Highlights of research being conducted by NIDDK-supported scientists
RNAi-based Therapeutic Strategies for Metabolic and Inflammatory Diseases
Dr. Michael Czech
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Dr. Michael Czech is a Professor and Chair of Molecular Medicine and a Professor of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School in Worcester, Massachusetts. He received his Ph.D. in Biochemistry from Brown University. Dr. Czech has published nearly 300 papers and has served, or is presently serving, on the editorial boards of dozens of journals. He has also served on the NIH endocrinology study section and on the Howard Hughes Medical Institute review panel. Dr. Czech was the recipient of the Grodsky Basic Research Award from the Juvenile Diabetes Research Foundation International in 1997, the 1998 Elliot P. Joslin Medal in diabetes research, and the 2000 Banting Medal of the American Diabetes Association. The following are highlights from the scientific presentation that Dr. Czech gave to the NIDDK’s Advisory Council in May 2008.
Understanding the process by which the body slowly becomes resistant to the hormone insulin, as is the case in type 2 diabetes, is critical to developing effective therapeutics for the disease. Recent research has revealed a link between insulin resistance and the inflammatory response of the immune system. As the body takes in excess calories, fat cells, known as adipocytes, increase in size to store the extra fat. Eventually, the adipocytes become overloaded and begin to release molecules that attract inflammatory cells, specifically macrophages. Macrophages are important in initiating the inflammatory response; they engulf foreign pathogens, such as bacteria or yeast, and secrete molecules that affect the behavior of other immune system cells and that attract additional inflammatory cells to the site of the pathogen. However, inflammation does not only occur when there is an obvious infection. A chronic state of inflammation can occur when macrophages are continually recruited to adipocytes, as in the case of obesity. In this state and with the adipocyte’s ability to store fat exceeded, the muscle begins to take up the excess fat. The build up of fat in the muscle disrupts the ability of insulin to stimulate the transport of glucose (sugar) from the blood into muscle, leading to insulin resistance.
Dr. Czech’s presentation moved from an initial fundamental discovery to an innovative strategy for its clinical application. He discussed his approach to understanding how cells become resistant to insulin and the role of the inflammatory response in insulin resistance. He shared how his laboratory has utilized a revolutionary technique—ribonucleic acid (RNA) interference, or RNAi—to identify novel molecules critical to these processes. Dr. Czech and his team are exploring the use of this technique as a potential therapy for insulin resistance. Dr. Czech also remarked that this research was made possible by NIDDK’s Diabetes Genome Anatomy Project (DGAP), a unique and multi-dimensional initiative for basic research in diabetes. DGAP was designed to facilitate interactions and coordinate a number of investigators at multiple institutions, with projects aimed at understanding the interface between insulin action, insulin resistance, and the genetics of type 2 diabetes.
Using RNAi To Identify Novel Proteins in Insulin Resistance
Dr. Czech and his colleagues sought to identify proteins that mediate the interactions between adipocytes and macrophages and to understand their role in the balance of blood glucose and fat levels. Such information could reveal new drug targets to break the link between obesity and insulin resistance. To uncover these proteins, the scientists used a technique based on the phenomenon of RNA interference. This technique involves designing small molecules, known as small interfering RNAs or siRNAs, that reduce levels of a specific protein by interacting—or interfering—with the genetic material that encodes the protein, to prevent the protein from being made. The scientists thus could design specific siRNAs to reduce the level of a protein and see whether insulin-mediated glucose uptake was affected. With this technique, they screened hundreds of different proteins in mouse adipocytes to determine whether any had a role in insulin action.
Several specific proteins were identified that Dr. Czech and his colleagues never expected to be involved in insulin resistance. One of these is called MAP4K4 (shorthand for mitogen activated protein 4 kinase). Dr. Czech’s laboratory subsequently demonstrated that MAP4K4 blocks insulin-stimulated glucose transport through a mechanism that also involves an inflammatory response protein called TNF-alpha. This positions MAP4K4 at the interface between adipocytes, where MAP4K4 can be found, and macrophages, which secrete TNF-alpha. MAP4K4 is also located in other types of cells, and Dr. Czech’s laboratory and others have identified additional roles for this protein in muscle and in macrophages, placing MAP4K4 in three key tissues involved in insulin resistance in obesity.
