BACKGROUND-Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown.

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SYNOPSIS Vascular injury and chronic arterial diseases result in exposure of vascular smooth muscle cells (VSMCs) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with platelet-derived growth factor (PDGF)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and upregulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1, or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 led to a remarkable stabilization α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix and abrogated VSMC hyperproliferation and migration. Interestingly, treatment of cells with PDGF prevented protein damage and cell death due to exposure to the lipid peroxidation product, 4-hydroxynonenal. These results demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under conditions of high oxidative stress found to occur in vascular lesions.

Background Cardiac hypertrophy and heart failure are associated with metabolic dysregulation and a state of chronic energy deficiency. Although several disparate changes in individual metabolic pathways have been described, there has been no global assessment of metabolomic changes in hypertrophic and failing hearts in vivo. Here, we investigated the impact of pressure overload and infarction on myocardial metabolism. Methods and Results Male C57BL/6J mice were subjected to transverse aortic constriction (TAC) or permanent coronary occlusion (myocardial infarction; MI). A combination of LC/MS/MS and GC/MS techniques was used to measure 288 metabolites in these hearts. Both TAC and MI were associated with profound changes in myocardial metabolism affecting up to 40% of all metabolites measured. Prominent changes in branched amino acids acids (BCAAs) were observed after 1 week of TAC and 5 days after MI. Changes in BCAAs after MI were associated with myocardial insulin resistance. Longer duration of TAC and MI led to a decrease in purines, acylcarnitines, fatty acids and several lysolipid and sphingolipid species, but a marked increase in pyrimidines as well as ascorbate, heme and other indices of oxidative stress. Cardiac remodeling and contractile dysfunction in hypertrophied hearts were associated also with large increases in myocardial, but not plasma, levels of the polyamines putrescine and spermidine as well as the collagen breakdown product prolylhydroxyproline. Conclusions These findings reveal extensive metabolic remodeling common to both hypertrophic and failing hearts that are indicative of extensive extracellular matrix remodeling, insulin resistance and perturbations in amino acid, lipid and nucleotide metabolism.

Rationale Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. Objective The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. Methods and Results In db/db mice and in high fat-fed wild-type (WT) C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS-TG mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with WT mice, high fat-fed eNOS-TG mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS-TG mice showed higher levels of PPAR-α and PPAR–γ gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. Conclusions These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.

In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput.

SUMMARY Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here, we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1–4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2 conjugating enzyme Ubc4, the Dsc1 RING E3 ligase and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower V̇(O₂), V̇(CO₂), and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity.