Relationship between serum phosphorus and mortality in non-dialysis chronic kidney disease patients: evidence from NHANES 2001-2018.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease (CKD), associated with higher mortality in dialysis patients. Its impact in non-dialysis patients, especially those with preserved kidney function, remains uncertain. METHODS: A prospective cohort study was conducted using data from the National Health and Nutrition Examination Survey (2001-2008). Serum phosphorus was analyzed as a continuous variable, or categorized into three groups: < 3.5 mg/dL, 3.5 to < 4.5 mg/dL, and ≥ 4.5 mg/dL. Cox proportional hazards models were used to analyze the association between phosphorus with all-cause and cardiovascular disease (CVD) mortality, with or without adjustment for age, sex, race, hemoglobin, estimated glomerular filtration rate (eGFR), serum albumin, serum calcium, 25(OH)D, obesity, hypertension, diabetes, and CVD. RESULTS: A total of 7694 participants were included in the analysis, representing 28 million CKD patients in the United States. During mean 92 months of follow up, 2708 all-cause deaths (including 969 CVD deaths) were observed. Per 1 mg/dL increase in phosphorus was associated with a 13% and 24% increased risk of all-cause mortality (hazard ratio [HR], 1.13; 95%CI, 1.02-1.24) and CVD mortality (HR, 1.24; 95%CI, 1.07-1.45), respectively. Compared with the < 3.5 mg/dL, phosphorus ≥ 4.5 mg/dL was associated with a 28% and 57% increased risk of all-cause mortality (HR, 1.28; 95%CI, 1.05-1.55) and CVD mortality (HR, 1.57; 95CI, 1.19-2.08), respectively. In participants with eGFR < 60 ml/min/1.73m2, elevated phosphorus (≥ 4.5 mg/ dL) were significantly associated with increased risk of all-cause mortality (HR, 1.36; 95%CI, 1.07-1.72). No significant association was observed in eGFR ≥ 60 ml/min/1.73m2 group (HR, 1.31; 95%CI, 0.86-1.99). This correlation does not differ significantly between subgroups defined by eGFR level (P for interaction = 0.889). CONCLUSION: Serum phosphorus above 4.5 mg/dL is significantly associated with a 28% and 57% increased risk of all-cause and CVD death in non-dialysis CKD patients, respectively. This relationship still demonstrated in patients with eGFR < 60 ml/min/1.73m2. However, for population with eGFR ≥ 60 ml/min/1.73m2, further verification is needed.

GBP2 upregulated in LPS-stimulated macrophages-derived exosomes accelerates septic lung injury by activating epithelial cell NLRP3 signaling.

Macrophages infiltration is a crucial factor causing Sepsis-associated acute lung injury (ALI). Accumulating evidence suggests macrophages-alveolar epithelial cells communication is proven to be critical in ALI. However, little is known regarding how activated macrophages regulated sepsis-associated ALI. To explore the role of macrophages-alveolar epithelial cells communication in the ALI process, our data revealed that Lipopolysaccharides-induced macrophages-derived exosomes (L-Exo) induced sepsis-associated ALI and caused alveolar epithelial cells damage. Moreover, