Sirtuin1 inhibits calcium oxalate crystal-induced kidney injury by regulating TLR4 signaling and macrophage-mediated inflammatory activation.

Sirtuin1 (Sirt1) activation significantly attenuated calcium oxalate (CaOx) crystal deposition and renal inflammatory injury by regulating renal immune microenvironment. Here, to elucidate the molecular mechanism underlying the therapeutic effects of Sirt1 on macrophage related inflammation and tubular epithelial cells (TECs) necrosis, we constructed a macrophage and CaOx monohydrate (COM)-stimulated tubular cell co-culture system to mimic immune microenvironment in kidney and established a mouse model of CaOx nephrocalcinosis in wild-type and myeloid-specific Sirt1 knockout mice. Target prediction analyses of Gene Expression Omnibus Datasets showed that only miR-34b-5p is regulated by lipopolysaccharides and upregulated by SRT1720 and targets the TLR4 3'-untranslated region. In vitro, SRT1720 suppressed TLR4 expression and M1 macrophage polarization and decreased reactive oxygen species (ROS) production and mitochondrial damage in COM-stimulated TECs by targeting miR-34b-5p. Mechanically, Sirt1 promoted miR-34b-5p expression by suppressing the tri-methylation of H3K27, which directly bound to the miR-34b-5p promoter and abolished the miR-34b-5p transcription. Furthermore, loss of Sirt1 aggravated CaOx nephrocalcinosis-induced inflammatory and oxidative kidney injury, while AgomiR-34b reversed these effects. Therefore, our data suggested that Sirt1 inhibited TLR4 signaling and M1 macrophage polarization and decreased inflammatory and oxidative injury of TECs in vitro and in vivo.

The ADRENAL score: A comprehensive scoring system for standardized evaluation of adrenal tumor.

Objectives: To propose an original and standardized scoring system to quantify the functional and anatomical characteristics of adrenal tumor. Materials and methods: Four groups of consecutive adrenalectomies (n = 458) with heterogeneity in tumor characteristics and surgical approaches, including 212 laparoscopic cases (Group 1) and 105 robotic cases (Group 2) from The First Affiliated Hospital of Nanchang University, 28 robotic cases from Temple University Hospital (Group 3) and 113 laparoscopic cases from The First Affiliated Hospital of Guangxi Medical University (Group 4). All patients were followed up for 4.5 to 5.5 years. Six parameters including functional status or suspicion of malignancy, tumor size, relationship to adjacent organs, intratumoral enhancement on CT, nearness of the tumor to major vessels and body mass index were assessed and scored on a 0, 1 and 2 points scale. Correlation between the sum of the 6 scores and tumor laterality (ADRENAL score) verse operative time (OT), estimated blood loss (EBL), perioperative complications, transfusion, conversion and length of hospital stay was analyzed. Results: ADRENAL score was a strong predictor of both OT and EBL in all four groups (p < 0.05 for all tests). In Group 2 and 4, higher ADRENAL score seemed to correlate with longer hospital stay. No statistically significant correlation between ADRENAL score and complication, transfusion or conversion was noted yet. Conclusions: ADRENAL score appears to be a valid predictor of surgical outcomes. It may provide a common reference for adrenal surgery training program, preoperative risk assessment and stratified comparative analysis of adrenal surgeries via different techniques and approaches.

International Alliance of Urolithiasis (IAU) guidelines on the metabolic evaluation and medical management of urolithiasis.

The aim of this study was to construct the fourth in a series of guidelines on the treatment of urolithiasis by the International Alliance of Urolithiasis (IAU) that by providing a clinical framework for the metabolic evaluation, prevention, and follow-up of patients with urolithiasis based on the best available published literature. All recommendations were summarized following a systematic review and assessment of the literature in the PubMed database from January 1976 to June 2022. Each generated recommendation was graded using a modified GRADE methodology. Guideline recommendations were developed that addressed the following topics: initial evaluation, metabolic testing, dietary measures, medical management, and follow-up of recurrent stone formers. It was emphasized by the Panel that prevention of new stone formation is as important as the surgical removal of the stones. Although general preventive measures may be effective in reducing stone recurrence rates in some patients, specific medical and dietary management should be well considered and eventually applied in an individualized manner based on the outcomes of metabolic work-up, stone analysis and some certain patient related factors. A detailed follow-up of each case is essential depending on the metabolic activity of each individual patient.

