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As well in light as in transmission electron microscopy can be seen that the renal stem/progenitor cell niche shows a special arrangement of two different kinds of stem/progenitor cells. Epithelial cells are found in the tip of an ureteric bud derived CD ampulla encircled by a special basal lamina. Mesenchymal cells are separated from them by a striking interstitial interface. Specimens fixed by conventional glutaraldehyde solution show that the interface looks bright and unremarkable. In contrast, fixation of specimens with glutaraldehyde in combination with cupromeronic blue, ruthenium red, or tannic acid illustrates that the interface contains a remarkable network of extracellular matrix spanning between epithelial and mesenchymal stem/progenitor cells. After unpacking this particular extracellular matrix for electron microscopy, elaboration of related functions such as structural composition of contained molecules, binding of morphogenetic factors, and influence on parenchyma development is under current experimental work.

Repair of parenchyma by stem/progenitor cells is seen as a possible alternative to cure acute and chronic renal failure in future. To learn about this therapeutic purpose, the formation of nephrons during organ growth is under focus of present research. This process is triggered by numerous morphogenetic interactions between epithelial and mesenchymal cells within the renal stem/progenitor cell niche. Recent data demonstrate that an astonishingly wide interstitial interface separates both types of stem/progenitor cells probably controlling coordinated cell-to-cell communication. Since conventional fixation by glutaraldehyde (GA) does not declare in transmission electron microscopy the spatial separation, improved contrasting procedures were applied. As a consequence, the embryonic cortex of neonatal rabbit kidneys was fixed in solutions containing glutaraldehyde in combination with cupromeronic blue, ruthenium red or tannic acid. To obtain a comparable view to the renal stem/progenitor cell niche, the specimens had to be orientated along the cortico-medullary axis of lining collecting ducts. Analysis of tissue samples fixed with GA, in combination with cupromeronic blue, demonstrates demasked extracellular matrix. Numerous braces of proteoglycans cover, as well, the basal lamina of epithelial stem/progenitor cells as projections of mesenchymal stem/progenitor cells crossing the interstitial interface. Fixation with GA containing ruthenium red or tannic acid illustrates strands of extracellular matrix that originate from the basal lamina of epithelial stem/progenitor cells and line through the interstitial interface. Thus, for the first time, improved contrasting techniques make it possible to analyze in detail a microheterogeneous composition of the interstitial interface within the renal stem/progenitor cell niche.

Stem/progenitor cells are promising candidates for the regeneration of parenchyma in acute and chronic renal failure. After an implantation stem/progenitor cells must migrate through the interstitial space to concentrate at the site of damage. However, information is lacking to what extent the interstitial interface is influencing the development of stem/progenitor cells into nephron structures. In consequence, tubule regeneration within an artificial polyester interstitium was analyzed by electron microscopy in comparison with the interstitial interface of matured tubules and the interstitium within the renal stem/progenitor cell niche. The experiments demonstrate that fixation of specimens with glutaraldehyde (GA) is leading in all cases to inconspicuously looking interstitial interfaces. In contrast, fixation of regenerating tubules in GA containing ruthenium red and tannic acid shows a dense network of fibers lining along the basal lamina. In contrast, matured tubules reveal after ruthenium red label an extremely thickened basal lamina, while only a punctate pattern is obtained after tannic acid treatment. Finally, within the renal stem/progenitor cell niche ruthenium red and tannic acid label reveals large amounts of extracellular matrix spanning through the interstitium. Thus, fixation of tissue in GA containing ruthenium red and tannic acid exhibits an unexpectedly regional heterogeneity of the renal interstitial interface. This fact has to be considered for an optimal therapeutic repair of parenchyma, since contacts between stem/progenitor cells with the interstitial interface influence further development., (Copyright © 2012 Wiley Periodicals, Inc.)

