms confirmed a two-open, two-closed state for the spontaneous channel at normal pH and the strong voltage dependence of the open probability. Although the single channel conductance did not change by modifying pH cis , the change in pH largely decreased the open probability of the channel. This effect was also voltage-dependent, being greater at a more positive V h such that the Boltzmann distribution of P o (V h ) was displaced to the left, as indicated by the decreased slope as a function of reducing pH cis . This suggests a placement of the titration site in the conductance pore of the channel. Conversely, an increase in pH cis increased the polycystin-2 mediated K ϩ currents also associated with an increased NP o but not the single channel conductance. The data further indicate that the changes induced by pH on polycystin-2 ion channel activity were not elicited by modifying the H ϩ chemical gradient but instead by a regulatory site in the channel protein because a decrease in pH such that the pH gradient was eliminated (pH cis ϭ pH trans ϭ 6.4) was without effect in reversing the inhibitory effect of reducing the cis pH alone. An effect of pH cis on polycystin-2 channel regulation was also observed with the purified protein from the in vitro translated material, confirming a pH-sensitive site in the channel protein itself. Taken together, these data suggest that an H ϩ ion (protonation) regulatory site, only accessible from the intracellular (cis) side of the channel where changes were made, controls the polycystin-2 cation channel from human placenta. The orientation of the reconstituted polycystin-2 channel was confirmed previously (2)  body to the cis side or amiloride to the trans side of the reconstitution chamber induced the expected blocking effect on either side of the channel (2).
To assess the affinity of the protonation site for pH regulation of polycystin-2, P o versus pH cis data were fitted to a Henderson-Hasselbach type equation (Fig. 7), indicating the presence of an equilibrium constant (pK a ) of ϳ6.4, which was confirmed independently by applying a kinetic model for the protonation reaction on the single channel currents at different pHs (Woodhull model , Fig. 7). The data are in agreement with the presence of a regulatory site in the channel protein, which is only accessible from the cytoplasmic side of the channel, thus suggesting that pH regulation of polycystin-2 is largely intracellular. This is supported by the fact that changes in cis, but not trans, pH modified the polycystin-2 channel currents. Only one of five experiments where pH was lowered from the trans side showed a detectable change in channel function (data not shown). Thus, although the extracellular domain of the channel may also be sensitive to pH (which will require further experimentation), it is unlikely that this effect is associated with the same site described in this report, and it may be speculated that it is instead associated with changes in surface potential as reported previously for other channels (15,16). Interestingly, polycystin-L, a channel homolog of polycystin-2 (17), and the ADPKD-causing truncated R742X-polycystin-2 (7) were also blocked by a reduction in cytoplasmic pH, further suggesting a homologous topology in the ion channel, consistent with a pH regulatory site most likely present in the pore conduction structure of the channel protein. However, the L-type calcium channel, which shares homology with polycystin-2 (18) and is also sensitive to changes in pH (15), may have a regulatory site external to the conduction pore of the channel (16).
The human syncytiotrophoblast, the most apical membrane barrier of the human placenta, provides the only natural epithelium where polycystin-2 function has been determined (2). As a permeable selective barrier for the transfer of various solutes between mother and fetus (19), the human syncytiotrophoblast may help maintain the fetal electrolytic homeostasis. As a non-selective cation channel, polycystin-2 may serve several purposes, including Na ϩ absorption, K ϩ secretion, and the regulation of Ca 2ϩ entry into the fetal environment. The particular nature of the epithelial hST, however, namely being syncytial, further requires specific and highly controlled regulatory mechanisms, essential for the maintenance of the hydro-electrolytic homeostasis of both the maternal blood and the fetal environments. The pH regulation of the polycystin-2 channel activity suggests the importance of H ϩ ions as second messengers whose presence may contribute to the control of hydroelectrolytic homeostasis. Further studies will be required to assess the metabolic role of this novel regulatory pathway in vivo and its role in the dysfunctional aspects associated with ADPKD.