Role of ROS-Induced NLRP3 Inflammasome Activation in the Formation of Calcium Oxalate Nephrolithiasis.

Calcium oxalate nephrolithiasis is a common and highly recurrent disease in urology; however, its precise pathogenesis is still unknown. Recent research has shown that renal inflammatory injury as a result of the cell-crystal reaction plays a crucial role in the development of calcium oxalate kidney stones. An increasing amount of research have confirmed that inflammation mediated by the cell-crystal reaction can lead to inflammatory injury of renal cells, promote the intracellular expression of NADPH oxidase, induce extensive production of reactive oxygen species, activate NLRP3 inflammasome, discharge a great number of inflammatory factors, trigger inflammatory cascading reactions, promote the aggregation, nucleation and growth process of calcium salt crystals, and ultimately lead to the development of intrarenal crystals and even stones. The renal tubular epithelial cells (RTECs)-crystal reaction, macrophage-crystal reaction, calcifying nanoparticles, endoplasmic reticulum stress, autophagy activation, and other regulatory factors and mechanisms are involved in this process.

Erratum: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis: Erratum.

[This corrects the article DOI: 10.7150/thno.44054.].

XIST Inhibition Attenuates Calcium Oxalate Nephrocalcinosis-Induced Renal Inflammation and Oxidative Injury via the miR-223/NLRP3 Pathway.

The roles of the lncRNA X inactive specific transcript (XIST) in many diseases, including cancers and inflammatory sickness, have been previously elucidated. However, renal calculus remained poorly understood. In this study, we revealed the potential effects of XIST on kidney stones that were exerted via inflammatory response and oxidative stress mechanisms. We established a glyoxylate-induced calcium oxalate (CaOx) stone mouse model and exposed HK-2 cells to calcium oxalate monohydrate (COM). The interactions among XIST, miR-223-3p, and NOD-like receptor protein 3 (NLRP3) and their respective effects were determined by RNAs and protein expression, luciferase activity, and immunohistochemistry (IHC) assays. Cell necrosis, reactive oxygen species (ROS) generation, and inflammatory responses were detected after silencing XIST, activating and inhibiting miR-223-3p, and both knocking down XIST and activating miR-223-3p in vitro and in vivo. The XIST, NLRP3, caspase-1, and IL-1ß levels were notably increased in kidney samples from glyoxylate-induced CaOx stone model mice. XIST knockdown significantly suppressed the inflammatory damage and ROS production and further attenuated oxalate crystal deposition. miRNA-223-3p mimics also exerted the same effects. Moreover, we verified the interactions among XIST, miRNA-223-3p and NLRP3, and the subsequent effects. Our results suggest that the lncRNA XIST participates in the formation and progression of renal calculus by interacting with miR-223-3p and the NLRP3/Caspase-1/IL-1ß pathway to mediate the inflammatory response and ROS production.

Regulation of endoplasmic reticulum stress on the damage and apoptosis of renal tubular epithelial cells induced by calcium oxalate crystals.

