Dale R. Abrahamson - US grants

The glycoprotein laminin is a major component of all basement membranes, but its ultrastructural distribution is uncertain. The structural modifications that occur within basement membranes during disease are also largely unknown. The goals of this project are to (1) determine the normal ultrastructural distribution of laminin within renal glomerular basement membranes (GBM). A new post-embedding method of immunocytochemistry will be used. Conjugates of affinity purified sheep or rabbit anti-laminin IgG (Alpha L) and biotin will be intravenously injected into rats and kidneys will be fixed in situ. Ultrathin sections of Lowicryl and epoxy-embedded tissue will then be stained with a particulate tracer, avidin-colloidal gold. Biotin conjugates of monoclonal Alpha L against pepsin and elastase resistant laminin fragments will also be injected and localized. These results will then be compared with those obtained with the enzymatic tracer, horseradish peroxidase (HRP)-Alpha L. These techniques will also be extended to localize within the GBM affinity purified and monoclonal anti-type IV collagen IgG. (2) To trace GBM development and turnover, Alpha L-biotin and Alpha L-HRP will be injected into newborn rats and kidneys will be examined at intervals from 1 hr to 18 months thereafter. Double label experiments will also be conducted to determine whether laminin is added only to new capillary loops or to existing GBM as well. (3) A cross-reacting, laminin-like molecule in rat kidney extracts will be immunopurified, examined by rotary shadowing, tested as an autoantigen in rats, and used for further ultrastructural studies. (4) In an accelerated model of glomerulonephritis, rats that have been preimmunized against rabbit Alpha L will receive injections of Alpha L. The resulting proteinuria and ultrastructural changes will be correlated with the titles of rat anti-Alpha L. This experiment differs significantly from previous work because it utilizes antibodies solely against laminin to create pathological changes. (5) Nephrotoxic nephritis (NTN) will be induced in rats by the injection of rabbit anti-kidney IgG. The distribution of laminin during the heterologous and autologous phases of NTN will then be determined by injecting Alpha L, Alpha L-biotin, and AlphaL-HRP. These will serve as specific GBM probes that can be visualized by immunofluorescent and immunoelectron microscopy. Of particular interest will be whether infiltrating neutrophils, monocytes, and lymphocytes affect the distribution of GBM laminin. This research will provide new information on the fundamental structure of basement membranes during health and disease.

Metanephric kidney glomerular capillary walls develop from tbe formation of microvasculature, close apposition of immature endothelial and visceral epithelial cells (podocytes), and fusion of a dual basement membrane between these two cell layers. With subsequent capillary loop expansion, additional basement membrane, derived mainly from podocytes, is spliced into the fused GBM. Although many of the morphological features of glomerular development have been described, very little is known about the exact origins of the microvasculature and how basement membrane assembly occurs. The three specific aims in this renewal application will begin to define the cellular and molecular mechanisms operative in glomerulogenesis in vivo. (1) The origins of the glomerular endothelium and mesangium will be determined. This will be accomplished by grafting fetal mouse kidneys from normal/transgenic donors into transgenic/normal host mice. In situ hybridization will define the glomerular cell lineages within these grafts. (2) Binding sites within the developing GBM for certain laminin domains are hypothesized to be important for basement membrane fusion and splicing. This idea will be tested by domain-specific mAb affinity isolation of proteolytic laminin fragments, injection of these into newborn rodents, and immunoelectron microscopy. In addition, a laminin cleaving enzyme identified in developing glomeruli will be characterized and its role in GBM assembly determined. (3) Using antibodies against recombinant laminin cloned from a fetal rat kidney expression library, the cellular origin, distribution, and fate of authentic kidney laminin within the developing glomerulus will be defined. These experiments will provide fundamentally new information on mechanisms of glomerular capillary growth and GBM assembly.

scanning transmission electron microscopy; transmission electron microscopy; scanning electron microscopy; biomedical facility; bioimaging /biomedical imaging; immunoelectron microscopy;

