Chronic kidney disease (CKD) has been considered as a major health problem in the world. Increasing uric acid (UA) could induce vascular endothelial injury, which is closely related to microinflammation, oxidative stress, and disorders of lipids metabolism. However, the specific mechanism that UA induces vascular endothelial cells injury in early CKD remains unknown.Human umbilical vein endothelial cells (HUVECs) were cultured and subjected to different concentrations of UA for different periods. Early CKD rat model with elevated serum UA was established. Western blotting and quantitative real-time PCR (qPCR) were applied for measuring protein and mRNA expression of different cytokines. The animals were sacrificed and blood samples were collected for measurement of creatinine, UA, IL-1β, TNF-α, and ICAM-1. Renal tissues were pathologically examined by periodic acid-Schiff (PAS) or hematoxylin-eosin (HE) staining.+ efflux. In vivo results showed that UA caused the vascular endothelial injury by activating NLRP3/IL-1β pathway. While allopurinol could reduce UA level and may have protective effects on cardiovascular system.The expression of IL-1β, ICAM-1, NLRP3 complexes, and activation of NLRP3 inflammasome could be induced by UA, but the changes induced by UA were partially reversed by siRNA NLRP3 or caspase 1 inhibitor. Furthermore, we identified that UA regulated the activation of NLRP3 inflammasome by activating ROS and K+ efflux and further induce vascular endothelial cells injury in early stages of CKD.UA could regulate NLRP3/IL-1β signaling pathway through ROS activation and K In recent years, chronic kidney disease (CKD) has become one of the most recognized public health problems worldwide , 2. Hype+) efflux also participate in the activation of NLRP3 inflammasome [+ efflux are still not well known.The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a multiprotein complex comprising NLRP3 receptor protein, apoptosis-associated speck-like protein (ASC) and protease caspase 1 . The incammasome . Therefo+ efflux in vitro, and further validated them in vivo. The aim of this study is to provide a new perspective for clinical drug therapy.Collectively, human umbilical vein endothelial cells (HUVECs) were used to investigate the mechanisms that UA activates NLRP3/IL-1β and further induces vascular endothelial cells injury through ROS activation and K2. When the adhering cells reached confluence, passage by trypsin digestion was conducted. After 3 to 5 passages, cells were treated by different concentrations of UA  for 24 h, then cells were collected for western blotting and quantitative real-time PCR (qPCR). A suitable concentration of UA was chosen to detect the influence of different incubation time  on the expression of IL-1β and ICAM-1 by western blotting and qPCR.HUVECs were isolated by collagenase A perfusion from umbilical cords  and cultured as described . The celTotal proteins in cells or cells culture supernatant were prepared and quantified. Equal contents of protein were loaded on an SDS-PAGE and then transferred electrophoretically to PVDF membranes . After blocking with TBST (5% milk), the membranes were incubated with primary antibody obtained from Abcam  at 4 °C overnight. After washing, the membranes were incubated with secondary antibody (1:2000) in TBST for 1 h at room temperature. ECL Plus detection system  was used for immunodetection, and the density of the bands was detected by Image J software.-ΔΔCt method.Total RNA was extracted through TRIzol reagent  and then was reverse-transcribed into cDNA using the Primer Script RT reagent kit . qPCR was performed with SYBR Premix Ex Taq™ II kit . The primers used for IL-1β, caspase 1, NLRP3, and ASC were listed in Table n = 5), but nephrectomy was not applied. Animals in Group C  and D  were fed with potassium oxonate (OXO) or/and allopurinol. Animals in Group A and B  were gavaged with drinking water with the same amount as in Group C and D.The animal model was established as described . BrieflyAfter 10 weeks of gavage treatment, animals were euthanized with prolonged exposure to isofluorane inhalation  and the renal tissues were collected. Renal tissues were fixed by 4% paraformaldehyde for 48 h, embedded in paraffin, and cut into 4 μm thick sections. After deparaffinization and rehydration, the sections were stained separately with periodic acid-Schiff (PAS) staining and hematoxylin-eosin (HE) staining to observe the pathological changes in tubules, glomeruli, and enal interstitial by microscope at 200× magnification.After 10 weeks of gavage treatment, animals were sacrificed and the blood samples were collected. After centrifugation, the blood samples were used for measurement of UA and creatinine. Measurement of UA and creatinine in serum was performed using an automated clinical chemistry analyzer .HUVECs were treated by either UA (20 mg/dL) or normal saline for 24 h. Then cells were loaded with 5 μM working solution at 37 °C for 10 min and washed twice with warm buffer according to the experimental protocol of ROS assay kit (Invitrogen). After another 10 min incubation, cells were analyzed with a fluorescence microscope .The blood samples were obtained as described above, and the measurements of IL-1β, intercellular adhesion molecule-1 (ICAM-1), and tumor necrosis factor-α (TNF-α) were conducted through ELISA kits .t-test for comparison between two groups or one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparison was performed for differences analysis. P-value < 0.05 was viewed as statistically significant difference.Data were shown as the mean ± SD, and analyzed using GraphPad Prism 6.0 . The unpaired two-tailed After treatment with different concentrations of UA or different incubation time, the expression of ICAM-1 and IL-1β in HUVECs was detected by western blotting and qPCR , ROS scavenger , or mitochondrial ROS inhibitor (Mito-TEMPO). Then the expression of NLRP3 and its downstream factors, caspase 1, IL-1β and ICAM-1, was measured. As shown, the ROS level in cells was significantly promoted by UA after 24 h incubation ; Group D, in which animals received right nephrectomy and then gavaged with OXO  and allopurinol .During the experiment, no dead rat, and no glomerular, tubule, and interstitial lesions in all groups Fig. a. The seUA is produced during the metabolism of nucleotides and adenosine triphosphate (ATP), it is the end product of purine metabolism in humans which excreted mainly by kidney , 19. WhiIt was reported that endothelial cells damage induced by inflammatory factors plays a key role in the pathogenesis of vascular diseases , 21. NLR+ concentration significantly influences the activation of NLRP3 inflammasome [+ concentration of healthy cells is ~ 140–150 mM, which does not induce NLRP3 activation [+ concentration by 50% [+ for blocking K+ efflux, and observed the inhibition of NLRP3 inflammasome activation. Interestingly, the activation of apoptosome also were correlated with the K+ concentration [+ efflux in the activation of NLRP3 inflammasome remains unclear, but low intracellular K+ concentration induces ROS production and vice versa [+ efflux.Some reports indicated that cytoplasmic Kammasome , 25. Thetivation , 27. ATPntration . ROS proce versa –30. Ther+ efflux. Therefore, UA may induce the vascular endothelial injury by activating NLRP3/IL-1β pathway. These findings provide a new insight into the adverse effect of UA on vascular endothelial cells, and new promising therapeutic targets aiming at early-stage CKD may be developed by regulating NLRP3/IL-1β pathway.In the present study, UA could activate NLRP3 inflammasome and increase the expression of some inflammatory factors including ICAM-1 and IL-1β in both in vitro and in vivo early CKD model. We further indicated that UA activated NLRP3 inflammasome by activating ROS or regulating K


The neural crest (NC), which has been referred to as the fourth germ layer, comprises a multipotent cell population which will specify diverse cells and tissues, including craniofacial cartilage and bones, melanocytes, the adrenal medulla and the peripheral nervous system. These cell fates are known to be determined by gene regulatory networks (GRNs) acting at various stages of NC development, such as induction, specification, and migration. Although transcription factor hierarchies and some of their interplay with morphogenetic signaling pathways have been characterized, the full complexity of activities required for regulated development remains uncharted. Deregulation of these pathways may contribute to tumorigenesis, as in the case of neuroblastoma, a frequently lethal embryonic cancer thought to arise from the sympathoadrenal lineage of the NC. In this “Hypothesis and Theory” article, we utilize the next generation sequencing data from neuroblastoma cells and tumors to evaluate the possible influences of Wnt signaling on NC GRNs and on neuroblastoma cell lineages. We propose that Wnt signaling is a major determinant of regulatory networks that underlie mesenchymal/neural crest cell (NCC)-like cell identities through PRRX1 and YAP/TAZ transcription factors. Furthermore, Wnt may also co-operate with Hedgehog signaling in driving proneural differentiation programmes along the adrenergic (ADRN) lineage. Elucidation of Signaling Regulatory Networks can augment and complement GRNs in characterizing cell identities, which may in turn contribute to the design of improved therapeutics tailored to primary and relapsing neuroblastoma. MYCN is crucial for NC cell fate  is a frequently lethal pediatric tumor, with 75% of NBs occurring in children under 5 years of age. About half of the tumors arise in the adrenal medulla, with the remainder originating in the paraspinal sympathetic ganglia in the abdomen or chest, or in pelvic ganglia. This distribution reflects the probable developmental origin of NB in the sympathoadrenal (SA) lineage of the neural crest (NC). Clinically, NB encompasses low-risk disease which responds well to treatment and may even spontaneously regress, and high-risk disease, representing about 40% of total cases, which frequently relapse and have less than 50% survival. This clinical heterogeneity is thought to reflect both the complex molecular etiology as well as the cellular heterogeneity of tumors Brodeur, . Whilst In this theoretical analysis, we will consider the potential regulatory influences and interactions of the canonical Wnt signaling pathway in determining phenotypes in the NC and neuroblastoma. Although this signaling pathway is known to be critical in regulating stemness, cell fate, differentiation and proliferation  and G-protein dependent calcium release  transcription factors  receptors. Wnt ligands are secreted, lipid-modified glycoproteins conserved in all metazoan animals, able to act as morphogens over short distances , and the E3-ubiquitin ligase β-TrCP. This complex regulates cytoplasmic β-catenin turnover through phosphorylation and proteosomal turnover. The LRP-Axin interaction leads to inactivation of the destruction complex, permitting β-catenin stabilization and nuclear availability. The TCF/LEF family of transcription factors are then able to utilize β-catenin as a transcriptional co-activator and instigate target gene expression  that govern NC development. Transcriptional analysis and molecular loss and gain-of-function studies facilitate a GRN model of the NC, which, encompassing transcriptional regulators and signaling molecules, can explain the formation and maintenance of NC lineages , in the neural plate border module, determining the boundaries of NC, neural and non-neural ectoderm. These, in turn, upregulate NC specifier genes, which initiate the EMT programme  to prepare for delamination of NCCs as well as maintain an undifferentiated state (premigratory module). Following EMT, the NCCs acquire a migratory programme enabling long-distance migration guided by environmental signals and maintained by a TF network . The sympathoadrenergic lineage follows a ventral migratory pattern and aggregate at the dorsal aorta and go on to form the primary sympathetic ganglia and colonize the adrenal medulla. BMPs released by the wall of the dorsal aorta induce SA speciation, activating ASCL1 and PHOX2B, which switches on other SA-lineage specific transcription factors, for example, ASCL1 upregulates PHOX2A, which in turn upregulates TH and DBH, enzymes characteristic of the SA lineage.The aforementioned signaling pathways orchestrate cell behavior MYCN is expressed at high levels in chick NC progenitors in early NC development but surprisingly is not apparent in highly proliferative migrating NCCs  and PRRX1. The ADRN cells were more tumorigenic in nude mice than MES cells, although MES cells were more chemoresistant and were enriched in relapsed tumors  NBs as a result of Wnt/β-catenin signaling  NB cell line -C and SH-SY5Y cell lines, increased proliferation was not observed. Instead, we found an EMT-like transition, evidenced by increased cell migration and induction of SNAI1/2 and TWIST1/2, and neural differentiation accompanied by increased expression of marker genes such as NGFR, NTRK1, NEFM and NEFL/NF68. Meta-analysis of the expression of our 90 Wnt target genes in primary tumor gene expression databases revealed: (i) four gene subsets within the 90 genes, and (ii) dramatically different prognostic outcomes associated with each of the four gene subsets (R = >0.76) in NB, and that these genes were more highly expressed in intermediate risk NB, rather than low- or high-risk NB. WMG-3 was overexpressed in the high-risk NB cluster, dominated by MNA tumors. Conversely, WMG-2 showed a strong positive correlation to Hedgehog signaling (R = >0.84) associated with differentiation . WMG-2 genes were, concordantly, strongly repressed in high-risk MNA NB  Wnt-induced transcriptomic changes by RNA sequencing in SK-N-BE(2)-C, an  subsets . By convCollectively, studies from our laboratory and from others allude to an underappreciated role for Wnt signaling in NC and NB biology. In particular, the induction of EMT-like changes and the correlation of a subset of our Wnt target genes (WMG-1) with Hippo-YAP/TAZ pathway gene signatures, seemingly characteristic of the MES lineage , are affected. Interestingly, given that the presumptive cell of origin for NBs is along the sympathoadrenal lineage, there is also strong induction for neural plate border specifiers MSX1, DLX5 and DLX6. This suggests that either SK-N-BE(2)-C cells have retained early transcriptional plasticity, or more intriguingly, that NBs may arise prior to sympathoadrenal differentiation. This notion is supported by developmental experiments showing ectopic MYCN expression in the neural plate border resulting in a shift towards a CNS-like state , but interestingly displaying an inverse pattern to that of MES. The functional significance of this pattern could be underpinned, for example, by FZD3, which has been shown to be required for maintenance of dividing sympathetic neuroblasts and correlates with ADRN identity -C cells. As we had reported Wnt3a/Rspo2 treatment leading to EMT -C cells, we found that WMG-1 genes, high expression of which is characteristic of intermediate risk NB . Expression of WMG-2 was also increased, although in a less uniform way, whereas WMG-3 and WMG-4 genes appeared to be partitioned in their response, with two subsets of genes regulated in an opposite fashion (PRRX1 induction in SK-N-BE(2)-C. We conducted the same analysis on Wnt pathway genes (KEGG and other known members) and observed increased expression of Wnt pathway receptor and transcriptional regulator genes accompanying PRRX1 induction , which were both implicated in MES/NCC-like identity. As these were not identified in our RNA sequencing timepoint (6 h), we assessed 96 h treatments of SK-N-BE(2)-C and SH-SY5Y cell lines by qPCR. Both PRRX1 and WWTR1 were upregulated by greater than 2-fold in both cell lines  cell fates. As a first step to analyzing this possibility, we determined the expression pattern of the ADRN signature genes in the four prognostic NB clusters defined by the expression of our NB Wnt target genes -C and SH-SY5Y cell lines, including induction of PHOX2A/2B, TFAP2B and EYA1 were all markedly upregulated in SK-N-BE(2)-C cells. In SH-SY5Y cells, these factors were already generally higher, but again induction of EYA1 was apparent following Wnt3a/Rspo2 treatment.Since we previously demonstrated that Wnt3a/Rspo2 treatment can prompt neural differentiation of NB cell lines, we evaluated whether long-term (96 h) treatments with Wnt ligands may influence transcription factors that contribute to the establishment of the ADRN, or noradrenergic, cell lineage. As shown in Taken together these analyses demonstrate that the ADRN signature can be partitioned into two discrete subsets, which we have termed ADRN-LR and ADRN-HR, with contrasting expression in low-risk and high-risk NBs. The role of Wnt in differentiation of sympathoadrenergic neurons is further underlined by a report by Bodmer et al.  that WntThe understanding of gene expression programmes, contingent on transcription factor hierarchies and chromatin level plasticity, in particular, epigenetic changes at super-enhancer elements, continues to facilitate our understanding of cellular identity. Notable advances have been made in ascertaining regulatory circuits in NCCs as well as in neuroblastoma recently, yet much remains to be determined about the contributions of autocrine, paracrine and juxtacrine signaling pathways that, together with transcription factors and epigenetic states, form a regulatory triumvirate.MSX1 and DLX5/6, as well as migratory factors such as SNAI2 and TWIST1/2. Further, we highlight the possible interplay between canonical Wnt signaling with Hippo-YAP/TAZ and Hedgehog signaling, emphasizing the need for integrative analysis of signaling inputs for both NC and NB biology. Our transcriptomic signatures facilitate this, but also underline the need for parallel studies using diverse cell types and signaling effectors.Our analysis of Wnt signaling strongly suggests that this pathway is a major determinant of transcription factor circuitries, and thereby lineage identity, in neuroblastoma. This is further supported by comparisons with NC GRNs, with Wnt signaling regulating early NC specifiers such as With regard to NB, our study begins to reconcile some of the apparent contradictions about the influence of Wnt signaling. The determination of MES/NCC-like identity by Wnt is consistent with reports that resistance to chemotherapy and stemness may be linked to Wnt .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.


Furthermore, the number of IL‐10+ regulatory T cells was observed to be increased in patients with high levels of TSLP. We found that TSLP values greater than 350 pg·mL−1 were associated with a higher mortality rate and longer stays in intensive care (sensitivity of 89% and specificity of 79%). In patients with low levels of neutrophils, the area under curve was only 0.71 . Our findings suggest that the serum levels of TSLP may be suitable as a biomarker for prediction of prognosis in a subgroup of patients with sepsis who are exhibiting hyperleukocytosis and a high neutrophil ratio.Thymic stromal lymphopoietin (TSLP) has been identified as a crucial inflammatory cytokine in immune homeostasis. Previous studies have reported conflicting effects of TSLP on sepsis in mice, and the effect of TSLP on sepsis in humans has not been investigated. In this study, we used the ELISA to measure serum levels of TSLP in patients with sepsis, and used flow cytometry and ELISA to evaluate the proinflammatory phenotype of circulating immune cells. In addition, we used quantitative RT‐PCR to examine the expression of proinflammatory cytokines  between patients with high and low levels of TSLP. Flow cytometry analysis was performed to evaluate the phagocytic and respiratory burst of circulating neutrophils. A significant increase in the production of proinflammatory cytokines by nonclassical monocytes and the number of interferon (IFN)‐γ +CD162+ monocytes, CD14+CD16+ monocytes and CD4+ T cells, which might be correlated with higher mortality of patients with sepsis with hyperleukocytosis or high neutrophil ratio.Elevated serum levels of thymic stromal lymphopoietin lead to an increase in the production of proinflammatory cytokines in CD14 AUCarea under curveCLPcecal ligation and punctureDCdendritic cellFITCfluorescein isothiocyanateFoxp3forkhead box P3HKSAStaphylococcus aureusheat‐killed HLhyperleukocytosisHNRhigh neutrophil ratioICUintensive care unitIFNinterferonILinterleukinPBMCperipheral blood mononuclear cellSDstandard deviationSIRSsystemic inflammation response syndromeThT helperTNFtumor necrosis factorTregregulatory TTSLPthymic stromal lymphopoietinTSLPRTSLP receptor−/− mice. Dextran sodium sulfate–induced colitis was more severe in TSLP receptor (TSLPR)−/− mouse than in wild‐type mouse. The ambivalent effect of TSLP on different tissues is unclear, and further investigations in humans revealed two isoforms Thymic stromal lymphopoietin (TSLP) has been identified as a crucial inflammatory cytokine in immune homeostasis. TSLP is known to trigger dendritic cells (DCs) and promote differentiation of T helper (Th) type 2 cells, and is linked to the pathogenesis of some allergic diseases, including asthma and atopic dermatitis −/− mouse Previous studies about the interrelationship between TSLP and sepsis have revealed the elevated levels of TSLP in a colorectal ligation and puncture animal model. However, the effect of TSLP in sepsis mice was opposite in two studies on a cecal ligation and puncture (CLP) mouse model. In the first study, the administration of a neutralizing antibody to TSLP in mice with sepsis resulted in decreased mortality and weakened the host immune response In this study, we demonstrate elevated levels of TSLP in patients with sepsis and reveal the negative correlation between serum levels of TSLP in patients with sepsis with a high ratio of neutrophils and mortality. The findings of this study reveal the insufficient infiltration of neutrophils in infectious lesions, as observed in animal experiments. Furthermore, we show that TSLP mainly stimulated the activation of monocytes and induced their proinflammatory phenotype rather than T lymphocytes and neutrophils among the circulating immune cells. These findings suggest that TSLP may serve as a potential biomarker for the prediction of mortality in a subgroup of patients with sepsis, particularly in patients with a high ratio of neutrophils.9 L−1, and HNR was defined as the percentage of neutrophils greater than 75%. The patients with a high level of TSLP were defined as more than the average level of TSLP in the corresponding patient subset. The patients with a low level of TSLP were defined as less than the average level of TSLP in the corresponding patient subset. The inclusion criteria were as follows: (a) age between 20 and 60 years; (b) no severe cardiopulmonary failure during serum collection, including mechanical ventilation, support of extracorporeal membrane oxygenation and administration of vasoactive drug for maintenance of blood pressure; (c) procalcitonin > 0.5 μg·L−1; and (d) no neoplastic, allergic and autoimmune diseases. The exclusion criteria included occurrence of cardiac arrest, possibility or definite diagnosis of viral and other atypical pathogenic infections, pregnant women and use of immunosuppressants and glucocorticoids. We then measured the functional characterization and frequency of inflammatory cells in all patients who suffered from sepsis regardless of the number of leukocytes and ratio of neutrophils, and compared the difference between patients with higher and lower levels of TSLP in the functional characterization and frequency of inflammatory cells. The study conformed to the Declaration of Helsinki and was approved by the medical ethics committee of the First Affiliated Hospital of Nanchang University. All patients enrolled in this study signed informed consent forms.Patient enrollment was performed from 2016 to 2018 in our institution. All patients were diagnosed with sepsis. The sepsis was identified according to two or more systemic inflammation response syndrome (SIRS) criteria, including changes in white blood cell count, temperature, respiratory rate and heart rate reflecting inflammation, in combination with life‐threatening organ dysfunction based on the Third Consensus Definition for Sepsis and Septic Shock (Sepsis‐3) −1 of cell culture fluid). All ELISA detection kits were purchased from Abcam Co.  for the measurement of cytokines secreted from PBMCs isolated from patients, including the human interleukin (IL)‐1β ELISA Kit , human IL‐6 ELISA Kit , human matrix metalloproteinase‐9 ELISA Kit , human tumor necrosis factor (TNF)‐α ELISA Kit (ab181421) and human IL‐10 ELISA Kit .Blood samples from patients were collected, and serum was isolated for the measurement of TSLP levels. Serum TSLP levels were measured according to ELISA method using a human TSLP ELISA detection kit . All samples were measured in duplicates, and the mean concentration was calculated. ELISA was also used for the measurement of cytokine levels in the supernatant fluid collected from the cultured peripheral blood mononuclear cells (PBMCs) subjected to stimulation with lipopolysaccharide ‐labeled bacteria [heat‐killed Staphylococcus aureus (HKSA)] at multiplicity of infection of 10 : 1 for 60 min or with PMA (800 ng·mL−1) for 15 min at 37 °C. This was followed by the addition of 25 mL of a 2800 ng·mL−1 solution of hydroethidine  and incubation for 5 min at 37 °C. Erythrocytes were excluded with lysis buffer  before analysis by flow cytometry . The number of neutrophils with phagocytic function in 1‐mL samples of whole blood was calculated by multiplying absolute neutrophil counts (determined using automated blood counter) with the percentage of phagocytosis  and dividing the obtained value by 100. Respiratory burst function, which occurs after phagocytosis, was subsequently calculated by multiplying the number of phagocytosing neutrophils  by the percentage of respiratory burst  and dividing the obtained value by 100.The flow cytometry was used for the analysis of phagocytosis and respiratory burst of neutrophils as described previously 5 cells·mL−1, and 100–200 μL of cell suspension was stimulated with cell stimulation cocktail . After stimulation in vitro, these cells were permeabilized with fixation and permeabilization buffer kit (BD Biosciences) and stained for 30 min at 4 °C in flow tubes with directly conjugated antibodies. The cells were fixed with 4% paraformaldehyde. The antibodies used for the flow cytometry analysis included anti‐human CD4‐allophycocyanin , anti‐human IFN‐γ‐PeCy7 , anti‐human IL‐4 , anti‐human forkhead box P3 (Foxp3)‐Alexa Fluor 488  and anti‐human IL‐10‐phycoerythrin . The cells were separated with a multicolor flow cytometer , and the fluorescence‐activated cell signaling data were analyzed by flowjo 10.0 software .We obtained PBMCs from patients with high or low levels of TSLP by density centrifugation using Ficoll‐Paque and cultured these cells in RPMI 1640 supplemented with 10% FBS. Cells were resuspended at a density of 0.5 × 102-ΔΔCt method.Total RNA was purified from cells using RNeasy Kit . cDNA synthesis was performed using SuperScript III reverse transcriptase  and random hexamers. Real‐time PCRs were carried out using the SYBR Green PCR kit and the primers listed in Table spss statistics 20 . Sensitivity, specificity, positive predictive value and negative predictive value were calculated, as described by the TDR Diagnostics Evaluation Expert Panel. Patients were stratified according to the serum levels of TSLP  for ex vivo investigation. Chi‐square test was used in comparison of two‐category data between patients with or without HL and HNR. Fisher’s exact test was used when the smallest expected count is less than 5 in the data instead of classical chi‐square test. The area under curve (AUC) was calculated in patients with or without HL and HNR according to the cutoff value. The sensitivity, specificity, positive predictive value and negative predictive value were calculated according to the best cutoff value in the diagnosis evaluation test. The corresponding best cutoff value used for AUC value and diagnostic evaluation test was determined according the Youden index (specificity + sensitivity − 1). The largest Youden index indicated that the corresponding cutoff value is the best.Data were entered into an Excel spreadsheet . Analysis was conducted in Excel and t‐test using spss statistics 20: *P < 0.05, **P < 0.01, ***P < 0.001.Other quantitative data were represented by means ± standard deviation (SD). Statistical significance was determined by the Student’s −1 in the total population. The average value of TSLP was 341.9 pg·mL−1 in those patients with HL and HNR. The average value of TSLP was 330.3 pg·mL−1 in patients without HL and HNR. The baseline characteristics are presented in Table The average value of TSLP was 337.1 pg·mL+CD16+ (intermediate) and CD14−CD162+  monocytes from patients with sepsis with high levels of TSLP  produced significantly increased levels of inflammatory cytokines, including IL‐1β, IL‐6, matrix metalloproteinase‐9 and TNF‐α, as compared with those isolated from patients with low TSLP levels (Fig. 2+CD16− monocytes from patients with high and low TSLP levels (Fig. + Th2 and Foxp3+ Treg cells in CD4+ T lymphocytes obtained from the two groups of patients (Fig. + Treg to total Treg cells (Fig. The protein TSLP acts as an inflammatory cytokine and exerts specific effects on circulating immune cells from patients with sepsis with high TSLP levels. Therefore, we evaluated the inflammatory phenotype of the circulating immune cells by flow cytometry and ELISA in patients with sepsis. Figure To evaluate the effect of TSLP on the function of neutrophils in patients with sepsis, we performed flow cytometry analysis and explored the functional changes in neutrophils in all sepsis regardless of whether these patients suffered from HNR and HL. We found that the number of phagocytic neutrophils Fig. A,B and t+CD16+, CD14−CD162+ and CD142+CD16− monocytes from PBMCs and CD4+ T cells to measure the expression of proinflammatory cytokines. As a result, we found an increase in the expression of TNF‐α and IFN‐γ in CD4+ T cells obtained from patients with high TSLP levels (Fig. + T cells obtained from patients with high levels of TSLP as compared with those obtained from patients with low levels of TSLP (Fig. +CD16+ and CD14−CD162+ monocytes obtained from patients with high levels of TSLP (Fig. We directly measured the expression of cytokines in PBMCs from patients with high or low levels of TSLP. A significant increase in the expression of IL‐1β, IFN‐γ, TNF‐α and IL‐6 was observed in patients with high TSLP levels compared with patients with low TSLP levels, regardless of the number of leukocytes and ratio of neutrophils Fig. A. We isoels Fig. B. In addSLP Fig. B. FurtheSLP Fig. C,D. No dSLP Fig. E. These −1 in patients with HL and HNR and 327 pg·mL−1 in patients without HL and high ratio of neutrophils. The AUC of the corresponding cutoff values was 0.85 and 0.71, respectively. The sensitivity was 89% and 62% and the specificity was 79% and 68% in patients with or without HL and HNR, respectively. The diagnostic efficiency of TSLP levels was significantly better in patients with higher absolute number of leukocytes and elevated ratio of circulating neutrophils (Fig. We performed the diagnostic test evaluation to evaluate whether TSLP levels predict mortality in patients with sepsis. We found the cutoff value according to the diagnostic test evaluation to evaluate mortality risk between patients with or without HL and HNR. For mortality prediction, the cutoff value was 350 pg·mLThe protein TSLP is a proinflammatory cytokine derived from epithelial tissues involved in inflammatory diseases through its binding to TSLPR. The TSLP/TSLPR signaling results in the activation of various innate and adaptive immune cells, including DCs, Treg, Th2 and Th1, indicating the complex influence of TSLP on inflammation S. aureus. The increase in the neutrophil function is dependent on the complement activation, and this study revealed an unrecognized, non‐Th2‐associated, downstream signaling pathway of TSLP/TSLPR involved in the host defense and infectious inflammation. TSLP could enhance the antibacterial effect through the engagement of complement C5 and induction of excessive reactive oxygen species. This effect could result in a dual effect on sepsis‐induced systemic inflammation, including promotion of neutrophil function for the phagocytosis of drug‐resistant bacterial strains and aggravation in the production of proinflammatory cytokines and formation of inflammatory cascade. In the study on the antibacterial effect of TSLP, stimulation with HKSA enhanced the expression of TSLPR and reduced the number of colony‐forming units, but the neutrophils were derived from healthy controls, and the enhanced antibacterial function was likely associated with the activation of the toll‐like receptor‐2 signaling pathway. However, during the sepsis infection in humans, the innate immune signaling pathway is activated by a variety of pathogen‐associated molecular patterns and damage‐associated molecular patterns S. aureus may incompletely simulate sepsis and may not meet the complexities involved in sepsis. Therefore, the TSLP‐induced antimicrobial effect dependent on neutrophils may fail to work in the case of other bacterial strains that participate in sepsis. In this study, we found that the function of neutrophils from patients with a higher level of TSLP have no discrimination when compared with the patients with a lower level. Neutrophils from patients were potentially exhausted and showed dysfunction. Neutrophils from patients with sepsis and severe systemic inflammatory syndrome were shown to be immature and less efficient in bacterial phagocytosis and production of reactive oxygen species highCD24high, was shown to be significantly decreased because of the downregulation of the membrane CD24, and this effect was associated with the lack of neutrophil function in these patients, leading to an impairment in CD24‐mediated apoptosis of neutrophils and formation of a neutrophil extracellular trap in vitro experiments may disappear because of the functional defects in neutrophils from patients with sepsis. On the contrary, the high TSLP level would activate the immune cells and boost the release of proinflammatory cytokines, which may enhance the proinflammatory phenotype and deteriorate the inflammatory cascade response.The TSLPR is widely expressed on various cells, including neutrophil, the key cell type involved in the killing of bacteria and control of acute infection Human TSLPR is shown to be preferentially coexpressed with IL‐7Rα on myeloid‐derived monocytes and DCs. TSLP could promote the differentiation of myeloid‐derived DCs and Th17 response in hepatitis C virus–infected hepatocytes −/− mouse, an increase in mortality was observed after CLP. The expression of IL‐6 and IL‐12 in peritoneal myeloid cells was significantly increased in CLP‐induced Lys‐Cre+;tslpfl/fl mouse as compared with Lys‐Cre−;tslpfl/fl mouse. The lack of TSLP/TSLPR may result in the dysregulation of inflammation in terms of the release of cytokines and proinflammatory mediators. Another study showed that the administration of the neutralizing antibody to TSLP may improve survival and facilitate the killing of bacteria in a septic mouse, as observed with an increase in the level of peritoneal TSLP. This study suggests that anti‐TSLP failed to inhibit neutrophil infiltration or induce normal production of proinflammatory cytokines to control infection in the septic mouse.In sepsis, excessive systemic inflammation may lead to multiple organ dysfunction and worsen prognosis. TSLP serves as a classical proinflammatory cytokine that has potential effects on patients with sepsis with severe SIRS. In two previous animal studies, opposite effects of TSLP in septic mice were observed + monocytes, CD142+CD16− monocytes are primary myeloid‐derived cells associated with chemokine receptors such as CCR2 and CD62L + T cells from patients with high levels of TSLP similarly produced more proinflammatory cytokines but less anti‐inflammatory cytokines such as IL‐10. However, it has been proved that TSLP could aggravate phagocytosis and reactive oxygen species–producing abilities of neutrophils in previous studies Our study revealed the correlation between the levels of TSLP and prognosis in patients with sepsis. We found that the nonclassical and intermediate subsets of monocytes from patients with high levels of TSLP showed the characteristic proinflammatory phenotype. The two subsets of monocytes were preferentially mobilized during inflammation and expanded in response to macrophage colony‐stimulating factor. In comparison with CD16In this study, we hypothesized that higher levels of TSLP might result in poor prognosis, and explored the correlation between prognosis and levels of TSLP. We found that in patients with HNR and HL, high levels of TSLP would result in poor prognosis. We identified the cutoff value for the prediction of mortality and verified the cutoff value in patients. The cutoff value was higher in patients with HL and HNR, consistent with the aforementioned result that high TSLP level showed positive correlation with poor prognosis only in patients with HL and HNR.This study also has some limitations. First, considering that this study is only a preliminary observational human study, the further mechanisms involved in the influence of TSLP on severe bacterial infection and sepsis. Although we found the circulating myeloid‐derived cells from patients with a higher level of TSLP have more significant proinflammatory phenotype, the underlying mechanisms need to be investigated in further animal experiments and basic research. Second, although our data about neutrophils cannot completely illustrate and interpret these functions of neutrophils, further evaluation about the neutrophils in the patients with higher TSLP is necessary in the future. Third, the sample size in the diagnosis evaluation test is relatively small. More reliable conclusions should be drawn in a trial of a larger, prospectively validated disease cohort.Our study provides a potential biomarker for the prediction of prognosis in a subgroup of patients with sepsis. We revealed the inflamed phenotype of immune cells in patients with sepsis with high levels of TSLP. We propose that the cutoff value of serum levels of TSLP could be used for risk assessment in the patients with sepsis with HNR and HL according to diagnostic test evaluation and may have potential value in the management of sepsis.The authors declare no conflict of interest.QY and HX conceived the study and designed the experiments. YL and HW contributed to the data collection, performed the data analysis and interpreted the results. QY wrote the manuscript. HX contributed to the critical revision of this article. All authors read and approved the final manuscript.


In this review, we summarize the evidence of LLPC generation and maintenance and provide novel paradigms of LLPC maturation.Antibody secreting cells (ASCs) are terminally differentiated cells of the humoral immune response and must adapt morphologically, transcriptionally, and metabolically to maintain high-rates of antibody (Ab) secretion. ASCs differentiate from activated B cells in lymph nodes and transiently circulate in the blood. Most of the circulating ASCs undergo apoptosis, but a small fraction of early ASCs migrate to the bone marrow (BM) and eventually mature into long-lived plasma cells (LLPCs). LLPC survival is controlled both intrinsically and extrinsically. Their differentiation and maintenance programs are governed by many intrinsic mechanisms involving anti-apoptosis, autophagy, and metabolism. The extrinsic factors involved in LLPC generation include BM stromal cells, cytokines, and chemokines, such as APRIL, IL-6, and CXCL12. In humans, the BM CD19 A key aspect of the adaptive immune response is the rapid production of high-affinity antibodies (Abs). This antibody production is the function of an antibody secreting cell (ASC), which arises from naïve (or memory) B cells as they encounter antigen, activate, proliferate, and differentiate. In draining lymph nodes, the B cell to ASC differentiation can occur either in the germinal center (GC) reactions  or outside GC, as a part of an extrafollicular response –3 Figur. DiffereIn the blood and secondary lymph organs (SLOs), the ASC presence is transient (days to a few weeks) during primary and secondary immune responses –7. AfterIn the circulation, the majority of circulating ASCs undergo apoptosis; yet a specialized fraction further matures into long-lived plasma cells (LLPCs) as they migrate to BM or other tissues  Figures. LLPCs aMany studies show that ASCs derived from GC reactions, rather than extrafollicular responses, have the potential for LLPC formation and survival , 18–21. Some investigators show that LLPCs can be generated independently of B cell maturation  –32. HoweIn this review, we focus on the molecular and cellular processes involved in the adaptation of ASCs after exiting the GC into the medullary cords and efferent lymphatics and upon entering the blood, and eventually the BM niche . ThroughThe prime directive of ASCs is to synthesize and secrete Abs that provide protection against microorganisms. ASCs produce large amounts of Abs, typically at a rate of between ~10 and 1,000  Ab molecules per cell per second, which corresponds to ~0.2–22 pg/cell/day –38. To pTo accommodate the production of copious amounts of proteins, ASCs expand their cytoplasm, endoplasmic reticulum (ER), and Golgi apparatus during differentiation and maturation, enabling increased capacity for Ab synthesis and secretion  Figure . Early AAs ASCs differentiate and mature, they alter their surface protein expression profile. These proteins can be used as the phenotypic markers for identifying subsets within the ASC population. Notably, there is no universally accepted or “standardized” phenotype to identify ASCs—in humans or in mice (which make it difficult to compare across studies). Nevertheless, human ASCs in general lose their surface immunoglobulins and can typically be identified based on the expression of one or more of the following cell surface markers: CD19, CD27, CD38, CD138, and BCMA –46 Figu. In miceintCD27hiCD38hi (gated on CD3−CD14−IgD− cells)  and 3  are the most abundant, and population 5  resembles LLPCs by surface markers (+), and 5 (CD19−). Based on RNAseq analysis, pops 2 and 3 are very similar transcriptionally whereas pop 5 is quite distinct  . However− cells) , 50  . Of the  markers . The othdistinct . More in−/− mice (The exit of ASCs from SLOs into the blood is guided by the expression of their S1P receptor 1 (S1P1) , 52. Sub−/− mice .It still remains unclear how the expression of chemokine receptors on ASCs is regulated, although transcription factor c-Myb may be involved . In the − ASCs as mature subsets , which include pops A and B that did not comprise of the long-lived specificities  , 70, 71.Prdm1)  , XBP1, aPrdm1) , 77–79. Prdm1) , 70, witPrdm1) , 80–82. Prdm1) , 61. AlsPrdm1) . FinallyPrdm1) .In addition to these two reciprocally-regulated groups of transcription factors, a number of other factors have been found differently expressed by ASCs in distinct compartments . Examples include anti-apoptotic factors, such as Mcl-1, Bcl-2, BclxL, Bim, and BCLw, which are increased, and pro-apoptotic factors, such as Bax and BID, which are decreased, to promote ASC maintenance –87. ImpoDuring differentiation, ASCs adapt and utilize various signaling pathways to accommodate the need for high-rate Ab production and maintenance of homeostasis . Thus, aAtg5, Atg9, and Atg13, are upregulated in ASCs and likely play a role as LLPCs mature (in vitro (Antibody secreting cells (ASCs) must also adapt their metabolism to produce Abs during circadian nutrient fluctuations while maintaining cellular homeostasis. As terminally-differentiated cells, ASCs do not actively remodel their cytoplasm through cell division but rely on autophagy to recycle their protein aggregates and organelles in order to optimize energy for survival. Multiple autophagy genes, such as s mature , 66, 93.in vitro . WhetherAnother important signaling pathway for ASCs is the mTOR. The mTOR kinase is a major regulator of many cellular processes, including survival and proliferation. mTOR signaling, mainly through the mTORC1 signaling complex (mTORC1), regulates the biosynthesis of cellular macromolecules, including proteins, nucleic acids, and fatty acids, as well as glycolysis and organelle biosynthesis , 96. BliAs ASCs evolve into LLPCs, major metabolic changes occur. In addition to differences in mTOR signaling that functionally distinguishes LLPCs from SLPCs, a sundry of metabolic mechanisms differs between these two cell types. LLPCs have higher glucose uptake than SLPCs, and utilize glucose for Ab glycosylation . MoreoveIgH mRNA is modulated post-transcriptionally (which requires Blimp-1)  , 61. ExpBlimp-1) , 99. HowRNA-binding proteins (RBPs) are involved in regulating the B cell programs and thus, can also influence ASC differentiation , 102. AnOur recent integrated transcriptomic and proteomic analysis distinguishing early minted ASCs and BM LLPCs identified a novel RBP, hnRNP A1, as a potential post-transcriptional modulator . BlockinEzh2, a histone methyltransferase that catalyzes trimethylation of histone H3 (H3K27me3), has been shown to epigenetically regulate gene expression during ASC differentiation. Ezh2 expression is upregulated during ASC differentiation, and Ezh2-deficient ASCs exert reduced levels of the UPR, glucose metabolism, and mitochondrial respiration, resulting in decreased Ab secretion  , 124, eoMesenchymal stromal/stem cells (MSCs) are the principal organizer of the BM microniche involved in supporting LLPC survival. Evidence shows that this support is mediated through cell-cell contacts and/or soluble signals , 94–145.+ MSCs are obligatory to form and maintain the survival niche  from BM MSCs provide additional evidence that MSC-ASC cell-cell contacts are non-essential . Both MSin vitro . Althougin vitro   . These s humans)   . BCMA is T cells . Moreove T cells . Molecul T cells , 176.neg ASCs could eventually upregulate SDC-1 in our in vitro BM mimetic cultures , otherwise known as CD138, a heparan sulfate proteoglycan (HSPG), has been shown to be a pro-survival factor for ASCs  Figure . While Bcultures . Not surin vitro BM mimetic cultures ; this interaction leads to dimerization and activation of the signal transducing protein gp130 , a transmembrane protein important for signal transduction following cytokine engagement through activation of the Jak-1/STAT and Akt/MAPK/ERK-1/2 signal transduction pathways, to eventually upregulate STAT3 –186. In in vivo  and its ligand CXCL12 (SDF-1) play an essential role in directing blood ASCs to BM , 54, 55.in vivo , or merein vivo . One stuin vitro . NonetheAdditional survival factors reported to date include TGF-β1 (as mentioned previously) , 154, IGex vivo . Previous systems supported human ASCs with co-cultures of supportive cells for a few days or from in vitro generated ASCs , of the Ab secreted from circulating ASCs can easily be assessed prior to serum level. Additionally, durability of the vaccines may also be assessed in vitro using the iBMM.The ability to support human blood and BM ASC survival In summary, the generation of LLPCs is complex and involves both intrinsic and extrinsic factors. B cell differentiation to an ASC is immensely intricate involving GC and non-GC reactions together with upregulating the ASC programs. Although ASC differentiation is necessary it is not sufficient for LLPC generation, which involves BM survival factors to maintain ASC Ab secretion as well as other extrinsic elements in the microniche that appear to fundamentally transform early minted ASCs into a LLPC phenotypes. The novel iBMM culture system provides tools for studying LLPC generation and maintenance. Coupling with the recent advances in transcriptomics, at both a high-throughput scale and single-cell resolution, this system has the potential to answer questions about the intrinsic and extrinsic factors that regulate differentiation, maturation, and longevity of human LLPCs. Despite the progress in understanding the extrinsic cues that support ASC survival and Ab secretion, more studies are needed to characterize the complex maturation and maintenance of LLPCs.DN, CJ, and FL wrote the manuscript. IS provided important editorial input.FL founded MicroB-plex, Inc. The remaining 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.


