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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 (, 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 disease. Hyperoxaluria- crystal-inducing agent. This dosage is similar to the inducing causing agents in rat model, including EG, HLP, and NaOx, dose of 0.75% EG in drinking water used previously in the rat were employed as lithogenic agents. The present animal urolithiasis model.10,11 model effectively produces crystals in the Drosophila urinary system. In addition to being cost effective, it is convenient to Crystal identification observe the crystals and perform genetic manipulations. We Qualitative analysis using energy-dispersive X-ray spectros- expect that this model is also adaptable to other lithogenic copy (EDS or EDX) is a powerful tool in microanalysis. agents (such as melamine-tainted infant formula) and will be Elemental analysis in scanning electron microscopy (SEM) is useful for investigations of the effects of new treatment agents performed by measuring the energy and intensity distribu- for the prevention of CaOx crystal formation. tion of the X-ray signal generated by a focused electron beam.23 In addition to use of the polarized light microscope RESULTS for assessing crystal refraction, SEM and EDS were also Crystal-inducing agents used to identify the relative elemental composition of the A pilot study has been performed with many different crystals. After removal of the Malpighian tubule tissue lithogenic agents to establish our Drosophila model. In order with lysis buffer containing 10% proteinase K (Invitrogen, to compare the effects of several experimental CaOx crystal Carlsbad, CA), SEM reveals the crystal deposition inside the

370 Kidney International (2011) 80, 369–377 Y-H Chen et al.: Drosophila model for stone disease original article

Malpighian tubule system Salt, water, and nitrogenus wastes Hemolymph

Rectum Hindgut Intestine Enlarged initial segment Ureter Main segment Transitional segment Midgut 500 µm

Control EG HLP NaOx

Monohydrate CaOx Dihydrate CaOx

– + ++ +++

Figure 1 | Hyperoxaluria-causing agent-induced calcium oxalate (CaOx) crystal deposition in Malpighian tubules. (a) A drawing of the Malpighian tubules. Drosophila has four tubules; the anterior pair and the posterior pair. Each tubule has distinct morphologic regions: initial, transitional, main segments, and lower tubule. The two tubules in each pair merge together at ureters and connect to the gut at the midgut/hindgut boundary. (b) Representative polarized microscopy photos for ethylene glycol (EG)-, hydroxyl-L-proline (HLP), and sodium oxalate (NaOx)-induced CaOx crystal formations in Malpighian tubules. (c) Monohydrate CaOx crystals and dihydrate CaOx crystals. (d) The different degrees (, þ , þþ, and þþþ) of CaOx crystal deposition in the Malpighian tubules for semiquantification of crystal formation (arbitrarily crystal score: 0 ¼ no, 1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong crystal formation).

Malpighian tubules, and the EDS analysis successfully Drosophila life span identifies the crystal composition. The predominant compo- Renal stones lead to chronic kidney disease in humans and nents are found to be carbon (C) (weight: 29.6±8.3%; may be associated with an increased mortality rate. As it is atom: 42.8±8.7%), oxygen (O) (weight: 39.6±5.7%; difficult to evaluate the levels of creatinine and urea nitrogen atom: 43.5±6.7%), and Ca (weight: 30.8±8.9%; atom: as well as symptoms, behaviors,24 and clinical characteristics 13.7±4.9%; Figure 2). The results of this microanalysis in our Drosophila model, the relationship between different confirm that the crystal composition is predominately CaOx lithogenic agent-induced crystal formations and the life span (chemical formula: CaC2O4 or Ca(COO)2). of Drosophila were measured. Survival studies were performed

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to determine the impact of lithogenic agents on life span and Inhibition of crystal formation and life-span extension mortality. The control insects had mean and maximum life Recent metabolic studies suggest that high doses of span of 40.5 and 66 days, respectively. The mean life span potassium citrate (K-Citrate) may be effective in reducing was significantly reduced by administration of EG, HLP, and the risk of formation of stones risk because of alkali load and NaOx (Figure 3a). Furthermore, administration of EG, HLP, the citraturic response.4 In a rat model of EG-induced CaOx and NaOx had the effect of elevating the incidence of crystal nephrolithiasis, oral K-Citrate was found to be effective in formation in a dose-dependent manner (Figure 3b). These data preventing CaOx stone formation.25 In the present study, we confirm that high-dose administration of EG, HLP, and NaOx next investigated the effect of K-Citrate granules (Gentle causes significant reduction of the life span of Drosophila. Pharma, Yunlin, Taiwan) for the prevention of crystal formation in Drosophila. K-Citrate granules are used as an Table 1 | Incidence of crystal formation for different effective treatment in humans. The results of this investiga- lithogenic agents in Drosophila tion indicate that administration of K-Citrate inhibits EG- induced CaOx crystal formation in a dose-dependent Crystal formation (%) manner. The crystal deposit scores dropped significantly in Male Female Total study groups treated with K-Citrate (Figure 4a). In parallel, Control 6.6±3.6 46.8±3.4 24.3±3.0 administration of K-Citrate was found to significantly ameliorate the EG-induced reduction of life span of the EG 0.1% 54.8±9.4* 67.0±4.9* 61.3±2.8* treated insects (Figure 4b). Administration of K-Citrate 0.5% 94.2±1.5* 98.4±0.1* 97.7±1.4* successfully reduces the quantity of CaOx crystals and 0.75% 100.0±0.0* 100.0±0.0* 100.0±0.0* modulates the integrity of the crystals. Smaller crystals ( þ ± ± ± 1% 100.0 0.0* 100.0 0.0* 100.0 0.0* to þþ; weak to moderate) of powdery shape were formed HLP mainly in the vicinity of the ‘ureter’ region of the Malpighian 0.01% 4.6±7.5 90.3±8.3* 38.5±3.1* tubules (Figure 4c). Additionally, administration of K-Citrate 0.1% 9.0±5.7 91.2±8.2* 42.4±9.9* significantly inhibited both HLP- and NaOx-induced crystal ± ± ± 1% 15.3 2.8* 91.6 11.7* 48.7 7.5* formations. However, administration of K-Citrate failed to NaOx ameliorate NaOx-induced reduction of life span (Figure 4d). 0.01% 49.6±3.5* 98.0±3.4* 73.8±4.3* 0.05% 91.0±5.3* 96.0±3.5* 93.7±4.5* Melamine-induced crystal formation Abbreviations: EG, ethylene glycol; HLP, hydroxyl-L-proline; NaOx, sodium oxalate. In addition to hyperoxaluria-causing agents, melamine- *A probability, P-value of o0.05, was considered significant compared with control. Values were expressed as means±s.e.m. Statistical evaluation was performed using contaminated milk formula has been found to cause infant one-way analysis of variance (ANOVA) followed by Bonferroni test. nephrolithiasis. We also tested the effect of melamine on

60 µm Electron image 1 60 µm Electron image 1 60 µm Electron image 1 Spectrum 1 Spectrum 1 Spectrum 2

0 12345678 9 0123456789 0123456789 Full scale 868 cts cursor: 0.000 keV Full scale 868 cts cursor: 0.000 keV Full scale 868 cts cursor: 0.000 keV

Element Weight (%) Atom (%) Element Weight (%) Atom (%) Element Weight (%) Atom (%) C 22.33 33.91 C 38.62 51.35 C 27.86 43.11 O 44.86 51.15 O 40.33 40.26 O 33.58 39.01 Ca 32.81 14.93 Ca 21.06 8.39 Ca 38.56 17.88 Totals 100.00 100.00 Totals 100.00 100.00 Totals 100.00 100.00

Figure 2 | Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS or EDX) microanalysis for calcium oxalate (CaOx) crystals. Representative SEM images and EDX spectrums of a grain present at the top of Malpighian tubules under ethylene glycol (EG) treatment. After removing Malpighian tubule tissue with lysis buffer, SEM shows internalization view. Surface shows adherence with protruding crystals. EDS spectra were recorded at 20 kV. The inset photo shows the polarized microscopy image of the crystal sample, the arrow shows the location where the beam was focused; EDS spectra obtained with the beam focused at points in the crystal sample. The predominant components were found to be carbon (C), oxygen (O), and calcium (Ca). Scale bar ¼ 60 mm.

372 Kidney International (2011) 80, 369–377 Y-H Chen et al.: Drosophila model for stone disease original article

Control 1.0 1.0 Control 1.0 EG 0.1% HLP 0.01% Control EG 0.5% HLP 0.1% NaOx 0.01% EG 0.75% HLP 1% NaOx 0.05% 0.8 EG 1% 0.8 0.8

0.6 0.6 0.6 Survival 0.4 Survival 0.4 Survival 0.4

0.2 0.2 0.2

0.0 0.0 0.0 010203040 50 60 70 0 10203040 50 60 70 0 10203040 50 60 70 Age (days) Age (days) Age (days)

