Overview

All animals must excrete the waste products of metabolism and maintain a constant body composition despite changes in the external environment. In higher animals, these functions are performed by specialized excretory organs. The excretory organs range from a single excretory cell in the nematode Caenorhabditis elegans to Malpighian tubules in insects; nephridia in annelids; rectal glands in sharks; and kidneys in amphibians, birds, and mammals. Although many of these organisms are separated by hundreds of millions of years of evolution, their excretory organs often show a striking degree of conservation of form and function. For example, the Malpighian tubules of fruit flies (Drosophila) consist of blind-ended epithelial tubes that emerge from the hindgut and project into the coelomic cavity. Fruit flies consume a diet that is rich in potassium (not unlike some dialysis patients!), so a major function of Malpighian tubules is elimination of excess potassium. This process is accomplished by ion transporters, such as bumetanide-sensitive Na+-K+-2Cl− co-transporters, which are not unlike those found in mammalian secretory epithelia (1). Fluid is secreted into the early Malpighian tubule and selectively reabsorbed in the late tubule, analogous to filtration-reabsorption in the mammalian nephron. Even the genes that are required for the embryonic development of excretory organs are conserved between highly disparate species. Such similarities raise the possibility that experimentally tractable organisms, such as Drosophila, might serve as useful models for understanding kidney biology in higher organisms. The purpose of this Frontiers in Nephrology miniseries is to illustrate how studies in nonmammalian model organisms can provide unique insights into kidney development and disease in humans. Two invertebrate models, Drosophila melanogaster and Caenorhabditis elegans, and two vertebrate models, Xenopus and zebrafish, are discussed. Some of the features and advantages of these models are summarized in Table 1. These model organisms have in common small adult size, short generation time, high fecundity, and experimental accessibility. Their embryos develop externally, and some are optically transparent, which makes them ideal for observations of the development of internal organs such as the kidney. Another major advantage is that the genomes of model organisms have been completely or partially sequenced (Table 1). Using comparative genomics, homologues of human genes can be identified in other species, which frequently reveals new insights into their functions. Combining classical methods of experimental embryology, such as tissue grafting and microinjection, with the modern tools of molecular biology, such as transgenesis and RNA interference, yields a powerful approach for elucidating the molecular mechanisms of epithelial induction, branching morphogenesis, cell lineage, tubulogenesis, and other processes that are common to excretory organ development in many species. In the first article, Jung et al. discuss Malpighian tubule development in Drosophila (2). The simple structure of Malpighian tubules permits detailed analyses of tubule elongation and epithelial remodeling that are difficult to perform in the mammalian kidney. Drosophila is the classic organism for genetic studies because of the large number of mutant lines and phenotypic markers that are available and the relative ease with which genetic crosses can be performed. Flies with mutations that affect Malpighian tubule development can be identified, and the mutated genes can be cloned. Genetic crosses can be performed to determine whether genes reside in common developmental pathways. Transgenic flies can be created using mobile elements (P-elements) and used to study the functions of cloned genes or to rescue mutant phenotypes. In their review, Jung et al. illustrate how these techniques have been applied to identify transcription factors and signaling pathways that are important for Malpighian tubule development. Many of the factors and pathways are conserved in the mammalian kidney, which suggests that they play fundamental roles in epithelial morphogenesis. As additional evidence for evolutionary conservation, the article discusses a recent study that showed that some cells that compose the Malpighian tubules originate from a mesenchymal-to-epithelial transition, similar to the development of the mammalian nephron. Studies in nonmammalian model organisms can also provide important insights into the molecular pathogenesis of human renal diseases. One of the best examples is the discovery of the link between cilia and autosomal dominant polycystic kidney disease (ADPKD) that was first made in C. elegans. C. elegans is a free-living nematode that is amenable to classical genetic crosses as well as molecular genetics techniques such as transgenics and RNA interference. Other advantages of this model organism include small size, transparency, complete fate map, and stereotyped development. In the second article, Barr reviews her seminal work showing that C. elegans has homologues of polycystin-1 and polycystin-2, the proteins encoded by the human ADPKD genes (3). Her studies showed that the worm homologues, named LOV-1 and PKD-2, are expressed in the primary cilia of sensory neurons that are required for mating behavior. These results suggest that the ADPKD genes encode ciliary proteins that are involved in cell sensing, a hypothesis that has been confirmed in mammals. In her review, Barr shows how comparative genomics has also been used to study disease genes for other renal disorders, including Bardet-Biedl syndrome and nephronophthisis. Although studies of model organisms typically focus on the similarities between species, examining the differences is also important. Barr notes that C. elegans lacks homologues of the ARPKD genes PKHD1 and cystin. Understanding why PKHD1 and cystin are expressed in mammals but not in lower metazoans may provide clues to the functions of these genes. The embryonic development of the kidney progresses through three stages: pronephros, mesonephros, and metanephros (4). The pronephros is the definitive kidney of jawless fish, the mesonephros forms the adult kidney in amphibians, and the metanephros is found in birds and mammals. Because of its simple structure and small size, the pronephros of larval amphibians and fish has emerged as an important experimental model for studying kidney development. In the third article, Jones discusses the frog Xenopus laevis as a model for the development of the pronephros (5). A major advantage of Xenopus is the availability of a precise cell fate map. Progenitor cells that will form the pronephros can be identified at the blastocyst stage and microinjected with mRNA to express wild-type or mutant proteins. Jones shows how this approach has been used to identify the functions of the transcription factors Lim-1, WT-1, Pax-8, and HNF-1β in nephron patterning and tubulogenesis. Another classical technique in Xenopus is tissue grafting. Cells can be transplanted from one region of the embryo to another or removed and cultured in vitro. In her article, Jones shows how she has used this approach to identify growth factors that induce pronephric tubules and glomeruli. She also explains that Xenopus laevis is less useful for genetic screens because of its pseudotetraploid genome and that the diploid frog X. tropicalis has recently been introduced to circumvent this limitation. In the last article, Drummond reviews the use of zebrafish for studies of pronephric development (6). Unlike Xenopus larvae, in which filtration occurs into the coelomic cavity and ciliated nephrostomes funnel fluid from the coelomic cavity into the pronephric tubules, zebrafish larvae have a closed nephron that is more similar to the mammalian nephron. The zebrafish pronephros consists of a single midline glomerulus that drains into two pronephric tubules, one on each side of the body. Because the larvae are transparent, mutants that affect pronephric development can be identified easily. Drummond’s group has analyzed a large series of zebrafish mutants that have morphologic abnormalities in the glomerulus or pronephric tubules. Cloning of the mutated genes often reveals homology to known genes in humans. For example, noi (no isthmus), a mutation in which the pronephric tubules fail to develop, was identified as a homologue of Pax-2, a transcription factor that is required for formation of the mammalian kidney (7). Drummond explains how gene knockdowns can be readily achieved in zebrafish using morpholino antisense oligonucleotides. This technique permits rapid screening of cloned genes for their effects on kidney development. Large-scale mutagenesis in zebrafish has confirmed the roles of PKD2 and HNF-1β as well as identified novel genes that are involved in cyst formation. Because of its single glomerulus, the zebrafish pronephros is also an ideal model for studies of glomerular development. As an example, Drummond discusses the mechanism of vasculogenesis. As the articles in this Frontiers series amply demonstrate, many genes that are important for kidney development and function, as well as the pathways in which they act, are evolutionarily conserved. Consequently, researchers who are interested in mammalian kidney biology can select from a variety of model organisms on the basis of their unique experimental advantages and use them to investigate the functions of mammalian genes and proteins. Together with transgenic and knockout mice, nonmammalian model organisms are an important addition for elucidating the molecular mechanisms underlying kidney development and human renal diseases.Peter Igarashi, M.D., is Professor, Chief of the Division of Nephrology, and Robert T. Hayes Distinguished Chair in Nephrology in Honor of Dr. Floyd C. Rector, Jr., at the University of Texas Southwestern Medical Center at Dallas.Table 1: Selected characteristics of nonmammalian model organismsa