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Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy.

ABSTRACT: A previous report shows that emodin extracted from the Chinese herbs rhubarb and giant knotweed rhizome can ameliorate the anticancer drug cisplatin-induced injury of HEK293 cells. In this study, we investigated whether and how emodin could protect renal tubular epithelial cells against cisplatin-induced nephrotoxicity in vitro.The viability and apoptosis of normal rat renal tubular epithelial cells (NRK-52E) were detected using formazan assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were measured with Western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.Cisplatin (10-50 μmol/L) dose-dependently induced cell damage and apoptosis in NRK-52E cells, whereas emodin (10 and 100 μmol/L) significantly ameliorated cisplatin-induced cell damage, apoptosis and caspase-3 cleavage. Emodin dose-dependently increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Furthermore, the protective effects of emodin were abolished by bafilomycin A1 (10 nmol/L), and mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) not only abolished emodin-induced autophagy activation, but also emodin-induced anti-apoptotic effects.Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the AMPK/mTOR signaling pathways and activating autophagy. Emodin may have therapeutic potential for the prevention of cisplatin-induced nephrotoxicity.

SUBMITTER: Liu H

PROVIDER: S-EPMC4753365 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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Project description:Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation, and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102's cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have a therapeutic potential for the treatment of AKI.

Project description:BackgroundWe have recently demonstrated that amniotic fluid stem cells (AFSC) express renal progenitor markers and can be differentiated in vitro into renal lineage cell types, viz, juxtaglomerular and renal proximal tubular epithelial-like cells. Here, we have evaluated the therapeutic efficacy of AFSC in a cisplatin-induced rat model of acute renal failure (ARF) and investigated the underlying mechanisms responsible for their renoprotective effects.MethodsARF was induced in Wistar rats by intra-peritoneal injection of cisplatin (7 mg/kg). Five days after cisplatin injection, rats were randomized into two groups and injected with either AFSC or normal saline intravenously. On days 8 and 12 after cisplatin injection, the blood biochemical parameters, histopathological changes, apoptosis and expression of pro-apoptotic, anti-apoptotic, and autophagy-related proteins in renal tissues were studied in both groups of rats. To further confirm whether the protective effects of AFSC on cisplatin-induced apoptosis were dependent on autophagy, chloroquine, an autophagy inhibitor, was administered by the intra-peritoneal route.ResultsAdministration of AFSC in ARF rats resulted in improvement of renal function and attenuation of renal damage as reflected by significant decrease in blood urea nitrogen, serum creatinine levels, tubular cell apoptosis as assessed by Bax/Bcl2 ratio, and expression of the pro-apoptotic proteins, viz, PUMA, Bax, cleaved caspase-3, and cleaved caspase-9, as compared to the saline-treated group. Furthermore, in the AFSC-treated group as compared to the saline-treated group, there was a significant increase in the activation of autophagy as evident by increased expression of LC3-II, ATG5, ATG7, Beclin1, and phospho-AMPK levels with a concomitant decrease in phospho-p70S6K and p62 expression levels. Chloroquine administration led to significant reduction in the anti-apoptotic effects of the AFSC therapy and further deterioration in the renal structure and function caused by cisplatin.ConclusionAFSC led to amelioration of cisplatin-induced ARF which was mediated by inhibition of apoptosis and activation of autophagy. The protective effects of AFSC were blunted by chloroquine, an inhibitor of autophagy, highlighting that activation of autophagy is an important mechanism of action for the protective role of AFSC in cisplatin-induced renal injury.

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Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy.

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