Cyanidin-3-glucoside inhibits ferroptosis in renal tubular cells after ischemia/reperfusion injury via the AMPK pathway.

BACKGROUND: Ferroptosis, which is characterized by lipid peroxidation and iron accumulation, is closely associated with the pathogenesis of acute renal injury (AKI). Cyanidin-3-glucoside (C3G), a typical flavonoid that has anti-inflammatory and antioxidant effects on ischemia‒reperfusion (I/R) injury, can induce AMP-activated protein kinase (AMPK) activation. This study aimed to show that C3G exerts nephroprotective effects against I/R-AKI related ferroptosis by regulating the AMPK pathway. METHODS: Hypoxia/reoxygenation (H/R)-induced HK-2 cells and I/R-AKI mice were treated with C3G with or without inhibiting AMPK. The level of intracellular free iron, the expression of the ferroptosis-related proteins acyl-CoA synthetase long chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4), and the levels of the lipid peroxidation markers 4-hydroxynonenal (4-HNE), lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were examined. RESULTS: We observed the inhibitory effect of C3G on ferroptosis in vitro and in vivo, which was characterized by the reversion of excessive intracellular free iron accumulation, a decrease in 4-HNE, lipid ROS, MDA levels and ACSL4 expression, and an increase in GPX4 expression and glutathione (GSH) levels. Notably, the inhibition of AMPK by CC significantly abrogated the nephroprotective effect of C3G on I/R-AKI models in vivo and in vitro. CONCLUSION: Our results provide new insight into the nephroprotective effect of C3G on acute I/R-AKI by inhibiting ferroptosis by activating the AMPK pathway.

Inhibition of Xanthine Oxidase Protects against Sepsis-Induced Acute Kidney Injury by Ameliorating Renal Hypoxia.

Xanthine oxidase (XO) utilizes molecular oxygen as a substrate to convert purine substrates into uric acid, superoxide, and hydrogen peroxide, which is one of the main enzyme pathways to produce reactive oxygen species (ROS) during septic inflammation and oxidative stress. However, it is not clear whether XO inhibition can improve sepsis-induced renal hypoxia in sepsis-induced acute kidney injury (SI-AKI) mice. In this study, pretreatment with febuxostat, an XO-specific inhibitor, or kidney knockdown of XO by shRNA in vivo significantly improved the prognosis of SI-AKI, not only by reducing the levels of blood urea nitrogen, serum creatinine, tumor necrosis factor-α, interleukin-6, and interleukin-1ß in peripheral blood but also by improving histological damage and apoptosis, reducing the production of ROS, and infiltrating neutrophils and macrophages in the kidney. More importantly, we found that pharmacological and genetic inhibition of XO significantly improved renal hypoxia in SI-AKI mice by a hypoxia probe via fluorescence staining. This effect was further confirmed by the decrease in hypoxia-inducible factor-1α expression in the kidneys of mice with pharmacological and genetic inhibition of XO. In vitro, the change in XO activity induced by lipopolysaccharide was related to the change in hypoxia in HK-2 cells. Febuxostat and XO siRNA significantly relieved the hypoxia of HK-2 cells cultured in 2% oxygen and reversed the decrease in cell viability induced by lipopolysaccharide. Our results provide novel insights into the nephroprotection of XO inhibition in SI-AKI, improving cell hypoxia by inhibiting XO activity and reducing apoptosis, inflammation, and oxidative stress.

Polydatin Attenuates Cisplatin-Induced Acute Kidney Injury by Inhibiting Ferroptosis.

Cisplatin is widely used in the treatment of solid tumors, but its application is greatly limited due to its nephrotoxicity; thus, there is still no effective medicine for the treatment of cisplatin-induced acute kidney injury (Cis-AKI). We previously identified that polydatin (PD) exerts nephroprotective effects by antioxidative stress in AKI models. Recent evidence suggests that oxidative stress-induced molecular events overlap with the process of ferroptosis and that there are common molecular targets, such as glutathione (GSH) depletion and lipid peroxidation. Nevertheless, whether the nephroprotective effect of PD is related to anti-ferroptosis remains unclear. In this study, the inhibitory effect of PD on ferroptosis was observed in both cisplatin-treated HK-2 cells (20 µM) in vitro and a Cis-AKI mouse model (20 mg/kg, intraperitoneally) in vivo, characterized by the reversion of excessive intracellular free iron accumulation and reactive oxygen species (ROS) generation, a decrease in malondialdehyde (MDA) content and GSH depletion, and an increase in glutathione peroxidase-4 (GPx4) activity. Remarkably, PD dose-dependently alleviated cell death induced by the system Xc- inhibitor erastin (10 µM), and the effect of the 40 µM dose of PD was more obvious than that of ferrostatin-1 (1 µM) and deferoxamine (DFO, 100 µM), classical ferroptosis inhibitors. Our results provide insight into nephroprotection with PD in Cis-AKI by inhibiting ferroptosis via maintenance of the system Xc--GSH-GPx4 axis and iron metabolism.

