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Oxidative damage to the kidneys is a primary factor in the occurrence of kidney stones. This study explores the inhibitory effect of Porphyra yezoensis polysaccharides (PYP) on oxalate-induced renal injury by detecting levels of oxidative damage, expression of adhesion molecules, and damage to intracellular organelles and revealed the molecular mechanism by molecular biology methods. Additionally, we validated the role of PYP in vivo using a crystallization model of hyperoxalate-induced rats. PYP effectively scavenged the overproduction of reactive oxygen species (ROS) in HK-2 cells, inhibited the adhesion of calcium oxalate (CaOx) crystals on the cell surface, unblocked the cell cycle, restored the depolarization of the mitochondrial membrane potential, and inhibited cell death. PYP upregulated the expression of antioxidant proteins, including Nrf2, HO-1, SOD, and CAT, while decreasing the expression of Keap-1, thereby activating the Keap1/Nrf2 signaling pathway. PYP inhibited CaOx deposition in renal tubules in the rat crystallization model, significantly reduced high oxalate-induced renal injury, decreased the levels of the cell surface adhesion proteins, improved renal function in rats, and ultimately inhibited the formation of kidney stones. Therefore, PYP, which has crystallization inhibition and antioxidant properties, may be a therapeutic option for the treatment of kidney stones.

Objective: Renal epithelial cell injury and cell-crystal interaction are closely related to kidney stone formation., Methods: This study aims to explore the inhibition of endocytosis of nano-sized calcium oxalate monohydrate (nano-COM) crystals and the cell protection of corn silk polysaccharides (CCSPs) with different carboxyl contents (3.92, 7.75, 12.90, and 16.38%). The nano-COM crystals protected or unprotected by CCSPs were co-cultured with human renal proximal tubular epithelial cells (HK-2), and then the changes in the endocytosis of nano-COM and cell biochemical indicators were detected., Results: CCSPs could inhibit the endocytosis of nano-COM by HK-2 cells and reduce the accumulation of nano-COM in the cells. Under the protection of CCSPs, cell morphology is restored, intracellular superoxide dismutase levels are increased, lipid peroxidation product malondialdehyde release is decreased, and mitochondrial membrane potential and lysosomal integrity are increased. The release of Ca 2+ ions in the cell, the level of cell autophagy, and the rate of cell apoptosis and necrosis are also reduced. CCSPs with higher carboxyl content have better cell protection abilities., Conclusion: CCSPs could inhibit the endocytosis of nano-COM crystals and reduce cell oxidative damage. CCSP3, with the highest carboxyl content, shows the best biological activity., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Background : The damage to renal tubular epithelial cells is closely related to the formation of kidney stones. At present, research on drugs that can protect cells from damage remains limited. Methods : This study aims to explore the protective effects of four different sulfate groups (-OSO 3 - ) of Laminaria polysaccharides (SLPs) on human kidney proximal tubular epithelial (HK-2) cells and determine the difference in the endocytosis of nano-sized calcium oxalate monohydrate (COM) crystals before and after protection. COM with a size of 230 ± 80 nm was used to damage HK-2 cells to establish a damage model. The protection capability of SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO 3 - contents of 0.73, 15, 23, and 31%, respectively, against COM crystal damage and the effect of SLPs on the endocytosis of COM crystals were studied. Results : Compared with that of the SLP-unprotected COM-injured group, the cell viability of the SLP-protected group was improved, healing capability was enhanced, cell morphology was restored, production of reactive oxygen species was reduced, mitochondrial membrane potential and lysosome integrity were increased, intracellular Ca 2+ level and autophagy were decreased, cell mortality was reduced, and internalized COM crystals were lessened. The capability of SLPs to protect cells from damage and inhibit the endocytosis of crystals in cells enhanced with an increase in the -OSO 3 - content of SLPs. Conclusions : SLPs with a high -OSO 3 - content may become a potential green drug for preventing the formation of kidney stones., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

BRAF inhibitors are commonly used in targeted therapies for melanoma patients harboring BRAF V600E mutant. Despite the benefit of vemurafenib therapy, acquired resistance during or after treatment remains a major obstacle in BRAF V600E mutant melanoma. Here we found that RSK2 is overexpressed in melanoma cells and the high expression of RSK2 indicates poor overall survival (OS) in melanoma patients. Overexpression of RSK2 leads to vemurafenib resistance, and the deletion of RSK2 inhibits cell proliferation and sensitizes melanoma cells to vemurafenib. Mechanistically, RSK2 enhances the phosphorylation of FOXO1 by interacting with FOXO1 and promoting its subsequent degradation, leading to upregulation of cyclin D1 in melanoma cells. These results not only reveal the presence of a RSK2-FOXO1-cyclin D1 signaling pathway in melanoma, but also provide a potential therapeutic strategy to enhance the efficacy of vemurafenib against cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wu, Li, Jiang, Li, Shi, Sun, Li and Cheng.)

Objective: Desmodium styracifolium is the best traditional medicine for treating kidney calculi in China. This study is aimed at increasing the carboxyl (-COOH) content of D. styracifolium polysaccharide (DSP0) and further increasing its antistone activity., Methods: DSP0 was carboxymethylated with chloroacetic acid at varying degrees. Then, oxalate-damaged HK-2 cells were repaired with modified polysaccharide, and the changes in biochemical indices before and after repair were detected., Results: Three modified polysaccharides with 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3) -COOH are obtained. Compared with DSP0 (-COOH content = 1.17%), CDSPs have stronger antioxidant activity in vitro and can improve the vitality of damaged HK-2 cells. CDSPs repair the cell morphology and cytoskeleton, increase the cell healing ability, reduce reactive oxygen species and nitric oxide levels, increase mitochondrial membrane potential, limit autophagy level to a low level, reduce the eversion of phosphatidylserine in the cell membrane, weaken the inhibition of oxalate on DNA synthesis, restore cell cycle to normal state, promote cell proliferation, and reduce apoptosis/necrosis., Conclusion: The carboxymethylation modification of DSP0 can improve its antioxidant activity and enhance its ability to repair damaged HK-2 cells. Among them, CDSP2 with medium -COOH content has the highest activity of repairing cells, whereas CDSP3 with the highest -COOH content has the highest antioxidant activity. This difference may be related to the active environment of polysaccharide and conformation of the polysaccharide and cell signal pathway. This result suggests that Desmodium styracifolium polysaccharide with increased -COOH content may have improved potential treatment and prevention of kidney calculi., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Gu-Hua Tang et al.)

Objective: Injury of renal tubular epithelial cells (HK-2) is an important cause of kidney stone formation. In this article, the repairing effect of polysaccharide (PCP0) extracted from the traditional Chinese medicine Poria cocos and its carboxymethylated derivatives on damaged HK-2 cells was studied, and the differences in adhesion and endocytosis of the cells to nanometer calcium oxalate monohydrate (COM) before and after repair were explored., Methods: Sodium oxalate (2.8 mmol/L) was used to damage HK-2 cells to establish a damage model, and then Poria cocos polysaccharides (PCPs) with different carboxyl (COOH) contents were used to repair the damaged cells. The changes in the biochemical indicators of the cells before and after the repair and the changes in the ability to adhere to and internalize nano-COM were detected., Results: The natural PCPs (PCP0, COOH content = 2.56%) were carboxymethylated, and three carboxylated modified Poria cocos with 7.48% (PCP1), 12.07% (PCP2), and 17.18% (PCP3) COOH contents were obtained. PCPs could repair the damaged HK-2 cells, and the cell viability was enhanced after repair. The cell morphology was gradually repaired, the proliferation and healing rate were increased. The ROS production was reduced, and the polarity of the mitochondrial membrane potential was restored. The level of intracellular Ca 2+ ions decreased, and the autophagy response was weakened., Conclusion: The cells repaired by PCPs inhibited the adhesion to nano-COM and simultaneously promoted the endocytosis of nano-COM. The endocytic crystals mainly accumulated in the lysosome. Inhibiting adhesion and increasing endocytosis could reduce the nucleation, growth, and aggregation of cell surface crystals, thereby inhibiting the formation of kidney stones. With the increase of COOH content in PCPs, its ability to repair damaged cells, inhibit crystal adhesion, and promote crystal endocytosis all increased, that is, PCP3 with the highest COOH content showed the best ability to inhibit stone formation., (Copyright © 2022. Published by Elsevier B.V.)

Objective: The formation of kidney stone is closely related to cell injury and crystal adhesion., Method: The sulfur trioxide-pyridine method was used to sulfate raw Undaria pinnatifida polysaccharide (UPP) with a molecular weight (Mw) of 8.33 kDa. Four polysaccharides with the sulfate group (-OSO 3 - ) contents of 1.59% (UPP0), 6.03% (UPP1), 20.83% (UPP2), and 36.39% (UPP3) were obtained. The antioxidant activity of the four UPPs, the difference in oxidative damage inflicted by nano-CaOx monohydrate (nano-COM) on human proximal tubular epithelial (HK-2) cells before and after protection by UPPs, and the inhibitory effect on nano-COM adhesion were explored., Results: Structural characterization showed that sulfation was successful. As the -OSO 3 - content in the UPPs was increased, the antioxidant activity and capability of the UPPs to regulate the growth of calcium oxalate (CaOx) crystals gradually increased. The damage caused by nano-COM crystals to HK-2 cells under protection by UPPs was weakened. This effect enhanced cell viability, enabled the maintenance of good cell morphology, reduced reactive oxygen species (ROS) levels, and inhibited the decrease in mitochondrial membrane potential, as well as decreased the eversion of phosphatidylserine (PS) and the expression of the adhesion proteins osteopontin (OPN), heat shock protein (HSP 90), and Annexin A1 (ANXA1). The adhesion of nano-COM to HK-2 cells was inhibited under the protection by UPPs., Conclusion: UPP3 with the highest content of -OSO 3 - presented the best antioxidant activity and crystal regulation ability, while UPP2 with the second highest -OSO 3 - content showed optimal cell protection ability and crystal adhesion inhibition ability. The biological activity of UPPs was regulated by Mw and -OSO 3 - content. UPP2 with moderate -OSO 3 - content may become a potential drug for preventing CaOx stones., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Background: The interaction between urinary microcrystals and renal epithelial cells is closely related to kidney stone formation. However, the mechanism of cell state changes that affect crystal-cell interaction remains unclear., Methods: This study investigated the relationship between the sulfate group (-OSO 3 - ) content in Porphyra yezoensis polysaccharide (PYP) and the ability to repair damaged cells, as well as the changes in cell adhesion and endocytosis of nano-calcium oxalate monohydrate (COM) crystals before and after PYP repair of damaged renal tubular epithelial cells. The sulfur trioxide-pyridine method was used to sulfate PYP (-OSO 3 - content of 14.14%), and two kinds of sulfated PYPs with -OSO 3 - content of 20.28% (SPYP1) and 27.14% (SPYP2) were obtained. The above three PYPs were used to repair oxalate-damaged human proximal tubular epithelial cells (HK-2), and the changes in the biochemical indicators of the cells before and after the repair and the changes in cell adhesion and endocytosis of nano-COM crystals were detected., Results: After repair by PYPs, the cell viability increased, the number of reactive oxygen species decreased, and the reduction of mitochondrial membrane potential and the release of intracellular Ca 2+ were suppressed. The cells repaired by PYPs inhibited the adhesion of nano-COM crystals while promoting the endocytosis of the adhered crystals. The endocytosed crystals mainly accumulated in the lysosome. The ability of PYPs to repair cell damage, inhibit crystal adhesion, and promote crystal endocytosis was enhanced when the -OSO 3 - content increased. Among them, SPYP2 with the highest -OSO 3 - content showed the best biological activity., Conclusion: SPYP2 showed the best ability to repair damaged cells, followed by SPYP1 and PYP. SPYP2 may become a potential green drug that inhibits the formation and recurrence of calcium oxalate stones., Competing Interests: The authors declare that they have no competing interests., (© 2021 Sun et al.)

The nucleation, growth and aggregation of calcium oxalate (CaOx) crystals and the oxidative damage of renal tubular epithelial cells are the key factors to induce kidney stones. In this study, degraded Porphyra yezoensis polysaccharide (PYP0) with 14.14% sulfate group (-OSO 3 - ) content was modified via the sulfur trioxide-pyridine method to obtain three kinds of sulfated P. yezoensis polysaccharides (PYPs), namely, PYPS1, PYPS2, and PYPS3, with -OSO 3 - group contents of 17.11%, 20.28%, and 27.14% respectively. Fourier transform infrared spectroscopy, 1 H NMR, and 13 C NMR analyses showed that the -OSO 3 - groups replaced the hydroxyl groups at the C2, C4, and C6 positions on (1 → 3)-linked β-D-galactose, the basic structural skeleton unit of PYP0. The antioxidant activity of the PYPSs increased after sulfation, and their scavenging capacity for OH and DPPH free radicals was enhanced with the increase in their -OSO 3 - group content. Calcium oxalate (CaOx) crystal growth experiments showed that sulfated PYPs promoted the conversion of the thermodynamically stable and sharp CaOx monohydrate (COM) crystals into the thermodynamically unstable and round CaOx dihydrate crystals. With the increase in the -OSO 3 - group content of the polysaccharides, the concentration of soluble Ca 2+ ions in the supernatant increased and the amount of CaOx precipitate decreased. PYPs were nontoxic to human kidney proximal tubular epithelial cells (HK-2) and could protect HK-2 from oxidative damage caused by nano-COM and reduce the level of reactive oxygen species in cells. PYPS3, which had the highest degree of sulfation, had the best protective capability. The results of this work showed that sulfation improved the biological activity of PYPs. This study could provide inspiration for the development of new drugs for the prevention and treatment of kidney stones., (Copyright © 2021. Published by Elsevier B.V.)

The original Laminaria polysaccharide (LP0) was sulfated using the sulfur trioxide-pyridine method, and four sulfated Laminaria polysaccharides (SLPs) were obtained, namely, SLP1, SLP2, SLP3, and SLP4. The sulfated (-OSO 3 - ) contents were 8.58%, 15.1%, 22.8%, and 31.3%, respectively. The structures of the polysaccharides were characterized using a Fourier transform infrared (FT-IR) spectrometer and nuclear magnetic resonance (NMR) techniques. SLPs showed better antioxidant activity than LP0, increased the concentration of soluble Ca 2+ in the solution, reduced the amount of CaOx precipitation and degree of CaOx crystal aggregation, induced COD crystal formation, and protected HK-2 cells from damage caused by nanometer calcium oxalate crystals. These effects can inhibit the formation of CaOx kidney stones. The biological activity of the polysaccharides increased with the content of -OSO 3 - , that is, the biological activities of the polysaccharides had the following order: LP0 < SLP1 < SLP2 < SLP3 < SLP4. These results reveal that SLPs with high -OSO 3 - contents are potential drugs for effectively inhibiting the formation of CaOx stones., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Wei-Bo Huang et al.)

The purpose of this study was to explore the repair effect of carboxymethyl-modified corn silk polysaccharide (CSP) on oxidatively damaged renal epithelial cells and the difference in adhesion between cells and calcium oxalate crystals. The CSP was degraded and modified through carboxymethylation. An oxidatively damaged cell model was constructed by oxalate damage to human kidney proximal tubular epithelial (HK-2) cells. Then, the damaged cells were repaired by modified polysaccharides, and the changes in biochemical indexes and adhesion ability between cells and crystals before and after repair were detected. Four modified polysaccharides with carboxyl group (-COOH) contents of 3.92% (CSP0), 7.75% (CCSP1), 12.90% (CCSP2), and 16.38% (CCSP3) were obtained. Compared with CSP0, CCSPs had stronger antioxidant activity, could repair damaged HK-2 cells, and could reduce phosphorylated serine eversion on the cell membrane, the expression of osteopontin (OPN) and Annexin A1, and crystal adhesion. However, its effect on the expression of hyaluronic acid synthase was not substantial. The carboxymethyl modification of the CSP can improve its ability to repair cells and inhibit crystal adhesion and aggregation. A high carboxymethylation degree results in strong polysaccharide activity. CCSPs are expected to reduce the risk of kidney stone formation and recurrence.

We developed a new ligand and adjuvant dual-assisted room temperature colloidal method for the synthesis of highly luminescent and stable Cs 4 PbBr 6 nanoparticles, in which acetone, oleamine (OM) and oleic acid (OA) were used as precursors, while water and dimethyl sulfoxide (DMSO) were used as adjuvants. In this process, we explored the influencing factors of process parameters (such as the amount of water, the standing time of precursors, and the molar ratio of raw materials), and found that Cs 4 PbBr 6 synthesized by water + DMSO can not only change the morphology and promote crystallization but also improve the lattice strain, reduce the lattice defects and optimize the passivation effect, so as to improve the luminescence properties. Simultaneously, we also found that the pc-LED made of Cs 4 PbBr 6 can still emit bright green light after 4344 h of operation, showing excellent stability and making it promising for solid-state lighting application. This method also provides an important reference value for solving the hydrolysis property of perovskites., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

We demonstrate the influence mechanism on the optical property of Cs 4 PbBr 6 during purification of solution with different protonated levels and polarities. During the purification process, organic groups originating from oleic acid (OA) and PbBr impurity on the surface of Cs 4 PbBr 6 nanocrystals can be removed using high polarity aprotic and protonic solvents, and the number of Br vacancies ( V Br ) can be reduced. The protonic polar solvent can not only etch the organic groups on the surface of nanocrystals, causing surface reconstruction and particle growth of nanocrystals, but also enter into the lattice of Cs 4 PbBr 6 and react with the embedded CsPbBr 3 . However, aprotic polar solvent decreases the particle size of Cs 4 PbBr 6 nanocrystals with the increase in the solvent polarity. The optical properties of Cs 4 PbBr 6 can be effectively improved using aprotic polar solvents as a purification solvent, which is very significant to improve the luminescence efficiency of perovskites., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

The protective effects of Porphyra yezoensis polysaccharides (PYPs) with molecular weights of 576.2 (PYP1), 105.4 (PYP2), 22.47 (PYP3), and 3.89 kDa (PYP4) on the oxidative damage of human kidney proximal tubular epithelial (HK-2) cells and the differences in adherence and endocytosis of HK-2 cells to calcium oxalate monohydrate crystals before and after protection were investigated. Results showed that PYPs can effectively reduce the oxidative damage of oxalic acid to HK-2 cells. Under the preprotection of PYPs, cell viability increased, cell morphology improved, reactive oxygen species levels decreased, mitochondrial membrane potential increased, S phase cell arrest was inhibited, the cell apoptosis rate decreased, phosphatidylserine exposure reduced, the number of crystals adhered to the cell surface reduced, but the ability of cells to endocytose crystals enhanced. The lower the molecular weight, the better the protective effect of PYP. The results in this article indicated that PYPs can reduce the risk of kidney stone formation by protecting renal epithelial cells from oxidative damage and reducing calcium oxalate crystal adhesion, and PYP4 with the lowest molecular weight may be a potential drug for preventing kidney stone formation., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Qian-Long Peng et al.)

Three carboxymethylated Poria cocos polysaccharides (PCP-C1, PCP-C2, and PCP-C3) with -COOH contents of 6.13%, 10.24%, and 16.22%, respectively, were obtained by carboxymethylation of the original polysaccharide (PCP-C0), which has a molecular weight of 4 kDa and a carboxyl (-COOH) content of 2.54%. The structure of the PCP-Cs was characterized by FT-IR, 1 H NMR, and 13 C NMR spectra. The four PCP-Cs exhibited antioxidant activity, and their ability to scavenge radicals (hydroxyl and DPPH) and chelate ferrous ions was positively correlated with the degree of carboxymethylation. As the content of -COOH groups in the PCP-Cs increases, their ability to regulate the growth of calcium oxalate (CaOx) crystals was enhanced, thus inhibiting the growth of calcium oxalate monohydrate (COM) crystals and inducing the formation of more calcium oxalate dihydrate (COD) crystals. The formed CaOx crystal was more round and blunt, the absolute value of the Zeta potential on the crystal surface increased, and the aggregation between crystals was inhibited. Thermogravimetric analysis curves showed that the proportions of PCP-C0, PCP-C1, PCP-C2, and PCP-C3 incorporated into the crystal were 20.52%, 15.60%, 10.65%, and 9.78%, respectively, in the presence of 0.4 g/L PCP-Cs. PCP-C protection resisted oxidative damages of human kidney proximal tubular epithelial cells (HK-2) caused by oxalate, resulting in increased cell viability and superoxide dismutase activity and decreased reactive oxygen species levels, malondialdehyde content, and 8-hydroxy-deoxyguanosine expression. Hence, PCP-Cs, especially PCP-C3, can inhibit the formation of CaOx crystals and may have the potential to be an alternative antistone drug., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Chuang-Ye Li et al.)

An oxidative damage model of human proximal renal epithelial cells (HK-2) was established using oxalate damage. The repair effects of Astragalus polysaccharide (APS) and selenized APS (Se-APS) on damaged HK-2 cells were investigated. Differences in the adhesion and endocytosis of HK-2 cells to calcium oxalate dihydrate crystals with a size of approximately 100 nm before and after APS and Se-APS repair were also explored. The results showed that after being repaired by APS and Se-APS, HK-2 cells exhibited increased cell viability, restored cell morphology, reduced reactive oxygen species level, increased mitochondrial membrane potential, reduced phosphatidylserine eversion, and osteopontin expression. Moreover, the amount of adherent crystals on the cell surface decreased, but the amount of endocytic crystals increased. At the same concentration, Se-APS exhibited better repair effects on the damaged HK-2 cells than APS. All these findings revealed that Se-APS may be a potential drug candidate for inhibiting the formation of kidney stones.

Mineralization crystallization is considered to be the initial stage of stone formation. However, the formation of crystals and subsequent cell damage have rarely been investigated. An oxidatively damaged cell model was established using oxalic acid to injure human proximal tubular epithelial cells (HK-2). Subsequently, CaOx crystallization was induced by adding 2.0 mmol/L sodium oxalate solution. We compared the synergistic effects of PYPs with molecular weights of 49.54 kDa (PYP1) and 4.02 kDa (PYP2) and K 3 Cit on the inhibition of CaOx crystallization and studied the nucleation, growth, and retention process of CaOx crystals on the cell surface and the subsequent damage of the formed crystals to the cells. Normal HK-2 cells mainly induced the formation of CaOx dihydrate (COD), whereas the damaged cells mainly induced the formation of CaOx monohydrate (COM) crystals. Under the protection of PYPs, the state of cells was improved, and the proportion of COD crystals in the formed crystals increased. Small-molecular-weight PYP2 exhibited better abilities of inhibiting CaOx crystallization and improving cell state compared with PYP1. Under the synergistic effects of PYPs and K 3 Cit, the number of formed crystals was obviously reduced, and the size was obviously decreased. PYPs can repair damaged cells and inhibit the conversion of COD phase to COM phase. K 3 Cit can obviously inhibit the nucleation of CaOx crystal and reduce the amount of crystal formation. The repair of damaged cells by PYPs and the synergistic inhibition of CaOx crystallization by PYPs and K 3 Cit reduce cell damage and crystal formation on the cell surface. By simultaneously repairing damaged cells and inhibiting crystallization, this strategy is expected to exert a desirable effect in preventing the formation and recurrence of stones., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.

We studied the protection of degraded Porphyra yezoensis polysaccharide (PYP) on human proximal tubular epithelial cells (HK-2) from cytotoxicity of nano-calcium oxalate monohydrate (COM) crystal, and the regulation of adhesion and endocytosis of the COM crystal. Four degraded fractions, namely, PYP1, PYP2, PYP3, and PYP4, were successfully obtained, with molecular weights (Mws) of 576.2, 49.5, 12.6, and 4.02 kDa, respectively. PYP protection reduced the crystal toxicity, prevented the destruction of cell morphology and cytoskeleton, inhibited the production of reactive oxygen species and the decline of lysosomal integrity, and reduced the expression of osteopontin and transmembrane protein (CD44). PYPi inhibited the adhesion and endocytosis of HK-2 cells by nano-COM. Endocytic COM crystals were accumulated in the lysosomes. With decreasing molecular weight, the ability of PYP to reduce cell damage and inhibit cell adhesion and endocytosis increased. PYP4, which has the smallest molecular weight, weaker intramolecular hydrogen bonds and more reducing groups, showed the best biological activity. PYPi can reduce the oxidative damage of the crystal to the cell, inhibit the adhesion and endocytosis of the crystal, and reduce the risk of kidney stone formation. Therefore, PYP, especially PYP4, has potential for use as a green drug to inhibit the formation and recurrence of calcium oxalate stones.

Background: Hydroxyapatite (HAP) is a common component of most idiopathic calcium oxalate (CaOx) stones and is often used as a nidus to induce the formation of CaOx kidney stones., Methods: This work comparatively studies the cytotoxicity of four kinds of HAP crystals with different sizes (40 nm to 2 μm), namely, HAP-40 nm, HAP-70 nm, HAP-1 μm, and HAP-2 μm, on human renal proximal tubular epithelial cells (HK-2)., Results: HAP crystals reduce the viability and membrane integrity of HK-2 cells in a concentration-dependent manner and consequently cause cytoskeleton damage, cell swelling, increased intracellular reactive oxygen species level, decreased mitochondrial membrane potential, increased intracellular calcium concentration, blocked cell cycle and stagnation in G0/G1 phase, and increased cell necrosis rate. HAP toxicity to HK-2 cells increases with a decrease in crystal size., Conclusion: Cell damage caused by HAP crystals increases the risk of kidney stone formation., Competing Interests: The authors declare that they have no competing interests., (© 2020 Sun et al.)

The regulation on calcium oxalate (CaOx) crystallization and protective effect on human proximal tubular epithelial cells (HK-2) of four green tea polysaccharides (TPSs) with molecular weights of 10.88 (TPS0), 8.16 (TPS1), 4.82 (TPS2), and 2.3 kDa (TPS3) were comparatively studied. XRD, Fourier transform infrared spectroscopy, and scanning electron microscopy results revealed that TPS1, TPS2, and TPS3 can increase the percentage of the dihydrate crystalline phase in CaOx crystals and reduce the size of CaOx monohydrate crystals. TPSs increased the absolute value of the zeta potential of CaOx crystal and inhibited crystal nucleation and aggregation. The nucleation inhibition rates of TPS1, TPS2, and TPS3 to CaOx crystallization were 56.67%, 75.52%, and 52.92%, respectively, and their aggregation inhibition rates were 22.34%, 47.59%, and 21.59%, respectively. TPS preprotection can alleviate the oxidative damage of HK-2 cells caused by oxalate, increase cell viability, protect cell morphology, and reduce lactate dehydrogenase release and reactive oxygen species levels. The degraded TSPs, especially TPS2 with moderate molecular weight, may be used as a green drug to inhibit stone formation., Competing Interests: There are no conflicts of interest to declare., (Copyright © 2020 Hong Liu et al.)

Astragalus polysaccharide (APS) was modified using the Na 2 SeO 3 /HNO 3 method to obtain selenized APS (Se-APS) with a selenium content of 1.75 mg/g. The structure and physicochemical properties of APS and Se-APS were investigated through transmission electron microscopy-energy dispersive spectroscopy mapping, fourier transform infrared spectroscopy, nuclear magnetic resonance, nano-zetasizer analysis, atomic force microscopy, and scanning electron microscopy. APS and Se-APS did not exhibit toxic effects on human kidney proximal tubular epithelial (HK-2) cells and were able to remove hydroxyl and DPPH radicals, alleviate the damage caused by calcium oxalate (CaOx) monohydrate (COM) crystals to HK-2 cells, reduce intracellular reactive oxygen species levels, and restore cell viability and morphology. Both APS and Se-APS could inhibit COM growth, induce calcium oxalate dihydrate formation, and increase the absolute zeta potential of the crystals to inhibit crystal aggregation. However, the ability of Se-APS to regulate CaOx crystals and protect the cells from COM-induced damage was better than that of APS. These results suggested that Se-APS might be a candidate drug for the treatment and prevention of kidney stones., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Endocytosis is a protective mechanism of renal epithelial cells to eliminate retained crystals. This research investigated the endocytosis of 100 nm calcium oxalate monohydrate crystals in human kidney proximal tubular epithelial (HK-2) cells before and after repair by four kinds of tea polysaccharides with molecular weights (MWs) of 10.88 (TPS0), 8.16 (TPS1), 4.82 (TPS2), and 2.31 kDa (TPS3), respectively. When HK-2 cells were repaired by TPSs after oxalic acid injury, the cell viability, wound healing ability, mitochondrial membrane potential, percentage of cells with endocytosed crystals, and dissolution rate of the endocytosed crystals increased; the cell morphology recovered; and the reactive oxygen level and lactate dehydrogenase release decreased. Most of the endocytosed crystals were found in the lysosomes. The repair effects of the four TPSs were ranked in the following order: TPS2>TPS1>TPS3>TPS0. TPS2 with moderate MW presented the optimal repair ability and strongest ability to promote endocytosis., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Chuang-Ye Li et al.)

Vascular calcification (VC) is a common pathological manifestation in patients with cardiovascular diseases, leading to high mortality in patients with chronic kidney diseases. The deposition of hydroxyapatite (HAP) crystals on vascular smooth muscle cells leads to cell damage, which promotes osteogenic transformation. In this study, four different molecular weights (MWs ) of Porphyra yezoensis polysaccharides (PYP1, PYP2, PYP3, and PYP4 with MWs of 576, 49.5, 12.6, and 4.02 kDa, respectively) were used to coat HAP, and the differences in toxicity and calcification of HAP on A7R5 cells before and after coating were studied. The results showed that PYPs could effectively reduce HAP damage to the A7R5 cells. Under the protection of PYPs, cell viability increased and lactate dehydrogenase release, active oxygen level, and cell necrosis rate decreased; also, the amount of the HAP crystals adhering to cell surfaces and entering cells decreased. PYPs with low molecular weights presented better protective effects than high-molecular-weight PYPs. PYPs also inhibited the osteogenic transformation of the A7R5 cells induced by HAP and decreased alkaline phosphatase (ALP) activity and expressions of bone/chondrocyte phenotype genes (runt-related factor 2, ALP, osteopontin, and osteocalcin). In the adenine-induced chronic renal failure (CRF) mouse VC model, PYP4 was found to obviously inhibit the aortic calcium level, and it also inhibited the serum creatinine, serum phosphorus and serum BUN levels. PYP4 (least molecular weight) showed the best inhibitory effect on calcification and may be considered as a candidate drug with therapeutic potential for inhibiting cellular damage and osteoblast differentiation induced by the HAP crystals.

