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Construction of Nephron by Fusion of Adult Glomeruli to Ureteric Buds with Type V Collagen

Yusuke Murasawa, Inst. Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai,, Tsukuba, Ibaraki, 305-8572, Japan and Pi Chao Wang, Institute of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai,, Tsukuba, Ibaraki, 305-8572, Japan.

Introduction: Although tissue engineering of artificial organs such as skin or cartilage have been developed greatly, organs such as kidney or liver have not been completed yet. Human kidney consists of one million of nephrons which is the basic unit for the filtration of blood, excretion of urine, secretion of cytokine and re-absorption of electrolytes. Every nephron comprising glomerulus and tubule with complicated structure and full of blood capillaries can hardly be re-constructed by current techniques. In this study, we constructed nephrons by fusion adult glomeruli to ureteric buds with a newly dynamic scaffold-type V collagen fiber under micro-gravity. We firstly proved that type V collagen fiber can not only provide a dynamic scaffold for renal glomerular endothelial cells but also can induce other ECM (excellular matrix) such as type IV collagen and fibronectin which are the main compoments for glomerular basement membrane. By using type V collagen fiber, we challenged a new concept different from the known tissue engineering in which stable scaffold such as collagen I was used. In our new experiment, adult mouse glomeruli and embyonic tubular could be fused to constitute a new nephron in vitro and blood capillary could be induced around glomeruli when both developmental and aging tissues were co-cultured on typle V collagen fiber under microgravity. Materilas and Methods: Glomeruli were isolated from kidney of adult mice more than 6 weeks, and ureteric buds of metanephros were isolated from fetal mice of E11, E13, E15 and E17. Type V collagen molecules was extracted from porcine cornea by pepsin treatment at acidity condition, and then neutralized with urea and NaCl to reconstitute type V collagen fiber. Adult glomeruli cultured in mebiol-gel were immersed in type V collagen fiber and then added to ureteric buds treated with collagenase and type V collagen, followed by subjecting to microgravity. Media consisting of epithelial medium and endothelial medium with a ratio of 1:1 was used for renal tissue culture. The culture condition is 37 C with 5% of carbon-dioxide. Type I collagen was used as a control scaffold for all experiments. Contrast microscopy was performed to observe the morphological change of glomeruli and metanephros after 1-2 week culture. Immunohistochemistry was performed on frozen sections of the cultured tissue. To distinguish tubule from glomeruli, Dolichos biflorus agglutinin was used to stain ureteric buds. Anti-type V collagen was used to detect the localization and amount of type V collagen fiber in the cultured tissue by immunohistochemistry and western blot analysis. Results: At first day of tissue culture of adult glomeruli with ureteric buds under microgravity, it is interesting to find that ureteric buds aggregated together immediately. Three days after tissue culture, adult glomeruli successfully fused to the tips of ureteric buds when culturing with type V collagen fiber in the media of epithelial and endothelial medium. Surprisingly, microgravity improved vasculogenesis of the blood capillaries of glomeruli even extra vasculogenesis factor such as VEGF was not added. The immunohistochemistry results revealed that type V collagen fiber formed a belt structure surrounding of ureteric bud and extending to the glomeruli so as to attract glomeruli close to the tip of ureteric buds, and finally caused the fusion of glomeruli to ureteric buds. Type V collagen fiber was observed locating at the outside of ureteric buds initially and then shifting to the outside of immature glomeruli, while type IV collagen was at the inside of mature glomeruli at the later stage. When comparing to the tissues cultured on type I collagen, only those cultured on type V collagen formed blood capillaries among the mesenchymal tissue which eventually invade to the inside of glomeruli, and were likely to function of blood filtration. In conclusion, we successfully constructed nephrons by fusing the adult glomeruli to the embryonic ureteric buds by using a dynamic scaffold . Such technique may be useful to the application of kidney re-generation of kidney in the future . Fig. Adult glomeruli (arrows) fused to the tip of developmental ureteric buds. .