Treating kidney failure in septic shock
Prof Tom Evans & Dr Susan Lindsay
Glasgow University
2004 – 2007 Postdoctoral Fellowship: Using a novel three-dimensional cell culture model to investigate sepsis-induced renal failure.
Prof Tom Evans is Professor of Molecular Microbiology in the Division of Immunology, Infection and Inflammation at Glasgow University.
Dr Susan Lindsay is a Dr Hadwen Trust Research Fellow at Glasgow University.
Septic shock is the most common cause of death in hospital intensive care units. Patients with sepsis deteriorate rapidly, and commonly suffer acute renal failure (ARF), resulting in death. At present the underlying pathophysiological mechanisms involved are not clear, there is no medicine to prevent or treat acute kidney failure.
There is some prospect for treatment of this condition with agents that promote renal tubule repair, such as hepatocyte growth factor (HGF). This project will use a three-dimensional model of human renal tubule cells to investigate the pathophysiology of sepsis-induced renal failure and the mechanisms of HGF action. The primary purpose is to define better treatment for renal failure in sepsis using a human tissue culture model, and thus enable a reduction in the use of animals in sepsis research.
Sepsis research on animals is categorised by the Home Office as having the potential to cause substantially severe levels of pain and suffering, as well as death. Sepsis experiments involve rodents, rabbits, sheep, dogs, pigs and baboons. Multiple organ failure is induced by injecting bacteria or bacterial products, or sometimes the animal’s bowel is punctured to allow gut bacteria to enter the bloodstream. Numerous sepsis treatments have been developed that are highly successful in experimental animals, but all have failed in human patients.
Dr Hadwen Trust Research Fellow, Dr Susan Lindsay, is characterising a novel three-dimensional model of human renal proximal tubules. This model is being used instead of animal experiments to study the pathophysiological effects of sepsis-related cytokines and anoxia, and to explore the possible therapeutic use of HGF in treatment of acute renal failure in sepsis.
Publications
Langley C, Brock C, Brouwer G et al (2005). Opportunities to replace the use of animals in sepsis research. The report and recommendations of a Focus on Alternatives workshop. Alternatives to Laboratory Animals (ATLA) 33:641-8.
PubMed Link
Langley G, Evans T, Holgate ST and Jones A (2007). Replacing animal experiments: choices, chances and challenges. BioEssays 29:918-926.
PubMed Link
Sepsis at the cell level
Work in progress report by Professor Tom Evans
The discovery of antibiotics revolutionised the practice of medicine, allowing formerly fatal bacterial infections to be readily treated. However, despite antibiotics, bloodstream infections with bacteria still remain a serious medical problem. These infections can produce a syndrome called sepsis that has a mortality of about 20%, despite antibiotic treatment, and accounts for about 14,000 deaths in hospitals every year in England and Wales [1]. Sepsis produces complex changes in the functions of organs such as the kidneys and lungs that typically lead to failure of these different organ systems.
How does sepsis result in multiple organ failure? Research over the last twenty years has shown that bacterial infections trigger the host to produce a variety of chemical mediators. In small amounts these enhance our ability to combat infection, but when produced in large amounts can result in the development of sepsis. Bacteria can trigger the release of these mediators early in an infection that continue to produce deleterious effects even if the bacteria are subsequently killed with antibiotics. We have been researching for some years how this host response causes organ damage, in the hope that this could lead to better therapies of this serious medical condition.
One of the challenges in sepsis research is the complexity of the syndrome, which involves different organ systems that can interact in an interdependent fashion. Animal models have been used extensively in this kind of research and have contributed to a better understanding of the mechanisms underlying the complex changes observed. However, animals often behave in a very different fashion to humans with sepsis and animal models have been poorly predictive of the clinical utility of new therapies that have been developed, many of which failed in clinical trial despite promising results in animal models. Moreover, the severity of the insult to the animal following infection is an important ethical consideration.
In work supported by the Dr Hadwen Trust, we have been exploring the use of human cell culture models to gain better understanding of the reasons for sepsis-induced organ failure. We have focused on kidney cells, since renal failure is common in sepsis and has a poor prognosis.
Using material from kidneys removed at surgery for clinical indications, we have been able to grow human kidney cells in culture. Usually this is done by growing them on a flat plastic surface to which the cells adhere. Although such two-dimensional models of tissue culture are very useful, they lack the more complex architecture found in a real organ. We have found that we can induce these cells to grow in three-dimensions by casting them in a collagen ‘gel’.
With the correct culture conditions, these cells form cysts and even tubules, resembling those of the normal human kidney. These three-dimensional cultures can be used to investigate the effects of the mediators released in sepsis on the processes of damage and repair of kidney tubules. The analysis of complex three-dimensional cultures also requires specialized microscopy to enable clear images in one optical plane of the specimen, as shown in the accompanying figure.
Figure: High power microscopic views of human kidney cells grown in three-dimensional culture. The images show phase-contrast views (left panel), cells stained for an actin-associated protein ezrin that is found in the brush border of the tubule (green, centre panel), and merged images to the right.
One of the mediators in which we are particularly interested is nitric oxide (NO) [2]. NO is a key mediator of many physiological processes and is produced in excess by many cells in sepsis. Some of its effects may be beneficial, others deleterious. We have found that NO alters the function of a protein called vasodilator-stimulated phosphoprotein (VASP) within human kidney cells. VASP regulates how cells move and interact with one another. In particular, it is found at the leading edge of moving cells – the lamellipodium. NO causes VASP to disappear from this leading edge, resulting in the cells rounding up and slowing down [3]. Movement of renal cells is essential to regenerate kidney tubule following damage. Thus, the effect of NO on VASP may be important in preventing renal repair in sepsis which if countered could improve renal regeneration and hence patient outcome. Our initial studies have been in two-dimensional culture models that we are now extending to our three-dimensional system.
These studies are some way off providing tangible benefits to patients with sepsis. However, we believe strongly that these kinds of approaches in sepsis research are important in developing new therapies for this condition, while replacing some of the numbers of animals used in such research.
Professor Tom Evans, who wrote this article, is Professor of Molecular Microbiology in the Division of Immunology, Infection and Inflammation at the University of Glasgow. His research interests include sepsis and the resultant stereotyped host response, which includes the recognition of invading microbes by elements of the innate immune system, mediators produced as part of this response, and the resulting tissue damage that can occur.
References
1. Harrison DA, Welch CA & Eddleston JM (2006). The epidemiology of severe sepsis in England, Wales and Northern Ireland, 1996 to 2004: secondary analysis of a high quality clinical database, the ICNARC Case Mix Programme Database. Crit Care 10:R42
PubMed Link
2. Evans TJ & Choen J (1996). Mediators: nitric oxide and other toxic oxygen species. Curr Top Microbiology 216: 189-207.
3. Lindsay SL, Ramsey S, Aitchison M et al (2007). Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239. J Cell Sci 120:3022-3021.
PubMed Link