Zebrafish (Danio rerio): A potential model for nephroprotective drug screening

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Review Article

Zebrafish (Danio rerio): A potential model for nephroprotective drug screening Pallavi Sharma, Supriya Sharma, Vikram Patial, Damanpreet Singh*, Yogendra Shantaram Padwad Regulatory Research Centre, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India

article info

abstract

Article history:

Zebrafish (Danio rerio) has emerged as a potential vertebrate model for high throughput

Received 19 June 2014

screening in drug discovery and development process. Easy breeding, short maturation

Accepted 8 November 2014

time, transparent embryos for easy observation, good regeneration capability, well char-

Available online xxx

acterization of developmental stages, low maintenance cost, high productivity, less ethical constrains, etc. are a few unique characteristics which make zebrafish an attractive model

Keywords:

for biomedical research. Apart from these, zebrafish possesses many anatomical, physio-

Embryo

logical and genetical similarities with higher mammals. Many pathological disorders have

Nephrotoxicity

been successfully studied in zebrafish like cancer, cardiovascular, renal diseases, immu-

Pronephros

nological, hematological disorders, etc. The pronephros of zebrafish imitates mammalian

Renal

kidney at structural, functional and cellular level and thus allows informative nephro-

Zebrafish

logical research. The present review highlights the use of zebrafish as a model to screen nephroprotective molecules, to enable better understanding of nephrotoxicity and thus to target new therapies. Copyright © 2014, Reed Elsevier India Pvt. Ltd. All rights reserved.

1.

Introduction

Zebrafish (Danio rerio), is a fresh water vertebrate belonging to family Cyprinidae. It is native of Himalayan region and is commonly found in India, Pakistan, Nepal, Bhutan and other parts of Southeast Asia. It is about 3e5 cm long, and derives its name from the longitudinal pigmented stripes (resembling zebra stripes) throughout its body. The short maturation time, easy breeding, good regeneration capability, well characterization of developmental stages, low maintenance cost, high

productivity, less ethical constraints, etc. makes the zebrafish an ideal tool for drug development, toxicity testing and high throughput screening of lead molecules.1,2 Furthermore, due to transparency in its embryo, they have been extensively used to study genetic manipulations.3e5 Zebrafish is becoming an emerging animal model, as in vivo drug discovery assays can be performed easily on their embryonic and larval stages in multi well plates economically.6e9 Zebrafish has been used since early 1970s as an experimental model for studying vertebrate development, developmental biology and genetic diseases.10,11 Thereafter extensive experimental studies have

* Corresponding author. Tel.: þ91 9417923132. E-mail addresses: [email protected], [email protected] (D. Singh). http://dx.doi.org/10.1016/j.cqn.2014.11.002 2211-9477/Copyright © 2014, Reed Elsevier India Pvt. Ltd. All rights reserved.

Please cite this article in press as: Sharma P, et al., Zebrafish (Danio rerio): A potential model for nephroprotective drug screening, Clinical Queries: Nephrology (2014), http://dx.doi.org/10.1016/j.cqn.2014.11.002

29 49

53 55 65 66 63

hpf: hours post fertilization.

Larvae (0e96 hpf) Larvae (0e96 hpf) 6. 7.

Gentamycin Cisplatin

2.3 nle9.2 nl 4.6 nl

Malformed kidney, decreased gfr Curved and cystic kidney, altered cell morphology and cell death Glomerular and tubular lesions, decreased renal function Disorganized arrangement of renal cells, decrease in dextran clearance Gut abnormalities, Malformation of pronephros, edema in pericardial sac, Reduced tactile sensitivity Decreased gfr, tubular obstruction Decreased renal function 1e10 ppm 0e45 mM 50 mM 200 mM 1e2000 ppm Aristolochic acid Acetaaminophen Citrinin Patulin Sodium benzoate Embryo (0e31 hpf) Embryo (12e60 hpf) Embryo (0e96 hpf) Embryo (0e96 hpf) Larvae (0e144 hpf) 1. 2. 3. 4. 5

Inference Concentration administered Toxicant Developmental stage

Table 1 e Renotoxic compounds studied on various stages of zebrafish.

