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.     

Stromal Protein Ecm1 Regulates Ureteric Bud Patterning and Branching

The interactions between the nephrogenic mesenchyme and the ureteric bud during kidney development are well documented. While recent studies have shed some light on the importance of the stroma during renal development, many of the signals generated in the stroma, the genetic pathways and interaction networks involving the stroma are yet to be identified. Our previous studies demonstrate that retinoids are crucial for branching of the ureteric bud and for patterning of the cortical stroma. In the present study we demonstrate that autocrine retinoic acid (RA) signaling in stromal cells is critical for their survival and patterning, and show that Extracellular matrix 1, Ecm1, a gene that in humans causes irritable bowel syndrome and lipoid proteinosis, is a novel RA-regulated target in the developing kidney, which is secreted from the cortical stromal cells surrounding the cap mesenchyme and ureteric bud. Our studies suggest that Ecm1 is required in the ureteric bud for regulating the distribution of Ret which is normally restricted to the tips, as inhibition of Ecm1 results in an expanded domain of Ret expression and reduced numbers of branches. We propose a model in which retinoid signaling in the stroma activates expression of Ecm1, which in turn down-regulates Ret expression in the ureteric bud cleft, where bifurcation normally occurs and normal branching progresses.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme during Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.     

An epithelial precursor is regulated by the ureteric bud and by the renal stroma

Kidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin alpha(5)) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma.     

Cessation of renal morphogenesis in mice

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time, the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.     

c-kit delineates a distinct domain of progenitors in the developing kidney

Early inductive events in mammalian nephrogenesis depend on an interaction between the ureteric bud and the metanephric mesenchyme. However, mounting evidence points towards an involvement of additional cell types--such as stromal cells and angioblasts--in growth and patterning of the nephron. In this study, through analysis of the stem cell factor (SCF)/c-kit ligand receptor pair, we describe an additional distinct cell population in the early developing kidney. While SCF is restricted to the ureteric bud, c-kit-positive cells are located within the renal interstitium, but are negative for Foxd1, an established marker of stromal cells. In fact, the c-kit-positive domain is continuous with a central mesodermal cell mass ventral and lateral to the dorsal aorta, while Foxd1-expressing stromal cells are continuous with a dorsal perisomitic cell population suggesting distinct intraembryonic origins for these cell types. A subset of c-kit-positive cells expresses Flk-1 and podocalyxin, suggesting that this cell population includes angioblasts and their progenitors. c-kit activation is not required for the survival of these cells in vivo, because white spotting (c-kit(W/W)) mice, carrying a natural inactivating mutation of c-kit, display normal intrarenal distribution of the c-kit-positive cells at E13.5. In addition, early kidney development in these mutants is preserved up to the stage when anemia compromises global embryonic development. In contrast, under defined conditions in organ cultures of metanephric kidneys, c-kit-positive cells, including the Flk-1-positive subset, undergo apoptosis after treatment with STI-571, an inhibitor of c-kit tyrosine phosphorylation. This is associated with reductions in ureteric bud branching and nephron number. Conversely, exogenous SCF expands the c-kit-positive population, including Flk-1-positive angioblasts, and accelerates kidney development in vitro. These data suggest that ureteric bud-derived SCF elicits growth-promoting effects in the metanephric kidney by expanding one or more components of the interstitial c-kit-positive progenitor pool.     

Cell lineages in the embryonic kidney: their inductive interactions and signalling molecules.

The first signalling genes acting in the inductive interactions in the kidney have now been identified. Differentiation of the permanent kidney or the metanephros is critically dependent on inductive signalling between the nephrogenic mesenchyme and ureteric bud epithelium. Further inductive interactions occur between developing nephrons, interstitial stroma, endothelial cells and neurones. Glial-cell-line-derived neurotrophic factor is a signal for the ureteric bud initiation and branching, and Wnt4 is an autocrine epithelializing signal at the pretubular stage of nephron formation. The signals for renal angiogenesis and innervation are less well defined, but seem to include vascular endothelial growth factor and neurotrophins, at least. The ureteric-bud-derived signal for induction of the nephrogenic mesenchyme (to bring the cells to the condensate stage) is not yet known, but fibroblast growth factor 2 is a good candidate. None of the signalling genes identified from the embryonic kidney is specific to the organ, which raises some general questions. How do the organs develop from similar rudiments to various patterns with different cell types and functions? Does the information for organ-specific differentiation pathways retain in the epithelial or mesenchymal compartment? The present, rather fragmentary molecular data would favour the view that similar molecules acting in different combinations and developmental sequences, rather than few organ-specific master genes, could be responsible for the divergence of patterning.     

Midkine Promotes Selective Expansion of the Nephrogenic Mesenchyme During Kidney Organogenesis

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.Key Words: growth factor, proliferation, apoptosis, ureteric bud, branching morphogenesis, epithelial progenitor, development, signaling     

Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis

The kidney of the Gpc3-/ mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Here, we identify cellular and molecular mechanisms underlying this renal dysplasia. Glypican-3 (GPC3) deficiency was associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation was increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggested that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogated the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converted BMP7-dependent inhibition to stimulation, and enhanced the stimulatory effects of KGF. Similar comparative differences were found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. We propose that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis.     

Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney

During kidney development and in response to inductive signals, the metanephric mesenchyme aggregates, becomes polarized, and generates much of the epithelia of the nephron. As such, the metanephric mesenchyme is a renal progenitor cell population that must be replenished as epithelial derivatives are continuously generated. The molecular mechanisms that maintain the undifferentiated state of the metanephric mesenchymal precursor cells have not yet been identified. In this paper, we report that functional inactivation of the homeobox gene Six2 results in premature and ectopic differentiation of mesenchymal cells into epithelia and depletion of the progenitor cell population within the metanephric mesenchyme. Failure to renew the mesenchymal cells results in severe renal hypoplasia. Gain of Six2 function in cortical metanephric mesenchymal cells was sufficient to prevent their epithelial differentiation in an organ culture assay. We propose that in the developing kidney, Six2 activity is required for maintaining the mesenchymal progenitor population in an undifferentiated state by opposing the inductive signals emanating from the ureteric bud.     

The murine Fgfrl1 receptor is essential for the development of the metanephric kidney

Fgfrl1 is a novel member of the fibroblast growth factor receptor family. Its extracellular domain resembles the four conventional Fgfrs, while its intracellular domain lacks the tyrosine kinase domain necessary for Fgf mediated signal transduction. During embryonic development Fgfrl1 is expressed in the musculoskeletal system, in the lung, the pancreas and the metanephric kidney. Targeted disruption of the Fgfrl1 gene leads to the perinatal death of the mice due to a hypoplastic diaphragm, which is unable to inflate the lungs. Here we show that Fgfrl1-/- embryos also fail to develop the metanephric kidney. While the rest of the urogenital system, including bladder, ureter and sexual organs, develops normally, a dramatic reduction of ureteric branching morphogenesis and a lack of mesenchymal-to-epithelial transition in the nephrogenic mesenchyme result in severe renal dysgenesis. The failure of nephron induction might be explained by the absence of the tubulogenic markers Wnt4, Fgf8, Pax8 and Lim1 at E12.5 of the mutant animals. We also observed a loss of Pax2 positive nephron precursor cells and an increase of apoptosis in the cortical zone of the remnant kidney. Fgfrl1 is therefore essential for mesenchymal differentiation in the early steps of nephrogenesis.     

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.     

Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney

Semaphorins, originally identified as axon guidance molecules, have also been implicated in angiogenesis, function of the immune system and cancerous growth. Here we show that deletion of Plexin B2 (Plxnb2), a semaphorin receptor that is expressed both in the pretubular aggregates and the ureteric epithelium in the developing kidney, results in renal hypoplasia and occasional double ureters. The rate of cell proliferation in the ureteric epithelium and consequently the number of ureteric tips are reduced in the kidneys lacking Plexin B2 (Plxnb2-/-). Semaphorin 4C, a ligand for Plexin B2, stimulates branching of the ureteric epithelium in wild type and Plxnb2+/- kidney explants, but not in Plxnb2-/- explants. As shown by co-immunoprecipitation Plexin B2 interacts with the Ret receptor tyrosine kinase, the receptor of Glial-cell-line-derived neurotrophic factor (Gdnf), in embryonic kidneys. Isolated Plxnb2-/- ureteric buds fail to respond to Gdnf by branching, but this response is rescued by Fibroblast growth factor 7 and Follistatin as well as by the metanephric mesenchyme. The differentiation of the nephrogenic mesenchyme, its morphology and the rate of apoptosis in the Plxnb2-/- kidneys are normal. Plexin B2 is co-expressed with Plexin B1 (Plxnb1) in the kidney. The double homozygous Plxnb1-Plxnb2-deficient mice show high embryonic lethality prior to onset of nephrogenesis. The only double homozygous embryo surviving to E12 showed hypoplastic kidneys with ureteric branches and differentiating mesenchyme. Taken together, our results show that Sema4C-Plexin B2 signalling regulates ureteric branching, possibly through modulation of Gdnf signalling by interaction with Ret, and suggest non-redundant roles for Plexin B1 and Plexin B2 in kidney development.     

