Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals
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- Hovater, M.B., Olteanu, D., Hanson, E.L. et al. Purinergic Signalling (2008) 4: 155. doi:10.1007/s11302-007-9072-0
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Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpkTg737) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca2+. Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na+) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.
KeywordsATP secretion Nucleotide secretion Purinergic signaling Kidney Renal collecting duct Cilia
Oak Ridge polycystic kidney
autosomal recessive polycystic kidney disease
- Tg737 gene
Madin-Darby canine kidney
cortical collecting duct
arbitrary light units
transient receptor potential
regulatory volume decrease
Monociliated ductal epithelial cells are receiving much attention due to their remodeling in polycystic kidney diseases, role in other cystic diseases of the kidney and other tissues, and in sensory physiology [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12]. Cilia and flagella from lower organisms have important roles in sensory physiology in response to flow, touch, chemical and osmotic stimuli [1, 2, 3, 4, 5, 15, 16]. MDCK cells and other cell and tissue models of the renal collecting duct have been essential in characterizing cilium-derived cell calcium (Ca2+) signals [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. It appears likely that this cilium-affected Ca2+ signal is derived from Ca2+ entry from extracellular stores and Ca2+ release from intracellular stores; the latter, perhaps, an ER cisternae near the basal body immediately beneath the monocilium [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. Previously thought to be a vestigial organelle [1, 2, 3, 4, 5] or morphological marker for the cortical collecting duct (CCD) principal cell (PC) , the apical central monocilium is likely a sensor for ductal epithelia .
Recently, our laboratory has shown that epithelial sodium channel (ENaC)-mediated sodium (Na+) absorption is upregulated in mutant cilium-deficient orpk CCD PC monolayers versus genetically rescued cilium-competent controls . ENaC activity is present under open-circuit voltage and short-circuit current measurements in rescued cell monolayers with a well-formed cilium, but the electrical signals were three- to sixfold less than mutant monolayers . One of our working hypotheses is that an inhibitory signal is lost (when the monocilium is not well formed) that is responsible for tonic attenuation of ENaC function . Indeed, flow-induced Ca2+ signals have been shown recently by Praetorius and Leipziger not to be due to the cilium of MDCK cells per se but to autocrine ATP signaling that is stimulated by pressure pulses and responsible for Ca2+ spark and wave signal formation . Immature MDCK cells without discernible cilia and mature MDCK cells with cilia responded similarly . Alternatively, Satlin and coworkers have compelling data that monocilia do confer flow-based Ca2+ signals in isolated perfused CCDs from control mice versus mutant Tg737orpk mice . In multiple different preparations from tissue to renal epithelia to heterologous cells, flow-induced Ca2+ signals have been observed [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. However, the concept that an autacoid might mediate these cilium-specific effects has not been addressed.
Herein, we show that the fully formed monocilium does confer a more robust Ca2+ signal in rescued cell monolayers versus mutant cell monolayers that are deficient in well-formed cilia. This finding agrees with the majority of the literature examining this phenotype [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. However and more importantly, we show that stimulated or regulated ATP release is impaired when the monocilium is malformed. Ionomycin-, hypotonicity-, and mechanically induced ATP release are more robust in cilium-competent monolayers versus cilium-deficient monolayers. Varying the order of stimuli also revealed that cell Ca2+ influences the mechanically induced secreted ATP pool and that hypotonic cell swelling and other mechanical stimuli trigger the release of the same ATP pool. Finally, the flow-induced Ca2+ signal in this cell model requires autocrine ATP release and signaling, as it was blocked by the ATPase/ADPase scavenger, apyrase. These data may reconcile the different conclusions within the Praetorius and Leipziger study  and the study of Satlin and coworkers . To our knowledge, this is the first report linking the sensory apical central and nonmotile cilium to ATP secretion.