140 60 140 60 140 60 CaOx formation CaOx formation CaOx formation Mean lifespan 120 Mean lifespan 50 120 Mean lifespan 50 120 50 ) s 100 * * * 100 100 * y

40 40 40 da * * ( *

80 80 80 an

* p * * 30 * 30 * 30 60 60 60 * 20 20 20 40 * 40 40 Mean lifes Mean lifespan (days) Mean lifespan Mean lifespan (days) Mean lifespan

CaOx crystal (%) formation 10 10 10 CaOx crystal (%) formation 20 20 * CaOx crystal (%) formation 20

0 0 0 0 0 0 0 0.1 0.5 0.75 1 0 0.01 0.1 1 0 0.01 0.5 EG (%) HLP (%) NaOx (%) Figure 3 | High-dose hyperoxaluria-causing agents induced Drosophila life-span reduction and increased incidence of crystal formation. (a) Cumulative survival distributions by administration of hyperoxaluria-causing agents ethylene glycol (EG), hydroxyl-L-proline (HLP), and sodium oxalate (NaOx). Flies treated with hyperoxaluria-causing agents showed significant life-span reduction in a dose-dependent manner compared with control (nD150 for each group, Po0.0001). (b) The relation of mean life span and incidence of calcium oxalate (CaOx) crystal formation in male Drosophila treated with hyperoxaluria-causing agents (nD100 for each group, *Po0.01 compared with control). In this and the following figures, only male flies were used, with their numbers indicated in parentheses for each experiment. crystal formation in Drosophila. The results indicate that treatments known to be effective in humans, and (3) the administration of melamine caused crystal formation in a drugs tested should be administered orally. In our investiga- dose-dependent manner (Supplementary Figure S1b online). tion, the physical characteristics of calculi removed from The crystals also appeared after ingestion of melamine human and Drosophila are found to be identical. Crystal in the Malpighian tubules of Drosophila when viewed formation in Drosophila is found to be responsive to with polarized light (Supplementary Figure S1a online). K-Citrate therapy, as it is in the case of human calcium- Administration of K-Citrate was found to significantly related urolithiasis. ameliorate the EG-induced reduction of life span. However, Administration of EG to rats is the most common method administration of K-Citrate failed to reduce the quantity of for inducing CaOx renal stones in vivo.28 Other methods for crystals (Supplementary Figure S1c online). As CaOx is not experimentally inducing CaOx renal stones include adding the major crystals induced by melamine,26 the predominant HLP to drinking water or food, implantation of osmotic components of melamine-induced crystals and the potential mini-pumps filled with oxalate, resection of ileum in crystal inhibitors warrant further investigation. combination with oxalate feeding, and intraperitoneal administration of oxalate or HLP.29–32 Knight et al.33 have DISCUSSION reported the data for HLP ingestion and urinary oxalate and The data provide us with evidence that the in vivo CaOx glycolate excretion in humans. Their results revealed that the stone disease can be efficiently studied using a Drosophila kidney absorbs significant quantities of HLP and glycolate. model. Like the previously employed rat model in combina- The Malpighian tubule has proved to be a good model for tion with EG administration, our Drosophila model has some human renal disease, and to act as an organotypic proven effective for inducing the CaOx crystal formation ‘testbed’ for mammalian genes.18 However, how similar is process responsible for production of CaOx renal stones. Our Drosophila metabolism to mammalian in the pathway where novel Drosophila animal model satisfies the following criteria HLP is a precursor to glyoxylate, and the major site of proposed by Wolkowski-Tyl et al.:27 (1) the test animals metabolism of HLP in flies versus mammals, are still unclear. should develop uroliths rapidly and reproducibly, (2) the Although the principal precursor of oxalate is believed to symptoms should be ameliorated or prevented by drug be glyoxylate, pathways in humans and flies resulting in

Kidney International (2011) 80, 369–377 373 original article Y-H Chen et al.: Drosophila model for stone disease

120 Control 1.0 EG 100 * + K-Citrate 0.01% + K-Citrate 0.1% 0.8 + K-Citrate 1% 80 + K-Citrate 2% 0.6 60 # #

Survival 0.4 40 Crystal (%) formation 20 0.2

0 0.0 Control EG 0.01 0.5 123 0 102030405060 70 80 EG + K-citrate (%) Age (days)

120 60

50 100 * # 40 80 * # 30 * 40 20 Mean lifespan (days) Mean lifespan Crystal (%) formation 20 * 10 #

