Growth Factors and Morphogenesis

FIGURE 16-14

Uninduced mesenchyme

Condensing cells ogcP

OOO O

S-shaped body

Condensing cells

S-shaped body

Basement membrane degrading proteinases

Integrin receptors for interstitial matrix

<—Proteinases

Cell-surface receptors for proteinases (uPA-R, ? for MMPs)

of integrin-mediated basement membrane initiated signaling

Basement membrane degrading proteinases

Integrin receptors for interstitial matrix

<—Proteinases

Cell-surface receptors for proteinases (uPA-R, ? for MMPs)

of integrin-mediated basement membrane initiated signaling

FIGURE 16-14

Kidney morphogenesis. Schematics demonstrate the development of the ureteric bud and metanephric mesenchyme during kidney organogenesis. During embryogenesis, mutual inductive events between the metanephric mesenchyme and the ureteric bud give rise to primordial structures that differentiate and fuse to form functional nephrons [74-76]. Although the process has been described morphologically, the nature and identity of molecules involved in the signaling and regulation of these events remain unclear. A, Diagram of branching tubulogenesis of the ureteric bud during kidney organogenesis. The ureteric bud is induced by the metanephric mesenchyme to branch and elongate to form the urinary collecting system [74-76]. B, Model of cellular events involved in ureteric bud branching. To branch and elongate, the ureteric bud must digest its way through its own basement membrane, a highly complicated complex of extracellular matrix proteins. It is believed that this is accomplished by cellular projections, "invadopodia," which allow for localized sites of proteolytic activity at their tips [77-81]. C, Mesenchymal cell compaction. The metanephric mesenchyme not only induces ureteric bud branching but is also induced by the ureteric bud to epithelialize and differentiate into the proximal through distal tubule [74-76]. (From Stuart and Nigam [80] and Stuart et al. [81]; with permission.)

Basic research

Renal development

Tubulogenesis in vitro

Renal injury and repair

Urogenital abnormalities

Hypertension

Artificial kidneys

Applied research

Renal cystic diseases

Renal diseases

FIGURE 16-15

Potential of in vitro tubulogenesis research. Flow chart indicates relevance of in vitro models of kidney epithelial cell branching tubulogenesis to basic and applied areas of kidney research. While results from such studies provide critical insight into kidney development, this model system might also contribute to the elucidation of mechanisms involved in kidney injury and repair for a number of diseases, including tubular epithelial cell regeneration secondary to acute renal failure. Moreover, these models of branching tubulo-genesis could lead to therapies that utilize tubular engineering as artificial renal replacement therapy [82].

Growth factor

Motogenesis j-N

Morphogenesis Antiapoptosis

Cell proliferation

Cell movement

Cell organization

Cell survival

Remodeling of cell substratum

Cellular response to growth factors. Schematic representation of the pleiotrophic effects of growth factors, which share several properties and are believed to be important in the development and morphogenesis of organs and tissues, such as those of the kidney. Among these properties are the ability to regulate or activate numerous cellular signaling responses, including proliferation (mitogenesis), motility (motogenesis), and differentiation (morphogenesis). These characteristics allow growth factors to play critical roles in a number of complex biological functions, including embryogenesis, angiogenesis, tissue regeneration, and malignant transformation [83].

FIGURE 16-17

Motogenic effect of growth factors—hepatocyte growth factor (HGF) induces cell "scattering." During development or regeneration the recruitment of cells to areas of new growth is vital. Growth factors have the ability to induce cell movement. Here, subconfluent monolayers of either Madin-Darby canine kidney (MDCK) C, D, or murine inner medullary collecting duct (mIMCD) A, B, cells were grown for 24 hours in the absence, A, C, or presence B, D, of 20 ng/mL HGF. Treatment of either

type of cultured renal epithelial cell with HGF induced the dissociation of islands of cells into individual cells. This phenomenon is referred to as scattering. HGF was originally identified as scatter factor, based on its ability to induce the scattering of MDCK cells [83]. Now, it is known that HGF and its receptor, the transmembrane tyrosine kinase c-met, play important roles in development, regeneration, and carcinogenesis [83]. (From Cantley et al. [84]; with permission.)

FIGURE 16-18

Three-dimensional extracellular matrix gel tubulogenesis model. Model of the three-dimensional gel culture system used to study the branching and tubulogenesis of renal epithelial cells. Analyzing the role of single factors (ie, extracellular matrix, growth factors, cell-signaling processes) involved in ureteric bud branching tubulo-genesis in the context of the developing embryonic kidney is an extremely daunting task, but a number of model systems have been devised that allow for such investigation [77, 79, 85]. The simplest model exploits the ability of isolated kidney epithelial cells suspended in gels composed of extracellular matrix proteins to form branching tubular structures in response to growth factors. For example, Madin-Darby canine kidney (MDCK) cells suspended in gels of type I collagen undergo branching tubulogenesis reminiscent of ureteric bud branching morphogenesis in vivo [77, 79]. Although the results obtained from such studies in vitro might not correlate directly with events in vivo, this simple, straightforward system allows one to easily manipulate individual components (eg, growth factors, extracellular matrix components) involved in the generation of branching epithelial tubules and has provided crucial insights into the potential roles that these various factors play in epithelial cell branching morphogenesis [77, 79, 84-87].