Materials and methods
The collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk) mouse model of autosomal recessive polycystic kidney disease (ARPKD) and the genetically rescued cells with the wild-type orpkTg737 gene were a generous gift from Dr. Bradley Yoder (University of Alabama at Birmingham). The “mutant 1” cell clone (94D pcDNA 3.1 cells), the “rescued 1” cell clone (94D BAP737-1 cells), and the “rescued 2” cell clone (94D 737-2 cells) were handled identically and were grown under G418 selection on 6.5-mm diameter (Corning Costar) or 12-mm diameter filter supports (Millicell CM) and bathed in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 HAM with L-glutamine and 15 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) (Sigma) . This medium was supplemented with 5% fetal bovine serum (FBS) and contained (per 1,000 ml) dexamethasone (100 μl/l of 2 mg/ml stock), interferon-γ (25 μl of 800 U/μl stock), T3 (10 μl/l of 13 mg/ml stock), G418 (500 μl of 400 μg/ul stock), penicillin-streptomycin (10 ml of 100X stock), and ITS (10 ml of 0.5 mg/ml insulin transferrin selenium concentrated stocks). The cells continued to be bathed on both sides of the filter until a monolayer tight to fluid formed. Measurement of the resistance of the filters was used as an indicator of the formation of a mature monolayer. Once the monolayers were formed for at least 12 h, the cells were then acceptable for the experimental assay.
In addition to these original clones developed in the Yoder laboratory, we also generated two new clonal lines from the original 94D mixed mutant CCD cell cultures, namely “mutant 2” and “mutant 3.” The only difference between these two subsequent clones and the original “mutant 1” clone is the fact that the “mutant 1” clone is stably transfected with an “empty” pcDNA 3.1 vector that confers G418 resistance. “Mutant 2,” “mutant 3,” and the mixed mutant cultures are grown in a medium without G418. We also studied a “rescued B2” clone that was stably transfected and rescued with the wild-type Tg737 gene but we derived from a different mutant CCD originally, 94E. The generation of these additional clones was described in a previous publication .
Bioluminescence detection of ATP released from epithelial monolayers
In every assay performed, there was an initial basal ATP measurement followed by subsequent ATP measurements in response to different stimuli added in different sequences. ATP release assays were performed mainly on well-polarized cell monolayers grown in clear polyester 6.5-mm diameter filter supports. Mutant versus rescued cell monolayers were studied side by side in each ATP release protocol. The use of luciferase-luciferin to indirectly measure the ATP concentration has been published previously in detail . Every drug prior to experimentation was tested to ensure that they did not interfere with the luciferase enzyme activity [34, 35, 36, 37]. There was also no change in cell viability with any of these maneuvers as has also been reported previously [34, 35, 36, 37]. Each experiment began with a basal ATP measurement or the addition of Opti-MEM I medium (GIBCO-BRL) with 1 mg/ml luciferase-luciferin reagent (Sigma) being added to the apical or basolateral side of the filter’s monolayer. Basolateral ATP release was negligible; therefore, all data reported are apical or luminal ATP secretion. Basal levels of ATP release were measured for 6 min in 15-s, nonintegrated photon collection periods in a TD-20/20 Luminometer (Turner Designs) [34, 35, 36, 37]. In different orders of addition, we used the following stimuli: (1) ionomycin (2 μM) (to increase the intracellular calcium concentration), (2) distilled water with 1 mg/ml luciferase-luciferin (to dilute the osmotic strength of the Opti-MEM I medium), and (3) intentionally harsh pipetting in the center of the apical surface of the cell monolayer (to induce a mechanical stimulus on the apical membrane). Normally, addition of drug or distilled water (or the same volume of medium as a volume addition control) is performed or dispensed very slowly along the wall of the plastic support that holds the permeable filter so as not to disrupt the tight, confluent monolayer. Therefore, by quickly pipetting the medium up and down onto the monolayers, a mechanical stimulation is induced. Luminescence was measured for 6 min after each stimulus. All experiments ended with the addition of hexokinase to eliminate any ATP left in the medium. All assays were performed at room temperature.
