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Lack of P2X7 Receptors Protects against Renal Fibrosis after Pyelonephritis with α-Hemolysin–Producing Escherichia coli

2019; Elsevier BV; Volume: 189; Issue: 6 Linguagem: Inglês

10.1016/j.ajpath.2019.02.013

1525-2191

Jacob R. Therkildsen, Mette G. Christensen, Stine Julie Tingskov, Julia Wehmöller, Rikke Nørregaard, Helle A. Prætorius,

Pediatric Urology and Nephrology Studies

Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X7+/+ and P2X7−/− mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X7−/− mice and mice exposed to the P2X7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X7+/+ and P2X7−/− mice. Interestingly, lack of P2X7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X7−/− mice. Hence, this study suggests the P2X7 receptor to be an appealing antifibrotic target after renal infections. Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X7+/+ and P2X7−/− mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X7−/− mice and mice exposed to the P2X7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X7+/+ and P2X7−/− mice. Interestingly, lack of P2X7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X7−/− mice. Hence, this study suggests the P2X7 receptor to be an appealing antifibrotic target after renal infections. The Gram-negative Escherichia coli is usually a commensal component of the normal intestinal flora. However, E. coli is also responsible for a large number of extraintestinal infections1Cavalieri S.J. Bohach G.A. Snyder I.S. Escherichia coli α-hemolysin: characteristics and probable role in pathogenicity.Microbiol Rev. 1984; 48: 326-343Crossref PubMed Google Scholar and is particularly prone to induce urinary tract infections (UTIs)2Foxman B. 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Mice that lacked the P2X7 receptor or were continuously treated with a P2X7 receptor antagonist showed a markedly lower degree of fibrosis. This effect is likely to result from reduced macrophage infiltration in the P2X7-deficient kidney and, thus, secondary fibrosis. P2X7−/− mice back bred over 15 generations into a BALB/cJ background were kept and bred at the Institute of Biomedicine, Aarhus University (Aarhus, Denmark). The P2X7−/− mice were matched with either P2X7+/+ littermates from heterozygous breeding or age-matched BALB/cJ mice from Janvier Labs (Saint-Berthevin, France). The P2X7−/− mice were originally developed by GlaxoSmithKline (London, UK). Animal experiments with the P2X7 antagonist, brilliant blue G (BBG), were performed on BALB/cJ mice from Janvier Labs. All animal experiments were conducted in line with the Federation of European Laboratory Animal Science Associations guidelines and the Danish national legislations. Permission to conduct these experiments was obtained from the Danish Animal Experiments Inspectorate (Dyreforsøgstilsynet, Glostrup, Denmark; 2014-15-0201-00316). Female P2X7+/+ and P2X7−/− mice or female BALB/cJ mice were injected with 50 mg/kg per day of saline (for up to 14 days). The mice were anesthetized by s.c. injection of ketamine (0.1 mg/g body weight) and xylazine (0.01 mg/g body weight). BBG was chosen as an antagonist because of its blue color, which allows us to detect the plasma value of the compound, as previously described.34Greve A.S. Skals M. Fagerberg S.K. Tonnus W. Ellermann-Eriksen S. Evans R.J. Linkermann A. Praetorius H.A. P2X1, P2X4, and P2X7 receptor knock out mice expose differential outcome of sepsis induced by α-haemolysin producing Escherichia coli.Front Cell Infect Microbiol. 2017; 7: 113Crossref PubMed Scopus (33) Google Scholar The abdomen was shaved and the mice were placed on their backs on a heating plate (38°C), resulting in a rectal temperature of approximately 36.5°C. After disinfection with iodine, a small sagittal insertion was placed in the skin above the urinary bladder. The urinary bladder was carefully located, punctured, and emptied of its content (urine was kept for culture). Bacteria (108) diluted in sterile 0.9% NaCl (20 μL) were injected into the urinary bladder. Both the peritoneum and skin were sutured with Ethilon polyamide 6.0 (Abena, Aabenraa, Denmark), and the mice were administered buprenorphine hydrochloride [Temgesic (Indivior, Berkshire, UK); 0.1 μg/g body weight] subcutaneously. By this procedure, the mice developed fulminant pyelonephritis after 24 hours, as determined in a separate concentration-response experiment (24 hours) and culture of homogenized kidneys. Pyelonephritis was confirmed on day 5. The infection was quantified by scoring blood plates streaked with equal samples of homogenized renal tissue, as follows: 0, no bacterial growth; 1, 10% of the plate. The infection status for experiments lasting for 14 days was assessed by spot urine sample at day 5 after surgery. Under anesthesia, the abdomen was opened and the left kidney was ligated and removed for mincing and culture. After this procedure, a blood sample was collected with a heparinized syringe from the inferior vena cava, immediately centrifuged at 2938 × g for 10 minutes, and divided into plasma and erythrocyte fractions. Plasma (50 μL) was stored at −80°C for cytokine measurements. Finally, the animals were perfusion fixed with 4% paraformaldehyde through the left ventricle of the heart at 0.2 bar. The hemolytic E. coli strain ARD6 (O6:K13:H1), obtained from Statens Serum Institute (Copenhagen, Denmark), was grown on agar plates containing lysogeny broth and kept for up to 1 month at 4°C. A fresh preparation of E. coli was cultured overnight from one colony in 4 mL lysogeny broth medium (37°C, 250 rpm) for every experiment. The overnight culture was centrifuged and resuspended in sterile saline twice. The preparation contained approximately 10% dead cells, assessed by a cell viability kit (BD Biosciences, Kongens Lyngby, Denmark) and flow cytometry (Accuri C6; BD Biosciences). Cytokine levels (IL-1β, IL-6, keratinocyte chemoattractant, and tumor necrosis factor-α) in urine and plasma samples were measured (CBA Flex Set; BD Biosciences) by flow cytometry (Accuri C6), following the manufacturer's manual (BD Cytometric Bead Array, Mouse/Rat Soluble Protein Master Buffer Kit; BD Biosciences). Fibrosis was quantified by identifying the number of fibrotic areas in picrosirius red (Sigma-Aldrich, Copenhagen, Denmark) stained kidney tissue (divided by area) by light microscopy (blinded counting, J.R.T.). To ensure that the fibrotic areas, in fact, were collagen and not artifacts, the identified areas were checked using plane-polarizing filters. Renal cortex was isolated and homogenized (240 seconds, 50 Hz in TissueLyser LT; Qiagen, Hilden, Germany) in a dissecting buffer (0.3 mol/L sucrose, 25 mmol/L imidazole, and 1 mmol/L EDTA, pH 7.2) containing the following: phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO) and complete miniprotease inhibitor cocktail tablets (serine, cysteine, and metalloprotease inhibitor; Roche, Basel Switzerland). The homogenized tissue was centrifuged at 4500 × g at 4°C for 10 minutes. The supernatant was added Laemmli buffer containing 2% SDS, and the protein content was measured by a Pierce BCA protein assay kit (Roche, Basel, Switzerland). The protein samples were size separated on a 12% Criterion TGX Precast Gel (Bio-Rad Laboratories, Copenhagen, Denmark) and electroblotted onto a nitrocellulose membrane (Hybond ECL; GE Healthcare, Hatfield, UK). The blots were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS) with Tween 20 (80 mmol/L Na2HPO4, 20 mmol/L NaH2PO4, 100 mmol/L NaCl, and 10% Tween 20, pH 7.4). After washing with PBS with Tween 20, the blots were incubated overnight at 4°C with