No description available.

Nephrons develop from a specialized group of mesenchyme cells known as the nephron progenitors. Nephron progenitors can very dynamic as they can self-renew, migrate, and change their cell morphology. These alterations are essential for orientating and organizing select cells for progression through various stages of nephrogenesis. However, the underlying mechanisms that drive these dynamic morphological changes are not fully understood. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are associated with poor nephron function and chronic kidney disease. Despite these findings, the underlying mechanisms of Shroom3 function and how genetic mutations contribute to abnormal nephron formation are unclear. Here, we investigated functional roles for Shroom3 in the nephron progenitor population by analyzing E13.5 and E18.5 Wildtype and Shroom3 deficient mice (termed Shroom3-/-). First, using in-situ hybridization (ISH) and immunofluorescence (IF), we confirm Shroom3 expression in select nephron progenitors. Next, we demonstrated abnormal cell shape and abnormal nephron progenitor cell clustering using H&E staining and Pax2 immunofluorescence. We showed a reduction in nephron progenitor cell numbers and decreased cell length in E13.5 Shroom3-/- kidneys. Using markers of cell orientation, we discovered altered cell orientation in some but not all nephron progenitor cells. While analyzing the cell cytoskeleton, we also demonstrated the abnormal distribution of F-actin in Shroom-/- nephron progenitors. Lastly, immunofluorescence and transmission electron microscopy analysis of Shroom3-/- nephron progenitors confirmed the abnormal shape and reduced filopodia-like thin actin-based membrane protrusions. Our findings conclude that Shroom3 is essential for maintaining and regulating nephron progenitor cell morphology. Taken together, these findings could help explain why Shroom3 mutations are highly associated with kidney disease. / Thesis / Master of Science in Medical Sciences (MSMS)

No description available.

The aim of this thesis is to provide a modelling approach and simulation framework that allows for emergent dynamics in multi-nephron systems to be studied. The ultimate intent of this research is to provide an approach to renal modelling that is capable of predicting whole-kidney function from the dynamics of individual nephrons, and can therefore be of practical use to clinicians. The contributions of this thesis are: / • A modelling approach—hierarchical dynamical networks—which combines complex networks and graph automata into a single modelling framework. This approach explicitly captures the structure and interactions in multi-nephron systems, and decouples the structure and behaviour of the model. This approach allows emergent dynamics to be easily explored and analysed. / • The development of a multi-nephron model that produces valid behaviour and renders the simulation of whole-kidney function from the dynamics of individual nephrons computationally tractable. Using this model, the emergent effects of the couplings and interactions between nephrons can be investigated. / • An investigation into the dynamics of multi-nephron systems that focuses on whole-system and hierarchical properties rather than the dynamics of individual nephrons. As part of this investigation, the dynamics of a 72-nephron system are analysed—a system significantly larger than existing multi-nephron models. / • A study of whole-system stability in response to localised impairments in nephron function. This is the first study of the emergent dynamics of impaired nephron function, and serves as an illustration of how the emergent dynamics produced by renal diseases may be predicted and analysed. The impaired multinephron systems are shown to exhibit very stable behaviour, which we contend is a feature of both the model and the kidney proper. / • The computational cost of the model is shown to be low enough that the simulation of whole-kidney function is feasible for the first time. It is also demonstrated that simulations can be easily distributed across multiple computers, resulting in a significant gain in performance. An implementation of the model that supports parallel and distributed execution is presented, based on the Join Calculus. / • In order to predict whole-kidney function, a whole-kidney model must be constructed. This thesis proposes two approaches for automatically generating such models. / I conclude that the modelling and analysis techniques presented in this thesis allow for emergent dynamics to be studied in large multinephron systems. This work demonstrates that, for the first time, simulation of whole-kidney function from the dynamics of individual nephrons is tractable. Furthermore, the work provides a basis for predicting emergent effects of localised renal disease. With the continued development of this model, we hope that significant insight will be gained into the onset, progression and treatment of renal diseases.

