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

The zebrafish, Danio rerio, is a relevant vertebrate model for biomedical research and translational studies because of its broad genetic conservation with humans. In recent years, scientists have formulated a growing list of zebrafish kidney disease paradigms, the study of which 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, which makes 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.

ppargc1a controls nephron segmentation during zebrafish embryonic kidney ontogeny.

Nephron segmentation involves a concert of genetic and molecular signals that are not fully understood. Through a chemical screen, we discovered that alteration of peroxisome proliferator-activated receptor (PPAR) signaling disrupts nephron segmentation in the zebrafish embryonic kidney (Poureetezadi et al., 2016). Here, we show that the PPAR co-activator ppargc1a directs renal progenitor fate. ppargc1a mutants form a small distal late (DL) segment and an expanded proximal straight tubule (PST) segment. ppargc1a promotes DL fate by regulating the transcription factor tbx2b, and restricts expression of the transcription factor sim1a to inhibit PST fate. Interestingly, sim1a restricts ppargc1a expression to promote the PST, and PST development is fully restored in ppargc1a/sim1a-deficient embryos, suggesting Ppargc1a and Sim1a counterbalance each other in an antagonistic fashion to delineate the PST segment boundary during nephrogenesis. Taken together, our data reveal new roles for Ppargc1a during development, which have implications for understanding renal birth defects.

Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development.

Kidney formation involves patterning events that induce renal progenitors to form nephrons with an intricate composition of multiple segments. Here, we performed a chemical genetic screen using zebrafish and discovered that prostaglandins, lipid mediators involved in many physiological functions, influenced pronephros segmentation. Modulating levels of prostaglandin E2 (PGE2) or PGB2 restricted distal segment formation and expanded a proximal segment lineage. Perturbation of prostaglandin synthesis by manipulating Cox1 or Cox2 activity altered distal segment formation and was rescued by exogenous PGE2. Disruption of the PGE2 receptors Ptger2a and Ptger4a similarly affected the distal segments. Further, changes in Cox activity or PGE2 levels affected expression of the transcription factors irx3b and sim1a that mitigate pronephros segment patterning. These findings show for the first time that PGE2 is a regulator of nephron formation in the zebrafish embryonic kidney, thus revealing that prostaglandin signaling may have implications for renal birth defects and other diseases.

A manual small molecule screen approaching high-throughput using zebrafish embryos.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

Production of haploid zebrafish embryos by in vitro fertilization.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity--a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Congenital and Acute Kidney Disease: Translational Research Insights from Zebrafish Chemical Genetics.

Today, acute kidney injury (AKI) and congenital anomalies of the kidney and urinary tract (CAKUT) represent major issues in healthcare. Both AKI and CAKUT can lead to end stage renal disease (ESRD) that requires life-long medical care with renal replacement therapy. Renal replacement by dialysis is intensive, and kidney transplantation is restricted by organ availability. These limitations, along with the growing epidemic of patients affected by kidney disease, highlight the significant need to identify alternative ways to treat renal injury and birth defects. Drug discovery is one promising avenue of current research. Here, we discuss zebrafish chemical genetics and its latent potency as a method to rapidly identify small molecule therapeutics to accelerate recovery after AKI. Specifically, we review two groundbreaking studies that have recently provided a template to screen for compounds that expand the renal progenitor field in development that were capable of treating AKI in both the zebrafish and the mouse. These new findings demonstrate that drug discovery using zebrafish can be used for relevant translational research to identify clinical interventions for renal conditions in humans.