Evidence for Basolateral P2Y₆ Receptors along the Rat Proximal Tubule: Functional and Molecular Characterization

Isolation of Rat Nephron Segments

The left kidneys of male Sprague-Dawley rats anesthetized with sodium pentobarbital (Nembutal; 50 mg/kg body wt, administered intraperitoneally) were perfused, via the renal artery, with 5 ml of Hepes-buffered saline solution (HBSS; 140 mM NaCl, 5 mM KCl, 0.8 mM MgSO₄, 0.33 mM Na₂HPO₄, 0.44 mM NaH₂PO₄, 1.0 mM MgCl₂, 1 mM CaCl₂, 10 mM Hepes, 5 mM NaOH, 5 mM glucose [pH 7.4]), followed by the same volume of a 0.16% collagenase solution (Serva; Boehringer Mannheim, Mannheim, Germany). Thin cortico-medullary slices were then incubated in 0.12% collagenase solution for 20 min at 30°C before segments were isolated in microdissection medium (HBSS to which 0.1% bovine serum albumin had been added) at 4°C. For proximal tubules, only non-collagenase-treated kidneys were used (isolated from rats of 90 to 100 g body wt), because collagenase treatment adversely affects responses to calcium-mobilizing agents. Pyruvate (1 mM), glycine (4 mM), and glutamate (2 mM) were included in the proximal tubule microdissection medium. All tubule samples were then stored in a droplet of microdissection medium (2 μl) at 0 to 4°C until use.

The following segments were studied: nonlocalized proximal convoluted tubules (PCT) taken from the kidney cortex or, when specified, S1 segments identified on the basis of functional criteria (see the Results section), proximal straight tubules taken from medullary rays along the S3 portion of the proximal tubules, type II thin descending limbs (DTL) of juxtamedullary nephrons, thin ascending limbs (ATL), cortical thick ascending limbs, medullary thick ascending limbs (MTAL), CCD, outer medullary collecting ducts (OMCD), and IMCD. DTL, MTAL, and OMCD were all isolated from the inner stripe of the outer medulla.

Extraction of mRNA

RNA was extracted from microdissected segments by using a micromethod (30) adapted from the guanidinium thiocyanate-phenol/chloroform method (31). Briefly, pools of individual segments were photographed for length determinations before being transferred, with 5 to 10 μl of microdissection medium, into 400 μl of denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate [pH 7.0], 0.5% sarcosyl, 0.1 mM β-mercaptoethanol, 20 μg of yeast tRNA). After phenol/chloroform extraction and isopropyl alcohol precipitation, the final pellet was vacuum-dried and resuspended in RNA dilution buffer [10 mM Tris-HCl [pH 7.6], 1 mM ethylenediaminetetraacetate, 2 mM dithiothreitol, 40 U/ml ribonuclease inhibitor (RNasin; Promega, Madison, WI)]. It was previously determined that the yield of this extraction procedure was >90% (30).

RT-PCR

Specific primers were selected from the sequence of the rat P2Y₆ receptor cDNA and from the sequence of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA by using Oligo Primer analysis software (Medprobe, Oslo, Norway). The sequences of the P2Y₆ receptor primers were 5′-TGCTTGGGTGGTATGTGGAGTC-3′ (positions 868 to 889) for the forward primer and 5′-TGGAAAGGCAGGAAGCTGATAAC-3′ (positions 1206 to 1184) for the reverse primer. The sequences of the GAPDH primers were 5′-GCCATCAATGACCCCTTCAT-3′ (positions 154 to 173) for the forward primer and 5′-GAGGGGGCAGAGATGATGAC-3′ (positions 434 to 415) for the reverse primer.

For each segment, mRNA extracted from 1-mm tubule length (approximately 60 pg of mRNA) was reverse-transcribed for 50 min at 42°C with 0.5 μg of oligo(dT)_12-18 primer, using a first-strand cDNA synthesis kit for RT-PCR (Superscript II RNase H^- reverse transcriptase; Invitrogen Ltd., Paisley, UK). After denaturation at 95°C for 3 min, the resulting product was used as a template with the PCR Core System I (Promega). For the P2Y₆ receptor, 40 PCR cycles were performed under the following conditions: 95°C for 30 s, 62°C for 1 min (annealing), and 72°C for 1 min (extension) for five cycles; 95°C for 30 s, 56°C for 1 min, and 72°C for 1 min for 34 cycles, with a last cycle with a 10-min extension stage. For GAPDH, only 30 cycles were performed, as follows: 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min for 29 cycles, plus a last cycle with a 10-min extension stage.