C1q TNF-related protein-3 (CTRP3) is effective at preventing high-fat diet-induced fatty liver; Recent studies have shown that overexpression of C1q TNF-related protein-3 (CTRP3) in mice fed a high alcohol diet can protect the animal from developing fatty liver disease, and therefore may be a possible treatment for alcoholic fatty liver disease (ALD). However, the possible effects of overexpression of CTRP3 on other tissue has not been widely investigated. If overexpression of CTRP3 proves to be harmful to other tissues, its use as a treatment for ALD would come into question. This study utilized kidney tissue from mice that were fed a high fat diet for 13-14 weeks. The feeding started when the mice were 7 weeks old and continued for 9 weeks. The mice were divided into 4 categories: wild-type/low fat diet, wild-type/high fat diet, transgenic/low fat diet, and transgenic/high fat diet. The kidneys were harvested and fixed in 4% paraformaldehyde and subsequently paraffin embedded. Sections were cut at 4µ and stained using three different staining techniques: standard H&E, Periodic Acid Schiff (PAS), and Masson’s Trichrome Staining. These three methods were utilized to better visualize possible effects on the tissue, i.e. changes in connective tissue deposition or basement membrane thicknesses, etc. Light microscopic examination of the tissues to date has revealed abnormalities in some of the kidney tubules in the transgenic high fat diet group. These same abnormalities have not been observed in the other treatment groups. This study is still in its early stages and much more in-depth investigation is needed to determine which of the tubules of the nephron are affected, and what this effect is. If this study confirms that overexpression of CTRP3 coupled with a high fat diet is harmful to kidney tissues, the use of CTRP3 in the treatment of ALD would require careful monitoring of the patient’s diet.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18503号 / 医博第3923号 / 新制||医||1005(附属図書館) / 31389 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 斎藤 通紀, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die Homöostase wird maßgeblich durch die Niere, bestehend aus Millionen funktioneller Untereinheiten, den Nephronen, aufrechtherhalten. Chronisch geschädigte Nephrone führen zur Entwicklung einer terminalen Nierenerkrankung (TNE). Die Erzeugung renaler Zellen aus humanen pluripotenten Stammzellen (hPSCs) stellt eine vielversprechende Strategie zur regenerativen Therapie und Behandlung von TNE dar. In der vorliegenden Arbeit wurde ein Protokoll zur Differenzierung von renalen Vorläufern (RV) aus hPSCs entwickelt, welches nephronale Zelltypen und Strukturen in vitro und ex vivo erzeugte. Eine selektierte Kombination von Faktoren wurde in diesem 8-Tage-Protokoll genutzt, um die schrittweise Differenzierung der hPSCs zu lenken, indem die embryonale Organogenese der Niere abgebildet wurde. Am Tag 6 der Differenzierung konnten SIX2+/CITED1+ Zellen des metanephrischen Mesenchyms und HOXB7+/GRHL2+ Zellen, welche auf Vorläufer der Ureterknospe hindeuten, nachgewiesen werden. Diese entwickelten sich am Tag 8 weiter zu LGR5+/JAG1+/WT1+ renalen Vesikelzellen. Weiterführende Kultivierung in drei verschiedenen induktiven Medien führte zu WT1+/PODXL+/SYNPO+ Podozytenvorläufern, PDGFRß+/DESMIN+/αSMA+ Mesangialzellen und epithelialen Zellen des proximalen und distalen Tubulus sowie des Sammelrohrs. Außerdem bildeten die Tag-8-Vorläuferzellen spontan 3D renale Organoide aus. Die RV induzierten tubuläre Strukturen an einer Luft-Flüssigkeits-Grenzfläche und integrierten sich in embryonale Nierenaggregate. Zusammenfassend konnte demnach ein Protokoll entwickelt werden, welches entstehenden Nephronen ähnliche RV generierte, die innerhalb von 14 Tagen in spezialisierte nephronale Zelltypen differenzierten. Diese einfache Methode, um renale Zellen aus einem gemeinsamen Vorläuferpool in einer 2D -Kultur zu erzeugen, schafft die Grundlage für eine Produktion im größeren Maßstab, sowie für Modellsysteme in toxikologischen Untersuchungen oder Zelltherapien. / Kidneys are the central organ for homeostasis for our body systems and composed of around a million functional units, the nephrons. Chronically damaged nephrons deteriorate progressively towards end stage renal disease (ESRD), owing to the limited regenerative capacity of adult mammalian kidneys. The generation of renal cells from human pluripotent stem cells (hPSCs) is a promising strategy to develop regenerative therapies for ESRD. In this study, we established a protocol to differentiate hPSCs to renal progenitors (RP), capable of producing nephronal cell types and structures in vitro and ex vivo. An effective combination of factors obtained after intensive screening, was used to create an 8-day-protocol that steered hPSCs to the renal lineage by a step-wise process outlining the embryonic milestones in kidney organogenesis. Six days after growth factor treatment, a mixture of SIX2+/CITED1+ cells representing metanephric mesenchyme and an HOXB7+/GRHL2+ population indicative of ureteric bud progenitors was obtained that developed into LGR5+/JAG1+/WT1+ renal vesicle cells by the day 8. Prolonged cultivation of these day 8 cells in three inductive media resulted in generation of WT1+/PODXL+/SYNPO+ podocyte-precursors, PDGFRß+/DESMIN+/αSMA+-mesangial cells and fractions of proximal, distal and collecting duct tubular epithelial cells in vitro. Moreover, day 8 cells differentiate spontaneously into renal organoids in culture. The hPSC-derived RP gave rise to tubular structures upon culture as a pellet in air-liquid interface and integrated into embryonic kidney re-aggregations. Thus, we demonstrate that our protocol generates RP reminiscent of nascent nephrons, which can be coaxed into specialized nephronal cell types in vitro after 14 days from hPSCs. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, applicable for drug testing or cell therapies.

The mammalian kidney and reproductive systems both derive from a common embryological origin, the intermediate mesoderm. Abnormal intermediate mesoderm development can result in congenital abnormalities of the urogenital system, yet the molecular mechanisms that govern intermediate mesoderm development are incompletely defined. The spatial and temporal expression of the proteins BMP2 and 4 and their receptor ALK3, in urogenital tissue, suggests a function for BMP-ALK3 signaling in the intermediate mesoderm. It was found that Alk3IM null kidneys display renal hypoplasia, associated with a decrease in kidney size and nephron number. The phenotype of renal hypoplasia in Alk3IM nulls was associated with early decreased number of developing nephron structures and secondary defects in branching morphogenesis. While neither apoptosis nor cell proliferation differed in metanephric mesenchyme cells in Alk3IM nulls, markers of renal progenitor cells were decreased in mutant animals. It was observed that Alk3 expression in the intermediate mesoderm also controls mesonephric tubule number. Alk3IM nulls had fewer mesonephric tubules and fewer derivative Leydig cells. The reduction in Leydig cells resulted in decreased levels in serum testosterone and defects in seminal vesicle formation and fertility. Alk3 expression was also required for normal development of the corpus epididymis. The morphological defects in nephrogenesis were associated with decreased phospho-p38 MAPK expression and in the testis with decreased Phospho-SMAD1/5/8. These results elucidated a requirement for Alk3 signaling in controlling progenitor cells derived from the intermediate mesoderm.

The mammalian kidney and reproductive systems both derive from a common embryological origin, the intermediate mesoderm. Abnormal intermediate mesoderm development can result in congenital abnormalities of the urogenital system, yet the molecular mechanisms that govern intermediate mesoderm development are incompletely defined. The spatial and temporal expression of the proteins BMP2 and 4 and their receptor ALK3, in urogenital tissue, suggests a function for BMP-ALK3 signaling in the intermediate mesoderm. It was found that Alk3IM null kidneys display renal hypoplasia, associated with a decrease in kidney size and nephron number. The phenotype of renal hypoplasia in Alk3IM nulls was associated with early decreased number of developing nephron structures and secondary defects in branching morphogenesis. While neither apoptosis nor cell proliferation differed in metanephric mesenchyme cells in Alk3IM nulls, markers of renal progenitor cells were decreased in mutant animals. It was observed that Alk3 expression in the intermediate mesoderm also controls mesonephric tubule number. Alk3IM nulls had fewer mesonephric tubules and fewer derivative Leydig cells. The reduction in Leydig cells resulted in decreased levels in serum testosterone and defects in seminal vesicle formation and fertility. Alk3 expression was also required for normal development of the corpus epididymis. The morphological defects in nephrogenesis were associated with decreased phospho-p38 MAPK expression and in the testis with decreased Phospho-SMAD1/5/8. These results elucidated a requirement for Alk3 signaling in controlling progenitor cells derived from the intermediate mesoderm.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22692号 / 医博第4636号 / 新制||医||1045(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 柳田 素子, 教授 斎藤 通紀, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM