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Am J Physiol Renal Physiol 292: F1537-F1547, 2007. First published January 30, 2007; doi:10.1152/ajprenal.00440.2006
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Multiple P2X receptors are involved in the modulation of apoptosis in human mesangial cells: evidence for a role of P2X4

Anna Solini,1 Eleonora Santini,1 Daniele Chimenti,1 Paola Chiozzi,3 Federico Pratesi,1 Sabina Cuccato,1 Simonetta Falzoni,3 Roberto Lupi,2 Ele Ferrannini,1 Giuseppe Pugliese,4 and Francesco Di Virgilio3,5

1Department of Internal Medicine, 2Section of Diabetes and Metabolic Diseases, University of Pisa, Pisa; 3Department of Experimental and Diagnostic Medicine, University of Ferrara; 4Department of Clinical Sciences, "La Sapienza" University, Rome; and 5Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara, Italy

Submitted 6 November 2006 ; accepted in final form 21 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Apoptosis, a normal event in renal tissue homeostasis, has been considered as a major mechanism for either resolution of glomerular hypercellularity in glomerulonephritis or loss of cellularity and progression to glomerulosclerosis in chronic renal disease. This study was aimed at investigating the role of extracellular ATP (eATP) in mediating apoptosis in human mesangial cells (HMC) and identifying the subtype(s) of purinergic receptors involved. eATP, but not uridin-5'-triphosphate (UTP), caused dose-dependent modifications of cellular morphology, as assessed by contrast-phase microscopy, and late apoptosis, as measured by Annexin V/propidium iodide-based flow cytometry and caspase-3 activation. Both phenomena were prevented by the P2X antagonist oxidized-ATP. 2', 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) was less effective than ATP, whereas 1[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl] -4-phenylpiperazine (KN62), a selective inhibitor of human P2X7, prevented morphological changes but potentiated apoptosis induced by BzATP. P2X7 was barely expressed in HMC and showed a relatively scarce functional activity, as assessed by monitoring nucleotide-induced intracellular calcium surge and plasma membrane depolarization by Fura-2/AM and bis[1,3-diethylthiobarbiturate]trimethineoxonal uptake, respectively. These data indicated a negligible role of P2X7 in eATP-mediated apoptosis and pointed to the involvement of other P2X receptor(s). Molecular and inhibitor studies suggested a main role for P2X4 receptor in nucleotide-induced apoptosis in HMC, indicating a relevant role for purinergic signaling in regulating death rate in these cells.

extracellular ATP; purinergic receptors; mesangium

APOPTOSIS IS A DISTINGUISHED form of scheduled, genetically determined cell death of eukaryotic cells resulting in DNA fragmentation, condensation of nuclear chromatin, cell shrinkage, and activation of a number of biochemical pathways (31). This form of cell loss is involved in normal tissue homeostasis; in fact, this is maintained through a balance between cell proliferation and death, sharing common molecular mechanisms (15). Apoptosis has also been implicated in pathological conditions such as cancer, autoimmune diseases, and degenerative disorders leading to tissue fibrosis (6).

Among many other autocrine/paracrine factors, extracellular ATP (eATP) can induce apoptosis through ligation of both subtypes of purinergic receptors, P2X and P2Y, although at high concentrations (5). However, the receptors of the P2X family, particularly P2X7, seem to play a more important role in the induction of apoptosis than the P2Y subtypes (29).

As in other tissues, cell loss through apoptosis participates in maintaining renal tissue homeostasis (22). In addition, it has been considered as a major mechanism for either resolution of glomerular hypercellularity in glomerulonephritis (3) or loss of cellularity and progression to glomerulosclerosis in chronic renal disease (18). In fact, cell death rate was shown to increase in several forms of human and experimental renal disease, including diabetic nephropathy, and to correlate with loss of renal function and structure (27). Both mesangial cells (14) and podocytes (28) have been shown to undergo apoptosis when challenged with high glucose-containing media.

ATP was found to be released by rat mesangial cells and to act on them in an autocrine manner through purinergic receptors (26), whereas no information is currently available on the human mesangium. Both P2X (P2X2, P2X3, P2X4, P2X5, and P2X7) and P2Y (P2Y2, P2Y4, and P2Y6) receptors were found to be expressed in rat mesangial cells (8, 20) and to exert opposite effects on cell turnover and extracellular matrix production. The P2Y receptors (likely P2Y2 and P2Y4) promote mesangial cell proliferation (8, 24) and inhibit matrix production (26). Conversely, the P2X receptors were shown to induce mesangial cell death by apoptosis and necrosis (8, 23) and to upregulate matrix synthesis (26). These effects are likely mediated by P2X7, being mimicked by exposure to 2', 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) and reduced by coincubation with oxidized ATP (oATP). However, both these compounds are not specific for P2X7, since they also bind other P2X receptors. In addition, P2X7 (as P2X2, P2X3) seems to be barely expressed in rat mesangial cells under both normal and high glucose conditions (26), although its level was found to be increased in glomeruli of animal models of diabetes and hypertension (34). Conversely, P2X4 and P2X5 showed a much higher expression level than P2X7; their function is still unclear, but one of them, P2X4, shares some properties with P2X7 (11).

The aims of this study were 1) to investigate the role of eATP in mediating apoptoptic cell death in human mesangial cells (HCM) and 2) to identify the subtype(s) of P2 receptors involved in this effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study design. HMC were probed with the purinergic receptor (P2X and P2Y) agonist ATP or the P2X receptor agonist BzATP or the P2Y receptor agonist uridine 5'-triphosphate (UTP), at different concentrations for different times, in the presence or absence of oATP, a selective P2X antagonist, or 1[N,O-bis(5-isoquinolinesulfonyl)- N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), a specific inhibitor of human P2X7. Then, based on the results of these experiments, we tested the effect of a selective P2X1–4 inhibitor, 2 ',3'-O-(2,4,6 trinitrophenyl) adenosine 5-triphosphate (TNP-ATP). Purinergic agonists and antagonists were purchased from Sigma (St. Louis, MO). The following parameters were assessed under the above experimental conditions: 1) morphological changes by contrast-phase mycroscopy, 2) apoptosis rate by annexin V/propidium iodide-based flow cytometry and caspase-3 activation, 3) plasma membrane potential alterations by bis [1,3-diethylthiobarbiturate] trimethineoxonal (bisoxonol) uptake, 4) intracellular calcium by fura-2/AM uptake, and 5) expression pattern of P2X and P2Y receptors by RT-PCR, Western blot, and immunofluorescence analysis.

Cell culture. HMC were obtained from normal kidney cortexes of patients undergoing unilateral nephrectomy for renal cell carcinoma. Patients were aware that this biological material could be used for scientific purposes (i.e., cell isolation and culture for performing experimental studies) and gave their written informed consent to allow this utilization. We only used parts of the cortex with normal glomerular morphology, as evaluated by routine procedures. Glomeruli from the cortex of human kidney tissue were isolated by a gradual sieving procedure and plated out onto gelatin-coated wells. After 15–30 days, outgrowth of mesangial cells was observed, and these were selectively collected by a cell scraper and separately expanded. The presence of endothelial or epithelial cells was excluded by antibodies against specific markers. Cells were initially cultured in RPMI 1640 medium (GIBCO BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/l glutamine, then shifted to DMEM (containing 100 mg/dl glucose) at passage 3 to avoid that the modestly elevated glucose concentration of the RPMI 1640 medium (200 mg/dl) could positively affect cell death rate. Subcultures were performed using trypsin/EDTA and cells were plated in 75-cm2 flasks. For experimental procedures, cells between passage 5 and 10 were stimulated with the purinergic agonists and antagonists for different incubation times, as above indicated.

Morphological changes. Cells were incubated with increasing concentrations of ATP or BzATP, alone or in combination with oATP or KN62, or left untreated. At the end of the different incubation times (5 min, 15 min, 1 h, and 2 h), they were rinsed and observed with a x40 objective.

Apoptosis. Cells were incubated with ATP, BzATP, and UTP for various time periods. TNF-{alpha}, a powerful apoptosis-inducing agent, was used as the positive control. Translocation of phosphatidylserine to the outer layer of the plasma membrane (marker of early apoptosis) was detected by annexin V/propidium iodide based flow cytometry (32). For this purpose, trypsinized and washed mesangial cells were stained with 10 µl of fluorescein-iso-thiocyanate-labeled-annexin V (Caltag Laboratories, Burlingame, CA) in 100 µl annexin buffer (10 mM HEPES, 140 mM NaCl, 5 mM CaCl2, pH 7.4) and incubated for 10 min in the dark. Then, the cells were supplemented with 500 µl of annexin buffer+10 µg/ml propidium iodide and immediately analysed by flow cytometry. Fluorescence histograms were recorded with a FACScan flow cytometer (Becton Dickinson Labware, Bedford, MA) equipped with a 488-nm argon ion laser. Late apoptosis was evaluated analyzing the cell content of DNA according to the methods described by Nicoletti et al. (17). Briefly, mesangial cells were detached from the flask, washed twice with D-PBS, and fixed in 70% ice-cold ethanol for 30 min on ice. Cells were then washed once with D-PBS and resuspended in DNA staining solution (D-PBS, 20 µg/ml propidium iodide, 200 µg/ml RNase A) and incubated for 30 min at room temperature in the dark. Apoptotic cell nuclei containing hypodiploid DNA were expressed as a percentage of the total population. Measurements were performed using the FACScan flow cytometer indicated above.

