Kidney failure in sepsis
|
|
2004 – 2008 Dr Hadwen Trust Postdoctoral Fellowship: Using a three-dimensional cell culture model to investigate sepsis-induced renal failureProf T Evans & Dr S LindsayGlasgow University |
Tom Evans is Professor of Molecular Microbiology in the Division of Immunology, Infection and Inflammation at Glasgow University.
For the duration of this grant, Dr Susan Lindsay was a Dr Hadwen Trust Research Fellow at Glasgow University.
Overwhelming bacterial infection in patients, termed sepsis, is a serious and life-threatening problem. Despite advances in antibiotics, sepsis remains 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, and there is no medicine to prevent or treat acute kidney failure.
The Dr Hadwen Trust supported work in Prof Tom Evans laboratory to develop three-dimensional models of human kidney tubules to investigate the pathophysiology of sepsis-induced renal failure. Ultimately it is hoped such human tissue culture models will be used to define better treatments for renal failure in sepsis, and thus help to replace the use of animals in sepsis research.
Research into sepsis has been extensive over the past 20 years, but so far has not resulted in particularly effective novel therapies. 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. Species used include 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. Thus there is clearly an ethical and scientific argument for better non-animal models.
Prof Evans and Dr Hadwen Trust research fellow, Dr Susan Lindsay, established cultures of human renal proximal tubular cells (PTECs) from fresh donated kidney tissue in two- and three-dimensional models. Human renal proximal tubular cells cultured in a collagen gel formed into large 3D hollow structures or cysts. The addition of various growth factors was explored, and hepatocyte growth factor (HGF) was found to be the most potent, producing some branching and morphological differentiation of cysts into tubule-like structures. See Figure.
These renal cell culture models can be used to investigate the process of damage and repair of kidney tubules in sepsis. Changes in renal tubules in sepsis are complex but a key feature is the shedding of renal tubule cells into the tubule lumen, resulting in blockage of the nephron. Cell shedding is mediated by nitric oxide, which is produced in large amounts in sepsis in response to cytokines. Our researchers investigated the effects of nitric oxide (NO) and cytokines characteristic of sepsis in the human cell culture models.
NO production in human renal cells was found to alter the function of a protein, called vasodilator-stimulated phosphoprotein (VASP) that regulates renal epithelial cell movement. NO causes VASP phosphorylation, resulting in the retraction of lamellipodia, the rounding of cells and slowing of cell movement. Movement of renal cells is essential to repair of kidney tubules following damage, so this effect of NO on VASP may be important in preventing renal repair in sepsis. Finding ways of countering this effect could improve renal regeneration and patient outcome.
Exposing cell cultures to pro-inflammatory cytokines typical of sepsis produced considerable morphological changes to kidney cells in 2D cultures, including changes in cell polarity. However 3D cell cultures proved to be surprisingly resistant to the effects of cytokines. Further research will be necessary to elucidate the additional factors producing these effects.
The project has gone a long way towards establishing three-dimensional kidney cell culture system for future studies to define the pathophysiological changes in sepsis and how novel treatments might be of benefit. This in turn will help to replace certain animal experiments in this area. These cell culture models allow experimental manipulation to a far greater degree than possible in animal models, and can be used to gain important insights into sepsis. The work has elicited much interest from scientists in Europe and China.
Summary
- Overwhelming bacterial infection, called sepsis, is a life-threatening condition. Patients with sepsis commonly suffer kidney failure, for which there is no effective treatment.
- Sepsis experiments on animals can cause substantially suffering, and may use rodents, rabbits, sheep, dogs, pigs and baboons.
- Numerous sepsis treatments developed in experimental animals have failed in human patients. Thus there is a clear need for better non-animal models.
- A Dr Hadwen Trust project developed two- and three-dimensional cell culture models of human kidney to replace animal studies of kidney failure in sepsis.
- Human kidney cells cultured in a collagen gel and treated with growth factors developed into 3D structures resembling human kidney tubules.
- The kidney cell culture models were used to investigate the effects of nitric oxide and cytokines produced in sepsis, shedding new light on the process of damage and repair of kidney tubules in sepsis.
- A better understanding of the processes that underlie kidney damage in sepsis will lead to more effective therapies for this life-threatening condition.
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.
Langley G, Evans T, Holgate ST et al (2007). Replacing animal experiments: choices, chances and challenges. BioEssays 29:918-926.
Lindsay SL, Ramsey S, Aitchinson M et al (2007). Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239. J Cell Sci 120:3011-21.