Developing RNAi as a Potential Therapeutic
Once a role had been identified for MAP4K4 in inflammation and insulin-dependent glucose uptake, Dr. Czech wanted to explore whether targeting this protein, using the power of RNAi, could have therapeutic potential for diabetes. The investigators decided to target levels of MAP4K4 protein in macrophages, rather than in muscle or adipocytes, because they hypothesized that insulin resistance results from the stimulation of inflammation by MAP4K4 in macrophages. In addition, because macrophages—and inflammation—are involved in many diseases, such as rheumatoid arthritis, colitis, inflammatory bowel disease, cardiovascular disease, and atherosclerosis, developing a strategy for therapy in the macrophage might be applied to many other diseases.
Dr. Czech explained that RNA interference as a potential therapeutic may have several advantages over traditional small molecule drugs, which interact with proteins. In traditional drug development there are a relatively limited number of proteins that can be targeted, as the small molecules normally tested as drug candidates are only effective if they can bind (attach) to the targeted protein. By contrast, RNAi works by interfering with genetic material encoding proteins, not the proteins themselves, and scientists think that there may be fewer structural constraints for this type of interaction. With RNAi, therefore, levels of any protein encoded in the human genome could theoretically be targeted and reduced. Second, traditional small molecule drugs can sometimes bind non-targeted proteins. Because siRNAs are extremely specific in their targets, off-target—and potentially toxic—effects can be minimized. Additionally, siRNAs are made from materials that are native to the body and have not shown toxicity thus far in animal models.
As Dr. Czech noted, an ideal therapeutic would be delivered orally for the patient’s ease. An orally delivered drug faces many obstacles on its way to the target tissue: it needs to pass through the acidic environment of the stomach, be absorbed by the gut, and enter the bloodstream. An ideal therapeutic would be specifically delivered to the targeted tissues, thereby avoiding any toxicity due to misdelivery. To address these challenges, Dr. Czech took advantage of special cells called “M cells,” which are located within the small intestine, and devised a way to get siRNA to these cells.
The M cells constantly sample the digestive cavity of the intestine looking for particles like bacteria and yeast that may have been ingested. Upon finding these, M cells are able to bind the particles, internalize them, and expel them where nearby macrophages are waiting to devour them. With this system, Dr. Czech utilized the normal biology of the intestine to efficiently direct his RNAi therapeutic to the macrophages.
Dr. Czech’s laboratory needed to generate a safe vehicle to deliver the siRNA to an animal being studied. Their efforts led to the development of hollow, porous, tiny (micron-sized) shells made of a substance called beta1,3-D-glucan, which is recognized by proteins on both the M cells and the macrophages, permitting these cells to take in the shells. Beta1,3-D-glucan is a non-toxic material made by yeast cells and has been sold as a human dietary supplement for many years. Layering the siRNA within the hollow center of the shell allows five to six layers of siRNA to be put into each of these particles. Therefore, the scientists could use multiple combinations of siRNA at one time and target several different genes, or use one siRNA at a higher dose. Dr. Czech and his colleagues termed these shell particles “GeRPs” or Glucan-encapsulated siRNA particles.
Proof of Principle: Using RNAi To Target MAP4K4 in Animals
This technology required multiple tests to determine whether it could be used as a potential therapeutic in animals. To begin, Dr. Czech and his colleagues needed to ascertain whether the macrophages would even ingest the GeRPs— the first step in this strategy. To do this, the scientists added a fluorescent label to the GeRPs and gave them orally to mice. Using a fluorescence microscope, they were able to see that the macrophages had taken in the GeRPs and that a single macrophage could ingest multiple GeRPs. Another exciting aspect of this technology is that it harnesses the macrophages in the gut to transport the GeRPs. These macrophages are part of the body’s lymphatic system, which enables them to travel throughout the body. This prompted Dr. Czech and his colleagues to assess if they could find GeRPs inside macrophages located in various tissues of the mouse body. After 8 days of feeding the mice GeRPs, the scientists observed the fluorescent GeRPs in the lungs, liver, and spleen. From this result, Dr. Czech and his laboratory concluded that they are able to target multiple tissues in the mouse body with this technology.
Dr. Czech’s next step was to examine whether GeRPs with siRNA directed to MAP4K4 led to a reduction in the levels of MAP4K4 proteins within the tissues of the mice. In spleen, liver, and lung, the scientists were able to see a reduction in the levels of MAP4K4 as they had hoped. Did this reduction in MAP4K4 protein levels affect the inflammatory response though, as Dr. Czech had predicted? The scientists again fed the mice GeRPs with siRNAs to MAP4K4 and then gave the animals a toxic chemical that mimics a bacterial infection in order to stimulate the inflammatory response. When mice without the siRNAs were given this chemical, their macrophages stimulated an excessive inflammatory response, leading to a very large release of the inflammatory protein TNF-alpha, which was fatal to the animals. However, by feeding the mice siRNA to MAP4K4, Dr. Czech and his colleagues were able to block this storm of TNF-alpha, halting the inflammatory response to the chemical, and protecting the mice. This exciting result demonstrated that the orally administered siRNAs were not only delivered to the correct cell, the macrophage, and carried to multiple tissues, but that these siRNAs also targeted MAP4K4 specifically and altered the mouse inflammatory response.
Does using this technology to target MAP4K4 reduce inflammation in fat tissue and affect insulin-mediated glucose transport into cells? For these preliminary experiments, Dr. Czech and colleagues used obese mice that are highly insulin-resistant and delivered MAP4K4 siRNA-containing GeRPs to the mice by injection. The scientists looked at various tissues to determine the location of GeRP-filled macrophages and evaluated whether the mice were still resistant to insulin with a test called a “glucose tolerance test.” They found, to their surprise, that the fat tissue of these mice was the main tissue that had macrophages with GeRPs in them. This indicated that, in these obese mice, the primary inflamed tissue is the fat tissue— macrophages are largely recruited to this tissue. Dr. Czech and his laboratory also observed a decrease in levels of MAP4K4 protein in the macrophages recovered in this tissue. In addition, these mice were better able to metabolize glucose, indicating that the insulin resistance of these obese mice could be ameliorated. These experiments suggested that delivery of MAP4K4 siRNA to obese mice could have a profound effect on glucose metabolism throughout the body.
Conclusion
Dr. Czech’s presentation illustrated the power of RNAi technology to identify novel proteins involved in insulin resistance. These proteins are potential targets for drug therapy, as they are found at the interface between fat cells, muscle, and the inflammatory response. One particular protein, MAP4K4, is especially interesting due to its location in all of these tissues. In addition, Dr. Czech showed his laboratory’s approach to using siRNAs as a therapeutic modality. By targeting siRNA to MAP4K4 within the macrophages of a mouse with an innovative delivery vehicle, the scientists were able to both block the inflammatory response and alter the insulin resistance in obese mice. Thus, Dr. Czech and his colleagues have developed a technology to deliver RNAi in vivo in mice. They plan to build on the studies to determine whether the therapeutic has a similar result in other animals. Dr. Czech’s research reveals the exciting potential for a new method of therapy for numerous diseases, including type 2 diabetes.
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Food Intake and Body Weight: Regulation by Apo A-IV in the Brain
Dr. Patrick Tso
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Dr. Patrick Tso is Professor of Pathology, Associate Director of the Cincinnati Obesity Research Center, and Director of the Center for Lipid and Atherosclerosis Research at the University of Cincinnati College of Medicine. Additionally, he is the Director of the Cincinnati Mouse Metabolic Phenotyping Center, funded by NIDDK. Dr Tso is a highly respected researcher in the area of lipid (fat) metabolism, a field in which he has worked for over 20 years. At the September 2008 meeting of the NIDDK Advisory Council, Dr. Tso shared insights from his exciting research on how food intake and body weight are regulated by apolipoprotein A-IV (apo A-IV). The following are highlights of his presentation.
Feeling Full after a High-Fat Meal: The Discovery That Apo A-IV Regulates Food Intake
How does a high-fat meal make one feel full? Dr. Tso described his laboratory’s research to understand what causes satiety and the insights that have emerged from these studies about the role of a small biologic factor called apo A-IV, which is made in humans and animals. Intrigued by the dramatic increase in intestinal apo A-IV production known to occur after ingestion of dietary fat, Dr. Tso and one of his post-doctoral fellows, Dr. Kazuma Fujimoto, investigated a potential role for apo A-IV in satiety. The researchers conducted their experiments in rodents, and began with a focus on a body fluid called lymph, which varies in composition depending upon what food has been ingested. After a fatty meal, lymph from the abdomen contains an abundance of apo A-IV along with fat absorbed from the food. To assess whether this fluid might curtail food intake, they compared different samples of lymph, some with fat and some without. After administering the lymph samples intravenously into fasting rats, they assessed how much the rats subsequently ate. In describing this study, Dr. Tso shared an anecdote about the experimental design. He recounted that Dr. Fujimoto was concerned that the rats might be too “worried” to eat if a person was nearby. So, Dr. Fujimoto decided to speak to each rat for 15 minutes every day (in his native Japanese) to help the animals feel comfortable around him. This procedure evidently worked, as the rats did eat—but those who had been given the fat-containing lymph ate significantly less. Thus, something in the lymph was signaling that a relatively small meal would be sufficient. The researchers then investigated which component of the lymph might be causing this satiety effect: the fat, or the apo A-IV. After a series of additional experiments, they discovered that it was apo A-IV.
