John D. Imig - US grants

nitric oxide; renal hypertension; cytochrome P450; muscle tone; kidney metabolism; renal tubule; eicosanoid metabolism; vascular smooth muscle; blood pressure; calcium; vascular resistance; arachidonate; hemoprotein biosynthesis; hemoprotein metabolism; kidney circulation; saluretic; kidney function; spontaneous hypertensive rat;

nitric oxide; renal hypertension; cytochrome P450; muscle tone; kidney metabolism; renal tubule; eicosanoid metabolism; vascular smooth muscle; blood pressure; calcium; vascular resistance; arachidonate; hemoprotein biosynthesis; hemoprotein metabolism; kidney circulation; saluretic; kidney function; spontaneous hypertensive rat;

DESCRIPTION: (Adapted from the application) The overall hypothesis to be tested in this proposal is that increases in production of lipoxygenase metabolites and decreases in production of epoxygenase metabolites contribute to enhanced vascular tone of preglomerular vessels and elevation in blood pressure associated with increased circulating levels of angiotensin II. Rats will be prepared with chronic infusion of angiotensin II and the development of hypertension followed. At selected time intervals, the metabolites of epoxygenase and lipoxygenase will be measured. The reactivity of renal microvasculatures will be evaluated and assayed for the production of the various metabolites of arachidonic acid. In addition, in vitro juxtamedullary nephron techniques combined with video microscopy will be used to determine preglomerular vascular tone in vitro; the various contributions of metabolites lipoxygenase and epoxygenase will be assessed using pharmacological inhibition of these probes both in the acute and chronic situation. Results of these studies will further define the renin-angiotensin system and determine the role of arachidonic acid metabolism in the development of angiotensin-dependent hypertension.

DESCRIPTION (provided by the applicant): Hypertension that develops following the long-term administration of initially subpressor doses of angiotensin II in rats has many of the same renal and vascular changes that are associated with human essential hypertension. In the past five years, we have provided compelling evidence that CYP45O-derived epoxyeicosatrienoic acids (EETs) have anti-hypertensive properties and play a part in the maintenance of renal microvascular function. A novel approach to increase EET levels is to inhibit epoxide hydrolase enzymes that are responsible for the conversion of the biologically active EETs to dihydroxyeicosatrienoic acids (DHETs) that are void of effects on the preglomerular vasculature. Recently, a role for soluble epoxide hydrolase (sEH) in the long-term control of arterial blood pressure and the pathogenesis of experimental hypertension has been proposed. Preliminary studies in our laboratory demonstrate increased kidney sEH protein expression during angiotensin II hypertension. In addition, we have observed a decrease in arterial blood pressure in angiotensin II hypertension following administration of a highly selective sEH inhibitor. Based on these observations, we hypothesis that during the development of hypertension increased kidney sEH will decrease kidney EET levels which contributes to increased renal microvascular reactivity, blood pressure and renal vascular injury. We will directly determine the involvement of epoxide hydrolases to the excessive preglomerular vasoconstriction during the development of angiotensin II hypertension using the juxtamedullary nephron preparation. We will also determine renal microvascular EET and DHET levels and the regulation of kidney epoxide hydrolase enzymes during hypertension. The proposed studies will employ newly developed highly selective epoxide hydrolase inhibitors to determine their ability to lower arterial blood pressure and improve renal microvascular function in angiotensin II hypertension. Collectively, these studies will provide a comprehensive understanding of epoxide hydrolases in the long-term regulation of blood pressure and renal hemodynamic function during angiotensin II hypertension.

End stage renal disease (ESRD) is manifest in hypertension and the progression of renal failure is accelerated by a high salt diet. In human patients and angiotensin II salt-sensitive hypertensive animal models, endothelial dysfunction and increased renal vascular resistance are observed as hypertension progresses and ESRD becomes evident. Another common feature of salt-sensitive hypertension is the inability of the kidney to properly increase epoxyeicosatrienoic acid (EET) levels. Others and we have provided compelling evidence that CYP450-derived EETs have anti-hypertensive properties and are endothelial-derived hyperpolarizing factors (EDHF) in the kidney. EETs also possess anti-inflammatory actions that could protect the kidney vasculature from injury during hypertension. Cytokine suppression of kidney EET production is a mechanism that could explain endothelial dysfunction and glomerular injury associated with salt-sensitive hypertension. Based on these observations, we hypothesis that a failure to properly increase kidney EET levels in response to high dietary salt contributes to endothelial dysfunction, glomerular injury, and salt-sensitivity in angiotensin II hypertension. We will determine the effects of salt diet, cytokines and arterial blood pressure on EDHF regulation, afferent arteriolar endothelial function and glomerular injury in salt-sensitive hypertension. The proposed studies will employ newly developed highly selective epoxide hydrolase inhibitors that increase EET levels to determine their ability to lower arterial blood pressure and improve renal microvascular function in angiotensin II salt-sensitive hypertension. Collectively, the proposed experiments in this application will provide novel information on the interaction between cytokines and EET levels in the long-term regulation of blood pressure and renal microvascular and glomerular function during angiotensin II salt-sensitive hypertension.

DESCRIPTION (provided by applicant): A major cause of morbidity and mortality is the progression of organ damage associated with cardiovascular diseases. For instance, the incidence of stroke and costs associated with stroke damage continues to have a devastating impact on public health despite numerous treatments aimed at cardiovascular risk factors. Recent findings indicate that increasing levels of epoxides of arachidonic acid (EETs) appear to be new and excellent means to treat cardiovascular diseases. We demonstrated that in vivo inhibition of soluble epoxide hydrolase (SEH) resulted in higher levels of EETs and kidney protection in animal models of hypertension. More recently, we have conducted preliminary studies that suggest that SEH inhibitors can protect the brain from cerebral ischemic damage. Therefore, we will test the hypothesis that SEH inhibitors will have stroke damage protection that is due to decreased cerebral vascular injury and platelet aggregation. First, we will evaluate the ability of a newly developed orally active SEH inhibitor to prevent stroke damage associated with cardiovascular disease. Secondly, we will determine the effect of SEH inhibition on cerebral artery structure, endothelial function and platelet function in stroke-prone spontaneously hypertensive (SHRSP) rats. In the Phase II work, Arete Therapeutics will use the collected information to progress toward clinical trials in the area of stroke and possible new therapeutic avenues.

End stage renal disease (ESRD) is manifest in hypertension and the progression of renal failure is accelerated by a high salt diet. In human patients and angiotensin II salt-sensitive hypertensive animal models, endothelial dysfunction and increased renal vascular resistance are observed as hypertension progresses and ESRD becomes evident. Another common feature of salt-sensitive hypertension is the inability of the kidney to properly increase epoxyeicosatrienoic acid (EET) levels. Others and we have provided compelling evidence that CYP450-derived EETs have anti-hypertensive properties and are endothelial-derived hyperpolarizing factors (EDHF) in the kidney. EETs also possess anti-inflammatory actions that could protect the kidney vasculature from injury during hypertension. Cytokine suppression of kidney EET production is a mechanism that could explain endothelial dysfunction and glomerular injury associated with salt-sensitive hypertension. Based on these observations, we hypothesis that a failure to properly increase kidney EET levels in response to high dietary salt contributes to endothelial dysfunction, glomerular injury, and salt-sensitivity in angiotensin II hypertension. We will determine the effects of salt diet, cytokines and arterial blood pressure on EDHF regulation, afferent arteriolar endothelial function and glomerular injury in salt-sensitive hypertension. The proposed studies will employ newly developed highly selective epoxide hydrolase inhibitors that increase EET levels to determine their ability to lower arterial blood pressure and improve renal microvascular function in angiotensin II salt-sensitive hypertension. Collectively, the proposed experiments in this application will provide novel information on the interaction between cytokines and EET levels in the long-term regulation of blood pressure and renal microvascular and glomerular function during angiotensin II salt-sensitive hypertension.

DESCRIPTION (provided by applicant): The World Health Organization has recently labeled obesity as one of the top global health problems. One of the primary reasons for the increase in chronic kidney disease is the increase in obesity related type 2 diabetes and its co-existence with hypertension. The clustering of cardiovascular risk factors such as inflammation, endothelial dysfunction and insulin resistance are intertwined in obesity and hypertension. This triad of risk factors is largely responsible for the progression of chronic kidney disease in obesity and hypertension or what is now known as Cardiometabolic Syndrome. TNF- is an important cytokine secreted by adipose tissue that can increase cardiovascular risk and end organ damage in obesity. We have recently demonstrated that TNF- can decrease Cyp2c derived epoxyeicosatrienoic acids (EETs). These Cyp2c derived epoxyeicosanoids have renal and cardiovascular protective properties. A signaling pathway that could possible link TNF- and epoxides to endothelial dysfunction and renal damage is NFB activation of cell adhesion molecules such as MCP-1. The contribution of TNF-, epoxyeicosanoids, and NFB to the progression of chronic kidney disease in obesity and hypertension has not been elucidated. Therefore, we will test the hypothesis that increased TNF- , decreased epoxides and increased MCP-1 contribute to renal vascular injury in obesity and hypertension. We will determine the effects of high fat diet induced obesity on afferent arteriolar function, blood pressure and renal injury in hypertensive animals. The current proposal will define the contribution of TNF-, EETs, NFB and MCP-1 and interactions between these pathways in obesity and hypertension. This proposal incorporates novel metabolic oxylipid profiling, RT-PCR arrays and targeted analysis of cellular mechanisms that will allow for unique insight concerning mechanisms that contribute to the progression of kidney disease in obesity and hypertension. Collectively, the proposed experiments in this application will provide novel mechanistic insight on the progression of chronic kidney disease in obesity and hypertension. Project Narrative: The World Health Organization has recently labeled obesity as one of the top global health issues. One of the primary reasons for the increase in chronic kidney disease is the increase in obesity related diabetes and its co- existence with hypertension. This project will test the hypothesis that interactions between fatty acid metabolites and inflammatory molecules accelerate the progression of chronic kidney disease in obesity and hypertension. On the whole, the proposed experiments in this application will provide novel insight and therapeutic targets for the treatment of chronic kidney disease in obesity and hypertension.

