Growth
factors and metanephrogenesis
MARC
R. HAMMERMAN,
SHARON
A. ROGERS,
AND GABRIELLA
RYAN
Renal Division, Departments of Internal Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis Missouri 63110 Hammerman, Marc R., Sharon A. Rogers, and Gabriella Ryan. factors and metanephrogenesis. Am. J. Physid. 262 (Renal Fluid Electrolyte Physiol. 31): F523-F532 1992.-The formation of all organs during embryogenesis,including kidney, is dependent on the timed and sequentialexpressionof a number of polypeptide growth factors. Synthesis and actions of one or more membersof the insulin-like growth factor, epidermal growth factor/transforming growth factor-a, transforming growth factor-@,platelet-derived growth factor, fibroblast growth factor, and Growth
nerve growth
factor
families
have been characterized
in the developing
metanephric kidney. Studiesoriginating from a numberof laboratorieshave defined the localization of growth factor mRNAs, receptors and peptides, have delineated patterns of growth factor synthesis, and have established the growth factor dependency of embryonic kidney development. The results of these investigations will be summarizedin this editorial review and integrated within the broader context of growth factor cellular physiology and growth
factor expression
in nonrenal
systems.
epidermalgrowth factor; fibroblast growth factor; insulin-like growth factor; nerve growth factor; platelet-derived growth factor; transforming growth factor-a; transforming growth factor-p MANY POLYPEPTIDE GROWTH FACTORS can be isolated
from plasma and have “endocrine” functions analogous to those of circulating hormones. However, it is clear that the majority, if not all polypeptide growth factors, also exert actions on the cell in which they are produced or in cells immediately adjacent to the sites of their productions and in this regard function as “autocrine,” “paracrine,” or “juxtacrine” (39) agents. Although growth factor expression has been studied in almost all organs, the kidney provides an excellent model for the characterization of intraorgan autocrine/paracrine/ juxtacrine systems. Compelling evidence exists that a number of growth factors produced within renal tissue exert actions on adjacent cells. The polypeptide growth factors/growth factor families, the synthesis of which within kidney of adult animals is best characterized, are the insulin-like growth factors (IGFs) (28), epidermal growth factor/transforming growth factor-a! (EGF/ TGF-cu) (21), transforming growth factor-p (TGF-p), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) (40, 64, 69), and nerve growth factor (NGF) (38, 43, 67, 68). In addition to their roles in the physiology of adult animals, polypeptide growth factors regulate growth and development during embryogenesis. These agents control the differentiation of germ layers early in vertebrate embryogenesis and, at a later stage in development, they modulate the growth and differentiative processes of organogenesis (41). The evidence supporting this latter role originates from two types of investigations as follows: 1) studies that map the distribution of growth 0363-6127/92
factor mRNA, peptide, and growth factor receptors as a function of developmental stage; and 2) studies designed to perturb growth and differentiation of tissue anlagen grown in vitro by altering the growth factor environment. During the past several years we and others have investigated the role of polypeptide growth factors in the growth and development of kidney tissue using the approaches outlined above. The foundations for these studies have been large bodies of existing and evolving knowledge defining renal developmental anatomy, the cellular biology of kidney development, and the roles that growth factors play in normal physiology and during organogenesis of nonrenal structures. The purpose of this editorial review is to summarize what is known about polypeptide growth factor participation in formation of the kidney within the context of knowledge relating to processes of nephrogenesis in particular, and organogenesis in general, and relating to the cellular and molecular biology of polypeptide growth factors. EMBRYONICKIDNEYDEVELOPMENT
It is customary to distinguish between three spatially and temporally different embryonic renal excretory organs. These are the pronephros, the mesonephros, and the metanephros. The pronephros is the most primitive of these organs, and it is functional during embryogenesis only in some of the lower vertebrates. The mesonephros, located further caudally, is functional during embryogenesis in higher fishes and amphibians. The metanephros, located most caudally, serves as the permanent kidney of
$2.00 Copyright 0 1992 the American Physiological
Society
F523
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
