Glomerulogenesis and De Novo Nephrogenesis in Medaka Fish: An Evolutionary Approach

Outline
Introduction 3
Medaka as a Model Animal for Kidney Research 4
- General Advantages of Using Medaka 4
- Notable Differences Between Humans (Mammals) and Medaka 4
- An Example of Medaka Mutant Models for Kidney Disease 4
Kidney Development in Medaka 5
- Gross Morphology of Medaka Kidney 5
- Histological Anatomy of the Kidney 6
- Nephrogenic Stages in Medaka Kidney 7
- Three-dimensional Images of Nephrogenesis in Developing Mesonephros 8
- wt1 Expression as a Marker of Nephrogenesis 10
Kidney Regeneration in Medaka 11
- Nephron Repair in Mammals 11
- Renal Damage with Gentamicin in Medaka 11
- Renal Repair in Medaka 11
- De Novo Nephrogenesis in Medaka 11
- Recapitulation of wt1 Expression in Repair and De Novo Nephrogenesis of Medaka Kidney 15
Perspectives 15
- Stem Cells ? 15
- wt1 Transgenic Fish 16
Conclusion 16

Medaka has the pronephros at early larval stages and thereafter the mesonephros develops in the tissues around the pronephric tubule and duct. A marked increase in mesonephric nephrons continues until 2–3 months after hatching, and consequently the mesonephros consists of 200–300 nephrons on each side. The nephrogenic processes can be histologically featured in the developing mesonephros as three distinguishable stages: mesenchymal condensation, formation of a nephrogenic body, and maturation of nephron. Appearance of mesenchymal condensates and nephrogenic bodies in the mesenchymal tissue indicates that the de novo nephrogenesis takes place actively. Since these nephron precursors are positive for wt1 expression, wt1 could be a good marker of de novo nephrogenesis. The program for nephron development can be reactivated in medaka during adulthood by artificial injury with chemicals. Intraperitoneal administration of gentamicin, damaging tubules, ducts and the glomeruli, leads to a significant increase in the mesenchymal condensates and nephrogenic bodies in the injured kidney, which can be also recognized as wt1 -positive cell masses. Thus, unlike mammals, medaka is capable of regenerating kidney through de novo nephrogenesis, possibly by recruiting renal stem cells retained in the mesenchymal tissue of the adult kidney.

Introduction

The kidney plays a pivotal role in controlling humoral conditions, such as electrolytes and osmolarity, as well as blood pressure. Abnormalities of renal function sometimes result in the serious conditions, acute and chronic renal failure, for which dialysis is required. Worldwide, millions of people suffer from end-stage renal failure. Kidney diseases are associated with various systemic diseases including diabetes and autoimmune diseases. The number of kidney disease patients needing dialysis treatment is increasing and is becoming a major issue in the cost of medical care. Therefore, the development of treatments for kidney diseases is an issue that has to be resolved urgently.

Kidney regeneration in humans is one of the major subjects in the development of new therapeutic strategies in renal medicine . A well-established characteristic of the adult mammalian kidney is its ability to recover from acute renal failure. This repair response is thought to occur through repopulation of the existing nephron cells . The formation of nephrons is terminated at embryonic or neonate stages, but does not take place in the human adult kidney. It is thought that stem cells for nephrogenesis are not present or are dormant in the human adult kidney. In contrast to the mammalian kidney, the teleost fish kidney is thought to retain the stem cells in adulthood and can reactivate de novo nephrogenesis . Convincing evidence of de novo nephrogenesis in adult fish was provided by Elger et al., who reported that partial nephrectomy in the skate fish (Elasmobranch Leucoraja erinacea ) induces proliferation of renal progenitor cells and leads to the formation of a nephrogenic zone . Intriguingly, unilateral nephrectomy of mammalian adult kidneys results in compensatory renal hypertrophy due to cellular hypertrophy but not due to the de novo nephrogenesis .

Studies using fish as a model organism have pioneered a new research field in kidney regeneration. However, analysis of the normal development and regeneration process of kidney in fish is required in order to exploit the fish system to develop a regenerative medical approach for human kidney diseases. This chapter introduces the present knowledge on kidney development and regeneration in medaka fish.

Medaka as a Model Animal for Kidney Research

General Advantages of Using Medaka

Medaka ( Oryzias latipes ) is a small, egg-laying freshwater teleost ( Fig. 1.1A ) that is widely used as a laboratory animal . Medaka is native to East Asia including Japan, Korea and China. It becomes sexually mature (about 3 cm in body length) within 2–3 months after hatching and spawns daily and year-round under artificial conditions. The transparency of eggs is a distinct advantage for embryological observation and manipulation. In addition, most internal organs including the kidney are visible through the transparent body wall in adult fish if using see-through medaka (STIII, Fig. 1.1B ) . The recent publication of the medaka draft genome sequence provides valuable resources on medaka genomic information which facilitate molecular genetics. In comparison to higher vertebrates, the organs in fish are like a minimalist version, using far fewer cells to fulfill the equivalent function in the organism. The kidney of the larva consists of a single pair of nephrons, which facilitates research into kidney development in zebrafish pronephros . With these biological characteristics and abundant research resources, medaka is comparable to zebrafish as a model fish. The medaka mesonephros (adult kidney) would be suitable for studies on renal regeneration in fish, as described below, because of its clear histology and small number of nephrons.

Notable Differences Between Humans (Mammals) and Medaka

Mammalian kidneys form from three successive structures, the pronephros, the mesonephros and the metanephros , whereas most fish including medaka have only the first two forms . Previous findings suggest that medaka develops more advanced kidneys, which can be referred to as opisthonephros instead of mesonephros: in contrast to some primitive species of teleosts exhibiting segmentally arranged nephrons, medaka mesonephros develops higher order nephron generation with a complex arrangement .

Renal cilia in the lumen of the tubules consist of 9 + 0 axonemes without dynein arms in mammals, are immotile and are called primary cilia . Recent advances in the understanding of the pathogenesis of polycystic kidney disease (PKD) have proposed the function of immotile primary cilia in the renal tubules: they serve as passive mechanosensors to detect fluid flow rate in the tubule and thereby sense the lumen size . It is thought that abnormality of their mechanoreceptor functions results in impaired regulation of the renal lumen size and eventually leads to PKD. In contrast to the mammalian kidney, the renal cilia in the fish consist of 9 + 2 axonemes with dynein arms, which are features of motile cilia (see Fig. 1.4D ). In medaka as well as zebrafish, the maintenance of the renal lumen size requires active beating of the renal cilia, which produce a driving force for intratubular urine flow; loss or reduced motility of the cilia causes PKD . Defective ciliary motility causes primary ciliary dyskinesia in humans, as manifested in reversed organ laterality, recurrent respiratory infections and dysmotile sperm flagella, but not PKD. In medaka, however, defects in ciliary motility cause PKD, which has recently been shown in mutants of Kintonun/PF13, which function in preassembly of axonemal dyneins , as well as of Dnai2/mii/joi, which constitutes the outer dynein arms of the axomene (authors’ own unpublished data and personal communication with Kobayashi). Medaka kintonun and mii/joi mutants exhibit organ laterality defects and PKD, while human patients having a mutation in these orthologous genes show defective left–right polarity formation but not the PKD phenotype .

An Example of Medaka Mutant Models for Kidney Disease

A lot of medaka and zebrafish mutants are used as human renal disease models, many of which are related to PKD and are good sources for identifying the genes of human genetic diseases . The medaka pc mutant ( Fig. 1.1C, D ) develops numerous cysts in the kidney and its slowly progressive nature leads to massive enlargement of the kidney in adulthood ( Fig. 1.1E ) . A recent study revealed that loss of glis3 causes renal cyst formation in pc mutant medaka . Further analyses in mice and other reports have shown that Glis3/GLIS3, if lost, causes PKD in mammals ( Fig. 1.1F ). Thus, the PKD phenotype of medaka pc mutant is relevant to human patients with GLIS3 mutation despite the species difference in the putative function of the renal cilia.

Kidney Development in Medaka

Gross Morphology of Medaka Kidney

Medaka has a pair of kidneys that are located retroperitoneally, extending from the bases of the pectoral fins to the caudal reaches of the abdominal cavity ( Fig. 1.1B ). Their large anterior portions, containing most of the nephrons, are much larger than those of zebrafish, whereas the caudal portions are smaller ( Fig. 1.2A ). Medaka hatchlings have functional pronephros consisting of a single pair of nephrons ( Fig. 1.2B, C ). The kidney size of the fry increases as mesonephric nephrons develop. The number of nephrons, when represented as corresponding to both mature and immature glomeruli, reaches approximately 200–300 in each kidney within 2–3 months after hatching ( Fig. 1.2D ). Adult medaka kidneys contain both pronephros and mesonephros, unlike kidneys of some fish species which lose pronephros after mesonephros starts functioning . The bean-shaped cranial portion is larger than the caudal portion and this proportion persists throughout life.

Histological Anatomy of the Kidney

A medaka hatchling has only pronephros ( Fig. 1.2B, C ), which consists of a single pair of nephrons having the butterfly-shaped glomus external to the kidney capsule and the tubule connecting the glomus with the duct extending to the urinary bladder ( Fig. 1.3A ). (Medaka pronephros has glomus but not glomerulus in the sense that the multiple blood vessels go into the single renal corpuscle.)

Mesonephric nephrons distribute throughout the kidney with no particular distinction between medulla and cortex ( Fig. 1.3B ). The entire kidney is composed of both nephrons and interstitial lymphoid tissue. Three segmental structures of nephron are recognized by hematoxylin & eosin staining of kidney section ( Fig. 1.3C, D ). The glomerulus is typically a round cell mass covered by a flat epithelium of Bowman’s capsule. The proximal segment of the tubule is lined by tall columnar epithelial cells possessing a brush border on the apical surface ( Fig. 1.3C, D ), which is positive for periodic acid–Schiff (PAS) staining ( Fig. 1.3E ) or Locus tetragonolobus lectin staining ( Fig. 1.3F ). The distal tubular segment has a wide lumen lined by low columnar epithelial cells ( Fig. 1.3C ).

Under the transmission electron microscope, three important structures are evident in the glomerulus: the capillary blood vessels containing erythrocytes, the capillary endothelium, the glomerular basement membrane (GBM) and the podocytes forming the outer layer of the glomerulus (see Fig. 1.8A, B ). The proximal tubule consists of a thick layer of epithelial cells with a brush border on the apical surface and numerous mitochondria on the basal side ( Fig. 1.4A ). The distal tubule has a scanty brush border of microvilli on the apical side and numerous mitochondria, but no vesicles of the apical endocytotic apparatus ( Fig. 1.4B ).