been carried out on zebrafish since 1980s, focusing mainly on its developmental stages, rendering it as a useful model for pharmacological research.12,13 It has been found that 70% of human disease gene homologs are present in zebrafish, which makes it an ideal model imitating human pathological condition.14 It has also been emerged as an important tool in cancer research. BRAF gene melanoma model of zebrafish was first generated by Patton et al15 involving insertion of transgenic human BRAF gene to zebrafish embryo that resulted in its expression up regulation, and then screened for formation of skin lesions called “Nevi” that could form melanoma. Zebrafish also shares human innate and cellular immunity components that make it essential for understanding differential immune responses.16 Many studies have been carried out on zebrafish to explore host pathogen interaction using Enterococcus faecalis (responsible for nosocomial infections)17 and Mycobacterium marinum (responsible for causing tuberculosis in fishes and granulomas in humans).18 Zebrafish has also developed as an ideal experimental model for hematological disorders like, hypo chromic anemia (mutant has been cloned),19,20 acute lymphoblastic leukemia like c-myc model21 and T-ALL (T-Acute Lymphoblastic Leukemia) model by over expression of NOTCH-1 in zebrafish embryos.22 Zebrafish model has been developed as an useful tool for cardiovascular diseases like, dilated cardiomyopathies,23 myocardial infarction,24 arrhythmia, etc.25 The important property of zebrafish i.e. good regeneration capability, make it as a valuable model to study injury and repair processes.26 Nephropathy is any kind of damage to the kidney that affects its normal physiology. Long term use of therapeutic agents like, analgesics, chemotherapeutics, industrial waste, pollutants, heavy metals, and diseases like, diabetes mellitus and hypertension are the main causes of nephropathy.27,28 Nephropathy results in induction of several renal complications and currently remained as a major health problem worldwide. Continuous research is in progress to develop renoprotective molecules for better disease outcome. The process involves screening of large number of molecules in preclinical studies to select the most efficacious and safest molecule for future clinical testing and use. Presently rodent model is used as a gold standard for screening in preclinical testing. But still there is a continuous interest in the development of better, reproducible and cheap animal models of nephropathy for high throughput screening of reno-targeted lead molecules, disease pathogenic studies, etc. Zebrafish remained as an ideal model for renal diseases and has been used as a model for polycystic kidney disease (PKD), nephronophthisis, acute kidney injury (AKI), ciliopathies and many more. Several agents have been reported to induce renal toxicity in zebrafish enabling it to be potential model of different types of renal diseases (Table 1). These agent interact several cellular pathways to induce renal damage in zebrafish (Fig. 2). Currently it has been used as a potential model to screen nephroprotective components.29,30 In several studies it has been utilized as an animal model to screen nephroprotective components. Treatment with fullerene nanoparticles DF-1 (having antioxidant properties), 3 h pre and 15 min post ionizing radiations exposure prevented renal damage in zebrafish embryo.31 Similarly, restoration of renal system both physiologically and morphologically was also observed by Rapamycin (2e100 nM) and

Reference

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S no.

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Please cite this article in press as: Sharma P, et al., Zebrafish (Danio rerio): A potential model for nephroprotective drug screening, Clinical Queries: Nephrology (2014), http://dx.doi.org/10.1016/j.cqn.2014.11.002

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Fig. 1 e (A): Zebrafish matured larval pronephric kidney (B) Functional regions in zebrafish pronephric nephron.

Roscovitin (5e100 Мm), when added to morpholino oligonucleotides treated zebrafish in water at 24 hpf. Hence, making zebrafish a potential model for screening molecules against ciliopathic renal disease.32 Adult zebrafish when exposed to Listeria monocytogens, showed colony forming bacteria in zebrafish kidney, interestingly the formation was prevented by zinc treatment (0.25 mg/L). The study indicated the potential of zebrafish as a screening model for antimicrobial compounds for renal infection.33 The present review highlights the use of zebrafish in renal development and toxicity to enable better

Fig. 2 e Proposed mechanism of renal failure by inducers. ROS: Reactive oxygen species; TNFa: Tumor necrosis factor alpha, ILb: interleukin 1b and COX; Cyclooxygenase.

understanding of this model for nephrotoxicity studies, and thus to target new therapies.

2.

