The goal of this R21 application is to develop a three-dimensional (3D) model of the glomerular filtration barrier that incorporates the key cellular components, podocytes and endothelial cells, and matrix that models the glomerular basement membrane (GBM). Using a novel method, we have developed a collagen-based hydrogel scaffold to replicate the basement membrane composition and structure, and have inserted this scaffold into an apparatus that permits the growth of apposing monolayers of podocytes and endothelial cells. The specific aims of this application will develop and characterize this 3D model, and will test requirements for the formation of mature cellular structures: foot processes, slit diaphragms, and fenestrae. Specific aim 1 will examine the role of cell matrix interactions and paracrine signaling events on the development of mature cell phenotypes. These studies will optimize thickness of the scaffold and incorporate relevant GBM peptides for native cell attachments. Hydrogel scaffold remodeling will be evaluated by Western analysis of native GBM proteins and HPLC quantitation of released di-tyramine adducts from degraded scaffold material. Cell phenotype will be analyzed by confocal immunofluorescence and electron microscopy. Specific aim 2 will focus on the physical properties of the acellular hydrogel scaffold and testing its ability to withstand pressure and shear stress loads using unconfined compression testing, and break tolerance testing in a flow chamber. In addition, the structure of the scaffold will be optimized to accommodate forces that replicate in vivo conditions through scaffold material concentration and crosslink density. Specific aim 3 then will integrate the cell optimization work in aim 1 with the scaffold optimization work in aim 2, by placing the 3D cell culture model into a flow chamber and testing its responses to the physical forces of filtration. These studies will allow integrated analysis of cell-cell, cell-matrix and hydrodynamic forces on the formation of foot processes, slit diaphragms and endothelial fenestrae. Our long-term goal is to develop a system that replicates all the "biochemical" (cell-cell and cell-matrix) and "mechanical" (fluid flow and pressure) signals that exist in the filtration barrier, as well as the integration of these signals into a system that recreates the in vivo dynamics of filtration barrier structure and function. We envision that this model could be used to test biological and pathological mechanisms of endothelial-podocyte interactions and model altered physical forces characteristic of glomerular disease. PUBLIC HEALTH RELEVANCE: We are designing an in vitro model system that can be used to test in a controlled manner the role of specific disease-inducing events on the structure and function of the kidney's filtration barrier. By determining which biochemical or mechanical stresses lead to abnormal cellular behavior will allow us to focus on these processes in disease, which may lay the ground work for new directions in therapy. These studies will allow us to understand not only how individual components of the filtration barrier work, but also how they interact with regard to both normal kidney function and how it is injured in disease. [unreadable] [unreadable] [unreadable]
NIH ExPorter
NIH ExPorter