Notch pathway activation can replace the requirement for Wnt4 and Wnt9b in mesenchymal-to-epithelial transition of nephron stem cells

The primary excretory organ in vertebrates is the kidney, which is responsible for blood filtration, solute homeostasis and pH balance. These functions are carried out by specialized epithelial cells organized into tubules called nephrons. Each of these cell types arise during embryonic development from a mesenchymal stem cell pool through a process of mesenchymal-to-epithelial transition (MET) that requires sequential action of specific Wnt signals. Induction by Wnt9b directs cells to exit the stem cell niche and express Wnt4, which is both necessary and sufficient for the formation of epithelia. Without either factor, MET fails, nephrons do not form and newborn mice die owing to kidney failure. Ectopic Notch activation in stem cells induces mass differentiation and exhaustion of the stem cell pool. To investigate whether this reflected an interaction between Notch and Wnt, we employed a novel gene manipulation strategy in cultured embryonic kidneys. We show that Notch activation is capable of inducing MET in the absence of both Wnt4 and Wnt9b. Following MET, the presence of Notch directs cells primarily to the proximal tubule fate. Only nephron stem cells have the ability to undergo MET in response to Wnt or Notch, as activation in the closely related stromal mesenchyme has no inductive effect. These data demonstrate that stem cells for renal epithelia are uniquely poised to undergo MET, and that Notch activation can replace key inductive Wnt signals in this process. After MET, Notch provides an instructive signal directing cells towards the proximal tubule lineage at the expense of other renal epithelial fates.     

Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarize to establish secretory function

Organs are made up of cells from separate origins, whose development and differentiation must be integrated to produce a physiologically coherent structure. For example, during the development of the kidney, a series of interactions between the epithelial mesonephric duct and the surrounding metanephric mesenchyme leads to the formation of renal tubules. Cells of the metanephric mesenchyme first induce branching of the mesonephric duct to form the ureteric buds, and they then respond to signals derived from them. As a result, mesenchymal cells are recruited to the buds, where they undergo a mesenchymal-to-epithelial transition as they condense to form nephrons. In contrast, the simple renal tubules of invertebrates, such as insect Malpighian tubules (MpTs), have always been thought to arise from single tissue primordia, epithelial buds that grow by cell division and enlargement and from which a range of specialized subtypes differentiate. Here, we reveal unexpected parallels between the development of Drosophila MpTs and vertebrate nephrogenesis by showing that the MpTs also derive from two cell populations: ectodermal epithelial buds and the surrounding mesenchymal mesoderm. The mesenchymal cells are recruited to the growing tubules, where they undergo a mesenchymal-to-epithelial transition as they integrate and subsequently differentiate as a physiologically distinctive subset of tubule cells, the stellate cells. Strikingly, the normal incorporation of stellate cells and the later physiological activity of the mature tubules depend on the activity of hibris, an ortholog of mammalian NEPHRIN.     

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.     

Chromatin dynamics in kidney development and function

Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little is known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. A better understanding of the renal cell-specific epigenetic modifications and the interaction of different cell types to form this highly complex organ will not only help to better understand developmental defects and early loss of kidney function in children, but also help to understand and improve chronic disease progression, cell regeneration and renal aging.     

pVHL and PTEN tumour suppressor proteins cooperatively suppress kidney cyst formation

In patients with von Hippel-Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.     

Growth and death in the developing mammalian kidney: signals, receptors and conversations.

Because the kidney (metanephros) starts to function before completing development, its patterning and morphogenesis need to be closely integrated with its growth. This is achieved by blast cells at the kidney periphery generating new nephrons that link up to the extending collecting-duct arborisation, while earlier-formed and more internal nephrons are maturing and beginning to filter serum. This pattern of development requires that cell division and apoptosis be co-ordinated in the various kidney compartments (collecting-ducts, blast cells, metanephric mesenchyme, nephrons and vascular system). The underlying regulatory networks for cell proliferation are beginning to be unravelled, mainly through expression studies, mutation analysis and experimentation in vitro. This article summarises current knowledge of kidney growth and apoptosis, and analyses some of the 80 or so ligand-receptor pairings that seem to sustain development and growth. It also points to some unanswered questions, the most intriguing being what role does apoptosis play during normal kidney development?     

Basolateral EGF Receptor Sorting Regulated by Functionally Distinct Mechanisms in Renal Epithelial Cells

Proliferation of epithelial tissues is controlled by polarized distribution of signaling receptors including the EGF receptor (EGFR). In kidney, EGFRs are segregated from soluble ligands present in apical fluid of nephrons by selective targeting to basolateral membranes. We have shown previously that the epithelial‐specific clathrin adaptor AP1B mediates basolateral EGFR sorting in established epithelia. Here we show that protein kinase C (PKC)‐dependent phosphorylation of Thr654 regulates EGFR polarity as epithelial cells form new cell–cell junctional complexes. The AP1B‐dependent pathway does not override a PKC‐resistant T654A mutation, and conversely AP1B‐defective EGFRs sort basolaterally by a PKC‐dependent mechanism, in polarizing cells. Surprisingly, EGFR mutations that interfere with these different sorting pathways also produce very distinct phenotypes in three‐dimensional organotypic cultures. Thus EGFRs execute different functions depending on the basolateral sorting route. Many renal disorders have defects in cell polarity and the notion that apically mislocalized EGFRs promote proliferation is still an attractive model to explain many aspects of polycystic kidney disease. Our data suggest EGFR also integrates various aspects of polarity by switching between different basolateral sorting programs in developing epithelial cells. Fundamental knowledge of basic mechanisms governing EGFR sorting therefore provides new insights into pathogenesis and advances drug discovery for these renal disorders.     