F524
EDITORIAL
the amniotes (Fig. 1). The pronephros, mesonephros, and metanephros each consist of paired organs derived from the intermediate mesoderm that is located immediately lateral to the somites. Pronephric tubules empty into the primary nephric ducts. These ducts are appropriated by the developing mesonephros and, after degeneration of the pronephric tubules, are designated mesonephric
REVIEW
ducts. These ducts empty into the cloaca. At a later stage in development, paired outgrowths develop from the mesonephric ducts near their cloaca1 ends (Fig. 2). This occurs during the 5th wk of gestation in humans, during day 12 of embryonic rat development and during day 11 of embryonic mouse development (61). These so-called metanephric diverticula, or ureteric buds, collect about their
Fig. 1. Paired mesonephroi (Mes) and metanephroi (Met) dissected from a Fig. 2. Photomicrograph of histological sections originating from !3-day-old (28) sections of embryos. Ureteric buds are marked with and transverse condensed metanephric blastema. Fig. 3. Photomicrographs of histological sections originating from 13-day-old and transverse (3B) sections illustrating ureteric buds (U) and surrounding
15-day-old rat embryo. rat embry an arrow and have collected rat embryos. metanephric
about
Shown arc parasagittal blastema (MB).
them (3A)
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
EDITORIAL
distal ends intermediate mesoderm caudal to the mesonephros. This mesoderm gives rise to the excretory tubules of the metanephros and for that reason is designated metanephrogenic mesenchyme, or metanephric blastema (Fig. 3). Numerous outgrowths arise from the distal end of the ureteric bud, which push radially into the surrounding mass of metanephric blastema. These outgrowths become hollow and give rise to the collecting ducts of the kidneys. The proximal ends of the ureteric bud give rise to the ureter and renal pelvis. The metanephric blastema differentiates into all of the tubular structures of the adult nephron with the exception of the collecting system. Differentiation of the metanephric blastema and of the ureteric bud is dependent on an inductive event(s) that occurs when the ureteric bud encounters the metanephric blastema. The nature of this event(s) is unknown. There is disagreement as to whether humoral agents trigger differentiation or whether cell-cell contact is also required for this process to be initiated (61). Following induction of the metanephros, tubulogenesis proceeds through a histologically defined sequence of stages. Initially the metanephric blastema undergoes condensation into a rounded structure, the cells in which proliferate as part of a growth stage. Following the growth phase, mitotic activity decreases, and the tubular anlage assumes a comma shape. Cells farthest from the collecting duct become elongated, and some become funnel shaped. This is the site of formation of a slit that represents the first sign of the glomerular crevice. While glomerulogenesis is taking place, a second slit forms at the opposite distal pole, and the tubular anlage assumes an S shape and joins the collecting system (Fig. 4) (61). The agents that control and regulate the postinductive growth and differentiation of the metanephros are unknown. It is proposed that a number of cell adhesion molecules, components of the extracellular matrix, and polypeptide growth factors coordinate the differentiative events (1, 8). The physiology of this process of coordination is not well delineated. However, the nature of potential interactions between growth factors, adhesion moleproteins can be inferred from cules, and matrix observations made in nonrenal systems. For example, TGF-P is known to enhance gene expression for extracellular matrix components (5). In addition, matrix components such as proteoglycans modulate growth factor activities (58). Vascularization of the nephron results from the interaction of a pair of vessels arising from the dorsal aorta with the S-shaped body. These vessels invade the metanephric blastema in close association with the branching ureteric bud. The endothelial cells of the vessels migrate in apposition to the condensing metanephric blastema and into the lower crevice of the S-shaped glomerular anlage (Fig. 4). At this point migration ceases, and the endothelial basement membrane fuses to that of epithelial podocytes to create the glomerular basement membrane (61). REGULATION DEVELOPMENT
OF
METANEPHRIC BY POLYPEPTIDE
GROWTH GROWTH
AND FACTORS
An enlarging body of experimental findings document the participation of several growth factors in the metanephrogenic process. These findings are most easily
REVIEW
Fig. 4. Semi-schematic illustration of the early nologically) of the nephron from condensation Reproduced with permission (61).
