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.

Diabetic nephropathy is the most serious complication in diabetes mellitus. It is known that oxidative stress and inflammation play a central role in the development of diabetic nephropathy. In this study, we investigated that ferulic acid (FA) known as anti-oxidative agent could effect on diabetic nephropathy by anti-oxidative and anti-inflammatory mechanism. We examined the effects of FA in obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats. We treated FA to experimental rats from 26 to 45 weeks of age. We evaluated ACR, MDA and MCP-1 in 24 h urine and examined renal histopathology and morphologic change in extracted kidneys from rats. Also, we evaluated the ROS production and MCP-1 levels in cultured podocyte after FA treatment. In the FA-treated OLETF rats, blood glucose was significantly decreased and serum adiponectin levels were increased. Urinary ACR was significantly reduced in FA-treated OLETF rats compared with diabetic OLETF rats. In renal histopathology, FA-treated OLETF rats showed decreased glomerular basement membrane thickness, glomerular volume, and mesangial matrix expansion. FA treatment decreased oxidative stress markers and MCP-1 levels in 24 h urine of rats and supernatants of cultured podocyte. In conclusion, it was suggested that FA have protective and therapeutic effects on diabetic nephropathy by reducing oxidative stress and inflammation.

Kim BH, et al. Yonsei Med J. 2016 May;57(3):664-673. https://doi.org/10.3349/ymj.2016.57.3.664

CCL2/C–C chemokine receptor 2 (CCR2) signalling is suggested to play a significant role in various kidney diseases including diabetic nephropathy. We investigated the renoprotective effect of a CCR2 antagonist, RS102895, on the development of diabetic nephropathy in a type 2 diabetic mouse model. Six-week-old diabetic db/db and non-diabetic db/m mice were fed either normal chow or chow mixed with 2 mg/kg/day of RS102895 for 9 weeks. We investigated the effects of CCR2 antagonism on blood glucose, blood pressure, albuminuria and the structure and ultrastructure of the kidney. Diabetes-induced albuminuria was significantly improved after CCR2 antagonist treatment, and glucose intolerance was improved in the RS102895-treated diabetic mice. RS102895 did not affect blood pressure, body weight or kidney weight. Mesangial expansion, glomerular basement membrane thickening and increased desmin staining in the diabetic kidney were significantly improved after RS102895 treatment. The up-regulation of vascular endothelial growth factor mRNA expression and the down-regulation of nephrin mRNA expression were markedly improved in the kidneys of RS102895-treated diabetic mice. Increased renal CD68 and arginase II and urinary malondialdehyde in diabetes were effectively attenuated by RS102895 treatment. Blockade of CCL2/CCR2 signalling by RS102895 ameliorates diabetic nephropathy not only by improving blood glucose levels but also by preventing CCL2/CCR2 signalling from altering renal nephrin and VEGF expressions through blocking macrophage infiltration, inflammation and oxidative stress in type 2 diabetic mice.

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β₁-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β₁ markedly depolarized mitochondrial membrane potential. TGF-β₁-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β₁. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β₁. These results suggest that TGF-β₁-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.