{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:05:59Z","timestamp":1785542759071,"version":"3.56.0"},"reference-count":53,"publisher":"PeerJ","license":[{"start":{"date-parts":[[2021,8,23]],"date-time":"2021-08-23T00:00:00Z","timestamp":1629676800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001321","name":"National Research Foundation","doi-asserted-by":"crossref","award":["CPRR13090332589"],"award-info":[{"award-number":["CPRR13090332589"]}],"id":[{"id":"10.13039\/501100001321","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Health and Welfare SETA and the University of Pretoria"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:p>Diclofenac caused the death of millions of vultures on the Asian subcontinent. Other non-steroidal anti-inflammatory drugs (NSAIDs) have since also been shown to be toxic to vultures with the exception of meloxicam. For this study, we evaluated the effect of diclofenac on renal uric acid transport and glomerulus filtration in an acute toxicity model. In a two-phase study with the same birds, healthy chickens (a validated model species) were treated intravenously with para-amino hippuric acid (PAH) and iohexol (IOH) in combination in phase 1. In phase 2, the same PAH and IOH combination was then combined with diclofenac (10 mg\/kg). In both phases, blood and faeces were sequentially collected. In phase 1, the birds showed no signs of ill health. Moreover, PAH, IOH and uric acid clearance was rapid. In phase 2, two chickens showed early signs of hyperuricemia 8 hours after exposure and died approximately 24h later. Necropsy showed classic signs of renal damage and gout. Diclofenac had a rapid plasma half-life of elimination of less than 2 hours indicating that toxicity was likely due to an irreversible destruction of a physiological process. All the birds in phase 2 had decreased uric acid, PAH and IOH clearance in comparison to phase 1. The decrease in PAH clearance was variable between the birds (average of 71%) but was near 98% reduced in the two birds that died. It is concluded that diclofenac alters both renal perfusion and renal plasma flow, with death associated with tubular secretion being reduced to negligible functionality for a prolonged period. This would support previous<jats:italic>in vitro<\/jats:italic>findings of early cell death from ROS accumulation. However, further evaluation is needed to elucidate this final step.<\/jats:p>","DOI":"10.7717\/peerj.12002","type":"journal-article","created":{"date-parts":[[2021,8,23]],"date-time":"2021-08-23T08:31:36Z","timestamp":1629707496000},"page":"e12002","source":"Crossref","is-referenced-by-count":14,"title":["Diclofenac toxicity in susceptible bird species results from a combination of reduced glomerular filtration and plasma flow with subsequent renal tubular necrosis"],"prefix":"10.7717","volume":"9","author":[{"given":"Bono","family":"Nethathe","sequence":"first","affiliation":[{"name":"Department of Paraclinical Science, University of Pretoria, Onderstepoort, Pretoria, South Africa"},{"name":"Department of Food Science and Technology, University of Venda for Science and Technology, Thohoyandou, Limpopo, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Chipangura","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Science, University of Pretoria, Onderstepoort, Pretoria, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ibrahim Zubairu","family":"Hassan","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Science, University of Pretoria, Onderstepoort, Pretoria, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neil","family":"Duncan","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of Pretoria, Onderstepoort, Pretoria, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmanuel Oluwasegun","family":"Adawaren","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Science, University of Pretoria, Onderstepoort, Pretoria, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren","family":"Havenga","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Physiology, University of Pretoria, Onderstepoort, Pretoria, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vinny","family":"Naidoo","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Science, University of Pretoria, Onderstepoort, Pretoria, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"4443","published-online":{"date-parts":[[2021,8,23]]},"reference":[{"key":"10.7717\/peerj.12002\/ref-1","first-page":"41","article-title":"Percentage of faecal excretion of meloxicam in the Cape vultures (Gyps corprotheres)","volume":"215","author":"Adawaren","year":"2019","journal-title":"Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology"},{"key":"10.7717\/peerj.12002\/ref-2","doi-asserted-by":"publisher","first-page":"147","DOI":"10.1016\/j.etap.2018.07.001","article-title":"The use of liver slices from the Cape vulture (Gyps coprotheres) to better understand the role of liver toxicity of non-steroidal anti-inflammatory drugs (NSAIDs) in vultures","volume":"62","author":"Adawaren","year":"2018","journal-title":"Environmental Toxicology and Pharmacology"},{"issue":"5","key":"10.7717\/peerj.12002\/ref-3","doi-asserted-by":"publisher","first-page":"411","DOI":"10.1254\/jphs.CRJ06006X","article-title":"Organic anion transporter family: current knowledge","volume":"100","author":"Anzai","year":"2006","journal-title":"Journal of Pharmacological Sciences"},{"issue":"6","key":"10.7717\/peerj.12002\/ref-4","doi-asserted-by":"publisher","first-page":"1193","DOI":"10.3382\/ps.0591193","article-title":"Measurement of uric acid levels in chicken cecal contents","volume":"59","author":"Beck","year":"1980","journal-title":"Poultry Science"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-5","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1152\/ajplegacy.1960.198.3.575","article-title":"Effect of drugs that alter uric acid excretion in man on uric acid clearance in the chicken","volume":"198","author":"Berger","year":"1960","journal-title":"American Journal of Physiology-Legacy Content"},{"key":"10.7717\/peerj.12002\/ref-6","first-page":"113","article-title":"Inhibitors of cyclooxygenases: mechanisms, selectivity and uses","volume":"57","author":"Botting","year":"2006","journal-title":"Journal of Physiology and Pharmacology"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-7","doi-asserted-by":"publisher","first-page":"184","DOI":"10.2165\/00003088-199733030-00003","article-title":"Clinical pharmacokinetics of diclofenac","volume":"33","author":"Davies","year":"1997","journal-title":"Clinical Pharmacokinetics"},{"key":"10.7717\/peerj.12002\/ref-8","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/j.jchromb.2019.04.017","article-title":"Development and validation of an ultra-high performance liquid chromatography\u2013tandem mass spectrometry method for the simultaneous determination of iohexol, p-aminohippuric acid and creatinine in porcine and broiler chicken plasma","volume":"1117","author":"Dhondt","year":"2019","journal-title":"Journal of Chromatography B"},{"issue":"22","key":"10.7717\/peerj.12002\/ref-9","doi-asserted-by":"publisher","first-page":"4305","DOI":"10.1242\/jeb.01879","article-title":"Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture","volume":"208","author":"Dudas","year":"2005","journal-title":"Journal of Experimental Biology"},{"issue":"1","key":"10.7717\/peerj.12002\/ref-10","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1186\/1297-9686-45-16","article-title":"Epigenetics and phenotypic variability: some interesting insights from birds","volume":"45","author":"Fr\u00e9sard","year":"2013","journal-title":"Genetics Selection Evolution"},{"issue":"1540","key":"10.7717\/peerj.12002\/ref-11","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1098\/rstb.2009.0267","article-title":"Phenotypic plasticity in development and evolution: facts and concepts","volume":"365","author":"Fusco","year":"2010","journal-title":"Philosophical Transactions of the Royal Society B: Biological Sciences"},{"key":"10.7717\/peerj.12002\/ref-12","first-page":"45","volume-title":"Drugs and the pharmaceutical sciences, vol 15, pharmacokinetics, 2nd edition, revised and expanded","author":"Gibaldi","year":"1982"},{"issue":"5","key":"10.7717\/peerj.12002\/ref-13","doi-asserted-by":"publisher","first-page":"793","DOI":"10.1111\/j.0021-8901.2004.00954.x","article-title":"Diclofenac poisoning as a cause of vulture population declines across the Indian subcontinent","volume":"41","author":"Green","year":"2004","journal-title":"Journal of Applied Ecology"},{"issue":"9","key":"10.7717\/peerj.12002\/ref-14","doi-asserted-by":"publisher","first-page":"1346","DOI":"10.1002\/jps.2600650921","article-title":"Influence of cholestasis on drug elimination: pharmacokinetics","volume":"65","author":"Harrison","year":"1976","journal-title":"Journal of Pharmaceutical Sciences"},{"key":"10.7717\/peerj.12002\/ref-15","article-title":"A morphological study of the kidney and renal portal system of the Cape griffon vulture (Gyps coprotheres)","author":"Havenga","year":"2016","journal-title":"Doctoral dissertation"},{"issue":"1656","key":"10.7717\/peerj.12002\/ref-16","doi-asserted-by":"publisher","first-page":"20130583","DOI":"10.1098\/rstb.2013.0583","article-title":"Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals","volume":"369","author":"Hutchinson","year":"2014","journal-title":"Philosophical Transactions of the Royal Society B: Biological Sciences"},{"key":"10.7717\/peerj.12002\/ref-17","article-title":"The IUCN Red List of threatened species. IUCN, Gland, Switzerland","author":"IUCN (International Union for Conservation of Nature)","year":"2017"},{"issue":"5","key":"10.7717\/peerj.12002\/ref-18","doi-asserted-by":"publisher","first-page":"237","DOI":"10.4103\/0253-7613.58515","article-title":"Diclofenac-induced biochemical and histopathological changes in white leghorn birds (Gallus domesticus)","volume":"41","author":"Jain","year":"2009","journal-title":"Indian Journal of Pharmacology"},{"key":"10.7717\/peerj.12002\/ref-19","doi-asserted-by":"publisher","first-page":"709","DOI":"10.1248\/bpb.b13-00967","article-title":"Total purine and purine base content of common foodstuffs for facilitating nutritional therapy for gout and hyperuricemia","volume":"37","author":"Kaneko","year":"2014","journal-title":"Biological and Pharmaceutical Bulletin"},{"key":"10.7717\/peerj.12002\/ref-20","first-page":"626","article-title":"NSAIDs, Antirheumatic Drugs, Nonopiod Analgesics and Drugs used in Gout. Chapter 36","volume-title":"Basic and Clinical Pharmacology","author":"Katzung","year":"2009"},{"issue":"2","key":"10.7717\/peerj.12002\/ref-21","doi-asserted-by":"publisher","first-page":"534","DOI":"10.1124\/jpet.102.037580","article-title":"Interactions of human organic anion transporters and human organic cation transporters with nonsteroidal anti-inflammatory drugs","volume":"303","author":"Khamdang","year":"2002","journal-title":"Journal of Pharmacology and Experimental Therapeutics"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-22","doi-asserted-by":"publisher","first-page":"653","DOI":"10.1515\/aoas-2016-0032","article-title":"Avian crop function\u2013a review","volume":"16","author":"Kiero\u0144czyk","year":"2016","journal-title":"Annals of Animal Science"},{"issue":"2","key":"10.7717\/peerj.12002\/ref-23","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1016\/j.rvsc.2004.11.011","article-title":"Quantitative evaluation of renal function in healthy Beagle puppies and mature dogs","volume":"79","author":"Laroute","year":"2005","journal-title":"Research in Veterinary Science"},{"key":"10.7717\/peerj.12002\/ref-24","article-title":"Effect of cytochrome p450 inhibition on pharmacokinetics and toxicity of diclofenac in chickens: unravelling toxicity in Gyps vultures (Masters dissertation, University of Pretoria)","author":"Locke","year":"2020"},{"issue":"1","key":"10.7717\/peerj.12002\/ref-25","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1242\/jeb.104.1.15","article-title":"Renal acid excretion in the domestic fowl","volume":"104","author":"Long","year":"1983","journal-title":"Journal of Experimental Biology"},{"key":"10.7717\/peerj.12002\/ref-26","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1016\/B978-0-12-370491-7.00030-1","article-title":"Avian Clinical Biochemistry","volume-title":"Clinical Bio-chemistry of Domestic Animals, 6th edition","author":"Lumeij","year":"2008"},{"issue":"10","key":"10.7717\/peerj.12002\/ref-27","doi-asserted-by":"publisher","first-page":"2106","DOI":"10.3382\/ps.0622106","article-title":"A simple spectrophotometric method for the direct determination of uric acid in avian excreta","volume":"62","author":"Marquardt","year":"1983","journal-title":"Poultry Science"},{"issue":"4","key":"10.7717\/peerj.12002\/ref-28","doi-asserted-by":"publisher","first-page":"533","DOI":"10.1111\/j.1469-185X.2009.00086.x","article-title":"The integration of digestion and osmoregulation in the avian gut","volume":"84","author":"McWhorter","year":"2009","journal-title":"Biological