Methods To Generate And Evaluate Zebrafish Models Of Human Kidney Diseases Part 1

ABSTRACT 

Kidney-related disorders affect millions of people worldwide. A survey of chronic kidney disease (CKD) patients showed that the burden of kidney diseases is increasing every year. The global burden of disease (GBD) study 2017 ranked CKD as the 12th leading cause of death worldwide. Hence, the identification of the causes of kidney diseases, the development of accurate diagnostic methods, and novel therapeutics are highly relevant. Model organisms that faithfully recapitulate human diseases play important roles in understanding the disease process and provide valuable ground to find their cure. Zebrafish is an excellent model to study the development, pathophysiology, and molecular aspects of human kidney diseases. In this review, we summarize various genetic and experimental manipulations that can be carried out in zebrafish to better understand the pathophysiology of human kidney diseases. We suggest that these methods will be helpful in the development of potential therapies to treat kidney diseases. 

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David.deng

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KEYWORDS: CKD, AKI, a zebrafish model of human kidney diseases, pronephros, renal pathology

Introduction

The kidney is one of the vital organs in vertebrates that removes waste products and maintains pH, ion, and metabolite concentrations of blood within the physiological range. The kidney secretes erythropoietin that regulates red blood cell production and activates vitamin D which helps bones to absorb calcium. There are about a million nephrons in each human kidney, which are the structural and functional units of this organ. Consequently, defects in nephrons affect kidney structure and functions. Dysfunction of the kidney can occur because of genetic mutations, infections, injury, medicines, or exposure to toxic compounds in the environment. Chronic diseases like diabetes, cardiovascular diseases, and hypertension are major contributors to the kidney disease burden (Levey et al., 2010). Impaired kidney function increases the risk of complications in other organ systems as well (Thomas et al., 2008). The common types of kidney diseases and their causes are outlined in Table 1.

Kidney diseases

Kidney diseases contribute to a significant fraction of the disease burden globally. Around 750 million people worldwide are affected by kidney-related disorders (Crews et al., 2019). CKD is most common among them with a global prevalence of 13.4% (Hill et al.,2016). The GBD study 2010 ranked CKD as the 18th leading cause of death worldwide, which had jumped to 12th by 2017 for causing the maximum number of deaths globally (Jha et al., 2013; Carney, 2020). According to the GBD study in 2017, about 697.5 million cases of CKD were reported worldwide, among which 1.2 million people died. The number of deaths due to CKD is estimated to rise to 4 million by 2040 in the worst-case scenario (Foreman et al., 2018). One-third of CKD patients live in two countries, China and India (Bikbov et al., 2020). The status of CKD among the Indian population is unclear due to the lack of accurate data collection systems. About 115 million cases of CKD were reported in India in 2017 (Bikbov et al., 2020). The most common diseases in the Indian population are diabetes and hypertension (Geldsetzer et al., 2018). As per the Indian Council of Medical Research (ICMR) report, the prevalence of diabetes and hypertension among the urban adult population is 28% and 21.4% respectively (Varma, 2015). Between 40% to 60% of CKD, cases occur because of these complications and their number is increasing rapidly (Rajapurkar et al., 2012). The International Society of Nephrology’s kidney disease data center reported a 16.8% prevalence of CDK among the Indian population (Ene-Iordache et al., 2016). The causes of CKD vary throughout India. Andhra Pradesh, Odisha, and Goa reported high levels of CKD of an unknown etiology known as chronic interstitial nephropathy (Varughese and Abraham, 2018). As the number of CKD patients is increasing worldwide at an alarming rate, there is an urgent need to carry out a thorough analysis of the root causes of common kidney diseases to find better ways for their prevention and cure. Model organisms that can mimic human physiology and diseased conditions can provide great avenues for addressing the above problems. Zebrafish have become a useful model to study development and diseases and provide promising ways to identify new therapeutic targets and drugs. In this review, we have discussed various methods to recapitulate human kidney diseases in zebrafish and how these models can be used to understand disease pathogenicity and its underlying molecular mechanisms. This may result in the generation of novel therapeutic approaches for the management and cure of kidney diseases.

