Denise K. Marciano - US grants

DESCRIPTION (provided by applicant): The aim of this project is to better understand the processes of early kidney development, focusing on the study of a protein called nephronectin and the integrin receptor to which it binds alpha8beta1. We know that the integrin alpha8beta1 is critical for kidney development in mice: mice lacking alpha8 beta1 do not develop kidneys while other organs are intact. Our goal is to investigate the specific role of nephronectin, as well as alpha8beta1, in kidney development using cultures of embryonic kidney. The numerous congenital kidney diseases that exist in humans underscore the importance of studying kidney development. In addition to the importance of kidney development in its own right, many parallels exist between molecules that are important for development and molecules that are important for kidney repair after an acute injury. This is particularly true of the early stages of kidney development, which this project focuses on.

DESCRIPTION (provided by applicant): As a physician-scientist in the field of nephrology, I have a strong interest in development and disease in the kidney. My long-term career goals are to understand cellular and molecular events in kidney development and translate this into clinically relevant therapeutics for acute kidney injury. A second goal for my career is to help elucidate the pathogenesis of heritable kidney diseases, such as polycystic diseases. Toward these goals, I have proposed a project that focuses on understanding the dynamics of cell-cell adhesion and organ morphogenesis in the developing kidney using a mouse model of polycystic disease. My research proposal, entitled The role of p120 catenin in metanephric kidney development, is constructed to provide a framework to achieve my goals. My preliminary data shows that newborn mice lacking p120ctn in nephrons develop kidney cysts and renal hypoplasia. I have proposed three specific aims to investigate the cellular and molecular mechanisms involved. First, I will test the hypothesis that loss of p120 catenin results in polycystic kidneys due to an alteration of cellular proliferation, apoptosis or cell polarity. This will be accomplished by biochemical and histological methods in mice lacking p120 catenin from nephron precursors. Employing similar methods, I also will test the hypothesis that loss of p120 catenin results in renal hypoplasia due to reduced nephron formation resulting from a defect in mesenchymal-to-epithelial transition during development. Second, I hypothesize that p120 catenin regulates cadherin levels and additional known intracellular signaling pathways, such as Rho family GTPases and NF(B signaling. I will test this using mouse genetics, immunohistochemistry, biochemistry and quantitative PCR. Third, I hypothesize that p120 catenin may regulate novel signaling pathways in the kidney that have not yet been elucidated. I propose to perform comparative gene expression profiling using microarrays to elucidate novel signaling pathways. Together this proposal is designed to complement my prior laboratory experiences and provide me with the technical and intellectual tools to become an independent investigator. PUBLIC HEALTH RELEVANCE: Defining the mechanisms of p120ctn's role in kidney development is important, not only because it will help elucidate the normal mechanisms of cell adhesion and epithelial tubulogenesis, but also because it may shed light on cystic disease pathogenesis. Furthermore, identification of the molecules and pathways that underlie metanephric development may increase understanding of renal regeneration and repair and may lead to improved strategies for enhanced recovery.

DESCRIPTION (provided by applicant): Role of Afadin Signaling in Nephron Tubulogenesis Project Summary/Abstract Formation of polarized epithelial tubules is central to the structure and function of the kidney. Despite its importance, there is a fundamental gap in understanding the earliest steps in tubulogenesis, namely how epithelia establish apical-basal polarity and generate a central lumen. Continued existence of this gap represents an important problem because, until it is filled, an understanding of developmental abnormalities of the kidney will remain largely incomprehensible. The objective of this application is to elucidate how apical- basal polarity is established and lumen formation initiated, focusing on the role of Afadin signaling in nephron formation. Afadin is an adaptor protein to the Nectin family of adhesion receptors. Our preliminary data identify Afadin as critical for establishing an apical surface and initiating lumen formation in developing mouse nephrons. To further elucidate the cellular and molecular mechanisms that initiate polarity and lumen formation, the proposed studies have three specific aims. The first aim is to characterize molecular steps of lumen formation in vivo and in great detail. These studies will generate a molecular timeline of lumen initiation through subcellular localization of polarity, trafficking, and junctional complexes, and through live imaging of lumen formation in mice. Once characterized, additional studies will begin to assign proteins to different stages in the pathway using mouse models. The second aim is to identify novel molecular mechanisms of apical polarity and lumen formation using an established 3D cell culture model. Specifically the proposed experiments will elucidate a detailed signaling pathway by which Nectins and Afadin initiate lumen formation. The third aim will examine the role of Afadin in tubule regeneration after ischemic injury, emphasizing similarities to the developmental process. Together these studies will delineate fundamental steps in polarity and lumen initiation and provide an improved framework for understanding developmental and acquired disorders of renal tubules.