DESCRIPTION (provided by applicant): Among the genes strongly induced by hypoxia-induced transcription factors (HIFs) and highly expressed during kidney microvascular development is VEGF, which encodes a potent endothelial mitogen and chemoattractant critical for embryonic vasculogenesis and angiogenesis. In developing kidney, glomerular podocytes are particularly rich sources of VEGF, which probably serves to attract endothelial precursors into vascular clefts of immature glomeruli, promote their mitosis and differentiation into glomerular endothelial cells, and assist with maintenance of their highly differentiated state through maturation. Moreover, we have recently localized expression of HIF mRNA transcripts and protein to developing podocytes of immature glomeruli. This leads to our central hypothesis: HIF stabilization coordinates synthesis of VEGF and its receptors, and governs kidney microvascular development. To address this hypothesis, we have designed three complementary specific aims. First, we will determine whether HIFs are differentially stabilized in podocyte and endothelial cell lines exposed to different oxygen tensions. Additionally, we will selectively knockdown separate HIF alpha subunits, HIF-1 beta, and HIF gene target mRNAs, using small interfering RNA (siRNA) technologies in vitro. Second, we will determine whether kidney vascular development is affected in hybrid nephrons containing ES cells with crippled HIF activation and VEGF signaling systems. Third, we will examine in vivo consequences of losses and gains of HIF alpha subunits, using Cre-lox technology, among other approaches. These results should provide fundamentally new information on mechanisms governing renal microvessel development and maintenance, which may have broad applicability to blood vessel formation in general.

Description: The kidney glomerular capillary wall is comprised of three elements: (1) an inner endothelial cell layer; (2) the glomerular basement membrane (GBM); and (3) an outer epithelial cell layer (podocytes). That this unique structure constitutes the glomerular filtration barrier has been well established, but how this structure develops during kidney organogenesis, is maintained in maturation normally, and becomes damaged in disease, is not fully understood. The purpose of our application is to establish a focused and complementary group of research projects that will define the development and assembly of the GBM and the molecular pathogenesis underlying selected glomerular diseases. Specifically, Project 1 will examine abnormal glomerular phenotypes in certain laminin and collagen IV mutant mice and rescue these phenotypes through metanephric grafting and inducible, cell-selective transgene expression strategies. Project 2 will study type IV collagen network formation and interaction with glomerular cell integrins using mass spectrometry, x-ray crystallography, and surface plasmon resonance, in combination with classic molecular and cellular methodologies. Project 3 will map the molecular location of pathogenic Goodpasture and Alport alloantigens on type IV collagen, examine the development of anti-GBM disease in a mouse expressing humanized type IV collagen, and in an Alport mouse model. Project 4 will examine the molecular regulation of extracellular matrix assembly and cell-matrix interactions in vivo in hydra and Zebrafish embryos as simplified model organisms, using antisense knock down and real-time imaging techniques, among other approaches. The Program is designed to promote extensive communication and sharing of resources among the Projects, which are also supported by an Administrative Core (A) and a Molecular Recognition Core (B). We anticipate that this Program will reveal fundamentally new information regarding glomerular structure in health and disease.

[unreadable] DESCRIPTION (provided by applicant): Five years of funding are requested to develop a Center of Biomedical Research Excellence at the ULKMC (KUMC) with a focus on the molecular regulation of cell development and differentiation. Five talented new faculty who share this research interest were selected with the goal of helping them become funded independent investigators. A PI, two co-PIs, an internal advisory committee (IAC), a group of mentoring faculty, and an external advisory committee (EAC) of distinguished investigators have been assembled to achieve this goal. The Center is also designed to improve the infrastructure and research environment at KUMC. The expected outcome of the program will be more research productivity as reflected in joint publications, funded R01 grants, and program project and research center grants based on a developmental biology theme. A mentor has been assigned to each initial junior faculty member, and individual mentoring plans and timetables have been developed. Central features of the mentoring plans include ongoing critical evaluation of the research projects by mentors and IAC, semiannual conferences with EAC members, and special training on statistics, manuscript and grant writing in workshops. The initial group will receive support for up to 3 years. Within this time frame, each member will be expected to compete successfully for NIH R01-type research support. Once funded externally, they will rotate off the grant financially to make room for the addition of new junior faculty, and we have firm commitments from Department Chairs/Center Directors who will provide these new faculty. Over 5 years, the grant will support 10-15 independent researchers. Another important feature is to establish 3 new scientific Core capabilities to provide additional research support for the Center's faculty. Together with the Administrative Core A, there will be Cores supporting transgenesis, gene targeting and genotyping (Core B), molecular profiling and recombin [unreadable] [unreadable]