This study simulated the high-phosphorus (Pi) environment in patients with chronic kidney disease. Nano-hydroxyapatite (HAP) crystals were used to damage rat aortic smooth muscle cells (A7R5) pre-damaged with different concentrations of Pi solution to compare the differences in HAP-induced calcification in A7R5 cells before and after injury by high-Pi condition. After the A7R5 cells were damaged by high-Pi environment, the following were observed. HAP resulted in declined cell viability and lysosomal integrity, release of lactate dehydrogenase, and increased reactive oxygen species production. The ability of high-Pi damaged cells to internalize HAP crystals declined; crystal adhesion and calcium deposition on the cell surface and alkaline phosphatase activities increased. Osteopontin expression and level of Runt-related transcription factor 2 were increased, and HAP-induced osteogenic transformation was enhanced. High-Pi condition promoted the adhesion of A7R5 cells to nano-HAP crystals and inhibited HAP endocytosis, increasing the risk of calcification., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Corn silk polysaccharide (CSP0; molecular weight = 124 kDa) was degraded by ultrasonication to obtain five degraded polysaccharides, namely, CSP1, CSP2, CSP3, CSP4, and CSP5, with molecular weights of 26.1, 12.2, 6.0, 3.5, and 2.0 kDa, respectively. The structures of these polysaccharides were characterized by FT-IR, 1 H NMR, and 13 C NMR analyses. The antioxidant activities, including scavenging ability for hydroxyl radicals and DPPH free radicals, chelation ability for Fe 2+ ions, and reducing ability of CSP increased with decreased molecular weight of CSPs within 6.0 to 124 kDa. However, antioxidant activity weakened when the molecular weight of CSPs reached 3.5 and 2 kDa. CSP3 with a molecular weight of 6.0 kDa exhibited the strongest antioxidant activity. After protection with 60  μ g/mL CSPs, the viability of human renal proximal tubular epithelial cells (HK-2) damaged by nano-COM crystals increased, the level of reactive oxygen species decreased, and the amount of COM crystal adhered onto the cell surface decreased. The ability of CSPs to protect cells from CaOx crystal damage was consistent with their antioxidant activity. CSPs can specifically combine with CaOx crystal to inhibit the conversion of calcium oxalate dihydrate crystal to calcium oxalate monohydrate crystal. All these results showed that the activity of CSPs was closely correlated with molecular weight. A very high or low molecular weight of CSPs was not conducive to their activity. CSPs, especially CSP3 with a molecular weight of 6.0 kDa, can be used as a potential antistone drug., Competing Interests: There are no conflicts of interest to declare., (Copyright © 2020 Jia-Yun Chen et al.)

Crystal adhesion is an important link in the formation of kidney stones. This study investigated and compared the adhesion differences between nano-calcium oxalate monohydrate (COM) and human renal proximal tubule epithelial (HK-2) cells before and after treatment with tea polysaccharides (TPSs) TPS0, TPS1, TPS2, and TPS3 with molecular weights of 10.88, 8.16, 4.82, and 2.31 kDa, respectively. TPS treatment effectively reduced the damage of COM to HK-2 cells, thereby resulting in increased cell activity, decreased release of lactate dehydrogenase, cell morphology recovery, decreased level of reactive oxygen species, increased mitochondrial membrane potential, increased lysosomal integrity, decreased expression of adhesion molecule osteopontin and eversion of phosphatidylserine, and decreased crystal adhesion. Among the TPSs, TPS2 with moderate molecular weight had the best protective effect on cells and the strongest effect on the inhibition of crystal adhesion. Thus, TPS2 may be a potential anticalculus drug., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Yao-Wang Zhao et al.)

Purpose : To determine the role of UCH-L1 in regulating ERα expression, and to evaluate whether therapeutic targeting of UCH-L1 can enhance the efficacy of anti-estrogen therapy against breast cancer with loss or reduction of ERα. Methods : Expressions of UCH-L1 and ERα were examined in breast cancer cells and patient specimens. The associations between UCH-L1 and ERα, therapeutic response and prognosis in breast cancer patients were analyzed using multiple databases. The molecular pathways by which UCH-L1 regulates ERα were analyzed using immunoblotting, qRT-PCR, immunoprecipitation, ubiquitination, luciferase and ChIP assays. The effects of UCH-L1 inhibition on the efficacy of tamoxifen in ERα (-) breast cancer cells were tested both in vivo and in vitro . Results : UCH-L1 expression was conversely correlated with ERα status in breast cancer, and the negative regulatory effect of UCH-L1 on ERα was mediated by the deubiquitinase-mediated stability of EGFR, which suppresses ERα transcription. High expression of UCH-L1 was associated with poor therapeutic response and prognosis in patients with breast cancer. Up-regulation of ERα caused by UCH-L1 inhibition could significantly enhance the efficacy of tamoxifen and fulvestrant in ERα (-) breast cancer both in vivo and in vitro . Conclusions : Our results reveal an important role of UCH-L1 in modulating ERα status and demonstrate the involvement of UCH-L1-EGFR signaling pathway, suggesting that UCH-L1 may serve as a novel adjuvant target for treatment of hormone therapy-insensitive breast cancers. Targeting UCH-L1 to sensitize ER negative breast cancer to anti-estrogen therapy might represent a new therapeutic strategy that warrants further exploration., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Vascular smooth muscle cell damage is a key step in inducing vascular calcification that yields hydroxyapatite (HAP) as a major product. The effect of the shape of HAP on the damage to vascular smooth muscle cells has yet to be investigated. In this study, we compared the differences in toxicity of four various morphological nano-HAP crystals, namely, H-Rod, H-Needle, H-Sphere, and H-Plate, in rat aortic smooth muscle cells (A7R5). The sizes of these crystals were 39 nm × 115 nm, 41 nm ×189 nm, 56 nm × 56 nm, and 91 nm × 192 nm, respectively. Results showed that all HAPs decreased cell viability, disorganized cell morphology, disrupted cell membranes, increased intracellular reactive oxygen species concentration, decreased mitochondrial membrane potential, decreased lysosome integrity, increased alkaline phosphatase activity, and increased intracellular calcium concentration, resulting in cell necrosis. The cytotoxicity of the four kinds of HAP was ranked as follows: H-Plate > H-Sphere > H-Needle > H-Rod. The cytotoxicity of each crystal was positively correlated with the following factors: large specific surface area, high electrical conductivity and low surface charge. HAP accelerated calcium deposits on the A7R5 cell surface and induced the expression of osteogenic proteins, such as BMP-2, Runx2, OCN, and ALP. The crystals with high cytotoxicity caused more calcium deposits on the cell surface, higher expression levels of osteogenic protein, and stronger osteogenic transformation abilities. These findings elucidated the relationship between crystal shape and cytotoxicity and provided theoretical references for decreasing the risks of vascular calcification.

This work investigated the effects of repairing injured renal proximal tubular epithelial (HK-2) cells by using three Astragalus polysaccharides (APS) with different molecular weights and the adhesion and endocytosis of HK-2 cells to the calcium oxalate dihydrate (COD) nanocrystals before and after repair to develop new products that can protect against kidney stones. HK-2 cells cultured in vitro were injured with 2.6 mmol/L oxalic acid to establish a damaged cell model. Three kinds of APS (APS0, APS1, and APS2 with molecular weights of 11.03, 4.72, and 2.60 kDa, respectively) were used to repair the damaged cells. The changes in the adhesion and endocytosis of 100 nm COD crystals to cells before and after the repair were detected. After the repair of HK-2 cells by the APS, the speed of wound healing of the damaged HK-2 cells increased, and the amount of phosphatidylserine (PS) ectropion decreased. In addition, the proportion of cells with adhered COD crystals decreased, whereas the proportion of cells with internalized crystals increased. As a result of the repair activity, APS can inhibit the adhesion and promote the endocytosis of COD nanocrystals to damaged cells. APS1, which had a moderate molecular weight, displayed the strongest abilities to repair the cells, inhibit adhesion, and promote endocytosis. Thus, APS, particularly APS1, may serve as potential green drugs for preventing kidney stones., (© 2019 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Group.)