Site of Action: The Brain
Dr. Tso next sought to discover where in the body apo A-IV exerts its effect to reduce food intake. Although apo A-IV was originally found to be produced in the intestine, substantial regulation of appetite occurs in the brain. Thus, Dr. Tso, with another post-doctoral fellow, Dr. Koji Fukagawa, explored whether apo A-IV could reduce food intake when infused directly into the brain. The answer was yes, as determined by further studies in rats.
Building on this research, Dr. Tso and another of his post-doctoral fellows, Dr. Min Liu, found that apo A-IV is not only produced in the intestine, but it is also synthesized in a part of the brain, the hypothalamus, known to play a crucial role in the control of food intake and body weight. In further experiments in rats, they demonstrated that excess apo A-IV in the brain, from infusions, reduces body weight in parallel to its effect on satiety.
From Feeding to Fullness: Elucidating Biologic Pathways in the Brain
Having illuminated the role of brain apo A-IV in regulating food intake, Dr. Tso and his colleagues next asked: What regulates apo A-IV? They first tested whether brain apo A-IV, like apo A-IV in the intestine, is affected by feeding and fasting. From studies in rats, the researchers found that apo A-IV levels in the brain substantially decreased after fasting, a result that is consistent with their findings regarding the role of apo A-IV in satiety; an animal that had not eaten for a day should not feel full. The researchers next explored the effects of different types of food on apo A-IV levels in the brain. When the rats, after fasting, were given their standard “chow,” apo A-IV levels in the brain did not change significantly. If the animals instead ate high-fat food, their brain apo A-IV levels greatly increased.
Dr. Tso and his laboratory also discovered that apo A-IV levels in the brain fluctuate with the circadian rhythm—the day/night cycles. Levels of this satiety-inducing factor were lowest at night, when rats typically eat, and peaked during the day, when rats normally do not eat. Thus, the daily rise and fall of apo A-IV levels mirrored the animals’ feeding patterns. The researchers then explored whether the changes in apo A-IV levels were caused by the cycles of light and dark per se, or by the concomitant cycles of feeding and fasting. When they shifted the rats’ meal times to the daylight hours (by providing food only during the day), the researchers found that apo A-IV levels changed, too. Dr. Tso concluded that it was the cycles of feeding and fasting that affected apo A-IV levels, rather than daylight and darkness. That is, under normal conditions, apo A-IV levels are low at night (when food is typically available), and the rats are thus able to eat. Their ingestion of food causes apo A-IV levels to rise by the morning hours, which in turn makes the rats too full to eat during the day. After not eating for a while, the apo A-IV levels fall again so that by night time, the rats become hungry and eat.
To further explore the pathway by which apo A-IV causes satiety, Dr. Tso and his research team investigated whether apo A-IV interacts with the hormone leptin. Mice deficient in leptin are strikingly obese, and this hormone also plays a critical role in body weight regulation in humans. The researchers measured apo A-IV levels, and the effects of fasting and feeding, in normal mice and mice that lacked leptin (as a result of a genetic mutation). In the leptin-deficient mice, levels of apo A-IV in the brain were lower than in the normal mice. Additionally, when leptin-deficient mice were given a high-fat meal, the levels of brain apo A-IV did not increase as in normal mice. The scientists then injected leptin into the deficient mice, and found that this led to a restoration of normal levels of apo A-IV. From these and other experiments, the researchers concluded that leptin regulates apo A-IV, and that leptin and apo A-IV interact to reduce food intake and body weight.
Dr. Tso’s research team then turned their attention to other factors in the brain known to regulate food intake, collectively referred to as the melanocortin system, to determine whether these factors interact with apo A-IV. Again using rodents as a model system, the researchers found that apo A-IV and a major component of the melanocortin system, called POMC, are present in the same brain cells, and both apo A-IV and POMC levels are low during fasting. Administering apo A-IV led to an elevation in POMC levels as well. This research, together with additional studies, demonstrated that apo A-IV also interacts with the melanocortin system to inhibit food intake.
