View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector http://www.kidney-international.org original article & 2011 International Society of Nephrology see commentary on page 327 Ethylene glycol induces calcium oxalate crystal deposition in Malpighian tubules: a Drosophila model for nephrolithiasis/urolithiasis Yung-Hsiang Chen1,4, Hsin-Ping Liu1,4, Huey-Yi Chen1,2, Fuu-Jen Tsai1,2, Chiao-Hui Chang1, Yuan-Ju Lee3, Wei-Yong Lin1 and Wen-Chi Chen1,2 1Graduate Institute of Integrated Medicine, Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan; 2Department of Urology, Department of Obstetrics and Gynecology, Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan and 3Department of Urology, National Taiwan University Hospital, Taipei, Taiwan Several animal species are used to study calcium oxalate Urolithiasis is a common human disorder affecting B9.6% urolithiasis; however, an ideal model has yet to be identified. of the population in Taiwan and a range of 10–12% of the We used Drosophila as a model organism and fed the flies population in industrialized countries.1,2 The progressive lithogenic agents such as ethylene glycol, hydroxyl-L-proline, increase of the social cost for treatment of urolithiasis may be and sodium oxalate. At different times, the Malpighian related to increased incidence of the disease and/or to an tubules, the kidney equivalent of insects, were dissected and increase in costs for diagnosing and treating renal stones. a polarized light microscope used to highlight the Many factors can induce urolithiasis or cause a predisposi- birefringent crystals. Scanning electron microscopy and tion to its development. Such factors include dehydration, energy-dispersive X-ray spectroscopy confirmed that the particular medications, alkaline urinary pH, hypercalciuria, crystal composition was predominately calcium oxalate. hyperoxaluria, and a family history of the disorder.3 In Furthermore, administration of potassium citrate successfully humans, calcium oxalate (CaOx) is the major component of reduced the quantity of and modulated the integrity of the uroliths, and CaOx stones constitute B80% of all stones.4 ethylene glycol-induced crystals. Thus, the Drosophila model Therefore, most investigations of urolithiasis have focused on of bio-mineralization produces crystals in the urinary system CaOx stones. Although several species may be used as animal through many lithogenic agents, permits observation of crystal models for the study of human CaOx stone disease, an ideal formation, and is amenable to genetic manipulation. This animal model has yet to be identified.5 The ideal animal model may mimic the etiology and clinical manifestations of model should mimic the true pathogenesis of the human calcium oxalate stone formation and aid in identification of disease and allow genetic manipulation with convenient thegeneticbasisofthisdisease. observation of results as well as being cost effective and easy 6 Kidney International (2011) 80, 369–377; doi:10.1038/ki.2011.80; to breed. published online 30 March 2011 Kidney stones in both humans and hyperoxaluric rats are KEYWORDS: calcium oxalate; Drosophila melanogaster; ethylene glycol; located on renal papillary surfaces and consist of an organic nephrolithiasis/urolithiasis; potassium citrate matrix and crystals of CaOx and/or calcium phosphate. Hyperoxaluria can induce CaOx nephrolithiasis in both humans and rats. Dietary factors play an important role in kidney stone formation.7,8 Oxalate metabolism is considered to be almost identical in rats and humans. Thus, there are many similarities between experimental nephrolithiasis induced in rats and human kidney stone formation. A rat model of CaOx nephrolithiasis can be used to investigate the mechanisms involved in human kidney stone formation.5,9 Thus, the disease is experimentally induced and the rats are Correspondence: Wen-Chi Chen or Wei-Yong Lin, Graduate Institute of generally made hyperoxaluric either by administration of 10,11 Integrated Medicine, Department of Urology, China Medical University and excess oxalate, exposure to the toxin ethylene glycol (EG), 12 13 Hospital, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan. hydroxyl-L-proline (HLP), sodium oxalate (NaOx), or E-mail: [email protected] or [email protected] various nutritional manipulations. The advantages of the rat 4These authors contributed equally to this study. model include a high rate of stone formation, convenient Received 29 March 2010; revised 9 January 2011; accepted 1 February animal breeding, and a well-known anatomy. However, the 2011; published online 30 March 2011 costs of breeding and care of experimental rats have been Kidney International (2011) 80, 369–377 369 original article Y-H Chen et al.: Drosophila model for stone disease increasing. In addition, the costs of performing gene formation models in Drosophila and to identify a simple and knockout experiments in rats are also high and occasionally convenient model with significant CaOx crystal deposition in accompanied by technical difficulties. Other problems that the Malpighian tubules, several lithogenic agents (including may arise with the use of rats may include cases of normal EG, HLP, and NaOx that were used in previous rat models) animals having natural inhibitors of abnormal mineralization were fed to individual insects of the Canton-S (CS) strain of and cases of uncharacterized genetic promoters of metabolic D. melanogaster. At different periods during the experiment, pathways related to stone formation or destruction. Malpighian tubules were dissected and a polarized light Dionne and Schneider14 have described two features of microscope was used to highlight the birefringent crystals of Drosophila melanogaster that make it particularly valuable as CaOx. Figure 1a shows a view of the classical morphology of a model organism. First, the tools available for studying Malpighian tubules. The CaOx birefringent crystals appeared Drosophila are very accommodating for the study of basic as early as 14–21 days after ingestion of EG, HLP, and NaOx biology. Second, Drosophila is a good model for researching in the Malpighian tubules of Drosophila when viewed with human disease, such as cancer, neurological disorders, polarized light (Figure 1b). Monohydrate CaOx crystals diabetes, and drug addiction, because any findings translate (clear or jewel-like gloss; six-sided prisms or various forms; well into medicine.14 All multicellular organisms have a Figure 1c) are more common than the typical dihydrate specialized organ for concentrating and excreting wastes from CaOx crystals (Maltese cross or envelope-shaped crystals the body. The kidneys in vertebrates and the Malpighian induced by feeding of a herbal formula; Figure 1d). Various tubules in Drosophila accomplish these functions. Mammals forms of monohydrate CaOx crystals shapes were also and Drosophila have similar features during renal tubular identified. Free crystals were extensive, with many incorpo- development.15,16 MacPherson et al. have reported that rated in casts. Their size is estimated to vary approximately Malpighian tubules act as a genetically tractable system in between 5 and 20 mm. Most crystals were identified within the regulation of ions and epithelial fluid transport.17 the ‘enlarged initial (distal) segment’ of the anterior Recently, investigations of the genomics of the Malpighian Malpighian tubules. Crystal formation was evaluated using tubules have revealed far more extensive roles for these a polarized light microscope. Figure 1d indicates the different structures. The tubules have the capability to actively excrete degrees (À, þ , þþ, and þþþ) of CaOx crystal a very broad range of organic solutes and xenobiotics such as deposition in the Malpighian tubules. Each blinded specimen insecticides.18 The major excretory epithelia in insects are the was evaluated by three investigators who assessed crystal Malpighian tubules and hindgut, which act in concert to formation using a crystal score of 0 ¼ none, 1 ¼ weak, form the functional kidney. Secretion of fluid and ions by 2 ¼ moderate, and 3 ¼ strong. To avoid interassay variability, Malpighian tubules may be followed by reabsorption of all samples used for this scoring method were observed from water, ions, or useful metabolites.19–21 samples under the same polarization conditions. The recently described models of animal stone disease may The optimized drug concentrations, crystal-inducing help us better understand and ultimately treat nephrolithiasis period, and incidence of crystal formation for different in humans. Given the number of similarities between lithogenic agents are shown in Table 1. We successfully treatment patterns for humans and animals, many urologic induced ‘moderate to strong’ CaOx crystal formation in human treatments are now being integrated into the Malpighian tubules of Drosophila with the hyperoxaluria- treatment of domestic animals.22 In our previous study, we causing agents, EG, HLP, and NaOx, in a dose-dependent used EG to induce kidney calculus formation in rats and manner. As EG caused the most consistent results for the identified examples of Chinese herbal formulas that can CaOx crystal deposition in the Malpighian tubules, we inhibit this effect.10,11 Herein, we report a novel Drosophila recommend using EG (0.5% in fly medium) as the major animal model for the study of stone