Effects of landfill refuse on the reductive dechlorination of pentachlorophenol and speciation transformation of heavy metals.

Landfill refuse is a mixture of inorganic minerals and organic matter that is capable of undergoing complexation and redox reactions due to its active functional groups. Organic matter often combines with minerals in landfill refuse and it remains unclear whether this combination involves electron transfer. Therefore, the effects of landfill refuse composition on reductive dechlorination and speciation transformation of heavy metals were investigated in this study. Results show that landfill refuse comprises protein- and humic-like substances, aliphatic structures, and a large number of hydroxyl, carboxyl, quinoid and other active functional group. The electron donating capacity (0.09-0.26 µmol/g(C)) of landfill refuse was found to be higher than its electron accepting capacity (0.03-0.23 µmol/g(C)), indicating that electron donating groups (hydroxyl) were the main redox-active moieties, facilitating the reductive dechlorination of pentachlorophenol (PCP) by microorganism. Fe2O3, FeO and SiO2 were the main inorganic minerals affecting PCP dechlorination. The speciation distribution of heavy metals in landfill refuse was determined by the BCR sequential extraction method. Results showed that Zn and Ni have high potential migration capacity, poor stability and the highest bioavailability, while Cr, Cu and Pb are relatively stable and have weak migration potential. The oxygen- and nitrogen-containing functional groups, aliphatic structures and aromatic carbon in landfill refuse can promote the transformation of Ni and Cr from an unstable to stable state. Protein-like substances exhibit a strong Cu binding ability, allowing Cu to combine with organic matter more easily than other assessed heavy metals. Both Fe2O3 and FeO affected the stability of Cu. FeO promoted the stabilization of Zn, whereas Fe2O3 and SiO2 promoted Cu instability. These results could provide some references for the treatment of organic chlorides and the stabilization of heavy metals in landfill refuse in China.

A fusion protein composed of the DSL domain of Dll1 and RGD motif protects cryptic stem cells in irradiation injury.

Intestine is vulnerable to irradiation injury, which induces cell death and compromises regeneration of intestinal crypts. It is well accepted that cryptic stem cells, which are responsible for cryptic regeneration under physiological and pathological conditions, are controlled by multiple cell-intrinsic and environmental signals such as Notch signaling. Therefore, in the present study, we tested whether a soluble Notch ligand tethered to endothelial cells-mD1R-the Delta-Serrate-Lag2 (DSL) domain of mouse Notch ligand Delta-like1 fused with a RGD motif could protect cryptic cells from irradiation-induced intestinal injury. The result showed that administration of mD1R, which activated Notch signaling in intestinal cells, ameliorated loss of body weight and reduction of cryptic structures in intestine after total body irradiation (TBI) in mice. Histological staining showed that injection of mD1R after TBI promoted cryptic cell proliferation and reduced cell apoptosis in crypts. Immunofluorescence staining and reverse transcription (RT)-PCR showed that mD1R increased the level of Lgr5, Bmi1, Olfactomedin-4 (OLFM4), and IRIG1 in crypts, suggesting a protective effect on cryptic stem and progenitor cells after irradiation. Moreover, we found that administration of mD1R increased the number of Paneth cells and the mRNA level of Defa1, and the number Alcian Blue+ Goblet cells decreased first and then increased after irradiation, suggesting that mD1R promoted the maturation of the intestinal crypt after irradiation injury. Our data suggested that mD1R could serve as a therapeutic agent for the treatment of irradiation-induced intestinal injury.

Polydatin ameliorates renal ischemia/reperfusion injury by decreasing apoptosis and oxidative stress through activating sonic hedgehog signaling pathway.