0 0

HLP HLP NaOx NaOx Control Control

HLP + K-Citrate HLP + K-Citrate NaOx + K-Citrate NaOx + K-Citrate Figure 4 | Inhibition of crystal formation and life-span extension by potassium citrate (K-Citrate) treatment. (a) Dose-dependent effects of K-Citrate on 0.5% ethylene glycol (EG)-induced calcium oxalate (CaOx) crystal formation (nD100 for each group, the results for least eight separate experiments are expressed as mean±s.d. *Po0.001 compared with control; #Po0.001 compared with EG-treated group). (b) Life-span extension of 0.5% EG-treated flies by K-Citrate treatment (nD150 for each group, Po0.0001). (c) Representative polarized microscopy photos for K-Citrate-remedied CaOx crystal formation in Malpighian tubules. Enlarged photo shows the powdered small crystals in the ‘ureter’ site. (d) Effects of K-Citrate on hydroxyl-L-proline (HLP)- and sodium oxalate (NaOx)-induced crystal formations (nD100 for each group, the results for least eight separate experiments are expressed as mean±s.d. *Po0.05 compared with control; #Po0.05 compared with HLP or NaOx-treated group); life-span extension of HLP-treated flies by K-Citrate treatment (nD150 for each group, Po0.05). glyoxylate synthesis are also not well defined; their metabo- crystals in the kidneys.34 The rat model has been criticized lism to oxalate in this tissue warrants further consideration. because of the side effect of metabolic acidosis induced In contrast, EG is broken down in vivo into four organic by administration of EG. Nevertheless, Green et al.35 stated acids: glycoaldehyde, glycolic acid, glyoxylic acid, and oxalic that metabolic acidosis does not always happen when the acid. The oxalic acid subsequently precipitates as CaOx renal function is preserved. Therefore, EG should be used

374 Kidney International (2011) 80, 369–377 Y-H Chen et al.: Drosophila model for stone disease original article

when the rat’s renal function is normal. Evaluation of the Because natural and experimental fly ‘urolithiasis’ is rat model may be complicated by impaired renal function similar to the human disease, the Drosophila model could due to drug or stone effects. As it is well established that be used to further define the pathogenesis of formation of EG leads to a high anion gap metabolic acidosis in patients, CaOx calculi. Other lithogenic factors in addition to we should be cautious in dismissing the development of a hyperoxaluria-causing agents need to be more clearly defined. metabolic acidosis in our model. It is entirely possible that For example, melamine-contaminated milk formula has been the negative correlation between EG dose and life span is found to cause infant nephrolithiasis in some areas of China. because of either toxic effects of the EG in Drosophila and/or Its combination with cyanuric acid causes crystallization in an EG dose-dependent-induced metabolic acidosis. The renal tubules. Recently, the assessment of melamine and positive correlation between EG dose and CaOx crystal cyanuric acid toxicity in cats and dogs has been reported.43–45 formation could be driven by the decline in ‘renal’ function, Our Drosophila model can provide another complementary as the severity of the acidosis or the degree of toxicity platform (as a substitute for ‘pets’) to avoid experimentation increases with an increase in EG dose. Given that K-Citrate is on domestic animals for further examinations of the equivalent to a base load (from an acid-base point of view) mechanisms of melamine-related urolithiasis. The model and increases citrate excretion (from a risk of kidney stone would also be useful in the evaluation of drugs, diets, or even formation point of view), it would be predicted that the herbal medicines46 that might be used to prevent the metabolic acidosis would be less severe (and life span formation or induce the dissolution of calculi. K-Citrate is increased) and CaOx crystal formation decreased with a well-known drug for the prevention of stone disease. K-Citrate administration. However, it can only adjust the thermodynamic stability from The function of all animal excretory systems is to rid the one side of