Flow-Induced [Ca²⁺]_i Increase Depends on Nucleotide Release and Subsequent Purinergic Signaling in the Intact Nephron

P2 Receptors in Mouse Medullary Thick Ascending Limb

We suggested that an increase of tubular flow triggers nucleotide release and subsequent stimulation of epithelial P2 receptors and increase of [Ca²⁺]_i. To test our hypothesis, we first needed to establish whether P2 receptors are functionally expressed in medullary thick ascending limb (mTAL). Generally, transporting epithelia express luminal and basolateral P2 receptors.⁶ Studies indicate prominent luminal expression of P2Y₂ receptors in collecting duct, which inhibit electrogenic Na⁺ absorption.^13,14 Therefore, we believed this receptor to be the prime candidate for extracellular nucleotide-mediated [Ca²⁺]_i signaling in mTAL. If this is the case, then the P2Y₂ receptor knockout (KO) mouse would be a suitable tool to investigate the functional expression of P2 receptor in mTAL. Figure 1 shows that both 100 μM luminal UTP and ATP trigger brisk increases of [Ca²⁺]_i of similar amplitude in wild-type (WT) but never in KO tubules (fura-2 ratio increase, luminal UTP, WT 0.71 ± 0.2 [n = 5], KO 0.02 ± 0.01 [n = 6]; luminal ATP, WT 0.60 ± 0.13 [n = 5], KO 0.02 ± 0.01 [n = 6]). Other nucleotides such as UDP and ADP in the same concentration were without effect (data not shown). This strongly suggests that the P2Y₂ receptor is the only P2 receptor expressed on the luminal side of mTAL.

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Figure 1.

Effect of luminal UTP (100 μM; A) and ATP (100 μM; B) on [Ca²⁺]_i in perfused mouse medullary thick ascending limb (mTAL). In wild-type (WT) tubules, both nucleotides elicited prompt and transient increases of [Ca²⁺]_i. Mice that were deplete of the P2Y₂ receptor never responded to either luminal ATP or UTP.

UTP also stimulated increases of [Ca²⁺]_i from the basolateral side (Figure 2). Again, this response was present only in tubules from WT mice (fura-2 ratio increase, basolateral UTP, WT 0.25 ± 0.06 [n = 18], KO 0.01 ± 0.01 [n = 13]). Paired experiments demonstrated that luminal UTP was more potent than basolateral UTP at increasing [Ca²⁺]_i in WT mTAL (luminal UTP, WT 0.71 ± 0.2 [n = 5] versus basolateral UTP, WT 0.24 ± 0.06 [n = 6]; P = 0.04). Basolateral ATP was also able to stimulate [Ca²⁺]_i increases in WT and KO tubules. The pattern of the basolateral ATP-induced [Ca²⁺]_i increase in WT tubules, however, was significantly different from that of basolateral UTP (Figure 3). Basolateral ATP increased [Ca²⁺]_i with an initial peak followed by a sustained [Ca²⁺]_i plateau in the continued presence of the agonist. The [Ca²⁺]_i plateau value measured 100 s after the basolateral nucleotide addition was significantly higher in tubules that were stimulated with ATP compared with UTP (0.19 ± 0.02 versus 0.01 ± 0.02, respectively; P = 0.001). An ATP-induced [Ca²⁺]_i plateau was also seen in KO tubules with an amplitude similar to WT (0.26 ± 0.08 versus 0.19 ± 0.08; n = 5; P = 0.462). In contrast, the initial basolateral ATP-induced [Ca²⁺]_i peak was significantly reduced in KO tubules (0.12 ± 0.04 versus 0.43 ± 0.12; n = 5; P = 0.03). These results show the presence of basolateral P2Y₂ receptors. Furthermore, the prevailing ATP-inducible [Ca²⁺]_i response in KO tubules unmasks other basolateral P2 receptors. The [Ca²⁺]_i plateau was completely dependent on basolateral Ca²⁺ (Figure 3). This supports that basolateral ATP stimulates basolateral Ca²⁺ influx and suggests this to occur via a Ca²⁺ permeable P2X receptor. In the entire series of experiments, the resting fura-2 ratio was slightly but statistically significantly lower in WT as compared with KO tubules (WT 0.95 ± 0.03 [n = 31]; KO 1.03 ± 0.03 [n = 34]). This indicates a small elevation of resting [Ca²⁺]_i in KO mTAL. Subsequently, we investigated the source of the UTP-stimulated [Ca²⁺]_i elevations. Luminal and basolateral UTP (100 μM) was tested in the bilateral absence of extracellular Ca²⁺ (Figure 4). Under these conditions, UTP still stimulated a transient [Ca²⁺]_i increase amounting to 0.35 ± 0.04 (n = 3) ratio units from the luminal side and 0.31 ± 0.15 (n = 3) ratio units from the basolateral side. In summary, these data strongly suggest that (1) mouse mTAL express luminal and basolateral P2Y₂ receptors, which trigger an increase of [Ca²⁺]_i via release of Ca²⁺ from internal stores and likely also activation of store-operated [Ca²⁺]_i entry; (2) luminal P2Y₂ receptors are significantly more potent at triggering [Ca²⁺]_i elevations than P2Y₂ receptors that are localized on the basolateral side; and (3) mTAL express an additional basolateral ATP-sensitive P2 receptor, which triggers a sustained [Ca²⁺]_i influx and plateau.

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Figure 2.

Effect of basolateral UTP (100 μM) on [Ca²⁺]_i in perfused mouse mTAL. In WT tubules, UTP caused a prompt increase of [Ca²⁺]_i. The basolateral UTP-induced [Ca²⁺]_i increase was always transient, similar to that observed by addition of luminal UTP and ATP. In mice that were deplete of the P2Y₂ receptor, basolateral UTP never elicited a response.

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Figure 3.

Effect of basolateral ATP (100 μM) on [Ca²⁺]_i in perfused mouse mTAL. In WT tubules, ATP induced a prompt increase of [Ca²⁺]_i. The basolateral ATP-induced [Ca²⁺]_i increase consisted of an initial peak followed by sustained elevation. In P2Y₂ receptor knockout (KO) mice, basolateral ATP triggered only a small initial [Ca²⁺]_i peak followed by a sustained [Ca²⁺]_i plateau. The left bottom panel summarizes these data (*statistical significance P < 0.05). The right bottom panel shows that the [Ca²⁺]_i plateau is absent without basolateral Ca²⁺.

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Figure 4.

Effect of luminal or basolateral UTP (100 μM) on [Ca²⁺]_i in perfused mouse mTAL in the absence of extracellular, bilateral Ca²⁺. UTP stimulated an increase of [Ca²⁺]_I, reflecting release from intracellular stores. Note that removal of basolateral (not luminal) Ca²⁺ reversibly reduced tubular resting [Ca²⁺]_i.

Flow-Induced [Ca²⁺]_i Response in Mouse mTAL

Next we investigated whether an increase of luminal flow is able to trigger [Ca²⁺]_i elevations in mTAL as seen in different cultured cells (MDCK) or intact rabbit or mouse cortical collecting duct. To this end, we followed the experience of the laboratory of Satlin.^2,15 After establishment of tubule perfusion, the pressure on the inflow side was reduced to 10 cmH₂O for 10 min. This led to a significant reduction of the tubular lumen as visible online in the original fluorescence image of the tubule. Subsequently, the hydrostatic pressure of the inflow was abruptly elevated (within 1 s) by 80 cmH₂O, leading to sudden distension of the tubule and concomitant elevation of tubular flow. Tubular distension was quantified in each tubule by measuring the area of tubular fura-2 fluorescence (pixels above threshold 345 nm). Figure 5 shows the effect of increasing inflow pressure on the tubular diameter and the corresponding [Ca²⁺]_i signal. Below the traces 6 fura-2 ratio images show the time course of this effect. Obviously, this pressure increase instantaneously (within the first seconds) elevated mTAL [Ca²⁺]_i in a biphasic manner with an initial peak followed by a decline of [Ca²⁺]_i within 1 to 3 min. After 3 min, tubular inflow pressure was reduced to 30 cmH₂O, resulting in a slow decline of tubular lumen. Frequently, single local [Ca²⁺]_i elevations were observed (image 5 and 6, Figure 5).

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Figure 5.

Effect of increasing the inflow perfusion pressure abruptly by 80 cmH₂O on [Ca²⁺]_i (top) and tubular geometry (middle). The numbered fura-2 ratio images in pseudo-color reflect the flow-induced elevations of [Ca²⁺]_i. Note that after the initial flow-induced increase of [Ca²⁺]_I, several single, confined [Ca²⁺]_i elevations are seen in images 5 and 6.

Flow Response Is Greatly Reduced in mTAL of P2Y₂ KO Mice

Then we studied whether this flow response was similar in P2Y₂ WT and KO tubules. Figure 6 summarizes the entire series of experiments and shows that the flow response in WT tubules is a robust phenomenon and could be stimulated in nearly all tested tubules. The mean [Ca²⁺]_i peak in WT tubules amounted to 0.44 ± 0.09 ratio units (n = 28). In clear contrast, the flow-induced increase of [Ca²⁺]_i was greatly reduced in KO tubules (0.16 ± 0.04 ratio units; n = 13; P < 0.05). The tubular distension as measured with pixel area above threshold approach was similar in both populations (18 ± 2.1% in WT, 22 ± 3.2% in KO). The actual diameters during the flow response changed in WT from 22.7 ± 0.6 to 25.6 ± 0.5 μm and in KO and from 22.8 ± 0.7 to 26.6 ± 0.7 μm. The tubular distensions showed a significant degree of variation. In Figure 6B, we plotted the magnitude of the induced [Ca²⁺]_i elevation as function of tubular distension for all WT and KO tubules. The data suggest that larger tubular distensions provoke bigger [Ca²⁺]_i signals in WT but not in KO tubules. This strongly indicates that the flow response that is induced in mouse mTAL depends on the presence of P2Y₂ receptors and suggests that increased flow promotes tubular release of nucleotides.

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Figure 6.

Flow-induced [Ca²⁺]_i increases in mTAL of P2Y₂ WT and KO mice. (A) Note that the flow-induced [Ca²⁺]_i increase is greatly reduced in P2Y₂ receptor KO tubules. (B) All flow-induced [Ca²⁺]_i increases as a function of the induced tubular distension in P2Y₂ receptor WT and KO mice. These data suggest that an increase of the mechanical stimulus increases the resulting [Ca²⁺]_i elevation in WT but not in KO tubules.

Effect of Bilateral Removal of Extracellular Ca²⁺ on the Flow Response

The flow response was investigated in the absence of Ca²⁺ on both sides of the epithelium. Figure 7 shows an original trace from a WT tubule in Ca²⁺-free medium. The luminal solution was kept Ca²⁺-free for 30 min before the pressure increase. Two minutes before the stimulus, basolateral Ca²⁺ was also removed. In WT mice, the flow response in the absence of extracellular Ca²⁺ (0.26 ± 0.08 ratio units; n = 8) was slightly but not significantly reduced as compared with WT controls (0.44 ± 0.09 ratio units; n = 28). These results indicate that a significant part of the flow response–mediated [Ca²⁺]_i increase can be triggered in the absence of extracellular Ca²⁺ and therefore must result from release of Ca²⁺ from internal stores.

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Figure 7.

(A) Effect of increasing the inflow perfusion pressure on tubular [Ca²⁺]_i (top) and geometry (middle) in the absence of luminal and basolateral Ca²⁺. Removal of luminal Ca²⁺ did not affect resting [Ca²⁺]_i. On the contrary, removal of basolateral Ca²⁺ always promptly and reversibly lowered [Ca²⁺]_i. (B) Summary of the different experimental series (*statistical significance P < 0.05).

