P2X7 Deficiency Attenuates Renal Injury in Experimental Glomerulonephritis
Simon R.J. Taylor*,
Clare M. Turner,,
James I. Elliott,
John McDaid,
Reiko Hewitt,
Jennifer Smith,
Matthew C. Pickering,
Darren L. Whitehouse||,
H. Terence Cook¶,
Geoffrey Burnstock**,
Charles D. Pusey,
Robert J. Unwin and
Frederick W.K. Tam
* MRC Clinical Sciences Centre, Imperial College Kidney and Transplant Institute, and Department of Molecular Genetics and Rheumatology, Division of Medicine, and ¶ Department of Pathology, Division of Investigative Science, Imperial College London, and Centre for Nephrology and ** Autonomic Neuroscience Centre, University College London (Royal Free Campus), London, United Kingdom; and || Department of Chemistry, Institutes for Pharmaceutical Discovery, LLC, Branford, Connecticut
Correspondence: Dr. Frederick W.K. Tam, Imperial College Kidney and Transplant Institute, Division of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12, UK. Phone: 020-8383-2354; Fax: 020-8383-2062; E-mail: f.tam{at}imperial.ac.uk
Received for publication June 1, 2008.
Accepted for publication January 22, 2009.
The P2X7 receptor is a ligand-gated cation channel that is normallyexpressed by a variety of immune cells, including macrophagesand lymphocytes. Because it leads to membrane blebbing, releaseof IL-1β, and cell death by apoptosis or necrosis, it isa potential therapeutic target for a variety of inflammatorydiseases. Although the P2X7 receptor is usually not detectablein normal renal tissue, we previously reported increased expressionof both mRNA and protein in mesangial cells and macrophagesinfiltrating the glomeruli in animal models of antibody-mediatedglomerulonephritis. In this study, we used P2X7-knockout micein the same experimental model of glomerulonephritis and foundthat P2X7 deficiency was significantly renoprotective comparedwith wild-type controls, evidenced by better renal function,a striking reduction in proteinuria, and decreased histologicglomerular injury. In addition, the selective P2X7 antagonistA-438079 prevented the development of antibody-mediated glomerulonephritisin rats. These results support a proinflammatory role for P2X7in immune-mediated renal injury and suggest that the P2X7 receptoris a potential therapeutic target.
Glomerulonephritis (GN) is a major cause of end-stage kidneydisease; current therapy usually involves relatively nonspecificimmunosuppression with often serious adverse effects.1 Glomerulardeposition of antibodies directed against exogenous antigensor autoantigens, leading to immune complex–mediated inflammationand tissue injury, has been well documented in both experimentaland clinical forms of GN.2
The rat model of nephrotoxic nephritis (NTN) has demonstratedthe importance of IL-1β in GN; renal levels of IL-1βare increased in this form of GN, and IL-1β has been shownto play an important role in glomerular crescent formation andin subsequent tubulointerstitial injury.3 Moreover, early andlate treatment with an IL-1 receptor antagonist prevents theprogression of crescentic GN.4,5 Crescentic GN is also lesssevere in IL-1β–/– or IL-18–/–mice, and treatment with caspase inhibitors reduces renal inflammationand apoptosis—all consistent with a central role for IL-1βin this experimental model of GN.6–8
The ATP-sensitive P2X7 receptor is a cation channel activatedby high concentrations of extracellular ATP.9 Stimulation ofthis receptor is proinflammatory, causing release of inflammatorycytokines such as IL-1β and IL-18 from macrophages, changesin plasma membrane lipid distribution, and cell death by necrosisor apoptosis.10,11 A central role for P2X7 in IL-1β secretionvia the Nacht Domain-, Leucine-Rich Repeat-, and PYD-ContainingProtein 3 (NALP3) inflammasome has been shown in P2X7-deficientmice.12,13 This receptor also has significant prothromboticeffects,14 causing release of tissue factor–bearing microparticles.15Indeed, P2X7 is already considered to be a possible therapeutictarget in inflammation, and antagonists are currently in PhaseII clinical trials for the treatment of rheumatoid arthritisand chronic obstructive pulmonary disease; however, the roleof this receptor in renal disease or injury is still unclear.16
We previously reported an increase in glomerular expressionof the P2X7 receptor (at the mRNA and protein levels) in ratsand mice with NTN induced by nephrotoxic globulin (NTG)—anestablished model of immune complex–mediated GN characterizedby proteinuria, glomerular thrombosis, and tubulointerstitialinjury—as well as in renal biopsy tissue from patientswith lupus nephritis.17,18 In this study, we used P2X7-deficientmice and the selective P2X7 antagonist A-438079 to examine inmore detail the role of P2X7 in the NTN model of GN.