Fura-2/AM imaging of cytosolic free calcium in a cell monolayer-based perfusion system
Fura-2/AM imaging was performed as described previously [38, 39, 40]. However, to remain faithful to the study of polarized cell monolayers, we designed a cell monolayer-based perfusion chamber system where 12-mm diameter Snapwell Transwell filter supports are inserted into a homemade perfusion chamber designed to accommodate this special filter support. Apical and basolateral perfusion are then performed separately through independent injection ports and separate ejection ports are subjected to vacuum. Response to changes in apical flow from 1 ml/min (a “slow” flow) to 5 ml/min (“high” flow) were performed to induce a flow-induced Ca2+ transient akin to that observed by many other laboratories. We assessed mutant and rescued cell monolayers in parallel during all protocols. We also assessed the flow-induced Ca2+ transient signal in the absence of apical extracellular Ca2+ and in the presence of gadolinium chloride and apyrase. It was not possible to calibrate the Fura-2 fluorescence signal to real free cytosolic Ca2+ values because of ionomycin contamination of the flow chamber. As such, fluorescence ratio values are shown. We give estimates of what the free Ca2+ may be based on previous calibrations of the same cells grown as nonpolarized cells.
All reagents and drugs were purchased from Sigma. Larger 12-mm diameter filter supports were obtained from Millipore. Smaller 6.5-mm diameter filter supports were obtained from Corning Costar. It should be noted that we are using less luciferase:luciferin detection reagent than in past studies [34, 35, 36, 37].
Basal ATP release or secretion is not different between mutant and rescued CCD PC monolayers
Ionomycin-stimulated ATP release is more robust in rescued versus mutant cell monolayers
Hypotonicity-induced ATP release is more robust in rescued versus mutant cell monolayers
Mechanically-induced ATP release is more robust in rescued versus mutant cell monolayers
An original physiological role for the apical central monocilium was that of a “mechanosensor.” Figure 5 validates this idea using ATP secretion as the biological endpoint. Repeated pipetting of isotonic medium containing the same amount of detection reagent revealed repeated stimulation of ATP release transients in cilium-competent monolayers. The responses in mutant cilium-deficient cell monolayers were greatly attenuated. This comparison was performed on the same day with the same preparation of luciferase:luciferin detection reagent. Figure 6 shows a similar experiment but with ionomycin pretreatment. Here, the classic ionomycin-induced ATP secretion phenotype is shown. It is a slow and monophasic increase in secreted ATP that is robust; however, the response of rescued monolayers is fourfold greater than mutant monolayers. Subsequent mechanical stimulation via repeated pipetting showed a more vigorous response in the cilium-competent monolayers versus the cilium-deficient monolayers. Figure 7 provides multiple compelling illustrations. First, the response to hypotonic challenge is shown and it is much more robust in rescued versus mutant cell monolayers. Interestingly, however, the repeated harsh pipetting stimulus is without effect in both rescued and mutant cells after the hypotonic challenge. In fact, the luminescence decreases modestly. These data suggest that these two mechanical stimuli may mobilize the same “releasable” pool of ATP. Figure 8 shows the relative effects of the three stimuli used in this study on rescued cilium-competent cell monolayers and on mutant cilium-deficient cell monolayers. Although greatly diminished because of the Y-axis scale, the mechanical stimulus triggered a fourfold greater ATP secretion transient in the rescued cell monolayers versus the mutant cell monolayers. Ionomycin also produced a fourfold greater sustained ATP release in rescued versus mutant monolayers. Hypotonic challenge also triggered a more robust response in the mutant cell monolayers. A large degree of the stimulated ATP release was inhibited by the broad specificity anion transport inhibitor, DIDS, suggesting that an ATP transport process may be mediating the release of ATP from intracellular pools. In the presence of DIDS inhibition, release is inhibited while degradation of released ATP proceeds unabated, leading to a sharp decline in the signal. In contrast, ionomycin-stimulated ATP release is attenuated by performing the luminescence experiment at 4°C (data not shown), suggesting a vesicular mechanism of release. Hexokinase is added at the end of every protocol to scavenge the ATP and abolish the luminescence signal.
Taken together, these data suggest that cell Ca2+ is critically important for priming the ATP release machinery. These data also suggest that the “releasable” pools of ATP are present in cilium-competent cell monolayers beneath the apical cell surface, while they may be impaired or missing in cilium-deficient cell monolayers. These ideas will be revisited and discussed below.