Basolateral and Luminal Extracellular Scavenging of ATP and P2 Receptor Blockage Inhibits the Flow Response

The results thus far suggest that an increase of tubular flow promotes release of nucleotides from the renal tubule. Next we examined the effect of apyrase on the flow response. Apyrase is known to scavenge extracellular ATP. These experiments were performed with the non-ratiometric Ca²⁺ dye fluo-4. Noteworthy, 7.5 U/ml apyrase reversibly reduced resting fluo-4 fluorescence by 12.3 ± 4.7% (n = 5, P < 0.05). Importantly, in all experiments, basolateral apyrase led to a near-complete inhibition of the flow-induced [Ca²⁺]_i increase as compared with the control response (F/F₀ increase without apyrase 1.8 ± 0.16 [n = 18]; with basolateral apyrase 1.09 ± 0.01 [n = 8]; P < 0.05; Figure 8). We also tested basolateral suramin, a nonspecific blocker of most P2 receptors. Addition of suramin (300 μM) basolaterally 3 min before stimulation also inhibited the flow response (F/F₀ increase with basolateral suramin 1.13 ± 0.03; n = 7; P < 0.05; Figure 8). Apyrase (7.5 U/ml) and suramin (300 μM) were also tested from the luminal side and were included in the luminal perfusate before starting fluo-4 AM loading. Both antagonists were present for 30 min before the stimulus was applied. Luminal apyrase and suramin inhibited the flow response (F/F₀ increase with luminal apyrase 1.34 ± 0.03 [n = 12; P < 0.05]; with luminal suramin 1.22 ± 0.11 [n = 9; P < 0.05]; Figure 8). These data provide additional evidence that flow stimulation provokes bilateral ATP release from the mTAL.

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Figure 8.

Effect of apyrase (7.5 U/ml) and suramin (300 μM) on flow-induced [Ca²⁺]_i increase in mouse mTAL measured with fluo-4. Note that luminal and basolateral apyrase or suramin inhibited the response (*statistical significance P < 0.05).

Length of Primary Cilium in mTAL of P2Y₂ WT and KO Mice

MDCK cells without primary cilium show no flow response,³ and intact renal tubules from orpk mice with shortened primary cilia display a reduced flow-stimulated increase of [Ca²⁺]_i.¹⁵ We therefore investigated whether the reduced flow response in mTAL of P2Y₂ KO mice coincides with reduced cilia length. The data in Figure 9 indicate that this is not the case. The length of the primary cilium was 3.8 ± 0.01 μm in WT (n = 4 mice and 22 cilia) and 3.9 ± 0.01 μm in KO tubules (n = 3 mice and 16 cilia). Figure 9 shows immunolabeling images of acetylated tubulin staining of the primary cilia from WT and KO mTAL tubules.

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Figure 9.

Length of primary cilia in mTAL from P2Y₂ receptor WT and KO mice. Confocal images of acetylated-tubulin–labeled mTAL showing the primary cilia on the luminal side of the tubule. The basolateral borders of the tubule are faintly visible and highlighted with a dashed line. The tubular lumen is collapsed. Length of primary cilium in WT is 3.8 ± 0.01 μm (n = 22) and in KO is 3.9 ± 0.01 μm (n = 16).