Mice lacking P2X7 develop normally, are of similar weight towild-type littermates used as controls, and have normal macroscopicand microscopic renal morphology and histology. At day 9 afterinjection of NTG, glomerular thrombosis (as indicated by periodicacid-Schiff [PAS]-positive fibrin)19 was reduced in P2X7–/–mice compared with controls (Figure 1, A through D). Quantificationof glomerular thrombosis revealed a 60% reduction in the P2X7–/–mice compared with controls (P < 0.01; Figure 1E). Consistentwith less severe histologic injury, P2X7–/– miceat day 8 had a 52% reduction of proteinuria (P < 0.05; Figure 1F)as well as a 38% reduction in serum creatinine levels measuredafter terminal bleeding on day 9 (P < 0.05; Figure 1G).
Figure 1. Glomerular thrombosis, proteinuria, and serum creatinine levels in wild-type and P2X7–/– mice. (A through D) Representative low- and high-power microscopy of PAS-stained sections of kidneys from wild-type (A and B) and P2X7–/– (C and D) mice 9 d after administration of nephrotoxic serum. (E) P2X7–/– mouse kidneys showed significantly reduced glomerular thrombosis compared with the wild-type, confirmed by quantification of thrombosis scores. (F and G) Proteinuria was also significantly reduced in P2X7–/– mice killed 9d after administration of nephrotoxic serum compared with controls (F), as were serum creatinine levels (G).
The Accelerated Nephrotoxic Nephritis (ANTN) model depends onboth the immune response to the injected sheep IgG before administrationof the NTG and deposition of the injected NTG within the kidney.To assess the former, we quantified circulating levels of mouseanti-sheep IgG, whereas, for the latter, we quantified glomerulardeposition of sheep IgG in each experimental group (SupplementalFigure 1). There were no differences in sheep IgG depositionbetween P2X7–/– and controls (Supplemental Figure1E), and circulating levels of mouse anti-sheep IgG were alsosimilar (Supplemental Figure 1G).
Although the immune response to sheep IgG did not differ betweenthe two experimental groups of mice, glomerular deposition ofmouse IgG was reduced by 26% in P2X7–/– mice comparedwith controls (P < 0.05; Supplemental Figure 1F); however,for every measure of disease activity, mice had significantlyhigher levels of activity for the same level of mouse anti-sheepIgG deposition, suggesting that this is not the explanationfor the reduced injury in the P2X7–/– mice (seeSupplemental Figure 2).
Macrophages are the major effector cells in human and experimentalGN.20 P2X7 has a central role in macrophage IL-1β secretionvia the NALP3 inflammasome.10 Monocyte chemoattractant protein1 (MCP-1) is expressed by macrophages, endothelial cells, mesangialcells, and epithelial cells,21 and this chemokine has been shownto be important in glomerular recruitment of macrophages andcrescent formation in NTN in WKY rats,22 as well as in mice.23We measured cytokine and chemokine levels in the urine usingspecific ELISAs. IL-1β levels proved too low for accuratedetection (data not shown), but MCP-1 levels were readily detectableand were reduced by 96% in P2X7–/– mice comparedwith controls (P < 0.0001; Figure 2A). To determine whetherdecreased MCP-1 levels in P2X7–/– mice were associatedwith reduced macrophage recruitment, we quantified glomerularmacrophage infiltration (Figure 2B). As expected, this demonstrateda significant reduction in macrophage numbers per glomerularcross-section in P2X7–/– mice compared with controls(28%; P < 0.001; Figure 2C). In contrast, interstitial macrophageswere not reduced in the P2X7–/– mice compared withcontrols (9.29 ± 1.13 versus 8.57 ± 1.05 macrophagesper high-power view, respectively).
Figure 2. Glomerular macrophage infiltration and urinary MCP-1 levels. (A) Urinary MCP-1 was significantly reduced in P2X7–/– mice compared with control (WT) mice. (B and C) Macrophage infiltration was similarly reduced in the P2X7–/– mice (B), with fewer CD68+ macrophages visible in glomerular cross-sections (GCS) from P2X7–/– than WT mice 9 d after administration of nephrotoxic serum (C). (D and E) In addition, C3 levels were unchanged between control and P2X7–/– mice (E), but fibrin deposition was significantly reduced (D).
