Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development.

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-b (TGF-b) superfamily (Roberts and Sporn, 1990). DNF is important for kidney development. First, the idney of newborn rodents contains high amounts of DNF mRNA (Suter-Crazzolara and Unsicker, 1994). DNF is expressed by the metanephrogenic mesenhyme (Hellmich et al., 1996; Suvanto et al., 1996). DNF 2/2 mice die shortly after birth, primarily due to dysgenesis of the kidney (Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996). The GDNF receptor Ret can be detected on the epithelium of the uretric bud, while the high affinity receptor GFRa-1 is exressed also in the mesenchyme (reviewed by Sariola nd Sainio, 1996). Mice with a disrupted Ret gene eveal a strikingly similar phenotype to GDNF 2/2 animals (Schuchardt et al., 1996). The proper function of the kidney depends on the coordinated development of renal vessels, nephrons, and collecting ducts. Several factors such as vascular endothelial growth factor (VEGF; Kloth et al., 1995) or retinoic acid (Kloth et al., 1998) are known to modulate renal vessel growth and differentiation. Here, we have

We have established a new test concept for in vitro pharmacological trials. Our model employs tissue explants to test compounds for toxicity which arises with the metabolic interactions among different cell types. Microsurgical preparation of tissue explants avoids the destruction of the organ-specific tissue architecture. Explants were mounted in tissue carriers to improve nutrition and handling of the sample. To allow for the omission of serum supplementation of the culture medium, explants were cultured under continuous medium flow. Test substances are applied considering the polar architecture of most tissues in vivo, for example, covering the apical aspect of epithelia. In principal, all tissues obtained from any species, including man, can be used in this system. A trial application was performed with vitreous body substitutes, substances used in ophthalmology. One compound had passed cell culture tests, but caused massive blood vessel deterioration in vivo. Using our test system based on the developing renal vessel system, we could confirm, within 24 hours, severe vessel damage which resembles the injury suffered by the rabbit retina. We demonstrate that an improved tissue culture assay is a suitable tool for the detection of toxicity that remained unidentified in cell culture tests.

While more and more humoral factors involved in nephrogenesis are being discovered, there is no detailed knowledge of the morphological structures at the interface of the nephron inducer and the surrounding mesenchyme. For that reason we examined this area in the cortex of neonatal rabbit kidneys by scanning electron-microscopical and transmission electron-microscopical techniques. Our interest was focused on the basal aspect of the collecting duct ampulla and the surrounding competent mesenchyme, where morphogenic signals are to be exchanged during nephron induction. Close contact between these two tissues involved in nephrogenesis is assumed to allow direct cellular contact or diffusion of soluble factors across a short distance. Our data, however, show the presence of a dense fibrillar meshwork around the collecting duct ampulla, spatially separating the inducer and the competent mesenchyme during nephron induction.

During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove's modified Dulbecco's medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams' medium E, McCoys 5A medium, and Dulbecco's modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.

An in vitro model was used to investigate the terminal differentiation mechanisms leading from embryonic to adult renal tissue. For these experiments the capsula fibrosa with adherent embryonic tissue was isolated from neonatal rabbit kidneys. These explants were mounted onto special tissue carriers and cultured in medium containing serum for 24 h. During that time collecting duct (CD) cells grew out and formed a monolayered epithelium covering the whole surface of the explant. The carriers were then transferred to perfusion culture containers to obtain an optimal degree of differentiation. A special type of container allowed us to continuously superfuse the epithelia with individual media on the luminal and basal sides. Using this method it became possible to culture embryonic CD epithelia in a fluid gradient for weeks. The epithelia were superfused with standard Iscove’s modified Dulbecco’s medium (IMDM) on the basal side, while IMDM containing additional NaCl was used on the luminal side. In controls IMDM was superfused on both the luminal and basal sides. It was found that the degree of differentiation in the CD epithelia is dependent on the influence of fluid gradient exposure. Perfusion culture under isotonic conditions revealed that less than 5% of cells were immunopositive for principal and intercalated cell features, while epithelia cultured in a luminal-basal gradient showed more than 80% positive cells. Immunoreactivity for characteristic markers started to develop after an unexpectedly long latent period of 3–6 days, then increased continuously during the following 5 days and reached a maximum on day 14. After switching back from the gradient to isotonic culture conditions the immunoreactivity for some markers decreased within 5 days, while other characteristic features remained stable. Thus, differentiation was not only under the control of growth factors but was also regulated by the electrolyte environment.

