Little fish, big catch: zebrafish as a model for kidney disease

Abstract

The zebrafish, Danio rerio, is a relevant vertebrate model for biomedical research and translational studies due to their broad genetic conservation with humans. In recent years, scientists have formulated a growing list of zebrafish kidney disease paradigms, whose study has contributed a multitude of insights into the basic biology of human conditions and even identified potential therapeutic agents. Conversely, there are also distinctive aspects of zebrafish biology lacking in higher vertebrates, such as the capacity to heal without lasting scar formation after tissue damage and the ability to generate nephrons throughout their lifespan, making the zebrafish uniquely suited to study regeneration in the context of the kidney. Here, we review several informative zebrafish models of kidney disease and discuss their future applications in nephrology.

Introduction

The zebrafish has become a premier, genetically tractable model organism for a diverse array of human disease studies over the past several decades¹. Central to their relevance and applicability for biological research is the marked genome conservation that exists between zebrafish and higher vertebrates, such that 71% of human genes have at least one zebrafish orthologue². Historically, zebrafish were used for environmental toxicology investigations, but eventually became appreciated as a powerhouse for identifying genes instrumental for development and disease^3,4. The appeal of the zebrafish model progressively led to the creation of a vast toolkit of methodologies for sophisticated cellular and molecular studies⁵. This tropical species of fish is ideal for experimental work due to a set of attributes which include, but are not limited to, their small adult size (~4–5 cm length from tip to tail) and ability to thrive in a lab environment, high fecundity characterized by the ability to reproduce weekly and spawn hundreds of embryos that develop ex utero, the optical transparency of the embryos during ontogeny, and the rapidity of organogenesis in which the assemblage of advanced structures like the brain, heart and liver occurs in the first week of life¹. Recent technological advances in transgenic engineering, genome editing, and chemical genetics have provided a bevy of high resolution methods that now enable even more intricate experimental pursuits and further facilitate disease modeling with zebrafish¹. Further, the ability to transition from the proverbial fish tank to the patient bedside has been realized, led by researchers using zebrafish chemical genetics to study hematopoietic stem cell (HSC) biology and their ensuing discovery that the small lipid prostaglandin, PGE₂, is a conserved regulator of HSC survival and proliferation—a finding that has directly led to clinical trials to improve the efficacy of umbilical cord blood transplantation for patients with leukemia and lymphoma⁶.

Within the realm of kidney biology, zebrafish have been established as an excellent model to study disease afflictions that alter nephron development and physiology⁷. Kidney research utilizing the zebrafish model was pioneered by a series of forward genetic screens that led to the collection of a rich cache of mutations affecting nephron form and function, whose subsequent study resulted in knowledge that increased our understanding of the mechanisms of ciliogenesis, polycystic kidney disease (PKD), and more^7–9. Detailed molecular characterization of renal anatomy in the zebrafish subsequently revealed the overall conservation of nephron segment pattern and cellular composition in both the embryonic and adult kidney structures compared to other vertebrates, including humans^10–12 (Figure 1). Further, it was discovered that the zebrafish kidney houses unique renal progenitors, which enable lifelong nephrogenesis and become more active in nephron formation in response to acute organ damage^11,13. This fascinating blend of conserved and distinctive renal features between zebrafish and other vertebrates has provided the opportunity for researchers to address fundamental disease pathologies and to discover ways to elicit regenerative responses. Here, we summarize advances in zebrafish models related to three kidney disease topics—acute kidney injury (AKI), glomerular disease, and several cystic diseases—and explore future applications of zebrafish in the field of nephrology.

Figure 1. The zebrafish pronephros shares genetic conservation with the human nephron.