Polydatin, a glucoside of resveratrol, recently has been demonstrated possibly to exert its biological effects by targeting sonic hedgehog (Shh). However, whether Shh signaling pathway is involved in the therapeutic effects of polydatin for renal ischemia/reperfusion (I/R) injury has not been evaluated. Our results showed that I/R induced the secretion of Shh, upregulated Patched and Smoothened, and enhanced the nuclear translocation and target gene transcription of Glioblastoma 1 in renal I/R injury models, which were further upregulated after the administration of polydatin significantly and in turn exerted prominent nephroprotective effects against cell apoptosis and oxidative stress. The treatment with cyclopamine (a specific inhibitor of Smoothened) or 5E1 (an anti-Shh antibody) not only markedly inhibited the activation of the Shh pathway, but also dramatically suppressed the nephroprotective effects of polydatin above-mentioned. These results advance our knowledge that polydatin can provide protection for kidneys against I/R injury by enhancing antioxidant capacity and decreasing cell apoptosis through activating Shh signaling pathway.

Nephroprotective Effects of Polydatin against Ischemia/Reperfusion Injury: A Role for the PI3K/Akt Signal Pathway.

Oxidative stress and inflammation are involved in the pathogenesis in renal ischemia/reperfusion (I/R) injury. It has been demonstrated that polydatin processed the antioxidative, anti-inflammatory, and nephroprotective properties. However, whether it has beneficial effects and the possible mechanisms on renal I/R injury remain unclear. In our present study I/R models were simulated both in vitro and in vivo. Compared with vehicle control, the administration of polydatin significantly improved the renal function, accelerated the mitogenic response and reduced cell apoptosis in renal I/R injury models, strongly suppressed the I/R-induced upregulation of the expression of tumor necrosis factor-α, interleukin-1ß, cyclooxygenase-2, inducible nitric oxide synthase, prostaglandin E-2, and nitric oxide levels, and dramatically decreased contents of malondialdehyde, but it increased the activity of superoxide dismutase, glutathione transferase, glutathione peroxidase and catalase, and the level of glutathione. Further investigation showed that polydatin upregulated the phosphorylation of Akt in kidneys of I/R injury dose-dependently. However, all beneficial effects of polydatin mentioned above were counteracted when we inhibited PI3K/Akt pathway with its specific inhibitor, wortmannin. Taken together, the present findings provide the first evidence demonstrating that PD exhibited prominent nephroprotective effects against renal I/R injury by antioxidative stress and inflammation through PI3-K/Akt-dependent molecular mechanisms.

The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02.

PURPOSE: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02. METHODS: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry. RESULTS: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5. CONCLUSION: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.

The effects of ABCG2 on the viability, proliferation and paracrine actions of kidney side population cells under oxygen-glucose deprivation.

Bcrp1/ABCG2 is exclusively expressed in side population (SP) cells, however, it has not been fully elucidated whether it has an impact on the viability, proliferation and paracrine actions in kidney SP cells under oxygen-glucose deprivation (OGD) followed by reoxygenation. In this study, we found that 2-h OGD did not injure SP cells (sub-lethal OGD) but induced SP cells proliferation 48 and 72 h after reoxygenation; whereas 4-h OGD markedly injured the cells (lethal OGD) and led to apoptosis 24-72 h after reoxygenation. Fumitremorgin C, an inhibitor of ABCG2, attenuated both the proliferation and viability of SP cells. Sub-lethal and lethal OGD induced the increase in the secretion of vascular endothelial growth factor, insulin-like growth factor 1, hepatocyte growth factor, and stromal cell-derived factor-1α in kidney SP cells, which was inhibited by Fumitremorgin C. Collectively, these findings provide evidence for a crucial role for the ABCG2 expression in the viability, proliferation and paracrine actions of kidney SP cells after OGD.

Treatment of cytomegalovirus infection after renal transplantation: experience from a single center in China.

BACKGROUND: We compared the efficacy and safety of 2 different treatments of CMV infection, including asymptomatic CMV replication and CMV disease. MATERIAL AND METHODS: 852 renal transplantation recipients, including asymptomatic CMV replication and CMV disease, received antiviral therapies of intravenous acyclovir or comprehensive anti-infection solution, mainly with intravenous ganciclovir. Effect, time, acute allograft rejection, and safety were analyzed during the antiviral therapy RESULTS: The total effective rates were higher with ganciclovir in both asymptomatic CMV replication (98.96% vs. 84.90%) and CMV disease (96.29% vs. 50.36%). Ganciclovir significantly shortened antiviral therapy duration in both asymptomatic CMV replication (15.0 ± 2.3 days vs. 16.0 ± 3.4 days) and CMV disease (19.7 ± 3.1 days vs. 21.5 ± 4.0 days). The acute allograft rejection incidences were significantly lower with ganciclovir in both asymptomatic CMV replication (8% vs. 14%) and CMV disease (11% vs. 22%). CMV-IEA was detected in renal grafts of patients with acute rejection. There was more CMV-associated acute rejection using acyclovir than using ganciclovir. Except for the higher incidence of anemia leucopenia and anemia with ganciclovir, the safety profiles of both drugs were similar. CONCLUSIONS: Comprehensive anti-infection solution, mainly with intravenous ganciclovir, can effectively treat CMV infection, shorten duration of therapy, and decrease acute rejection. The few adverse effects had negligible effects on use of ganciclovir.

ABCG2 protects kidney side population cells from hypoxia/reoxygenation injury through activation of the MEK/ERK pathway.

Breast cancer resistance protein 1 (BCRP1/ABCG2) is used to identify the side population (SP) within a population of cells, which is enriched for stem and progenitor cells in different tissues. Here, we investigated the role of extracellular signal-regulated kinase (ERK) 1/2 in the signaling mechanisms underlying ischemic/hypoxic conditions in kidney SP cells. Kidney SP cells were isolated using Hoechst 33342 dye-mediated fluorescein-activated cell sorting and then incubated under hypoxia/reoxygenation (H/R) with or without verapamil, a selective BCRP1/ABCG2 inhibitor. ABCG2 expression, ERK activity, cell viability, metabolic activity, and membrane damage were tested after H/R treatment. To evaluate the role of ERK 1/2 on the expression and function of ABCG2, the expression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which preferentially activates ERK, was upregulated by transfection with the recombinant sense expression vector pcDNA3.1-MEK and downregulated by pretreatment with U0126, a specific MEK inhibitor. We found that hypoxia activated ERK activity in the kidney SP cells but not in non-SP cells both in vitro and in vivo. Overexpression of MEK mimicked hypoxia-induced ABCG2 expression. Contrarily, U0126 inhibited hypoxia- and MEK-upregulated ABCG2 expression. Furthermore, H/R induced significant increases in nuclear, metabolic, and membrane damage in both SP cells and non-SP cells; however, this H/R-induced cytotoxicity was much more severe in non-SP cells than in SP cells. Notably, the viability of kidney SP cells was enhanced by MEK overexpression and inhibited by U0126. Verapamil treatment reversed MEK-induced viability of kidney SP cells. When administered systemically into animals with renal ischemia/reperfusion injury, the SP cells significantly improved renal function, accelerated mitogenic response, and reduced cell apoptosis. However, this improved therapeutic potential of SP cells was significantly reduced by pretreatment with verapamil. Collectively, these findings provide evidence for a crucial role for the MEK/ERK-ABCG2 pathway in protecting kidney SP cells from ischemic/hypoxic injury.

Interpretation of interlocking key issues of cancer stem cells in malignant solid tumors.

OBJECTIVE: In this review, several interlinking issues related to cancer stem cells (CSCs) in malignant solid tumors are sequentially discussed. METHODS: A literature search was performed using PubMed, Web of Science and the Cochrane library, combining the words CSCs, solid tumor, isolation, identification, origination, therapy, target and epithelial-mesenchymal transition. RESULTS: Because a primary problem is the isolation of CSCs, we first analyzed the advantages and disadvantages of recently used methods, which were mostly based on the physical or immunochemical characteristics of CSCs. Once CSCs are isolated, they should be identified by their stem cell properties. Here, we suggest how to establish a standard identification strategy. We also focused on the origination hypotheses of CSCs. The supporting molecular mechanisms for each theory were thoroughly analyzed and integrated. Especially, epithelial- mesenchymal transition is an increasingly recognized mechanism to generate CSCs that are endowed with a more invasive and metastatic phenotype. Finally, we discuss putative strategies of eliminating CSCs as effective cancer therapies. CONCLUSION: After several interlocking issues of CSCs are thoroughly clarified, these CSCs in solid malignant tumors may specifically be targeted, which raises a new hope for eliminating these tumors.

Several important in vitro improvements in the amplification, differentiation and tracing of fetal liver stem/progenitor cells.