We next investigated complement activation by measuring C3 depositionto determine whether this might explain reduced MCP-1 productionin P2X7–/– mice. We found no significant differencesbetween P2X7–/– and control mice (Figure 2E andSupplemental Figure 3, A and B), suggesting that differentialactivation of complement is not responsible for the reductionin MCP-1; however, fibrin deposition was reduced by 24% in P2X7–/–mice compared with control mice (P < 0.05; Figure 2D andSupplemental Figure 3, C and D).
A-438079 is a selective P2X7 antagonist in rat tissue in vitroand in in vivo models of pathologic nociception.24 To assessthe potential of P2X7 as a therapeutic target in vivo, we usedthis antagonist in the rat NTN model to determine whether pharmacologicinhibition of the P2X7 receptor can affect the course of NTN.Two dosages were used: a "low dosage" of 100 µmol/kg anda "high dosage" of 300 µmol/kg, based on previous reports.24At day 7 after injection of nephrotoxic serum (NTS), glomerularthrombosis (as indicated by PAS-positive fibrin)19 was not significantlyreduced in rats treated with low-dosage A-438079 but was reducedby 96% in rats treated with high-dosage A-438079 compared withvehicle-injected controls (P < 0.01; Figure 3A). Rats treatedwith high-dosage A-438079 also had a 90% reduction in proteinuriaat days 6 to 7 compared with controls (P < 0.001; Figure 3B).
Figure 3. Effect of early treatment with the P2X7 antagonist A-438079 on NTN in rats. (A) Rats treated with the 300-µmol/kg dose of A-438079 had a significant reduction in fibrinoid necrosis compared with vehicle-treated rats, whereas rats treated with the 100-µmol/kg dose showed only a trend toward lower levels of disease. (B) Similarly, rats treated with the higher dosage of A-438079 had a significant reduction in proteinuria compared with the vehicle-treated group, whereas rats treated with the lower dosage did not have significantly lower levels.
We then assessed whether P2X7 inhibition affected the immuneresponse to NTS administration. There were no significant differencesin rabbit IgG and rat IgG deposition between rats treated witheither dosage of A-438079 and controls (Supplemental Figure4). As expected from our results in P2X7-deficient mice, macrophageinfiltration of glomeruli was reduced in animals treated withhigh-dosage A-438079, the number of macrophages per glomerulusbeing reduced by 65% (P < 0.001; Figure 4). Similarly, renallevels of MCP-1 were reduced by 50% in animals treated withhigh-dosage A-438079 (P < 0.05; Supplemental Figure 5).
Figure 4. Effect of early treatment with the P2X7 antagonist A-438079 on infiltrating macrophages. Macrophage infiltration was assessed by immunoperoxidase staining using ED-1 mAb. (A) Glomeruli from vehicle-treated rats and those on the lower dosage of 100 µmol/kg A-438079 showed intense macrophage infiltration, whereas macrophage infiltration was significantly reduced in rats given the higher dosage of 300 µmol/kg of the P2X7 antagonist A-438079. (B) Macro-dosage of 300 µmol/kg of the P2X7 antagonist A-438079. (B) Macrophage infiltration as assessed by counting the number of infiltrating macrophages per GCS in 25 consecutive glomeruli confirmed these results.
These results clearly demonstrate that P2X7 deficiency protectsagainst the inflammatory damage that occurs in ANTN; P2X7-deficientmice develop less severe glomerular thrombosis and proteinuriaand have lower levels of serum creatinine when compared withtheir controls. Similarly, the selective P2X7 antagonist A-438079prevents the development of NTN in WKY rats in a dosage-dependentmanner. Both P2X7-deficient mice and rats treated with A-438079have reduced macrophage infiltration of renal tissue and expressmarkedly lower renal or urinary levels of the chemokine MCP-1.Interestingly, in a model of unilateral ureteric obstructionin another line of P2X7 gene–deficient mice, Goncalveset al.25 also observed reduced renal macrophage infiltration,as well as decreased TGF-β1 synthesis and collagen deposition.
Macrophages are important effectors of injury in GN. Accumulationof macrophages is linked closely to the severity of glomerularinjury,26 and macrophages are a major source of IL-1β andIL-18, both known to play an important role in crescent formationand tubulointerstitial injury.3,6,7 MCP-1 is a key moleculeresponsible for recruitment of macrophages to inflamed glomeruli;it is expressed by both macrophages and intrinsic renal cells.21
In line with our finding of decreased urinary MCP-1 in P2X7–/–mice in ANTN and reduced renal MCP-1 in WKY rats treated withthe selective P2X7 antagonist A-438079, others have reportedreduced tissue MCP-1 production in an adjuvant-induced modelof paw inflammation in P2X7–/– mice.27 Furthermore,the P2X7 agonist BzATP [3'-O-(4-benzoyl)benzoyl ATP] increasesMCP-1 expression in macrophage-like astrocytes28; therefore,reduced MCP-1 production—and urinary excretion—couldbe a consequence of P2X7 deficiency itself or secondary to adecrease in IL-1β secretion.
In conclusion, our findings indicate a significant and possiblykey proinflammatory role for the P2X7 receptor in autoimmunerenal injury and provide evidence that P2X7 antagonists canprevent the development of disease, confirming that P2X7 isan important novel therapeutic target in GN.
Animals
P2X7-deficient mice were a gift from GlaxoSmithKline and havebeen described in detail elsewhere.27 Mice were kept in a pathogen-freeenvironment, and experiments were performed according to localinstitutional guidelines. All mice were female and of 6 to 8wk of age. Mice were initially bred from P2X7+/– breedingpairs; we studied P2X7–/– mice and used P2X7+/+littermates as controls. Male WKY rats that weighed between180 and 220 g were used. Animals had free access to standardlaboratory diet and water.
Experimental Design
To induce ANTN, we immunized mice with a single intraperitonealinjection of 200 µg of sheep IgG (Sigma, Gillingham, UK)in a 50:50 mix with complete Freund's adjuvant (Sigma, Poole,UK). This was followed 5 d later by the intravenous administrationof 200 µl of sheep NTG via the tail vein. Preparationof NTG was performed as described previously.29 NTN was inducedin WKY rats by single intravenous injection of 0.1 ml of rabbitanti-rat NTS.30 In P2X7 antagonist experiments, the first doseof A-438079 was administered on the same day (at a dosage ofeither 100 or 300 µmol/kg), and further doses were giventwice daily for an additional 7 d. Mice were killed at 9 d andrats at 7 d after the administration of NTG or NTS, respectively.
Reagents
A-438079 has a molecular weight of 342.61; the chemical nameis 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridinehydrochloride (pIC50 = 6.5 for recombinant rat P2X7).31 Twodosages were chosen, 100 and 300 µmol/kg, based on previouslyreported data.30
Measurement of Disease
Mice and rats were housed in metabolic cages for 24-h collectionof urine. The protein concentration was measured using the sulfosalicylicacid method, in which 10 µl of 25% sulfosalicylic acidwas added to 100 µl of urine diluted to 1:100, and absorbancewas read at 450 nm. Serum creatinine was measured using an OlympusAU600 analyzer (Olympus, Watford, Hertfordshire, UK). Samplesof kidney were either unfixed or fixed in periodate-lysine-paraformaldehydeand snap-frozen in isopentane precooled in liquid nitrogen orfixed in Bouin's solution and then embedded in paraffin forsectioning.
IL-1β and MCP-1 Estimation
DuoSet ELISA kits (R&D Systems, Minneapolis, MN) were usedaccording to the manufacturer's instructions. The antibody suppliedin the IL-1β kit does not cross-react with pro–IL-1β.Sandwich ELISA was performed to assess the presence of rat MCP-1in renal tissue homogenate. Briefly, matched paired antibodiesfrom BD Pharmingen (San Diego, CA) were used according to themanufacturer's instructions. Absorbance was read at 450 nm usinga spectrophotometric ELISA plate reader (Anthos HTII; AnthosLabtec, Salzburg, Austria).
Histologic Analysis
Kidneys were fixed for 2 h in Bouin solution, transferred to70% ethanol, and embedded in paraffin. Sections were then stainedwith PAS. All analyses were performed blind. Glomerular thrombosiswas assessed by grading the degree of PAS-positive materialper glomerulus as follows: Grade 0, no PAS-positive materialwithin the glomerulus; grade 1, 0 to 25% of glomerulus thrombosed;grade 2, 25 to 50%; grade 3, 50 to 75%; and grade 4, 75 to 100%.The mean thrombosis score for 50 glomeruli was then calculated.
For detection of macrophages, slides were dewaxed and rehydrated,boiled with 0.01 M sodium citrate buffer for 15 min, and thenimmersed in 50% methanol containing 0.3% H2O2 to block endogenousperoxidase activity. Slides were washed with PBS and then incubatedwith 20% normal goat serum. The primary antibody was mAb ED-1(Serotec, Kidlington, Oxfordshire, UK) diluted 1:500 and incubatedovernight. Slides were then incubated with biotinylated goatanti-mouse (Dako, Ely, Cambridgeshire, UK) for 45 min, followedby avidin-peroxidase conjugate (Vector Labs, Orton Southgate,Peterborough, UK) for 30 min. Antibody binding was visualizedusing 3,3'diaminobenzidene (Sigma Aldrich, Gillingham, Dorset,UK) and counterstained with hematoxylin. Macrophages were countedin 25 glomerular cross-sections per sample, and the mean numberof macrophages was calculated.
For detection of rat MCP-1, staining slides were dewaxed andrehydrated, boiled with 0.01 M sodium citrate buffer for 15min, and then immersed in 0.3% H2O2 to block endogenous peroxidaseactivity. Slides were then washed with PBS and incubated withavidin for 15 min and then with biotin for 15 min, followedby 20% normal swine serum. The MCP-1 antibody (Santa Cruz Biotechnology,Santa Cruz, CA) was diluted 1:50 with 5% rat serum and incubatedovernight. Slides were then incubated with biotinylated swineanti-goat antibody (Dako, Ely, Cambridgeshire, UK), dilutedin 5% rat serum for 1 h, followed by extravidin peroxidase (SigmaAldrich) for 30 min. Antibody binding was visualized using 3,3'diaminobenzidene(Sigma Aldrich).
Direct Immunofluorescence Microscopy
Detection of glomerular mouse and sheep IgG in mouse tissueand rat and rabbit IgG in rat tissue was assessed on frozensections by direct immunofluorescence microscopy. Briefly, sectionswere fixed in acetone for 10 min, blocked with 10% normal goatserum, and then incubated with FITC-conjugated anti-IgG antibodies(Sigma Aldrich). FITC-labeled goat anti-mouse C3 (cat. no. 55500;Cappel/ICN, Aurora, OH) and FITC-labeled goat anti-mouse Fibrinogen(Nordic Immunology, Tilburg, The Netherlands) were used to detectC3 and fibrin deposition, respectively. For quantification ofglomerular immunofluorescence, sections were examined usingan Olympus BX4 fluorescence microscope (Olympus Optical, London,UK) at x20 magnification. Images were captured using a PhototonicScience Color Coolview camera (Photonic Sciences, Robertsbridge,UK) and analyzed using Image Pro Plus software (Media Cybernetics,Silver Spring, MD). For each section, 20 consecutive glomeruliwere analyzed and the mean fluorescence intensity was calculated.
Homogenization of Frozen Kidney Tissues
Snap-frozen renal tissue was homogenized and sonicated withhomogenizing buffer (20 mM Tris-HCL [pH 7.5], 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton-X-100, 2.5 mM sodium pyrophosphate,1 mM β-glycerophosphate, and 1 mM sodium orthovanadateplus 1% protease inhibitor cocktail; Sigma Aldrich) at a concentrationof 0.1 g of tissue with 1 ml of buffer. The homogenate was thencentrifuged to remove tissue debris. Protein concentration wasdetermined using the bicinchoninic acid assay kit (Pierce, Rockford,IL) according to the manufacturer's protocol. The supernatantwas aliquotted and stored at –20°C.
Statistical Analysis
All values described in the text and figures are expressed asmeans ± SEM. Statistical analysis was carried out byGraphPad Prism 5.01 (GraphPad Software, San Diego, CA). Datawere analyzed by the Mann-Whitney U test; P < 0.05 was consideredto be significant.
F.W.K.T. has received research project grants from Roche PaloAlto and Baxter Biosciences and has received travel grants fromAmgen, Genzyme, MSD, Novartis, and Roche to attend conferences.C.D.P. has received research project grants from Biogen andCelltech.
Acknowledgments
S.R.J.T. was supported by the Medical Research Council of theUnited Kingdom. This project was supported by a project grantfrom the Wellcome Trust.
We thank Dr. I.P. Chessell, Dr. J.C. Richardson, and J.P. Hatcherfor the use of P2X7–/– mice. The P2X7 antagonistA-438079 was a gift from Michael J. Fare (Institutes for PharmaceuticalDiscovery LLC).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
S.R.J.T. and C.M.T. contributed equally to this work.
Supplemental information for this article is available onlineat http://www.jasn.org/.
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Abstract
Introduction