Hydroxyapatite (HAP) is not only a common component of most idiopathic CaOx stones, but also the core of Randall's plaque. HAP is a nest that can induce the formation of Randall's plaques and even kidney stones. We studied the toxic effects and mechanisms of four different types of nano-HAP crystals (H-Sphere, 72.5 nm × 72.5 nm; H-Needle, 37.2 nm × 162.7 nm; H-Rod, 42.3 nm × 115.3 nm; and H-Plate, 145.5 nm × 272.9 nm) on human renal proximal tubular epithelial cells (HK-2). HAP crystals could cause oxidative stress that triggered a series of cell dysfunction problems, resulting in decreased cell viability, loss of cell membrane integrity, cell swelling, and cell necrosis. The toxic effect of HAP was mainly attributed to its entry into cell by endocytosis and its accumulation in the lysosomes, causing the level of intracellular reactive oxygen species (ROS) to rise, the mitochondrial membrane potential (Δψm) to decrease, the lysosomal integrity to be destroyed, and the cell cycle blocked during the G0/G1 phase. The cytotoxicity of the four kinds of HAP crystals was ranked as follows: H-Sphere > H-Needle > H-Rod > H-Plate. The cytotoxicity of each crystal was positively correlated with low absolute zeta potential, conduciveness to internalized morphology, large specific surface area and aspect ratio, and small particle size. These results indicated that nano-HAP could damage HK-2 cells, and the physical properties of HAP crystals play a vital effect in their cytotoxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The adhesion and endocytosis of renal epithelial cells to urinary microcrystals are closely related to kidney stone formation; however, the mechanism of cell state changes that affect adhesion and endocytosis remains unclear. In this study, a damaged cell model was established using oxalate to impair human kidney proximal tubular epithelial cells (HK-2). Then, we used four degraded Porphyra yezoensis polysaccharides (PYPs), namely, PYP1, PYP2, PYP3, and PYP4, with molecular weight of 576.2, 49.5, 12.6, and 4.02 kDa, respectively, to repair the damaged cells. After repairing the damaged HK-2 cells by PYPs, the cell morphology gradually recovered to near normal; the cell migration speed increased; the phosphatidylserine eversion ratio and osteopontin expression decreased; and the intracellular adenosine triphosphate was promoted. The adhesion and endocytosis of calcium oxalate monohydrate (COM) crystals were closely related to cell state. Normal cells could endocytose crystals more than the damaged cells, whereas damaged cells could adhere to crystals more than the normal cells. The cells repaired by PYPs inhibited the adhesion of nano-COM crystals and promoted the endocytosis of the adherent crystals, thereby reducing the adhesion of crystals on the cell surface under both effects. As the molecular weight of PYP decreased, the ability of PYP to repair cells, inhibit crystal adhesion, and promote endocytosis of cells was enhanced; that is, PYP4, which had the lowest molecular weight, exhibited the best biological activity. Therefore, PYPs, especially PYP4, may become an optional green drug to inhibit the formation and recurrence of calcium oxalate stones.

Renal epithelial cell injury is a key step in inducing kidney stone formation. However, research on the role of cell repair in the prevention and treatment of kidney stones is limited. In this study, the repair effect of degraded Porphyra yezoensis polysaccharide (PYP) on oxidative stress-mediated intracellular damage triggered by oxalate in human kidney proximal tubular epithelial (HK-2) cells was investigated, and the influence of molecular weight (Mw) on the repair ability of PYP was elucidated. Polysaccharides with different Mws were prepared by degrading PYP with hydrogen peroxide. Four degraded fractions, namely, PYP1, PYP2, PYP3, and PYP4, were successfully obtained with Mws of 576.2, 49.54, 12.65, and 4.020 kDa, respectively. A damaged cell model was established using 2.6 mmol L-1 oxalate to injure HK-2 cells. Various Mws of PYPs were used to repair the damaged cells. The repair mechanism of PYPs against oxalate-induced oxidative stress was examined by evaluating cell proliferation and physiological function recovery. Our study revealed that PYPs increased the viability of oxalate-injured HK-2 cells and restored their morphological characteristics and cytoskeleton. PYPs reduced the levels of oxalate-mediated lactase dehydrogenase release, reactive oxygen species generation, and intracellular Ca2+, the loss of mitochondrial membrane potential, the number of cells arrested in S phase, the expression of 8-hydroxy-desoxyguanosine and poly ADP ribose polymerase, lysosomal damage, and the number of apoptotic cells. The PYP fraction with low Mw presented an increased repair activity against cellular damage induced by oxalate. The resistance of the repaired renal cells to crystal adhesion and aggregation was stronger than that of the damaged cells. PYPs might inhibit the formation of kidney stones by repairing damaged cells and inhibiting crystal adhesion and aggregation. We concluded that PYP with low Mw could be used as a potential therapeutic agent against renal stone formation and recurrence.

This study investigated the repair effects of three Astragalus polysaccharides (APSs) with different molecular weights (Mws) on injured human renal proximal tubular epithelial (HK-2) cells to reveal the effect of Mw of polysaccharide on cell repair. A damage model was established by injuring HK-2 cells with 2.6 mM oxalate, and APS0, APS1, and APS2 with Mw of 11.03, 4.72, and 2.61 KDa were used to repair the damaged cells. After repair by APSs, the morphology of damaged HK-2 cells gradually returned to normal, the destruction of intercellular junctions recovered, intracellular reactive oxygen species production amount decreased, and their mitochondrial membrane potential increased. In addition, the cell cycle progression gradually normalized, lysosome integrity increased, and cell apoptotic rates obviously declined in the repaired cells. All three APSs could promote the expression of Keap1, Nrf2, SOD1, and CAT. In addition, the expression levels of inflammation markers containing MCP-1 and IL-6 decreased after APS repair. We deduced that APSs exert their repair function by activating the Nrf2-Keap1 signaling pathway and inhibiting inflammation. Among the APSs, APS1 with a moderate Mw provided the strongest repair effect. APSs may have a preventive effect on kidney stones.

Background: Hypomagnesemia was identified as a strong risk factor for cardiovascular disease in patients with chronic renal failure (CRF). However, the effects of magnesium (Mg) on vascular calcification (VC) have not been fully elucidated. Thus, we aim to determine the effects of Mg citrate (MgCit) on VC in CRF rats., Methods: Rats were divided into 5 groups: group 1 (normal diet), group 2 (normal diet with MgCit), group 3 (the VC model of CRF induced by 0.75% adenine and 0.9% phosphorus diet from day 1 to day 28), group 4 (group 3 treated with low-dose MgCit from day 1 to day 42), and group 5 (same as group 3 except the high-dose MgCit). All rats were killed at day 43 with collection of blood and aortas. Then, serum biochemical parameters, VC-related staining, calcium and P contents, alkaline phosphatase contents and activity, expression of alpha smooth muscle actin, and runt-related transcription factor 2 (RUNX2) in aortas were assessed., Results: Group 3 had extensive VC. The VC degree decreased in groups 4 and 5 in a dose-depended manner with reduced calcium content, P levels, alkaline phosphatase content and activity, and protein levels of RUNX2 and increased protein levels of alpha smooth muscle actin in aortas., Conclusions: MgCit exerted a protective role in VC in adenine-induced CRF rats; thus, it may be a potential drug for the prevention of VC in patients with CRF.

BaGd 2 O 4 :Eu 3+ scintillating phosphors by Pr 3+ -codoping were synthesized at 1300°C in air using a solid-state reaction method. The as-synthesized phosphors were characterized by X-ray diffraction (XRD), photoluminescence (PL) including excitation and emission spectra, radioluminescence (RL) spectra excited by X-ray and thermoluminescence (TL) spectra. Both the PL and RL spectra are composed of the featured trivalent europium (Eu 3+ ) without any praseodymium (Pr 3+ ) ions, and the PL and RL intensities as well as the lifetimes of BaGd 2 O 4 :Eu 3+ scintillating phosphors decrease dramatically with an increasing concentration of Pr 3+ ions. Finally, the TL spectra reveal the trap concentration of the existing defects decrease with an increasing concentration of Pr 3+ ions, while the relative TL intensity ratio of the high temperature band to the low temperature one increases with an increasing concentration of Pr 3+ ions, which results in the afterglow suppression of BaGd 2 O 4 :Eu 3+ scintillating phosphors., (© 2018 John Wiley & Sons, Ltd.)

This study aims at investigating the antioxidant activity and repair effect of green tea polysaccharide (TPS) with different molecular weights (Mw) on damaged human kidney proximal tubular epithelial cells (HK-2). Scavenging activities on hydroxyl radical ( · OH) and ABTS radical and reducing power of four kinds of TPS with Mw of 10.88 (TPS0), 8.16 (TPS1), 4.82 (TPS2), and 2.31 kDa (TPS3) were detected. A damaged cell model was established using 2.6 mmol/L oxalate to injure HK-2 cells. Then, different concentrations of TPSs were used to repair the damaged cells. Index changes of subcellular organelles of HK-2 cells were detected before and after repair. The four kinds of TPSs possessed radical scavenging activity and reducing power, wherein TPS2 with moderate Mw presented the strongest antioxidant activity. After repair by TPSs, cell morphology of damaged HK-2 cells was gradually restored to normal conditions. Reactive oxygen species production decreased, and mitochondrial membrane potential (Δ ψ m) of repaired cells increased. Cells of G1 phase arrest were inhibited, and cell proportion in the S phase increased. Lysosome integrity improved, and cell apoptotic rates significantly reduced in the repaired group. The four kinds of TPSs with varying Mw displayed antioxidant activity and repair effect on the mitochondria, lysosomes, and intracellular DNA. TPS2, with moderate Mw, showed the strongest antioxidant activity and repair effect; it may become a potential drug for prevention and treatment of kidney stones.

Natural Gracilaria lemaneiformis sulfated polysaccharide (GLP0, molecular weight = 622 kDa) was degraded by H 2 O 2 to obtain seven degraded fragments, namely, GLP1, GLP2, GLP3, GLP4, GLP5, GLP6, and GLP7, with molecular weights of 106, 49.6, 10.5, 6.14, 5.06, 3.71, and 2.42 kDa, respectively. FT-IR and NMR results indicated that H 2 O 2 degradation does not change the structure of GLP polysaccharides, whereas the content of the characteristic -OSO 3 H group (13.46% ± 0.10%) slightly increased than that of the natural polysaccharide (13.07%) after degradation. The repair effects of the polysaccharide fractions on oxalate-induced damaged human kidney proximal tubular epithelial cells (HK-2) were compared. When 60  μ g/mL of each polysaccharide was used to repair the damaged HK-2 cells, cell viability increased and the cell morphology was restored, as determined by HE staining. The amount of lactate dehydrogenase released decreased from 16.64% in the injured group to 7.55%-13.87% in the repair groups. The SOD activity increased, and the amount of MDA released decreased. Moreover, the mitochondrial membrane potential evidently increased. All polysaccharide fractions inhibited S phase arrest through the decreased percentage of cells in the S phase and the increased percentage of cells in the G2/M phase. These results reveal that all GLP fractions exhibited repair effect on oxalate-induced damaged HK-2 cells. The repair ability is closely correlated with the molecular weight of the fractions. GLP2 with molecular weight of about 49.6 kDa exhibited the strongest repair effect, and GLP with higher or lower molecular weight than 49.6 kDa showed decreased repair ability. Our results can provide references for inhibiting the formation of kidney stones and developing original anti-stone polysaccharide drugs.

Objective: This study aimed to investigate the differences and inhibitory effects of diethyl citrate (Et2Cit), sodium citrate (Na3Cit), and phosphonoformic acid (PFA) on calcification induced by high inorganic phosphate (Pi) contents in mouse aortic smooth muscle cells (MOVAS) and to develop drugs that can induce anticoagulation and inhibit vascular calcification (VC)., Methods: Alive and fixed MOVAS were assessed for 14 days in the presence of high Pi with increasing Et2Cit, Na3Cit, and PFA concentrations. Calcification on MOVAS was measured through Alizarin red staining and the deposited calcium amount; apoptosis was detected by annexin V staining; and cell transdifferentiation was examined by measuring smooth muscle lineage gene (α-SMA) expression and alkaline phosphatase activity., Results: Coincubation of MOVAS with Et2Cit, Na3Cit, and PFA significantly decreased Pi-induced VC in live MOVAS, and the apoptotic rate was reduced by low inhibitor concentrations. The 3 inhibitors could prevent the alkaline phosphatase activity induced by high Pi contents and increased the expression of α-smooth muscle actin genes. Thus, the transdifferentiation of MOVAS into osteoblast-like cells was blocked. Their inhibitory effects exhibited concentration dependence. The inhibitory effect of each inhibitor at the same concentration showed the following trend: PFA > Na3Cit > Et2Cit., Conclusions: Et2Cit, Na3Cit, and PFA prevented the calcification of MOVAS and inhibited the osteochondrocytic conversion of vascular smooth muscle cells. Thus, Et2Cit and Na3Cit as anticoagulants may alleviate VC in clinical applications.

Objective: This study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (Et 2 Cit) and sodium citrate (Na 3 Cit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification., Methods: The change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively., Results: Nano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca 2+ concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et 2 Cit or Na 3 Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et 2 Cit and Na 3 Cit could also chelate with Ca + to inhibit the intracellular Ca 2+ elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et 2 Cit and Na 3 Cit exhibited strong inhibitory effects. The inhibitory capacity of Na 3 Cit was stronger than that of Et 2 Cit at similar concentrations., Conclusion: Both Et 2 Cit and Na 3 Cit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. Et 2 Cit and Na 3 Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Comparative studies were conducted to investigate the time effect of cell injury induced by nano-sized (50nm) and micron-sized (10μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The effects of nano-/micron-sized COM and COD exposure on Vero cells were investigated by detecting the cell viability, cell morphology, LDH release, reactive oxygen species, mitochondrial membrane potential, cell cycle, and cell apoptosis, as well as the intracellular and extracellular crystal distribution. Nano-/micron-sized COM and COD exposure lead to subcellular organelle injury in varying degrees, but the injury sequence of various organelles differed. The time sequence of organelle injury presenting significant variation was described as follows: cell membrane injury (1h)

Renal epithelial cell injury is a key step in inducing kidney stone formation. This injury induced by crystallites with different shapes and aggregation states has been receiving minimal research attention. To compare the shape and aggregation effects of calcium oxalate crystals on their toxicity, we prepared calcium oxalate monohydrate (COM) crystals with the morphology of a hexagonal lozenge, a thin hexagonal lozenge, and their corresponding aggregates. We then compared their toxicities toward human kidney proximal tubular epithelial (HK-2) cells. All four shapes of COM crystals caused cell-membrane rupture, upregulated intracellular reactive oxygen, and decreased mitochondrial membrane potential. This series of phenomena ultimately led to necrotic cell death. The overall damage in cells was determined in terms of both exterior and interior damage. Crystals with a large Ca 2+ ion-rich (1̅01) active face showed the greatest toxicity in HK-2 cells and the largest extent of adhesion onto the cell surface. Crystals with sharp edges easily caused cell-membrane ruptures. The aggregation of sharp crystals aggravated cell injury, whereas the aggregation of blunt crystals weakened cell injury. Therefore, crystal shapes and aggregation states were important factors that affected crystal toxicity in renal epithelial cells. All of these findings elucidated the relationship between the physical properties of crystals and cytotoxicity and provided theoretical references for inhibiting stone formation., Competing Interests: The authors declare no competing financial interest.

N25, a novel histone deacetylase inhibitor, was created through structural modification of suberoylanilide hydroxamic acid. To evaluate the anti-tumor activity of N25 and clarify its molecular mechanism of inducing autophagy in glioma cells, we investigated its in vitro anti-proliferative effect and in vivo anticancer effect. Moreover, we detected whether N25 induces autophagy in glioma cells by transmission electron microscope and analyzed the protein expression level of HDAC3, Tip60, LC3 in glioma samples by western blot. We additionally analyzed the protein expression level of HDAC3, Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment with N25 in glioma cells. Our results showed that the anti-tumor activity of N25 in glioma cells is slightly stronger than SAHA both i n vitro and in vivo . We found that N25 induced autophagy, and HDAC3 was significantly elevated and Tip60 and LC3 significantly decreased in glioma samples compared with normal brain tissues. Nevertheless, N25 inhibited HDAC3 and up-regulated the protein expression of Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment of glioma cells with N25. In conclusion, these data suggest that N25 has striking anti-tumor activity in part due to inhibition of HDAC3. Additionally, N25 may induce autophagy through inhibiting HDAC3.

Renal epithelial cell injury causes crystal retention and leads to renal stone formation. However, the effects of crystal shape on cell injury and stone risk remain unclear. This study compared the cytotoxicity degrees of calcium oxalate dihydrate (COD) crystals having different shapes toward human kidney proximal tubular epithelial (HK-2) cells to reveal the effect of crystal shape on cell injury and to elucidate the pathological mechanism of calcium oxalate kidney stones. The effects of exposure to cross-shaped (COD-CS), flower-like (COD-FL), bipyramid (COD-BD), and elongated-bipyramid (COD-EBD) COD crystals on HK-2 cells were investigated by examining the cell viability, cell membrane integrity, cell morphology change, intracellular reactive oxygen species, mitochondrial membrane potential (Δψm), and apoptotic and/or necrotic rate. Crystals with large (100) faces (COD-EBD) and sharp edges (COD-CS) showed higher toxicity than COD-BD and COD-FL, respectively. COD crystal exposure caused cell membrane rupture, upregulated intracellular reactive oxygen, and decreased Δψm. This series of phenomena ultimately led to a high apoptotic rate and a low necrotic rate. Crystals with large active faces have a large contact area with epithelial cell surface, and crystals with sharp edges can easily scratch epithelial cells; these factors could promote crystal adhesion and aggregation, thus increasing stone risk.

Urinary crystals with various sizes are present in healthy individuals and patients with kidney stone; however, the cellular uptake mechanism of calcium oxalate of various sizes has not been elucidated. This study aims to compare the internalization of nano-/micron-sized (50 nm, 100 nm, and 1 μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The internalization and adhesion of COM and COD crystals to Vero cells were enhanced with decreasing crystal size. Cell death rate was positively related to the amount of adhered and internalized crystals and exhibited higher correlation with internalization than that with adhesion. Vero cells mainly internalized nano-sized COM and COD crystals through clathrin-mediated pathways as well as micron-sized crystals through macropinocytosis. The internalized COM and COD crystals were distributed in the lysosomes and destroyed lysosomal integrity to some extent. The results of this study indicated that the size of crystal affected cellular uptake mechanism, and may provide an enlightenment for finding potential inhibitors of crystal uptake, thereby decreasing cell injury and the occurrence of kidney stones., Competing Interests: The authors declare no competing financial interests.

Self-activated phosphors are capable of generating optical emissions from the internal ion groups of host lattice before externally introducing luminescent ions. However, numerous self-activated phosphors only show luminescence at low temperature due to the thermally activated energy migration among ion groups at room temperature, severely confining their application conditions. In this letter, we propose a strategy to converting the low-temperature luminescence to a room-temperature one through changing the synthesis conditions to induce structural distortions and thus to limit energy migration. Room-temperature self-activated luminescence of Ca 2 Nb 2 O 7 was accordingly achieved in submicroplates synthesized using the sol-gel method. By further coupling the blue broadband emission from Ca 2 Nb 2 O 7 submicroplates with the characteristic luminescence of Ln 3+ (Pr 3+ , Sm 3+ , and Dy 3+ ) dopants, multicolor emissions were successively tuned through adjusting the concentration of Ln 3+ . Our results are expected to expand the scope of designing room-temperature self-activated phosphors and tuning multicolor emission., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study aims to investigate the repair effect of subcellular structure injuries of the HK-2 cells of four degraded seaweed polysaccharides (DSPs), namely, the degraded Porphyra yezoensis , Gracilaria lemaneiformis , Sargassum fusiform , and Undaria pinnatifida polysaccharides. The four DSPs have similar molecular weight, but with different content of sulfate groups (i.e., 17.9%, 13.3%, 8.2%, and 5.5%, resp.). The damaged model was established using 2.8 mmol/L oxalate to injure HK-2 cells, and 60  μ g/mL of various DSPs was used to repair the damaged cells. With the increase of sulfate group content in DSPs, the scavenging activity of radicals and their reducing power were all improved. Four kinds of DSPs have repair effect on the subcellular organelles of damaged HK-2 cells. After being repaired by DSPs, the release amount of lactate dehydrogenase was decreased, the integrity of cell membrane and lysosome increased, the Δ ψ m increased, the cell of G1 phase arrest was inhibited, the proportion of S phase increased, and cell apoptotic and necrosis rates were significantly reduced. The greater the content of sulfate group is, the stronger is the repair ability of the polysaccharide. These DSPs, particularly the polysaccharide with higher sulfate group content, may be a potential drug for the prevention and cure of kidney stones.

Urinary crystals in normal and kidney stone patients often differ in crystal sizes and surface structures, but the effects of different crystal properties on renal tubular epithelial cells remain unclear. This study aimed to compare the cytotoxicity of micron/nano-calcium oxalate monohydrate (COM) crystals with sizes of 50 nm, 200 nm, 1 μm, 3 μm, and 10 μm to African green monkey renal epithelial (Vero) cells, to reveal the effect of crystal size and surface structure on cell injury, and to investigate the pathological mechanism of calcium oxalate kidney stones. Cell viability, cellular biochemical parameters, and internalized crystal amount in Vero cells were closely associated with the size of COM crystals. At the same concentration (200 μg/mL), COM-1 μm induced the most serious injury to Vero cells and caused the most significant change to cellular biochemical parameters, which were related to the specific porous structure and highest internalized amount in Vero cells. By contrast, COM-50 nm and COM-200 nm crystals lost their small size effect because of serious aggregation and weakened their toxicity to cells. COM-3 μm and COM-10 μm crystals were too large for cells to completely internalize; these crystals also exhibited a low specific surface area and thus weakened their toxicity. The excessive expression of intracellular ROS and reduction of the free-radical scavenger SOD were the main reasons for cell injury and eventually caused necrotic cell death. Crystal size, surface structure, aggregation, and internalization amount were closely related to the cytotoxicity of COM crystals.

This study aimed to investigate the protective effects of degraded soybean polysaccharides (DSP) on oxidatively damaged African green monkey kidney epithelial (Vero) cells. Low DSP concentration (10 μg/mL) elicited an evident protective effect on H 2 O 2 -induced cell injury (0.3 mmol/L). The cell viabilities of the H 2 O 2 -treated group and the DSP-protected group were 57.3 and 93.1%, respectively. The cell viability decreased to 88.3% when the dosage was increased to 100 μg/mL. DSP protected Vero cells from H 2 O 2 -mediated oxidative damage by enhancing cellular superoxide dismutase activity and total antioxidant capacity and by decreasing malonaldehyde content and lactate dehydrogenase release. The H 2 O 2 -treated cells stimulated the aggregation of calcium oxalate monohydrate crystals. DSP could also reduce the crystal size, decrease the attached crystal content, and prevent the cell aggregation by alleviating oxidative injury and lipid peroxidation, enhancing antioxidant capacity, and decreasing hyaluronan expression on cellular surfaces. The internalization ability of the injured cells was improved after these cells were exposed to DSP solution. The regulation ability of DSP-repaired cells on calcium oxalate dihydrate formation, crystal attachment, aggregation, and internalization was lower than that of normal cells but was higher than that of the injured cells. DSP may be a potential green drug to prevent calcium oxalate (CaOx) stone formation because DSP could protect cells from oxidative damage and inhibit CaOx crystal formation.

Background: Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear., Methods: African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin-eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry., Results: The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased., Conclusion: Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone formation.