OBJECTIVE: We previously isolated fetal liver stem/progenitor cells (FLSPCs), but there is an urgent need to properly amplify FLSPCs, effectively induce FLSPCs differentiation, and steadily trace FLSPCs for in vivo therapeutic investigation. METHODS: FLSPCs were maintained in vitro as adherent culture or soft agar culture for large-scale amplification. To direct the differentiation of FLSPCs into hepatocytes, FLSPCs were randomly divided into four groups: control, 1% DMSO-treated, 20 ng/ml HGF-treated and 1% DMSO+20 ng/ml HGF-treated. To trace FLSPCs, the GFP gene was introduced into FLSPCs by liposome-mediated transfection. RESULTS: For amplifying FLSPCs, the soft agar culture were more suitable than the adherent culture, because the soft agar culture obtained more homogeneous cells. These cells were with high nuclear:cytoplasmic ratio, few cell organelles, high expression of CD90.1 and CD49f, and strong alkaline phosphatase staining. For inducing FLSPCs differentiation, treatment with HGF+DMSO was most effective (P<0.05), which was strongly supported by the typical morphological change and the significant decrease of OV-6 positive cells (P<0.01). In addition, the time of indocyanine green elimination, the percentage of glycogen synthetic cells, and the expressions of ALB, G-6-P, CK-8, CK-18 and CYP450-3A1 in HGF+DMSO-treated group were higher than in any other group. For tracing FLSPCs, after the selection of stable FLSPC transfectants, GFP expression continued over successive generations. CONCLUSIONS: FLSPCs can properly self-renew in soft agar culture and effectively differentiate into hepatocyte-like cells by HGF+DMSO induction, and they can be reliably traced by GFP expression.

Neuroprotective effects of curculigoside against NMDA-induced neuronal excitoxicity in vitro.

Glutamate is an important excitatory neurotransmitter in the central nervous system. Excessive accumulation of glutamate can cause excitotoxicity, which plays a key role in spinal cord injury, traumatic brain injury, stroke, and neurodegenerative diseases. Curculigoside (CCGS) is a major bioactive compound isolated from the rhizome of Curculigo orchioides Gaertn. CCGS has an extensive biological effect and has been used in Traditional Chinese Medicine. However, little is known about the neuroprotective effects of CCGS on glutamate-induced excitotoxicity. This study aims to evaluate the neuroprotective effects of CCGS in cultured cortical neurons. The results indicated that treatment with 1 and 10 µM CCGS evidently prevented N-methyl-d-aspartate (NMDA)-induced neuronal cell loss and reduced the number of apoptotic and necrotic cells in a time- and concentration-dependent manner. The neuroprotective effects of CCGS are related to down regulating the apoptotic protein levels and reducing the production of intracellular reactive oxygen species in cultured cortical neurons. These findings give a new insight into the development of natural anti-excitotoxicity agents.

Simultaneous blockade of the CD40/CD40L and NF-κB pathways prolonged islet allograft survival.

Activation of NF-κB pathway and co-stimulatory system CD40/CD40L promotes the inflammation, which plays a key role in the failure of islet graft. Therefore, the purpose of this study was to determine if simultaneous blockade of CD40/CD40L and IκB/NF-κB pathways could protect islet graft. Streptozocin-induced diabetic Wistar rats were transplanted intraportally with 2000 IEQ islets isolated from Sprague-Dawley rats. The rats were divided into five groups: nontreatment group, AdGFP-treated group, Ad-IκBα-treated group, Ad-sCD40LIg-treated group, and Ad-IκBα-IRES(2) -sCD40L-treated group. The islet graft mean survival time (MST), insulin expression of islet grafts, and the levels of cytokines in peripheral blood, were measured for the animals in each group. Our study confirmed that islet cells transfected with low doses of adenovirus could achieve high transfection efficiency, and would not affect the function of islet cells (P > 0.05). Splenocytes cultured with Ad-IκBα-IRES2-CD40L-transfected islets resulted in homospecific hyporesponsiveness. The islet graft MST (>100 d) in the Ad-IκBα-IRES2-sCD40L-treated group was dramatically prolonged compared with that in the nontreatment group (7.1 ± 1.16 d). In addition, TNF-α, IL-1ß, and IFN-γ were diminished in the Ad-IκBα-IRES2-sCD40L-treated group, which was commensurate with the reduced cellular infiltration (P < 0.01). Simultaneous blockade of the CD40/CD40L and IκB/NF-κB pathways could effectively extend the survival of islet grafts.