F525
development (A-F, chroto the S-shaped body.
understood within the context of the cellular biology of participating growth factors. Observations made in kidney can be integrated with those originating in nonrenal systems to gain insight into general mechanisms of growth factor action during embryogenesis. IGF-I and IGF-II. IGF-I is a single-chain insulin-like polypeptide that is 70 amino acids in length. It circulates in tight noncovalent association with specific carrier proteins. The functions of the carrier proteins are incompletely defined. In vitro, one or another carrier protein has been shown to function as both agonist and antagonist for the biological activity of IGF-I. It originally was thought that IGF-I in circulation mediated growth-promoting actions of growth hormone (GH) as part of a GH-IGF-I endocrine axis. However, presently, it is recognized that IGF-I is produced in several tissues including kidney in a GH-dependent manner as part of GHIGF-I autocrine or paracrine axes. Local production of IGF-I is mediated by factors in addition to GH. In addition to its role in adult physiology, IGF-I is thought to participate during embryogenesis in growth and development of a number of organ systems. GH is not a stimulus
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
F526
EDITORIAL
Fig. 5. Photograph of histological sections originating from 13-day-old rat embryos grown in organ culture for 5 days. Shown is condensing metanephric blastema (GB) surrounding the ureteric bud (U) (day I); pretubular condensates (PC) (day 2); a comma-shaped body (CB) (day 3); an S-shaped body (SB) with a glomerular crevice (GC) (day 4); and an avascular glomerulus (G) (day 5).
for IGF-I production during embryogenesis. However, one or more GH-like peptides may regulate IGF-I gene expression in this setting (18). IGF-II is slightly smaller than IGF-I (67 amino acids). It also circulates in tight noncovalent association with the carrier proteins that bind IGF-I. Because levels of IGF-II mRNA in all tissues, except neural tissue, and levels of circulating IGF-II fall precipitously in rodents following birth, it has been postulated that IGF-II functions predominantly as a fetal growth factor. However, no such fall occurs in humans, consistent with a role for IGF-II in adult physiology (18). Whereas the cell membrane receptor for IGF-I is similar in structure to the tetrameric insulin receptor, the IGF-II receptor is homologous to the cation-independent mannose 6-phosphate receptor. The IGF-II/mannose 6-phosphate receptor binds ligands that contain mannose 6-phosphate in addition to binding IGF-II (57). We have shown that two such ligands are capable of activating phospholipase C via this receptor. These are the precursor form of TGF-01 and the GH-like molecule, proliferin (53).
REVIEW
Mice homozygous for deletion of the IGF-II gene are dwarfed at birth, but normally developed. It is proposed that IGF-II is an indispensable participant in some early crucial proliferation events, but that subsequently growth becomes IGF-II independent. However, such a proposal cannot account for the pattern of IGF-II expression during embryogenesis that implies a role in organogenesis. Accordingly, it is suggested that there may be overlap or compensation of IGF-II function by other gene products affecting growth (16). IGF-II is expressed in human fetal kidney predominantly in undifferentiated stromal and blastemal cells. Differentiated epithelial structures show a relative absence of IGF-II expression (33). To examine the roles that endogenously produced IGFI and IGF-II play in growth and development during renal organogenesis, we removed metanephroi from 13-day-old rat embryos and grew them in organ culture using chemically defined, serum-free conditions. Growth of metanephroi in vitro recapitulates many events of nephrogenesis in vivo and has provided a useful model for kidney development (3). Individual metanephric kidneys were allowed to grow for up to 6 days in organ culture. Over this period of time the metanephroi increased in size and morphological complexity. The diameter of the long axis increased by m-50%, and the renal anlage adopted a morphology that was more kidneylike in appearance [see fig. 1 in Rogers et al. (55)]. The ureteric bud underwent extensive arborization during 6 days in culture, and elements of the nephron became differentiated within the metanephric blastema (see fig. 2 in Ref. 55). Metanephrogenesis proceeds in vitro through the stages described for this process in vivo (3). By 4-5 days in culture, nephrons with avascular glomeruli had differentiated within developing rat kidneys (Fig. 5). Since the origin of the glomerular blood vessels is extrametanephric, vascularization of the nephron does not occur in the organ culture system (3). Messenger RNAs for both IGFs were detected in the metanephroi by amplification of cDNA following reverse transcription of mRNA, using the polymerase chain reaction (Fig. 6). IGF-I and IGF-II contents of media were determined by specific radioimmunoassays following extraction and separation of IGFs by molecular weight IGF I