Conclusions—Apo A-IV
Dr. Tso’s research on apo A-IV has yielded novel insights into the regulation of food intake and satiety. In concluding his presentation, Dr. Tso noted that apo A-IV has other functions as well, related to fat metabolism and other biologic processes. By shedding light on the regulation of satiety, this research will also advance understanding of what could go awry in obesity, with implications for potential intervention approaches.
Dr. Tso acknowledged the contributions of the scientists who worked with him on these studies when they were post-doctoral fellows in his laboratory: Drs. Kazuma Fujimoto, Koji Fukagawa, and Min Liu. Additionally, Dr. Tso thanked his long-time collaborator on this research, Dr. Stephen Woods, who is also a Professor at the University of Cincinnati.
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Liver Disease in Alpha-1-Antitrypsin Deficiency: Organ-specific Complications Arise from a Misfolded Protein
Dr. David Perlmutter
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Dr. David Perlmutter is a Vira I. Heinz Professor and Chairman of Pediatrics at the University of Pittsburgh. He is also the Physician-in-Chief and Scientific Director of Children’s Hospital of Pittsburgh. Dr. Perlmutter has carried out basic research on alpha-1-antitrypsin deficiency for more than 20 years. His work has led to many new concepts about the underlying causes of liver disease in this genetic condition and has suggested several new concepts for approaches to prevent chronic liver injury, liver cancer, and lung disease that sometimes result from alpha-1-antitrypsin deficiency. Dr. Perlmutter spoke at the January 2008 meeting of the NIDDK Advisory Council to share some insights from his ongoing studies of alpha-1-antitrypsin deficiency. Following are highlights of that presentation.
Alpha-1-antitrypsin deficiency (a condition also referred to as Alpha-1) is a genetic disorder caused by defective production of the protein alpha-1-antitrypsin (alpha-1AT). It affects about 1 in every 1,800 live births.1 In normal individuals, alpha-1AT protein is produced in the liver and secreted into the bloodstream. Its main site of action is in the lungs, where it protects the delicate tissue from damage. People with Alpha-1 carry a mutation in the gene encoding alpha-1AT, which results in a protein that retains some of its biological function but is poorly secreted, and thus does not reach the lungs and may accumulate—sometimes forming large aggregates—within the liver.
Mutant alpha-1AT can cause two different medical problems: pulmonary complications such as emphysema may arise because the protein does not perform its function in the lungs; and liver complications such as inflammation and cancer may arise because the mutant protein can build up in the liver. While lung complications are hallmarks of Alpha-1, most patients do not develop serious liver disease; in fact, only 8 to 10 percent of the people with Alpha-1 will do so. This wide variation in the severity of liver symptoms among people with Alpha-1 strongly suggests that additional genetic and/or environmental variables contribute to the development of clinical liver disease. The identity of these factors is unknown. One hypothesis Dr. Perlmutter posed was whether “protected” individuals—those who carry the alpha-1AT mutation but do not develop liver disease—are somehow able to metabolize the mutant alpha-1AT, while patients who are susceptible to liver disease are not. The first questions Dr. Perlmutter addressed concerned the mechanisms by which this mutant protein was degraded in the liver, and whether these pathways were less effective in people whose livers have aggregates of mutant alpha-1AT.
Alpha-1AT Processing in the Liver
Using a series of experiments in cultured cells, Dr. Perlmutter and his colleagues found that a metabolic pathway known as the “autophagic pathway” was involved in the degradation of mutant alpha-1AT in the liver. Autophagy is the degradation of a cell’s own components by its internal digestive pathways—literally, autophagy is a process by which a cell eats part of itself. It is a tightly-regulated process that plays a part in normal cell growth and metabolism and helps to maintain a balance between the synthesis, degradation, and recycling of cellular components. It is also a major mechanism by which a cell under stress—starvation, for example—reallocates scarce nutrients to essential processes. The autophagic pathway seemed to be particularly important in the disposal of the very large aggregates of protein found when very high levels of the mutant protein were produced, as the aggregates of alpha-1AT were able to activate the autophagic response.
Other Pathways for Disposing of Mutant, Misshapen, or “Misfolded” Proteins
Dr. Perlmutter’s research team next turned to two other well-characterized cellular pathways activated in response to the accumulation of misfolded proteins in general to see if they were involved in the metabolism of mutant alpha-1AT. The first was the “unfolded protein response” pathway, which is activated in response to the presence of misfolded or defective proteins. When the researchers looked at markers for activation of the unfolded protein response pathway, however, they were unable to detect increased activity in the presence of mutant alpha-1AT. A second pathway, the “ER overload response” pathway, is activated when the endoplasmic reticulum—a specialized area within the cells where proteins are prepared for secretion—becomes “backed up” with proteins that cannot get out of the cell. In contrast to the unfolded protein response, the ER overload response pathway did show increased activity in the presence of the mutant alpha-1AT.
