Hypothesis: A New Role for the Renin-Angiotensin System in Ureteric Bud Branching

Branching morphogenesis in the developing mammalian kidney involves growth and branching of the ureteric bud (UB), leading to formation of its daughter collecting ducts, calyces, pelvis and ureters. Even subtle defects in the efficiency and/or accuracy of this process have profound effects on the ultimate development of the kidney and result in congenital abnormalities of the kidney and urinary tract. This review summarizes current knowledge regarding a number of genes known to regulate UB development and emphasizes an emerging role for the renin-angiotensin system (RAS) in renal branching morphogenesis. Mutations in the genes encoding components of the RAS in mice cause renal papillary hypoplasia, hydronephrosis, and urinary concentrating defect. These findings imply that UB-derived epithelia are targets for angiotensin (ANG) II actions during metanephric kidney development. Here, it is proposed that papillary hypoplasia in RAS-deficient mice is secondary to an intrinsic defect in the development of the renal medulla. This hypothesis is based on the following observations: (a) UB and surrounding stroma express angiotensinogen (AGT) and ANG II AT₁ receptors in vivo; (b) ANG II stimulates UB cell process extension, branching and cord formation in collagen gel cultures in vitro; and (c) AT₁ blockade inhibits ANG II-induced UB cell branching. It is further postulated that ANG II is a novel stroma-derived factor involved in stroma/UB cross-talk which regulates UB branching morphogenesis.Key Words: kidney development, branching morphogenesis, renin-angiotensin, stromal mesenchyme, ureteric bud     

Hypothesis: A New Role for the Renin-Angiotensin System in Ureteric Bud Branching

Branching morphogenesis in the developing mammalian kidney involves growth and branching of the ureteric bud (UB), leading to formation of its daughter collecting ducts, calyces, pelvis and ureters. Even subtle defects in the efficiency and/or accuracy of this process have profound effects on the ultimate development of the kidney and result in congenital abnormalities of the kidney and urinary tract. This review summarizes current knowledge regarding a number of genes known to regulate UB development and emphasizes an emerging role for the renin-angiotensin system (RAS) in renal branching morphogenesis. Mutations in the genes encoding components of the RAS in mice cause renal papillary hypoplasia, hydronephrosis, and urinary concentrating defect. These findings imply that UB-derived epithelia are targets for angiotensin (ANG) II actions during metanephric kidney development. Here, it is proposed that papillary hypoplasia in RAS-deficient mice is secondary to an intrinsic defect in the development of the renal medulla. This hypothesis is based on the following observations: a) UB and surrounding stroma express angiotensinogen (AGT) and ANG II AT1 receptors in vivo; b) ANG II stimulates UB cell process extension, branching and cord formation in collagen gel cultures in vitro; and c) AT1 blockade inhibits ANG II-induced UB cell branching. It is further postulated that ANG II is a novel stroma-derived factor involved in stroma/UB cross-talk which regulates UB branching morphogenesis.     

Regulation of prostate branching morphogenesis by activin A and follistatin

Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.     

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.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

Semaphorin3a inhibits ureteric bud branching morphogenesis

Class 3 semaphorins are guidance proteins involved in axon pathfinding, vascular patterning and lung branching morphogenesis in the developing mouse embryo. Semaphorin3a (Sema3a) is expressed in renal epithelia throughout kidney development, including podocytes and ureteric bud cells. However, the role of Sema3a in ureteric bud branching is unknown. Here we demonstrate that Sema3a plays a role in patterning the ureteric bud tree in both metanephric organ cultures and Sema3a mutant mice. In vitro ureteric bud injection with Sema3a antisense morpholino resulted in increased branching, whereas recombinant SEMA3A inhibited ureteric bud branching and decreased the number of developing glomeruli. Additional studies revealed that SEMA3A effects on ureteric bud branching involve downregulation of glial cell-line derived neurotrophic factor (GDNF) signaling, competition with vascular endothelial growth factor A (VEGF-A) and decreased activity of Akt survival pathways. Deletion of Sema3a in mice is associated with increased ureteric bud branching, confirming its inhibitory role in vivo. Collectively, these data suggest that Sema3a is an endogenous antagonist of ureteric bud branching and hence, plays a role in patterning the renal collecting system as a negative regulator.     

