Generation of Renal Tubules at the Interface of an Artificial Interstitium

Abstract

During kidney development a multitude of tubular portions is formed. Little knowledge is available by which cellbiological mechanism a cluster of embryonic cells is able to generate the threedimensional structure of a tubule. However, this know-how is most important in tissue engineering approaches such as the generation of an artificial kidney module or for the therapy of renal diseases using stem cells. To obtain cellbiological insights in parenchyme development we elaborate a new technique to generate under in vitro conditions renal tubules derived from the embryonic cortex of neonatal rabbits. The aim of the experiments is to establish a specific extracellular environment allowing optimal threedimensional development of renal tubules under serum-free culture conditions. In the present paper we demonstrate features of the renal stem cell niche and show their isolation as intact microcompartiments for advanced tissue culture. Perfusion culture in containers exhibiting a big dead fluid volume results in the development of a flat collecting duct (CD) epithelium at the surface of the tissue explant. In contrast, by fine-tuning the dead fluid volume within a perfusion culture container by an artificial interstitium made of a polyester fleece shows the generation of tubules. It is an up to date unknown morphogenetic information which tells the cells to form tubular structures.

The Interface Between Generating Renal Tubules and a Polyester Fleece in Comparison to the Interstitium of the Developing Kidney

“…7c). 19 Thus, label for collagen type III is missing in the zone of renal stem/progenitor cells, but it is co-localized with laminin c1 in the maturing zone along the shaft of the collecting duct ampulla. In consequence, the experiments clearly show that collagen type III is not the primary skeletonal element of the interstitium in developing renal parenchyma.…”

“…For soybean agglutinin-labeling (SBA, Vector, Burlingame, USA) the samples were exposed to fluorescein-isothiocyanate (FITC)-conjugated lectin diluted 1:2000 in blocking solution for 45 min as earlier described. 18 For immunohistochemical label monoclonal antibodies such as mab anti-laminin c1 (kindly provided by Dr. L. Sorokin, Lund, Sweden), 19 mab anti-P CD Amp 1, 27 mab anti-Na/K-ATPase alpha 5 (Developmental Studies Hybridoma Bank, Iowa City, USA) and mab anti-collagen type III (III-53, Calbiochem, Schwalbach, Germany) was applied in blocking solution. After washing with phosphatebuffered saline (PBS) containing 1% bovine serum albumin (BSA), the specimens were then incubated for 45 min with donkey-anti-mouse-IgG-fluoresceinisothiocyanate (FITC) or goat-anti-rat-IgG-rhodamine (Jackson Immunoresearch Laboratories, West Grove, USA) diluted 1:50 in this solution.…”

“…1b). 11,19 The sandwich set-up containing renal stem/progenitor cells was mounted then in a base ring of a Minusheet Ò tissue carrier. First, a polyester fleece measuring 13 mm in diameter was placed in the tissue carrier.…”

“…Renal stem/progenitor cells are placed between two layers of a polyester fleece to simulate an artificial interstitium. 19 The space between the fibers facilitates exchange of nutrition and respiratory gas. Transport of always fresh and chemically defined medium in a perfusion container guarantees a constant provision with nutrition and respiratory gas during long-term culture.…”

The tubulogenic effect of aldosterone is attributed to intact binding and intracellular response of the mineralocorticoid receptor

“…Previous publications of our group proved the feasibility of generating tubules derived from renal stem/progenitor cells at the interface of an artificial interstitium made of a polyester fleece within a perfusion culture container [18,19]. We found that the generation of renal tubules depends specifically on the presence of aldosterone.…”