(A) A picture taken of a live 24 hours post fertilization zebrafish embryo. (B) A schematic depicting the location of the pronephros in a 24 hours post fertilization embryo in relation to the somites. A dorsal view is expanded out where the G (glomerulus), N (neck), PCT (proximal convoluted tubule), PST (proximal straight tubule), DE (distal early), CS (corpuscle of Stannius), DL (distal late) and PD (pronephric duct) segments are labeled. (C) A diagram of the human nephron where the G (glomerulus, PCT (proximal convoluted tubule), PST (proximal straight tubule), TL (thin limb), TAL (thick ascending limb), MD (macula densa), DCT (distal convoluted tubule), CNT (connecting tubule) and CD (collecting duct). The segment colors were matched to indicate genetic conservation between the zebrafish pronephros and human nephron.

AKI Models in Zebrafish

AKI is a common malady defined by the abrupt impairment of renal function, typically a product of ischemic injury, exposure to nephrotoxic agents, or sepsis¹⁴. AKI is a heterogeneous condition that poses a major public health issue due to its high morbidity and mortality, leading to the deaths of approximately 2 million people worldwide annually¹⁴. As such, the identification of interventions to replenish nephron integrity after AKI has received considerable research attention.

The zebrafish embryonic kidney, the pronephros, is an established model of AKI that is conducive to research because of its simple composition of two nephrons¹⁵ (Figure 1). Administration of nephrotoxic compounds, such as gentamicin or cisplatin, to zebrafish embryos induces AKI in a dosage-dependent manner that recapitulates mammalian AKI^16,17. In the case of gentamicin, drug exposure causes flattening of the proximal tubule brush border, tubular and glomerular distension, lysosomal phospholipidosis, formation of debris in the nephron lumen and accumulation of leukocytes¹⁶. Likewise, cisplatin treatment leads to cellular vacuolization, flattening and denigration of tubular brush borders, and nephron distention among other symptoms typical of AKI¹⁶. Importantly, nephrotoxic induced-AKI in zebrafish triggers the loss of tubular cell polarity, which is typified by the mislocalization of the basolateral Na⁺/K⁺ ATPase pump to the apical membrane surface¹⁷. Treatment with gentamicin or cisplatin concludes with a severe decline of renal function and pericardial edema^16,17. Taken together, these studies established that cellular changes in the zebrafish pronephros closely mimic those in mammalian nephrons following exposure to nephrotoxic agents.

Due to the combination of these similarities and the diminutive size of the zebrafish embryo, subsequent studies have implemented chemical genetics, in which the effect of small molecules is evaluated on a process of interest, to identify restorative compounds to treat AKI. The proof-of-principle demonstration that zebrafish chemical genetics could discover potential AKI therapeutics has emerged from research on the histone deactylase (HDAC) inhibitor, 4-(phenylthio)-butanoic acid (PTBA), as well as several newly designed analogs of PTBA. Exposure to PTBA or its esterified analog, methyl-4-(phenylthio)butanoic (m4PTB), was found to increase renal progenitor numbers by stimulating proliferation during zebrafish pronephros development¹⁸. Ensuing work revealed that administration of m4PTB promoted nephron tubule proliferation in zebrafish embryos with gentamicin-induced AKI and in adult mice with ischemia-reperfusion-induced AKI by reducing the number of epithelial cells in G2/M arrest¹⁹. Additionally, nephron atrophy and interstitial fibrosis were also attenuated in the mouse model¹⁹. More recently, m4PTB administration was demonstrated to accelerate recovery and reduce renal fibrosis in mice following exposure to aristolochic acid, a plant extract that induces prolonged progressive cellular damage to proximal tubule epithelial cells²⁰. In this aristolochic acid-AKI model, enhanced recovery also correlated with the reduction of cell cycle arrest²⁰. These findings suggest that treatment with PTBA analogs can prompt renal proliferation in the context of abrupt as well as protracted injury, and highlight the promise of further research using zebrafish to identify candidate AKI therapeutics.

The adult zebrafish kidney, the mesonephros, is comprised of several hundred nephrons¹³, making it suitable for modeling more complex AKI diseases (Figure 2). Upon gentamicin insult, the zebrafish mesonephros regenerates damaged nephron tubular epithelial cells^13,21. This intricate process of regeneration involves partially overlapping waves of cell death and proliferation within the first week following injury²¹, and the elevated expression of renal developmental regulators such as the transcription factor pax2a—characteristics that are consistent with mammalian AKI pathology^13,21.

Figure 2. The zebrafish mesonephros undergoes continuous neonephrogenesis.

(A) A live image of an adult zebrafish with the location of the mesonephros depicted in light gray and an expanded out dorsal image of the mesonephros containing nephrons and collecting ducts. (B) A schematic depicting nephrons made up of a tubule and renal corpuscle branching off a collecting duct. Renal progenitors and an incipient nephron undergoing nephrogenesis are also depicted. (C) A histological cross section of an adult zebrafish kidney stained with hematoxylin and eosin with arrows pointing towards a RC (renal corpuscle), PT (proximal tubule), DT (distal tubule) and I (interstitial stroma).

However, distinct from humans and other mammals, adult zebrafish with AKI simultaneously create new functional nephrons, a process termed neonephrogenesis²² that typifies a number of other vertebrate fishes including the goldfish²³, skate²⁴ and medaka²⁵. Neonephrogenesis involves the formation of interstitial cell aggregates (Figure 2), distinguished through histological stains as basophilic in character, which grow in size via proliferation and elongate into functional nephrons between 2 and 3 weeks post injury ^11,13,21. Using transgenic reporter strains, these zebrafish neonephron aggregates have been distinguished based on the upregulated activity of the wt1b and lhx1a promoters in the aftermath of AKI^11,13. Important evidence for their fate was gathered through a series of elegant transplantation studies, in which the renal progenitors from Tg(lhx1a:EGFP) fish were demonstrated to generate new nephrons when introduced into a damaged host kidney¹¹. Further, serial transplantation to secondary or tertiary recipients were successful, suggesting that these renal progenitors may possess the capacity for self-renewal¹¹. Whether they are indeed bona fide stem cells has yet to be ascertained by experimental analysis. Nevertheless, the continued study of these zebrafish renal progenitors may elucidate avenues to stimulate regeneration following AKI.

Glomerular Disease Models in Zebrafish

A compelling number of renal diseases affect the glomerulus and more specifically, the podocyte epithelial cells. The zebrafish glomerulus and its podocytes are highly conserved with mammals, and these findings have inspired multiple investigations that utilize zebrafish as models for glomerular disease. For example, electron micrograph studies showed that zebrafish podocytes have extended foot processes and interdigitating foot processes in both embryonic and adult nephrons⁸. Furthermore, a podocyte-specific transgenic line Tg(podocin:GFP) illustrated that the 3-dimensional structure constructed from scanning electron micrograph visualization was morphologically similar to mammalian podocytes²⁶. Gene expression studies also suggest that zebrafish podocyte lineage development is conserved with mammals, based on the finding that they share the expression of many genes, such as those that encode slit diaphragm components²⁷. Several forward genetic screens have identified essential roles for previously unappreciated genes during podocyte development using zebrafish, and these exciting contributions have been summarized in another review⁷.

