Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress.

Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca(2+) increase due to depletion of luminal ER Ca(2+). Palmitate-induced ER Ca(2+) depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca(2+) pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca(2+) depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca(2+) loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca(2+) depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca(2+) depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.

Sterically stabilized anti-G(M3), anti-Le(x) immunoliposomes: targeting to B16BL6, HRT-18 cancer cells.

Various tumor-associated antigens have been identified as carbohydrates bound to lipids or to proteins expressed on tumor cell membranes. We prepared tumor-specific immunoliposomes by coupling anticarbohydrate antibodies, such as antiganglioside G(M3) antibody (DH2) or anti-Le(x) antibody (SH1), to polyethylene glycol (PEG)-coated liposomes. In vitro and in vivo targetability of anti-G(M3) and anti-Le(x) immunoliposomes to B16BL6 mouse melanoma cells and HRT-18 human colorectal adenocarcinoma cells were monitored with a fluorescence microscopy, and analyzed by biodistribution assay of the immunoliposome in mice bearing the tumor tissues. The antibody coupling to the PEG liposomes did not greatly diminish the circulation time of the liposome in the C57BL/6 mouse model. In vitro cytotoxicity of doxorubicin encapsulated in liposomes was enhanced by antibody coupling, but still behind free doxorubicin. However, in vivo antitumor therapeutic efficacy of doxorubicin encapsulated in the immunoliposomes was far greater than the free drug or in conventional liposomes. Doxorubicin encapsulated in anti-G(M3) immunoliposomes was able to reduce in vivo tumor growth and metastasis of B16BL6 mouse melanoma cells more greatly than any other formulations of the drug. This study suggests that tumor-associated antigens can be good target molecules for tumor-specific delivery of liposomal drugs or other synthetic drug delivery systems.