小熊猫肾脏和输尿管的组织学研究

小熊猫的肾脏呈蚕豆形,表面光滑不分叶,只有1个肾锥体和1个肾盏,无肾盂。肾脏皮质内可见皮质迷路和髓放线。皮质迷路内有近曲小管、远曲小管和肾小体等结构。髓放线内有近端小管直部和远端小管直部。髓质可分为外髓和内髓两个区域。外髓有较多的集合管断面,少量的远端小管直部和细段,较多的直小血管束。内髓部位有大量的细段和乳头管。各种泌尿小管之间有少量的疏松结缔组织构成的间质,间质内有丰富的毛细血管。输尿管横切面呈圆形或卵圆形,管腔呈不规则的裂隙状。管壁由粘膜、肌肉层和外膜组成。并与大熊猫肾脏和输尿管的组织结构作了比较研究。     

Similar developmental patterns in immunolocalisation of stem cell factor and KIT in bovine meso- and metanephros

The mesonephros is often regarded as a simplified version of the terminal renal organ, the metanephros. Both renal organs result from an epithelio-mesenchymal interaction between the Wolffian duct and the nephrogenic ridge. It appears that the epithelio-mesenchymal interaction makes use of similar signal cascades for both renal organs and that key events required for the development of the metanephros occur at earlier stages. In murine metanephroi, the stem cell factor (SCF)/-KIT-signal transduction pathway has recently been shown to regulate ureteric bud branching and epithelial cell differentiation. We immunohistochemically defined the time-sequence of KIT and SCF presence in both renal organs using bovine embryos/foetuses with crown rump length (CRL) of 1.7–24 cm. In the mesonephroi, epithelial cells with strong KIT staining were scattered in distal tubules, and SCF was expressed in the epithelial wall of corpuscles and proximal tubules. KIT positivity occurred in the metanephroi of embryos prior to SCF; KIT was predominantly localised at the ureteric bud tips in the nephrogenic zone. In foetuses of 13 cm and more CRL, the SCF/KIT profile of developmentally advanced nephrons mirrored the situation in the mesonephros. Epithelial cells with strong KIT staining were scattered in the cortical areas of distal tubules, while SCF was expressed in the epithelial wall of corpuscles and proximal tubules. Our morphological findings agree with a potential role of KIT at the ureteric bud tips and demonstrate a similar expression of KIT and SCF along the areas of developmentally advanced mesonephric and metanephric nephrons.     

Disruption of polycystin-1 function interferes with branching morphogenesis of the ureteric bud in developing mouse kidneys

The polycystic kidney disease (PKD1) gene-encoded protein, polycystin-1, is developmentally regulated, with highest expression levels seen in normal developing kidneys, where it is distributed in a punctate pattern at the basal surface of ureteric bud epithelia. Overexpression in ureteric epithelial cell membranes of an inhibitory pMyr-GFP-PKD1 fusion protein via a retroviral (VVC) delivery system and microinjection into the ureteric bud lumen of embryonic day 11 mouse metanephric kidneys resulted in disrupted branching morphogenesis. Using confocal quantitative analysis, significant reductions were measured in the numbers of ureteric bud branch points and tips, as well as in the total ureteric bud length, volume and area, while significant increases were seen as dilations of the terminal branches, where significant increases in outer diameter and volumes were measured. Microinjection of an activating 5TM-GFP-PKD1 fusion protein had an opposite effect and showed significant increases in ureteric bud length and area. These are the first studies to experimentally manipulate polycystin-1 expression by transduction in the embryonic mouse kidney and suggest that polycystin-1 plays a critical role in the regulation of epithelial morphogenesis during renal development.