solubility equilibrium. The solubility of a body of toxins and to maintain homeostatic balance. combination of potassium oxalate and calcium citrate is Although excretory organs in diverse animal species appear higher than the original ions from CaOx. Future trends in the superficially different, they are often built on the two treatment of CaOx stone disease should be focused on the common principles of filtration and tubular secretion/ other side of the solubility equilibrium, that is, the reabsorption. The Drosophila excretory system is composed adjustment of oxalic acid levels. Selectivity of the response of filtration Malpighian tubules.36,37 Various insects contain to different types of therapy implies that the Drosophila crystalline structures such as CaOx or calcium urate that are model may provide value in the development of new drugs. predominantly localized in the Malpighian tubules.38 In one Thus, because of its simplicity and specificity, the model notable example, the urine of plant-feeding caterpillars is appears to offer advantages for studies of the mechanism, loaded with granules and crystals of calcium carbonate and pathology, and treatment of calcium urolithiasis. CaOx that are derived from the diet. These compounds are Urolithiasis is usually associated with metabolic abnorm- thought to either bind to or eliminate excess calcium or, in alities that may include hypercalciuria, hyperphosphaturia, the case of CaOx, to neutralize and eliminate the oxalic hyperoxaluria, hypocitraturia, hyperuricosuria, cystinuria, a acid.38,39 Calcium salts have also been reported to be present low urinary volume, and defects of urinary acidification.47 in the shells of eggs of various insects, adding to their rigidity. The etiology of these metabolic abnormalities and of Whereas other calcium salts such as calcium carbonate, urolithiasis is multifactorial and involves interactions be- calcium urate, or CaOx that have been observed in a variety tween environmental, hormonal, and genetic determinants.48 of insects usually occur in the Malpighian tubules, calcium With the complete sequencing of the Drosophila genome, and tartrate crystals have been identified in the midgut of the the concurrent development of postgenomic technologies grape leafhopper. The accumulation of calcium salts in the such as microarrays, proteomics, metabolomics, and systems midgut of insects has been detected only in rare cases.39,40 biology, completely unexpected roles for the insect Mal- The primary excretory organs of insects are the Malpighian pighian tubule have emerged.49 In addition to the classical tubules and the rectum. The blind-ended Malpighian tubules role of osmoregulation, the Malpighian tubule is highly empty into the intestinal tract at the midgut/hindgut specialized for organic solute transport, as well as metabolism junction.41 The site of the initial solid phase has long been and detoxification. Our present Drosophila model may also the subject of debate. Our observations of the initial segment be used as a platform for studies of lithogenic genetic are in agreement with previous observations that interstitial factors50 that play roles in idiopathic hypercalciuria or crystals are located at, or adjacent to, the papillary tip. hyperoxaluria.51 Randall’s plaques were found to be common in the tubules A comparison of the characteristics52 of human and of insects with stone formations. On the other hand, Drosophila urolithiasis/nephrolithiasis is provided in Table 2. Malpighian tubules secrete primary urine that is isosmotic Although Drosophila can be used as a model for urolithiasis, to hemolymph and rich in KCl and/or NaCl. The processes of EG-induced crystal deposition in Drosophila and sponta- regulation of calcium and oxalate transport in Malpighian neous urolithiasis in humans have some differences. The tubules remains unclear.42 The molecular mechanisms induced urolithiasis was found to be more prevalent in underlying calcium and oxalate transport must be more human males and was accompanied by hypercalciuria and clearly defined. aciduria. Men are also more susceptible to stone formation