Identification of a Novel Pathway Involved in Alpha-1AT Metabolism
The researchers next asked whether there were any other, previously unknown pathways that might also be involved in a cell’s disposal of mutant, misfolded proteins. They reasoned that, when faced with a potentially toxic accumulation of mutant alpha-1AT, a cell may turn on or off certain genes to regulate various metabolic pathways, some of which would help it dispose of the mutant protein. Thus, the researchers engineered mice to produce mutant alpha-1AT in their livers in an inducible manner, and then analyzed the patterns of gene expression (the extent to which genes are turned on or off) in the mouse livers in the absence and presence of the mutant protein. When the mutant alpha-1AT was produced, the expression of 75 liver genes was increased, and the expression of 131 was decreased. Analysis of these response patterns found that these changes in expression involved genes that play a role in various cellular processes.
One gene whose expression was markedly increased in these mice in the presence of mutant alpha-1AT is the “regulator of G-protein signaling 16,” also known as RGS16. G-proteins are important mediators of intracellular signals, so changes in the expression of a gene that modulates G-protein activity could have potentially far-reaching effects on a cell. The increase in RGS16 gene expression was associated strongly with the presence of aggregates of the mutant alpha-1AT
in the mouse livers. Similar changes in RGS16 expression were seen in samples of human livers from individuals with Alpha-1.
RGS16 seems to be activated in response to the aggregation of mutant alpha-1AT that characterizes Alpha-1 in individuals with liver disease. Therefore, it may be an excellent marker for the distinct form of metabolic stress seen in these patients. RGS16 may also represent a key player in a novel pathway through which autophagy is regulated, making it a potential target for the development of future therapeutic strategies. Future research will further characterize the role played by RGS16 in modulating cellular metabolism in the presence of mutant alpha-1AT.
A New Model System To Study Alpha-1
Dr. Perlmutter next described an innovative series of experiments using a model organism to study Alpha-1, the roundworm Caenorhabditis elegans. C. elegans is a small (about 1 mm long), transparent worm that is used extensively by biomedical researchers. This organism offers a number of benefits as a disease model, both biological and practical. Its genome has been fully sequenced and its genes and their functions are similar to those of mammals. It is relatively easy to work with, reproducing every 3 days and generating many offspring, and it is transparent—facilitating observation of its inner workings. There are also substantial existing genetic and molecular tools that researchers can employ when using this organism.
Dr. Perlmutter’s collaborators, Drs. Gary Silverman and Stephen Pak, constructed fusions of various alpha-1AT genes with a gene encoding “green fluorescent protein,” a marker often used by biologists to allow easy visualization of a protein. When they inserted the normal alpha-1AT gene, fused with green fluorescent protein, into the intestinal cells of worms, they saw green fluorescence in the interior of the intestinal tract, indicating that the protein was being properly secreted out of the cells. (In C. elegans, the intestine performs many of the functions of the liver.) In worms that produced fusions with the mutant alpha-1AT gene, the green fluorescence was retained within the cell in globules, indicating a failure to secrete, and intracellular aggregation of the protein. Additionally, worms expressing the mutant gene exhibited arrested development at the larval stage, and did not live as long as normal worms or worms expressing the normal alpha-1AT.
But what is responsible for the physiological manifestation of the mutant alpha-1AT? To answer this question, the researchers used a slightly different mutant of alpha-1AT that is non-functional and accumulates within the cells, but does not form aggregates. When this alternate mutant was inserted into worms, there was no growth arrest at the larval stage in these worms. This finding indicates that some of the biological effects seen in worms with the original mutant alpha-1AT require not only the retention of the protein within the liver cells, but also the formation of protein aggregates within cells.
Dr. Perlmutter outlined the next steps in the research he is doing with Drs. Silverman and Pak: the adaptation of the worm model for high-throughput screening for genetic modifiers of disease severity and for potential drug candidates. He described technology that could automatically sort through and characterize large numbers of these tiny worms. Such an approach would allow the rapid screening of hundreds of potential genetic and/or pharmacologic approaches to address the problems seen in Alpha-1.