IGF II
241
I
bp
I
130
Metanephros
Fig. 6. Insulin-like growth factors (IGF) I and II mRNAs are present in the rat metanephros. Shown is a 5% polyacrylamide gel stained with ethidium bromide. Left: molecular size markers expressed in base pairs (bp). RLght: sizes of IGF-I (241 bp) and IGF-II (285 bp) cDNAs amplified using the polymerase chain reaction. Reproduced from J. Cell Bill. 113: 1447-1453, 1991; by copyright permission of the Rockefeller University Press (54).
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
EDITORIAL *Or
OIGFI
_
q
IGFII
T
T
70 60 ? ; 50 Q E40 30 P 20 10 !&
: 1
2
3 DAYS
IN
4
5
6
CULTURE
Fig. 7. Levels of IGF-I and IGF-II in media removed from developing rat metanephroi. Volumes of the media were 1 ml/explant. Reproduced from J. Cell Biol. 113: 1447-1453, 1991; by copyright permission of the Rockefeller University Press (54).
using reverse-phase, high-performance liquid chromatography (HPLC). Immunoreactive IGF-I and IGF-II were produced by the renal anlagen and released into culture media. Levels of these peptides were relatively constant during the 6 days in culture and averaged 5.5 X 10eg M IGF-I and 8.3 x 10eg M IGF-II in media removed from metanephroi after 24 h of contact (Fig. 7). Such levels exceed the inhibition constant (Ki) for binding of IGFs to most receptors, including those in renal tissue (29). In contrast to the presence of peptides in supernatants, no IGF binding protein activity was detected (54). IGF-I produced by the metanephroi was indistinguishable from recombinant human IGF-I in a radioreceptor assay that measured binding of 1251-IGF-I to isolated canine renal proximal tubular basolateral membranes. IGF-II produced by the metanephroi was indistinguishable from human recombinant IGF-II in a biological assay that measured mannose 6-phosphate-activated phospholipase C activation in proximal tubular basolatera1 membranes (54). Metanephric kidneys were cultured in the absence or presence of highly specific anti-IGF-I or anti-IGF-II antibodies or appropriate control additions. Growth and development of metanephroi, as evaluated by gross visual inspection (Fig. 8) and by histological examination of tissues (54), were prevented by both anti-IGF-I and antiIGF-II antibodies. Similarly, anti-IGF-II receptor antibodies prevented growth and development in vitro (Fig. 8). These observations demonstrate production of IGF-I and IGF-II by developing rat metanephroi in organ culture. Each of the peptides is necessary for growth and development of the renal anlage to take place in vitro. Our findings suggest that both IGFs are produced within the developing metanephros in vivo and promote renal organogenesis (54). EGF/TGF-a. EGF and TGF-a are members of the same growth factor family and are thought to act via the same membrane receptor (13, 20). EGF is a 53-amino acid peptide. Tissue levels are highest in salivary glands of adult mammals, followed by kidney. Although the relative protein abundance is 2,000:1, relative prepro-EGF mRNA abundance is 2: 1 in submaxillary gland compared with whole kidney. In humans, EGF was first purified from urine. The high levels in urine (5 x lo-lo M) com-
REVIEW
F527
pared with blood (
factors and metanephrogenesis
MARC
R. HAMMERMAN,
SHARON
A. ROGERS,
AND GABRIELLA
RYAN
Renal Division, Departments of Internal Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis Missouri 63110 Hammerman, Marc R., Sharon A. Rogers, and Gabriella Ryan. factors and metanephrogenesis. Am. J. Physid. 262 (Renal Fluid Electrolyte Physiol. 31): F523-F532 1992.-The formation of all organs during embryogenesis,including kidney, is dependent on the timed and sequentialexpressionof a number of polypeptide growth factors. Synthesis and actions of one or more membersof the insulin-like growth factor, epidermal growth factor/transforming growth factor-a, transforming growth factor-@,platelet-derived growth factor, fibroblast growth factor, and Growth
nerve growth
factor
families
have been characterized
in the developing
metanephric kidney. Studiesoriginating from a numberof laboratorieshave defined the localization of growth factor mRNAs, receptors and peptides, have delineated patterns of growth factor synthesis, and have established the growth factor dependency of embryonic kidney development. The results of these investigations will be summarizedin this editorial review and integrated within the broader context of growth factor cellular physiology and growth
factor expression
in nonrenal
systems.
epidermalgrowth factor; fibroblast growth factor; insulin-like growth factor; nerve growth factor; platelet-derived growth factor; transforming growth factor-a; transforming growth factor-p MANY POLYPEPTIDE GROWTH FACTORS can be isolated
from plasma and have “endocrine” functions analogous to those of circulating hormones. However, it is clear that the majority, if not all polypeptide growth factors, also exert actions on the cell in which they are produced or in cells immediately adjacent to the sites of their productions and in this regard function as “autocrine,” “paracrine,” or “juxtacrine” (39) agents. Although growth factor expression has been studied in almost all organs, the kidney provides an excellent model for the characterization of intraorgan autocrine/paracrine/ juxtacrine systems. Compelling evidence exists that a number of growth factors produced within renal tissue exert actions on adjacent cells. The polypeptide growth factors/growth factor families, the synthesis of which within kidney of adult animals is best characterized, are the insulin-like growth factors (IGFs) (28), epidermal growth factor/transforming growth factor-a! (EGF/ TGF-cu) (21), transforming growth factor-p (TGF-p), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) (40, 64, 69), and nerve growth factor (NGF) (38, 43, 67, 68). In addition to their roles in the physiology of adult animals, polypeptide growth factors regulate growth and development during embryogenesis. These agents control the differentiation of germ layers early in vertebrate embryogenesis and, at a later stage in development, they modulate the growth and differentiative processes of organogenesis (41). The evidence supporting this latter role originates from two types of investigations as follows: 1) studies that map the distribution of growth 0363-6127/92
factor mRNA, peptide, and growth factor receptors as a function of developmental stage; and 2) studies designed to perturb growth and differentiation of tissue anlagen grown in vitro by altering the growth factor environment. During the past several years we and others have investigated the role of polypeptide growth factors in the growth and development of kidney tissue using the approaches outlined above. The foundations for these studies have been large bodies of existing and evolving knowledge defining renal developmental anatomy, the cellular biology of kidney development, and the roles that growth factors play in normal physiology and during organogenesis of nonrenal structures. The purpose of this editorial review is to summarize what is known about polypeptide growth factor participation in formation of the kidney within the context of knowledge relating to processes of nephrogenesis in particular, and organogenesis in general, and relating to the cellular and molecular biology of polypeptide growth factors. EMBRYONICKIDNEYDEVELOPMENT
It is customary to distinguish between three spatially and temporally different embryonic renal excretory organs. These are the pronephros, the mesonephros, and the metanephros. The pronephros is the most primitive of these organs, and it is functional during embryogenesis only in some of the lower vertebrates. The mesonephros, located further caudally, is functional during embryogenesis in higher fishes and amphibians. The metanephros, located most caudally, serves as the permanent kidney of
$2.00 Copyright 0 1992 the American Physiological
Society
F523
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
F524
EDITORIAL
the amniotes (Fig. 1). The pronephros, mesonephros, and metanephros each consist of paired organs derived from the intermediate mesoderm that is located immediately lateral to the somites. Pronephric tubules empty into the primary nephric ducts. These ducts are appropriated by the developing mesonephros and, after degeneration of the pronephric tubules, are designated mesonephric