Flow-induced calcium signals are attenuated in cilium-deficient mutant monolayers versus cilium-competent monolayers
An original physiological role for the apical central monocilium was that of a mediator of flow- or touch-induced Ca2+ signals in MDCK cells by Praetorius and Spring [17, 18, 19]. Subsequent studies in heterologous cells, renal collecting duct cell models, and renal collecting ducts showed that an intact cilium is required for the flow-induced Ca2+ signal [20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. To be faithful to a polarized epithelial cell monolayer system used routinely for this study and other studies [32, 34, 35, 36, 37, 38, 39, 40], we devised a flow chamber where the apical versus basolateral sides of the monolayer could be perfused independently and at different flow rates. The 12-mm diameter Snapwell transwell filter can then be inserted into this chamber for selective perfusion and fluorescence imaging. With a constant low rate of perfusion of the basolateral surface of the monolayers, low versus high rates of perfusion were performed on the apical surface in rescued versus mutant monolayers.
In the presence of extracellular Ca2+, modification of the Ringer to mimic collecting duct tubular fluid and to disinhibit Ca2+ entry channels augmented cell Ca2+ significantly only in cilium-competent cell monolayers (Fig. 10a). A change in the flow rate from 1 ml/min to 5 ml/min again only augmented cell Ca2+ in the form of a brief Ca2+ transient with a “spike” and a “shoulder” (Fig. 10a). A typical phenotype is shown in Fig. 9. Mutant cell monolayers failed to respond to flow with a significant change in cell Ca2+ (Fig. 9 shows the typical degree of response to flow). In the absence of extracellular Ca2+, the same magnitude and type of Ca2+ entry “spike” was observed in rescued cilium-competent cell monolayers (Fig. 10b). However, the sustained “shoulder” of this response was missing in the absence of extracellular Ca2+. Any response from the mutant cell monolayers was insignificant and sluggish. In this light, the rescued cell monolayers showed a complete reversal when flow was slowed; however, the mutant cell monolayers did not. The reason for this lack of reversal is unclear, but we speculate that the cells can no longer “sense” flow and, therefore, display deregulation with regard to cell Ca2+ in this manner as well. In Fig. 10c, responses in the rescued cell monolayers were assessed in the presence of apical gadolinium chloride, a broad spectrum inhibitor of mechanosensitive ion channels and Ca2+ entry channels [30, 38]. Infusion of gadolinium chloride during the course of the apical perfusion in the presence of extracellular calcium and in low and high flow also quieted the flow-induced calcium signal (Fig. 10c). The rise in cell Ca2+ in response to the reduction of Na+ and Mg2+ was not blocked, suggesting that this Ca2+ entry mechanism is insensitive to gadolinium. These data suggest that a mechanosensitive Ca2+ entry channel is sensitive to gadolinium and plays a role in flow-induced Ca2+ entry. These data agree with the literature [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30].
Many studies, however, have also implicated autocrine and paracrine purinergic signaling as a major player in mechanically induced Ca2+ sparks and waves in monolayer and tissue preparations [33, 48, 49, 50, 51, 52, 53, 54]. Indeed, in immature and mature MDCK cells lacking or bearing monocilia, a pressure pulse-induced Ca2+ signal was observed in each preparation and the signal was abolished by antagonists to purinergic signaling . To test whether cilium-conferred autocrine ATP release and signaling was important for the cilium-dependent Ca2+ signal, we performed a similar apical flow protocol in the presence of the ATPase/ADPase, apyrase (Fig. 10c). Apyrase blocked the flow-induced Ca2+ signal. Apyrase also attenuated the rise in cell Ca2+ induced by lowering apical Na+ and Mg2+. Taken together, these data suggest that an autocrine ATP signal, released by the monolayer itself, contributes directly to the flow-induced and cilium-derived Ca2+ signal. With the more robust stimulated ATP release phenotypes and the flow-induced Ca2+ signals in the rescued monolayers versus mutant monolayers, we speculate that each may require the apical central monocilium as an integrating sensory organelle.