Caspase-3 activation. Caspase-3 activation was measured fluorimetrically with an ENZcHEK caspase-3 Assay Kit (Molecular Probes, Eugene, OR). Excitation and emission lenghts of 485 ± 5 and 530 ± 5 nm, respectively, were used. The fluorescence increase given by 100 µl of supernatant from 2 x 105 HMC incubated for 3 h with 5 mmol/l ATP at 37°C corresponded to 100 arbitrary units.

Intracellular calcium concentration. The intracellular concentration of free Ca2+ [Ca2+]i was assessed using the fluorescent calcium indicator fura-2. Washed cells (4 x 108/ml) were incubated in buffer solution for 30 min, at 37°C, with 2 µM fura-2/AM. The loaded cells were then washed by centrifugation at 780 g for 10 min to remove extracellular fura-2, incubated for another 15 min, and washed twice as described above. The cells were used immediately for fluorescence measurements using a Viktor-3 spectrofluorophotometer, with the dual excitation wavelength set of 340 and 380 nm and emission of 500 nm. During fluorescence measurements, cellular suspensions were stirred at 37°C. [Ca2+]i was calculated using an equation from Grynkiewicz et al. (7), where [Ca2+]i = Kd * Fmax/Fmin * (R – Rmin)/(Rmax – R). Rmin is the ratio of fluorescence intensities at 340 and 380 nm obtained at zero [Ca2+]i, Rmax is the ratio at saturating [Ca2+]i, Kd is the dissociation constant for Fura-2, and Fmin and Fmax are the fluorescence intensities at 380 nm ± calcium, respectively. Parallel experiments were run with a PerkinElmer LS50 spectrofluorimeter (PerkinElmer, Norwalk, CT), as previously reported (19).

Plasma membrane potential. Plasma membrane depolarization (PMDep) was measured with the fluorescent dye bis[1,3-diethylthiobarbiturate] trimethineoxonal (bis-oxonol, Invitrogen, San Giuliano Milanese, Italy). Briefly, when this dye moves from aqueous solution into a nonpolar environment, as when it binds to membranes or proteins, its fluorescence increases. Depolarization of the membranes increases the transfer of the dye anions from the external solution onto binding sites inside the cell, thus increasing the net fluorescence. Excitation and emission wavelenghts were 540 and 580 nM, respectively. Bis-oxonol was added at a concentration of 100 nM to saline medium containing 125 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgSO4, 1 mmol/l Na2HPO4, 5.5 mmol/l glucose, 5 mmol/l NaHCO3, and 20 mmol/l HEPES. All fluorescence measurements were done in a PerkinElmer LS50 spectrofluorimeter equipped with a thermostatically controlled cuvette holder and a magnetic stirrer, starting after a 5-min equilibration period.

P2X and P2Y mRNA expression. P2 receptors were identified in HMC by RT-PCR. Total RNA was isolated by using a RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The extraction yield was quantified spectrophotometrically and the RNAs obtained were normalized. A constant amount of total RNA (1 µg) was reverse transcribed at 42°C for 60 min in a total 20- µl reaction volume using a first-strand cDNA Syntesis Kit (Roche Diagnostics, Indianapolis, IN). The cDNA was incubated at 95°C for 5 min to inactivate the reverse transcriptase and served as a template DNA for 35 cycles of amplification using a Cycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). PCR was performed in a standard 25 µl reaction mixture consisting of 10 mM Tris·HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM dNTPs, 20 pmol of each sense and antisense primer, and 2.5 U of AmpliTaq DNA polymerase (Laboratoires Eurobio, Les Ulis Cedex, France). Primer sequences for P2X and P2Y receptors were used for the amplification reaction, as reported in Table 1, together with experimental conditions. Amplified PCR products were run on a 2% agarose gel containing 0.5 µg/ml ethidium bromide. The presence of a 548-bp band amplified with specific primers for beta-actin with the same cDNA was used as internal control.


