Mechanisms of epithelial development and neoplasia in the metanephric kidney.

Recent studies on the mechanisms of normal epithelial development in the kidney, and on the aetiology of renal neoplasms, are converging to reveal remarkably close relationships between the phenotypes and behaviours of normally-developing and neoplastic cells. Normal renal epithelia arise from two sources; those of the collecting duct system develop by arborisation of an initially-unbranched ureteric bud, in a manner similar to the development of other glandular organs, while epithelial nephrons develop via an unusual mesenchyme-to-epithelial transition. Both types of development require controlled proliferation, cell-cell and cell-matrix interactions, protease activity etc., but of the two tissues, the development of the nephrons is arguably the more complex. It includes many defined stages, signals and checkpoints that ensure that events happen at the right time, and that processes such as proliferation, apoptosis and differentiation are properly balanced. Detailed investigation of renal neoplasms has revealed some to be caused by mutations in molecules with known roles in normal nephrogenesis (e.g. Wilms' tumour and the WT-1 gene, renal cell carcinoma and the c-met receptor tyrosine kinase gene), some to be caused by mutations in genes expressed during normal development (e.g. renal cell carcinoma and the TSC-2 gene, renal cell carcinoma of the clear cell variety and the VHL gene). Furthermore, these and other tumours of unknown aetiology re-express genes such as Pax-2 that are expressed during the normal mesenchyme-to-epithelium transition but are shut off during terminal differentiation. Their re-appearance in tumours suggests that the cells have 'regressed' in an ontogenic sense, and their biology may therefore be understood most clearly by reference to the properties of normal developing cells rather than cells of a mature kidney.     

Development of the renal corpuscle during metamorphosis in the lamprey.

The renal corpuscle of the adult lamprey, Petromyzon marinus L., is formed during the programmed period of metamorphosis. Development is initiated early in this metamorphic period and is marked by the synchronous formation and growth of rudimentary nephron units (RNU) from longitudinal cord of nephrogenictissue extending from the posterior tip of the degenerating larval kidney to the cloaca and connected to the peritoneal epithelium. Detachment of the RNU from the peritoneum involves autolysis and cell death and is accompanied by their branching into five or six hexagonally-arranged nephrons which radiate from the original point of attachment. Differentiation of the epithelial cells at the proximal ends of the nephrons is preceded by the widening of lateral intercellular spaces, the formation of tubular lumina (primitive urinary spaces), the loss of apical cell junctions, and the development of a capillary network with its associated mesangium. With the extension of the capillaries and mesangium between the proximal ends of adjacent undifferentiated nephrons, visceral epithelial cells (podocytes), with long cell processes (trabeculae) and slit membranes, make their appearance. The urinary spaces resulting from this form of development are lined by the epithelium of the dilated ends of the nephrons (nephric capsules). The cells of these capsules differentiate mainly into podocytes, but a few parietal cells connect to the draining tubule. This method of development explains the unique form of the renal corpuscle in the adult lamprey. Despite the type of morphogenesis, this renal corpuscle possesses the fine-structural features seen in the renal corpuscles of other vertebrates.     

Expression of pigment-epithelium-derived factor during kidney development and aging

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development. Ana Luisa Pina and Marion Kubitza contributed equally to this work.     

FGF8 is required for cell survival at distinct stages of nephrogenesis and for regulation of gene expression in nascent nephrons

During kidney morphogenesis, the formation of nephrons begins when mesenchymal nephron progenitor cells aggregate and transform into epithelial vesicles that elongate and assume an S-shape. Cells in different regions of the S-shaped body subsequently differentiate into the morphologically and functionally distinct segments of the mature nephron. Here, we have used an allelic series of mutations to determine the role of the secreted signaling molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron formation is initiated, but the nascent nephrons do not express Wnt4 or Lim1, and nephrogenesis does not progress to the S-shaped body stage. Furthermore, the nephron progenitor cells that reside in the peripheral zone, the outermost region of the developing kidney, are progressively lost. When FGF8 signaling is severely reduced rather than eliminated, mesenchymal cells differentiate into S-shaped bodies. However, the cells within these structures that normally differentiate into the tubular segments of the mature nephron undergo apoptosis, resulting in the formation of kidneys with severely truncated nephrons consisting of renal corpuscles connected to collecting ducts by an abnormally short tubular segment. Thus, unlike other FGF family members, which regulate growth and branching morphogenesis of the collecting duct system, Fgf8 encodes a factor essential for gene regulation and cell survival at distinct steps in nephrogenesis.     

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.     

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.     

Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney

Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a co-receptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis.     

Insulin alters cell proliferation during the early development of rodent kidney.

The effects of insulin on early differentiated 15-day fetal mouse kidneys were assessed using an organotypic culture system. High concentrations (30 to 125 mU/ml) of the hormone drastically reduced (50%) the incorporation of 3H-thymidine in replicating cells without affecting either differentiation of forming nephrons or epithelio-mesenchymal relationships. When compared to insulin-like growth factor-I or the potent phorbol ester PMA, the action of insulin seemed to specifically deregulate some components of the transductional machinery controlling cell proliferation. This is opposed to the previous demonstration of a positive influence of insulin on cell proliferation in the human fetal kidney. The results suggest that the common definition of insulin as a fetal growth promoter may depend on the developmental stage of each organ, particularly for the mammalian kidney.     

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure¹. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury¹. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized^1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules^1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types³. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely^1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals^4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish⁶. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish^7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.     