? DESCRIPTION (provided by applicant): Diabetes and metabolic syndrome (MetS) afflicts close to 70 million Americans and diabetes accounts for close to half of all new cases of kidney failure. There is increasing interest in finding therapeutic targets and therapies that target multiple risk factors, thereby minimizing problems associated with multi-drug regimens in MetS and type 2 diabetic patients. The eicosanoid metabolome is altered in MetS and type 2 diabetes patients. The eicosanoid metabolome is altered in MetS and type 2 diabetes, and such alterations have been demonstrated to affect multiple factors including blood pressure, lipid levels, and insulin signaling. We have developed a novel chemical entity, 4-(phenyl-3-{3-[-(4-trifluoromethyl-phenyl)-ureido]-propyl}-pyrazol-1-yl)- benzenesulfonamide (PTUPB), that uniquely inhibits both soluble epoxide hydrolase (sEH) and cyclooxygenase (COX) and demonstrates potential as a therapeutic for MetS, type 2 diabetes and the associated kidney failure. Our long-term objective is to make significant steps towards an ultimate goal of an Investigational New Drug (IND) application for a novel chemical entity that uniquely alters eicosanoid metabolites and demonstrates potential as a therapeutic for MetS, type 2 diabetes. The overall objective of this application, which is the next step toward attainment of our long-term goal, is the pharmacological testing and the development and optimization of PTUPB-based COX-2/sEH inhibitors as a novel therapy for MetS and type 2 diabetes. Our central hypothesis is that inhibition of both COX-2 and sEH will uniquely alter eicosanoid metabolites to improve insulin signaling and renal function in MetS and type 2 diabetes. Our preliminary experiments demonstrate that the COX-2/sEH inhibitor, PTUPB has great therapeutic potential for treating multiple risk factors of MetS, type 2 diabetes and the associated kidney failure. Guided by strong preliminary data, our central hypothesis will be tested by pursuing three specific aims: 1) Optimize the PTUPB chemical scaffold to enhance the pharmacokinetic profile and the therapeutic potential; 2) Test the hypothesis that COX- 2/sEH inhibitors will manipulate eicosanoid metabolites to improve insulin signaling and pancreatic function, and decrease renal injury in MetS; 3) Test the hypothesis that COX-2/sEH inhibitors will manipulate eicosanoid metabolites to improve insulin signaling and pancreatic function, and decrease renal injury in type 2 diabetes. This project will conduct pharmacological testing of prototype small molecules in relevant animal models of MetS and type 2 diabetes. A major part of this proposal will be to utilize medicinal chemistry and computational approaches to optimize our early pre-therapeutic lead PTUPB. This contribution will be significant because it will open the door for identification and further development of COX-2/sEH inhibitors towards a therapeutic for diabetes and kidney disease.

Epidemiological and outcomes studies in patients, as well as studies in rodent models, reveal that renal ischemic kidney injury and unilateral obstructive uropathy brings on long-term consequences: hypertension and chronic kidney disease. Major pathophysiological contributors include impaired renal hemodynamics, endothelial dilator dysfunction, and endothelial cell inflammation. Because the renal microcirculation lacks efficient regenerative capacity, acute damage to the microcirculation can lead to long-term changes in renal hemodynamics that predispose patients to hypertension and chronic kidney disease. A class of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs) increase renal blood flow and improve endothelial cell function. Not known is the contribution of CYP2C epoxygenases, soluble epoxide hydrolase (sEH), and regioisomeric EETs to salt-sensitive hypertension and chronic kidney disease following obstructive uropathy and renal ischemic injury. We hypothesize that decreased endothelial EET levels result in endothelial dysfunction and impaired renal hemodynamics following renal ischemic injury or urinary tract obstruction. The immediate goals of this project are to determine the ability for endothelial EETs to improve endothelial- dependent afferent arteriolar dilation, to decrease endothelial inflammation, and to prevent salt-sensitive hypertension and chronic kidney disease following unilateral ureter obstruction (UUO) or ischemia/reperfusion (I/R) kidney injury. This project will utilize pharmacological as well as global and tissue-specific genetic manipulation of CYP2C, sEH, and EETs. We will obtain our immediate goals by completing three aims. Aim 1 will test the hypothesis that decreased EET levels or EET function contributes to the development of salt- sensitive hypertension and chronic kidney disease following UUO or I/R kidney injury. Aim 2 will test the hypothesis that increasing endothelial EET levels will improve renal microvascular endothelial function following UUO or I/R kidney injury to prevent salt-sensitive hypertension and chronic kidney disease. Aim 3 will test the hypothesis that pharmacological approaches to increase EET levels can prevent the long-term salt-sensitive hypertensive and chronic kidney injury following UUO or I/R kidney injury. Accordingly, our findings promise to advance the field forward by not only enhancing our understanding of the pathophysiological mechanisms whereby UUO or I/R kidney injury leads to chronic kidney disease but also leading to new therapeutic treatments.