Renal system in zebrafish

The zebrafish renal system has evolved as a potential model to study the development and functions of mammalian kidney due to anatomical and physiological similarities among them. There are three stages of kidney development in vertebrates that include pronephric (most basic and develops from intermediate mesoderm), mesonephric (main excretory organ in aquatic vertebrates and temporary in reptiles, birds and mammals) and metanephric (permanent kidney in birds, reptiles and mammals, developed after 10th week in human embryos from the inferior part of wolffian duct and replaces mesonephros). Among these, pronephros and mesonephros are well developed in zebrafish. The pronephros is the first stage of kidney development and functions as an excretory organ for the first 30 days of larval development, which subsequently provide support to the nephrons to form mesonephros. Nephrogenesis is well exhibited by mesonephros in adult zebrafish to form new nephrons from renal progenitor cells during growth and injury.34e36 The pronephros stage consists of two nephrons, a fused glomeruli (present at the embryo midline) and pronephric tubules (connected directly to the glomeruli through a neck segment). There is a pair of bilateral pronephric duct which transfers the blood filtrate outside the body of zebrafish.37e39 The tubular epithelium of

Please cite this article in press as: Sharma P, et al., Zebrafish (Danio rerio): A potential model for nephroprotective drug screening, Clinical Queries: Nephrology (2014), http://dx.doi.org/10.1016/j.cqn.2014.11.002

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zebrafish imitate mammalian kidney and consists of two proximal tubular segments (proximal convoluted tubule and proximal straight tubule) and two distal tubular segments (distal early and distal late) that structurally mimics mammalian kidney (Fig. 1A and B). Furthermore, the pronephros exhibits a well developed brush border epithelium and extensive columnar epithelial cells. Moreover, the cells typical of mammalian kidney such as fenestrated capillary endothelial cells, podocytes and polarized tubular epithelial cells are also present in glomerulus of zebrafish.39e42 The pronephric nephrons perform the vital renal functions like, ultrafiltration, tubular reabsorption and fluid excretion along with osmoregulation.43 The majority of anatomical and physiological renal features of zebrafish mimic currently used rodent animal model, indicating it to be a potential replacement due to several shortcomings of later in contrast to former. Renal system of rodents consists of paired kidneys. The basic structural and functional unit is a nephron having similarity with zebrafish nephron. The first part consists of glomerulus and Bowman's capsule which is attached to proximal convulated tubule, loop of Henle (ascending than descending loop of Henle) and distal convoluted tubules, as seen in zebrafish nephron (Fig. 1A and B). The structure of thick descending loop of Henle is very much similar to proximal convulated tubule while thin descending limb, thin ascending limb and thick ascending limb of loop of Henle is similar to distal convulated tubule.44 Kidney development in zebrafish can be studied in a short span of time as this whole process occurs during the first two days of embryonic development, whereas it takes a long duration in currently used rodent model. Pronephric duct is formed first by the conversion of fibroblastic cells into a polarized epithelial tube, followed by, nephron formation and ultimately glomerulus is formed.41,43 In addition to these structural and physiological similarities, several genetic similarities that are crucial for development of kidney in higher vertebrates are also expressed in zebrafish during pronephric development.45e47 Casper, a new strain of zebrafish, has been recently developed with adult bodies having transparent skin, which allow better visibility.48e50 Hence, the structural and physiological similarity to the mammalian system and the ease of genetic manipulation makes zebrafish an important tool for nephrological research.

3. Nephrotoxicity studies in zebrafish embryo Rats and mice are the most commonly used experimental animal models, but the use of these models face a lot of shortcomings, more importantly the position of renal structures, as they are not accessible easily. Moreover, only a small portion of renal tubules and vesicles is observable in the vicinity of the kidney surface. But however, zebrafish offers several experimental advantages over currently used rodent model because of its anatomical simplicity and transparent embryos facilitating microscopic observation along the whole length of kidney. Thus it has emerged a potential model to study renal system.51,52 Zebrafish embryos

have been utilized to develop as a screening model to study nephrotoxicity.

3.1.

Drug-induced nephrotoxicity

3.1.1.

Aristolochic acid-induced nephrotoxicity

Aristolochic acid (AA; nitrophenanthrene carboxylic acid), commonly used for the treatment of arthritis, gout and festering wounds has been applied to induce experimental nephrotoxicity in zebrafish embryo.53 The study was performed according to six experimental protocols [methods based on different combinations of drug exposure onset ranging from 18 to 24 h post fertilization (hpf) and at 3, 5 and 7 h durations]. The embryos were treated with AA (1 and 10 ppm solution). The results showed reduction in survival rates and defective kidney phenotypes such as curved kidney, cystic pronephric tubes and ducts, and resulted in increased atrophic glomeruli with increased exposure time (72 hpf) and AA concentration (10 ppm). Reduced glomerular filtration rate and accumulation of red blood cells in kidney was also observed after AA treatment, which ultimately resulted in cessation of blood circulation and embryonic death. The qRTPCR studies showed increased expression of proinflammatory genes like, cox2, mpo, and TNF-a that has been suggested to induce renal damage and accumulation of blood cells in renal glomerulus and tubules resulting in poor blood circulation. In situ hybridization experiment using wt1b and cmlc2 (cardiac myosin light chain 2) as riboprobes, indicated that renal system is more sensitive to AA in contrast to heart. The teratogenic effect of AA on the late stages of zebrafish embryonic development was also observed, whereas no such effects were seen in the early stages. This model can be utilized for studying renal and circulation abnormalities induced by inflammatory components. AA has also been reported to induce renal damage in rodent model. In salt-depleted Wistar rats it induced glycosuria, proteinuria and elevated serum creatinine levels. Histological studies showed tubular necrosis and tubular atrophy associated with interstitial fibrosis.54

3.1.2.

NSAIDs-induced nephrotoxicity

The zebrafish model has also been implicated to study the renotoxic effects of NSAIDs. In another study, the toxic effects of acetaminophen (AAP) were studied on green fluorescent kidney line Tg (wt1b: GFP) of zebrafish.55 The study was conducted according to nine experimental protocols [exposure methods based on drug exposure at different developmental stages (12e60 hpf) at different concentrations of AAP (0e45 mM) for different timelines (12e60 h)]. AAP treatment showed the disorganized mesenchymal cells and disrupted morphology of glomerular cells, when the sections from pronephros region (8e10 mm) were stained with hematoxylin and monoclonal antibody a6F. Furthermore, the TUNNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay showed mesenchymal cell death, suggesting activation of apoptotic pathways. The malformed kidney phenotypes increased with increase in exposure time (48e72 hpf) and dose (22.5e45 mM). It was observed that at same concentration (22.5 mM) and contact time (12 h), higher percentage of severe defects were observed at early developmental stages (12e24 hpf), whereas higher percentage of mild defects were

Please cite this article in press as: Sharma P, et al., Zebrafish (Danio rerio): A potential model for nephroprotective drug screening, Clinical Queries: Nephrology (2014), http://dx.doi.org/10.1016/j.cqn.2014.11.002

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observed at late developmental stages (60e72 hpf). The qRTPCR studies showed decrease in expression of cox2 and bcl2 and increase in p53 expression. The study revealed that reactive metabolite formed by AAP metabolism is involved in the activation of apoptotic and inflammatory pathways regulated by bcl-xL, bcl2, cox2 and p53. The assay can be used in future to screen novel compounds intended for drug-induced renal toxicity. AAP has also been reported to induce renal toxicity in rodent models. AAP at a dose of 600e800 mg/kg in male mice resulted in renal tubular necrosis.56 Similar renotoxic effects were observed in rats at 2.5 g/kg dose.57

3.1.3. Antibiotics and chemotherapeutics-induced nephrotoxicity The zebrafish model has also been implicated to study the nephrotoxic effects of antibiotics. Gentamycin and cisplatin have been reported to induce acute renal failure in zebrafish embryo49 Gentamicin (10 mg/mL) and cisplatin (1.5 mg/mL) were injected into the cardiac venous sinus of zebrafish embryo (50e55 h post fertilization) using a nanojet II injection device. The histological and functional changes in renal structure similar to that observed in mammalian model were observed. The histological examination of gentamycin treated embryos showed lysosomal phospholipidosis, loss of brush border epithelium, presence of debris in tubular lumen, distention of glomerular and tubular lumen. Similarly, cisplatin treatment resulted in cellular vacuolization and decrease in cell height of proximal pronephric tubule. Moreover, loss of ability to maintain fluid homeostasis, tubular obstruction and reduced glomerular filtration rate was observed. The study supported the use of zebrafish as a model for acute renal failure induced by antibiotics and chemotherapeutic agents. Several studies have proved that gentamycin is renotoxic in rodent models. When tested in rats it resulted in decreased glutathione level along with reduced activity of superoxide dismutase and glutathione peroxidase. Tubular necrosis and glomerular congestion were also observed with gentamycin treatment (100 mg/kg).58 Furthermore, cisplatin and gentamicin have been reported to cause tubular degeneration, necrosis, inflammation and accumulation of protein casts in the tubular lumen in rats along with swollen glomerulus and reduced bowmen space.59,60 Similarly, histological examination of gentamycin treated zebrafish embryos showed lysosomal phospholipidosis, loss of brush border epithelium, presence of debris in tubular lumen, distention of glomerular and tubular lumen. Cisplatin causes cellular vacuolization and decrease in cell height of proximal pronephric tubule. Moreover, loss of ability to maintain fluid homeostasis, tubular obstruction and a decline in glomerular filtration rate was observed. These studies confirmed zebrafish as a good alternative model than rodents for studying acute renal failure.

3.2.

Benzo(a)pyrene-induced nephrotoxicity

Lo et al studied the effect of benzo(a)pyrene on renal development in zebrafish embryo.61 Benzo(a)pyrene is produced during the manufacturing of fried, roasted and smoked foods. The embryos of transgenic zebrafish Tg(wt1b: GFP) were treated with different concentrations of bezo(a)pyrene at

5

different concentrations (2, 20 and 200 ppb) and the effects observed were compared to that of healthy untreated embryos. However, no major effects were observed on survival rates, but the diverse malformations like, enlarged glomerulus, partial development of pronephric ducts and bending of pronephric tubes were observed in the developing kidney. It is evident from this experiment that an exposure dose of 2e200 ppb of benzo(a)pyerene is suitable for studying renal defects in zebrafish. Benzo(a)pyerene-induced renal toxicity is a well established rodent model used to screen nephroprotective components. The compound shows altered renal histology and biochemical impairments in rodents.62

3.3.

Preservatives-induced nephrotoxicity

Sodium benzoate, a commonly used preservative and antimicrobial agent that has been reported to induce nephrotoxicity in zebrafish embroys.63 At low concentration (1e1000 ppm), 100% survival rate was observed in zebrafish embryo, which was found to be decreased with increased concentration of sodium benzoate (1100e1900 ppm). There was complete embryonic mortality at a concentration of 2000 ppm or more. However time-dependent decrease in survival rate of zebrafish was observed at 1000 ppm. Complete mortality was observed at 144 h of exposure. The treatment resulted in misalignment of muscle fibers, increased cholinergic receptors, motor neuron innervations, deformities in pronephros and defective formation of pronephric tubes. A part from renal abnormalities, sodium benzoate treatment resulted in dose-dependent gut abnormalities, edema in pericardial sac and imperfect hatching gland. Furthermore, sodium benzoate treated embryos exhibited reduced tactile sensitivity to touch induced movement at early larval stages assessed by motor function test. In the test a tactile stimulation of 30 stimuli within 5 s interval was imposed to zebrafish embryo and response to swam out of a given area was quantified. Thus, the study favors the use of zebrafish as an ideal model for studying the nephrotoxic effects of food and drug excipients. When tested in rats, sodium benzoate at a dose of 200 mg/kg causes alteration in renal functions with marked elevation in serum urea, uric acid and creatinine levels.64

3.4.

Fungal metabolites-induced nephrotoxicity

Wu et al tested citrinin (CTN) and patulin (PAT) for their nephrotoxic effect on zebrafish embryonic kidney.65 CTN and PAT are secondary fungal metabolites usually found in food and reported to cause organotoxicity in mature animals. In the study toxic effects and LC 50 (Lethal concentration 50) values were determined. Both, CTN and PAT treatment did not show any phenotypic defect in the renal morphology. However, the histological studies of CTN (15 mM) treated embryos (3 day old) showed cystic glomeruli and disorganized pronephric tubules, indicating developmental renal toxicity. Moreover, cyst formation in both glomeruli and ducts was observed at higher concentration of CTN (50 mM). Earlier studies reported that citrinin causes enlargement of Bowmen's capsule, degeneration of gomeruli and renal tubules and necrosis of renal tubular epithelium in mice, more or less similar to the changes observed in the zebrafish due to citrinin

Please cite this article in press as: Sharma P, et al., Zebrafish (Danio rerio): A potential model for nephroprotective drug screening, Clinical Queries: Nephrology (2014), http://dx.doi.org/10.1016/j.cqn.2014.11.002

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toxicity.66 In addition, PAT treatment showed bungling arrangement of renal cells. Dextran clearance ability of embryos was also found to be reduced, which was prevented by pentoxifylline treatment (competitive non selective phosphodiesterase inhibitor that improves blood flow by lowering blood viscosity), indicating reduction in the glomerular blood flow. CTN treatment caused an upregulation in gene expression of cox2 and TNF-a, but no effect was observed on IL-1b and IL-6 expression, indicating the involvement of prostaglandin and other inflammatory pathways in CTN-induced renal toxicity. The alteration in blood circulation and inflammatory pathways can be a putative mechanism underlying the observed renal damaging effects. The study supported zebrafish model to screen renoprotective components acting through hemodynamic mechanisms.

4.

Nephrotoxicity in adult zebrafish

Apart from embryo, adult zebrafish provides an additional tool to understand development of nephrons, nephrotoxicity and drug-induced renal complications. As discussed earlier, the nephron of adult zebrafish is almost identical to one present in embryonic kidneys except for presence of collecting ducts and branching.67 The final adult kidney of zebrafish is formed after mesonephros and it completely covers the pronephros.68,69 Currently adult zebrafish is used to study drug withdrawal effects,70 along with the studying drug efficacy and toxicity. The total dependence on drugs consumed over a certain period results in development of group of symptoms, mainly known as drug withdrawal effects. Many studies have been carried out to understand the effect of drug withdrawal in zebrafish model. Lopez-Patino et al reported the withdrawal effect of cocaine on zebrafish and observed anxiety and behavioral changes after withdrawal of drug. Similar studies have been carried out to study the adverse effects of NSAIDs withdrawal on renal system.71 In adult zebrafish, the main function of kidney is clearance of water due to which it is highly exposed to circulating drugs, which causes damage to liver and kidney both. The uniqueness of adult zebrafish renal system is its nephron regeneration ability also called neonephrogenesis.72 Diep et al used gentamicin as a nephrotoxicant in adult zebrafish to induce nephron damage to study neo-nephrogenesis.29 As zebrafish lack bone marrow, all major blood cell types and stem cells are produced in kidney. Therefore, whole kidney marrow (WKM) cells when isolated

from transgenic line of zebrafish and injected into donors, result in generation of new nephrons in donors after 18 days post transplantation. The study suggested that the progenitors in zebrafish have capability to form new nephrons after AKI and transplantation. Further studies are required on zebrafish to warrant the use of new regenerative therapies as a screening model prior to their clinical use in AKI, ciliopathy and PKD. Since, zebrafish shares homology with mammalian immune system and blood lineages, hence it is currently being used to study innate and adaptive immune responses. As described, unlike mammalians zebrafish lacks bone marrow for hematopoietic site. Its hematopoietic progenitor cells are developed in kidney marrow and then get differentiated into respective cell types. Transplantation experiments were carried out on adult zebrafish, where marrow cells were transplanted after g-irradiation.73,74 The study involved exposure of g-irradiation (978 cGy/min) to adult zebrafish and the irradiated adults were then transplanted with hematopoietic cells from kidney marrow. It resulted in increased count of hematopoietic cells in transplanted irradiated zebrafish. Therefore, it makes zebrafish a model organism to study decrease in immune response due to radiotherapy in cancer. A transplantation experiment has been done on zebrafish to allow deep tissue imaging and for analyzing stem cell and tumor biology.50

5. Genetic screening in zebrafish renal system Zebrafish embryo has been successfully used in several genetic screening techniques such as, morpholino or gripNA technology, microRNA injections and proteinuria detection. These are combined products of nucleic acid base and morpholine ring and as antisense synthetic short oligonucleotides, used for gene knockdown by inhibiting translation.75 The bases are antisense and due to presence of morpholine, enzyme degradation of these compounds is inhibited.76 In zebrafish morpholinos are used to study the restoration of function of genes involved in fin regeneration,77,78 renal edema by injecting morpholinos or mRNA in to zebrafish larvae for phenotypic analysis. The transgenic lines of zebrafish Tg(wt1b: EGFP) were used to study the glomerular filteration in diabetes mellitus which affect the kidney function in later stages of the disease, hence can be a potential model to screen molecules intended

Table 2 e Accounting genes in zebrafish screened for nephropathies. S.No.

