Direct visualisation of epithelial fluid transport at the subcellular scale

Epithelial cells are the specialized cells which form the barriers between tissues of the body and the inside or outside worlds. Different organs have different functions and different internal environments, which are set up and maintained by the transporting activities of epithelial cells. Epithelial cells form polarised sheets whose apical and basolateral "ends" face in opposite directions across the barrier. This polarity allows epithelial sheets to pump fluid in a directed fashion to maintain the normal physiology of individual tissues and the entire body. These fluid pumping processes are crucial to normal health and often found to be awry in various disease states. For example, disrupted fluid secretion in cystic fibrosis patients affects the function of their lungs, digestive system, and other organs all through this common pathway. In the eye, disturbed fluid regulation is believed to be responsible for glaucoma, cataract formation, macular degeneration, and retinal detachment. Between them, these diseases cause the majority of blindness worldwide.

The retinal pigment epithelium lies behind the neurons of the retina, and performs a series of functions to make sure vision is preserved by doing the "housekeeping" for the layers of nerve cells. The pigment epithelium is black to avoid reflections within the eyeball, and reaches out tiny fingers to wrap around and protect the vital photoreceptor cells which do the actual "seeing". When the epithelium is disrupted or injured, fluid can accumulate between the neurons and the epithelium like a blister, keeping the epithelium from helping the neurons to work properly. We still don't understand exactly how the epithelium works normally to prevent this from happening, and would like to know how to help the cells repair such damage by boosting their fluid pumping abilities in the right way.

A lot of what goes on when epithelia pump fluid happens between the cells, in a very confined space. Salt is pumped into these tiny spaces and water follows along to create secretion of fluid. Scientists are still not sure exactly how this is done to precisely match the balance of salts and water required for particular functions - from tears and sweat to urine and bile, these secretions can vary widely in their properties. The only trouble is, these tiny spaces which are so important are also too small to look inside very easily.

This project will develop a new tool which will help us understand epithelial fluid transport. It's actually a combination of tools, all being applied together, which will give us information about what the whole tissue is doing - from the large scale right down to the very small. Two new techniques are proposed (one an optical measurement, and one an optical stimulation technique) which will make measurements of salt and water movements in the tiny spaces we believe are crucial to fluid transport. By applying them together with measurements at the large scale, we can then perform experiments to see how the secretion of salt affects secretion of water, and vice versa. It's a bit of a chicken and egg problem, but attacking it by making many measurements at once should give us the answers we are looking for to understand what's going on.

If we understand fluid transport better we can make better choices in treating diseases resulting from fluid transport problems. In particular, this project will give hope to those with retinal disease which might be better treated by strategies we develop during this research project. We hope to make the system available to other researchers looking at fluid transport in other tissues too, so that the benefits of new technology can have the widest impact possible.

Transport of ions and water by epithelia is essential to homeostasis at the whole body, tissue, and cellular levels. Polarised epithelia use defined sets of ion transporters to accomplish directed movement of fluid transmurally. Most puzzling is the transport of apparently isotonic fluid, in the absence of detectable driving gradients. Several theories explain this by establishment of local ion and/or osmotic gradients within restricted intercellular spaces. These local gradients drive secretion by attracting water and solutes to produce vectorial fluid transport. The tiny geometries believed to be essential to this process have been historically difficult to probe.

Development of a system to address this problem is proposed, enabling direct measurement of water flux correlated with transcellular and paracellular pathways available to transported fluid. Retinal pigment epithelium (RPE), an important fluid transporting tissue, will be cultured on permeable membranes and probed using Raman microspectroscopy to image the movement of D2O ("heavy water") across the barrier. Measurements will be combined with perfusion and electrophysiology to manipulate the biophysics of epithelial transport, and watch the response in terms of water flux at the micron scale across the tissue. Included in the proposal is the deployment of "optogenetics" to introduce light-sensitive transport pathways into the epithelium, which will allow the system to be driven non-invasively with pulses of light to either shunt particular cells by opening cation channels across the membrane (Channelrhodospin-2), or by accumulating Cl- by activating an electrogenic anion pump (Halorhodopsin).

Applying these methods in concert with transmural electrodes to measure or impose voltage and current clamp will allow the process of epithelial fluid transport to be investigated at a level never before seen, and will unmask the fundamental processes by which isotonic fluid transport is obtained.

Epithelial fluid transport lies at the heart of bodily homeostasis - maintaining fluid and ion balance globally throughout the body and within individual organs or compartments. Some of the seminal discoveries in medicine have related serious disease to defects in epithelial transport, among them cystic fibrosis, kidney disease, gut defects, and ophthalmic diseases. This project will provide a technology base capable of increasing knowledge of these conditions and in many other contexts, by extending observations of fluid dynamics into the subcellular scale; illuminating fundamental biophysical processes which may then be manipulated for clinical benefit. The proposed investigation of retinal pigment epithelium has direct relevance to cutting-edge treatment of retinal diseases such as macular degeneration, retinal detachment, and retinitis pigmentosa. The ARMI retinal modelling collaboration is intended as a tool both for academic research and ultimately as an adjunct to clinical decision making, founding in a rational understanding of fundamental biophysics and cellular physiology. The project will therefore impact the knowledge base which informs the choice of clinical strategies, and potentially lead to the development of novel strategies superior to the current approaches to treating retinal disease.

As well as developing biomedical knowledge, the proposed project will yield a novel multimodal imaging platform which will be applicable to the study of any transporting epithelium or indeed any other tissue. In a broader context, the technology of ratiometric Raman imaging of water flux has potential application in other disciplines such as plant biology, soil science, oceanography, and other areas where water dynamics on a micro scale is important. The instrument proposed could be fitted in principle with any objective lens and used at scales both smaller and larger than that proposed while using fundamentally the same technology. Water flux imaging using D2O as a biocompatible tracer may also be useful in design and optimisation of bioreactors, microfluidic systems, and in evaluation of tissue perfusion in pharmacokinetic or similar studies, both in vitro and in vivo in human and animal subjects.