The metanephric kidney consists of two types of epithelia; the Wolffian duct-derived ureteric bud and the nephrogenic components that originate from mesenchymal-toepithelial transitions in the metanephric mesenchyme. The ureteric bud forms when inductive signals from the metanephric mesenchyme stimulates the evagination of an epithelial tube from the Wolffian duct into the mesenchyme. Reciprocal signalling between the ureteric bud and the metanephric mesenchyme regulates the branching of the ureteric bud and the induction of nephron formation. Inductive and inhibitory signalling of ureteric bud growth and branching has been shown by several protein families, however, the mechanical aspects of ureteric bud branching and nephrogenesis are largely unknown. I investigated the roles of Rac1-GTPase and Rho-kinase during kidney development. These proteins are important regulators of the cytoskeleton where Rac1 is a promoter of actin filament polymerisation and Rho-kinase directly stimulates the formation and contraction of actin-myosin stress fibres. Using a cell-permeable inhibitor, Rac1 was inhibited with no effects on nephron formation or subsequent segmentation and patterning. Inhibition of active Rac1 significantly reduced the level of ureteric bud branching and also resulted in lower proliferation rates. Rho-kinase was similarly targeted using two inhibitors. Rho-kinase inhibition had important effects on nephron formation and nephron maturation. Inhibition of Rhokinase resulted in decreased levels of nephron formation and severely morphologically abnormal nephrons. The formation of apical-basal polarity was disturbed as was the development of the visceral and parietal epithelia; precursors of the renal corpuscle. Inhibition of Rho-kinase led to abnormal formation of the proximal-distal axis and abnormal segmentation of the nephron. The effects of Rho-kinase inhibition were partially mimicked by direct targeting of actin-myosin contractions using a myosin-ATPase inhibitor. This demonstrated that Rho-kinase is necessary during multiple stages of nephrogenesis and maturation, at least in part, as a result of its ability to regulate actin-myosin contraction. These results show that Rac1 and Rho-kinase play important roles during several aspects of kidney development and highlights the significance of further investigating the mechanisms involved during kidney organogenesis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22879号 / 医博第4673号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 柳田 素子, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.

Renal tissue engineering and regenerative medicine represent a significant clinical objective because of the very limited prospect of cure after classical kidney treatment. Thus, approaches to isolate, manipulate and reintegrate structures or stimulating the selfregenerative potential of renal tissue are of special interest. Such new strategies go back to knowledge and further outcome of developmental biological research. An understanding of extracellular matrix (ECM) structure and composition forms thereby a particularly significant aspect in comprehending the complex dynamics of tissue regeneration. Consequently the reconstruction of these structures offers beneficial options for advanced cell and tissue culture technology and tissue engineering. In an effort to investigate the influence of natural extracellular structures and components on embryonic stem cell and renal embryonic tissue, methodologies which allow the easy application of exogenous signals on tissue in vitro on the one hand and the straight forward evaluation of decellularization methods on the other hand, were developed. Both systems can be used to investigate and modulate behaviour of biological systems and represent novel interesting tools for tissue engineering. The novel technique for culturing tissue in vitro allows the growing of embryonic renal explants in very low volumes of medium and optimized observability, which makes it predestined for testing additives. In particular, this novel culture set up provides an ideal opportunity to investigate renal development and structure formation. Further studies indicated that the set is universally applicable on all kinds of (embryonic) tissue. Following hereon, more than 20 different ECM components were tested for their impact on kidney development under 116 different culture conditions, including different concentrations and being either bound to the substrate or dissolved in the culture medium. This allowed to study the role of ECM constituents on renal structure formation. In ongoing projects, kidney rudiments are exposed to aligned matrix fibrils and hydrogels with first promising results. The insights gained thereof gave rise to a basis for the rational application of exogenous signals in regenerative kidney therapies. Additionally new strategies for decellularization of whole murine adult kidneys were explored by applying different chemical agents. The obtained whole matrices were analysed for their degree of decellularization and their residual content and composition. In a new straight forward approach, a dependency of ECM decellularization efficiency to the different agents used for decellularization could be shown. Moreover the capability of the ECM isolated from whole adult kidneys to direct stem cell differentiation towards renal cell linage phenotypes was proved. The data obtained within this thesis give an innovative impetus to the design of biomaterial scaffolds with defined and distinct properties, offering exciting options for tissue engineering and regenerative kidney therapies by exogenous cues.:Table of Contents LISTS OF FIGURES AND TABLES VI ACKNOWLEDGEMENTS..................................................................................VII ABSTRACT ............................................................................................................IX NOMENCLATURE ................................................................................................X 1 INTRODUCTION...................................................................................................1 2 FUNDAMENTALS..................................................................................................2 2.1 KIDNEY DEVELOPMENT AND REGENERATION ...............................................................................2 2.1.1 Function of the kidney............................................................................................2 2.1.2 Development of the metanephric kidney ................................................................2 2.1.3 Selfregenerative potential of the kidney.................................................................5 2.2 THE EXTRACELLULAR MATRIX AS BIOLOGICAL SCAFFOLD ...............................................................6 2.2.1 Molecular composition of the ECM........................................................................7 2.2.1.1 An overview of the main ECM components..................................................................................8 2.2.2 Cell/tissue-matrix interactions.............................................................................12 2.2.2.1 Biochemical signals....................................................................................................................13 2.2.2.2 Mechanical signals......................................................................................................................14 2.2.2.3 Structural signals........................................................................................................................15 2.3 TISSUE ENGINEERING FOR THERAPEUTIC PURPOSES .....................................................................15 2.3.1 An overview of tissue engineering and regenerative medicine.............................15 2.3.2 Biomaterials for tissue engineering and regenerative medicine...........................18 2.3.2.1 Decellularization approach as tool to extract natural matrices....................................................19 2.3.3 Tissue engineering and regenerative medicine in kidney treatment.....................19 2.4 ORGAN AND TISSUE CULTURE AS TOOL FOR TISSUE ENGINEERING...................................................22 2.4.1 Common organ culture systems............................................................................24 3 OBJECTIVES AND MOTIVATION...................................................................25 4 RESULTS AND DISCUSSION............................................................................27 4.1 A NOVEL, LOW-VOLUME METHOD FOR ORGAN CULTURE OF EMBRYONIC KIDNEYS THAT ALLOWS DEVELOPMENT OF CORTICO-MEDULLARY ANATOMICAL ORGANIZATION..............................................27 4.1.1 Additional evidences (to Appendix A) for stress reduction of kidney rudiments cultured in the novel system than those grown in conventional organ culture.....28 4.1.2 Additional evidences (to Appendix A) for corticomedullary zonation and improved development of kidney rudiments cultured in the novel system for a period of 12 days......................................................................................................................30 4.1.3 Additional evidences (to Appendix A) for the application of the glass based low volume culture system for other organs................................................................32 4.2 ECM MODULATED EARLY KIDNEY DEVELOPMENT IN ORGAN CULTURE ...........................................34 4.3 ESTABLISHING AND EVALUATING DECELLULARIZATION TECHNIQUES TO ISOLATE WHOLE KIDNEY ECMS FROM ADULT MURINE KIDNEYS................................................................................................37 4.4 THE ABILITY OF WHOLE DECELLULARIZED ECM CONSTRUCTS TO INFLUENCE MURINE EMBRYONIC STEM CELL DIFFERENTIATION AND RENAL TISSUE BEHAVIOUR IN A NEW STRAIGHT FORWARD APPROACH..........38 iv 5 SUMMARY AND OUTLOOK.............................................................................39 5.1 SUMMARY..........................................................................................................................39 5.2 OUTLOOK...........................................................................................................................42 6 BIBLIOGRAPHY.................................................................................................49 7 APPENDICES..........................................................................................................I 7.1 APPENDIX A: A NOVEL, LOW-VOLUME METHOD FOR ORGAN CULTURE OF EMBRYONIC KIDNEYS THAT ALLOWS DEVELOPMENT OF CORTICO-MEDULLARY ANATOMICAL ORGANIZATION......................I 7.2 APPENDIX B: ECM MODULATED EARLY KIDNEY DEVELOPMENT IN EMBRYONIC ORGAN CULTURE ....XIX 7.3 APPENDIX C: THE DEWAXED ECM: AN EASY METHOD TO ANALYZE CELL BEHAVIOUR ON DECELLULARIZED EXTRACELLULAR MATRICES.......................................................................XLIV 7.4 PUBLICATIONS AND SCIENTIFIC CONTRIBUTIONS......................................................................LXV 7.5 SELBSTSTÄNDIGKEITSERKLÄRUNG......................................................................................LXIX