REVIEW
ducts. These ducts empty into the cloaca. At a later stage in development, paired outgrowths develop from the mesonephric ducts near their cloaca1 ends (Fig. 2). This occurs during the 5th wk of gestation in humans, during day 12 of embryonic rat development and during day 11 of embryonic mouse development (61). These so-called metanephric diverticula, or ureteric buds, collect about their
Fig. 1. Paired mesonephroi (Mes) and metanephroi (Met) dissected from a Fig. 2. Photomicrograph of histological sections originating from !3-day-old (28) sections of embryos. Ureteric buds are marked with and transverse condensed metanephric blastema. Fig. 3. Photomicrographs of histological sections originating from 13-day-old and transverse (3B) sections illustrating ureteric buds (U) and surrounding
15-day-old rat embryo. rat embry an arrow and have collected rat embryos. metanephric
about
Shown arc parasagittal blastema (MB).
them (3A)
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
EDITORIAL
distal ends intermediate mesoderm caudal to the mesonephros. This mesoderm gives rise to the excretory tubules of the metanephros and for that reason is designated metanephrogenic mesenchyme, or metanephric blastema (Fig. 3). Numerous outgrowths arise from the distal end of the ureteric bud, which push radially into the surrounding mass of metanephric blastema. These outgrowths become hollow and give rise to the collecting ducts of the kidneys. The proximal ends of the ureteric bud give rise to the ureter and renal pelvis. The metanephric blastema differentiates into all of the tubular structures of the adult nephron with the exception of the collecting system. Differentiation of the metanephric blastema and of the ureteric bud is dependent on an inductive event(s) that occurs when the ureteric bud encounters the metanephric blastema. The nature of this event(s) is unknown. There is disagreement as to whether humoral agents trigger differentiation or whether cell-cell contact is also required for this process to be initiated (61). Following induction of the metanephros, tubulogenesis proceeds through a histologically defined sequence of stages. Initially the metanephric blastema undergoes condensation into a rounded structure, the cells in which proliferate as part of a growth stage. Following the growth phase, mitotic activity decreases, and the tubular anlage assumes a comma shape. Cells farthest from the collecting duct become elongated, and some become funnel shaped. This is the site of formation of a slit that represents the first sign of the glomerular crevice. While glomerulogenesis is taking place, a second slit forms at the opposite distal pole, and the tubular anlage assumes an S shape and joins the collecting system (Fig. 4) (61). The agents that control and regulate the postinductive growth and differentiation of the metanephros are unknown. It is proposed that a number of cell adhesion molecules, components of the extracellular matrix, and polypeptide growth factors coordinate the differentiative events (1, 8). The physiology of this process of coordination is not well delineated. However, the nature of potential interactions between growth factors, adhesion moleproteins can be inferred from cules, and matrix observations made in nonrenal systems. For example, TGF-P is known to enhance gene expression for extracellular matrix components (5). In addition, matrix components such as proteoglycans modulate growth factor activities (58). Vascularization of the nephron results from the interaction of a pair of vessels arising from the dorsal aorta with the S-shaped body. These vessels invade the metanephric blastema in close association with the branching ureteric bud. The endothelial cells of the vessels migrate in apposition to the condensing metanephric blastema and into the lower crevice of the S-shaped glomerular anlage (Fig. 4). At this point migration ceases, and the endothelial basement membrane fuses to that of epithelial podocytes to create the glomerular basement membrane (61). REGULATION DEVELOPMENT
OF
METANEPHRIC BY POLYPEPTIDE
GROWTH GROWTH
AND FACTORS
An enlarging body of experimental findings document the participation of several growth factors in the metanephrogenic process. These findings are most easily
REVIEW
Fig. 4. Semi-schematic illustration of the early nologically) of the nephron from condensation Reproduced with permission (61).