Flow and Pressure Increases in the Renal Tubule

The method to increase tubular flow involves the sudden elevation of the inflow pressure, which therefore must increase the tubular flow. We did not measure the tubular flow by collection of fluid. However, in each experiment, we quantified the degree of tubular distension, which inevitably occurs during this maneuver and is in part the consequence of the mechanical compliance of the tubule. The surrounding tissue in situ is likely to limit tubular distension. We observed an average of approximately 10 to 20% distension in very close similarity to values that were obtained with the same protocol in rabbit and mouse collecting duct^15,17 or in animals in which in vivo imaging of distal tubular dimensions was quantified after the infusion of, for example, loop diuretics.²⁰ It needs to be borne in mind that a complex mechanical stimulus is applied in our experiments. Increasing the pressure on the inflow side led to the acute, moderate distension of the tubule and at the same time increased the tubular flow. Throughout this article, we have used the term “flow response,” being aware that an equally suitable term could be “pressure or stretch response.” Our results need to be seen in contrast to those that were obtained in cultured cells that were grown on glass in an open chamber. In this “classical” system, an increase of inflow pressure does not cause distension of the epithelial layer but will primarily lead to increases of flow over the apical cell surface. This flow response is strictly dependent on the presence of luminal Ca²⁺ and shows a distinct time delay of approximately 35 s between the start of flow increase and the peak [Ca²⁺]_i elevation.¹ The response that we observed has a short time delay of a few seconds and is clearly inducible in the absence of bilateral Ca²⁺. These differences may suggest that the flow response in cultured cells reflects a different phenomenon to that described in the intact renal tubule.

The role of the primary cilium as a flow-sensing organelle was mentioned at the beginning of this article. Our results do not address whether cilia are required for the observed flow response in mouse mTAL. Noteworthy, the flow response is reduced in perfused collecting ducts of a mouse model (orpk) with shortened primary cilia.¹⁵ In conjunction with our findings, this may suggest a link between the primary cilium and nucleotide release. It will need to be established whether bending of the primary cilium is a mechanical trigger to release ATP from renal epithelium.

Purinergic Signaling in the Renal Tubule

Nonlytic cellular release of nucleotides is an established phenomenon in a wide variety of cells, including renal epithelia.^8,10,21 The mechanism of this release is not unraveled, and multiple modes have been suggested.²¹ Released nucleotides are likely perceived by the renal tubule because the entire tubular system expresses multiple luminal and basolateral P2 receptors.^6,22,23 P2 receptors are either metabotropic P2Y or ionotropic P2X receptor, and most of the 15 subtypes link to an increase of [Ca²⁺]_i. Frequently, multiple P2 receptors are expressed in the same cell type, which can cause major problems to pinpoint one relevant receptor for a given effect.^6,22 In mouse mTAL, we show luminal functional expression of only the P2Y₂ receptor, whereas the basolateral membrane contains the P2Y₂ and at least one other ATP-sensitive P2 receptor that is able to stimulate a sustained Ca²⁺ influx. Many tissues display cell-to-cell propagating [Ca²⁺]_i waves. In addition to gap junction–mediated [Ca²⁺]_i waves, traveling [Ca²⁺]_i signals are shown to be caused by extracellular nucleotides. This phenomenon is termed ATP-induced ATP release and underlies, for example, a propagation of a [Ca²⁺]_i wave in astrocytes or mammary epithelial cells.^21,24

Mice

Mouse handling complied with Danish animal welfare regulations. P2Y₂ receptor KO mice were provided as KO breeder pairs (on a B6D2 genetic background) by Dr. B.H. Koller (University of North Carlina, Chapel Hill, Chapel Hill, NC). B6D2 P2Y₂^−/− mice were crossed with the SV129 mouse strain, generating B6D2/SV129 P2Y₂^+/+ (WT) and B6D2/SV129 P2Y₂^−/− (KO) littermates. Genotyping was performed as described previously.²⁵

Tubule Perfusion

Experiments were performed using 4- to 7-wk-old P2Y₂ receptor WT and KO mice of either gender. Mice had free access to standard rodent diet and tap water. Mice were killed by decapitation. Both kidneys were removed and placed in ice-cold dissection solution (see Solutions and Chemicals) before slicing as described previously.²⁶ Slices were transferred into a dissection chamber cooled to 4°C and gassed with 5% CO₂/95% O₂. mTAL were dissected in dissection solution from the inner stripe of the outer medulla using fine forceps. Kidney tubules were transferred to a perfusion chamber on an inverted microscope. Isolated tubules were perfused using a system of concentric glass pipettes as described previously.²⁷ Care was taken to choose viable tubules by microscopic inspection and responsiveness to 5 mM basolateral Ca²⁺ at the end of each experiment. High extracellular Ca²⁺ stimulates the calcium-sensing receptor.²⁸ Only tubules with a brisk [Ca²⁺]_i elevation were included, and the response was similar in WT and KO tubules (data not shown).

