Time course of development of diabetic nephropathy and its reversal by copper chelation.

Globally, more than 200 million people are currently thought to have diabetes - it is the leading cause of terminal kidney disease in the UK, as well as the rest of the world. Diabetes causes kidney damage through diabetic kidney disease, medically termed Diabetic Nephropathy (DN). Unfortunately, about a third of all diabetic patients will ultimately lose their lives as a direct result of this DN. Identification of factors that contribute to the early development of DN could enable detection of patients most likely to get this disease with the consequence that treatment could be started earlier. Despite currently available treatments aimed at improving blood glucose and blood pressure control, many diabetic patients still go on to develop kidney failure, which, once established, is debilitating and requires expensive and difficult treatments, such as dialysis or a transplant. Although some progress has recently been made toward understanding DN, how and why it develops is still not fully worked out.
Our research aims to address these issues in two ways.
1) We have recently found that diabetes causes a build-up of copper to apparently toxic levels in the cortex of the kidneys (the outer layer of the kidney just under the capsule), where it appears to damage microscopic structures (termed glomeruli) that filter the blood and produce the urine. Currently, DN is diagnosed by detecting elevated levels of a protein, albumin, in urine, which occurs because the glomeruli have become leaky. However by the time albumin is detected in urine, extensive and potentially fatal kidney damage has already occurred. In addition, the presence of albumin in urine does not tell us anything about the processes that actually cause damage to the glomeruli. The development of DN in patients is also accompanied by increased copper levels in urine, as well as of two components of the glomeruli, heparan sulphate (HS) (a polysaccharide attached to proteins) and heparanase, an enzyme that breaks down HS. Our recent research indicates that the presence of HS or heparanase in urine may reflect the actual disease process at work. The First Aim of this project is to determine whether measurement of one or more of these substances could be useful for the early detection of DN, so that we might be able to detect patients at an earlier stage of kidney disease and therefore start treatment sooner, since early intervention will save lives.
2) We have identified an inexpensive drug, named TETA, that binds to the main form of copper ('divalent' copper) present in altered levels in the tissues of diabetic patients and removes it from the body into the urine in a non-toxic form. We think that this copper build-up is the probable cause of glomerular damage in diabetes, leading to DN. TETA has previously been found to be safe and effective for the treatment of patients with a rare form of copper overload (Wilson's Disease). We have undertaken studies in diabetic patients and animals, which have indicated that TETA is safe and effective for lowering copper levels in diabetic patients and can reverse diabetic damage in the heart and kidney. We have recently shown that TETA also restores HS levels in kidney glomeruli in a model of diabetes. The Second Aim of this project, therefore, is to determine whether measuring urine levels of HS, heparanase and other molecules known to be important in kidney function might complement albumin measurements in acting as indicators of the response of diabetic kidney disease to TETA treatment. The ultimate aim of this research is to uncover how TETA restores kidney function in diabetes. In conclusion, our work aims to identify ways of detecting kidney disease at an earlier stage than is currently possible and developing a way, through the use of TETA, to slow the progression of kidney disease. Our hope is that this will reduce the suffering and prolong the lives of diabetic people.

This project aims to determine the time-course of changes in key renal variables during the development of diabetic nephropathy (DN) and in response to the reversal of DN caused by TETA treatment. We and others have found that diabetes causes the build-up of catalytically active copper in the renal cortex and urine of streptozotocin-diabetic rats and we have subsequently shown that removal of copper by TETA (triethylenetetramine, a selective copper II chelator) reverses damage to the glomerular basement membrane, including its thickening and loss of heparan sulphate (HS). The development of diabetes in patients is known to be accompanied by increased urinary levels of copper as well as of HS and heparanase, although the temporal relationship between these effects is unknown.
In this study, we will use an established model of diabetes to follow animals for 16 weeks in 4 groups: control, diabetic, diabetic-TETA (from week 1), diabetic-TETA (from week 9) and to collect twice weekly urine samples in order to measure urinary copper by a validated inductively coupled plasma-mass spectrometry method; carboxymethyllysine (an advanced glycation end-product thought to bind copper), H2O2 as a marker of oxidative stress, HS, heparanase and albumin will also be measured using established plate-based assays. In addition, we have developed a liquid chromatography-MS assay that can quantify specific HS disaccharides; measurement of specific HSs could add further insight to the source of urinary HS. These data will be analysed to describe a mechanistic time-course of DN development and the effects of copper chelation on DN. We hypothesise that the order in which these markers appear will shed light on the underlying mechanisms of DN and could provide both an early indication of DN risk and an effective tool for guiding TETA therapy, either in determining an optimal population to receive treatment and/or to monitor therapeutic response.

Approximately 7.5% of the adult population in the UK suffer from diabetes, at an annual cost of £13.7 billion or ~10% of the NHS budget (http://www.diabetes.co.uk/cost-of-diabetes.html) and the number of people with this condition continues to increase. A major complication of diabetes is kidney damage leading to kidney failure that necessitates dialysis or transplantation. Unfortunately, the mechanism of progressive renal deterioration is unknown and despite the myriad of strategies and therapies geared to slowing progression of chronic kidney disease (CKD), one in five diabetic patients die from renal complications.
Our previous work has shown a significant reversal of diabetic kidney damage following administration of the copper (II) chelator, TETA. The work detailed in this application aims to investigate the mechanism of TETA-induced remission of diabetic renal damage and in addition, has the potential to uncover novel biomarkers of early kidney disease. The latter feature is of critical importance if we are to ease the financial and societal burden of diabetic kidney disease (DKD), since early detection could result in interventions to prevent either the onset or the progression of microalbuminuria.
The potential of early detection and reversal of DKD is very significant in terms of global morbidity, mortality and health care costs. In a recent NHS-sponsored study (http://www.kidneycare.nhs.uk/our_work_programmes/preparation/diabetes_with_kidney_disease_key_facts/), the authors concluded "that the health and economic burden of diabetic nephropathy is so great that even costly interventions will be worth exploring". TETA is an inexpensive compound that is relatively safe at therapeutic concentrations and one major follow-up to this study would be a full-scale clinical trial. Given the above, our work potentially will have a very broad and significant impact. If copper chelation proves a viable treatment for diabetic complications, then patients will be the immediate benefactors in terms of a reduction in their morbidity and mortality. Economically, the UK will benefit in terms of a reduction in the need for care to treat DKD and a reduction in the cost of drugs to treat complications of renal nephropathy, since TETA is an inexpensive and widely available drug. Additionally, if we discover urinary biomarkers as dependable indicators of early CKD, then our aim is to develop a robust diagnostic test (for instance based on mass spectrometry) that could be used in hospital clinical biochemistry departments to enable detection of these early stages of disease that currently are clinically silent. In itself, this would be of benefit nationally in terms of health and health care savings because early detection of kidney disease correlates with improved morbidity and mortality. This research has the potential to have a major impact on public health policy in terms of when to start screening for early markers of CKD and when to initiate prophylaxis with the copper chelator (if it proves to be effective as such) including potentially immediately after kidney transplants in diabetic patients. Finally, CKD is a major cause of loss of productivity in the UK economy, both in terms of the patients themselves and for their carers.