The resulting PCR products were resolved on a 2% (wt/vol) agarose gel containing 10 μg/ml ethidium bromide and were observed under ultraviolet illumination. For the P2Y₆ receptor, the nature of the PCR product (expected size, 339 bp) was confirmed by sequencing (Oswel DNA Sequencing Laboratories, Southampton, UK). In all experiments, the presence of possible contaminants was investigated using control RT-PCR assays of samples in which either mRNA had been excluded (blank) or reverse transcriptase had been omitted from the RT mixture.

Measurement of [Ca²⁺]_i

[Ca²⁺]_i was measured using previously described methods (32). Each tubular segment was loaded with fura2/acetoxymethyl ester (10 μM in microdissection medium; Molecular Probes, Eugene, OR) for 1 h at room temperature and was subsequently transferred to a superfusion chamber, where the ends of the segment were aspirated into two glass holding pipettes (to prevent possible perfusion of the luminal membrane). The segment was then superfused with HBSS, at a rate of 10 ml/min, for a 5-min equilibration period. Fura2 fluorescence was measured in a tubular portion of 20 to 30 cells, using a standard photometric configuration (model MSP 21; Zeiss, Jena, Germany), during superfusion with either HBSS alone or HBSS to which agonists had been added. All solutions were stored in individual reservoirs at room temperature until use; the temperature of each solution was increased to 37°C just before introduction into the perfusion chamber.

After subtraction of tubular autofluorescence from the fura2 fluorescence intensities at 340 and 380 nm, [Ca²⁺]_i was calculated using the equation presented by Grynkiewicz et al. (33), with a calcium dissociation constant for fura2 of 224 nM. The calibration parameters were determined from internal calibration assays using solutions containing 10 μM ionomycin and both 0 mM Ca²⁺ and 1 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (for the minimal ratio) or 3 mM Ca²⁺ (for the maximal ratio).

Measurement of Phosphoinositide Metabolism

The method used was previously described in detail (34,35). Briefly, well preserved pieces of proximal tubules (i.e., a mixture of S1, S2, and S3 cortical fragments) were sorted from tubule suspensions prepared, by sieving, from non-collagenase-treated rat kidney cortex (35). OMCD were microdissected from collagenase-treated rat kidneys, as described above. All tubules were labeled for 2 h in HBSS (2 mM Ca²⁺) containing 1 mCi/ml myo-[³H]inositol (Amersham, Buckinghamshire, UK), in a humid atmosphere at 37°C.

After labeling, segments were extensively rinsed at room temperature under stereomicroscopic observation, for removal of extracellular myo-[³H]inositol, and then batches of 10 segments (approximately 5-mm tubule length) were transferred, in 2 μl of HBSS, to glass test tubes containing 48 μl of HBSS (1 mM Ca²⁺). Incubations with agonists were initiated by the addition of 50 μl of HBSS (1 mM Ca²⁺) containing the agent (at double concentration) and LiCl (final concentration, 10 mM).

Reactions were terminated after 15 min by the addition of 940 μl of chloroform/methanol (1:2, vol/vol) and 200 μl of 5 mM Tris-ethylenediaminetetraacetate (pH 7.0). Water and chloroform (300 μl of each) were added to the reaction tube, which was then centrifuged for 5 min at 3000 × g at 4°C for separation of the phases. For each sample, the upper hydrophilic phase was removed for chromatography, whereas 500 μl of water and 500 μl of methanol were added to the remaining hydrophobic phase. After vortex-mixing and repeat centrifugation, the lower phase, containing phosphoinositides (PI), was recovered and evaporated to dryness in counting vials before measurement of radioactivity.

Radioactivities associated with free inositol, glycerophosphoinositol (GPI), and IP were separated by chromatography, as described previously (35). Samples were applied to columns containing 0.25 g of Dowex AG1-X8 resin, and free inositol, GPI, and IP were successively and respectively eluted with the following: (1) 3 mM Hepesbuffered water (pH 7.0), (2) 30 mM ammonium formate, and (3) 1 M ammonium formate and 0.1 M formic acid. The radioactivity contained in each eluate and in the PI extract was counted by β-emission spectrometry, after the addition of 15 ml of Aquasol-2 scintillation cocktail (Packard Bioscience Ltd., Pangbourne, UK) to each vial. IP production was expressed as a percentage of the total radioactivity present in the sample (inositol plus GPI plus IP plus PI), because it was previously demonstrated that there is a good correlation between IP formation expressed in this way and that expressed per unit of tubule length (35,36).