Video Imaging of Perfused Renal Tubules

The set-up comprised an inverted microscope (Axiovert 100TV; Zeiss, Jena, Germany) with the objective C-Apochromat, 63×/1.2NA (Zeiss), a monochromator (Polychrome IV; Till Photonics, Planegg, Germany), and a CCD camera (MicroMax, 5 MHz; Princeton Instruments, Princeton, NJ). Image acquisition and data analysis were performed with Metamorph/Metafluor (Universal Imaging, West Chester, PA). Measurement of [Ca²⁺]_i was performed with fura-2 or fluo-4. Tubules were incubated in 20 μM basolateral fura-2-AM or fluo-4-AM for 20 min at room temperature in control solution. In Ca²⁺-free conditions, fura-2 loading was performed without Ca²⁺ in the luminal perfusate. As a measure of [Ca²⁺]_i with fura-2, the fluorescence emission ratio at 345 nm/380 nm excitation was used (acquisition frequency 0.5 Hz). The fluo-4 measured [Ca²⁺]_i changes (excitation 488 nm, emission >510 nm) were expressed relative to baseline values. Cellular excitation light–induced damage²⁹ was controlled by neutral density filters in the excitation light path and a three-fold binning. Fluorescence was recorded from the entire tubule (approximately 150 μm). During dye loading, the tubule was continuously perfused. The experiment was started 5 to 10 min after washout of extracellular dye.

Immunohistochemistry

Freshly dissected mTAL were placed on cell-tak–coated (BD Biosciences, Erembodegem, Belgium) coverslips and washed in PBS. Tubules were fixed in 2.5% paraformaldehyde at room temperature. Tissue was washed and permeabilized with 0.3% Triton-X-100 PBS that contained BSA (15 g/L) for 15 min and incubated overnight with anti-acetylated tubulin (Sigma-Aldrich, Schnelldorf, Germany) at 4°C. After washing, sections were incubated with Alexa488-conjugated goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR) in PBS supplemented with BSA and Triton X-100. The preparations were inspected with a c-Apochromat 63×/1.2NA objective on a confocal microscope (LSM 410; Zeiss).

Solutions and Chemicals

Experiments were performed at 37°C with the following control solution (in mM): 145 NaCl, 1 MgCl₂, 1.3 Ca-gluconate, 5 d-glucose, 0.4 KH₂PO₄, 1.6 K₂HPO₄, and 5 HEPES. The Ca²⁺-free solution contained (in mM) 145 NaCl, 1 MgCl₂, 5 d-glucose, 0.4 KH₂PO₄, 1.6 K₂HPO₄, 5 EGTA, and 5 HEPES. The dissection solution contained (in mM) 118 NaCl, 24 NaHCO₃⁻, 1 MgCl₂, 1.3 Ca-gluconate, 5 d-glucose, 0.4 KH₂PO₄, and 1.6 K₂HPO₄. Solutions were titrated with NaOH to pH 7.4 (37°C).

Fura-2-AM and fluo-4-AM were obtained from Invitrogen (Taastrup, Denmark). All other chemicals were obtained from Sigma-Aldrich Denmark (Vallensbaek, Denmark) and Merck (Darmstadt, Germany). Agonist and antagonist solutions were prepared directly before the experiment.

Statistical Analyses

Data are shown as means ± SEM. For experimental series, the n reflects the number of tubules used. On average, two tubules were used from each mouse. Paired and unpaired t test were used to compare mean values within one experimental series. P < 0.05 was accepted to indicate statistical significance.