Similarities between human and zebrafish kidney

Kidney development in mammals is unique as it takes place through three distinct structures during embryogenesis: pronephros, mesonephros, and metanephros. The first two structures are transient in mammals and only the metanephros persists throughout life (Smyth et al., 2017; Jain, 2014). Pronephros is the functional kidney during embryonic development in lower vertebrates like amphibians and fishes, which is replaced by mesonephros as the functional kidney at later stages (Tahara et al., 1993; Diep et al., 2015). Table 2 describes the time duration of different forms of kidney in humans, mice, Xenopus, and zebrafish.

Although the overall complexity of the kidney increases as we move towards the higher forms, nephrons are the structural and functional units of all types of kidneys. Each nephron has three parts: renal corpuscle to filter blood, tubule to absorb and secrete solutes, and collecting duct to collect unwanted wastes for excretion. The tubular epithelium is patterned into different segments to carry out specific functions. Nephrons from all forms of kidney possess a similar segmentation pattern (Desgrange and Cereghini, 2015). The segmental organization of the nephron of zebrafish pronephros and mesonephros is similar to mammalian metanephros, as shown in Fig. 1. The zebrafish pronephric tubule is divided into proximal convoluted tubule (PCT), proximal straight tubule (PST), and distal early (DE) and distal late (DL) tubules, which are analogous to the segmentation pattern of mammalian metanephric nephrons. Zebrafish have an endocrine gland named corpuscles of Stannius (CS), which maintains calcium homeostasis (Krishnamurthy, 1976). The CS is formed by transdifferentiation of renal epithelial cells in between DE and DL segments, which gradually separate from the tubule and form the paired CS gland that is positioned retroperitoneally at the surface of the kidney in adult fish (Roberts and Ellis, 2012; Naylor et al., 2018). One of the major differences between zebrafish and mammalian nephrons is the lack of loop of Henle in zebrafish, which acts as a countercurrent multiplier to generate medullary osmotic gradient for water conservation. This segment does not have any utility in zebrafish as it is a freshwater fish (Elmonem et al., 2018). Each segment possesses distinct cell types and segment-specific gene expressions, which are conserved among vertebrates (Verlander, 1998; Desgrange and Cereghini, 2015). All kidney types follow similar pathways of development. The following four stages are successively involved in nephron development; A: induction of intermediate mesoderm to form renal primordium, B: epithelialization and growth of nephric duct, C: patterning of nephron into specialized segments, and D: vascularization of nephron for blood filtration (Drummond, 2003).

Methods to generate zebrafish models of human kidney diseases

Zebrafish possess one pair of pronephros which start to form after 12 hours post fertilization (hpf) and becomes fully functional by 48 hpf. Availability of whole genome sequence, 71% human genes having at least one zebrafish orthologue, ease of handling, transparent embryos, short generation time, efficient gene manipulation techniques, methods for transgenic fish generation and its rapid screening make zebrafish the most suitable model organism to study kidney abnormalities (Howe et al., 2013; Poureetezadi and Wingert, 2016). One of the crucial features of adult zebrafish is its ability to regenerate new nephrons by the process of neo-nephrogenesis in response to renal injury (Chambers and Wingert, 2016). Table 3 describes the comparisons between zebrafish and other common models organisms like mice and Xenopus. Genetic manipulations in zebrafish have shown that the novel genetic components of kidney development and function can be identified by using this model organism (Poureetezadi and Wingert, 2016). There are several methods to generate zebrafish models of human kidney diseases.