F525
development (A-F, chroto the S-shaped body.
understood within the context of the cellular biology of participating growth factors. Observations made in kidney can be integrated with those originating in nonrenal systems to gain insight into general mechanisms of growth factor action during embryogenesis. IGF-I and IGF-II. IGF-I is a single-chain insulin-like polypeptide that is 70 amino acids in length. It circulates in tight noncovalent association with specific carrier proteins. The functions of the carrier proteins are incompletely defined. In vitro, one or another carrier protein has been shown to function as both agonist and antagonist for the biological activity of IGF-I. It originally was thought that IGF-I in circulation mediated growth-promoting actions of growth hormone (GH) as part of a GH-IGF-I endocrine axis. However, presently, it is recognized that IGF-I is produced in several tissues including kidney in a GH-dependent manner as part of GHIGF-I autocrine or paracrine axes. Local production of IGF-I is mediated by factors in addition to GH. In addition to its role in adult physiology, IGF-I is thought to participate during embryogenesis in growth and development of a number of organ systems. GH is not a stimulus
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
F526
EDITORIAL
Fig. 5. Photograph of histological sections originating from 13-day-old rat embryos grown in organ culture for 5 days. Shown is condensing metanephric blastema (GB) surrounding the ureteric bud (U) (day I); pretubular condensates (PC) (day 2); a comma-shaped body (CB) (day 3); an S-shaped body (SB) with a glomerular crevice (GC) (day 4); and an avascular glomerulus (G) (day 5).
for IGF-I production during embryogenesis. However, one or more GH-like peptides may regulate IGF-I gene expression in this setting (18). IGF-II is slightly smaller than IGF-I (67 amino acids). It also circulates in tight noncovalent association with the carrier proteins that bind IGF-I. Because levels of IGF-II mRNA in all tissues, except neural tissue, and levels of circulating IGF-II fall precipitously in rodents following birth, it has been postulated that IGF-II functions predominantly as a fetal growth factor. However, no such fall occurs in humans, consistent with a role for IGF-II in adult physiology (18). Whereas the cell membrane receptor for IGF-I is similar in structure to the tetrameric insulin receptor, the IGF-II receptor is homologous to the cation-independent mannose 6-phosphate receptor. The IGF-II/mannose 6-phosphate receptor binds ligands that contain mannose 6-phosphate in addition to binding IGF-II (57). We have shown that two such ligands are capable of activating phospholipase C via this receptor. These are the precursor form of TGF-01 and the GH-like molecule, proliferin (53).
REVIEW
Mice homozygous for deletion of the IGF-II gene are dwarfed at birth, but normally developed. It is proposed that IGF-II is an indispensable participant in some early crucial proliferation events, but that subsequently growth becomes IGF-II independent. However, such a proposal cannot account for the pattern of IGF-II expression during embryogenesis that implies a role in organogenesis. Accordingly, it is suggested that there may be overlap or compensation of IGF-II function by other gene products affecting growth (16). IGF-II is expressed in human fetal kidney predominantly in undifferentiated stromal and blastemal cells. Differentiated epithelial structures show a relative absence of IGF-II expression (33). To examine the roles that endogenously produced IGFI and IGF-II play in growth and development during renal organogenesis, we removed metanephroi from 13-day-old rat embryos and grew them in organ culture using chemically defined, serum-free conditions. Growth of metanephroi in vitro recapitulates many events of nephrogenesis in vivo and has provided a useful model for kidney development (3). Individual metanephric kidneys were allowed to grow for up to 6 days in organ culture. Over this period of time the metanephroi increased in size and morphological complexity. The diameter of the long axis increased by m-50%, and the renal anlage adopted a morphology that was more kidneylike