Gene

Renal abnormalities

Disease

Stage

Reference

1 2 3 4 5 6 7

pkd1a/pkd1b inversin pkd2/curly up nphp3 nphp5 cep290 lhx1

Polycystic kidney disease (PKD) Nephronopthisis (NPHP) Polycystic kidney disease (PKD) Nephronopthisis (NPHP) Nephronopthisis (NPHP) Nephronopthisis (NPHP) Pronephric cysts

Embryo Embryo Embryo Embryo Embryo Embryo Embryo

95 85,86 95e99 72,85,86 87 88 100

8

pax2

Pronephros cysts and curved dorsal surface axis Curved ventral surface axis and pronephros cysts Pronephros cysts and curved dorsal surface axis Curved ventral surface axis and pronephros cysts Curved axis and pronephros cysts Curved ventral surface axis and pronephros cysts Enlargement of kidney, Malformed or absence of pronephric tubule Malformed or absence of pronephric tubule

Pronephric cysts

Embryo

101

Please cite this article in press as: Sharma P, et al., Zebrafish (Danio rerio): A potential model for nephroprotective drug screening, Clinical Queries: Nephrology (2014), http://dx.doi.org/10.1016/j.cqn.2014.11.002

c l i n i c a l q u e r i e s : n e p h r o l o g y x x x ( 2 0 1 4 ) 1 e9

for use in diabetic nephropathy. Mutation of genes in zebrafish proved to be helpful in understanding many diseases conditions as mutation in wt1 gene can lead to formation of Wilms' tumor, incongruity in development of kidney, etc.79e83 Similarly, Sun et al identified the mutation in 12 cystic kidney genes that are responsible for cyst formation in zebrafish.84 Several studies identified the specific genes responsible for the formation of cysts in medulla termed as “nephronopthisis”, curving of ventricular axis and loss of cilary formation (Table 2).72,85e89 Thus, it has been found that genes responsible for cilia formation and functioning in zebrafish can be promising candidates for studying PKD to screen protective molecules. Zebrafish has emerged as a suitable model for analysis of genetic disorders90 due to easy mutagenesis, gene disruption and replacement techniques, apoptosis, etc.91e93 Since apoptosis is one of the major inducer of renal damage and several protective molecules act by interacting on one or more site(s) in the pathways. Apoptosis can be easily detected in zebrafish embryo due to their transparency and easy labeling techniques.

6.

Limitations with the use of zebrafish

In spite of several advantages zebrafish still is not being used as a reliable source for confirmation studies in renoprotective drug research. This is due to its distant relationship to mammalian phylogeny, presence of double replica of mammalian genes, as a result of gene duplication and lack of drug metabolism studies. Although, it is nowadays being used for initial screening of drugs, further validation is necessary on higher mammals. Another disadvantage includes absence of well developed organs like lungs, mammary glands, pancreas etc. in zebrafish leading to its limited use in biomedical research.94

7.

Conclusion

Zebrafish emerged as an important experimental model for drug testing and have been implicated in various drug discovery processes for toxicity evaluation and high throughput screening. Zebrafish remained as an ideal model for renal diseases and has been used as a model for PKD, nephronophthisis, AKI, ciliopathies and many more. The renal system of zebrafish physiologically, anatomically and genetically mimics mammalian renal system. Zebrafish has been employed to study drug, food additives and microbial metabolites-induced nephrotoxicity. Zebrafish has shown several advantages over gold standard rodent model in terms of reproducibility, cost, ethical constraints, time span, etc. However few shortcomings have also been observed with the use of zebrafish as an experimental model like its distant relationship to mammalian phylogeny and presence of double replica of mammalian genes. It is expected that further experimental studies will generate the interest of scientific community to use zebrafish for the study of development and function of kidney and renal drug discovery processes. This will further refine the use of zebrafish as an experimental model.

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Conflicts of interest All authors have none to declare.

Acknowledgment The authors are thankful to the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P.), India for providing necessary facilities. The IHBT publication number of the manuscript is 3678.

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