Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2 +/-) mice had no evidence of increased renal disease, and Ampka2 -/-mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity. IntroductionComplications of diabetes, including retinopathy, neuropathy, and nephropathy, are responsible for considerable morbidity, and are experienced by a majority of individuals with diabetes over time. How elevated glucose levels initiate these complications remains unclear. The prevailing theory states that enhanced mitochondrial production of superoxide anion in response to elevated cellular glucose concentrations leads to pathological pathway activation and cell dysfunction (1, 2). Studies in cell culture systems demonstrated that addition of high glucose produced an increase in ROS that has been attributed to the mitochondrial electron transport chain (ETC). The currently accepted scheme is that chronically elevated glucose leads to increased ROS production by mitochondria, contributing to downstream cellular injury processes, and ultimately resulting in end-organ dysfunction and structural changes. Based on this theory, numerous strategies are being developed to interrupt the mitochondrial production of superoxide and inhibit downstream deleterious pathways.

The mechanisms underlying the association between obesity and progressive renal disease are not well understood. Exposure to a high-fat diet decreases levels of the cellular energy sensor AMPK in many organs, including the kidney, but whether AMPK contributes to the pathophysiology of kidney disease induced by a high-fat diet is unknown. In this study, we randomly assigned C57BL/6J mice to a standard or high-fat diet. After 1 week, mice fed a high-fat diet exhibited an increase in body weight, renal hypertrophy, an increase in urine H 2 O 2 and urine MCP-1, and a decrease in circulating adiponectin levels and renal AMPK activity. Urine ACR progressively increased after 4 weeks of a high-fat diet. After 12 weeks, kidneys of mice fed a high-fat diet demonstrated a marked increase in markers of fibrosis and inflammation, and AMPK activity remained significantly suppressed. To determine whether inhibition of AMPK activity explained these renal effects, we administered an AMPK activator along with a high-fat diet for 1 week. Although AMPK activation did not abrogate the weight gain, it reduced the renal hypertrophy, urine H 2 O 2 , and urine and renal MCP-1. In vitro, AMPK activation completely inhibited the induction of MCP-1 by palmitic acid in mesangial cells. In conclusion, these data suggest that the energy sensor AMPK mediates the early renal effects of a high-fat diet. Obesity has been dramatically increasing in the United States and worldwide. Between 1999 and 2004, 32.2% of all adults in the United States were obese (body mass index Ͼ 30 kg/m 2 ) 1 and the prevalence of obesity is expected to reach 50% by 2030. Obesity is the major risk factor for the metabolic syndrome, characterized by insulin resistance, dyslipidemia, and hypertension. 2 Obesity in association with insulin resistance contribute to the development of cardiovascular disease and diabetes. Furthermore, obesity is now being increasingly recognized as a major and independent risk factor for the development of kidney disease. [3][4][5] In the general population, obesity was the second most highly predictive factor to predict ESRD, even independent of diabetes and hypertension. 6 Clinical and experimental studies have demonstrated that the characteristic features of obesity-induced kidney injury include glomerular hypertrophy, thickening of the glomerular basement membrane, mesangial matrix expansion, and increased renal inflammation. [7][8][9][10] These alterations likely contribute to albuminuria, a progressive decline in renal function and ultimately glomerulosclerosis and tubulointerstitial fibrosis. 7-10 Despite the public health and clinical implications of the relationships between obesity and kidney injury, the signaling pathways leading to renal pathology with obesity are not well understood.

Rationale and Objectives-Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objective is to develop a robust microbubble (MB) ultrasound contrast agent platform to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index imaging processing method and corresponding quantifying method. The microbubbles and the imaging methods were evaluated in a mouse model of breast cancer in vivo.Materials and Methods-We utilized a cyclic RGD (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting α v β 3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine α v β 3 integrin protein and α v β 3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for α v β 3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted microbubbles were visualized using a low mechanical index contrast imaging pulse sequence, and quantified by intensity normalization and two-dimensional Fourier transform analysis, Results-The cRGD ligand concentration on the MB surface was ~8.2 × 10 6 molecules/MB. At a wall shear stress of 1.0 dynes/cm 2 , cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant α v β 3 integrin relative to non-targeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared to non-targeted MB and cRAD-MB. In addition, cRGD-MB, but not non-targeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. bEnd.3 was reduced by 80% after using anti-α v monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ~0.8 seven minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by pre-administration with an anti-α v blocking antibody.Conclusion-The results demonstrate the functionality of a novel microbubble contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of α v β 3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low mechanical index.

AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway.

Chronic kidney disease (CKD) is prevalent in 9.1% of the global population and is a significant public health problem associated with increased morbidity and mortality. CKD is associated with highly prevalent physiological and metabolic disturbances such as hypertension, obesity, insulin resistance, cardiovascular disease, and aging, which are also risk factors for CKD pathogenesis and progression. Podocytes and proximal tubular cells of the kidney strongly express AMP-activated protein kinase (AMPK). AMPK plays essential roles in glucose and lipid metabolism, cell survival, growth, and inflammation. Thus, metabolic disease-induced renal diseases like obesity-related and diabetic chronic kidney disease demonstrate dysregulated AMPK in the kidney. Activating AMPK ameliorates the pathological and phenotypical features of both diseases. As a metabolic sensor, AMPK regulates active tubular transport and helps renal cells to survive low energy states. AMPK also exerts a key role in mitochondrial homeostasis and is known to regulate autophagy in mammalian cells. While the nutrient-sensing role of AMPK is critical in determining the fate of renal cells, the role of AMPK in kidney autophagy and mitochondrial quality control leading to pathology in metabolic disease-related CKD is not very clear and needs further investigation. This review highlights the crucial role of AMPK in renal cell dysfunction associated with metabolic diseases and aims to expand therapeutic strategies by understanding the molecular and cellular processes underlying CKD.