Fiber at majority fruiting positions under CCMS were longer and stronger than those under ICMS, but CCMS increased the contribution of bolls on FB11+ to the cotton yield, which overall resulted in no significant change in fiber length and strength by management system at field level. The number of bolls at FP1−2 on FB1−5 under CCMS while the number of bolls on FB1−5 and at FP1−2 on FB1−5 were not significantly changed by soil fertility, resulting in diminished yield difference in soil fertility among ICMS relative to that of CCMS. The high soil fertility significantly increased seedcotton yield relative to low soil fertility, which was attributed to more number of bolls on FB11+ and higher seedcotton weight per boll at all fruiting positions. High soil fertility field not only recorded superior fiber quality on FB11+, but also increased the contribution of these bolls to the cotton yield relative to those in the low soil fertility field, resulting in no significant change in overall fiber quality among soil fertility. These findings demonstrate that by combining optimal management practices on infertile soils ICMS could minimize the yield differences due to soil fertility without sacrificing fiber quality.In order to increase cotton productivity and optimize fiber quality on limited arable land, an integrated crop management system (ICMS), which combined with some optimal management practices, is projected to replace the conventional crop management system (CCMS) for cotton production in the Yangtze River valley. The seedcotton yield and fiber quality with respect to fruiting position under ICMS and CCMS were investigated in 2012 and 2013 in two fields differing in soil fertility. Reduced bolls on fruiting branches 1–10 (FB Gossypium hirsutum L.) is an important cash crop and provides raw material for producing textiles. However, with increasing population and greater demand for food in China, a large area of fertile land previously cultivated to cotton has been occupied by food crops  at Dafeng, Jiangsu, China . The soil at the experimental site was typical sandy loam, and two soil fertility levels were identified based on the soil nutrient status Table . Siza 3,A CCMS and an ICMS was applied to two fields with different soil fertility levels in a 2 × 2 factorial design, with three replicates of the four treatments. CCMS followed a longstanding management practice which is utilized by the majority of farmers in the Yangtze River valley , FB6−10 (fruiting branches 6 through 10), FB11−15 (fruiting branches 11 through 15) and FB16+ (the 16th and upper fruiting branches). Similarly, plants were divided horizontally into two primary zones, the first and second fruiting positions closest to the main stem (FP1−2) and the third and greater fruiting positions (FP3+). Fifteen successive plants from three central rows in each replicate plot of two management systems in the low and high soil fertility fields were randomly tagged at maturity to determine the number of fruiting branches and positions, and the number of mature bolls (>2 cm in diameter) on the eight primary zones per unit area.Vertically, plants were divided into four primary zones, i.e., FB3, FB8, FB13 were chosen as representative samples for zones FB1−5, FB6−10, FB11−15, respectively. Bolls from FB18 and FB16 were chosen to represent FB16+ of CCMS and ICMS, respectively. White blooms in each position were tagged with a jeweler tag, labeling the flowering and boll opening date, which encompasses the entire boll development period. All tagged, open bolls from each primary zone were harvested by hand. The seedcotton was ginned, and fiber was sent to the Cotton Quality Supervision, Inspection, and Testing Center of the China Ministry of Agriculture for quality analysis. Fiber qualities including fiber length, strength, and micronaire of each lint sample were measured with the Uster HVI MF100 cotton fiber quality analyzer .Bolls from FB−2 s−1 light intensity, 65 ± 5% relative humidity, 32 ± 2°C leaf temperature and 380 ± 5 ppm CO2 between 9:30 and 11:00 a.m. on cloudless days.Net photosynthetic rate was measured on main stem leaves of representative fruiting branches on July 15, August 15 and September 15 in 2012 and 2013 in each field. Measurements were made using a Li-6400XT portable photosynthetic system  under 1,500 μmol mThe effects of year (Y), soil fertility (S), management system (M) and their interactions on environmental conditions, morphological indices and net photosynthetic rates were analyzed using a full factorial three-way ANOVA. While, The effects of year (Y), soil fertility (S), management system (M), fruiting positions (FP) and their interactions on seedcotton yield, yield components, and fiber quality with respect to fruiting positions were analyzed using a full factorial four-way ANOVA. All statistical analyses in our study were performed using IBM SPSS Statistics 20.0 .The flowering date, time required for boll maturation and the mean daily temperature of bolls with respect to fruiting position varied significantly across years, but they were not affected by the interactions between year and any of other factors Table . The floThe flowering date, duration of boll development, and environmental conditions experienced by bolls at different fruiting positions were significantly affected by year and management system, but only the flowering date and time required for boll maturation were significantly affected by soil fertility Table . Among mAll the morphological indices of cotton measured in the present study varied significantly across years, but there was no interaction between year and any other effects Table . The varThe morphological indices of cotton were significantly affected by soil fertility and management system but no S × M interaction was observed Table . Among mSeedcotton yield, boll number and seedcotton weight per boll with respect to fruiting position varied significantly across years, but no interactions between year and any other effect were observed Table . The var1−10 and at FP1−2 on FB11−15 than CCMS. In contrast, the number of bolls on FB16+ under ICMS was significantly lower than for the CCMS treatment , and M × FP interaction, but not by S × M and S × FP interactions Table . Among mnt Table . Intereson Table , and seeon Table .11+) were more important contributors to overall yield production under CCMS and/or in the high soil fertility field than those of ICMS and/or in the low soil fertility field. The average contribution of the bolls on FB11+ to the total yield under CCMS was 11.13% higher than that under ICMS and the average contribution of the bolls on FB11+ to the total yield in the high soil fertility field was 3.48% higher than that in the low soil fertility field , soil fertility, and management system, but only the net photosynthetic rate of leaves on FB1−5 and FB6−10 was significantly affected by S × M interaction   Table . DecreasThe nutrient status of the high soil fertility field contributed to a greater number of bolls Table  and highThere was no significant difference in fiber length, strength and micronaire between the low and high soil fertility fields at the field level; similar results were observed by Blaise et al. , who rep1−10 and at FP1−2 on FB11−15 under CCMS could not be fully compensated for by increased bolls on FB16+, resulting in lower overall seedcotton yield under CCMS relative to that under ICMS.The boll reduction on FB1−2 on FB1−5 under CCMS while the number of bolls on FB1−5 and at FP1−2 on FB6−10 under ICMS were not significantly changed by soil fertility, resulting in diminished yield differences in soil fertility among ICMS relative to that of CCMS.The number of bolls at FP11+) and the seedcotton weight per boll at all fruiting positions.High soil fertility enhanced seedcotton yield by increasing the number of bolls on upper fruiting branches (FB1−10) to the total yield.Fiber length and strength at the majority of fruiting positions under CCMS was superior to those under ICMS. However, there was no significant difference in fiber length and strength between CCMS and ICMS at the field level, which was attributed to the elevated contribution of bolls on lower fruiting branches (FB11+) to the total yield. Thus, fiber length, strength and micronaire were not changed by soil fertility at the field level.High soil fertility increased not only fiber length, strength, and micronaire, but also the contribution of bolls on upper fruiting branches (FBICMS maybe have a greater capacity to tolerate extreme climate events than CCMS, but this needs to be further verified in the future.In our study, we evaluated the responses of seedcotton yield and fiber quality as a function of fruiting positions, management systems, and soil fertility levels. Our results clearly demonstrated that:ZZ, YM, and BC conceived the idea and led the study design. XZ carried out the experiment, performed analysis and wrote the paper. HY assisted in plant sampling and laboratory analysis. RZ, JS and BI assisted manuscript writing and editing. All authors reviewed the manuscript.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.


Background. Major Depressive Disorder (MDD) is the most common psychiatric disorder with high prevalence and disease burden. Biological treatments of MDD over the last several decades include a wide range of antidepressants and neurostimulation therapies. While recent meta-analyses have explored the efficacy and tolerability of antidepressants, the changing trends of biological treatments have not been evaluated. Our study measured the indices of change, expectations, and popularity of biological treatments of MDD between 1988 and 2017. Methods. We performed a scientometric analysis to identify all relevant publications related to biological treatments of MDD from 1988 to 2017. We searched the Web of Science websites for publications from 1 January 1988 to 31 December 2017. We included publications of fluoxetine, paroxetine, citalopram, sertraline, amitriptyline, fluvoxamine, escitalopram, venlafaxine, duloxetine, milnacipran, desvenlafaxine, levomilnacipran, clomipramine, nortriptyline, bupropion, trazodone, nefazodone, mirtazapine, agomelatine, vortioxetine, vilazodone, electroconvulsive therapy (ECT), repetitive transcranial magnetic stimulation (rTMS), vagus nerve stimulation (VNS), deep brain stimulation (DBS), and transcranial direct current stimulation (tDCS). We excluded grey literature, conference proceedings, books/book chapters, and publications with low quality as well as publications not related to medicine or human health. The primary outcomes assessed were indices of change, expectations, and popularity. Results. Of 489,496 publications identified, we included 355,116 publications in this scientometric analysis. For the index of change, fluoxetine, sertraline and ECT demonstrated a positive index of change in 6 consecutive periods. Other neurostimulation therapies including rTMS, VNS, DBS and tDCS had shown a positive index of change since 1998. We calculated the index of change of popularity index (PI), which indicates that from 2013 to 2017, the number of publications on tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs) were reduced by 85.0% and 81.3% respectively, as compared with the period 2008–2012. For the index of expectation, fluoxetine and ECT showed the highest index of expectations in six consecutive periods and remained the highest in 2013–2017. For popularity, the three antidepressants with highest PI were fluoxetine (4.01), paroxetine (2.09), and sertraline (1.66); the three antidepressants with lowest PI were desvenlafaxine (0.08), vilazodone (0.04) and levomilnacipran (0.03). Among neurostimulation therapies, ECT has the highest PI (2.55), and tDCS the lowest PI (0.14). The PI of SSRI remained the highest among all biological treatments of MDD in 2013–2017. In contrast, the PI of ECT was reduced by approximately 50% during the period 2008 to2012 than that in the period 2013 to 2017. Conclusions. This scientometric analysis represents comprehensive evidence on the popularity and change in prospects of biological treatments for MDD from 1988 to 2017. The popularity of SSRI peaked between 1998 and 2002, when their efficacy, tolerability and safety profile allowed them to replace the TCAs and MAOIs. While the newer neurostimulation therapies are gaining momentum, the popularity of ECT has sustained.  Depression is the most common psychiatric disorder with the aggregate point, one-year and lifetime prevalence of 12.9%, 7.2%, and 10.8% respectively . DepressOver the last several decades, biological treatments of MDD have made significant progress. The mechanism of action for all available antidepressants is mainly based on the monoamine mechanisms . TricyclRecently, Cipriani et al. (2018) performed a network meta-analysis to evaluate the efficacy of 21 antidepressant drugs except for neurostimulation therapies . All antPrevious scientometric analyses in psychiatry focused on illicit drug addiction , child pTherefore, we performed a scientometric analysis to measure the index of popularity, change, and expectations of 23 antidepressants and 5 neurostimulation therapies including electroconvulsive therapy (ECT), repetitive transcranial magnetic stimulation (rTMS), transcranial direct current stimulation (tDCS), vagus nerve stimulation (VNS) and deep brain stimulation (DBS) at 5-year intervals from 1988–2017. We hypothesized that there would be no change in the index of popularity, change, and expectations of 23 antidepressants and 5 neurostimulation therapies at 5-year intervals from 1993 to 2017 as compared to baseline period 1998–1992.The Web of Science (WOS) was used to search scientific articles related to depression published between 1 January 1988 to 31 December 2017. We decided to choose the WOS based on the following reasons: 1) the WOS included articles published in higher quality journals compared to other databases ; 2) PubMed focuses on biomedical studies only and resulted in selection bias while the WOS includes articles from different fields  [The keywords “Depress*” (including depression and depressive) OR “antidepressant*” (including antidepressant and antidepressants) OR “tricyclic-antidepressant” OR “TCA”, OR “selective serotonin reuptake inhibitor” OR “SSRI”, OR “serotonin noradrenaline reuptake inhibitor” OR “SNRI”, OR “monoamine oxidase inhibitor” OR “MAOI”, OR ‘noradrenaline/norepinephrine-dopamine reuptake inhibitor” OR “NDRI” OR “noradrenergic specific serotonergic antidepressant” OR “NASSA”, OR “serotonin antagonist and reuptake inhibitor” OR “SARI”, OR “electroconvulsive therapy” OR “ECT” OR “repetitive transcranial magnetic stimulation” or “rTMS” OR “vagus nerve stimulation” OR “VNS” OR “deep brain stimulation” OR “DBS” OR transcranial direct current stimulation” OR “tDCS” were used to search all articles and reviews from 1988 to 2017 that contained these words in the title, abstract or keywords see . We focuTwenty-three antidepressants were searched in various kinds of literature: six SSRIs ; five SNRIs ; three TCAs ; one NDRI (bupropion); two SARIs (trazodone and nefazodone), one NaSSA (mirtazapine), and three new antidepressants . We included five neurostimulation therapies: ECT, rTMS, VNS, DBS, and tDCS. The name of each antidepressant and therapy above was entered in the search box with the combination of the above keywords and “depress”. In addition to the terms related to the primary field of depression or depressive disorder, we applied the sub-specialty areas: Psychiatry, Neuroscience, Neurology, Psychology, Pharmacology and Pharmacy. These sub-specialties are based on the research areas defined by the WOS. Data including the publication year, total papers published per year per biological treatment and the number of papers published per biological treatment were extracted. In the evaluation of biological treatments for MDD, we applied the following indicators ,21,22.The PI is the share of papers on a specific topic (the name of an antidepressant or neurostimulation therapy) relative to all articles in the field of MDD in a period of time. The index of change is the percentage change in the number of publications of an antidepressant or neurostimulation therapy during a period of five years compared to the previous five years. This index reflects the change of interest on a biological treatment for MDD. indicating that the increase in the number of publications (in percentage) on a biological treatment in the whole field of MDD during the current period as compared to previous period.The index of expectations or top journal selectivity index is the ratio of the number of articles of an antidepressant or neurostimulation therapy in the top 20 journals relative to the total number of articles in biological treatments for MDD. It reflects the level of interest on the particular biological treatment of MDD in the top journals. We used the WOS to select the 20 top journals for each antidepressant or therapy in consecutive five-year periods see . With the 23 antidepressants, the criteria for selection of an antidepressant into further analysis was the level of its PI during 1988–2017. If the PI was higher than 0.5 , an antiEthical approval was not required for this study as it does not involve direct involvement of research participants.The search identified a total of 489,496 publications. We excluded 134,380 publications that did not meet inclusion criteria: 1) research article or review; 2) non-English papers; 3) anonymous author . We inclThe PI indicates the biological treatments which were the subject of the highest number of publications for the treatment of MDD in the period 1988–2017. Of the 23 antidepressants included in the search, eight antidepressants have a PI larger than 0.5%. The PI of major classes of antidepressants and neurostimulation therapies in four sub-specialties are presented in The PI of 23 antidepressants and neurostimulation therapies are presented in n = 16,331)  see .The PI for major classes of antidepressants and neurostimulation therapies for the treatment of MDD between 1988 and 2017 is shown in During the same period, the PI of ECT had reduced gradually from −17.5% during 1993–1997 to −48.1% during 2013–2017. The index of change of SSRI and SNRI had increased during 1993–1997 and 1998–2002. Nevertheless, the growth rate has reduced since the period 2003–2007, and index of change took a negative direction in the subsequent periods. The key findings of this scientometric review are summarized as follows: The popularity of SSRIs remained the highest among all biological treatments of MDD in 2013–2017. Three SSRIs, fluoxetine, paroxetine and sertraline were the subject of the highest number of publications. By comparison with other antidepressants, the popularity of fluoxetine and sertraline had increased in the past 30 years with a positive index of change in 6 consecutive periods. The popularity of the older antidepressants such as the TCAs had declined since 1998. For neurostimulation therapy, the popularity of ECT had increased in the past 30 years with a positive index of change in 6 consecutive periods. The PI of rTMS, VNS, DBS and tDCS were higher in the field of Neurosciences and Neurology compared to Psychiatry. Although the PI of other neurostimulation therapies including rTMS, tDCS and DBS had increased, they could not replace ECT. The popularity of SSRIs reached its peak in 1998–2002. The index of change of journal articles on the biological treatment of MDD started to decline since 2003. Three antidepressants, fluoxetine, paroxetine and sertraline had the highest PI. The Canadian Network for Mood and Anxiety Treatments (CANMAT) guidelines recommend all SSRIs as first-line treatment with level 1 evidence (evidence from at least 1 RCT) . SSRIs, Three antidepressants with lowest PI are desvenlafaxine, vilazodone and levomilnacipran. Desvenlafaxine is a synthetic form of the isolated major active metabolite of venlafaxine. Its effectiveness is comparable to the parent drug, venlafaxine which is an established SNRI. It is not surprising that desvenlafaxine does not obtain much popularity because it does not offer additional therapeutic advantages as compared to the parent drug. Furthermore, its parent drug, venlafaxine was found to be one of the antidepressants with the most side effects in a network meta-analysis . VenlafaThe popularity of ECT has increased in the past 30 years with a positive index of change over 6 consecutive periods. ECT remains one of the most effective treatments for MDD, with response rates as high as 80%, and remission rates 50% or higher. It has the level 1 evidence (evidence from at least 1 RCT) in acute efficacy, maintenance efficacy, safety and tolerability . The NatThe popularity of other neurostimulation therapies including rTMS, tDCS and DBS had increased, but these new therapies were not able to replace ECT. The NICE guidelines (UK) reported that ECT was found to be more clinically effective , and cosThere are several reasons to explain the decline in the publication of journal articles regarding the biological treatment of MDD from 2003 onwards. First, there have been significant advances in psychopharmacology which have offered better treatment options for those suffering from MDD. However, the field has now reached a steady state and improvements brought about by new antidepressants are expected to be less significant than those achieved 30 years ago . Second,n = 355,116), there was a high possibility that these articles were indexed in other databases including PubMed, Embase, PsychInfo and Cochrane Library. Hence, articles included in this study are representative of the field.This study inherited the general limitations of the scientometric analysis. First, the scientometric indices could not differentiate between publications characterizing an antidepressant positively or negatively ,22. FurtThis scientometric analysis represents the most comprehensive evidence on the popularity and change in prospects of 23 antidepressants and 5 neurostimulation therapies in the treatment of MDD from 1988 to 2017. Among 23 antidepressants, fluoxetine, paroxetine and sertraline were the subjects of the highest number of publications. The popularity of SSRI reached its peak in 1998–2002 while TCA and MAOI were replaced by other antidepressants and neurostimulation therapies. Among 5 neurostimulation therapies, the popularity of ECT had increased in the past 30 years with a positive index of change in six consecutive periods. Other antidepressants and neurostimulation therapies have not yet been able to replace ECT.


Here, we used RNA sequencing to screen the most likely regulators of NKA signaling and found that the liver kinase B1(LKB1)/adenosine monophosphate (AMP)-activated protein kinase (AMPK)/ mammalian target of rapamycin (mTOR) pathway was the most abundantly enriched pathway in HUA. AMPK is a key regulator of cell energy metabolism; hence, we examined the effect of AMPK on HUA-induced dysregulation of NKA signaling and cell injury. We first detected AMPK activation in high uric acid (UA)-stimulated proximal tubular epithelial cells (PTECs). We further found that sustained treatment with the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), but not the AMPK inhibitor Compound C, significantly alleviated UA-induced reductions in NKA activity and NKA α1 subunit expression on the cell membrane by reducing NKA degradation in lysosomes; sustained AICAR treatment also significantly alleviated activation of the NKA downstream molecules Src and interleukin-1β (IL-1β) in PTECs. AICAR further alleviated high UA-induced apoptosis, autophagy, and mitochondrial dysfunction. Although AMPK activation by metformin did not reduce serum UA levels in hyperuricemic rats, it significantly alleviated HUA-induced renal tubular injury and NKA signaling impairment in vivo with effects similar to those of febuxostat. Our study suggests that AMPK activation may temporarily compensate for HUA-induced renal injury. Sustained AMPK activation could reduce lysosomal NKA degradation and maintain NKA function, thus alleviating NKA downstream inflammation and protecting tubular cells from high UA-induced renal tubular injury.One of the mechanisms in hyperuricemia (HUA)-induced renal tubular injury is the impairment of Na +-K+-ATPase (NKA) signaling pathway, which regulates uric acid transportation through the kidneys, is a crucial feature of renal damage progression. The team have now shown that NKA is regulated by the AMP-activated protein kinase (AMPK) pathway, and that AMPK is enriched during the initial phases of hyperuricemia. Studies on rat models indicated that sustained AMPK activation restored NKA signaling, limiting damage from hyperuricemia.High serum levels of uric acid cause kidney tissue damage through cellular processes that have now been identified by researchers in China. Uric acid is a common component of urine, but causes damage if it is present in high levels in the blood (hyperuricemia). While investigating the mechanisms behind hyperuricemia, Zhibin Ye and co-workers at Fudan University in Shanghai recently showed that impairment of the Na Renal tubulointerstitial injuries are frequently detected in urate nephropathy4. The renal proximal tubule is responsible for almost all renal urate transport and is the primary location for urate reabsorption5. We previously found that impaired activity and subcellular expression of Na+-K+-ATPase (NKA), the driver of urate transport on the basolateral side of proximal tubular epithelial cells (PTECs), lead to increased cellular autophagy; apoptosis; mitochondrial dysfunction; reduced intracellular ATP levels; sequential NKA-dependent activation of Src, Nod-like receptor pyrin domain-containing protein 3 (NLRP3), and interleukin-1β (IL-1β); and deregulation of urate transporters in HUA-induced renal tubular injury. These effects suggest that cellular metabolic disorder of such an ATPase could transfer stress signals to cause downstream inflammatory injury, leading to the deregulation of urate transportation and ultimately enhancing the renal injury of hyperuricemia6. However, whether alleviation of cell metabolic disorder can attenuate NKA-dependent HUA-induced renal tubular injury is currently unknown.Hyperuricemia (HUA) has recently been recognized as an independent risk factor for renal injury7. AMPK activation via phosphorylation at Thr172 reduces energy consumption and cell activity and increases energy production 8, thus regulating the synthesis and decomposition of metabolic pathways to guarantee the cellular energy supply9. The roles of multiple signaling pathways downstream of AMPK in the pathogenesis of renal disease have been explored9. Moreover, AMPK regulates multiple ion channels in renal tubular epithelial cells10. In fact, an AMPK activator has been shown to preserve basolateral localization of NKA in Madin-Darby canine kidney cells subjected to energy depletion11 and to activate NKA activity in skeletal muscle cells12; however, the effect of AMPK on NKA signaling in HUA-induced renal tubular injury is currently unknown. Therefore, we hypothesized that AMPK might alleviate HUA-induced renal tubular NKA dysfunction and signaling impairment and thus subsequently reduce HUA-induced NKA-dependent inflammation and renal tubular cell injury. We first studied the effect of UA on AMPK activation in cultured PTECs. We then examined the effect of AMPK on NKA activity, NKA α subunit subcellular expression, downstream Src-NLRP3-IL-1β signaling, autophagy, and mitochondrial function and examined possible pathways involved. We also evaluated and compared the AMPK-activating effects of metformin with those of the UA-lowering agent febuxostat in an HUA rat model.To screen possible target pathways involved in HUA-induced renal tubular injury, we performed RNA-seq on high uric acid (UA)-treated and untreated PTECs and found that adenosine monophosphate (AMP)-activated protein kinase (AMPK) was highly elevated upon UA treatment. AMPK is the most sensitive cell energy metabolism regulatorHuman primary renal PTECs and cell culture medium were purchased from ScienCell . UA and oxonic acid (OA) were purchased from Sigma . An IL-1β ELISA kit was purchased from eBioscience . A Cell Surface Protein Isolation Kit was purchased from Pierce . Anti-NKA α1, Src/phosphorylated (p)-Src, AMPK/pAMPK, CIAS1/NALP3, peroxisome proliferator-activated receptor γ-coactivator-1α (PGC-1α), beclin-1, SQSTM1/p62, lysosome-associated membrane protein 2 (LAMP2), and uncoupling protein 2 (UCP2) antibodies and a reactive oxygen species (ROS) production assay kit were obtained from Abcam . Anti-mammalian target of rapamycin (mTOR)/pmTOR and forkhead box O3a (FoxO3a)/pFoxO3a antibodies were obtained from Cell Signaling Technology . An anti sirtuin type 1 (Sirt1) antibody was obtained from Millipore . An anti-IL-1β antibody was obtained from Santa Cruz Biotechnology . An anti-LC3 antibody was obtained from Novus . Secondary antibodies were purchased from Sinobio . An NKA activity kit, a mitochondrial isolation kit, a mitochondrial membrane potential (MMP) assay kit, an aldolase assay kit and respiratory complex I, II, III, IV, and V activity kits were purchased from Genmed . LysoTracker Red was purchased from Beyotime . UA, AMP, and ATP measurement kits were purchased from Jiwei Biological Technology . An annexin V/propidium iodide (PI) flow cytometry assay kit was obtained from Becton Dickinson . mtDNA and 18SRNA primers were obtained from Invitrogen . A Cell Counting Kit-8 (CCK-8) kit was purchased from Dojindo Laboratories . Febuxostat was kindly provided by Jiangsu Hengrui Medicine Co., Ltd. . Metformin was purchased from MedChemExpress .n = 5 in each group): (1) the Cont group, (2) the OA group (750 mg/kg/day OA for 8 weeks), and (3) the OA + Feb group (750 mg/kg/day OA for 8 weeks + 3 mg/kg/day Feb for 4 weeks starting at the 5th week), and 4) the OA + Met group (750 mg/kg/day OA for 8 weeks + 250 mg/kg/day Met for 4 weeks starting at the 5th week). The rats were sacrificed at the end of the 8th week. Twenty-four hour urine samples were collected from the rats with metabolic cages. Serum UA (SUA), urinary UA (UUA), blood urea nitrogen (BUN), serum creatinine (Scr), urinary creatinine (Ucr), serum sodium (SNa), urinary sodium (UNa), serum potassium (SK) and urinary potassium (UK) concentrations were determined by an enzymatic colorimetric assay method using a fully automatic chemistry analyzer . Urinary albumin (UALB) was measured using the sulfosalicylic acid method. The urinary albumin to creatinine ratio (UACR) was calculated as UALB/Ucr. Fractional excretion of uric acid (FEUA) was calculated as (UUA × Scr)/(SUA × Ucr) × 100 and expressed as a percentage. Fractional excretion of sodium (FENa) was calculated as (UNa × Scr)/(SNa × Ucr) × 100 and expressed as a percentage. Fractional excretion of potassium (FEK) was calculated as (UK × Scr)/(SK × Ucr) × 100 and expressed as a percentage.Twenty 8-week-old male-specific pathogen-free SD rats weighing 200 to 250 g were bred at the animal center of Shanghai Rat & Mouse Biotech Co., Ltd. The rats were divided into four groups  or Masson’s trichrome stain for light microscopy. The rest of the tissues were stored at −80 °C for immunoblotting assays. All animal procedures were performed in accordance with the National Institutes of Health guidelines  and were approved by the Animal Care and Use Committee of Shanghai Rat & Mouse Biotech Co., Ltd.5 preseeded PTECs. After 0, 24, and 48 h of incubation, the supernatants were discarded, and 200 μL of TRIzol reagent  was added to each of the triplicates for the three groups.UA-containing (100 μg/mL) cell culture medium was added to stimulate 2 × 10Total RNA was extracted by using TRIzol and a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol with DNase treatment. RNA quality was evaluated with an Agilent 2100QC Bioanalyzer (Agilent Technologies). All RNA samples had RNA integrity numbers (RINs) greater than 8.0. Briefly, 2 μg of total RNA was subjected to ribosomal RNA removal using a Ribo-Zero Magnetic kit , 300 ng of rRNA-depleted RNA was fragmented, and cDNA was synthesized using random primers  and SuperScript II polymerase (Invitrogen). Illumina TruSeq adaptors were added to the second strand and further amplified with P5/P7 primers. Fragments 350–500 bp in length were recovered with a gel extraction kit. All libraries were quantified and pooled at concentrations of 2 nM for sequencing on a NovaSeq system .The raw read data from nine PTEC samples were filtered with Trimmomatic v0.22, mapped to the UCSC Hg19 human genome and annotated with the RefSeq database using TopHat v2.0.0. The mapping results were then processed using Cufflinks v2.0.4 for transcript assembly. The FPKM (Fragments per Kilobase per million mapped reads) value for each RefSeq-annotated gene was calculated and then transformed to a log2 scale. To avoid infinite values, a value of 1 was added to the FPKM value for each gene before log2 transformation.http://www.r-project.org/). Other analyses, such as expression heatmap analysis, Pearson correlation analysis, Student’s t-test, principal component analysis (PCA), and linear discriminant analysis (LDA), were performed using R (v3.3.3).Hierarchical clustering analysis (HCA) was performed using the Ward linkage method based on a distance matrix of the Pearson correlation of the samples in R  and Reactome pathway databases using Gene Set Enrichment Analysis (GSEA v3.0 beta).13. Cell viability, apoptosis, MMP, and respiratory complex activity were analyzed according to the kit manufacturers’ instructions. Primers and probe sets for human mtDNA and 18SRNA were designed from known sequences in GenBank (Table Human primary PTEC culture and soluble UA preparation were performed as previously described13 with antibody dilutions of 1:2000 (beclin-1 and LC3), 1:1000 , and 1:500 .Western blot analysis was conducted as described previously14. NKA activity specifically refers to ouabain-sensitive NKA activity and was defined as the amount of NKA required to oxidize 1 mol of NADH into nicotinamide adenine dinucleotide (NAD) at 37 °C and pH 7.5 per milligram of protein per minute. All procedures were performed according to the manufacturer’s instructions.NKA activity in cultured PTECs was measured with an NKA activity kitg for 10 min at 4 °C. The supernatant was collected and subjected to NKA activity measurement. NKA