Spatial expression of the kallikrein-kinin system during nephrogenesis

During nephrogenesis, new nephrons are induced in the periphery of the kidney, while maturing nephrons occupy a deeper position in the renal cortex. This centrifugal pattern of maturation is characterized by nephron patterning, establishment of proximal-distal segment identity, tubular and glomerular growth and differentiation, and acquisition of specialized functions. All of these processes are coordinated in time and space with renal vasculogenesis, glomerulogenesis and regional hemodynamic changes. The end-result ensures that tubular structure and function are tightly coordinated with glomerular filtration during normal kidney development. To achieve this delicate task of glomerulotubular balance, the developing kidney produces growth factors and vasoactive hormones that act in a paracrine manner to regulate nephrovascular growth, differentiation and physiological functions. One such paracrine system is the kallikrein-kinin system (KKS), which generates bradykinin (BK) from the cleavage of kininogen by kallikrein. BK activates a G-protein coupled receptor, B2R, to regulate renal blood flow and salt and water excretion. The developing kidney expresses an endogenous KKS. Expression of the KKS components and B2R is intimately coordinated with the terminal differentiation of the distal nephron. Kallikrein marks the onset of connecting tubule development, whereas kininogen and B2R map to the developing ureteric bud branches and maturing collecting ducts.Gene targeting studies indicate that the fetal KKS plays an important role in the maintenance of terminal epithelial cell differentiation.     

Bmp7 Maintains Undifferentiated Kidney Progenitor Population and Determines Nephron Numbers at Birth

The number of nephrons, the functional units of the kidney, varies among individuals. A low nephron number at birth is associated with a risk of hypertension and the progression of renal insufficiency. The molecular mechanisms determining nephron number during embryogenesis have not yet been clarified. Germline knockout of bone morphogenetic protein 7 (Bmp7) results in massive apoptosis of the kidney progenitor cells and defects in early stages of nephrogenesis. This phenotype has precluded analysis of Bmp7 function in the later stage of nephrogenesis. In this study, utilization of conditional null allele of Bmp7 in combination with systemic inducible Cre deleter mice enabled us to analyze Bmp7 function at desired time points during kidney development, and to discover the novel function of Bmp7 to inhibit the precocious differentiation of the progenitor cells to nephron. Systemic knockout of Bmp7 in vivo after the initiation of kidney development results in the precocious differentiation of the kidney progenitor cells to nephron, in addition to the prominent apoptosis of progenitor cells. We also confirmed that in vitro knockout of Bmp7 in kidney explant culture results in the accelerated differentiation of progenitor population. Finally we utilized colony-forming assays and demonstrated that Bmp7 inhibits epithelialization and differentiation of the kidney progenitor cells. These results indicate that the function of Bmp7 to inhibit the precocious differentiation of the progenitor cells together with its anti-apoptotic effect on progenitor cells coordinately maintains renal progenitor pool in undifferentiated status, and determines the nephron number at birth.     

Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development

Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.     

VEGF spatially directs angiogenesis during metanephric development in vitro

Vascular endothelial growth factor (VEGF) is required for endothelial cell differentiation, vasculogenesis, and normal glomerular vascularization. To examine whether VEGF plays a role as a chemoattractant for the developing kidney vasculature, avascular metanephric kidneys from rat embryos (E14) were cocultured with endothelial cells. To determine whether VEGF directly provides chemoattractive guidance for migration, we examined migration of endothelial cells toward VEGF-coated beads. Mouse glomerular endothelial cells expressing beta-galactosidase (MGEC) were isolated from Flk-1(+/-) heterozygous mice and passaged 4-12 times. Upon 24 h culture on collagen I gels MGEC formed a lattice or capillary-like network. Embryonic metanephroi were cocultured with MGEC on collagen I gels for 1-6 days in defined media, stained for beta-galactosidase, and examined by light microscopy. Metanephric organs induced a rearrangement of the endothelial cell lattice and attracted MGEC. MGEC invaded the metanephric organs forming capillary-like structures within and surrounding the forming nephrons. This process was accelerated and amplified by low oxygen (3% O(2)) and was prevented by anti-VEGF neutralizing antibodies. MGECs migrated toward VEGF-coated beads, whereas PBS-coated beads did not alter MGEC networks. We conclude that VEGF produced by the differentiating nephrons acts as a chemoattractant providing spatial direction to developing capillaries toward forming nephrons during metanephric development in vitro.     

Development of embryonic stem cells in recombinant kidneys

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms’ tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.     

Morphogenesis and molecular mechanisms involved in human kidney development

The development of the human kidney is a complex process that requires interactions between epithelial and mesenchymal cells, eventually leading to the coordinated growth and differentiation of multiple highly specialized stromal, vascular, and epithelial cell types. The application of molecular biology and immunocytochemistry to the study of cell types involved in renal morphogenesis is leading to a better understanding of nephrogenesis, which requires a fine balance of many factors that can be disturbed by various prenatal events in humans. The aim of this paper is to review human kidney organogenesis, with particular emphasis on the sequence of morphological events, on the immunohistochemical peculiarities of nephron progenitor populations and on the molecular pathways regulating the process of mesenchymal to epithelial transition. Kidney development can be subdivided into five steps: (i) the primary ureteric bud (UB); (ii) the cap mesenchyme; (iii) the mesenchymal-epithelial transition; (iv) glomerulogenesis and tubulogenesis; (v) the interstitial cells. Complex correlations between morphological and molecular events from the origin of the UB and its branching to the metanephric mesenchyme, ending with the maturation of nephrons, have been reported in different animals, including mammals. Marked differences, observed among different species in the origin and the duration of nephrogenesis, suggest that morphological and molecular events may be different in different animal species and mammals. Further studies must be carried out in humans to verify at the morphological, immunohistochemical, and molecular levels if the outcome in humans parallels that previously described in other species.