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  		Table 1. Amplification primer sequences and experimental conditions used in the competitive RT-PCR assessment of P2X and P2Y receptor subtypes in human mesangial cells

 
P2X receptor protein expression and localization. For Western blot analysis, cells were resuspended in a buffer containing 300 mM sucrose, 1 mM MgSO4,1 mM K2HPO4, 5,5 mM glucose, 20 mM HEPES (pH 7,4), 1 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, 0,2 µM deoxyribonuclease, and 0,2 µg of ribonuclease and lysed by ultrasons. To identify P2X7, proteins were separated on a 7.5% SDS-PAGE and blotted overnight on nitrocellulose paper (20 µg of proteins were loaded in each lane). Blocking was performed with TBS buffer (10 mM Tris·HCl, 150 mM NaCl, pH 7.6) containing 5% BSA. The primary anti-P2X7 antibody was used at a dilution of 1:500 (Chemicon International, Temecula, CA). Neutravidin diluted 1:4,000 in Tween-Tris buffer was used as internal control. The immunocomplexes were revealed with peroxidase-labeled protein A developed by chemoluminescence using the ECL kit (Amersham Pharmacia Biotechnology, Milano, Italy). To identify P2X4, proteins were incubated with a blocking solution (10% nonfat milk powder in TBST: 20 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 2 h at room temperature, then probed with the primary anti-P2X4 antibody (Alomone Laboratories, Jerusalem, Israel), used at a dilution of 1:200 for 2 h at room temperature. After two 5-min washes in blocking solution, membranes were incubated with protein A peroxidase-conjugated secondary antibody (1:3,000) for 1 h at room temperature. After five rinses of 5 min each, signals were detected with an enhanced chemiluminescence detection system (ECL, Amersham Biosciences, Milan, Italy).

For immunofluorescence, cells were seeded on glass coverslips, rinsed with PBS, and fixed with paraformaldehyde (2% in PBS). After 2 h, they were permeabilized with Triton X-100 (0.1% in PBS) and incubated for 20 min in FCS (2% in PBS), rinsed and incubated at 4°C overnight with the rabbit polyclonal anti-P2X7 (kindly provided by Dr. G. Buell, Serono Research Laboratories, Geneva, Switzerland) or anti-P2X4 serum (Alomone Laboratories). Cell were then rinsed three times with PBS and incubated for 30 min with an anti-rabbit Ig FITC-labeled antibody (1:50 dilution in PBS). At the end of this incubation, coverslips were rinsed three times and analysed with a TE-300 Nikon (Nikon, Tokyo, Japan) fluorescence microscope.

Statistical analysis. Data are expressed as means ± SD. Analysis was performed by one- or two-way ANOVA and Bonferroni-Dunn as ANOVA post hoc test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The first step of the experimental protocol was to test the effect of extracellular nucleotides on cell morphology. Untreated control cells are reported in Fig. 1, A and E. ATP stimulation caused dose-dependent cell shrinkage which left behind filopodia-like cellular projections dotted with varicosities (Fig. 1, B and C), which at the magnification used in these experiments is difficult to identify as dilatations or sites of cell adhesion to the substrate. Although ATP is well known to cause formation of true varicosities (or small blebs) in neurites of retinal cholinergic neurons (19) and other cell types, this is much less straightforward in mesangial cells or fibroblasts. In HMC, cell shrinkage and varicosity formation appeared shortly after addition of ATP, disappeared in a few minutes and were fully prevented by preincubating cells with the P2X antagonist oATP (Fig. 1D). BzATP induced some morphological alterations only at high concentrations (above 1 mM) and to a lower extent than ATP (Fig. 1, F and G); however, these changes in cell morphology were abolished by the specific human P2X7 inhibitor KN62 (Fig. 1H).


Figure 1
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  		Fig. 1. ATP and BzATP-dependent shape changes and formation of plasma membrane varicosities in HMC. Cell monolayers were incubated at 37°C in DMEM for different times (up to a maximum of 2 h) in the absence (A and E) or presence of 1 and 3 mM ATP (B and C after 5-min incubation) or 1 and 3 mM BzATP (F and G after 5-min incubation). Cells were also incubated with 3 mM ATP (D after 20-min incubation) or BzATP (H after 20-min incubation) after preincubation with the P2X antagonist oATP (1 mM) or the specific human P2X7 blocker KN62 (50 nM), respectively. At the end of the incubation time, monolayers were rinsed and viewed with a x40 objective.