Reviews"},{"issue":"4","key":"10.7717\/peerj.12002\/ref-29","doi-asserted-by":"publisher","first-page":"707","DOI":"10.7589\/0090-3558-41.4.707","article-title":"Pathology and proposed pathophysiology of diclofenac poisoning in free-living and experimentally exposed oriental white-backed vultures (Gyps bengalensis)","volume":"41","author":"Meteyer","year":"2005","journal-title":"Journal of Wildlife Diseases"},{"issue":"6","key":"10.7717\/peerj.12002\/ref-30","doi-asserted-by":"crossref","first-page":"1308","DOI":"10.3382\/ps.2011-01929","article-title":"An initial safety assessment of hepatotoxic and nephrotoxic potential of intramuscular ketoprofen at single repetitive dose level in broiler chickens","volume":"91","author":"Mohan","year":"2012","journal-title":"Poultry Science"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-31","doi-asserted-by":"publisher","first-page":"260","DOI":"10.1016\/j.etap.2007.06.003","article-title":"Validating the domestic fowl as a model to investigate the pathophysiology of diclofenac in Gyps vultures","volume":"24","author":"Naidoo","year":"2007","journal-title":"Environmental Toxicology and Pharmacology"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-32","first-page":"269","article-title":"Diclofenac toxicity in Gyps vulture is associated with decreased uric acid excretion and not renal portal vasoconstriction","volume":"149","author":"Naidoo","year":"2009","journal-title":"Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-33","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1016\/j.yrtph.2009.01.010","article-title":"Veterinary diclofenac threatens Africa\u2019s endangered vulture species","volume":"53","author":"Naidoo","year":"2009","journal-title":"Regulatory Toxicology and Pharmacology"},{"issue":"6975","key":"10.7717\/peerj.12002\/ref-34","doi-asserted-by":"publisher","first-page":"630","DOI":"10.1038\/nature02317","article-title":"Diclofenac residues as the cause of vulture population decline in Pakistan","volume":"427","author":"Oaks","year":"2004","journal-title":"Nature"},{"issue":"11","key":"10.7717\/peerj.12002\/ref-35","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1056\/NEJM198809153191106","article-title":"Clinical implications of prostaglandin and thromboxane A2 formation","volume":"319","author":"Oates","year":"1988","journal-title":"New England Journal of Medicine"},{"issue":"11","key":"10.7717\/peerj.12002\/ref-36","doi-asserted-by":"publisher","first-page":"2341","DOI":"10.1897\/08-123.1","article-title":"Apparent tolerance of turkey vultures (Cathartes aura) to the non-steroidal anti-inflammatory drug diclofenac","volume":"27","author":"Rattner","year":"2008","journal-title":"Environmental Toxicology and Chemistry: An International Journal"},{"issue":"2","key":"10.7717\/peerj.12002\/ref-37","doi-asserted-by":"publisher","first-page":"142","DOI":"10.1016\/j.etap.2006.02.004","article-title":"Comparative toxicity studies in birds using nimesulide and diclofenac sodium","volume":"22","author":"Reddy","year":"2006","journal-title":"Environmental Toxicology and Pharmacology"},{"key":"10.7717\/peerj.12002\/ref-38","volume-title":"Groups to Animals","author":"Rodrigues","year":"2007"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-39","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1007\/s004240000297","article-title":"The multispecific organic anion transporter (OAT) family","volume":"440","author":"Sekine","year":"2000","journal-title":"Pfl\u00fcgers Archiv"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-40","doi-asserted-by":"publisher","first-page":"282","DOI":"10.1017\/S0959270913000609","article-title":"Diclofenac is toxic to the Steppe Eagle Aquila nipalensis: widening the diversity of raptors threatened by NSAID misuse in South Asia","volume":"24","author":"Sharma","year":"2014","journal-title":"Bird Conservation International"},{"issue":"1","key":"10.7717\/peerj.12002\/ref-41","doi-asserted-by":"crossref","first-page":"F46","DOI":"10.1152\/ajprenal.1981.240.1.F46","article-title":"Renal venous portal contribution to PAH and uric acid clearance in the chicken","volume":"240","author":"Shideman","year":"1981","journal-title":"American Journal of Physiology-Renal Physiology"},{"issue":"11","key":"10.7717\/peerj.12002\/ref-42","doi-asserted-by":"publisher","first-page":"2828","DOI":"10.1097\/01.ASN.0000143473.64430.AC","article-title":"Contribution of multidrug resistance protein 2 (MRP2\/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter","volume":"15","author":"Smeets","year":"2004","journal-title":"Journal of the American Society of Nephrology"},{"key":"10.7717\/peerj.12002\/ref-43","doi-asserted-by":"publisher","first-page":"109129","DOI":"10.1016\/j.cbpc.2021.109129","article-title":"iTRAQ-based quantitative proteomic analysis reveals the toxic mechanism of diclofenac sodium on the kidney of broiler chicken","volume":"249","author":"Sun","year":"2021","journal-title":"Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology"},{"issue":"2","key":"10.7717\/peerj.12002\/ref-44","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1098\/rsbl.2005.0425","article-title":"Toxicity of diclofenac to Gyps vultures","volume":"2","author":"Swan","year":"2006","journal-title":"Biology Letters"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-45","doi-asserted-by":"publisher","first-page":"e66","DOI":"10.1371\/journal.pbio.0040066","article-title":"Removing the threat of diclofenac to critically endangered Asian vultures","volume":"4","author":"Swan","year":"2006","journal-title":"PLOS Biology"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-46","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1016\/j.taap.2004.10.016","article-title":"Organic anion transporter (Slc22a) family members as mediators of toxicity","volume":"204","author":"Sweet","year":"2005","journal-title":"Toxicology and Applied Pharmacology"},{"key":"10.7717\/peerj.12002\/ref-47","first-page":"23","article-title":"Renal organic cation and anion transport: from physiology to genes","author":"Sweet","year":"2010"},{"issue":"7","key":"10.7717\/peerj.12002\/ref-48","doi-asserted-by":"publisher","first-page":"1933","DOI":"10.1002\/art.39115","article-title":"Food sources of protein and risk of incident gout in the Singapore Chinese Health Study","volume":"67","author":"Teng","year":"2015","journal-title":"Arthritis & Rheumatology"},{"issue":"3","key":"10.7717\/peerj.12002\/ref-49","doi-asserted-by":"publisher","first-page":"233","DOI":"10.1007\/BF02041396","article-title":"Effect of nonsteroidal anti-inflammatory drugs on the renal excretion of uric acid","volume":"2","author":"Tiitinen","year":"1983","journal-title":"Clinical Rheumatology"},{"issue":"1","key":"10.7717\/peerj.12002\/ref-50","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/bdd.693","article-title":"Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology","volume":"31","author":"Van Wert","year":"2010","journal-title":"Biopharmaceutics & Drug Disposition"},{"key":"10.7717\/peerj.12002\/ref-51","first-page":"511","article-title":"Renal physiology","volume-title":"Textbook of veterinary physiology 2nd edition","author":"Verlande","year":"1997"},{"issue":"11-12","key":"10.7717\/peerj.12002\/ref-52","doi-asserted-by":"publisher","first-page":"868","DOI":"10.1515\/znc-1995-11-1220","article-title":"Phylogeny of Old and New World vultures (Aves: Accipitridae and Cathartidae) inferred from nucleotide sequences of the mitochondrial cytochrome b gene","volume":"50","author":"Wink","year":"1995","journal-title":"Zeitschrift F\u00fcr Naturforschung C"},{"issue":"38","key":"10.7717\/peerj.12002\/ref-53","doi-asserted-by":"publisher","first-page":"15904","DOI":"10.1073\/pnas.1112694108","article-title":"Fossil evidence of avian crops from the Early Cretaceous of China","volume":"108","author":"Zheng","year":"2011","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"}],"container-title":["PeerJ"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/peerj.com\/articles\/12002.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/peerj.com\/articles\/12002.xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/peerj.com\/articles\/12002.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/peerj.com\/articles\/12002.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,9,7]],"date-time":"2024-09-07T02:29:50Z","timestamp":1725676190000},"score":1,"resource":{"primary":{"URL":"https:\/\/peerj.com\/articles\/12002"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,23]]},"references-count":53,"alternative-id":["10.7717\/peerj.12002"],"URL":"https:\/\/doi.org\/10.7717\/peerj.12002","archive":["CLOCKSS","LOCKSS","Portico"],"relation":{},"ISSN":["2167-8359"],"issn-type":[{"value":"2167-8359","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,23]]},"article-number":"e12002"}}