primary antibodies against fibronectin (Ab2413; Abcam, Cambridge, UK), kidney injury molecule-1 (AF1817; R&D Systems, Minneapolis, MN) or α-smooth muscle actin (M0851; Dako A/S, Glostrup, Denmark). The antigen-antibody complex was visualized with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature (P447, P448, and P449; diluted 1:4000; Dako A/S) using an enhanced chemiluminescence system (Amersham ECL Plus, Little Chalfort, UK). All Western blot analyses were normalized to total protein, as measured using Stain-Free technology (Bio-Rad Laboratories).35Gurtler A. Kunz N. Gomolka M. Hornhardt S. Friedl A.A. McDonald K. Kohn J.E. Posch A. Stain-Free technology as a normalization tool in Western blot analysis.Anal Biochem. 2013; 433: 105-111Crossref PubMed Scopus (269) Google Scholar The organs were dehydrated after 1 hour in 4% paraformaldehyde by replacing the liquid every 2 hours with 70% ethanol solution, followed by 96% ethanol and 99% ethanol and finally by xylene. Then, the organs were embedded in paraffin, cut into sections (2 or 5 μm thick) with a Leica 2165 rotating microtome (Leica, Wetzlar, Germany), and fixed on objective glass (Thermo Fisher Scientific, Waltham, MA). For quantification of fibrosis, sections (5 μm thick) were dewaxed in xylene and rehydrated again in graded ethanol, stained, and inspected by light microscopy (×10 magnification; Leica DMRE; Leica) equipped with a Leica DM300 digital camera. For immunohistochemistry, tissue sections (2 μm thick) were dewaxed in xylene, rehydrated again in graded ethanol, and stained, followed by heat-induced epitope retrieval. The sections were boiled in a TEG buffer for 10 minutes in a microwave, followed by cooling on ice. Reactive aldehyde residues, produced during the antigen retrieval, were then blocked by incubation in PBS with addition of 50 mmol/L NH4Cl for 30 minutes. Unspecific antibody binding was blocked by incubation in PBS with 1% bovine serum albumin (BSA), 0.2% gelatin, and 0.05% saporin (10 minutes). The sections were labeled with either the primary antibody, NIMP-R14 (ab2557; diluted 1:500; Abcam), or anti-CD68 (ab125212; diluted 1:900; Abcam), diluted in PBS with 0.1% BSA and 0.3% Triton X-100 for 1 hour at 21°C, followed by 4°C overnight. The tissue was washed with PBS with 1% BSA, 0.2% gelatin, and 0.05% saporin (10 minutes). For immunofluorescence, Alexa 488–conjugated, goat anti-rat IgG (Invitrogen, Carlsbad, CA) was used as secondary antibody, diluted 1:1000 in PBS with 0.1% BSA and 0.3% Triton X-100 at room temperature. Coverslips were mounted with a hydrophilic mounting medium containing antifading reagent (Glycergel; Dako A/S). The sections were imaged on a Nikon TE-2000 microscope (Nikon, Tokyo, Japan) equipped with monochromator and super–video graphics array charge-coupled device (Visitech International, Sunderland, UK) with a plan fluo20/numerical aperture 0.45 objective. For peroxidation staining on hematoxylin and eosin, sections were incubated for 1 hour with horseradish peroxidase–conjugated secondary antibody in PBS with 0.1% BSA and 0.3% Triton X-100, washed, and visualized with diaminobenzidine in 35% H2O2 for 10 minutes. The sections were counterstained with Mayer's hematoxylin and rinsed in running tap water before mounting with coverslips using Eukitt (CellPath, Newtown, UK). The sections were inspected by light microscopy (Leica DMRE), equipped with a Leica DM300 digital camera. Data are presented as means ± SEM. Data were tested for normal distribution by Kolmogorov-Smirnov test. When normally distributed, the data were tested by two-way analysis of variance with multiple comparison design or t-test, in the case in which only two groups were compared. In the cases in which the results were not normally distributed, the data were tested by Kruskal-Wallis test, followed by Dunn's post hoc test. Kaplan-Meier plots were used to assess mortality rates, and the data in Figure 1B are tested by a χ2 test. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA). The murine pyelonephritis model was established to provide fulminant infection of the renal parenchyma after 24 hours of infection. Mice with spot urine positive for E. coli after 24 hours of inoculation with HlyA-producing E. coli in the urinary bladder were followed up for either 5 or 14 days. Pyelonephritis on day 5 after infection was confirmed as growth of HlyA-producing E. coli after seeding homogenized kidney on a blood plate. The degree of pyelonephritis was similar in P2X7+/+ and P2X7−/− mice (Figure 1). Figure 1A shows representative blood plates of homogenized renal tissue. The degree of infection was similar in P2X7+/+ (n = 17) and P2X7−/− (n = 14) mice (P = 0.7575) (Figure 1B). Mice contaminated with bacteria other than HlyA-producing E. coli were excluded from the study (four mice), as were mice that had cleared their UTI during the first 5 days of observation (10 P2X7+/+ and 17 P2X7−/− mice). Figure 2 illustrates the survival of the included experimental groups, for a total of 131 mice. Less than 20% of the mice died during the 5 days of observation, and the few occurrences happened early and were likely associated with the surgical procedure. The mortality was similar in P2X7+/+ and P2X7−/− mice (P = 0.1627). The P2X7 receptor is strongly associated with activation and secretion of IL-1β,26Kahlenberg J.M. Dubyak G.R. Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release.Am J Physiol Cell Physiol. 2004; 286: C1100-C1108Crossref PubMed Scopus (283) Google Scholar and one would expect P2X7 receptor–deficient mice to exhibit lower levels of proinflammatory cytokines. This is certainly not the case in a murine model of urosepsis. On the contrary, the P2X7−/− mice show a massive cytokine storm and, indeed, exceedingly high IL-1β compared with control mice.34Greve A.S. Skals M. Fagerberg S.K. Tonnus W. Ellermann-Eriksen S. Evans R.J. Linkermann A. Praetorius H.A. P2X1, P2X4, and P2X7 receptor knock out mice expose differential outcome of sepsis induced by α-haemolysin producing Escherichia coli.Front Cell Infect Microbiol. 2017; 7: 113Crossref PubMed Scopus (33) Google Scholar This was likely the result of activation of the caspase 8 pathway during sepsis, induced by HlyA-producing E. coli.34Greve A.S. Skals M. Fagerberg S.K. Tonnus W. Ellermann-Eriksen S. Evans R.J. Linkermann A. Praetorius H.A. P2X1, P2X4, and P2X7 receptor knock out mice expose differential outcome of sepsis induced by α-haemolysin producing Escherichia coli.Front Cell Infect Microbiol. 2017; 7: 113Crossref PubMed Scopus (33) Google Scholar Figure 3 shows the levels of proinflammatory cytokines in plasma and urine, collected 5 days after infection. In P2X7+/+ mice, pyelonephritis caused a marked increase in IL-6 in both urine and plasma (Figure 3), whereas IL-1β only was statistically significant in urine (Figure 3). Surprisingly, the surgery in itself was enough to trigger a substantial increase in both IL-1β and IL-6 in the P2X7−/− mice. The surgery causes this difference because P2X7−/− mice that have not been exposed to surgery have normal circulating proinflammatory cytokines.34Greve A.S. Skals M. Fagerberg S.K. Tonnus W. Ellermann-Eriksen S. Evans R.J. Linkermann A. Praetorius H.A. P2X1, P2X4, and P2X7 receptor knock out mice expose differential outcome of sepsis induced by α-haemolysin producing Escherichia coli.Front Cell Infect Microbiol. 2017; 7: 113Crossref PubMed Scopus (33) Google Scholar However, the level of proinflammatory cytokines in the P2X7+/+ and P2X7−/− mice exposed to HlyA-producing E. coli was not statistically different. This means that any difference there may be between the P2X7+/+ and P2X7−/− mice was not explain