FIGURE 16-19

FIGURE 16-19

An example of the branching tubulogenesis of renal epithelial cells cultured in three-dimensional extracellular matrix gels. Microdissected mouse embryonic kidneys (11.5 to 12.5 days) were cocultured with A, murine inner medullary collecting duct

(mIMCD) or, B, Madin-Darby canine kidney (MDCK) cells suspended in gels of rat-tail collagen (type I). Embryonic kidneys (EK) induced the formation of branching tubular structures in both mIMCD and MDCK cells after 48 hours of incubation at 37oC. EKs produce a number of growth factors, including hepatocyte growth factor, transforming growth factor-alpha, insulin-like growth factor, and transforming growth factor-^, which have been shown to effect tubulo-genic activity [86-93]. Interestingly, many of these same growth factors have been shown to be effective in the recovery of renal function after acute ischemic insult [21-30]. (From Barros et al. [87]; with permission.)

Pregnant SV40-transgenic mouse

Pregnant SV40-transgenic mouse

Isolate embryos

Dissect out embryonic kidney

Isolate metanephric mesenchyme

Isolate ureteric bud

Culture to obtain immortalized cells

FIGURE 16-20

Development of cell lines derived from embryonic kidney. Flow chart of the establishment of ureteric bud and metanephric mesenchymal cell lines from day 11.5 mouse embryo. Although the results obtained from the analysis of kidney epithelial cells— Madin-Darby canine kidney (MDCK) or murine inner medullary collecting duct (mIMCD) seeded in three-dimensional extracellular matrix gels has been invaluable in furthering our understanding of the mechanisms of epithelial cell branching tubulogenesis, questions can be raised about the applicability to embryonic development of results using cells derived from terminally differentiated adult kidney epithelial cells [94]. Therefore, kidney epithelial cell lines have been established that appear to be derived from the ureteric bud and metanephric mesenchyme of the developing embryonic kidney of SV-40 transgenic mice [94, 95]. These mice have been used to establish a variety of "immortal" cell lines.

FIGURE 16-21

Ureteric bud cells undergo branching tubulogenesis in three-dimensional extracellular matrix gels. Cell line derived from ureteric bud (UB) and metanephric mesenchyme from day 11.5 mouse embryonic kidney undergo branching tubulogenesis in three-dimensional extracellular matrix gels. Here, UB cells have been induced to form branching tubular structures in response to "conditioned" media collected from the culture of metanephric mesenchymal cells. During normal kidney morphogenesis, these two embryonic cell types undergo a mutually inductive process that ultimately leads to the formation of functional nephrons [74-76]. This model system illustrates this process, ureteric bud cells being induced by factors secreted from metanephric mes-enchymal cells. Thus, this system could represent the simplest in vitro model with the greatest relevance to early kidney development [94]. A, UB cells grown for 1 week in the presence of conditioned media collected from cells cultured from the metanephric mesenchyme. Note the formation of multicellular cords. B, After 2 weeks' growth under the same conditions, UB cells have formed more substantial tubules, now with clear lumens. C, Interestingly, after 2 weeks of culture in a three-dimensional gel composed entirely of growth factor-reduced Matrigel, ureteric bud cells have not formed cords or tubules, only multicellular cysts. Thus, changing the matrix composition can alter the morphology from tubules to cysts, indicating that this model might also be relevant to renal cystic disease, much of which is of developmental origin. (From Sakurai et al. [94]; with permission.)

Free HGF and empty c-Met receptor

HGF binding to c-Met receptor

Dimerization of c-Met receptor and activation of Gab 1

Free HGF and empty c-Met receptor

HGF binding to c-Met receptor

Dimerization of c-Met receptor and activation of Gab 1

FIGURE 16-22

Growth factor binding

Up-regulation of proteases Mitogenic response Motogenic response Alteration of cytoskeleton Other responses

Branching morphogenesis

Up-regulation of proteases Mitogenic response Motogenic response Alteration of cytoskeleton Other responses

FIGURE 16-22

Signalling pathway of hepatocyte growth factor action. Diagram of the proposed intracellular signaling pathway involved in hepatocyte growth factor (HGF)-mediated tubulogenesis. Although HGF is perhaps the best-characterized of the growth factors involved in epithelial cell-branching tubulogenesis, very little of its mechanism of action is understood. However, recent evidence has shown that the HGF receptor (c-Met) is associated with Gab-1, a docking protein believed to be involved in signal transduction [96]. Thus, on binding to c-Met, HGF activates Gab-1-mediated signal transduction, which, by an unknown mechanism, affects changes in cell shape and cell movement or cell-cell-cell-matrix interactions. Ultimately, these alterations lead to epithelial cell-branching tubulogenesis.

FIGURE 16-23

Mechanism of growth factor action. Proposed model for the generalized response of epithelial cells to growth factors, which the depends on their environment. Epithelial cells constantly monitor their surrounding environment via extracellular receptors (ie, inte-grin receptors) and respond accordingly to growth factor stimulation. If the cells are in the appropriate environment, growth factor binding induces cellular responses necessary for branching tubulo-genesis. There are increases in the levels of extracellular proteinases and of structural and functional changes in the cytoarchitecture that enable the cells to form branching tubule structures.

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