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BACKGROUND Palmitate, a common saturated free fatty acid, is increased in patients with diabetic nephropathy (DN). Excessive palmitate in kidney is known to cause proteinuria and fibrosis. Several studies have demonstrated that paclitaxel has anti-fibrotic and anti-inflammatory effects on kidney disease. However, whether paclitaxel can relieve podocyte injury is unclear. MATERIAL AND METHODS Immortalized mouse podocytes were used as an in vitro system. Palmitate was used to induce podocyte injury. Podocytes were divided into 4 groups: bovine serum albumin, palmitate, palmitate+1 nM paclitaxel, and palmitate+5 nM paclitaxel. The effects of paclitaxel on palmitate-induced podocyte injury were analyzed by western blot and real-time PCR. Intracellular reactive oxygen species (ROS) generation and podocyte cytoskeletons were analyzed using CM-H2DCF-DA and phalloidin staining. RESULTS Paclitaxel restored downregulated expression of nephrin and synaptopodin and upregulated VEGF expression after injury induced by palmitate. Remarkably, palmitate-induced actin cytoskeleton rearrangement in podocytes was repaired by paclitaxel. Four endoplasmic reticulum stress markers, ATF-6alpha, Bip, CHOP, and spliced xBP1, were significantly increased in palmitate-treated podocytes compared with control podocytes. Such increases were decreased by paclitaxel treatment. Palmitate-induced ROS generation was ameliorated by paclitaxel. Elevated Nox4 expression was also improved by paclitaxel. Paclitaxel alleviated the expression levels of the antioxidant molecules, Nrf-2, HO-1, SOD-1, and SOD-2. The paclitaxel effects were accompanied by inhibition of the inflammatory cytokines, MCP-1, TNF-alpha, TNF-R2, and TLR4, as well as attenuation of the apoptosis markers, Bax, Bcl-2, and Caspase-3. Furthermore, paclitaxel suppressed the palmitate-induced fibrosis molecules, fibronectin and TGF-ß1. CONCLUSIONS This study suggests that paclitaxel could be a therapeutic agent for treating palmitate-induced podocyte injury in DN.

Amphiregulin (AREG) is a transmembrane glycoprotein recently implicated in kidney fibrosis. Previously, we reported that the AREG-targeting Self-Assembled-Micelle inhibitory RNA (SAMiRNA-AREG) alleviated fibrosis by stably silencing the AREG gene, and reduced the side effects of conventional siRNA treatment of pulmonary fibrosis. However, the therapeutic effect of SAMiRNA-AREG in renal fibrosis has not been studied until now. We used two animal models of renal fibrosis generated by a unilateral ureteral obstruction (UUO) and an adenine diet (AD) to investigate whether SAMiRNA-AREG inhibited renal fibrosis. To investigate the delivery of SAMiRNA-AREG to the kidney, Cy5-labeled SAMiRNA-AREG was injected into UUO- and AD-induced renal fibrosis models. In both kidney disease models, SAMiRNA-AREG was delivered primarily to the damaged kidney. We also confirmed the protective effect of SAMiRNA-AREG in renal fibrosis models. SAMiRNA-AREG markedly decreased the UUO- and AD-induced AREG mRNA expression. Furthermore, the mRNA expression of fibrosis markers, including α-smooth muscle actin, fibronectin, α1(I) collagen, and α1(III) collagen in the UUO and AD-induced kidneys, was diminished in the SAMiRNA-AREG-treated mice. The transcription of inflammatory markers (tumor necrosis factor-α and monocyte chemoattractant protein-1) and adhesion markers (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) was attenuated. The hematoxylin and eosin, Masson’s trichrome, and immunohistochemical staining results showed that SAMiRNA-AREG decreased renal fibrosis, AREG expression, and epidermal growth factor receptor (EGFR) phosphorylation in the UUO- and AD-induced models. Moreover, we studied the effects of SAMiRNA-AREG in response to TGF-β1 in mouse and human proximal tubule cells, and mouse fibroblasts. TGF-β1-induced extracellular matrix production and myofibroblast differentiation were attenuated by SAMiRNA-AREG. Finally, we confirmed that upregulated AREG in the UUO or AD models was mainly localized in the distal tubules. In conclusion, SAMiRNA-AREG represents a novel siRNA therapeutic for renal fibrosis by suppressing EGFR signals.

MYH9, a widely expressed gene encoding nonmuscle myosin heavy chain, is also expressed in podocytes and is associated with glomerular pathophysiology. However, the mechanisms underlying MYH9-related glomerular diseases associated with proteinuria are poorly understood. Therefore, we investigated the role and mechanism of MYH9 in diabetic kidney injury. MYH9 expression was decreased in glomeruli from diabetic patients and animals and in podocytes treated with Ang II in vitro. Ang II treatment and siRNA-mediated MYH9 knockdown in podocytes resulted in actin cytoskeleton reorganization, reduced cell adhesion, actin-associated protein downregulation, and increased albumin permeability. Ang II treatment increased NOX4 expression and ROS generation. The Ang II receptor blocker losartan and the ROS scavenger NAC restored MYH9 expression in Ang II-treated podocytes, attenuated disrupted actin cytoskeleton and decreased albumin permeability. Furthermore, MYH9 overexpression in podocytes restored the effects of Ang II on the actin cytoskeleton and actin-associated proteins. Ang II-mediated TRPC6 activation reduced MYH9 expression. These results suggest that Ang II-mediated MYH9 depletion in diabetic nephropathy may increase filtration barrier permeability by inducing structural and functional podocyte injury through TRPC6-mediated Ca2+ influx by NOX4-mediated ROS generation. These findings reveal a novel MYH9 function in maintaining urinary filtration barrier integrity. MYH9 may be a potential target for treating diabetic nephropathy.