More recently, models of podocyte attrition have been created using targeted cell ablation (Figure 3). One manner by which to accomplish this is to engineer transgenic animals that express the Escherichia coli nitroreductase gene (NTR), downstream of a tissue-specific promoter. The cells expressing NTR can be ablated at a time point of interest by introducing the prodrug metronidazole (MTZ) into the living system. When taken up into the target cells, the MTZ is converted by the NTR protein into a cytotoxin that crosslinks DNA leading to cell death. Two cornerstone studies conducted such an experiment in zebrafish to model the loss of podocyte function^28,29. In each study the podocyte-specific enhancer for the podocin gene was placed upstream of the NTR gene and different fluorescent reporter genes were placed downstream of the NTR sequence to enable visual identification of the podocytes that expressed NTR^28,29. Upon MTZ treatment of adult²⁸ or larval^28,29 transgenic fish, there was a reduction in the number of fluorescent podocytes, which was confirmed to be a result of apoptosis by caspase-3 and TUNEL staining^28,29. In both cases, transgenic NTR fish treated with MTZ developed pericardial edema and fluid accumulation in other tissues, findings which resemble nephrotic syndrome and/or kidney failure in humans^28,29. It was also shown that vitamin D binding protein (VDBP), a large protein which is typically prevented from entering the nephron by the podocytes, was present in the nephrons of NTR-MTZ transgenic fish, indicating severe disruption of podocyte function and proteinuria²⁸. Interestingly, following MTZ exposure in larval fish, it was shown using transmission electron microscopy that podocyte foot processes with correct split diaphram morphology were regained 4 days after washing out the MTZ prodrug²⁹. Further, 7 days after MTZ washout, foot processes appeared to be completely repaired²⁹. A select group of cells were also seen to incorporate BrdU, implying podocyte proliferation²⁹. Examination of the adult mesonephros in Tg(pod:NTR-mCherry/wt1b:GFP) after MTZ-induced injury revealed that partially damaged glomeruli expressed wt1b:GFP, suggesting a response to injury that involved expression of developmental factors²⁸. In sum, these experiments suggest that podocyte regeneration is occurring in the zebrafish NTR-MTZ model. Additional studies are needed to determine whether podocyte repopulation occurs via the activities of stem cell progenitors or the proliferation of existing podocytes. Together, zebrafish transgenic approaches offer an intriguing opportunity to model podocyte development and regeneration.

Figure 3. Transgenic expression of nitroreductase for targeted ablation of podocytes.

(A) A diagram showing the transgenic cassette that encodes a fusion of the NTR (nitroreductase) and GFP (green fluorescent protein) genes under control of the podocin promoter, flanked by Tol2 sites for recombination. (B) A schematic showing MTZ (metronidazole)-induced injury (red X) of the podocytes (green) in a glomerulus and subsequently regenerated podocytes (blue).

Cystic Disease Models in Zebrafish

Cystic kidney diseases are an important cause of end stage renal disease (ESRD). Many genetic mutations can lead to the development of cystic kidney disease, and progressive involvement of the renal parenchyma by cysts can lead to kidney failure in some patients, as well as multiple extrarenal manifestations associated with increased morbidity and mortality in affected individuals. A typical underlying cause for these diseases is a defect in primary cilia, which are non-motile organelles that act as mechanosensors and function in signal transduction. Known as ciliopathies, these afflictions have been extensively modeled with the zebrafish, and a recent detailed review of this literature has been compiled elsewhere³⁰. Here, we specifically discuss two major categories of cystic kidney diseases and how they have been modeled with zebrafish to highlight the advantages of this research area.

Autosomal dominant polycystic kidney disease models

Polycystic kidney disease (PKD) occurs as an autosomal dominant (ADPKD) or autosomal recessive (ARPKD) condition³¹. ADPKD is associated with mutations in the genes polycystin-1 (PKD1) and polycystin-2 (PKD2)³¹. The PKD1 and PKD2 proteins interact to create a dynamic complex that controls the levels of intracellular Ca⁺ in the primary cilium³¹. Genetic knockdown of pkd1 or pkd2 during zebrafish development causes axis curvature, abnormal left-right asymmetry, hydrocephalus, kidney cysts and pericardial edema, which are in line with the phenotype observed in Pkd2−/− mice^32,33. These studies established the parallel between pronephric cysts in the zebrafish embryo and cyst phenotypes in mammals. With this foundation established, subsequent PKD insights were revealed through a chemical genetic screen in which the pan-HDAC inhibitor trichostatin A was found to suppress cyst formation in pkd2 knockdowns and ift172^9,34 mutant zebrafish (the latter discussed further in the next subsection)³⁵. Further, HDAC inhibitor treatment reduced cyst formation and the decline of renal function in Pkd1 deficient mice³⁵ and in Pkd2−/− mouse embryos³⁶. Thus, the use of zebrafish chemical genetics led to the discovery of an essential and conserved role for HDACs in PKD pathogenesis. Interestingly, subsequent studies in cell culture and a murine model of Pkd1 deficiency found that HDAC inhibition downregulated proliferation in cystic epithelial cells³⁷. Also, they identified the molecular mechanism at work, such that HDAC inhibition causes the upregulation of the transcriptional regulator inhibitor of differentiation 2 (Id2), which in turn blocks proliferation through p21 upregulation and/or suppression of S-phase entry through the Rb-E2F pathway³⁷.

