The Zebrafish Model to Study the Role of microRNAs in Glomerular Function and Disease

Abstract

The zebrafish physiology of many organs including the kidney is very similar to that of human beings. Zebrafish develop from a fertilized egg to free-swimming larvae in only 48 h. The zebrafish larvae's correlate for the human kidney is the pronephros consisting of two nephrons with glomeruli fused at the embryo mid-line ventral to the dorsal aorta [1] ( Figure 1a).The glomerular filtration barrier of the pronephros is ultrastructurally almost indistinguishable from a mammalian glomerulus with interdigitating podocyte foot processes, a thin glomerular basement membrane and a fenestrated glomerular endothelium [2]. These structures can be found in 120 h old zebrafish larvae on ultrastructural level (Figures 1b and 1c). To generate these pictures zebrafish larvae were fixed in solution D and embedded in EPON. Ultrathin sectioning from head to tail was performed with a microtome until the glomerular region was found and sections transferred onto copper slit grids that were stained with uranyl acetate and lead citrate. Imaging was done with a transmission electron microscope.The pronephros tubular epithelium is composed of two proximal tubule segments, a proximal straight tubule as well as the early and late distal tubus [3]. The tubular system expresses a brush border of microvilli on the apical side for reabsorption processes (Figure 1d). The glomerular filtration of the zebrafish pronephros begins around 48 h post fertilization (hpf) [4]. Taking into consideration the structural and functional similarities and the fact that zebrafish gene expression can easily be influenced by specific knockdown and overexpression techniques using microinjections of morpholinos (MOs), mRNAs, microRNAs (miRs), small RNAs or CRISPR/Cas9 technology, the zebrafish serves as an ideal model to study glomerular diseases [5]. We and others successfully used the zebrafish model to screen for novel genes involved in kidney diseases [6][7][8]. On the other hand, gene mutations identified from human genetic studies can be verified as causative factors of proteinuric kidney disease in the zebrafish model. An upcoming field of research interest is the role of miRs in disease processes. MiRs regulate gene expression posttranscriptional by binding to the 3'-untranslated region (3' UTR) of a target mRNA and inhibiting translation [9,10]. Different miRs have been found to be enriched in human kidneys include miR-192, miR-194, miR-204, miR-215 and miR-216 [11]. MiRs are also essential for podocyte homeostasis [12][13][14][15] and can be secreted in body fluids [16,17].To antagonize the function of miRs, several antagomirs have been engineered and are commercially available. On the contrary, miR effects can be enhanced with miR mimics, which are chemically modified short double-stranded RNA sequences.Delivery of miRs in the zebrafish larvae system can be accomplished by microinjection of miR mimics in one-to-four cell stage or at 48 hpf by cardinal vein (c.v.) injection. After injections in egg stage the miR mimic is equally distributed in all zebra...

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

“…With their seed sequence they bind specifically to target mRNAs, thereby blocking translation and/or facilitating mRNA degradation 1 . In the recent past it could have been shown that microRNAs play a pivotal role in kidney development, physiology and pathology [2][3][4] .…”