Conclusions
In a subset of patients with Alpha-1, accumulation of aggregates of the mutant protein in the liver causes damage and increases the risk of cancer. The risk for liver disease is heavily influenced by genetic and/or environmental factors that may impact various degradation pathways and other protective cellular responses. Dr. Perlmutter and his colleagues discovered that the autophagic pathway appears to play a particularly important role in disposing of the mutant protein. Finally, Dr. Perlmutter’s development of a novel worm model amenable to high-throughput screening may expedite the identification of genetic modifiers and new therapeutic agents.
Dr. Perlmutter acknowledged the contributions of his collaborators in research, Drs. Silverman and Pak. Gary Silverman, M.D., Ph.D. is The Twenty Five Club Endowed Professor of Pediatrics, Professor of Cell Biology and Physiology at University of Pittsburgh School of Medicine and Chief of Newborn Medicine at University of Pittsburgh Medical Center. Stephen Pak, Ph.D. is Assistant Professor of Pediatrics at University of Pittsburgh School of Medicine. Drs. Silverman and Pak have worked with Dr. Perlmutter to characterize the C. elegans model organism in order to elucidate the role of cellular signaling molecules in regulating cell metabolism.
1 Perlmutter DH, et al: Molecular pathogenesis of alpha-1-antitrypsin deficiency-associated liver disease: a meeting review. Hepatology 45: 1313-1323, 2007.
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More Intensive Dialysis Does Not Improve Outcomes among Patients with Acute Kidney Injury
Dr. Paul M. Palevsky
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Dr. Paul M. Palevsky is a Professor of Medicine in the Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, and is Section Chief of the Renal Section at the VA Pittsburgh Healthcare System. His research interests focus on the prevention and treatment of acute kidney injury and the management of kidney replacement therapy in acute and chronic kidney disease. In May of 2008, Dr. Palevsky described the findings of the Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network Study in a featured presentation at the annual American Thoracic Society International Conference in Toronto, Canada. The following summary is based on that presentation. The results of the study were subsequently published in the July 3, 2008, issue of the New England Journal of Medicine.
Acute kidney injury (also called “acute renal failure”) is a serious medical condition characterized by a relatively rapid loss of kidney function, usually over a period of several hours or days. The resulting inability to excrete nitrogenous waste products and maintain fluid and electrolyte balance poses urgent health problems for patients and their physicians. Acute kidney injury may arise from a number of causes, most commonly sepsis (a serious, whole-body inflammatory reaction caused by infection), decreased blood pressure, or kidney damage from drugs or other toxins. It is a relatively common complication among hospitalized patients; it affects between 2 and 7 percent of all hospitalized patients.1 Even though a significant fraction of patients with acute kidney injury will regain kidney function, many do not, and this medical condition is associated with high in-hospital mortality rates ranging from 50 to 80 percent among the critically ill.1
There is no effective drug therapy to reverse acute kidney injury. The goal of treatment is to prevent fluid and waste from building up in the body while waiting for the kidneys to resume functioning. Treatment involves hemodialysis and other forms of life-sustaining therapy to replace lost kidney function. Dialysis removes waste products from the blood, and it also helps control blood pressure and keeps the proper electrolyte balance.
Although dialysis has been used to treat acute kidney injury for over 60 years, it is still not clear when it is best to initiate therapy, which method of dialysis is best to use, and what dose of dialysis to deliver. Several recent, small studies had suggested that increased frequency or intensity of dialysis might improve survival in patients with acute kidney injury. However, the results of these studies have not been definitive. This uncertainty raises the possibility that some patients may be receiving a sub-optimal dose or frequency of dialysis, or that other patients may be receiving excessive dialysis that may carry no clinical benefit and may, in fact, expose them to unnecessary risk. In order to investigate this issue, the NIDDK partnered with the U.S. Department of Veterans Affairs to launch a clinical trial comparing “standard” with “intensive” dialysis in patients with acute kidney injury.
Design of the ATN Study
The VA/NIH Acute Renal Failure Trial Network (ATN) Study was designed to determine whether higher-dose (intensive) dialysis would reduce the death rate, shorten the duration of the illness, and decrease the number of complications in other organs among patients with acute kidney injury, as compared to standard-dose dialysis. It enrolled over 1,100 critically ill patients—defined as patients with acute kidney injury as well as either sepsis or the failure of at least one other organ. Notably, the trial did not enroll patients with chronic kidney disease. These patients were not studied in this trial because the causes and progression of their acute kidney injury are different from that seen in people without underlying chronic kidney disease.
