Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals. Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application

Renal stem cells: fact or science fiction?

The kidney is widely regarded as an organ without regenerative abilities. However, in recent years this dogma has been challenged on the basis of observations of kidney recovery following acute injury, and the identification of renal populations that demonstrate stem cell characteristics in various species. It is currently speculated that the human kidney can regenerate in some contexts, but the mechanisms of renal regeneration remain poorly understood. Numerous controversies surround the potency, behaviour and origins of the cell types that are proposed to perform kidney regeneration. The present review explores the current understanding of renal stem cells and kidney regeneration events, and examines the future challenges in using these insights to create new clinical treatments for kidney disease

Repopulating Decellularized Kidney Scaffolds: An Avenue for Ex Vivo Organ Generation

Recent research has shown that fully developed organs can be decellularized, resulting in a complex scaffold and extracellular matrix (ECM) network capable of being populated with other cells. This work has resulted in a growing field in bioengineering focused on the isolation, characterization, and modification of organ derived acellular scaffolds and their potential to sustain and interact with new cell populations, a process termed reseeding. In this review, we cover contemporary advancements in the bioengineering of kidney scaffolds including novel work showing that reseeded donor scaffolds can be transplanted and can function in recipients using animal models. Several major areas of the field are taken into consideration, including the decellularization process, characterization of acellular and reseeded scaffolds, culture conditions, and cell sources. Finally, we discuss future avenues based on the advent of 3D bioprinting and recent developments in kidney organoid cultures as well as animal models of renal genesis. The ongoing mergers and collaborations between these fields hold the potential to produce functional kidneys that can be generated ex vivo and utilized for kidney transplantations in patients suffering with renal disease

Zebrafish as a Model to Study Retinoic Acid Signaling in Development and Disease

Retinoic acid (RA) is a metabolite of vitamin A (retinol) that plays various roles in development to influence differentiation, patterning, and organogenesis. RA also serves as a crucial homeostatic regulator in adult tissues. The role of RA and its associated pathways are well conserved from zebrafish to humans in both development and disease. This makes the zebrafish a natural model for further interrogation into the functions of RA and RA-associated maladies for the sake of basic research, as well as human health. In this review, we explore both foundational and recent studies using zebrafish as a translational model for investigating RA from the molecular to the organismal scale

New zebrafish model for monitoring proximal tubule physiology in genetic and acquired renal Fanconi syndromes

Inherited and acquired disorders that affect proximal tubule endocytosis and lysosomal processing manifest with improper loss of solutes and proteins. The zebrafish pronephros is conserved with humans and is used to model numerous renal conditions, but has few quantitative measures for proximal tubule function. Here, Chen et al. developed a high-throughput assay to quantify proteinuria and lysosomal processing in transgenic zebrafish by labeling vitamin D protein, allowing for precise reporting of proximal tubule function

gldc Is Essential for Renal Progenitor Patterning during Kidney Development

The glycine cleavage system (GCS) is a complex located on the mitochondrial membrane that is responsible for regulating glycine levels and contributing one-carbon units to folate metabolism. Congenital mutations in GCS components, such as glycine decarboxylase (gldc), cause an elevation in glycine levels and the rare disease, nonketotic hyperglycinemia (NKH). NKH patients suffer from pleiotropic symptoms including seizures, lethargy, mental retardation, and early death. Therefore, it is imperative to fully elucidate the pathological effects of gldc dysfunction and glycine accumulation during development. Here, we describe a zebrafish model of gldc deficiency that recapitulates phenotypes seen in humans and mice. gldc deficient embryos displayed impaired fluid homeostasis suggesting renal abnormalities, as well as aberrant craniofacial morphology and neural development defects. Whole mount in situ hybridization (WISH) revealed that gldc transcripts were highly expressed in the embryonic kidney, as seen in mouse and human repository data, and that formation of several nephron segments was disrupted in gldc deficient embryos, including proximal and distal tubule populations. These kidney defects were caused by alterations in renal progenitor populations, revealing that the proper function of Gldc is essential for the patterning of this organ. Additionally, further analysis of the urogenital tract revealed altered collecting duct and cloaca morphology in gldc deficient embryos. Finally, to gain insight into the molecular mechanisms underlying these disruptions, we examined the effects of exogenous glycine treatment and observed analogous renal and cloacal defects. Taken together, these studies indicate for the first time that gldc function serves an essential role in regulating renal progenitor development by modulating glycine levels