Statistical Analyses

All data are presented as mean ± SEM. Statistical comparisons were made using t tests for paired and unpaired samples and ANOVA, as appropriate. Statistical significance was taken as P < 0.05.

Chemicals

UDP, ADP, and ATP (disodium salts), as well as arterenol bitartrate (norepinephrine), carbamyl choline chloride (carbachol), angiotensin II, and 8-arginine vasopressin, were purchased from Sigma (Poole, Dorset, UK). Because commercially available UDP contains as much as 10% UTP, the UDP stock solution was treated for 1 h with hexokinase (10 U/ml), in the presence of 25 mM glucose.

Distribution of P2Y₆ Receptor mRNA along the Rat Nephron

For each segment, P2Y₆ receptor mRNA expression was investigated in three or four extracts. Results obtained from one extract are illustrated in Figure 1. Strong signals were repeatedly observed in both the proximal tubule (PCT and proximal straight tubule) and the thick ascending limb (cortical thick ascending limb and MTAL), and less intense bands were observed in the DTL, CCD, and OMCD. No signal could be observed in the ATL or IMCD (Figure 1). The absence of detectable P2Y₆ signal in the ATL was not attributable to its thin diameter (and thus a smaller amount of mRNA in each PCR tube), because comparable GAPDH signals were obtained for the DTL, ATL, and OMCD (Figure 1B).

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Figure 1.

P2Y₆ receptor mRNA analysis. (A) Sample gel from one reverse transcription (RT)-PCR experiment, showing the distribution of P2Y₆ receptor mRNA in isolated segments of the rat nephron. PCT, proximal convoluted tubule; PST, proximal straight tubule; ATL, thin ascending limb; MTAL, medullary thick ascending limb; CTAL, cortical thick ascending limb; CCD, cortical collecting duct; IMCD, inner medullary collecting duct. + and -, samples assayed in the presence and absence of reverse transcriptase, respectively. (B) Comparison of P2Y₆ receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in whole-kidney (WK), thin descending limb of Henle's loop (DTL), thin ascending limb of Henle's loop (ATL), and outer medullary collecting duct (OMCD) extracts. For whole-kidney samples, 1 μg of total RNA (approximately 20 ng of mRNA) was used for RT-PCR; for DTL, ATL, and OMCD samples, we used 3 μl of extract (i.e., the mRNA extracted from 1-mm tubule length) in each tube. Note that the GAPDH signal was greater in whole-kidney samples than in tubule samples, whereas signals in ATL, DTL, and OMCD samples were comparable. This finding indicates that GAPDH signals did not reach saturation in the tubule samples and that comparable amounts of mRNA were used for RT-PCR for DTL, ATL, and OMCD samples.

Effects of UDP on [Ca²⁺]_i in Isolated Nephron Segments

RT-PCR data indicated that P2Y₆ receptor mRNA is present in segments of the renal tubule, but they provide no information regarding expression of the protein. In the absence of an antibody against the P2Y₆ receptor, we measured UDP-stimulated changes in [Ca²⁺]_i in most segments containing mRNA, i.e., the proximal tubule, the DTL, the MTAL, and the OMCD. As shown in Figure 2A, significant increases in [Ca²⁺]_i occurred in the proximal tubule. In contrast, the other segments were poorly responsive or unresponsive, although they were sensitive to other PLC-stimulating agonists used as controls (ATP or UTP for DTL and OMCD and angiotensin II for MTAL; results not shown). Compared with the results we obtained previously with ATP and ADP (23), the response to UDP in the proximal tubule was characterized by more intra- and interanimal variation (mean [Ca²⁺]_i increase, 365 ± 40 nM; n = 33). Nine of 42 tubules were observed to be insensitive to UDP, although they responded normally to either ATP (441 ± 74 nM, n = 4) or ADP (460 ± 81 nM, n = 5). The effect of UDP dose-dependently increased between 10 and 30 μM (Figure 2B). At all concentrations tested, the response was monophasic, with a single sharp peak before [Ca²⁺]_i returned to resting values (Figure 2C). In contrast, the responses to ATP and ADP were biphasic, consisting of a peak and then a lower plateau phase that was sustained throughout exposure to the agonist. Similarly, the responses to the other agonists used in this set of experiments (carbachol and norepinephrine) were biphasic (Figures 2C, 3, and 4).

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Figure 2.