Hs2st mediated kidney mesenchyme induction regulates early ureteric bud branching

Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFβ superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st^−/− kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st^−/− UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development.     

Matrix Metalloproteinases MMP2 and MMP9 Are Produced in Early Stages of Kidney Morphogenesis but Only MMP9 Is Required for Renal Organogenesis In Vitro

We analyzed matrix metalloproteinase (MMP) production by 11-d embryonic mouse kidneys and the effects of these enzymes on subsequent renal organogenesis. In vivo, immunolocalization of metalloproteinases by laser scanning confocal microscopy and zymograms of kidney lysates showed that the mesenchyme of embryonic kidneys synthesized both MMP9 and MMP2 enzymes. In vitro, embryonic kidneys also secreted both enzymes when cultured in a medium devoid of hormone, growth factor, and serum for 24 h during which T-shaped branching of the ureter bud appeared. We then evaluated the role of MMP2 and MMP9 in kidney morphogenesis by adding anti-MMP2 or anti-MMP9 IgGs to the culture medium of 11-d kidneys for 24 or 72 h. Although it inhibited activity of the mouse enzyme, anti-MMP2 IgGs had no effect on kidney morphogenesis. In contrast, anti-MMP9 IgGs with enzyme-blocking activity impaired renal morphogenesis, in a concentration-dependent manner, by inhibiting T-shaped branching and further divisions of the ureter bud. This effect was irreversible, still observed after inductive events and reproduced by exogenous tissue inhibitor of metalloproteinase 1 (TIMP1), the natural inhibitor of MMP9. These data provide the first demonstration of MMP9 and MMP2 production in vivo by 11-d embryonic kidneys and further show that MMP9 is required in vitro for branching morphogenesis of the ureter bud.     

Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.     

Renal branching morphogenesis: concepts, questions, and recent advances

The ureteric bud (UB) is an outgrowth of the Wolffian duct, which undergoes a complex process of growth, branching, and remodeling, to eventually give rise to the entire urinary collecting system during kidney development. Understanding the mechanisms that control this process is a fascinating problem in basic developmental biology, and also has considerable medical significance. Over the past decade, there has been significant progress in our understanding of renal branching morphogenesis and its regulation, and this review focuses on several areas in which there have been recent advances. The first section deals with the normal process of UB branching morphogenesis, and methods that have been developed to better observe and describe it. The next section discusses a number of experimental methodologies, both established and novel, that make kidney development in the mouse a powerful and attractive experimental system. The third section discusses some of the cellular processes that are likely to underlie UB branching morphogenesis, as well as recent data on cell lineages within the growing UB. The fourth section summarizes our understanding of the roles of two groups of growth factors that appear to be particularly important for the regulation of UB outgrowth and branching: GDNF and FGFs, which stimulate this process via tyrosine kinase receptors, and members of the TGFbeta family, including BMP4 and Activin A, which generally inhibit UB formation and branching.     

Canonical WNT/beta-catenin signaling is required for ureteric branching

WNT/beta-catenin signaling has an established role in nephron formation during kidney development. Yet, the role of beta-catenin during ureteric morphogenesis in vivo is undefined. We generated a murine genetic model of beta-catenin deficiency targeted to the ureteric bud cell lineage. Newborn mutant mice demonstrated bilateral renal aplasia or renal dysplasia. Analysis of the embryologic events leading to this phenotype revealed that abnormal ureteric branching at E12.5 precedes histologic abnormalities at E13.5. Microarray analysis of E12.5 kidney tissue identified decreased Emx2 and Lim1 expression among a small subset of renal patterning genes disrupted at the stage of abnormal branching. These alterations are followed by decreased expression of genes downstream of Emx2, including Lim1, Pax2, and the ureteric tip markers, c-ret and Wnt 11. Together, these data demonstrate that beta-catenin performs essential functions during renal branching morphogenesis via control of a hierarchy of genes that control ureteric branching.     

Branching morphogenesis and kidney disease

Branching morphogenesis in the kidney is a tightly regulated, complex process and its disruption potentially can lead to a broad spectrum of diseases, ranging from rare hereditary syndromes to common conditions such as hypertension and chronic kidney failure. This review synthesizes data on branching during kidney development derived from in vitro and in vivo rodent studies and to apply them to human diseases. It discusses how the broad organization of molecular interactions during kidney development might provide a mechanistic framework for understanding disorders related to aberrant branching.     

Growth and branching morphogenesis of rat collecting duct anlagen in the absence of metanephrogenic mesenchyme

The growth and differentiation of the epithelium in many tissues is mediated by interactions with the adjacent mesenchyme, but the mechanisms responsible remain undefined. To identify the factors involved in the growth and branching morphogenesis of ureteric bud, which is the collecting duct anlagen, buds from 13-gestation-day rat embryos were separated from the metanephrogenic mesenchyme and explanted to culture dishes coated with gelled type I collagen in a defined medium. Under these conditions buds attached to the substrate and grew out without indication of cell senescence. When buds were instead suspended in gelled type I collagen, branching morphogenesis was observed despite the absence of mesenchyme although it was not as extensive as in vivo. Since growth occurred much more slowly in culture than expected, culture conditions were varied in attempts to accelerate the process. Despite extensive screening of matrices and growth factors, only epidermal and endothelial cell growth factors stimulated growth to a significant extent. Transforming growth factor-beta, on the other hand, was a potent inhibitor of growth. Homogenates from tumors that caricature metanephrogenic mesenchyme were highly mitogenic for bud cells and, thus, will be a source of material for characterizing regulatory factors involved in renal growth. These studies show that growth and branching morphogenesis of the ureteric bud can occur without direct cell-cell interactions with the metanephrogenic mesenchyme and that matrices and factors secreted by the mesenchyme may mediated these activities in vivo.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.     

Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development

Reciprocal cell-cell interactions between the ureteric epithelium and the metanephric mesenchyme are needed to drive growth and differentiation of the embryonic kidney to completion. Branching morphogenesis of the Wolffian duct derived ureteric bud is integral in the generation of ureteric tips and the elaboration of the collecting duct system. Wnt11, a member of the Wnt superfamily of secreted glycoproteins, which have important regulatory functions during vertebrate embryonic development, is specifically expressed in the tips of the branching ureteric epithelium. In this work, we explore the role of Wnt11 in ureteric branching and use a targeted mutation of the Wnt11 locus as an entrance point into investigating the genetic control of collecting duct morphogenesis. Mutation of the Wnt11 gene results in ureteric branching morphogenesis defects and consequent kidney hypoplasia in newborn mice. Wnt11 functions, in part, by maintaining normal expression levels of the gene encoding glial cell-derived neurotrophic factor (Gdnf). Gdnf encodes a mesenchymally produced ligand for the Ret tyrosine kinase receptor that is crucial for normal ureteric branching. Conversely, Wnt11 expression is reduced in the absence of Ret/Gdnf signaling. Consistent with the idea that reciprocal interaction between Wnt11 and Ret/Gdnf regulates the branching process, Wnt11 and Ret mutations synergistically interact in ureteric branching morphogenesis. Based on these observations, we conclude that Wnt11 and Ret/Gdnf cooperate in a positive autoregulatory feedback loop to coordinate ureteric branching by maintaining an appropriate balance of Wnt11-expressing ureteric epithelium and Gdnf-expressing mesenchyme to ensure continued metanephric development.