How well do plasma cholesterol and triglyceride levels correlate with measurement of organ dysfunction across various AMDCC mouse strains?
The role of lipids in the pathogenesis, progression, and complications of atherosclerosis and neuropathy is well established. However, the potential role of lipids in the pathogenesis and progression of nephropathy is less well established and/or appreciated. We will test the hypothesis that there are significant correlations between plasma cholesterol and/or triglyceride level and diabetic nephropathy. As a positive control we will also determine the correlation between plasma lipids and extent of atherosclerosis. Our specific aims are: 1) To examine the association of plasma total cholesterol level with the development of nephropathy in diabetic mice; 2) To examine the association of plasma triglyceride level with the development of nephropathy in diabetic mice; 3) To examine the association of plasma total cholesterol and triglyceride levels with the development of atherosclerosis in diabetic mice.

Liver X Receptor in Diabetic Nephropathy and Cardiomyopathy
Despite major advances in the treatment of diabetes, many patients continue to experience diabetes-related pathologies in major organ systems. Diabetic renal and cardiovascular disease remains leading causes of premature mortality in patients with diabetes. In addition, women with diabetes have 40% greater risk for death from all causes, and two times the risk for fatal and nonfatal vascular events, than men with the disease. Therefore, new strategies that target novel pathophysiological causes of microvascular and macrovascular disease in both men and women are urgently needed. We propose to generate pilot data to determine if targeting the Liver X Receptor (LXR) holds therapeutic promise for prevention and treatment of renal and cardiac disease in diabetic male and female mice. Our hypothesis is that in diabetes there is down regulation of LXR expression and activity in the kidney which results in increased cholesterol accumulation, activation of the inflammatory response, and endoplasmic reticulum stress, cellular processes that playa critical role in the pathogenesis of diabetic renal disease. Renal disease by itself induces or accelerates cardiovascular disease. We suggest that downregulation of LXR expression and activity in the cardiac tissue also further mediates cardiac disease. In Specific Aim 1 we will treat male and female a) nondiabetic control db-m mice and b) diabetic db-db mice with an LXR agonist. At the end of a 12 week treatment period with the LXR agonist DMHCA (1-3) we will determine effects of LXR agonist on: 1) renal function including glomerular filtration rate, albuminuria, and renal histopathology, 2) cardiac function including in vivo echo, cardiac histopathology, and markers of fibrosis and hypertrophy; 3) perform high resolution and label-free imaging for lipid accumulation, inflammation, oxidative stress, and fibrosis with CARS-TPE-SHG-FLIM microscopy. We will perform detailed biochemical studies to determine effects of the LXR agonist on i) cholesterol and lipid composition, ii) inflammation, and iii) endoplasmic reticulum stress. In Specific Aim 2 we will perform mechanistic studies in a) human podocytes and b) human cardiac myocytes to determine the effects of 1) LXRa or 2) LXRß overexpression. In cells grown with i) control media, ii) high glucose media, iii) media with fatty acids, and iv) media with high glucose and fatty acids, we will determine effects of LXRa or LXRß overexpression on a) cholesterol and lipid composition, b) inflammation, and c) endoplasmic reticulum stress. We will further check the ability of LXRa or LXRß overexpression in resisting i) cholesterol induced inflammation and ER stress in cells loaded with acetylated LDL in the presence of ACAT inhibitor, ii) thapsigargin induced ER stress, and iii) LPS induced inflammation.

Molecular Mechanisms of Kidney Disease in Diabetes and Obesity
The prevalence of obesity, insulin resistance, and diabetes mellitus is increasing and becoming the leading causes of renal disease. In spite of all the beneficial interventions implemented in patients with diabetes renal disease still progresses in most of these patients. Additional treatment modalities that modulate the pathogenic pathways involved in obesity and diabetic nephropathy are therefore needed to slow the progression of renal disease. In this regard recent studies in rodent models of diabetes indicate that a) nuclear receptors including the farnesoid X receptor (FXR) and liver X receptor (LXR), b) transcriptional factors including the sterol regulatory element binding proteins (SREBPs), and c) G protein coupled receptors including TGR5 have highly beneficial actions to prevent or slow down the progression of renal disease in obesity and diabetes. However it is not known whether these pathways are altered in human kidney disease in obesity and diabetes and whether the expression levels and activity of these pathways correlate with renal histopathology. This pilot grant will therefore address this important question. In Specific Aim 1 we will extract mRNA, miRNA, and protein from fixed and paraffin embedded human kidney biopsy samples from subjects with diabetic nephropathy (DN), obesity related glomerulopathy (ORG), and normal kidneys from samples are obtained from the Department of Pathology, Columbia University, NY, USA, and Department of Pathology, Tel Aviv University, Tel Aviv, Israel. We will analyze pathways related to a) fibrosis, b) oxidative stress, c) inflammation, and d) lipid metabolism, as well as e) nuclear receptors, f) transcription factors, and g) G protein coupled receptors that have been demonstrated to regulate these important pathogenic factors.In Specific Aim 2 we will correlate these molecular and metabolic pathways with estimated GFR, proteinuria, and histopathology for each of the biopsy samples.In Specific Aim 3 we will study the similarities and differences of gene expression secondary to a) type 2 diabetes mellitus and b) obesity without T2DM to study the effect of obesity in the mechanism of diabetic nephropathy.

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