in appearance [see fig. 1 in Rogers et al. (55)]. The ureteric bud underwent extensive arborization during 6 days in culture, and elements of the nephron became differentiated within the metanephric blastema (see fig. 2 in Ref. 55). Metanephrogenesis proceeds in vitro through the stages described for this process in vivo (3). By 4-5 days in culture, nephrons with avascular glomeruli had differentiated within developing rat kidneys (Fig. 5). Since the origin of the glomerular blood vessels is extrametanephric, vascularization of the nephron does not occur in the organ culture system (3). Messenger RNAs for both IGFs were detected in the metanephroi by amplification of cDNA following reverse transcription of mRNA, using the polymerase chain reaction (Fig. 6). IGF-I and IGF-II contents of media were determined by specific radioimmunoassays following extraction and separation of IGFs by molecular weight IGF I
IGF II
241
I
bp
I
130
Metanephros
Fig. 6. Insulin-like growth factors (IGF) I and II mRNAs are present in the rat metanephros. Shown is a 5% polyacrylamide gel stained with ethidium bromide. Left: molecular size markers expressed in base pairs (bp). RLght: sizes of IGF-I (241 bp) and IGF-II (285 bp) cDNAs amplified using the polymerase chain reaction. Reproduced from J. Cell Bill. 113: 1447-1453, 1991; by copyright permission of the Rockefeller University Press (54).
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 22, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
EDITORIAL *Or
OIGFI
_
q
IGFII
T
T
70 60 ? ; 50 Q E40 30 P 20 10 !&
: 1
2
3 DAYS
IN
4
5
6
CULTURE
Fig. 7. Levels of IGF-I and IGF-II in media removed from developing rat metanephroi. Volumes of the media were 1 ml/explant. Reproduced from J. Cell Biol. 113: 1447-1453, 1991; by copyright permission of the Rockefeller University Press (54).
using reverse-phase, high-performance liquid chromatography (HPLC). Immunoreactive IGF-I and IGF-II were produced by the renal anlagen and released into culture media. Levels of these peptides were relatively constant during the 6 days in culture and averaged 5.5 X 10eg M IGF-I and 8.3 x 10eg M IGF-II in media removed from metanephroi after 24 h of contact (Fig. 7). Such levels exceed the inhibition constant (Ki) for binding of IGFs to most receptors, including those in renal tissue (29). In contrast to the presence of peptides in supernatants, no IGF binding protein activity was detected (54). IGF-I produced by the metanephroi was indistinguishable from recombinant human IGF-I in a radioreceptor assay that measured binding of 1251-IGF-I to isolated canine renal proximal tubular basolateral membranes. IGF-II produced by the metanephroi was indistinguishable from human recombinant IGF-II in a biological assay that measured mannose 6-phosphate-activated phospholipase C activation in proximal tubular basolatera1 membranes (54). Metanephric kidneys were cultured in the absence or presence of highly specific anti-IGF-I or anti-IGF-II antibodies or appropriate control additions. Growth and development of metanephroi, as evaluated by gross visual inspection (Fig. 8) and by histological examination of tissues (54), were prevented by both anti-IGF-I and antiIGF-II antibodies. Similarly, anti-IGF-II receptor antibodies prevented growth and development in vitro (Fig. 8). These observations demonstrate production of IGF-I and IGF-II by developing rat metanephroi in organ culture. Each of the peptides is necessary for growth and development of the renal anlage to take place in vitro. Our findings suggest that both IGFs are produced within the developing metanephros in vivo and promote renal organogenesis (54). EGF/TGF-a. EGF and TGF-a are members of the same growth factor family and are thought to act via the same membrane receptor (13, 20). EGF is a 53-amino acid peptide. Tissue levels are highest in salivary glands of adult mammals, followed by kidney. Although the relative protein abundance is 2,000:1, relative prepro-EGF mRNA abundance is 2: 1 in submaxillary gland compared with whole kidney. In humans, EGF was first purified from urine. The high levels in urine (5 x lo-lo M) com-
REVIEW
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pared with blood (
