Genetic Renal Diseases: The Emerging Role of Zebrafish Models

Abstract

The structural and functional similarity of the larval zebrafish pronephros to the human nephron, together with the recent development of easier and more precise techniques to manipulate the zebrafish genome have motivated many researchers to model human renal diseases in the zebrafish. Over the last few years, great advances have been made, not only in the modeling techniques of genetic diseases in the zebrafish, but also in how to validate and exploit these models, crossing the bridge towards more informative explanations of disease pathophysiology and better designed therapeutic interventions in a cost-effective in vivo system. Here, we review the significant progress in these areas giving special attention to the renal phenotype evaluation techniques. We further discuss the future applications of such models, particularly their role in revealing new genetic diseases of the kidney and their potential use in personalized medicine.

Keywords: CRISPR; genetic renal diseases; morpholino; new therapies; pathophysiology; pronephros; zebrafish.

Figure 1

Anatomy, patterning, and histology of the mammalian adult nephron and zebrafish larval pronephros. The segmented nephron distribution of genes expressed in the mammalian nephron (A) and zebrafish pronephros at 48 h post fertilization (hpf) (B), shows major similarities between different segments of both nephrons [7,8,9,10,11,12]. All gene symbols are in accordance with the Hugo Gene Nomenclature Committee (HGNC) guidelines. Hematoxylin and eosin stained images of cut sections of the human metanephros (C) and zebrafish pronephros at the level of the glomerulus and proximal tubules in 4 days post fertilization (dpf) larvae (D) showing basic similar architecture. Abbreviations: C, cloaca; CD, collecting duct; DCT, distal convoluted tubule; DE, distal early tubule; DL, distal late tubule; DT, distal tubule; G, glomerulus; GIT, gastrointestinal tract; NC, notochord; PCT, proximal convoluted tubule; PD, pronephric duct; PST, proximal straight tubule; PT, proximal tubule; TAL, thick ascending limb of Henle; TL, thin limb of Henle.

Figure 2

Reverse genetics in zebrafish using morpholinos and CRISPR-Cas9. (A) Morpholino antisense oligonucleotides (MOs): Morpholinos are synthetic single stranded nucleic acid analogues with a methylenemorpholine ring backbone replacing the sugars normally present in nucleic acids. The designed MO is injected at the 1-4 cell stage embryo, binds specifically to its target mRNA or pre-mRNA. Depending on whether the MO binds to the translation start site or a splice donor or acceptor site, it will either block protein translation or cause alternate splicing to produce a defective message that is either degraded, resulting in loss of protein expression, or still present in which case it will produce a defective protein. The resulting phenotype typically lasts for a few days. (B) Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9: The bacterial endonuclease enzyme is a large protein encoded by the cas9 gene. Specificity of the DNA strand cleavage is dependent on the pairing between the single guide RNA (spacer domain) and the complementary DNA target (protospacer domain). The Cas9 protein has also a domain that binds to a short sequence of target DNA, named the protospacer adjacent motif (PAM), which is found directly downstream of the target sequence in the genomic DNA, on the non-target strand. Because the spacer domain sequence provides at least 20 nucleotides of specificity in addition to the specificity of the PAM sequence, the CRISPR-Cas9 system can uniquely cleave DNA at a highly specific target site [6,34]. The cleaved DNA is then left to the non-homologous end-joining repair machinery, which can result in random deletions or insertions and loss of a functional allele. Alternatively, if a synthesized DNA template is introduced, homology-directed repair results in the generation of an engineered mutant allele at the break site [35].

Figure 3

Evaluation of glomerular function in the zebrafish. (A) 70-kDa rhodamine labelled dextran is injected in zebrafish larvae at 72 hpf (hours post fertilization). Immediately after injection (0 hpi, hours post injection), the success of intravascular injection is confirmed through observing the fluorescent dye in all capillaries including those situated in the retinal vascular bed (white arrows). At 24 hpi, the fluorescence signal intensity is quantified in fixed diameter circles in the retinal vascular bed using image-processing software, such as ImageJ. In wild type larvae, glomerular function is preserved and fluorescence accumulates in the retinal vascular bed as expected, while in the cystinosis mutant (ctns^−/−) larvae, the glomerular barrier is defective [48] and the 70-kDa dextran is lost in urine, thus the fluorescence intensity is significantly reduced (bars from left to right = 500 µm, 200 µm, and 200 µm). (B) FITC labelled inulin is injected at 96 hpf. Initial images are obtained immediately after injection (0 hpi) and 4 h later (4 hpi). The intensity of fluorescence is quantified over the cardinal vein at the 14th, 15th, and 16th somites (yellow lines). The average is determined for each fish and for each time point, then glomerular filtration rate (GFR) is expressed as the percentage decline of fluorescence over the 4 h incubation period (bars = 500 µm), white arrows refer to the site of the cloaca. (C) The VDBP-GFP transgenic zebrafish line at 72, 96, 120, and 144 hpf. The fluorescence intensity naturally accumulates in the retinal vascular bed over time with the increased production of the vitamin D binding protein (bars = 200 µm).

Figure 4

Evaluation of proximal tubular endocytosis. (A) Evaluation of megalin localization: Transverse confocal fluorescence images of the proximal pronephric region of wild type (wt) and cystinosis mutant larvae (5 dpf) showing endogenous megalin distribution with an anti-megalin antibody. In the wild type zebrafish, megalin is localized predominantly at the luminal brush border of the pronephric tubules, while in the cystinosis zebrafish, megalin abundance is significantly reduced in the brush border and it is mainly trapped in multiple subapical and cytoplasmic vacuoles, demonstrating defective endosomal trafficking in the cystinosis zebrafish (bars = 5 µm). (B) Transverse fluorescent images of the proximal pronephric region in wt and ocrl mutant zebrafish larvae after 2.5 h of 10-kDa Alexa488-conjugated dextran injection at 72 hpf. In wild type dextran is normally reabsorbed at the proximal tubular level, while in the Lowe syndrome model dextran reabsorption is almost completely absent (bars = 5 µm). White dashed lines represent the outline of the proximal tubule.