Engineering a composite basement membrane for in vitro modelling of the glomerular filtration barrier

The function of the kidneys is to remove the body's waste products from the blood stream by filtering the blood. The part of the kidney which does this filtration is called the glomerulus. Each glomerulus consists of a ball of small blood vessels known as capillaries. The wall of these capillaries has unique adaptations enabling it to function as a selective filter. Although a lot is known from microscopy studies about the structure of the glomerular capillary wall, little is known about how it actually works. This is important as there are a number of diseases which can damage the filter leading to loss of proteins in the urine and eventually to kidney failure. Affected people need life-long dialysis treatment or a kidney transplant. However, if the filters recover the kidneys can function properly again. At the moment animal experiments are often required to understand effects of the disease on the kidneys and to examine how chemicals such as drugs are removed from the body by the kidneys. The glomerular capillary wall consists of layers of 2 specialised cells, endothelial cells and podocytes. These 2 cell types produce a thin matrix layer between them (the glomerular basement membrane containing extracellular matrix proteins such as collagen) to form a 3-layer structure. Until recently these cells have not been studied in detail because they have been difficult to keep alive in the laboratory. We have recently developed a technique to solve this problem by using introducing a new gene into the cells which allows them to be kept alive indefinitely. These means that we can study the cells in much greater detail. Previously we have studied these cells separately grown in plastic tissue culture flasks. We want to be able to grow layers of the 2 types of cell together, as they are in real life, to produce a model of the glomerular filtration barrier in the laboratory. We now have the two cell types we need to make this model but we don't yet have a good substitute for the basement membrane. In this project we will engineer a basement membrane suitable for use in the these models of the glomerular filtration barrier. This will be a composite of a very fine mesh to give structural support and a very thin layer of collagen. To produce and integrate these components we will need to use the latest available technologies. The fine mesh will be made of nickel by a process called micro-photo electro forming. This results in a high precision mesh with exactly defined aperture size. The collagen layer will be composed of nanofibres produced by a process known as electro-spinning. The collagen layer can be directly deposited on the nickel mesh. The composite mesh will be optimised to produce the thinnest basement membrane (as close to that in real life) as possible and to support attachment and growth of endothelial cells and podocytes. Once the best membrane has been defined it will incorporated, with the 2 cell types, into 3-layer models of the glomerular filtration barrier. These models will include important aspects of the conditions found in the glomerulus including the forces generated by blood flow. Hence we will produce a model which most accurately reflects behaviour of the glomerular capillary wall. We will be able to use this model to understand in more detail how the glomerular filter works and to determine what goes wrong in various diseases which can effect the kidneys. This will enable us to devise treatments to protect and repair the glomeruli, holding promise that patients found to have protein in their urine on screening tests could receive treatment before their kidney damage becomes serious. Furthermore these models will be useful to test the effects of drugs on the filters and to see how easily drugs are likely to be removed from the body by the kidneys. These models will increasingly replace the use of animal experiments to address these kinds of questions.

Glomerular filtration depends on the specialised three-layer glomerular capillary wall consisting of glomerular endothelial cells (GEnC), glomerular basement membrane (GBM) and podocytes. However the exact structural and functional mechanisms allowing selective filtration have not yet been elucidated, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised GEnC and podocytes. We are now using these cells to develop sophisticated in vitro models which will enable detailed studies of the glomerular filtration barrier (GFB), reducing the need for animal experiments. However a major problem with currently available coculture systems, is the thickness (minimum available 10microns) and bioincompatibility of the supports separating the two cell types in a three-layer structure. This project will address this problem by using state-of-the-art techniques to engineer a composite membrane composed of a nickel mesh and extracellular matrix (ECM) components. The mesh is produced by a high-precision technique of micro-photo electroforming and it provides structural support allowing a much thinner composite membrane than one made from ECM alone. The matrix component will be produced by electrospinning of sheets of collagen nanofibres. Mesh characteristics and nanofibre composition (in combinations of types I and IV collagen and chitosan) will be optimised. This will produce a composite with an overall thickness of less than 3micron and with at least 50% of the area composed entirely of ECM nanofibres. This composite will have permeability characteristics much nearer to the GBM in vivo, will be biodegradable and allow cell-cell communication via soluble mediators as well as being much thinner than existing alternatives. These characteristics will be verified by applying the composite membranes in existing coculture systems with GEnC and podocytes to reproduce the three-layer GFB.