Page 1 ofArticles 33 in PresS. Am J Physiol Renal Physiol (January 30, 2007). doi:10.1152/ajprenal.00440.2006
Multiple P2X receptors are involved in the modulation of apoptosis in human mesangial cells: evidence for a role of P2X4
Anna Solini1, Eleonora Santini1, Daniele Chimenti1, Paola Chiozzi3, Federico Pratesi1, Sabina Cuccato1, Simonetta Falzoni3, Roberto Lupi,2, Ele Ferrannini1, Giuseppe Pugliese4, and Francesco Di Virgilio3,5
1
Department of Internal Medicine, University of Pisa, Italy;
2
Section of Diabetes and Metabolic Diseases, University of Pisa, Italy;
3
Department of Experimental and Diagnostic Medicine, University of Ferrara, Italy;
4
Department of Clinical Sciences, "La Sapienza" University, Rome, Italy;
5
Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Italy.
Running head: P2X receptor-mediated mesangial cell apoptosis.
Corresponding author: Anna Solini, M.D. Ph.D. Department of Internal Medicine, University of Pisa Via Roma, 67 I-56100 Pisa, Italy Phone: +39-050-993482 Fax: +39-050-553235 E-mail:
[email protected]
Copyright © 2007 by the American Physiological Society.
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1 Abstract 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 signalling in regulating death rate in these cells.
Keywords: extracellular ATP, purinergic receptors, apoptosis, mesangium.
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2 Introduction 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, though 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
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3 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 co-incubation 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), though 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 (a) to investigate the role of eATP in mediating apoptoptic cell death in human mesangial cells (HCM); and (b) to identify the subtype(s) of P2 receptors involved in this effect.
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4 Materials and Methods 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 concentration 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: (a) morphological changes by contrast-phase mycroscopy; (b) apoptosis rate by Annexin V/propidium iodide based flow cytometry and caspase-3 activation; (c) plasma membrane potential alterations by bis [1,3-diethylthiobarbiturate] trimethineoxonal (bisoxonol) uptake; (d) intracellular calcium by Fura-2/AM uptake; (e) 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 the 3rd
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5 passage 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/ethylenediaminetetraacetic acid (EDTA) and cells were plated in 75 cm2 flasks. For experimental procedures, cells between 5th and 10th passage 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 hour and 2 hours) they were rinsed and observed with a 40x objective. Apoptosis - Cells were incubated with ATP, BzATP and UTP for various time periods. TNF , a powerful apoptosis inducing agent, was used as 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, USA ) equipped with a 488 nm argon ion laser. Late apoptosis was evaluated analysing 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.
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6 Caspase-3 activation - Caspase-3 activation was measured fluorimetrically with the ENZcHEK caspase-3 Assay Kit (Molecular Probes, Eugene, OR, USA ). Excitat ion and emission lenghts of 485±5 nm and 530±5 nm, respectively, were used. The fluorescence increase given by 100 µL of supernatant from 2x105 HMC incubated for 3 hours 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 x 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 nm and 380 nm and emission of 500 nm. During fluorescence measurements, cellular suspension 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 minus and plus calcium, respectively. Parallel experiments were run with a Perkin-Elmer LS50 spectrofluorimeter (Perkin-Elmer, Norwalk, CT, USA ), 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 non-polar 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
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7 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 Perkin-Elmer LS50 spectrofluorimeter equipped with a thermostatically controlled cuvette holder and a magnetic stirrer, starting after 5 min of 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 (QUIAGEN, 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 1st-strand TM cDNA Syntesis Kit (Roche Diagnostics Corporation, 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 the CyclerTM Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA ). PCR was performed in a standard 25µL -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 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 -actin with the same cDNA was used as internal control. 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
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8 anti-P2X7 antibody was used at a dilution of 1:500 (Chemicon International, Temecula, CA, USA ). Neutravidin diluted 1:4000 in Tween-Tris buffer was used as internal control. The immunocomplexes were revealed with peroxidase-labelled protein A developed by chemoluminescence using the ECL kit (Amersham Pharmacia Biotech., 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 hours at room temperature, then probed with the primary anti-P2X4 antibody (Alomone Laboratories, Israel), used at a dilution of 1:200 for 2 hours 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 hour at room temperature. After five rinses of 5 min each, signals were detected with an enhanced chemiluminescence detection system (ECL, Amersham Biosciences, Milano, Italy). For immunofluorescence,cells were seeded on glass coverslips, rinsed with PBS and fixed with paraformaldehyde (2% in PBS). After 2 hours, 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, Israel). Cell were then rinsed three times with PBS and incubated for 30 min with an anti-rabbit Ig FITC-labelled antibody (1:50 dilution in PBS). At the end of this incubation, coverslips were rinsed 3 times and analysed with a TE-300 Nikon (Nikon Co., Tokio, Japan) fluorescence microscope. Statistical analysis – Data are expressed as mean±SD. Analysis was performed by one-way or two-way ANOVA and Bonferroni-Dunn as ANOVA Post Hoc test.
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9 Results The first step of the experimental protocol was to test the effect of extracellular nucleotides on cell morphology. Untreated control cells are reported in Figure 1A and 1E. ATP stimulation caused dose-dependent cell shrinkage which left behind filopodia-like cellular projections dotted with varicosities (Figure 1B and C), which at the magnification used in these experiments is difficult to identify as dilatations or sites of cell adhesion to the substrate. Though 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 few min and were fully prevented by preincubating cells with the P2X antagonist oATP (Figure 1D). BzATP induced some morphological alterations only at high concentrations (above 1 mM) and to a lower extent than ATP (Figure 1F and G); however, these changes in cell morphology were abolished by the specific human P2X7 inhibitor KN62 (Figure 1H). We then tested the effect of extracellular nucleotides in inducing early and late apoptosis. After 3 or 6 hours 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 was unable to trigger apoptosis at these early time points. Conversely, after 18 hours of incubation, ATP at the optimal concentration of 1 mM caused apoptosis in about 15% of HMC vs 4% in untreated cells (Figure 2C and 2A, p