 
We then tested the effect of extracellular nucleotides in inducing early and late apoptosis. After 3 or 6 h of incubation in the presence of the nucleotide there was no sign of cell death (data not shown). This is likely to be a cell-specific rather than a stimulus (ATP)-specific feature, since also TNF-{alpha} was unable to trigger apoptosis at these early time points. Conversely, after 18 h of incubation, ATP at the optimal concentration of 1 mM caused apoptosis in about 15% of HMC vs. 4% in untreated cells (Fig. 2, C and A, P < 0.0001). The relatively small percentage of HMC undergoing ATP-stimulated apoptosis suggests that under these experimental conditions only a subpopulation of the HMC is susceptible to apoptosis. This was confirmed by the very similar level of apoptosis caused by TNF-{alpha} (Fig. 2B, P < 0.0001 vs. untreated), already known to cause death of these cells.


Figure 2
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  		Fig. 2. ATP and BzATP trigger delayed apoptosis in HMC. HMC (1 x 105 cells) were left untreated (A) or incubated with 100 ng/ml TNF-{alpha} (positive control, B), 1 mM ATP (C), 1 mM ATP + 1 mM oATP (D), 1 mM BzATP (E), 1 mM BzATP + 1 mM oATP (F), 1 mM BzATP + 50 nM KN62 (G), or 1 mM UTP (H) for 18 h. Representative graphics (A–H) and quantification (%, means ± SD, n = 6; I) of delayed apoptosis under the above experimental conditions.

 
The protective effect of oATP (Fig. 2D, P < 0.0001 vs. ATP alone) and the lack of effect of UTP (Fig. 2H) suggested the participation of P2X rather than P2Y receptors. Among the P2X receptors, P2X7 is the prototypical cytotoxic nucleotide receptor. However, BzATP, a powerful P2X7 agonist, mimicked the ATP effect only at very high concentrations (Fig. 2E, P < 0.0001 vs. untreated); at lower concentrations, usually sufficient to maximally activate the P2X7 receptor, BzATP was ineffective (data not shown). Furthermore, BzATP-stimulated apoptosis was prevented by the nonspecific P2X7 receptor blocker oATP (Fig. 2F, P < 0.0001 vs. BzATP alone) but not by the highly specific human P2X7 receptor antagonist KN62, which even potentiated it (Fig. 2G, P < 0.0001 vs. BzATP alone). Figure 2I summarizes the effects of purinergic agonists and antagonists on late apoptosis. Similarly, although both ATP and BzATP dose dependently stimulated caspase-3 release (P < 0.0001 for both main factors), BzATP was less effective than ATP (P < 0.0001 for the interaction dose x nucleotide; Table 2).


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  		Table 2. Caspase-3 concentrations in conditioned media from unstimulated cells and cells incubated with increasing concentrations of ATP and BzATP

 
To assess the level of P2X7 activity, we monitored [Ca2+]i and PMDep, the earliest detectable changes in nucleotide-stimulated cells. ATP induced a dose-dependent increase in [Ca2+]i (P < 0.0001 vs. unstimulated), whereas the effect of BzATP was trivial (Fig. 3A). Moreover, the fast Ca2+ release from intracellular stores induced by ATP was not followed by the transmembrane Ca2+ influx tipically observed following the activation of receptors coupled to Ca2+ mobilization from intracellular stores (Fig. 3B). In the presence of EGTA, this response was strongly attenuated (Fig. 3C). ATP also induced a dose-dependent collapse of plasma membrane potential, as indicated by the increasing uptake of the lipophilic indicator bis-oxonol (P < 0.0001 vs. untreated by Bonferroni-Dunn test). Surprisingly, BzATP caused a negligible drop in this potential, suggesting an absent/low expression of P2X7, a plasma membrane channel whose activation is well known to cause a dramatic increase in transmembrane ion fluxes (Fig. 3D).


Figure 3
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  		Fig. 3. ATP and BzATP-induced intracellular calcium concentration [Ca2+]i increase and plasma membrane depolarization (PMDep) in HMC. A: effect of increasing concentrations of ATP (filled bars) and BzATP (gray bars) and 100 nM ionomycin on [Ca 2+]i. Cells were treated as described in MATERIALS AND METHODS. Data are means ± SD of 6 experiments. Iono, positive control with calcium ionopohore. B: representative [Ca2+]i tracing in response to 1 mM ATP and 100 nM iono. A peak immediately follows the stimulation with extracellular nucleotides, with return to baseline within few seconds. C: representative [Ca 2+]i tracing under the same conditions in the presence of EGTA. D: ATP (triangles) and BzATP (squares) dose dependency curves of PMDep. Cells were treated as described in MATERIALS AND METHODS. PMDep is expressed as percentage of the maximum triggered by 30 mmol/l KCl. Data are means ± SD of 6 experiments; where not shown, SD are smaller than the symbols.

 
We then investigated the expression of the P2X7 receptor at the message and protein level. In keeping with the negligible functional activity, we found that the P2X7 receptor was barely expressed by HMC: mRNA was almost undetectable, even after stimulation with BzATP for 1 h (Fig. 4A); similarly, the typical 75-kDa band in Western blotting (Fig. 4B) was weak and did not increase significantly upon challenge with TNF-{alpha} (5 ± 4% above the unstimulated state, P = NS). Immunofluorescence analysis with a polyclonal anti-P2X7 antibody revealed plasma membrane ring-like aggregates reminiscent of similar structures previously described in other cell types and putatively assigned to P2X7 (25), but staining was weak and several cells were frankly negative (Fig. 4C); therefore, functional and molecular data suggest that P2X7 has a negligible role in ATP-mediated apoptosis, and point to participation of different P2 receptor(s) in this process.


Figure 4
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  		Fig. 4. Detection of P2X7 expression by RT-PCR (A) and Western blot (B) and immunolocalization by immunofluorescence (C) in HMC. A, lane 1: unstimulated HMC; lane 2: HMC treated with 1 mM ATP; lane 3: HMC treated with 1 mM BzATP; lane 4: positive control (FB2 cells). B, lane 1: human skin fibroblasts; lane 2: unstimulated HMC; lane 3: HMC treated with 100 ng/ml TNF-{alpha} for 24 h. C, 1: negative control (secondary antibody only); 2, 3, and 4: P2X7, where present, is preferentially located at the periphery of the cell and forms ring-like structures after challenge with 0.5 mM BzATP.

 
To identify the P2 receptor(s) responsible for this phenomenon, we analyzed the expression pattern of the other P2 receptors. HMC expressed the message for the following P2X receptor subtypes, in addition to P2X7: P2X2, P2X3, P2X4, P2X6 (Fig. 5A). Among P2Y receptors, P2Y2 and P2Y6 were found to be expressed in HMC; however, neither ATP nor BzATP exerted any appreciable effect on their expression (Fig. 5B; P2Y2: 5 ± 3% above unstimulated state with ATP and 7 ± 2% above unstimulated state with BzATP by densitometric analysis, P = NS; P2Y6: 1 ± 3% below unstimulated state with ATP and 0.5 ± 4% below unstimulated state with BzATP, P = NS).


Figure 5
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  		Fig. 5. Detection of P2X and P2Y receptor expression in HMC by RT-PCR. A: in addition to P2X7, HMC express P2X2, P2X3, P2X4, and P2X6 subtypes. B: unstimulated HMC express P2Y2, and P2Y6 subtypes (lane 1); none of these receptor expression was upregulated either by ATP (lane 2) or BzATP (lane 3).

 
Since UTP, an agonist at the P2Y2 and P2Y6 receptors, was not an inducer of apoptosis in HMC, we ruled out a role for these two receptors. Among other P2X receptors expressed by HCM, we focused our attention on P2X4, since this receptor is more sensitive to BzATP than P2X3 and, particularly, P2X2, and shares some functional features with P2X7 (11, 33); thus it may be a trigger of apoptosis in these cells. To test the involvement of this receptor, in the absence of selective P2X4 blockers, we used TNP-ATP. As shown in Fig. 6, TNP-ATP prevented the proapoptotic effect of BzATP, whether alone or in the presence of KN62 (P < 0.0001 vs. BzATP by Bonferroni-Dunn for both comparisons). Figure 6E summarizes the effects of TNP-ATP on late apoptosis induced by BzATP ± KN62. Among the P2X receptors, expression of the P2X4 message, low in quiescent cells, was the only one strongly upregulated by challenge with either ATP or BzATP (Fig. 7A, 66 ± 3% above unstimulated state with ATP and 69 ± 2% above unstimulated state with BzATP, P = 0.0003 by one-way ANOVA for both). Conversely, P2X4 protein expression was clearly detectable in unstimulated cells and was not changed by these treatments (Fig. 7B, 1 ± 3 and 1 ± 2% with 0.25 and 1 mM ATP and 0 ± 3 and 1 ± 2% with 0.25 and 1 mM BzATP above unstimulated state, P = NS). Immunofluorescence showed diffuse staining, both in the cell bodies and in the plasma membrane of unstimulated cells (Fig. 7C).