The collecting duct (CD) epithelium of the mammalian kidney is an extraordinary structure with respect to its functional changes during development and its heterogeneous composition when matured. All of the different nephron epithelia of the mammalian kidney consist of one single cell type. In contrast, the differentiated CD is composed of at least three distinct cell types [principal, alpha intercalated-, and beta intercalated cells] that are responsible for the multiple physiological functions of this kidney compartment. During development the function of the CD changes: initially, the CD ampulla serves as an embryonic inducer, while the matured epithelium plays a key role in maintaining the homeostasis of body fluids. At present the process of CD maturation is not well understood. Neither the time course of development nor the morphogenic factors leading to the heterogeneously composed epithelium are known. In the present study the differentiation of the CD epithelium was investigated using newly developed monoclonal antibodies and well-characterized antisera. The morphological changes induced during differentiation were monitored by immunohistochemistry and scanning electron microscopy. The experiments were performed on neonatal and adult rabbit kidneys. Results obtained by light microscopical techniques and scanning electron microscopy revealed that the ampullary tip can be distinguished from the ampullary neck, as well as from the maturing CD. A number of proteins that were not detectable in the ampulla were detected in the neonatal CD and were found at even higher concentrations in the adult CD (PCD8, chloride/bicarbonate exchanger). Other proteins (PCD9) were downregulated during differentiation. For the first time the transient character of the differentiation stage of the neonatal CD could be demonstrated unequivocally. Furthermore, considerable heterogeneity in protein expression patterns (PCD6 and PCD9) was demonstrated within the beta IC cell population of the mature CD.

During kidney organogenesis the development of renal vessels must be synchronized with the maturation of nephrons and the collecting duct system. Several reports showed that hormones and mitogenic peptides as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) are involved in this regulatory process. It is a known fact that bFGF receptors are expressed by differentiating tubular epithelium and mesenchyme, but little information is available about the function of bFGF in kidney organogenesis. The role of bFGF during kidney development was investigated using an organotypic culture system and immunohistological techniques. Renal cortex explants were prepared from the kidneys of neonatal rabbits with a microsurgical method, retaining the natural tissue composition. The explants were cultured serum free under continuous medium perfusion. Our results indicate a new and unexpected role of bFGF during the differentiation process. When bFGF alone was applied, vessels could no longer be detected. The inhibitory influence of bFGF could be overcome by addition of VEGF or hormones such as retinoic acid and aldosterone/vitamin D3. The combination of these factors with bFGF resulted in the expression of small vessel-like structures. We conclude that bFGF has a morphogenic rather than a mitogenic function during kidney vessel development.

During kidney development the embryonic collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into functional principal (P) and intercalated (IC) cells. Aldosterone is able to modulate this differentiation. Consequently we investigated whether increased concentrations of extracellular NaCl or Na gluconate may also have an influence on the development of individual CD cell features. Embryonic CD epithelia were isolated from neonatal rabbit kidneys, placed on tissue carriers and cultured in gradient containers, which were constantly perfused with medium for 13 days. Isotonic culture conditions could be mimicked, when on both the luminal and basal side standard Iscove's Modified Dulbecco's Medium (IMDM) was used. In another set of experiments, gradient culture was performed. Standard IMDM was applied on the basal side and IMDM supplemented with 12 mM NaCl and 17 mM Na gluconate on the luminal side. This adaptation of IMDM led to the same Na concentrations as found in the serum of neonatal rabbits. The development of CD cell features was monitored by cellular markers such as the monoclonal antibodies (Mabs) 703 and 503 recognizing P and IC cell features respectively. Epithelia cultured under isotonic conditions showed less than 5% Mab 703- and 503-immunopositive cells. In contrast, epithelia cultured in a luminal-basal medium gradient revealed more than 80% positive cells. Immunoreactivity started to develop after a long lag period of 4 days, then increased continously during the following 5 days and reached a maximum at day 14. When the medium gradient was then changed to an isotonic environment for another 5 days immunoreactivity for Mab 703 remained stable, while the number of Mab 503-positive cells was found to be decreased to 10%. Thus, the extracellular electrolyte environment not only induces but also preserves individual cell features.

During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.