Introduction and General Research
Background
The members of the Blazer-Yost laboratory are investigating how hormones, diet and genetics regulate salt and fluid homeostasis. Slight imbalances in ionic balance contribute to the development of essential hypertension (www.Americanheart.org) and play a critical role in the progression of diseases such as polycystic kidney disease (http://kidney.niddk.nih.gov/kudiseases/pubs/polycystic/index.htm), diabetic hypertension (www.diabetes.org), cystic fibrosis (www.cff.org) and gall stone formation. It is only by understanding the normal regulatory pathways that aberrations which cause disease can be elucidated.
Control of whole body salt and fluid balance resides primarily in the kidney and most of the projects in our laboratory use defined renal cell lines to study regulatory mechanisms. However, in collaboration with our local colleagues at the IU School of Medicine as well as with investigators around the US and Europe, we have examined electrolyte balance in cysts from intrahepatic bile ducts and gall bladder epithelia as well as in whole animal models of renal disease and hypertension.
The independent projects in our laboratory intersect at a common point - regulation of ion channels in high resistance epithelia. The major channel that we study is the amiloride sensitive epithelial Na+ channel, ENaC. This channel in found on the apical membrane of transporting epithelial cells and is regulated by a variety of hormone, autocrine and paracrine factors. The number of effectors which independently regulate this channel underscores the importance of this entity in controlling salt and water homeostasis. Genetic alterations leading to mutations which increase or decrease channel activity have been shown to be involved in the development of hypertension or hypotension, respectively.
It has been hypothesized that the channel or channel regulatory components may be major factors in the development of essential hypertension. Essential hypertension affects over 25% of adults in Western societies and is the prime factor contributing to heart disease - the major cause of death in these countries (American Heart Association). Since a substantial portion of the risk for developing hypertension is thought to be genetic, polymorphisms in the ENaC itself or in the intracellular signaling factors that regulate the channel may be key to understanding and better treating this devastating age-related disease. Thus far, the search for channel polymorphisms which could predispose people to essential hypertension has been disappointing. Therefore, understanding channel regulation is of utmost importance in the search for improved therapeutic interventions which can extend life expectancies in developed countries.
There are three hormones which positively regulate the activity of ENaC. Aldosterone is a steroid hormone which exerts relatively long-term effects via new protein synthesis. ADH and insulin are both peptide hormones which exert immediate, short-term effects on channel activity. The natriferic actions of each of these hormones are additive to one another suggesting that the intracellular pathways are at least partially distinct. The signaling components linking receptor binding to final channel regulation remain incompletely characterized for each of the hormonal systems. Based on recent experiments from our laboratory, we have devised working models which form the basis for hypothetical predictions which are currently being tested.
Although many unknowns remain, it appears that the phosphoinositide pathway plays a major role in aldosterone and insulin-stimulated transport while the cAMP/PKA pathway is modulated in response to ADH. Unpublished data suggest that these generalizations are likely to be over-simplifications. For example, the two pathways likely intersect and form complex regulation of signal turn-on as well as turn-off in response to hormonal stimulation. In addition to the complexity inherent in intersecting signaling pathways is the finding that the PKA pathway also positively regulates the Cl- channel, CFTR (cystic fibrosis transmembrane regulator). Both ENaC and CFTR are found in the apical membrane of various types of epithelial cells including renal, pulmonary and intestinal cells.
Recent Translational Studies
Translational research in the Blazer-Yost laboratory involves several approaches to understanding and treating polycystic kidney disease (PKD). PKD is a devastating disorder that causes the growth of large fluid filled cysts predominately in the kidney and liver, ultimately compromising organ function. The major form of this disease is relatively common and occurs at a rate of approximately 1 in 800 people. Although the cysts are present early in life, the disease is often asymptomatic until the fourth decade. PKD usually leads to renal failure in patients in their mid 50’s. The primary treatment is renal transplantation and there are currently no pharmaceutical agents approved for treatment of PKD. Drugs in clinical trials include chemotherapeutic and immunosuppressive agents and drugs that interfere with hormone action. All of these have relatively serious side effects that may interfere with long-term treatment.
Through a series of unexpected and serendipitous events, the experiments in the Blazer-Yost laboratory have uncovered possible FDA approved drugs that may be useful for halting the progression of PKD. These observations arose during experiments that were being conducted in collaboration with scientists at GlaxoSmithKline to determine why insulin sensitizing agents called Peroxisome Proliferator Activated Receptor gamma (PPARg) agonists caused fluid retention. The Blazer-Yost laboratory disproved a common theory as to the mechanism of action of the fluid retention and in the process made the unexpected finding that the PPARg agonists inhibit a transport mechanism that is responsible for the cyst growth in PKD.
The original studies were performed in tissue culture cell line. Currently the Blazer-Yost laboratory is testing the hypothesis the insulin sensitizing agents can stop the growth of the cysts in PKD by using rodent models of the disease. These studies are being conducted in collaboration with scientists at the IU School of Medicine and at the Mayo Clinic. The animal studies conducted thus far are very positive with PPARg agonist therapy resulting in rodents with smaller cysts in both the kidney and liver. The animal studies extend the tissue culture findings and provide an important proof of principle showing that the insulin sensitizing agents could be an effective treatment for PKD.
The two PPARg agonists used in the animal studies are rosiglitazone (Avandia) and pioglitazone (Actos). These drugs are FDA approved and have been in long term clinical use for the treatment of type II diabetes. The animal studies will provide the basis for potential human clinical trials using PKD patients. These drugs have been proven relatively safe in long-term treatment of diabetic patients which makes them an ideal choice as pharmaceutical agents for a disease like PKD that will require life-long treatment to prevent the growth of cysts.
Nanotoxicology Studies
Another project in the laboratory is examining the effects of carbon nanoparticles on barrier epithelial cells. The cellular layers lining the kidney tubules, the intestinal tract and the airways are all considered "barrier epithelia" because they separate the external and internal milieu. The epithelia protect the body from insults of all kinds including carbon naoparticles. Carbon nanoparticles (CNPs) are important components of the rapidly expanding nanotechnology field. Due to their size and unique electrical, mechanical, and thermal properties, they have found widespread application in electronic, aerospace, medical, agricultural, pharmaceutical, and other industries. Consequently, mass production and widespread application of nanoparticles continues to rise and, along with it, the likelihood of occupational and environmental exposure and potential for exposure-related inflammation, human illness, and dysfunction. Depending on the manufacturing process, CNPs are released to the air and water and ultimately contaminate soil and food products.
Despite recent efforts to characterize potential health hazards, the overall understanding of the biological effects of CNP exposure is far from complete. Three of the most common types are single wall carbon nanotubes (SWNT), multiwall carbon nanotubes (MWNT), and fullerenes (C60). SWNT consist of covalently bound carbon atoms arranged in a long, thin tube-like structure with a diameter of approximately 1.4 nm. MWCNT have a similar structure, but multiple layers of graphene sheets are concentrically rolled up for their formation with a diameter in the range of 10-50 nm. C60, also known as fullerenes or “buckyballs,” typically consist of 60 carbon atoms covalently linked together to form a spherical molecule
Together with colleagues at the IU School of Medicine, we are conducting studies to determine functional, structural, and proteomic changes induced by application of physiologically relevant concentrations of CNPs to barrier epithelial cells. Electrophysiological studies are used to determine the effect of CNPs on transepithelial electrical resistance (TEER), a measure of barrier integrity, and hormone responsiveness. Quantitative proteomic studies were conducted to correlate the observed structural and functional studies with CNP-induced changes in the expressed cellular proteome.