Glomerular development is marked by a sequence of type IV collagen isotype replacements in the GBM. Specifically, collagen IV a1a2a1 network, present in the earliest glomerular basement membrane (GBM) of immature nephrons, is replaced in mature GBM by collagen IV a3a4a5 network. Our central hypothesis is: GBM collagen IV network switching is critical for development and maintenance of both endothelial cells and podocytes. We speculate that: cells survey their evolving type IV collagen networks through integrins;synthesis of type IV collagen and its cognate integrin receptors are linked;and when receptors engage abnormal collagen networks, or if receptor expression is incorrect, altered signals are conveyed. This abnormal signaling then thwarts normal basement membrane protein synthesis, leading to further defects in glomerular structure and function. Our aims are: Aim 1) To examine possible defects in GBM-integrin signaling in Col4a3 mutant mice, a model of Alport disease. We will (a) define the GBM composition from Col4a3 mutants;(b) characterize integrin expression during progression of Alport disease;and (c) inventory changes in glomerular cell proteins between normal and Alport mice using a proteomics approach. Aim 2) To characterize glomeruli of Col4a1 heterozygous mice, which are microalbuminuric with thin GBM. We will (a) define the GBM composition from Col4a1 mutants;(b) characterize their integrins and (c) compare their glomerular proteomes with wild type. Aim 3) To determine the contribution of integrins a1 [unreadable]1 and a2[unreadable]1 on collagen IV network formation. Integrin a1[unreadable]1is anti-fibrotic whereas a2[unreadable]1 is pro-fibrotic. We will (a) cross integrin a1-null mice onto Col4a3 and Col4a1 backgrounds expecting increased fibrosis;and (b) cross integrin a2-null mice onto the same, expecting amelioration. We will also test for GBM assembly perturbations through in vivo administration of heterotrimeric triple helical collagen peptides containing integrin binding sites and by injection of antibodies against the type IV collagen integrin binding sites. Aim 4) Determine whether GBM protein synthesis and integrin expression are linked. This will be tested in glomerular cell cultures by qRT-PCR, Western blotting, siRNA knockdowns, and confocal immunofluorescence and immunoelectron microscopy. Together, these studies will provide fundamentally new information on the dynamic reciprocity between glomerular cells and collagen IV networks and how integrins mediate this critically important interrelationship.

DESCRIPTION (provided by applicant): The broad, long term objective of this application is to obtain funds to purchase a new JEOL JEM-1400 transmission electron microscope (TEM) for the Electron Microscopy Research Laboratory (EMRL) at the University of Kansas Medical Center (KUMC). This core facility has been in operation since 1972, and has received annual financial support from KUMC continuously. The EMRL has superb, experienced technical staff and contains all of the necessary ancillary equipment to carry out a wide range of experiments using TEM and scanning electron microscopy (SEM). However, the only operative TEM instrument available is a JEOL JEM-100CX II that is more than 24 years old. This microscope is now obsolete. Several NIH-funded teams researching a diverse set of health-related topics currently rely on the EMRL at KUMC. These include projects that address mechanisms of (a) normal kidney glomerular capillary development and defects leading to loss of filtration barrier properties;(b) formation of neuronal synapses;(c) intestinal dysmotility and bacterial overgrowth in cystic fibrosis;(d) damage induced by oxidative stress in aging muscle and brain;(e) somatic cell-gonadal germ cell interactions critical for fertility;(f) composition and functions of the bacterial cytoskeleton important for cell division;(g) pathogenesis of HIV-AIDS in a primate model;and (f) anti-cancer activity of select fatty acids on human breast cancer cells. Progress on all of these projects will be enhanced with the availability of a reliable, modern TEM, and throughput will also be increased with access to the requested AMT digital camera system and 5 sample grid holder.

Core A: Administrative and Mentoring Core The Administrative and Mentoring Core A of the COBRE will provide administative leadership and coordinate the overall research efforts of the Center, provide financial accounting, clerical and other program management support, organize a group of established senior investigators who will serve as Mentors for newly hired faculty working in the area of developmental biology, manage and document the junior faculty mentoring process, increase the research infrastructure at KUMC, offer a source of expert outside consultation, insure the maintenance of cooperative collaborative interactions between all components of the COBRE and other research groups at KUMC, including the newly funded CTSA, and establish a Scientific Writing Training Program to benefit all members of the Center.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This Core integrates and enhances the capabilities of 2 exisiting cores at KUMC: the Electron Microscope Research Laboratory (EMRL) and the Confocal Imaging Facilty, both of which have assisted investigators at KUMC for many years. The High Resolution Imaging Core provides cost-effective and time-saving services for the preparation and viewing of cell and tissue samples by routine confocal and scanning and transmission electron microscopy. More than 20% of the senior faculty and almost all of the junior faculty in this COBRE have steady needs for high resolution microscopy. This Core also offers expanded service carried out by expert technicians which includes immunolocalization technology for both light and electron microscopy, as well as laser capture microdissection.

This Core integrates and enhances the capabilities of two existing scientific cores at KUMC: the Electron Microscope Research Laboratory (EMRL) and the Confocal Imaging Facility (CIF), both of which have assisted investigators at KUMC for many years. The High Resolution Imaging Core will provide cost effective and time-saving services for the preparation and viewing of samples by routine confocal and scanning and transmission electron microscope imaging. More than 20% of the senior faculty and all of the COBRE Beginning Investigators have steady needs for high resolution microscopy. This Core will offer new, expanded service carried out by expert technicians that will include immunolocalization technology for both light and electron microscopy.

OVERALL PROJECT SUMMARY ABSTRACT Funds are requested to renew a Center of Biomedical Research Excellence (COBRE) at the University of Kansas Medical Center (KUMC) as a Phase III, five year award. This COBRE has a research focus on the regulation of cell development and differentiation, which may provide new, therapeutic insights into a host of developmental abnormalities and diseases. During the initial 10 year term, we will have trained 15 Beginning Investigators and most of them are becoming successful, independent researchers. An important feature of this application is the maintenance of 3 scientific Cores that provide research support to all of the Center faculty as well as to KUMC as a whole. Together with Administrative Core A, there are Cores supporting transgenesis in mice and rats (Core B), molecular profiling (next generation sequencing, microarray, and bioinformatics, Core C), and high resolution imaging (confocal and electron microscopy, Core D). The second major component of this application is the operation of a Pilot Projects Program in developmental biology, which represents a natural extension of a pilot program initiated in Phase I and II that has a proven, successful track record. The outstanding combination of scientific talent, strong research environment, and substantial institutional commitment to the scientific Cores and Pilot Projects Program ensure that this COBRE will continue to foster the development of a thematic, multidisciplinary research effort and enhance the ability of KUMC researchers to compete for extramural support.

CORE D: HIGH RESOLUTION IMAGING CORE PROJECT SUMMARY This Core integrates and enhances the capabilities of two existing Cores at KUMC: the Electron Microscope Research Laboratory (EMRL) and the Confocal Imaging Facility (CIF), both of which have assisted KUMC investigators for many years. The High Resolution Imaging Core provides cost-effective and time-saving services for the preparation and viewing of samples by confocal and scanning and transmission electron microscopy. Most of the members of this COBRE have steady needs for high resolution microscopy, and this Core has kept pace with advances in technology and equipment. A new super resolution microscope (requested in a separate and pending S10 application to NIGMS) will be housed in space assigned to this COBRE-supported Core and staffed by Core personnel.

Core A Abstract The Administrative Core A of the COBRE will provide administrative leadership and coordinate the overall research efforts of the Center, provide financial accounting, clerical and other program management support, help increase the research infrastructure at KUMC through Core support, offer a source of expert outside consultation, and insure the maintenance of cooperative collaborative interactions between all of the components of the COBRE and other research groups at KUMC.