Abstract
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 Ca2+ increase due to depletion of luminal ER Ca2+. Palmitate-induced ER Ca2+ depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca2+ 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 Ca2+ depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca2+ loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca2+ 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 Ca2+ depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.
Highlights
IntroductionPalmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was blocked by mitoTEMPO
These data suggest that oxidative stress caused by saturated free fatty-acid (FFA) leads to mitochondrial dysfunction and endoplasmic reticulum (ER) Ca2+ depletion through FAT/CD36 and phospholipase C (PLC) signaling, possibly contributing to podocyte injury
We report in this study on mouse podocytes that palmitate, but not oleate, (1) induced mitochondrial and cytosolic reactive oxygen species (ROS) production and apoptotic cell death, (2) impaired mitochondrial energy metabolism, (3) caused alterations of mitochondrial and ER morphology, (4) decreased the ER Ca2+ concentration, (5) elicited ER stress responses and (6) caused cytoskeletal rearrangement and albumin permeability in podocyte monolayers
Summary
Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca2+ depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury. Cell death and detachment of podocytes lead to proteinuria, an early prognostic symptom of chronic kidney disease (CKD).[1] Epidemiologic studies have shown that the majority of CDKs in patients are caused by glomerular disorders with diabetic nephropathy (DN).[2,3] One of the major pathogenic mediators in type 2 diabetes and its complications is dyslipidemia, resulting in high saturated free fatty-acid (FFA) concentrations. ER Ca2+ depletion by mitoROS S Xu et al cytochrome c release and apoptosis.[8,11,12] The role of unsaturated FFA in this process has been controversial, but accumulating evidence suggests a protective function counteracting the cytotoxic effect of saturated FFA.[8,13,14]
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