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Table 2 | Characteristics of human and Drosophila urolithiasis/ MATERIALS AND METHODS nephrolithiasis Fly stocks and rearing conditions I. Similarities between human and Drosophila urolithiasis/nephrolithiasis Wild-type flies, D. melanogaster CS, were used in these experiments. A. Overall prevalence and incidence (for male) of urolithiasis rate Flies were reared in plastic vials containing standard fly medium (B10%) (yeast, corn syrup, sugar, and agar), at 25 1C, 50–60% humidity, with B. Dietary factors play an important role in stone/crystal formation a 12-h light–dark cycle. C. The stone/crystal can be ameliorated or prevented by oral drug treatment D. Familiar physiology function of kidneys and Malpighian tubules Lithogenesis of flies The experimental model of CaOx crystal formation was produced in II. Differences between human and Drosophila urolithiasis/nephrolithiasis both male and female CS Drosophila as described below. Different A. Increased prevalence in male of humans but in female of Drosophila concentrations of EG, HLP, NaOx, or various nutritional manipula- B. The symptom, complication, and mortality are inconsistent tions were added in the fly medium (wt/vol). After 3 weeks, the flies C. Distinct drug pharmacokinetics and pharmacodynamics (nD100 for each group) were killed under CO narcotization, and D. Discrepant anatomy conformation between kidneys and Malpighian 2 tubules the Malpighian tubules were dissected, removed, and processed for polarized light microscopy examination. The crystals were also processed for further SEM and EDS studies. The K-Citrate granules were kindly provided by Gentle Pharma. than women, and hypercalciuria is perhaps the most Polarized light microscopy common biochemical abnormality in patients with calcium After the CaOx crystal induction period, the Malpighian tubules urolithiasis. Calcium salts have also been reported in the eggs were dissected and immediately observed under normal and of various insects and add to the rigidity of the shell. polarized white light with an Olympus BX51 optical microscope Therefore, the male Drosophila rather than female may (Tokyo, Japan). The relevant aspects were photographed and the represent a more stable model for CaOx stone disease. On the scales were obtained with the projection of a micrometric slide other hand, pathological changes in the kidneys, which under the same conditions utilized in the illustrations. include renal injury and dysfunction, can lead to retention of crystals.32 As Drosophila is not appropriate for investigation Electron microscopy and EDS microanalysis of renal functions, appropriate evaluation methods must be Microanalyses were performed with a JEOL JSM-6700F SEM further established. Additionally, whether the drug dosages (Tokyo, Japan), with EDS, operated at an accelerated voltage of 2 scaled for administration to Drosophila can provide an 20 kV. Pieces (12 12 mm ) of the slides containing the samples approximation for the optimum therapeutic range for were fixed on a carbon support with carbon tapes. In order to humans has yet to be clarified. Furthermore, because dietary improve the image contrast, carbon was evaporated to form a thin (few nanometers) layer over the sample. factors play an important role in the crystal formation, the Drosophila medium should be conscientiously controlled to Fly collection and life-span assay prevent contamination with potential lithogenic factors such To set up life-span assays, new emergents were collected under light as melamine-containing dairy products. CO2 anesthesia. Foam plugs, instead of cotton plugs, were used and Some limitations should be considered in this study. the food vials were kept horizontally to avoid weaker flies being The translation of our obtained results using the proposed accidentally stuck to food or cotton plugs. Survivors in each vial model to the humans is rather difficult. There are two were counted and dead flies were removed daily. Survivorship was main concerns. One is that the absorption, metabolism, compared and tested for significance with log-rank tests. Life-span and excretion of a given substance using an insect model curves were from pooled counts of a large number of vials (nD150). can be totally different to those of mammals, and conse- quently the results may not be comparable. The second Statistical analyses aspect is related to the composition of fluids in Malpighian One-way analysis of variance was applied to detect overall tubules of insects and the urine composition of mammals. differences among the groups; for all multiple comparisons, Bonferroni correction was applied. Significantly different groups Obviously, the crystallization of CaOx in a fluid strongly were compared pairwise using the Mann–Whitney U-test for crystal depends not only on its supersaturation, but also on the scores. For comparison between two life-span curves, we determined other components of such fluids and on their ionic strength. P-value in the log-rank test. All statistics were done using the As Drosophila is not appropriate for investigation of renal SigmaStat software (SPSS; Systat Software, San Jose, CA). functions, appropriate evaluation methods must be further established. DISCLOSURE In summary, from the study of fruitfly, we have made well All the authors declared no competing interests. progress toward validation of an original research method for ACKNOWLEDGMENTS studying in vivo CaOx stone disease. The use of hyper- This study was supported by grants NSC 97-2320-B-039-022-MY3 oxaluria-causing agents for inducing CaOx crystal deposition and NSC 98-2314-B-039-023-MY3 from the National Science Council, in the Malpighian tubules of Drosophila has the potential to Taiwan; grants DOH98-TD-F-113-098011 and DOH100-TD-B-111-004s allow Drosophila to be used as a novel cost-effective from the Department of Health, Taiwan; Executive Yuan, Taiwan; urolithiasis model. and grant CMU97-CMC-003 from China Medical University Hospital,

