Overview: Nonmammalian Organisms for Studies of Kidney Development and Disease
Peter Igarashi
Department of Internal Medicine and Division of Basic Science, University of Texas Southwestern Medical Center, Dallas, Texas
Address correspondence to: Dr. Peter Igarashi, Division of Nephrology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8856. Phone: 214-648-2754; Fax: 214-648-207; E-mail: peter.igarashi{at}utsouthwestern.edu
All animals must excrete the waste products of metabolism andmaintain a constant body composition despite changes in theexternal environment. In higher animals, these functions areperformed by specialized excretory organs. The excretory organsrange from a single excretory cell in the nematode Caenorhabditiselegans to Malpighian tubules in insects; nephridia in annelids;rectal glands in sharks; and kidneys in amphibians, birds, andmammals. Although many of these organisms are separated by hundredsof millions of years of evolution, their excretory organs oftenshow 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 thehindgut and project into the coelomic cavity. Fruit flies consumea diet that is rich in potassium (not unlike some dialysis patients!),so a major function of Malpighian tubules is elimination ofexcess 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 andselectively reabsorbed in the late tubule, analogous to filtration-reabsorptionin the mammalian nephron. Even the genes that are required forthe embryonic development of excretory organs are conservedbetween highly disparate species. Such similarities raise thepossibility that experimentally tractable organisms, such asDrosophila, might serve as useful models for understanding kidneybiology in higher organisms.
The purpose of this Frontiers in Nephrology miniseries is toillustrate how studies in nonmammalian model organisms can provideunique insights into kidney development and disease in humans.Two invertebrate models, Drosophila melanogaster and Caenorhabditiselegans, and two vertebrate models, Xenopus and zebrafish, arediscussed. Some of the features and advantages of these modelsare summarized in Table 1. These model organisms have in commonsmall adult size, short generation time, high fecundity, andexperimental accessibility. Their embryos develop externally,and some are optically transparent, which makes them ideal forobservations of the development of internal organs such as thekidney. Another major advantage is that the genomes of modelorganisms have been completely or partially sequenced (Table 1).Using comparative genomics, homologues of human genes canbe identified in other species, which frequently reveals newinsights into their functions. Combining classical methods ofexperimental embryology, such as tissue grafting and microinjection,with the modern tools of molecular biology, such as transgenesisand RNA interference, yields a powerful approach for elucidatingthe molecular mechanisms of epithelial induction, branchingmorphogenesis, cell lineage, tubulogenesis, and other processesthat are common to excretory organ development in many species.
Table 1. Selected characteristics of nonmammalian model organismsa
In the first article, Jung et al. discuss Malpighian tubuledevelopment in Drosophila (2). The simple structure of Malpighiantubules permits detailed analyses of tubule elongation and epithelialremodeling that are difficult to perform in the mammalian kidney.Drosophila is the classic organism for genetic studies becauseof the large number of mutant lines and phenotypic markers thatare available and the relative ease with which genetic crossescan be performed. Flies with mutations that affect Malpighiantubule development can be identified, and the mutated genescan be cloned. Genetic crosses can be performed to determinewhether genes reside in common developmental pathways. Transgenicflies can be created using mobile elements (P-elements) andused to study the functions of cloned genes or to rescue mutantphenotypes. In their review, Jung et al. illustrate how thesetechniques have been applied to identify transcription factorsand signaling pathways that are important for Malpighian tubuledevelopment. Many of the factors and pathways are conservedin the mammalian kidney, which suggests that they play fundamentalroles in epithelial morphogenesis. As additional evidence forevolutionary conservation, the article discusses a recent studythat showed that some cells that compose the Malpighian tubulesoriginate from a mesenchymal-to-epithelial transition, similarto the development of the mammalian nephron.
Studies in nonmammalian model organisms can also provide importantinsights into the molecular pathogenesis of human renal diseases.One of the best examples is the discovery of the link betweencilia and autosomal dominant polycystic kidney disease (ADPKD)that was first made in C. elegans. C. elegans is a free-livingnematode that is amenable to classical genetic crosses as wellas molecular genetics techniques such as transgenics and RNAinterference. Other advantages of this model organism includesmall size, transparency, complete fate map, and stereotypeddevelopment. In the second article, Barr reviews her seminalwork showing that C. elegans has homologues of polycystin-1and polycystin-2, the proteins encoded by the human ADPKD genes(3). Her studies showed that the worm homologues, named LOV-1and PKD-2, are expressed in the primary cilia of sensory neuronsthat are required for mating behavior. These results suggestthat the ADPKD genes encode ciliary proteins that are involvedin cell sensing, a hypothesis that has been confirmed in mammals.In her review, Barr shows how comparative genomics has alsobeen used to study disease genes for other renal disorders,including Bardet-Biedl syndrome and nephronophthisis. Althoughstudies of model organisms typically focus on the similaritiesbetween species, examining the differences is also important.Barr notes that C. elegans lacks homologues of the ARPKD genesPKHD1 and cystin. Understanding why PKHD1 and cystin are expressedin mammals but not in lower metazoans may provide clues to thefunctions of these genes.
The embryonic development of the kidney progresses through threestages: pronephros, mesonephros, and metanephros (4). The pronephrosis the definitive kidney of jawless fish, the mesonephros formsthe adult kidney in amphibians, and the metanephros is foundin birds and mammals. Because of its simple structure and smallsize, the pronephros of larval amphibians and fish has emergedas an important experimental model for studying kidney development.In the third article, Jones discusses the frog Xenopus laevisas a model for the development of the pronephros (5). A majoradvantage of Xenopus is the availability of a precise cell fatemap. Progenitor cells that will form the pronephros can be identifiedat the blastocyst stage and microinjected with mRNA to expresswild-type or mutant proteins. Jones shows how this approachhas been used to identify the functions of the transcriptionfactors Lim-1, WT-1, Pax-8, and HNF-1 in nephron patterningand tubulogenesis. Another classical technique in Xenopus istissue grafting. Cells can be transplanted from one region ofthe embryo to another or removed and cultured in vitro. In herarticle, Jones shows how she has used this approach to identifygrowth factors that induce pronephric tubules and glomeruli.She also explains that Xenopus laevis is less useful for geneticscreens because of its pseudotetraploid genome and that thediploid frog X. tropicalis has recently been introduced to circumventthis limitation.
In the last article, Drummond reviews the use of zebrafish forstudies of pronephric development (6). Unlike Xenopus larvae,in which filtration occurs into the coelomic cavity and ciliatednephrostomes funnel fluid from the coelomic cavity into thepronephric tubules, zebrafish larvae have a closed nephron thatis more similar to the mammalian nephron. The zebrafish pronephrosconsists of a single midline glomerulus that drains into twopronephric tubules, one on each side of the body. Because thelarvae are transparent, mutants that affect pronephric developmentcan be identified easily. Drummond’s group has analyzeda large series of zebrafish mutants that have morphologic abnormalitiesin the glomerulus or pronephric tubules. Cloning of the mutatedgenes often reveals homology to known genes in humans. For example,noi (no isthmus), a mutation in which the pronephric tubulesfail to develop, was identified as a homologue of Pax-2, a transcriptionfactor that is required for formation of the mammalian kidney(7). Drummond explains how gene knockdowns can be readily achievedin zebrafish using morpholino antisense oligonucleotides. Thistechnique permits rapid screening of cloned genes for theireffects on kidney development. Large-scale mutagenesis in zebrafishhas confirmed the roles of PKD2 and HNF-1 as well as identifiednovel genes that are involved in cyst formation. Because ofits single glomerulus, the zebrafish pronephros is also an idealmodel 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 evolutionarilyconserved. Consequently, researchers who are interested in mammaliankidney biology can select from a variety of model organismson the basis of their unique experimental advantages and usethem to investigate the functions of mammalian genes and proteins.Together with transgenic and knockout mice, nonmammalian modelorganisms are an important addition for elucidating the molecularmechanisms 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.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Jung AC, Denholm B, Skaer H, Affolter M: Renal tubule development in Drosophila: A closer look at the cellular level.
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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. 
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