Determining the role of coagulation proteases in the development of renal fibrosis.

The normal response to kidney damage is inflammation, followed by expansion of a group of cells, called myofibroblasts that produce proteins which form scar tissue. In some situations, the scar tissue is dissolved, and normal kidney function returns. However, sometimes, particularly after repeated damage, the scar tissue becomes excessive, resulting in permanent, sometimes ongoing scarring and loss of kidney function leading to chronic kidney disease, a major cause of early death worldwide. This is called kidney fibrosis. Whilst we have numerous treatments for particular types of kidney injury (such as blood pressure treatments), there are currently no known treatments that can stop or reverse fibrosis. This process also occurs in kidneys after transplantation, contributing to loss of kidney transplant function and, ultimately, failure of the transplant.

In other organs, proteins involved in blood clotting have been shown to be involved in fibrosis. These proteins have functions besides clotting and in fibrosis they are thought to be working not by stimulating blood to clot, but by stimulating enhanced myofibroblast activity through a number of possible mechanisms including the recruitment of myofibroblast precursors, the transformation of these precursors into myofibroblasts, or the laying down of scar tissue by myofibroblasts. Which of these effects is most important is still not certain. In my time in the laboratory last year, I investigated the development of kidney fibrosis after exposing mice to a toxin called aristolochic acid (AA). This toxin is the most common cause of toxic kidney fibrosis in humans worldwide, so discoveries in mice may have direct relevance for human disease. I found evidence that the same clotting proteins as have been shown to be important in liver, lung and heart fibrosis are involved in kidney fibrosis, and blocking their activity on certain cells can provide some protection against fibrosis.

In this project, I will build on my preliminary findings by pursuing three aims. First, I will examine the scar tissue that develops after exposure to AA in a special strain of mice which have an inhibitor of blood clotting proteins on the surface of specific cells. I already know that these mice develop less fibrosis than mice that don't have the inhibitor, so by comparing the patterns I will get clues about which specific cells are involved and how the clotting proteins may be influencing them. Next, I want to focus on differentiating whether bone marrow-derived or intrinsic kidney cells are most important for fibrosis, and then look in detail at one specific cell type, called monocytes, known to be involved in fibrosis in other models of kidney damage. Based on solid background data, I will investigate the role monocytes play in the development of fibrosis after exposure to AA and ask whether, by influencing their response to clotting proteins, I can alter the development of fibrosis in normal mice. Finally, I want to study how the clotting proteins influence behaviour of myofobroblast precursors and whether they interact with known cell signalling pathways that have been studied by my supervisors.

This work is important due to the global healthcare burden of chronic kidney disease. In addition, my supervisors have developed a new class of drug designed to inhibit clotting proteins with minimal effect on bleeding. I hope my project will provide the basis for future study of whether these novel drugs can stop fibrosis developing. In doing so, it may be possible to prevent or slow the progression of chronic kidney disease, so decreasing the impact of the disease on patients and improving quality of life, in addition to prolonging kidney transplant survival. This research may also have the potential to offer insights into the management of fibrosis in other organs, including the heart, lungs and liver, given the similar underlying mechanisms proposed.

The cellular and molecular basis of fibrogenesis, particularly in the kidneys, is incompletely understood, though has become an exciting field in recent years. There is a growing body of evidence for coagulation protease involvement in fibrogenesis in a number of tissues, but the origin of the cellular component, the myofibroblast, remains elusive. All the proposed myofibroblast precursors express either Tissue Factor (TF) or protease-activated receptors, consistent with the concept that coagulation proteases could be influencing any of these putative precursors.
During my ACF, I refined a protocol to induce fibrosis in C57BL/6 mice following repeated doses of a prevalent tubular toxin, aristolochic acid (AA). I demonstrated this was associated with upregulation of TF, and that transgenic mice expressing a membrane-tethered inhibitor of coagulation proteases, human Tissue Factor Pathway Inhibitor (hTFPI) on an alphaSMA promoter (alpha-TFPI-Tg) were more resistant to fibrosis than wild-type C57BL/6 mice.
My first objective is to describe the specific patterns of TF expression and innate immune activation seen during fibrogenesis in C57BL/6 mice and compare these patterns to those seen in alpha-TFPI-Tg mice.
I will then explore, using reciprocal bone marrow transplants and adoptive transfer of labelled monocytes, whether the resistance to fibrosis conferred by the transgene is conferred by renal parenchymal cells (e.g. resident fibroblasts or pericytes), or bone marrow-derived cells (e.g. monocytes or circulating fibrocytes).
In the third objective I will isolate and examine in vitro the cells implicated by the experiments above, investigating the pathways involved in transducing coagulation protease-mediated signals to fibrogenesis. This will be done by assessing for Ras/ERK activation and using PAR 1,2 and 4 inhibitors, k-Ras antisense oligonucleotides and siRNA to evaluate their impact on myofibroblast formation, proliferation and collagen production.

Chronic kidney disease (CKD), defined as a reduced glomerular filtration rate (GFR), affects approximately 2.6 million individuals in England alone, and the prevalence worldwide is increasing, with the number of patients requiring dialysis worldwide estimated to double by 2030. Although only approximately 1 in 50 of patients with CKD require renal replacement therapy (dialysis), all patients with reduced GFR suffer excess risk of stroke and myocardial dysfunction, so the overall health and economic burden of CKD is enormous, currently accounting for 1-2% of total NHS spending. Any development that can prevent or slow the progressive loss of kidney function as a consequence of fibrosis in some patients will have a potentially significant impact on mortality, morbidity, total health spending and quality of life.

Irrespective of the cause of CKD, abnormal deposition of fibrous tissue by cells called myofibroblasts is responsible for the progressive loss of kidney function. One of the most challenging aspects of management is preventing this process, which may ultimately lead to the need for renal replacement therapy such as dialysis or transplantation. Aside from treating the individual causes of damage (such as diabetes for instance), the mainstay of therapy to slow progressive loss of kidney function remains treatment of secondary phenomena, such as hypertension and proteinuria, which in themselves are injurious; there are no specific drugs targeting the fibrotic process itself, nor any that can reverse this process.

The overarching aim of the research in the two host laboratories is to maximise the lifespan of native or transplanted kidneys by understanding more about the cellular and molecular basis of the pathophysiology of renal fibrosis, in the hope that this knowledge will help develop new avenues for future therapeutic manipulation. This project will continue this trend. As such, it has the potential to have a significant impact on future studies in fibrosis.

Although focused on kidney patients, this work has the potential to be translated into other chronic diseases where fibrosis also contributes to progressive organ dysfunction. In particular, liver, lung and heart fibrosis have all also been linked to the proteins involved in coagulation, the coagulation proteases, so gaining a greater understanding of the role of protease-activated receptor (PAR)-mediated signalling, and the cells involved, could significantly impact on disease paradigms in these disciplines.