376 Kidney International (2011) 80, 369–377 Y-H Chen et al.: Drosophila model for stone disease original article

Taichung, Taiwan. We thank Miss Ching-Yi Lu, Yun-Chen Hsu, and 22. Robinson MR, Norris RD, Sur RL et al. Urolithiasis: not just a 2-legged Chi-Hsiang Wei for experimental assistance. animal disease. J Urol 2008; 179: 46–52. 23. Rio MC, de Oliveira BV, de Tomazella DP et al. Production of calcium oxalate crystals by the basidiomycete Moniliophthora perniciosa, the AUTHOR CONTRIBUTIONS causal agent of witches broom disease of Cacao. Curr Microbiol 2008; 56: Y-HC took the primary role in writing the manuscript and provided 363–370. statistical support. W-YL designed most of the experiments, 24. Dankert H, Wang L, Hoopfer ED et al. Automated monitoring and analysis with H-YC, F-JT, H-PL, C-HC, and Y-JL, and supervised the final of social behavior in Drosophila. Nat Methods 2009; 6: 297–303. experiments. W-CC obtained the funding and conceived, designed, 25. Yasui T, Sato M, Fujita K et al. Effects of citrate on renal stone formation and osteopontin expression in a rat urolithiasis model. Urol Res 2001; 29: and supervised the study. W-CC and W-YL provided the original 50–56. ideas for this study. 26. Chen WC, Wu SY, Liu HP et al. Identification of melamine/cyanuric acid- containing nephrolithiasis by infrared spectroscopy. J Clin Lab Anal 2010; SUPPLEMENTARY MATERIAL 24: 92–99. Figure S1. Melamine-induced crystal deposition in Malpighian 27. Wolkowski-Tyl R, Chin TY, Popp JA et al. Chemically induced urolithiasis in tubules. weanling rats. Am J Pathol 1982; 107: 419–421. Supplementary material is linked to the online version of the paper at 28. Hennequin C, Tardivel S, Medetognon J et al. A stable animal model of diet-induced calcium oxalate crystalluria. Urol Res 1998; 26: 57–63. http://www.nature.com/ki 29. Tawashi R, Cousineau M, Sharkawi M. Calcium oxalate crystal formation in the kidneys of rats injected with 4-hydroxy-L-proline. Urol Res 1980; 8: REFERENCES 121–127. 1. Lee YH, Huang WC, Tsai JY et al. Epidemiological studies on the 30. O’Connor RC, Worcester EM, Evan AP et al. Nephrolithiasis and prevalence of upper urinary calculi in Taiwan. Urol Int 2002; 68: nephrocalcinosis in rats with small bowel resection. Urol Res 2005; 33: 172–177. 105–115. 2. Moe OW. Kidney stones: pathophysiology and medical management. 31. Marengo SR, Chen DH, MacLennan GT et al. Minipump induced Lancet 2006; 367: 333–344. hyperoxaluria and crystal deposition in rats: a model for calcium oxalate 3. Broadus AE, Thier SO. Metabolic basis of renal-stone disease. N Engl J Med urolithiasis. J Urol 2004; 171: 1304–1308. 1979; 300: 839–845. 32. Khan SR. Renal tubular damage/dysfunction: key to the formation of 4. Tracy CR, Pearle MS. Update on the medical management of stone kidney stones. Urol Res 2006; 34: 86–91. disease. Curr Opin Urol 2009; 19: 200–204. 33. Knight J, Jiang J, Assimos DG et al. Hydroxyproline ingestion and urinary 5. Khan SR. Animal models of kidney stone formation: an analysis. World J oxalate and glycolate excretion. Kidney Int 2006; 70: 1929–1934. Urol 1997; 15: 236–243. 34. Leth PM, Gregersen M. Ethylene glycol poisoning. Forensic Sci Int 2005; 6. Dow JA. Model organisms and molecular genetics for endocrinology. 155: 179–184. Gen Comp Endocrinol 2007; 153: 3–12. 35. Green ML, Hatch M, Freel RW. Ethylene glycol induces hyperoxaluria 7. Borghi L, Schianchi T, Meschi T et al. Comparison of two diets for the without metabolic acidosis in rats. Am J Physiol Renal Physiol 2005; 289: prevention of recurrent stones in idiopathic hypercalciuria. N Engl J Med F536–F543. 2002; 346: 77–84. 36. O’Donnell MJ. Too much of a good thing: how insects cope with excess 8. Curhan GC, Willett WC, Speizer FE et al. Comparison of dietary calcium ions or toxins in the diet. J Exp Biol 2009; 212: 363–372. with supplemental calcium and other nutrients as factors affecting the 37. Denholm B, Skaer H. Bringing together components of the fly renal risk for kidney stones in women. Ann Intern Med 1997; 126: 497–504. system. Curr Opin Genet Dev 2009; 19: 526–532. 9. Evan AP, Bledsoe SB, Smith SB et al. Calcium oxalate crystal localization 38. Teigler DJ, Arnott HJ. Crystal development in the Malpighian tubules of and osteopontin immunostaining in genetic hypercalciuric stone-forming Bombyx mori (L.). Tissue Cell 1972; 4: 173–185. rats. Kidney Int 2004; 65: 154–161. 39. Boll S, Schmitt T, Burschka C et al. Calcium tartrate crystals in the midgut 10. Tsai CH, Chen YC, Chen LD et al. A traditional Chinese herbal antilithic of the grape leafhopper. J Chem Ecol 2005; 31: 2847–2856. formula, Wulingsan, effectively prevents the renal deposition of calcium 40. Waku Y, Sumimoto K. Metamorphosis of midgut epithelial cells in the oxalate crystal in ethylene glycol-fed rats. Urol Res 2008; 36: 17–24. silkworm (Bombyx Mori L.) with special regard to the calcium salt 11. Tsai CH, Pan TC, Lai MT et al. Prophylaxis of experimentally induced deposits in the cytoplasm I light microscopy. Tissue Cell 1971; 3: 127–136. calcium oxalate nephrolithiasis in rats by Zhulingtang, a traditional 41. Singer MA. Dietary protein-induced changes in excretory function: a Chinese herbal formula. Urol Int 2009; 82: 464–471. general animal design feature. Comp Biochem Physiol B Biochem Mol Biol 12. Khan SR, Glenton PA, Byer KJ. Modeling of hyperoxaluric calcium oxalate 2003; 136: 785–801. nephrolithiasis: experimental induction of hyperoxaluria by hydroxy-L- 42. Dube K, McDonald DG, O’Donnell MJ. Calcium transport by isolated proline. Kidney Int 2006; 70: 914–923. anterior and posterior Malpighian tubules of Drosophila melanogaster: 13. Poonkuzhali B, Saraswathi CP, Rajalakshmi K. Effect of uric acid on sodium roles of sequestration and secretion. J Insect Physiol 2000; 46: 1449–1460. oxalate-induced urolithiasis in rats–biochemical and histological 43. Puschner B, Poppenga RH, Lowenstine LJ et al. Assessment of melamine evidences. Indian J Exp Biol 1994; 32: 20–24. and cyanuric acid toxicity in cats. J Vet Diagn Invest 2007; 19: 616–624. 14. Dionne MS, Schneider DS. Models of infectious diseases in the fruit fly 44. Thompson ME, Lewin-Smith MR, Kalasinsky VF et al. Characterization of Drosophila melanogaster. Dis Model Mech 2008; 1: 43–49. melamine-containing and calcium oxalate crystals in three dogs with 15. Singh SR, Hou SX. Lessons learned about adult kidney stem cells from the suspected pet food-induced nephrotoxicosis. Vet Pathol 2008; 45: Malpighian tubules of Drosophila. J Am Soc Nephrol 2008; 19: 660–666. 417–426. 16. Jung AC, Denholm B, Skaer H et al. Renal tubule development in 45. Dobson RL, Motlagh S, Quijano M et al. Identification and characterization Drosophila: a closer look at the cellular level. J Am Soc Nephrol 2005; 16: of toxicity of contaminants in pet food leading to an outbreak of renal 322–328. toxicity in cats and dogs. Toxicol Sci 2008; 106: 251–262. 17. MacPherson MR, Pollock VP, Broderick KE et al. Model organisms: 46. Butterweck V, Khan SR. Herbal medicines in the management of urolithiasis: new insights into ion channel and transporter function. L-type calcium alternative or complementary? Planta Med 2009; 75: 1095–1103. channels regulate epithelial fluid transport in Drosophila melanogaster. 47. Stechman MJ, Loh NY, Thakker RV. Genetics of hypercalciuric Am J Physiol Cell Physiol 2001; 280: C394–C407. nephrolithiasis: renal stone disease. Ann NY Acad Sci 2007; 1116: 461–484. 18. Dow JA, Davies SA. The Malpighian tubule: rapid insights from post- 48. Khan SR, Canales BK. Genetic basis of renal cellular dysfunction and the genomic biology. J Insect Physiol 2006; 52: 365–378. formation of kidney stones. Urol Res 2009; 37: 169–180. 19. O’Donnell MJ, Maddrell SH. Fluid reabsorption and ion transport by the 49. Dow JA. Insights into the Malpighian tubule from functional genomics. lower Malpighian tubules of adult female Drosophila. J Exp Biol 1995; 198: J Exp Biol 2009; 212: 435–445. 1647–1653. 50. Sayer JA. The genetics of nephrolithiasis. Nephron Exp Nephrol 2008; 110: 20. O’Donnell MJ, Ianowski JP, Linton SM et al. Inorganic and organic anion e37–e43. transport by insect renal epithelia. Biochim Biophys Acta 2003; 1618: 51. Devuyst O, Pirson Y. Genetics of hypercalciuric stone forming diseases. 194–206. Kidney Int 2007; 72: 1065–1072. 21. Beyenbach KW. Transport mechanisms of diuresis in Malpighian tubules 52. Klausner JS, Osborne CA, Griffith DP. Canine struvite urolithiasis. Am J of insects. J Exp Biol 2003; 206: 3845–3856. Pathol 1981; 102: 457–458.

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