The development of the nephron is piloted by interactions between epithelial and surrounding mesenchymal stem/progenitor cells. Data show that an astonishingly wide interstitial space separates both kinds of stem/progenitor cells. A simple contrasting procedure was applied to visualize features that keep renal epithelial and mesenchymal stem/progenitor cells in distance. The kidney of neonatal rabbits was fixed in solutions containing glutaraldehyde (GA) in combination with alcian blue, lanthanum, ruthenium red, or tannic acid. To obtain a comparable view to the renal stem/progenitor cell niche, the tissue was exactly orientated along the axis of collecting ducts. Fixation with GA or in combination with alcian blue or lanthanum revealed an inconspicuous interstitial space. In contrast, fixation with GA containing ruthenium red exhibits strands of extracellular matrix lining from epithelial stem/progenitor cells through the interstitium up to the surface of mesenchymal stem/progenitor cells. Fixation with GA containing tannic acid shows that the basal lamina of epithelial stem/progenitor cells, the adjacent interstitial space and also the surface of mesenchymal stem/progenitor cells are connected over a net of extracellular matrix. The applied technique appears to be a suitable method to illuminate the interstitium in stem/progenitor cell niches of specialized tissues, the microenvironment of tumors and extension of degeneration.

The cell biological mechanism controlling the regeneration of renal tubules in renal failure after application of stem/progenitor cells is subject of actual research. Unsolved issues are the integration of stem/progenitor cells in a diseased organ environment, the differentiation into epithelial tissue and the formation of tubules in a spatial environment. Following this therapeutic strategy new biomaterials have to be found promoting spatial development of tubules. To obtain new information about the growth of tubules renal stem/progenitor cells from neonatal rabbit kidney were isolated and mounted in a tissue carrier between a selection of commercially available polyester fleeces. This procedure replaces coating by extracellular matrix proteins and creates an artificial interstitium supporting development of tubules. Perfusion culture was performed with chemically defined IMDM containing aldosterone as tubulogenic factor. Polyester fleeces were investigated by scanning electron microscopy. The spatial development of tubules was registered on whole-mount specimens and on cryosections labeled with SBA and antibodies indicating tubule differentiation. It is found that some polyester fleeces promote the spatial development of tubules between the fibers, whereat each of them produces its individual growth pattern.

Reciprocal exchange of morphogenetic proteins between epithelial and mesenchymal cells in a stem/progenitor cell niche results in formation of a nephron. To maintain diffusion of morphogenetic proteins, it is assumed that a close contact exists between involved cells. However, recent publications underline that both types of stem/progenitor cells are separated by a striking interface. To explore this microarchitecture in detail, neonatal rabbit kidneys were fixed in traditional glutaraldehyde (GA) solution for transmission electron microscopy. For contrast enhancing specimens were fixed in GA solution including cupromeronic blue, ruthenium red or tannic acid. To record same perspectives, embedded blocks of parenchyma were cut in exactly orientated vertical and transverse planes to lining collecting ducts. Electron microscopy of specimens fixed by traditional GA solution illustrates a spatial separation of stem/progenitor cells and an unobstrusively looking interface. In contrast, advanced fixation of specimens in GA solution including cupromeronic blue, ruthenium red and tannic acid unmasks earlier not visible extracellular matrix. In addition, projections of mesenchymal cells covered by matrix cross the interface to contact epithelial cells. Surprisingly, the end of a mesenchymal cell projection does not dangle but is enclosed in a fitting sleeve and connected via tunneling nanotubes with the plasma membrane of an epithelial cell. Regarding this complex ensemble the question is to what extent illustrated cell-cell connections and extracellular matrix are involved in communication and transmission of morphogenetic proteins during induction of a nephron. [ABSTRACT FROM AUTHOR]

A special feature of the renal stem/progenitor cell niche is its always close neighborhood to the capsule during organ development. To explore this link, neonatal kidney was investigated by histochemistry and transmission electron microscopy. For adequate contrasting, fixation of specimens was performed by glutaraldehyde including tannic acid. The immunohistochemical data illustrate that renal stem/progenitor cells are not distributed randomly but are positioned specially to the capsule. Epithelial stem/progenitor cells are found to be enclosed by the basal lamina at a collecting duct (CD) ampulla tip. Only few layers of mesenchymal cells are detected between epithelial cells and the capsule. Most impressive, numerous microfibers reacting with soybean agglutinin, anti-collagen I and III originate from the basal lamina at a CD ampulla tip and line between mesenchymal stem/progenitor cells to the inner side of the capsule. This specific arrangement holds together both types of stem/progenitor cells in a cage and fastens the niche as a whole at the capsule. Electron microscopy further illustrates that the stem/progenitor cell niche is in contact with a tunnel system widely spreading between atypical smooth muscle cells at the inner side of the capsule. It seems probable that stem/progenitor cells are supplied here by interstitial fluid. [ABSTRACT FROM AUTHOR]

Abstract: In regenerative medicine much attention is given to stem/progenitor cells for a future therapy of acute and chronic renal failure. However, up to date sound cell biological knowledge about nephron renewal in kidney is lacking. For that reason molecular mechanisms are under intense investigation leading from stem/progenitor cells to regenerated tubules. In this coherence new biomaterials and drug delivery systems have to be elaborated showing an intense stimulation on the renewal of parenchyma. To analyze tubule regeneration a powerful culture system is of fundamental importance. An advanced technique stimulates renal stem/progenitor cells to develop numerous tubules between layers of a polyester fleece. Use of chemically defined Iscove''s Modified Dulbecco''s Medium (IMDM) containing aldosterone (1×10−7 M) results in spatial development of renal tubules within 13days of perfusion culture. Immunohistochemistry exhibits that numerous features of a polarized epithelium are expressed in generated tubules. Transmission electron microscopy (TEM) illuminates that generated tubules contain a polarized epithelium with a tight junctional complex and an intact basal lamina at the basal aspect. Development of tubules depends on applied aldosterone concentration and cannot be mimicked by precursors of its synthesis pathway or by other steroid hormones. Antagonists such as spironolactone or canrenoate prevent the development of tubules. This result illuminates that the tubulogenic development is mediated via the mineralocorticoid receptor (MR). Application of geldanamycin, radicicol, quercetin or KNK 437 in combination with aldosterone blocks development of tubules by disturbing the contact between MR and heat shock proteins 90 and 70. In conclusion, for the first time generation of renal tubules can be simulated under controlled in-vitro conditions. Using this model the effect of numerous innovative biomaterials and drug delivery system can be critically analyzed. [Copyright &y& Elsevier]

Stem/progenitor cells are promising candidates for a therapy of renal failure. However, sound knowledge about implantation and regeneration is lacking. Therefore, mechanisms leading from stem/progenitor cells into tubules are under research. Renal stem/progenitor cells were isolated from neonatal rabbit kidney and mounted between layers of polyester fleece. It creates an artificial interstitium and replaces coating by extracellular matrix proteins. Tubulogenic development is induced by aldosterone. Electron microscopy illuminates growth of tubules in close vicinity to polyester fibers. Tubules contain a differentiated epithelium. The spatial extension of tubules opens a new strategy for testing morphogenic drugs and biocompatible fleece materials. [ABSTRACT FROM AUTHOR]

Abstract: The construction of an artificial kidney module by tissue engineering or the application of cell-based therapies for the treatment of renal failure requires exact information regarding the cellbiological mechanisms of parenchyme development in combination with different kinds of biomaterials. To learn more about these processes tissue cultures are frequently used experimental tools. However, apart from experiments with early kidney anlagen there is a lack of suitable in-vitro models regarding the generation and long-term maintenance of renal tubules. In the present paper we like to demonstrate an advanced culture technique, which allows to generate tubular elements derived from renal stem cells. For the growth of tubules it is essential to fine-tune the interface between the embryonic tissue and the dead fluid space within a perfusion culture container by offering a polyester artificial interstitium. Culture was performed in IMDM supplemented with hormones and growth factors but using serum-free conditions over 14 days. Formation of tissue was then analysed by immunohistochemistry and two-dimensional (2D) electrophoresis. Culture in pure IMDM leads to a complete loss of tissue formation. In contrast, application of aldosterone (A) induces the development of numerous polarised tubules. Surprisingly, addition of epidermal growth factor (EGF), a cocktail of insulin, transferrin and selenium (ITS), retinoic acid (RA), cholecalciferol (VitD3) or bovine pituitary extract (BPT) does not further improve development of tubules, but leads to intensive cell clustering and a decrease of tubule formation. 2D Western blots of developing tissue probed with soybean agglutinin (SBA) reveal a unique pattern of newly detected proteins. It is found that growth factors do not support but abolish protein spots upregulated by aldosterone. It remains to be investigated, which cellbiological effect stimulates the embryonic cells to develop tubules in competition to cell clusters at the interphase of an artificial interstitium. [Copyright &y& Elsevier]