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Aims: This study was designed to reveal the role and underlying mechanism of excessive autophagy mediated by ERS via the PERK-eIF2α pathway in the apoptosis and formation of CaOx kidney stones., Main Methods: Ethylene glycol (EG) was used to establish a rat model of CaOx kidney stones, and 100 mg/kg of ERS inhibitor 4-phenylbutyric acid (4-PBA) or 60 mg/kg of autophagy inhibitor chloroquine (CQ) was administered daily to the rats. Four weeks after administration, we collected blood and kidney tissues to analyze the occurrence of ERS and autophagy, apoptosis, renal function, renal tubular crystal deposition, and kidney damage, respectively., Key Findings: We observed that both 4-PBA and CQ treatment significantly inhibited the excessive autophagy and reduced apoptosis as well as decreasing p-PERK and p-eIF2α expressions. Meanwhile, the proportion of kidney weight, contents of creatinine and blood urea nitrogen, excretion of neutrophil gelatinase-associated lipocalin and kidney injury molecule 1, and renal tubular deposition were markedly down-regulated., Significance: The findings in this study suggested that ERS induced excessive autophagy via the PERK-eIF2α pathway, regulating cell damage and apoptosis. ERS-mediated inhibition of excessive autophagy effectively protected kidney function and prevented the apoptosis and formation of kidney stones., Competing Interests: Declaration of competing interest The manuscript is approved by all authors for publication and no conflict of interest exits in the submission of this manuscript., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: M2 macrophages have important roles in diseases such as tumours, cardiovascular diseases and renal diseases. This study aimed to determine the effects and protective mechanism of M2 macrophages against oxidative stress injury and apoptosis induced by calcium oxalate crystals (CaOx) in renal tubular epithelial cells (HK-2) under coculture conditions., Methods: THP-1 cells were induced to differentiate into M2 macrophages by using phorbol-12-myristate-13-acetate, IL-4 and IL-13. Morphological features were observed by microscopy. Phenotypic markers were identified by reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay (ELISA). HK-2 cells were treated with 0.5 mg/mL CaOx crystals and co-cultured with M2 macrophages or apocynin. The viability of HK-2 cells was detected by CCK-8 assay. The lactate dehydrogenase (LDH) activity of HK-2 cells was analysed using a microplate reader. The apoptosis of HK-2 cells was examined by flow cytometry and Hoechst 33258 staining. Reactive oxygen species (ROS) expression and mitochondrial membrane potential in HK-2 cells were detected by a fluorescence microplate reader. Western blot analysis was conducted to detect the expression of p47phox, Bcl-2, cleaved caspase-3, cytochrome c, p38 MAPK, phospho-p38 MAPK, Akt and phospho-Akt., Results: The results of morphology, reverse transcription-polymerase chain reaction, Western blot and ELISA showed that THP-1 cells were successfully polarised to M2 macrophages. The results of co-culture suggested that M2 macrophages or apocynin significantly increased the cell viability and decreased the LDH activity and apoptosis rate after HK-2 cells were challenged with CaOx crystals. The expression of the p47phox protein and the concentration of ROS were reduced, the release of mitochondrial membrane potential and the expression of the Bcl-2 protein were upregulated and the protein expression of cleaved caspase-3 and cytochrome c was downregulated. The expression of the phosphorylated form of p38 MAPK increased. Under coculture conditions with M2 macrophages, the Akt protein of HK-2 cells treated with CaOx crystals was dephosphorylated, but the phosphorylated form of Akt was not reduced by apocynin., Conclusions: M2 macrophages reduced the oxidative stress injury and apoptosis of HK-2 cells by downregulating the activation of NADPH oxidase, reducing the production of ROS, inhibiting the phosphorylation of p38 MAPK and enhancing the phosphorylation of Akt. We have revealed one of the possible mechanisms by which M2 macrophages reduce the formation of kidney stones., (© 2019 The Author(s) Published by S. Karger AG, Basel.)

This study developed an in vitro system by co-culturing HK-2 cells with different concentration of hydroxyapatite (HAP) and/or macrophage cells to simulate the internal environment of urolithiasis as far as possible, investigating the regulatory effects of macrophage cells on HAP-induced expression of relative inflammatory factors of HK-2 cells. The control group (H group) was only comprised of HK-2 cells. Experimental groups included co-culturing HK-2 cells and macrophage cells (H + M group), co-culturing HK-2 cells and HAP (H + A group), co-culturing macrophage cells and HAP (M + A group), and co-culturing HK-2 cells and macrophage cells with HAP (H + M + A group). In the H + A, M + A, and H + M + A group, we set the concentration of HAP as 5 μg/cm2 (A1) and 10 μg/cm2 (A2). After co-culturing for 2, 4, and 6 h, we detected the expression of CCL-2 in the liquid by ELISA. We tested the expression of LDH and ROS to evaluate the damage of HK-2 cells. We assessed the apoptosis of HK-2 cells using DAPI staining assay, flow cytometry, and the rate of BAX/BCL-2. Western Blotting detected OPN, Fetuin-A, BAX, and BCL-2 of HK-2 cells. The expression of CCL-2 in the medium of H + A1 and H + A2 group increased significantly compared with the control (P < 0.05); CCL-2 of M + A1 and M + A2 group was higher than the H + A1 and H + A2 group (P < 0.05). The expression of CCL-2 in H + M + A1 and H + M + A2 group was also higher than M + A1 and M + A2 group (P < 0.05). Compared with control, the expression of OPN, LDH release, the ratio of BAX/BCL-2, and the generation of ROS in HK-2 cells increased in a dose- and time-dependent manner. Compared with the control, the expression of Fetuin-A decreased in various degrees at different incubation periods. Especially when co-culturing for 6 h, Fetuin-A decreased most seriously in the H + M + A1 group. (1) The HAP can induce the HK-2 cells oxidative stress and inflammatory damage and apoptosis, when adding the macrophages to co-culture, macrophage cells can aggravate the damage and apoptosis of the HK-2 cells. (2) After the stimulation of HAP, the expression of OPN in HK-2 cells increased in a time- and dose-dependent manner; macrophage cells can aggravate the increase of OPN in HK-2 cells. (3) In the HAP and HK-2 cells co-cultured system, the low-level Fetuin-A of HK-2 cells may be related to the excessive consumption of Fetuin-A in the process of HAP-induced renal tubular epithelial cell excessive oxidative stress, inflammatory injury, and cell apoptosis. When adding macrophage cells to co-culture, Fetuin-A decreased even more seriously, it reminds us that macrophage cells can slightly regulate the expression of Fetuin-A in the HK-2 cells. [ABSTRACT FROM AUTHOR]