This study aimed to observe whether calcium oxalate (CaOx) crystals can induce the activation of endoplasmic reticulum (ER) stress in human renal cortex proximal tubule epithelial (HK-2) cells and to explore the regulatory of ER stress on the damage and apoptosis of HK-2 cells induced by CaOx crystals. We detected the optimal CaOx crystal concentration and intervention time by Western blot. ER stress modifiers tunicamycin (TM) and 4-phenylbutyric acid (4-PBA) were used to regulate the ER stress of HK-2 cells. The activities of ER stress marker proteins GRP78 and CHOP were evaluated by Western blot and immunohistochemistry. Western blot and TUNEL staining were used to detect cell apoptosis. We observed cell-crystal adhesion with an optical microscope. Lactate dehydrogenase (LDH) test kit and IL-1ß enzyme-linked immunosorbent assay kit were used to detect and evaluate HK-2 cell damage. We found that the expression of ER stress marker proteins GRP78 and CHOP gradually increased with the increase in CaOx crystal concentration and intervention time and reached the maximum at 2.0 mmol/L and 24 h. The use of ER stress modifiers TM and 4-PBA can effectively regulate the ER stress level induced by CaOx crystals, and the level of apoptosis is positively correlated with the level of ER stress. 4-PBA pretreatment remarkably reduced cell-crystal adhesion and the secretions of IL-1ß and LDH, whereas the results of TM pretreatment were the opposite. In summary, the damage and apoptosis of HK-2 cells induced by CaOx crystals are closely related to the level of ER stress. Inhibiting the ER stress of HK-2 cells can substantially reduce the cell damage and apoptosis induced by CaOx crystals.

Taurine suppresses ROS-dependent autophagy via activating Akt/mTOR signaling pathway in calcium oxalate crystals-induced renal tubular epithelial cell injury.

Oxidative stress and autophagy are the key promoters of calcium oxalate (CaOx) nephrolithiasis. Taurine is an antioxidant that plays a protective role in the pathogenesis of kidney disease. Previous studies found that taurine suppressed cellular oxidative stress, and inhibited autophagy activation. However, the effect of taurine on CaOx kidney stone formation remains unknown. In the present work, we explored the regulatory effects of taurine on CaOx crystals-induced HK-2 cell injury. Results showed that pretreatment with taurine significantly enhanced the viability of HK-2 cells and ameliorated kidney tissue injury induced by CaOx crystals. Taurine also markedly reduced the levels of inflammatory cytokines, apoptosis, and CaOx crystals deposition. Furthermore, we observed that taurine supplementation alleviated CaOx crystals-induced autophagy. Mechanism studies showed that taurine reduced oxidative stress via increasing SOD activity, reducing MDA concentration, alleviating mitochondrial oxidative injury, and decreasing the production of intracellular ROS. Taurine treatment also effectively activated Akt/mTOR signaling pathway in CaOx crystals-induced HK-2 cells both in vitro and in vivo. In summary, the current study shows that taurine inhibits ROS-dependent autophagy via activating Akt/mTOR signaling pathway in CaOx crystals-induced HK-2 cell and kidney injury, suggesting that taurine may serve as an effective therapeutic agent for the treatment of CaOx nephrolithiasis.

Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis.

Intrarenal calcium oxalate (CaOx) crystals induce renal tubular epithelial cells (TECs) injury and inflammation, which involve Toll-like receptor 4 (TLR4)/interferon regulatory factor 1 (IRF1) signaling. Additionally, infiltrating macrophages (Mϕs) might influence intrarenal CaOx crystals and CaOx-induced renal injury. Although the roles of nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating inflammation and macrophage polarization are well characterized, its potential mechanisms in regulating CaOx nephrocalcinosis remain undefined. Methods: We used a Gene Expression Omnibus dataset to analyze gene-expression profiles. Luciferase reporter, western blot, quantitative polymerase chain reaction, immunofluorescence staining, fluorescence in situ hybridization, positron emission tomography computed tomography imaging, flow cytometry, and chromatin immunoprecipitation assays were employed to study the mechanism of miR-93-TLR4/IRF1 regulation by Nrf2. Anti-inflammatory activity and regulation of macrophage polarization by Nrf2 were investigated in vitro and in vivo. Results: We found that stone-mediated kidney inflammation significantly affected stone growth, and that sulforaphane attenuated CaOx nephrocalcinosis-induced kidney injury and renal CaOx crystals deposition. Additionally, Nrf2 levels significantly increased and negatively correlated with TLR4 and IRF1 levels in a mouse model of CaOx nephrocalcinosis following sulforaphane treatment. Moreover, Nrf2 suppressed TLR4 and IRF1 levels and decreased M1-macrophage polarization which induced by supernatants from COM-stimulated TECs in vitro. In terms of mechanism, transcription factor analyses, microRNA microarray, and chromatin immunoprecipitation assays showed that Nrf2 exhibited positive transcriptional activation of miR-93-5p. In addition, Luciferase reporter, qRT-PCR, and western blot validated that miR-93-5p targets TLR4 and IRF1 mRNA. Furthermore, suppressed miR-93-5p expression partially reversed Nrf2-dependent TLR4/IRF1 downregulation. Conclusions: The results suggested that sulforaphane might promote M2Mϕ polarization and inhibit CaOx nephrocalcinosis-induced inflammatory injury to renal tubular epithelial cells via the Nrf2-miR-93-TLR4/IRF1 pathway in vitro and in vivo.