Figure 6
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  		Fig. 6. P2X4 antagonist TNP-ATP prevents BzATP- and BzATP + KN62-induced delayed apoptosis in HMC. HMC (1 x 105 cells) were left untreated (A) or incubated with 1 mM BzATP (B), 1 mM ATP + 50 nM KN62 (C), or 1 mM ATP + 50 nM KN62 + 10 µM TNP-ATP (D) for 18 h. Representative graphics (A–D) and quantification (%, mean ± SD, n = 6; E) of delayed apoptosis under the above experimental conditions.

 

Figure 7
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  		Fig. 7. Detection of P2X4 expression by RT-PCR (A) and Western blot (B) and immunolocalization by immunofluorescence (C) in HMC. A, lane 1: unstimulated cells; lane 2: cells treated with 1 mM ATP; lane 3: cells treated with 1 mM BzATP. B, lane 1: unstimulated cells; lane 2: cells treated with 0.25 mM ATP; lane 3: cells treated with 1 mM ATP; lane 4: cells treated with 0.25 mM BzATP; lane 5: cells treated with 1 mM BzATP; lane 6: HEK293 cells transfected to express the human P2X4 receptor; lane 7: wild-type HEK293 cells. C, 1: negative control (secondary antibody only); 2 and 3: staining with the anti-P2X4 antibody shows abundant expression of this receptor in HMC.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
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Apoptosis participates in maintaining tissue homeostasis under normal conditions as well as in response to tissue injury, leading to abnormal cellularity. In addition, it is considered to play a major role in progressive tissue damage, ultimately resulting in fibrosis and organ failure. This is the case also for chronic renal disease, in which glomerular cell apoptosis might promote tissue repair processes, leading to matrix deposition and glomerulosclerosis (10). Mesangial cells lay in a central anatomic position within the glomerulus and participate in the glomerular scarring process by producing increased amounts of ECM. Abnormal matrix-cell interaction, due to the expansion of the mesangial matrix and consequent disruption of physiological cell-cell interaction and availability of survival factors, could promote apoptosis (13), which involves mainly mesangial cells (14) and podocytes (28) and, in turn, might trigger further matrix deposition and scarring.

Regulatory mechanisms of mesangial cell and ECM turnover include a complex array of factors which control cell proliferation, survival, and apoptosis as well as matrix synthesis, assembly and degradation. Recently, eATP has emerged as an autocrine/paracrine factor which could operate under normal and pathological conditions to modulate these processes. It has been reported that eATP has both pro- and antiapoptotic effects in various cell types, such as the developing thymocytes and multipotent haemopoietic stem cells (2, 12, 21). The contradictory reports on the ability of eATP to trigger cell death or proliferation are probably due to differences in cell types and culture conditions, suggesting that eATP effects should be considered in a cell context-dependent manner. In mesangial cells, while P2Y receptors were shown to stimulate proliferation (8, 24) and to inhibit matrix production (26), the P2X receptors trigger cell death (8, 23) and increase matrix deposition (26). P2X7 was considered as the predominant subtype of P2X receptor involved in these actions, based on data with purinergic agonists and antagonists, such as BzATP and oATP, which, however, are not absolutely selective for the P2X7 receptor. On the other hand, P2X7, which is not expressed appreciably under normal conditions in the rat kidney, is upregulated following glomerular injury in rats with streptozotocin-induced diabetes mellitus or hypertension; under these circumstances, it is localized mainly in podocytes, and to a lesser extent in endothelial and mesangial cells (34). Moreover, although the exact function and regulation of this receptor remain unclear, its ability to cause inflammatory cytokine release and cell death suggests that an increased P2X7 expression might be involved in the pathogenesis of glomerular cell injury or repair.

We report here that, as previously shown in rat mesangial cells and other cell types, eATP induces apoptosis in HMC. However, a novel finding of this study is that, at variance with that previously described in many other tissues and cell types, the classic proapoptotic receptor P2X7 does not play a significant role in this process. We found very little expression of this receptor at the mRNA and protein level and negligible ATP- or BzATP-stimulated Ca2+ influx from the extracellular medium. Furthermore, depolarization of the plasma membrane and uptake of YO-PRO or lucifer yellow in response to challenge with BzATP (unpublished observations) were also absent. This is very strong evidence against activation of the P2X7 receptor as this receptor is well known to cause opening of a nonselective plasma membrane pore that causes massive ion fluxes and permeation of low molecular mass aqueous solutes. Low P2X7 expression and activity were not fully unexpected, as we and others previously showed that rat mesangial cells also express low P2X7 receptor levels (8, 26).