Patients were randomly assigned to receive intensive- or standard-dose dialysis. Patients who did not require medications to maintain their blood pressure were treated with conventional dialysis, either three times per week in the standard arm of the study or six times per week in the intensive arm. Patients with very low blood pressure who required medications to increase their blood pressure were treated with more gentle forms of dialysis, either a slower form of hemodialysis, three or six times per week, or a continuous form of dialysis, at a lower or higher dose, as randomly assigned. One important element in the design of the study was that patients were able to switch between forms of therapy as their clinical condition changed, while remaining within the lower or higher intensity treatment arms of the study. This approach reflects typical clinical practice in that it allowed physicians to adjust the method of dialysis as the patient’s condition changed, and was chosen so that the results of the trial would be more relevant to actual patient care.
Results of the ATN Study: Is More Better?
The primary question the trial was designed to answer was whether more intensive dialysis provided a clinical benefit. The first, and perhaps most important, clinical endpoint was patient survival. After 60 days, no significant difference in rates of death by any cause was found between the two groups of patients. Over this period, 289 of 561 patients (51.5 percent) in the standard-dose treatment group died, compared to 302 of 563 patients (53.6 percent) in the intensive treatment group. Mortality rates were similar in men and women and across racial and ethnic subgroups.
When the researchers assessed kidney function and other medical conditions, similar patterns were seen. A total of 102 patients (18.4 percent) in the standard-dose group had complete recovery of kidney function after 28 days, and 50 patients (9.0 percent) had partial recovery. By comparison, 85 patients (15.4 percent) in the intensive-treatment group had complete recovery of kidney function over the same time period, and 49 patients (8.9 percent) had partial recovery. A total of 92 patients (16.4 percent) undergoing less-intensive therapy were able to return home without requiring continued dialysis after 60 days, compared to 88 patients (15.7 percent) who underwent intensive therapy. None of these differences between groups was statistically significant. Rates of treatment-related complications across all groups were also similar.
In summary, the ATN Study found no significant differences between the two groups in recovery of kidney function, the rate of failure of organs other than kidneys, or the number of patients able to return home after recovery. In patients enrolled in this trial, there was no benefit to intensive dialysis.
Implications of the ATN Study
Although a few studies have suggested that increased frequency or intensity of hemodialysis might improve survival in patients with acute kidney injury, they have been small and conducted at single sites. In contrast, the ATN study enrolled over 1,100 patients from 17 Veterans Affairs medical centers and 10 university-affiliated medical centers across the U.S. The results of the larger ATN Study show that when it comes to dialysis in acute kidney injury, more is not better.
The results of the ATN study, however, should be interpreted carefully. One limitation of the ATN study concerns the exclusion from the trial of patients with advanced chronic kidney disease. Such patients make up a substantial proportion of people who develop acute kidney injury. Therefore, it may be inappropriate to extrapolate the ATN results to persons in whom acute kidney injury develops in the context of pre-existing chronic kidney disease. Further study will be necessary to resolve this longstanding question and address the optimal treatment of acute kidney injury in this population.
Conclusion
The results of the ATN study indicate that increasing dialysis treatments to five to six times per week does not confer an additional benefit beyond a standard three times per week regimen. However, this does not mean that dose of dialysis does not matter. The dose of dialysis targeted in the standard-treatment group was greater than what is often achieved in a typical clinical setting. The results also do not mean that higher doses of continuous therapies are never beneficial, only that routine use of higher-dose dialysis is unnecessary. Nevertheless, the findings of this study may spare patients from unnecessarily-intensive medical interventions. They also underscore the importance of continued research into other approaches to treating acute kidney injury. Future research efforts may include studies to identify biomarkers of kidney injury prior to renal failure, which could enable physicians to predict who is likely to develop acute kidney injury, to lessen its severity through earlier intervention, or to preempt this life-threatening condition altogether.
The NIDDK has begun a new initiative entitled “Identification and Evaluation of Biomarkers and Risk Assessment Tools for Chronic Kidney Disease and Acute Kidney Injury.” The goal of this initiative is to identify and validate biomarkers and risk assessment tools for kidney function, injury, and progression. Both existing and new biomarkers and risk assessment tools will be rigorously evaluated for clinical utility under this initiative. In addition to seeking new molecular markers in chronic kidney disease and acute kidney injury, the initiative will also examine whether these two conditions share common biomarkers.
1 Palevsky PM, et al: Intensity of renal support in critically ill patients with acute kidney injury. NEJM 359:7-20, 2008.
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