Atorvastatin inhibits renal inflammatory response induced by calcium oxalate crystals via inhibiting the activation of TLR4/NF-κB and NLRP3 inflammasome.

This study aimed to investigate the renal protective effect of atorvastatin (ATV) on the kidney inflammation induced by calcium oxalate (CaOx) crystals. A cell model of cell-crystal interactions and a rat model of CaOx kidney stone were established. The expressions of TLR4, NF-κB, NLRP3, and cleaved caspase-1 in cells and rat kidney tissues were detected using Western blot, immunohistochemical, and/or immunofluorescence. The concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS) in cells, and lactic acid dehydrogenase (LDH) in the culture medium were measured. The secreted levels of interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were examined by ELISA. The serum levels of creatinine (CRE) and blood urea nitrogen (BUN) were measured. von Kossa staining was used for the evaluation of renal lens deposition. The CaOx model group showed significantly decreased SOD level; increased concentrations of MDA; ROS and LDH; elevated expressions of TLR4, NF-κB, NLRP3, and cleaved caspase-1; and the elevated release of IL-1ß, IL-18, IL-6, and TNF- α as compared to the control group. The treatment with ATV significantly inhibited the formation of CaOx kidney stone by increasing the level of SOD; downregulating MDA, ROS, and LDH; inhibiting the expressions of TLR4, NF-κB, NLRP3 and cleaved caspase-1; and blocking the secretion of inflammatory cytokines. In addition, the serum levels of CRE and BUN, and the intrarenal crystal deposition were also significantly decreased in ATV-treated rats. In summary, oxidative stress, TLR4/NF-κB, and NLRP3 inflammasome pathways are involved in renal inflammatory responses induced by CaOx crystals. ATV treatment significantly suppressed oxidative stress, inhibited the activation of TLR4/NF-κB and NLRP3 inflammasome pathways, and decreased the release of inflammatory mediators, thereby ameliorating CaOx crystal-induced damage and crystal deposition in HK-2 cells and rat kidney tissues.

Resonance Raman spectroscopic and density functional theoretical study on microsolvated 2-Thiocytosine clusters with polar solvents.

Microsolvation effects on the excited state deactivation dynamics of 2-thiocytosine (2tC) were studied in hydrogen-bonded 2tC clusters with protic solvents using resonance Raman, FT-IR, FT-Raman, UV-vis spectroscopy combining with density functional theoretical calculation. Two protic solvents, water (H2O) and methanol (MeOH), and one aprotic solvent, acetonitrile (MeCN), were used to investigate the 2tC(H2O)1-5, 2tC(MeOH)1-5, and 2tC(MeCN)1-3 microsolvated clusters. In CH3OH and H2O solvents, most of the Raman shifts were due to the vibration modes of 2tC(solvent)n (solvent = H2O, CH3OH; n = 1-4) clusters via intermolecular NH⋯O hydrogen bonds (HB). The intermolecular >NH⋯O hydrogen bond interactions, which are the key constituents of stable thione structure of 2tC, revealed the spectra difference of 2tC in CH3CN, CH3OH and H2O. With the aid of electronic structural and vibration frequency calculations, the observed Raman spectra were assigned to the low energy isomers of 2tC(solvent)2 (solvent = H2O, CH3OH) clusters in water and methanol and 2tC(CH3CN) in acetonitrile solvents. 2tC(solvent)2 clusters in water and methanol may prohibit or promote excited state proton transfer reaction from sulfur atom to neighbor nitrogen atom due to the hydrogen bonding chain between 2tC and protic solvent molecules. Our experimental and theoretical studies confirmed that the hydrogen bond sites were located on the specified functional group SCNH of 2tC with solvent molecules.