In situ histone landscape of nephrogenesis

In the developing kidney, self-renewing progenitors respond to inductive signaling from the adjacent branching ureteric bud by undergoing mesenchyme-to-epithelium transition. Nascent nephrons subsequently undergo elongation, segmentation, and differentiation into a mature renal epithelium with diverse functions. Epigenetic mechanisms have been implicated in impacting cell fate decisions during nephrogenesis; however, the chromatin landscape of nephron progenitors and daughter differentiating cells are largely unknown. Here, we examined the spatiotemporal expression patterns of histone H3 methylation and histone methyltransferases in E15.5 mouse kidneys. Kidney sections were probed with antibodies against histone modifications, enzymes, and markers of progenitors and differentiation. The results revealed that: (1) nephron progenitor cells exhibit a broad histone methylation signature that comprises both “active” and “repressive” marks (H3K4me3/K9me3/K27me3/R2me2/R17me2); (2) nascent nephrons retain high H3K4me3 but show downregulation of H3K9/K27me3 and; (3) maturing epithelial tubules acquire high levels of H3K79me2/3. Consistent with respective histone marks, the H3K4 methyltransferase, Ash2l, is expressed in progenitors and nascent nephrons, whereas the H3K9/K27 methyltransferases, G9a/Ezh2, are more enriched in progenitors than nascent nephrons. We conclude that combinatorial histone signatures correlate with cell fate decisions during nephrogenesis.     

Nephron formation adopts a novel spatial topology at cessation of nephrogenesis

Nephron number in the mammalian kidney is known to vary dramatically, with postnatal renal function directly influenced by nephron complement. What determines final nephron number is poorly understood but nephron formation in the mouse kidney ceases within the first few days after birth, presumably due to the loss of all remaining nephron progenitors via epithelial differentiation. What initiates this event is not known. Indeed, whether nephron formation occurs in the same way at this time as during embryonic development has also not been examined. In this study, we investigate the key cellular compartments involved in nephron formation; the ureteric tip, cap mesenchyme and early nephrons; from postnatal day (P) 0 to 6 in the mouse. High resolution analyses of gene and protein expression indicate that loss of nephron progenitors precedes loss of ureteric tip identity, but show spatial shifts in the expression of cap mesenchyme genes during this time. In addition, cap mesenchymal volume and rate of proliferation decline prior to birth. Section-based 3D modeling and Optical Projection Tomography revealed a burst of ectopic nephron induction, with the formation of multiple (up to 5) nephrons per ureteric tip evident from P2. While the distal–proximal patterning of these nephrons occurred normally, their spatial relationship with the ureteric compartment was altered. We propose that this phase of nephron formation represents an acceleration of differentiation within the cap mesenchyme due to a displacement of signals within the nephrogenic niche.     

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.     

In situ histone landscape of nephrogenesis

In the developing kidney, self-renewing progenitors respond to inductive signaling from the adjacent branching ureteric bud by undergoing mesenchyme-to-epithelium transition. Nascent nephrons subsequently undergo elongation, segmentation, and differentiation into a mature renal epithelium with diverse functions. Epigenetic mechanisms have been implicated in impacting cell fate decisions during nephrogenesis; however, the chromatin landscape of nephron progenitors and daughter differentiating cells are largely unknown. Here, we examined the spatiotemporal expression patterns of histone H3 methylation and histone methyltransferases in E15.5 mouse kidneys. Kidney sections were probed with antibodies against histone modifications, enzymes, and markers of progenitors and differentiation. The results revealed that: (1) nephron progenitor cells exhibit a broad histone methylation signature that comprises both “active” and “repressive” marks (H3K4me3/K9me3/K27me3/R2me2/R17me2); (2) nascent nephrons retain high H3K4me3 but show downregulation of H3K9/K27me3 and; (3) maturing epithelial tubules acquire high levels of H3K79me2/3. Consistent with respective histone marks, the H3K4 methyltransferase, Ash2l, is expressed in progenitors and nascent nephrons, whereas the H3K9/K27 methyltransferases, G9a/Ezh2, are more enriched in progenitors than nascent nephrons. We conclude that combinatorial histone signatures correlate with cell fate decisions during nephrogenesis.     

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.     

The Function of MicroRNAs in Renal Development and Pathophysiology

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that modulate diverse biological processes predominantly by translation inhibition or induction of mRNA degradation. They are important regulatory elements involved in renal physiology and pathology. Dysregulation of miRNAs disrupts early kidney development, renal progenitor cell differentiation and the maintenance of mature nephrons. miRNAs are also reported to participate in various renal diseases, including chronic kidney disease, acute kidney injury, allograft acute rejection and renal cell carcinoma. Differentially regulated miRNAs may represent innovative biomarkers for diagnosis and prognosis. Therefore, determining the roles of miRNAs in different types of renal diseases will help to clarify the pathogenesis and facilitate the development of novel 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.     

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.