activity was obtained by measuring inorganic phosphate (Pi) release using an NKA activity kit  as per Forbush’s method15. The procedures were performed according to the manufacturer’s instructions. Readings were obtained at 660 nm. Enzyme-specific activity is expressed as 1 μmol of Pi released per milligram of protein per hour.Fifty micrograms of fresh rat renal cortex tissues were collected, 50 mL of 0.01 mol/L PBS (pH 7.4) was added, and the renal tissue was homogenized using a homogenizer. The samples were centrifuged at 8000 × 13 using mouse anti-NKA α1 (1:500) as the primary antibody. The colocalization of NKA α1 and LysoTracker Red was evaluated by immunocytochemistry as previously described16 and according to the manufacturer’s instructions using an anti-NKA α1 antibody diluted 1:200 in staining buffer. Fluorescence was detected using an LSM 510 Meta confocal laser-scanning microscope . Western blotting of NKA α1 on the renal cortex was conducted with an anti-NKA α1 (1:1000) antibody.The abundance of the endogenous NKA α1 subunit on PTEC membranes was analyzed by determining surface biotinylation using a Cell Surface Protein Isolation Kit according to the manufacturer’s instructions. Surface proteins were eluted and processed for Western blotting as described previouslyThe IL-1β and aldolase protein levels in culture supernatants were determined using commercial assay kits according to the manufacturers’ instructions. Cell lysates were collected, and intracellular ATP and AMP levels were measured using bioluminescence assay kits according to the manufacturer’s instructions.P < 0.05 was considered to indicate statistical significance.All data are expressed as the means ± standard deviations (SDs) unless otherwise specified. The statistical analysis was performed using SPSS v19.0 for Windows . Intergroup differences in continuous variables were assessed by multivariate analysis of variance (ANOVA). To identify the UA-targeted pathway, we performed transcriptomic analysis on UA-treated and untreated PTECs. A total of 905 differentially expressed genes (DEGs) (FDR < 0.05) were observed in the UA-treated cells compared to the control cells after 24 h. Reactome-based Gene Set Enrichment Analysis (GSEA) was used to reveal the signaling pathways induced by UA treatment Fig. . Functio17, we further examined the dose- and time-dependent effects of UA on energy metabolism molecules, including AMP/ATP , aldolase, LKB1, and AMPK. UA stimulation  for 48 h increased the intracellular AMP/ATP ratios  or the AMPK activator AICAR (0.1 mM) 1 h before the 48 h stimulation with UA (100 μg/mL) to inhibit or activate AMPK, respectively. UA significantly increased LKB1 Fig.  and AMPKWe then used NKA activity and intracellular staining assays to identify whether AMPK activation plays a protective or detrimental role in the UA-induced impairment of NKA expression and signaling. The results showed that UA significantly reduced intracellular ATP levels Fig.  and incrUA significantly induced Src activation  for 8 weeks and administered Feb (3 mg/kg/day) or Met (250 mg/kg/day) for 4 weeks starting at the 5th week. OA rats showed significantly increased serum and liver UA levels, reduced FEUA, FEK and 24 h UUA levels, and reduced renal cortex AMP/ATP ratios Fig. . OA rats6. In the current study, we further verified by GSEA that the LKB1/AMPK/mTOR pathway was the most abundantly enriched pathway in UA-stimulated PTECs. We also demonstrated that UA significantly activated AMPK in the renal cortex of HUA rats and in UA-stimulated PTECs. The impairment of NKA signaling and the occurrence of NKA-mediated mechanisms of cell injury, such as ROS production, autophagy, apoptosis, mitochondrial dysfunction, and the inflammatory response, could be significantly alleviated by sustained AMPK activation.In our previous study, we demonstrated the contribution of impaired NKA to HUA-induced renal tubular inflammation17. We measured both AMP/ATP ratios and aldolase levels and demonstrated that the AMP/ATP ratio time-dependently increased and remained elevated at 72 h, while aldolase was reduced time-dependently until 48 h but did not continue to decrease after 48 h. Our findings suggest that UA may trigger the activation of LKB1/AMPK by increasing the AMT/ATP ratio and/or reducing aldolase levels, a possibility that needs further exploration. Similar to our findings, other researchers have also observed the activation of AMPK after 30 min of stimulation with 10 mg/dL and 15 mg/dL UA in rat pancreatic β-cells18 and after 24 h of stimulation with 15 mg/dL UA in skeletal muscle cells19 and in human monocytic THP-1 cells20. However, 16 h of stimulation with 0.2 mg/mL monosodium urate (MSU) crystals has been found to inhibit AMPK activation in macrophages21 and in a gouty arthritis animal model21. We have also examined whether soluble urate, but not the crystal form of MSU, exerted NLRP3-IL-1β-associated inflammatory effects in renal tubular13 and mesangial cells22. In the present study, we similarly used soluble UA rather than the crystal form. It is possible that different urate forms  could affect AMPK activation differently; this possibility needs further investigation.We first screened possible targeted pathways involved in HUA-induced renal tubular injury using RNA-seq in a cell model, and the GSEA results revealed that the LKB1/AMPK/mTOR pathway was the most abundantly enriched pathway. We further validated and demonstrated the activation of AMPK in a high UA-stimulated in vitro PTEC model. LKB1 and AMPK activation increased gradually at first, peaked at 48 h, and started to decrease at 72 h under UA stimulation, suggesting a possible temporary compensatory response of LKB1/AMPK to counteract the detrimental effect of UA; however, at 72 h, AMPK activity started to decrease. LKB1 is activated by high AMP/ATP ratios, which are classic activators, and by the recently found aldolase6 and that AMPK activation is observed in HUA-induced renal tubular injury23. In our present study, we further noted that AMPK activation significantly increased NKA activity by restoring NKA cell surface expression following AICAR treatment, whereas the AMPK inhibitor Comp C exerted the opposite effects. In HUA rats, although metformin-mediated AMPK activation did not significantly reduce serum UA, as febuxostat-mediated activation did, metformin treatment significantly alleviated NKA signaling impairment and renal tubular injury, effects that were not inferior to those of febuxostat. This result suggested that the renal tubular protective effect of AMPK activation by metformin in vivo did not depend on reductions in serum UA. Although metformin failed to reduce high UCP2 levels in OA-treated rats, it significantly increased renal ATP production compared with febuxostat, suggesting that AMPK could provide more energy for renal tubules, and further exerted a protective effect through restoration of NKA expression on the membrane. Large amounts of ATP are needed to enable NKA function and to maintain normal NKA activity. Similar results have been observed in canine kidney cells, in which the AMPK inhibitor Comp C increased NKA cell internalization and degradation24, whereas the AMPK activator metformin protected basolateral NKA in tubular cells from ischemia-induced cytoplasmic transport and degradation11. In L6 muscle cells, AICAR also increases NKA activity and expression on the cell membrane via NKA dephosphorylation at Ser1812. However, in lung epithelial H441 cells, AICAR inhibits NKA activity26 and mediates NKA internalization and degradation29. It is possible that renal tubular cells and muscle cells are energy-demanding cells, whereas lung tubular cells consume less energy.We have previously demonstrated that the impairment of NKA function is one of the pivotal steps in HUA-induced renal tubular injury17; such sensing occurs on lysosomes and depends on the activation of LKB1 but can be triggered by aldolase during glucose deficiency17. We also noted that AMPK activation by AICAR did not reduce the AMP/ATP ratio, while Comp C significantly reduced the AMP/ATP ratio but did not exert any protective effect. We examined aldolase and LKB1 levels under UA stimulation and found that aldolase and LKB1 were activated, suggesting the activation of AMPK by aldolase/LKB1 under UA stimulation; however, this possibility needs further investigation. It is generally recognized that AMPK can inhibit mTOR complex 1 (mTORC1) activity30, releasing the inhibitory effect of mTORC1 and allowing autophagy to proceed31. Our GSEA analysis demonstrated enrichment of the LKB1/AMPK/mTOR pathway, and we observed reduced mTOR phosphorylation and increased autophagy in UA-stimulated cells accompanied by activation of AMPK. Although AICAR did not further change the extent of mTOR phosphorylation under UA stimulation, it significantly alleviated autophagy, while the AMPK inhibitor Comp C significantly increased mTOR phosphorylation and enhanced autophagy in high UA-stimulated PTECs. These findings suggest that mTOR is actively involved in the effect of AMPK on high UA-induced autophagy. Emerging evidence has identified a common location, the lysosome, as the site of activation for both mTORC1 and AMPK during the shift between starvation and nutrient repletion34. AMPK activation by metformin has been found to occur through the lysosomal pathway35. We also observed that UA increased the levels of the lysosome marker LAMP2 but that LAMP2 was significantly reduced by AICAR, suggesting that lysosomes might be the key organelles involved in the effect of AMPK on UA-induced NKA-associated injury. AMPK may regulate the stress signal for NKA degradation under UA stimulation, and mTOR may be a key regulator involved.As NKA is degraded in lysosomes and as AMPK reduces NKA expression in lysosomes, we studied mechanisms in vitro, examining apoptosis and autophagy levels in cells under HUA conditions with or without AMPK modulators. We observed that AMPK activation by AICAR significantly relieved high UA-induced apoptosis, autophagy and lysosomal marker changes. Recent studies have demonstrated that AMPK senses not only cellular glucose but also cellular stress and that this sensing of AMPK could be independent of the AMP/ATP ratio36, and we demonstrated that supplementation with NKA restored mitochondrial function in high UA-stimulated PTECs in our previous study6. In the current study, we demonstrated that AMPK activation by AICAR significantly relieved UA-induced mitochondrial dysfunction, as demonstrated by increased mtDNA, mitochondrial complex I and V (ATP Synthase) activity, and ATP production and reduced mitochondrial ROS, MMP and UCP2 expression. We also studied regulators of mitochondrial biogenesis and found that UA significantly increased Sirt1, PGC-1α, and FoxO3a phosphorylation. AMPK activation significantly alleviated the increases in Sirt1 and FoxO3a phosphorylation, while Comp C exerted no effect on Sirt1 and significantly enhanced HUA-induced FoxO3a phosphorylation, suggesting the involvement of Sirt1/FoxO3a in AMPK-mediated regulation of mitochondrial function under high UA stimulation in renal tubules. As FoxO3a is one of the key regulators of autophagy/apoptosis38, we suggest that AMPK may act through FoxO3a to enable crosstalk between mitochondria and lysosomes.In the classic Whittam model, NKA is coupled with mitochondrial functionIn summary, we found that high UA-induced activation of AMPK temporarily compensated for HUA-induced renal injuries, including translocation of membrane NKA to lysosomes, NKA downstream Src-NLRP3-IL-1β signaling, increased autophagy and apoptosis, and mitochondrial dysfunction. Sustained AMPK activation reduced lysosomal NKA degradation and downstream inflammation and alleviated UA-induced autophagy, apoptosis and mitochondrial dysfunction Fig. .Fig. 6Sc