Forward genetics

The forward genetics screen is used to identify genes that are associated with phenotypes of interest (Lawson and Wolfe, 2011). Both chemical and insertional mutagenesis are used in forward genetics screens (Patton and Zon, 2001). Chemical mutagenesis is a three-generation-based screening in which adult males are treated with chemical mutagens such as ethyl methane sulphonate (EMS), ethyl nitrosourea (ENU), or physical mutagens like gamma radiation to generate several mutations in germ cells (Varshney and Burgess, 2014). The fish carrying mutations are then crossed with wild-type females to generate F1 fishes which contain unique alleles of generated mutation. F1 fishes are out-crossed to generate F2 carriers that are then in-crossed to obtain their homozygous mutants in F3 generation. The offspring having the desired phenotypes are then isolated and used for genetic mapping and sequencing to identify the mutated gene (Patton and Zon, 2001). Other variations of this screen, such as using haploid and homozygous diploid, can help to reduce the time and effort needed to identify the phenotype and the causative genetic mutation (discussed in detail by Patton and Zon, 2001). Transgenic reporter lines expressing fluorescent proteins in the organ of interest can be combined with conventional forward genetic screening to easily screen mutants of interest. An ENU-based mutagenesis screen led to the identification of the lightbulb (lib) mutant zebrafish which show morphological and nephron segmentation defects similar to retinoic acid (RA) deficient zebrafish embryos. It was found that lib mutants have C to A transition at nucleotide 174 of aldh1a2, which is predicted to synthesize a truncated protein of 58 amino acid length, thus abrogating aldh1a2 function and affecting the synthesis of RA from retinaldehyde. Analysis of lib, neckless (nls) mutant having point mutation in the catalytic domain of aldh1a2 and DEAB (retinoic acid synthesis inhibitor) treated embryos revealed that the levels of RA modulate nephron segmentation by changing the spatial expression of segments specific transcription factors (Wingert and Davidson, 2011; Begemann et al., 2001; Mullins et al., 1994). Another mutant called Zeppelin helped to identify brac2 as a regulator of podocyte development (Kroeger et al., 2017).

Insertional mutagenesis is a transposon or anti-virus-based method that is used to insert DNA randomly at different genomic loci, and this foreign DNA then helps to identify the mutated gene (Amsterdam et al., 1999). The Hopkins Laboratory at MIT performed an insertional mutagenesis screen in zebrafish embryos using a retroviral vector. They identified 12 genes that were associated with defective kidney phenotypes showing cysts in the glomerular tubular region, among which four were linked with cilia formation (Sun et al., 2004). The drawbacks of the forward genetics approach are that the screening process is cumbersome and time-consuming. However, the advances in new sequencing technology have made the forward genetics approach much more efficient.

Gene knock-out using programmable nucleases

One of the major advantages of using zebrafish as a model organism is the availability of efficient genome editing techniques such as CRISPR/Cas, TALEN, and ZFN (Sertori et al., 2016). CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/ Cas9 is the most versatile and commonly used genome editing technology in zebrafish. A chimera of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) is designed as a single guide RNA (sgRNA), which along with Cas9 protein/mRNA are injected into zebrafish embryos at the one-cell stage (Varshney et al., 2016). The sgRNA binds to the 20bp target DNA sequence adjacent to a protospacer adjacent motif (PAM) NGG sequence in the genome, Cas9 then generates a double-strand cut in the target DNA sequence. This is then preferably repaired by non-homologous end joining, which is an error-prone repair mechanism that results in the generation of random insertion/deletion (indel) leading to changes in the reading frame of the coding sequence, and consequently, abrogates the functions of the gene of interest. Advanced methodologies have been developed in recent years to increase the efficacy of the CRISPR-based genome editing technique (Liu et al., 2019). It is possible to delete a large fragment of DNA using two or more sgRNAs at a time (Kim and Zhang, 2020). It is also possible to simultaneously target multiple genes efficiently in a single zebrafish embryo (Shah et al., 2016). Many studies have been conducted to generate zebrafish mutants to recapitulate human kidney diseases by using CRISPR/Cas9 technology. CRISPR-mediated knockout of ciliary membrane protein Arl13b resulted in the generation of mutants that mimic the “Joubert syndrome” (Cantagrel et al., 2008). It is reported that mutations in the human ELMO1 gene contribute to diabetic nephropathy. elmo1 mutant zebrafish exhibit phenotypes seen in hyperglycaemic embryos generated by knockdown of the pdx1 gene such as larger glomerulus, defective podocyte, shorter neck, and hyperfiltration, thus highlighting the conserved function of this gene (Sharma et al., 2016). Mutant magi2a zebrafish exhibit steroid-resistant nephrotic syndrome which is also observed in people with mutations in MAGI2 (Jobst-Schwan et al., 2019). These observations highlight the suitability of zebrafish as an organism to model human kidney diseases.

Another method to generate zebrafish mutants is Transcription Activator-Like Effector Nucleases (TALEN) based on genome editing. In this technique, the DNA binding domain TALE is made up of monomers, each monomer consisting of tandem repeats of 34 amino acid residues that bind to a particular nucleotide in the DNA sequence. These TALE sequences are derived from the pathogenic bacteria Xanthomonas which alters the transcription of genes in host cells (Boch and Bonas, 2010). The TALEN targeting construct consists of a nuclear localization signal, the DNA binding domain, and the FokI nuclease domain at their carboxyl termini. TALEN work in pairs with their binding sites located on opposite strands of DNA separated by a 12 to 25 bp spacer sequence. The constructs bind to the target site in the nucleus and generate a double-strand break, which is then repaired by a non-homologous end joining (Liu et al., 2014). The mutant zebrafish generated by CRISPR or TALEN can activate genes that compensate for the loss of genes of interest, thus making it difficult to uncover the gene function (Rossi et al., 2015). However, it is possible to identify these compensatory genes by transcriptome analysis using next-generation sequencing (NGS) methods, which can help to identify the function of the gene of interest. Identification of genes and molecular pathways that compensate for the loss of function of a gene can help find novel treatment methods for gene mutations that cause serious diseases (El-Brolosy and Stainier, 2017).

Gene knock-down using morpholino antisense oligos

Morpholinos are non-anionic oligonucleotides that are relatively stable as compared to their DNA or RNA oligomers (Summerton, 1999). The morpholino antisense oligonucleotides bind to the mRNA of a target protein at its translation initiation site and block its translation. Another type of morpholino antisense oligos can target splicing junctions and block pre-mRNA splicing (Summerton, 1999). Morpholino-antisense oligo effect is transient and can be used to study the early developmental role of the genes of interest. Injection of morpholino-antisense oligo is done at the 1-4 cell stage of zebrafish embryos and its effect can be seen up to 3-5 days post fertilization (pdf) (Bill et al., 2009). Morpholino antisense oligonucleotides often display off-target effects (Robu et al., 2007). Control experiments such as a rescue experiment should be performed by injecting morpholino along with mRNA that cannot be targeted by this antisense oligo to verify its specificity (Eisen and Smith, 2008). A photo-activated morpholino has been developed, whose activity can be controlled spatially and temporally by UV exposure (Tallafuss et al., 2012). Numerous studies have been carried out using morpholino antisense oligo-mediated gene knock-down to understand human kidney diseases. For example, the knock-down of the nephrocystin-3 gene led to the formation of cysts and hydrocephaly in zebrafish embryos in a similar way to nephronophthisis type-3 disease (Zhou et al., 2010).

Aminoglycoside antibiotics generally used to treat many life-threatening infections are known for their nephrotoxic and ototoxic effect (Mingeot-Leclercq and Tulkens, 1999). Gentamicin is a commonly used antibiotic that can induce acute kidney injury (AKI) in zebrafish. Gentamicin causes flattening of the brush border epithelium, loss of tubular epithelium, deformation of glomerulus structure, lysosomal phospholipidosis, and accumulation of leukocytes or cell debris in the tubular lumen, which mimics gentamicin overdose in humans (Cianciolo Cosentino et al., 2010). Zebrafish can regenerate and replace damaged nephrons. Gentamicin insult can be used to study renal regeneration in zebrafish (Kamei et al., 2015). Gentamicin injury induces a regeneration response which triggers kidney stem cells to undergo the stages of specification, proliferation, and differentiation to generate new nephrons. It takes around 14 to 21 days in adult fish to regenerate nephrons (Diep et al., 2011; McCampbell et al., 2015). Cisplatin, which is used as a chemotherapeutics drug to treat tumors, also has nephrotoxic effects, as observed in zebrafish (Hentschel et al., 2005). Etimicin is another aminoglycoside that can be used while mimicking low nephrotoxicity and ototoxicity in zebrafish embryos (Shao et al., 2020). Thus, AKI and renal regeneration can be studied in zebrafish with the help of these antibiotics.

Mechanical injury to the kidney

The zebrafish pronephros can be physically injured using resection, stabbing, or cryoinjury. Surgical injury can be performed by using fine tweezers to stab the desired area of the pronephros to create an AKI model. This technique was used to damage the pronephric duct close to the cloaca, which impaired fluid flow and led to cyst formation within 30 minutes (Kramer-Zucker, 2005). It was found that the reduction in fluid flow rate in pronephros generates back pressure at the fluid entry site, causing tubule luminal expansion and cyst formation. Another group of researchers made obstruction in the pronephric tubule at 50 hpf or in mesonephric tubules of 12-month-old zebrafish by using tweezers to pinch off the area near the distal collecting tubules (Hellman et al., 2010). They discovered that tubule scarring leads to an increase in cilia beating rate and an upregulation of the foxj1a transcription factor that regulates lipogenic gene expression. This suggests that injury to pronephric tubules generates a cilia-based mechanosensory signal to maintain nephron homeostasis.

Laser-induced kidney injury

Laser-mediated cell ablation is used as a tool to study renal injury and mimic AKI in zebrafish. Johnson et al., (2011) have described a method for laser-mediated ablation followed by tracking of regenerating nephrons. They injected 40 kDa dextran-FITC in the trunk somites of zebrafish embryos at 48-55 hpf to label the proximal tubule epithelial cells, which were then targeted for laser-mediated ablation at 72 hpf. The ablated embryos were then reinjected with rhodamine dextran to trace the proximal tubule epithelial cells. They found that a fully developed proximal tubule was formed on the 7th day following laser-mediated ablation (Johnson et al., 2011). Another alternative is to use a kidney-specific transgenic line to identify cells for laser-mediated ablation and monitor the behaviors of neighboring cells. A violet laser light of 405 nm wavelength was used to target the pronephric tubule of Tg(atp1a1a.4:GFP) zebrafish (Palmyre et al., 2014). As GFP excitation spectra lie in the range of blue to violet light, it absorbed 405 nm laser light, which acts as an energy sink to potentially induce injury at the target epithelial cells. This experiment led to the discovery that cell migration is the primary response of injured epithelia. Collective cell migration caused mechanical stretch that provided stimuli for cell proliferation to repair the injured tubule. To determine the time when the pronephric tubule of zebrafish acquires the ability of regeneration, Yakulov et al., (2018) used a 2-photon laser to ablate a small part of pronephric tubules in Tg(cldn2b:lyn-GFP) embryos at different time points and followed the regeneration process. They found that the ablation of pronephric tubules of 2-day-old embryos was rapidly repaired by migratory responses, whereas 1-day-old embryos did not have this ability. They carried out gene expression profiling of injured zebrafish embryos and found that cxcr4b and mica are involved in this repair process (Yakulov et al., 2018).

Chemical genetics

Small bioactive molecules can be used to interfere with protein function and understand their biological role. A large number of chemical libraries are commercially available or can be custom-made for probing protein functions. These small molecule libraries include kinase inhibitors, protease inhibitors, nuclear receptors, and ligands that can be used to identify the role of signaling pathways involved in organ development and function (Kawasumi and Nghiem, 2007). In the last few decades, zebrafish have emerged as a powerful vertebrate model organism for high-throughput chemical screening and phenotypic scoring (Kaufman et al., 2009). Cao et al., (2009) used the chemical screen approach to identify compounds that can reverse phenotypes caused by mutations in pkd2 (causal gene of PKD) and ift172 (a gene responsible for cilia formation). They uncovered that a pan-histone deacetylase (pan-HDAC) inhibitor trichostatin A (TSA) and a class-I specific HDAC inhibitor valproic acid (VPA) can suppress kidney cyst formation in pkd2 knock-out model (Cao et al., 2009). Thus, chemical genetics can be a useful tool to identify new drug candidates that can either reverse or suppress disease conditions.

Genetically inducible kidney injury models

A genetically inducible tissue ablation model can be used in zebrafish in which bacterial nitroreductase (NTR) is expressed under the control of a promoter of choice that drives the expression of NTR in a particular segment of the pronephros. NTR converts metronidazole into a cytotoxic metabolite that can cause death to NTR-expressing cells (Curado et al., 2008). Zhou and Hildebrandt (2012) used this technique to induce injury of podocytes by expressing NTR under the control of podocin promoter in the Tg(pod: NTR-mCherry) zebrafish line. They also developed a double transgenic line of VDBP-GFP (Vitamin D binding protein tagged with GFP) as a tracer of proteinuria along with the Tg(pod: NTR-mCherry) line. Treatment with metronidazole caused the injury of the podocyte resulting in whole-body edema and accumulation of VDBP-GFP in proximal tubules, mimicking the phenotype of the human nephrotic syndrome (Zhou and Hildebrandt, 2012).

Methods to evaluate pronephros development and function in zebrafish

We have described the methods to generate human kidney disease models by using zebrafish. The creation of a disease model using another organism is the initial step, which needs to be evaluated for its ability to faithfully recapitulate various aspects of a disease seen in humans. Zebrafish offers many advantages that can be used to quickly evaluate its ability to serve as a surrogate to understand human kidney diseases as discussed below.

Morphology based screening

Most often mutant zebrafish embryos show morphological differences, compared with the wild-type that can be easily judged by observations under the microscope. The transparency of zebrafish embryos and their ability to survive up to 5 days even with severe developmental defects is a major advantage in morphology-based screens. Common morphological changes observed in zebrafish embryos with defective pronephros include pericardial edema, pronephric cysts, curved body axis, and hydrocephalus (Poureetezadi and Wingert, 2016; Outtandy et al., 2019). Edema is one of the common signs of a defective kidney which is also seen in other organ deficiencies such as heart development (Hanke et al., 2013). Mutants with kidney defects may develop cysts because of other ver-proliferation of epithelial cells, as can be easily observed under a microscope (Zhao and Malicki, 2007; Yamaguchi et al., 2006). A curved body axis is often seen in mutants having pronephric cilia defects. Mutant zebrafish such as Locke (lok), shen yantf214a(shy), garbustm304 (grb), and zatortg238a (zar) are some other examples showing curved body axis (Zhao and Malicki, 2007). Zebrafish mutants with kidney defects show multiple morphological defects in the same embryo. The knock-down of two polycystin genes pkd1a and pkd1b led to the orally curved body axis, hydrocephaly, cartilage, and craniofacial defects with low frequency of pronephric cysts (Mangos et al., 2010). The mutants of intraflagellar transport proteins ift57, ift88, and ift172, where cilia were defective, had ventrally curved body axis (Lunt et al., 2009). Thus, zebrafish mutants with defective kidneys exhibit many morphological features that can be easily identified.

For more info: david.deng@wecistanche.com  WhatApp:86 13632399501