Autophagy-endoplasmic reticulum stress inhibition mechanism of superoxide dismutase in the formation of calcium oxalate kidney stones.

BACKGROUND: Nephrolithiasis is a common disease in urology, and its pathogenesis is associated with various factors. Recent studies have shown that reactive oxygen species (ROS) can promote autophagy in the formation of kidney stones and exacerbate kidney injury. Endoplasmic reticulum stress (ERS), a key factor in regulating intracellular environmental homeostasis, is also directly related to ROS production. Therefore, this study aimed to investigate the regulatory effect of superoxide dismutase (SOD) on autophagy-ERS response during the formation of calcium oxalate (CaOx) kidney stones in rats. METHODS: Thirty-two rats were randomly divided into four groups (n = 8): normal control group, stone model group, stone model with atorvastatin group, and stone model with diethyldithiocarbamic acid (DETC) group. Rat models of CaOx kidney stones were established by intragastric administration of 0.75 % ethylene glycol for 4 weeks. Kidney/body weight was used to assess renal enlargement. Renal function was assessed by measuring serum SOD, creatinine (CRE), and blood urea nitrogen (BUN) levels. The expression of autophagy-related proteins LC3B and BECN1 was detected through immunohistochemical staining. Meanwhile, the expression of autophagy-ERS response-related proteins LC3B, BECN1, p62, GRP78, and CHOP was detected using Western blot and RT-PCR. Renal tubular injury markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (Kim-1) were determined through enzyme-linked immunosorbent assay. The apoptosis of renal tubular cells and the expression of their signature proteins cleaved Caspase-3, Bax and Bcl-2 were detected using Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Western blot assays, respectively. Crystal deposition and histological tissue injury were assessed through Von Kossa staining. RESULTS: Compared with the control group, the stone model group showed higher kidney/body weight ratio; evidently higher expression of autophagy-ERS response- and apoptosis-related proteins LC3B, BECN1, GRP78, CHOP, Bax and cleaved Caspase-3; and lower levels of p62, bcl-2 protein, and SOD. The stone model group also showed higher levels of apoptosis, serum CRE, BUN, NGAL, and Kim-1, as well as considerably greater crystal deposition and renal injury, than the control group. Atorvastatin reduced the levels of autophagy-ERS response, kidney injury, and crystal deposition, but they were increased by DETC. CONCLUSION: Enhanced SOD activity can protect the kidneys by reducing autophagy-ERS response and CaOx kidney stone formation. Atorvastatin may be a new option for the prevention and treatment of nephrolithiasis.

Effect of endoplasmic reticulum stress-mediated excessive autophagy on apoptosis and formation of kidney stones.

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.

Effect of M2 Macrophages on Injury and Apoptosis of Renal Tubular Epithelial Cells Induced by Calcium Oxalate Crystals.

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.

Calcifying nanoparticles induce cytotoxicity mediated by ROS-JNK signaling pathways.

Calcifying nanoparticles (CNPs) play an important role in kidney stone formation, but the mechanism(s) are unclear. CNPs were isolated and cultured from midstream urine of patients with kidney stones. CNP morphology and characteristics were examined by electron microscopy and electrophoresis analysis. Chemical composition was analyzed using energy-dispersive X-ray microanalysis and Western blotting. Human renal proximal convoluted tubule cell (HK-2) cultures were exposed to CNPs for 0, 12 and 72 h, and production of reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis levels were evaluated. CNPs isolated from patients showed classical morphology, the size range of CNPs were 15-500 nm and negative charge; they were found to contain fetuin-A. Exposure of HK-2 cells to CNPs induced ROS production, decreased mitochondrial membrane potential and decreased cell viability. Transmission electron microscopy showed that CNPs can enter the cell by phagocytosis, and micrographs revealed signs of apoptosis and autophagy. CNPs increased the proportion of apoptotic cells, down-regulated Bcl-2 expression and up-regulated Bax expression. CNPs also up-regulated expression of LC3-B, Beclin-1and p-JNK.CNPs are phagocytosed by HK-2 cells, leading to autophagy, apoptosis and ROS production, in part through activation of JNK signaling pathways. ROS and JNK pathways may contribute to CNP-induced cell injury and kidney stone formation.

Inhibition of Autophagy Attenuated Ethylene Glycol Induced Crystals Deposition and Renal Injury in a Rat Model of Nephrolithiasis.

BACKGROUND/AIMS: Nephrolithiasis is a common and frequently occurring disease, its exact pathogenesis is remains unclear. Emerging data suggest that autophagy plays a vital role in the pathophysiological processes of kidney diseases. Therefore, this study was designed to investigate the potential role of autophagy in the formation of calcium oxalate (CaOx) kidney stones in rat model. METHODS: Thirty-two rats were randomly divided into four groups (eight rats/group): untreated control group, stone model group, rapamycin-treated group, chloroquine-treated group. Rat models of CaOx nephrolithiasis was administration of 0.75% ethylene glycol (EG) in their drinking water for 4 weeks. Western blot and transmission electron microscope (TEM) were used to detect the expression of autophagy related protein LC3-II, BECN1 and p62 and autophagic vacuoles respectively. Renal function was evaluated by measuring the levels of serum CRE and BUN. Renal tubular injury markers NGAL and Kim-1 was determined by ELISA kits. Von Kossa staining was used to assess crystal deposits and histological tissue injury. TUNEL staining was employed to assess apoptosis of the renal tubular cell. RESULTS: Compare with the controls, the expression of autophagy related protein LC3-II, BECN1 and number of autophagic vacuoles were increased significantly, whereas the p62 protein level was decreased in the stone model group. The levels of apoptosis, serum CRE and BUN, NGAL and Kim-1 in the stone model group were increased compared with the control group and crystals deposition and renal injury were increased significantly. However, the levels of autophagy, kidney injury and crystal deposition were decreased by chloroquine but increased by rapamycin. CONCLUSION: These findings suggested that rats were administration of ethylene glycol could lead to the formation of CaOx nephrolithiasis and autophagy activation. Inhibiting autophagy could be an effective therapeutic approach for decreasing the formation of nephrolithiasis.

Inhibition of autophagy-attenuated calcium oxalate crystal-induced renal tubular epithelial cell injury in vivo and in vitro.

Accumulating evidence suggests that autophagy is involved in the pathophysiological processes of kidney diseases. However, the role of autophagy in the formation of calcium oxalate (CaOx) nephrolithiasis remains unclear. In this study, we investigated the effects of autophagy on renal tubular epithelial cell injury induced by CaOx crystals in vivo and in vitro. We first observed that the expression levels of LC3-II and BECN1 and number of autophagic vacuoles were markedly increased in the renal tissue of CaOx stone patients. We subsequently found that exposure of HK-2 cells to CaOx crystals could increase LC3-II and BECN1 expression as well as the number of GFP-LC3 dots and autophagic vacuoles in a dose- and time-dependent manner. In addition, our results suggest that CaOx crystals induced autophagy, at least in part, via activation of the reactive oxygen species (ROS) pathway in HK-2 cells. Furthermore, inhibition of autophagy using 3-methyladenine or siRNA knockdown of BECN1 attenuated CaOx crystal-induced HK-2 cells injury. However, enhancing autophagic activity with rapamycin exerted an opposite effect. Taken together, our results demonstrate that autophagy is essential for CaOx crystal-induced renal tubular epithelial cell injury and that inhibition of autophagy could be a novel therapeutic strategy for CaOx nephrolithiasis.

The effects of HAP and macrophage cells to the expression of inflammatory factors and apoptosis in HK-2 cells of vitro co-cultured system.

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.