Pattern of UDP-induced intracellular calcium concentration ([Ca²⁺]_i) changes along the nephron. (A) [Ca²⁺]_i increases measured in nonlocalized PCT, DTL, MTAL, and OMCD. Bars correspond to mean ± SEM [Ca²⁺]_i increases (peak — basal levels) calculated for several tubules (numbers in parentheses) from at least four different rats. (B) Dose-dependent effects of UDP on [Ca²⁺]_i in PCT (mean ± SEM, n in parentheses). (C) Representative trace comparing the effects of UDP and ATP on [Ca²⁺]_i in PCT.

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Figure 3.

Representative traces, illustrating the effects of 30 μM UDP on [Ca²⁺]_i in different portions of the proximal tubule (S1, S2, and S3 segments). S3 segments were isolated from the cortex on the basis of anatomic criteria (see the Materials and Methods section); as explained in the Results section, S1 and S2 segments were identified on the basis of their different responsiveness to carbachol (Carba). Norepinephrine (NEp) was used as an index of tubule functional integrity.

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Figure 4.

Representative trace, illustrating homologous desensitization of the calcium response to UDP after a single stimulation with the nucleotide (30 μM). Note that the responses to 30 μM ADP (an agonist specific for P2Y₁ receptors) and to 10 μM norepinephrine (NEp) were not blunted.

Because the proximal tubule segments used in these initial experiments were microdissected at random from the kidney cortex and were not anatomically localized, it was possible that the scatter of UDP-induced [Ca²⁺]_i changes was physiologic and reflected different sensitivities of S1, S2, and S3 segments of the proximal tubule to this nucleotide. Therefore, subsequent experiments were performed with localized segments of proximal tubules. As stated in the Materials and Methods section, S3 segments were identified by anatomic criteria that could not be applied to S1 segments, because it is very difficult to microdissect proximal tubules attached to glomeruli in non-collagenase-treated rat kidneys. As an alternative, we used a functional criterion based on results from previous studies, which demonstrated that changes in [Ca²⁺]_i in response to carbachol decrease from S1 to S3, whereas responses to norepinephrine remain constant (35). Therefore, responses to UDP, carbachol, and norepinephrine were always measured in the same segments, using carbachol as an index of localization and norepinephrine as an index of tubule viability. Figure 3 presents sample traces obtained for the S1, S2, and S3 segments. The mean UDP-induced responses were not statistically different between the S1 segment (Δ[Ca²⁺]_i = 340 ± 73 nM, n = 8) and the S3 segment (Δ[Ca²⁺]_i = 425 ± 30 nM, n = 10), a finding that suggests that the proximal tubule is responsive to UDP along its entire length.

Finally, we observed that two consecutive responses to UDP (30 μM) did not occur in the same proximal tubule, even when stimulations were separated by 15 to 20 min of superfusion with control medium (data not shown). We took advantage of this result to examine whether this desensitization was specific for UDP or also applied to other nucleotides that had been previously demonstrated to stimulate PLC in this tubule segment (23). As illustrated by the trace in Figure 4, previous stimulation by UDP (Δ[Ca²⁺]_i = 374 ± 116 nM, n = 9) prevented the effect of a second basolateral application of the nucleotide. However, these tubules remained sensitive to ADP (Δ[Ca²⁺]_i = 440 ± 118 nM, n = 6), ATP (Δ[Ca²⁺]_i = 333 ± 89 nM, n = 3), and norepinephrine (Δ[Ca²⁺]_i = 404 ± 63 nM, n = 5), all of which were tested within the period of desensitization to UDP.

Effects of UDP on PI Metabolism in the PCT

To confirm that the effects of UDP on [Ca²⁺]_i were mediated via G-protein-coupled receptor activation of the PLC pathway, we assessed the effects of this agent on PI signaling in isolated segments of the proximal tubule. Production of IP was also investigated in the OMCD, a segment that expresses P2Y₆ receptor mRNA (Figure 1) but weakly responds to basolateral UDP superfusion (Figure 2). As previously reported for the PCT (35), approximately 80% of the total radioactivity was incorporated as PI. In contrast, only 5% of the total radioactivity appeared in the PI fraction in the OMCD, as reported for the thick ascending limb of Henle's loop (36).

For both segments, the percentages of radioactivity present in the IP pool under different experimental conditions are presented in Table 1. In the PCT, UDP (30 μM) typically caused a fivefold increase in IP production, compared with basal values (P < 0.01). This increase was slightly, but not significantly, larger than that evoked by either ATP (used at the same concentration) or norepinephrine. In the OMCD, the P2Y₆ agonist also promoted a significant increase in the accumulation of IP (three- to fourfold, P < 0.05), as did ATP (sixfold, P < 0.01) and arginine vasopressin (sevenfold, P < 0.01).