Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development.

In this study, five different in vitro assays, which together recapitulate much of kidney development, were used to examine the role of the Rho-associated protein serine/threonine kinase (ROCK) in events central to ureteric bud (UB) and metanephric mesenchyme (MM) morphogenensis, in isolation and together. ROCK activity was found to be critical for (1) cell proliferation, growth, and development of the whole embryonic kidney in organ culture, (2) tip and stalk formation in cultures of isolated UBs, and (3) migration of MM cells (in a novel MM migration assay) during their condensation at UB tips (in a UB/MM recombination assay). Together, the data indicate selective involvement of Rho/ROCK in distinct morphogenetic processes necessary for kidney development and that the coordination of these events by Rho/ROCK provides a potential mechanism to regulate overall branching patterns, nephron formation, and thus, kidney architecture.

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney.

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.

Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney.

Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.

TGF-beta superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud.

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.

Developmental approaches to kidney tissue engineering.

Recent advances in our understanding of the developmental biology of the kidney, as well as the establishment of novel in vitro model systems, have potential implications for kidney tissue engineering. These advances include delineation of the roles of a number of growth factors in the developmental programs of branching morphogenesis and mesenchymal differentiation, a new understanding of the roles of the extracellular matrix, identification of potential "renal" stem cells, the ex vivo propagation and subsequent recombination of isolated components of the kidney, and successful transplantation of renal primordia into adult hosts. This review will examine these advances in the context of approaches to tissue engineering. Finally, novel approaches that synthesize advances in both cell-based and organ-based approaches are proposed.

A strategy for in vitro propagation of rat nephrons.

BACKGROUND: Recent advances in the understanding of the molecular biology of rodent renal development have lead to the ability to culture the components of the developing rat kidney-the ureteric bud (UB) and the metanephric mesenchyme (MM)-in isolation from one another. Here we here describe a method for subculturing and propagating either whole rat metanephric rudiments or isolated rat UBs. Exploiting the branching program intrinsic to the UB, propagated rat UBs can be recombined with fresh rat mesenchyme to form a large number of rat "neokidneys" derived from a single progenitor that may be amenable to site-specific modulation of function. METHODS: Whole rat metanephric rudiments or isolated rat UBs were cultured and subdivided through several generations. Both cultured progenitor and subsequent generations of isolated rat UBs were recombined with freshly isolated rat metanephric mesenchyme. The tubules of these rat neokidneys were examined for expression of epithelial markers. RESULTS: Isolated rat UBs and whole rat metanephric rudiments could be propagated through several generations and appeared morphologically identical to their progenitors. Generations of isolated rat UB could be recombined with fresh rat mesenchyme and the resultant neokidney displayed the same morphologic appearance as the whole rat kidney rudiment. The UB-derived and MM-derived portions of the tubules of these rat neokidneys appear contiguous. CONCLUSIONS: The recombination of cultured and propagated rat UB with rat mesenchyme yielded rat neokidneys with tubular structures that appeared morphologically identical to whole rat kidney. In vitro propagation of rat metanephric rudiments and recombination of rat UB and MM suggest the possibility of designing nephrons that possess specific desirable functions that can be propagated in vitro.