Project abstract: The glomerular basement membrane (GBM) forms a critical component of glomeruli, and is comprised of numerous proteins, many of whose individual functions are unknown. Much of the GBM is located between podocytes and endothelia, however, it also interacts directly with mesangial cells, a pericyte-like, contractile, support cell. Very little is known about the individual GBM proteins involved in this interaction and the role these interactions play in glomerular structure and function throughout development and in adulthood. In preliminary studies, we have identified a novel GBM-mesangial cell adhesion that contains the GBM protein nephronectin. We find that nephronectin is essential for development of the glomerular tuft, and also plays a role in maintenance of glomerular structure. We therefore propose to 1) determine the role of nephronectin in various stages of glomerulogenesis, 2) test its role in mediating stability of glomerular structure in mature kidneys, and 3) dissect the role of nephronectin-integrin signaling in cultured cells. Through these studies, we will greatly expand our understanding of GBM- mesangial cell interactions. Gaining molecular knowledge of these interactions and their function will be a critical step to understanding glomerular biology, and may contribute to directed therapies for glomerular disease.

Project Summary Pancreatic endocrine cells, including insulin-producing beta cells, acquire their fate in a step-wise manner during embryonic development. Understanding and recapitulating these steps has proven essential for directed differentiation of ES cells into beta cells. A number of academic and biopharma groups have reported improved efficiency of beta cell generation upon shifting culture methods from 2D to 3D cultures. This observation suggests that architecture of the cellular niche for beta cell generation is critical, however, this idea currently remains unexplored. We previously reported that when the pancreas first emerges, the endodermal epithelium undergoes transient stratification, followed by microlumen formation and fusion to generate a 3D network of interconnected epithelial tubes called the pancreatic `plexus' [1]. Interestingly, recent studies demonstrate that endocrine progenitors are born within this transient core plexus. It is unclear how the plexus architecture impacts the fate of pancreatic progenitors, including those of endocrine lineage. Since our initial proposal, we published the findings that Afadin is essential to pancreas morphogenesis and endocrine fate (Azizoglu et al., 2017). Here, we propose to elucidate the cellular and molecular mechanisms by which Afadin controls epithelial lumen formation and plexus morphogenesis. In previous work, we generated a mutant mouse with deletion of the junctional and cytoskeletal regulator Afadin (AfapancKO) that fails to resolve its transient plexus. Co-depletion of Afadin and RhoA (AfaRhoApancKO or AfaRhoDKO) exhibits multiple lumen defects. Surprisingly, it also produces an increase in endocrine cell numbers, including beta cells. RhoApancKO however, show no pancreatic defects. How Afadin and RhoA pathways interact remains unknown. One striking observation in both AfapancKO and AfaRhoDKO is that the core plexus persists. We propose that the progenitor pool and final endocrine mass is determined by the perdurance of the core plexus. How this occurs is the central question of this proposal. We hypothesize that Afadin and RhoA drive epithelial lumen morphogenesis (formation/extension/resolution) via regulation of cellular processes, such as vesicle trafficking (Aim 1), and cell division and/or cell migration (Aim 2). Further, we hypothesize that Afadin and RhoA control these processes by regulating cytoskeletal organization (Aim 3). Together, these processes coordinate to build a niche propitious for generation of endocrine cells. Completion of these studies will expand our knowledge of pancreatic development, and will lead to enhanced strategies for generating endocrine cells, including beta cells, which may contribute to novel treatments for type I diabetic patients.

Project Summary Kidney disease is the 9th leading cause of death in the U.S. Because few therapies exist to prevent or slow progression, over 700,000 patients have End Stage Renal Disease. These patients are treated with dialysis or renal transplant, the latter resulting in markedly superior survival. However, kidney donors are limited and there is an important unmet need for strategies that enhance renal repair or generate new nephrons for renal replacement. Pluripotent stem cell derived organoids display key features of differentiated kidney tubules and glomerular structures in vitro, and we have shown that they generate patterned nephrons in vivo displaying kidney functions such as filtration and glucose uptake by the proximal tubule. To develop this technology for renal replacement, stem cell derived tubules must be connected to host tubules for urinary output. Our recent work in the zebrafish demonstrated that FGF signaling acts as a chemotactic signal to recruit and polarize cells at sites of new nephron formation and canonical Wnt signaling is required for invasive cell rearrangement to connect tubule lumens. Additional signaling pathways including non-canonical wnt signaling are also likely to play a role in tubule interconnection. To fully explore the requirements for tubule interconnection we have established a synergistic, three-part discovery platform comprising 1) genetic analysis of in vivo new nephron addition in the regenerating zebrafish adult kidney, 2) in vitro 3D cell culture analysis of mammalian epithelial fusion, and 3) in vivo stem cell-derived kidney organoid engraftment to a host mouse collecting system. We will combine these approaches to analyze multiple steps of the tubule fusion process involving 1) recruitment of nephron progenitor cells to target epithelia, 2) removal of intervening ECM/basement membranes, 3) patterned collective cell invasion of target epithelia, and 4) establishment of a continuous patent new lumen to convey the nephron filtrate. These studies will provide important new insights about an essential but understudied cellular mechanism that will be required for in vivo engraftment of new kidney tissue-based renal regeneration therapies.