AimsObesity and hyperlipidaemia are associated with insulin resistance (IR); however, the mechanisms responsible remain incompletely understood. Pro-atherogenic hyperlipidaemic states are characterized by inflammation, oxidant stress, and pathophysiologic oxidized lipids, including ligands for the scavenger receptor CD36. Here we tested the hypothesis that the absence of CD36 protects mice from IR associated with diet-induced obesity and hyperlipidaemia.Methods and resultsAdipose tissue from CD36−/− mice demonstrated a less inflammatory phenotype and improved insulin signalling in vivo and at the level of the adipocyte and macrophage. The pathophysiologic ligand oxidized low-density lipoprotein (oxLDL) activated c-Jun N-terminal kinase (JNK) and disrupted insulin signalling in both adipocytes and macrophages in a CD36-dependent manner. Macrophages isolated from CD36−/− mice after high-fat diet feeding elicited less JNK activation and inhibition of insulin signalling in adipocytes after co-culture compared with wild-type macrophages.ConclusionThese data suggest that a CD36-dependent inflammatory paracrine loop between adipocytes and macrophages facilitates chronic inflammation and contributes to IR common in obesity and dyslipidaemia.

Pro-atherogenic, hyperlipidemic states demonstrate increases in circulating ligands for scavenger receptor CD36 (e.g. oxidized LDL (oxLDL)) and the Na/K-ATPase (e.g. cardiotonic steroids). These factors increase inflammation, oxidative stress, and progression of chronic kidney disease. We hypothesized that diet-induced obesity and hyperlipidemia potentiate a CD36/ Na/K-ATPase -dependent inflammatory paracrine loop between proximal tubule cells (PTC) and their associated macrophages and thereby facilitates development of chronic inflammation and tubulointerstitial fibrosis. ApoE−/− and apoE−/−/cd36−/− mice were fed a high-fat diet (HFD) for up to 32 weeks and examined for physiologic and histologic changes in renal function. Compared to apoE−/−, apoE−/−/cd36−/− mice had improved creatinine clearance and blood pressure which corresponded histologically to less glomerular and tubulointerstitial macrophage accumulation, foam cell formation, oxidant stress, and interstitial fibrosis. Co-IP and a cell surface fluorescence-based crosslinking assay showed CD36 and Na/K-ATPase α-1 co-localized in PTC and macrophages, and this association was increased by oxLDL or the cardiotonic steroid ouabain. OxLDL and ouabain also increased activation of Src and Lyn in PTC. Cell-free conditioned media from PTC treated with oxLDL or ouabain increased macrophage migration. OxLDL, ouabain, or plasma isolated from HFD-fed mice stimulated reactive oxygen species production in PTC which was inhibited by N-acetyl-cysteine, apocynin or Na/K-ATPase α-1 knockdown. These data suggest that ligands generated in hyperlipidemic states activate CD36 and the Na/K-ATPase, and potentiate an inflammatory signaling loop involving PTC and their associated macrophages which facilitates the development of chronic inflammation, oxidant stress, and fibrosis underlying the renal dysfunction common to pro-atherogenic, hyperlipidemic states.

Background Plasma levels of cardiotonic steroids (CTS) are elevated in volume-expanded states such as chronic kidney disease, but the role of these natriuretic hormones in subjects with heart failure (HF) is unclear. We sought to determine the prognostic role of the CTS marinobufagenin (MBG) in HF, particularly in relation to long-term outcomes. Methods and Results We first measured plasma MBG levels and performed comprehensive clinical, laboratory, and echocardiographic assessment in 245 HF patients. All-cause mortality, cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort, median [interquartile range] MBG was 583 [383-812] pM. Higher MBG was associated with higher myeloperoxidase (MPO, r=0.42, p<0.0001), BNP (r=0.25, p=0.001), and asymmetric dimethylarginine (ADMA, r=0.32, p<0.001). Elevated levels of MBG were associated with measures of worse right ventricular function (RV s’: r= −0.39, p<0.0001) and predicted increased risk of adverse clinical outcomes (MBG ≥574 pM: HR 1.58 [1.10-2.31], p=0.014) even after adjustment for age, gender, diabetes mellitus, and ischemic etiology. In mice, a left anterior descending coronary artery ligation model of heart failure lead to increases in MBG, while infusion of MBG into mice for 4 weeks lead to significant increases in MPO, ADMA, and cardiac fibrosis. Conclusions In the setting of heart failure, elevated plasma levels of MBG are associated with right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable models adjusting for established clinical and biochemical risk factors. Infusion of MBG appears to directly contribute to increased nitrative stress and cardiac fibrosis.

Cardiotonic steroids (CTS) are Na+/K+-ATPase (NKA) ligands that are elevated in volume-expanded states and associated with cardiac and renal dysfunction in both clinical and experimental settings. We test the hypothesis that the CTS telocinobufagin (TCB) promotes renal dysfunction in a process involving signaling through the NKA α-1 in the following studies. First, we infuse TCB (4 weeks at 0.1 µg/g/day) or a vehicle into mice expressing wild-type (WT) NKA α-1, as well as mice with a genetic reduction (~40%) of NKA α-1 (NKA α-1+/−). Continuous TCB infusion results in increased proteinuria and cystatin C in WT mice which are significantly attenuated in NKA α-1+/− mice (all p < 0.05), despite similar increases in blood pressure. In a series of in vitro experiments, 24-h treatment of HK2 renal proximal tubular cells with TCB results in significant dose-dependent increases in both Collagens 1 and 3 mRNA (2-fold increases at 10 nM, 5-fold increases at 100 nM, p < 0.05). Similar effects are seen in primary human renal mesangial cells. TCB treatment (100 nM) of SYF fibroblasts reconstituted with cSrc results in a 1.5-fold increase in Collagens 1 and 3 mRNA (p < 0.05), as well as increases in both Transforming Growth factor beta (TGFb, 1.5 fold, p < 0.05) and Connective Tissue Growth Factor (CTGF, 2 fold, p < 0.05), while these effects are absent in SYF cells without Src kinase. In a patient study of subjects with chronic kidney disease, TCB is elevated compared to healthy volunteers. These studies suggest that the pro-fibrotic effects of TCB in the kidney are mediated though the NKA-Src kinase signaling pathway and may have relevance to volume-overloaded conditions, such as chronic kidney disease where TCB is elevated.