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.

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system.

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.

Activin A is an endogenous inhibitor of ureteric bud outgrowth from the Wolffian duct.

Development of metanephric kidney begins with ureteric bud outgrowth from the Wolffian duct (WD). GDNF is believed to be a crucial positive signal in the budding process, but the negative regulation of this process remains unclear. Here, we examined the role of activin A, a member of TGF-beta family, in bud formation using an in vitro WD culture system. When cultured with the surrounding mesonephros, WDs formed many ectopic buds in response to GDNF. While the activin signaling pathway is normally active along the non-budding WD (as measured by expression of activin A and phospho-Smad2/3), activin A was absent and phospho-Smad2/3 was undetectable in the ectopic buds induced by GDNF. To examine the role of activin A in bud formation, we attempted to inactivate activin action. Interestingly, the addition of neutralizing anti-activin A antibody potentiated GDNF action. To further clarify the role of activin A, we also tested the effect of activin blockade on the WD cultured in the absence of mesonephros. WDs without mesonephros did not form ectopic buds even in the presence of GDNF. In contrast, blockade of activin action with a variety of agents acting through different mechanisms (natural antagonist, neutralizing antibodies, siRNA) enabled GDNF to induce ectopic buds. Inhibition of GDNF-induced bud formation by activin A was accompanied by inhibition of cell proliferation, reduced expression of Pax-2, and decreased phosphorylation of PI3-kinase and MAP kinase in the WD. Our data suggest that activin A is an endogenous inhibitor of bud formation and that cancellation of activin A autocrine action may be critical for the initiation of this process.

Decreased renal organic anion secretion and plasma accumulation of endogenous organic anions in OAT1 knock-out mice.

The "classical" organic anion secretory pathway of the renal proximal tubule is critical for the renal excretion of the prototypic organic anion, para-aminohippurate, as well as of a large number of commonly prescribed drugs among other significant substrates. Organic anion transporter 1 (OAT1), originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J. G., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), has physiological properties consistent with a role in this pathway. However, several other transporters (e.g. OAT2, OAT3, and MRP1) have also been proposed as important PAH transporters on the basis of in vitro studies; therefore, the relative contribution of OAT1 has remained unclear. We have now generated a colony of OAT1 knock-out mice, permitting elucidation of the role of OAT1 in the context of these other potentially functionally redundant transporters. We find that the knock-out mice manifest a profound loss of organic anion transport (e.g. para-aminohippurate) both ex vivo (in isolated renal slices) as well as in vivo (as indicated by loss of renal secretion). In the case of the organic anion, furosemide, loss of renal secretion in the knock-out results in impaired diuretic responsiveness to this drug. These results indicate a critical role for OAT1 in the functioning of the classical pathway. In addition, we have determined the levels of approximately 60 endogenous organic anions in the plasma and urine of wild-type and knock-out mice. This has led to identification of several compounds with significantly higher plasma concentrations and/or lower urinary concentrations in knock-out mice, suggesting the involvement of OAT1 in their renal secretion. We have also demonstrated in xenopus oocytes that some of these compounds interact with OAT1 in vitro. Thus, these latter compounds might represent physiological substrates of OAT1.

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.