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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]

Abstract: 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. [Copyright &y& Elsevier]

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]