The ability of both UTP and ATP to activate cell responses is characteristic of the pharmacological profile of P2Y receptors; thus, the failure of UTP to evoke an apoptotic response suggests that, as already demonstrated in several other tissue and cell types, these purinergic receptor subtypes have no cytotoxic properties (1, 9); moreover, apoptosis was fully blocked by oATP, which is known to have no inhibitory activity on P2Y receptors (16). On the other hand, apoptosis appeared to be definitely mediated by P2X receptors, given that it was abolished by the preferential P2X antagonist oATP. HMC expressed several P2X receptors besides P2X7, among which P2X4 is a good candidate as a trigger of apoptosis. First of all, this receptor interacts predominantly with eATP, being responsible for excitatory neurotransmission to drive many physiological functions such as immune responses and pain (30, 35). Second, even though weakly expressed in unstimulated cells, P2X4 gene expression, unique among all the purinergic receptors, is upregulated by both ATP and BZATP [in keeping with previous results showing that cell death evoked by eATP in different cell populations occurs with direct upregulation of several purinergic receptors (4)], is abundantly represented at the protein level, and is preferentially activated by BzATP. Third, it shares several electrophysiological features of P2X7 and the proximity of the p2x4 and p2x7 genes on chromosome 12 may indicate a close functional relationship between these two receptors. Fourth, nucleotide-stimulated apoptosis in HMC was inhibited by TNP-ATP, a blocker of P2X4, also active on P2X2 and P2X3 subtypes, both expressed in HMC. There are no agonists or antagonists currently known that are selective for P2X2 receptors; however, BzATP is scarcely active on P2X2 (36) and a role for P2X4 in apoptosis of human epithelial cells has been earlier proposed by Coutinho-Silva et al. (5). Finally, the observations that BzATP-induced apoptosis was further augmented by KN62 and that this unexpected effect was prevented by coincubation with TNP-ATP might suggest that P2X7 blockade by this inhibitor favored BzATP binding to proapoptotic P2X4. The alternative explanation that KN62 has a direct proapoptotic effect, possibly via the known inhibitory activity on calcium/calmodulin-dependent protein kinase II, was ruled out by control experiments showing that KN62 by itself does not induce cell death.

In response to ATP or BzATP stimulation, HMC undergo the typical changes previously observed in other cell types (shrinking, membrane blebbing, emission of neurite-like elongations). These morphological alterations occurred in the vast majority of HMC, but surprisingly they culminated in frank apoptotic death of a minority of cells, showing that although shrinking and blebbing are widely considered preapoptotic events, they are not always followed by death. It is likely that these striking changes in morphology reflect a nonspecific state of cell stress that, like the loss of membrane asymmetry signaled by exposure of phosphatidylserine on the outer leaflet of the plasma membrane, may be reversible. There is also a clear dissociation between the KN62 effect on the nucleotide-stimulated morphological changes and on apoptosis, as this inhibitor fully inhibited the former but not the latter. While this can be taken as further evidence of the fact that these responses can be independent of each other, it raises the questions of how many different intracellular pathways are activated by nucleotide receptors and how are they related. This is clearly a more relevant problem when, as in the case of HMC, apoptosis is a delayed response to P2 receptor stimulation.

In conclusion, our data indicate that signaling through P2X receptors participates in the modulation of apoptosis in HMC, thus suggesting a role for these receptors in the pathogenesis of chronic renal disease in which eATP release from resident or nonresident glomerular cells is increased by metabolic, hemodynamic, or inflammatory stimuli. Among P2Xs, P2X4 appeared to have a prominent role in supporting apoptosis. On the other hand, P2X7, the classic proapoptotic P2 receptor, played a negligible role. Low expression and functional activity of the P2X7 receptor seem to be a feature of both HMC and rat mesangial cells.


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This work was supported by grants from the Italian Ministry of Education, University and Scientific Research (MIUR), the Italian Association for Cancer Research (AIRC), the Italian Space Agency (ASI), the Fund for Investments in Basic Research (FIRB), and local funds from Pisa, Ferrara, and Rome Universities.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Solini, Dept. of Internal Medicine, Univ. of Pisa, Via Roma, 67 I-56100 Pisa, Italy (e-mail: a.solini{at}med.unipi.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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