Zebrafish models of other cystic kidney diseases

Complementary to work described above, insights into the cell biology of cystic kidney phenotypes have been provided from investigations on mutations isolated through zebrafish forward genetic screens^8,9. For example, it was discovered that pronephric renal cysts in zebrafish embryos also occur from defects in the intraflagellar transport (IFT) genes, ift57 and ift172^9,34. In genetic interaction studies, these IFT factors were shown to interact with a prickle1, a component of planar cell polarity (PCP) whose disruption also induced renal cysts in the zebrafish³⁴. This helped to further the understanding of how PCP signaling—which coordinates polarization among the plane of cells in an epithelial sheet—is linked to ciliogenesis³⁴.

Zebrafish studies have also been useful in several regards to examine the function of genes associated with nephronopthisis (NPHP), a disease that can cause cystic kidneys³⁸. NPHP is autosomal recessive and is the most common cause of ESRD in patients younger than 30 years of age³⁸. To date, there are at least 20 genes linked to NPHP, which like PKD1 and PKD2, localize to the primary cilia³⁹. In zebrafish, knockdown of the NPHP2 zebrafish ortholog inversin (invs) causes ventral axis curvature, defects in heart looping and pronephric cysts, consistent with what is seen in human and mice⁴⁰. The zebrafish invs mutant phenotype can be attenuated by expression of the murine orthologue, revealing yet another case of the conserved nature of kidney development between zebrafish and mammals⁴⁰. Like invs, genetic knockdown of zebrafish nphp3 and nphp5 using anti-sense morpholinos also generates abnormal axis curvature and cystic kidneys, again recapitulating what is seen in humans^41,42. These similarities create the possibility for further disease modeling of NPHP with zebrafish, such as through the application of chemical genetics.

As we continue to understand the genetic hierarchy that leads to predominant cystic kidney diseases and syndromic diseases with a PKD component, it is critical to delineate the underlying mechanisms and matching treatments. The zebrafish model is applicable in this regard because of these well-established molecular parallels with humans. Taken together, work with zebrafish models has led to valuable discoveries about PKD pathogenesis and identified putative drugs for PKD treatment.

Conclusions and Perspectives

Kidney research using the zebrafish has ushered in a new age of gene discovery and functional analysis for AKI, glomerular disease and cystic conditions using this animal due to the feasibility of creating models with forward and reverse genetic screens, transgenics, genome editing, and chemical genetics. With these Danio rerio models of kidney disease, we can continue to further our understanding of kidney disease and how to treat afflicted patients. As such, we postulate that these models will be critical for basic and translational research about kidney function and development. With ongoing advances in personalized medicine and genome wide sequencing, emergent data about the genetic make-up of patients will implicate candidate mutations that correlate with different disease states. The ability to rapidly and economically test gene function in zebrafish is likely to provide a worthwhile avenue to evaluate such candidates. Particularly promising future prospects for research with zebrafish kidney disease models entail the identification of pro-regenerative agents and chemical suppressors for heritable conditions (Figure 4). Since zebrafish chemical genetics allow for the rapid identification of compounds that can stimulate regeneration or suppress kidney disease phenotypes, these are likely to be powerful approaches for future study. Thus, despite the numerous contributions to nephrology from research with zebrafish kidney disease models, the possibilities for further insights gained through study of this little fish are immense indeed.

Figure 4. Developing new treatments to cure kidney disease.

(A) A schematic showing a kidney affected by AKI (acute kidney injury) (red X) and a kidney with PKD (polycystic kidney disease) (yellow circle) that are ameliorated through regeneration and/or chemical suppression (green masses) to become a repaired kidney.