As introduced, it is becoming abundantly clear that the monocilium in particular and cilia and flagella in general are sensory organelles [1, 2, 3, 4, 5]. In tissues where high flow is present (large airways, proximal tubule, arteries, arterioles, etc.), the monocilium or cilia may be flow sensors for the cell on which they are present. In tissues where low flow is present (bile duct, pancreatic duct, renal collecting duct, small airways, capillaries, venules, veins, etc.), the monocilium may be a chemosensor or an osmosensor. Our data suggest that it may both influence ATP secretion as well as be a chemosensor for the secreted ATP. Arguably, secreted nucleotides and nucleosides are most potent in a local microenvironment within a cellular or tissue microenvironment. Burnstock and colleagues have described this concept elegantly in past review where they described purinergic signaling as being potent and essential in the lumen of tubules and ducts of kidney and gut [62, 63]. A semiclosed system such as the lumen of a renal tubule or duct is ideal in this regard.
Along these lines, the effect of changes in flow has been studied extensively and elegantly. Touch, flow, shear stress, and cell swelling are potent regulators of ion transport [64, 65, 66, 67]. Ca2+ entry as well as Na+, K+, and Cl− transport are governed by mechanical stimuli [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 64, 65, 66, 67]. Mechanical stimuli are also well-known to trigger ATP release in many cell and tissue systems [48, 49, 50, 51, 52, 53, 54, 68]. The propagation of Ca2+ sparks and waves triggered by mechanical stimuli are thought to be mediated by at least two cellular mechanisms: (1) Ca2+-permeable gap junctions that link the cells together and (2) paracrine extracellular purinergic signaling that allows cells to communicate in a local environment . In fact, our data show that an increase in cell Ca2+ primes the “releasable” pools of ATP that are mobilized by hypotonic cell swelling and other mechanical stimuli. However, one can still observe both pipetting- and hypotonicity-induced ATP release without ionomycin pretreatment that is more robust in cilium-competent versus cilium-deficient cell monolayers. Moreover, we also found that we could not observe a mechanically induced ATP release signal after hypotonic cell swelling. This finding suggests that these two “mechanical” stimuli (albeit different) affect the same pool of releasable ATP. Unfortunately, we still need better tools to define each ATP release mechanism and pool. However, our work with low temperature inhibition of vesicle traffic and inhibition of anion transport properties with the broad specificity inhibitor, DIDS, suggests that both ATP transport mechanisms and exocytosis of ATP-filled vesicles contribute to secreted ATP phenotypes (EM Schwiebert et al., unpublished observations).
Recently, we found that ENaC-mediated Na+ absorption is upregulated markedly in cilium-deficient CCD PC monolayers derived from the Tg737orpk mouse . Of many hypotheses put forward to explain this pathophysiological phenotype, one prominent postulate was that the malformed cilium caused the loss of key inhibitory signals that are normally cilium-derived that limit ENaC activity. Indeed, in several different cellular systems, there is agreement that purinergic signaling inhibits ENaC function [69, 70, 71, 72, 73, 74]. Modulation of purinergic receptor-driven cell Ca2+ signaling may be a future putative therapeutic target (along with ENaC itself) to control hypertension that occurs in the majority of human ARPKD patients, especially the children that escape respiratory insufficiency soon after birth .
Finally, it is our hope that this work connects the seemingly disparate conclusions of Satlin and coworkers  and those of Leipziger, Praetorius, and colleagues . Taken together, our data suggest that a well-formed monocilium central to the apical membrane of a collecting duct principal cell is essential for a mature ATP secretion apparatus. One could conceive of this apparatus as a well-formed pool of ATP poised to be secreted in response to different stimuli or the appropriate presence of ATP release machinery (ATP-filled vesicles and/or ATP transport mechanisms). Our studies also suggest that the cilium-derived Ca2+ transient, induced by flow in our study or by other modes in other studies, requires an underlying autocrine ATP signal that is likely transduced by P2X and P2Y ATP receptors on or near the monocilium. Without a well-formed cilium at the apical surface, autocrine purinergic signaling, cilium-derived signaling, and modulation of downstream effectors become disrupted.
We acknowledge the support and assistance of Elisabeth Welty, our laboratory manager, in this work. We acknowledge the support of R01 DK067343 to EMS, R01 DK055007 to BKY, R21 DK071007 to PDB, and the P30 Recessive PKD Research and Translation Core Centers (DK074038). We acknowledge the support of the Department of Physiology and Biophysics for the stipend and benefits for MBH.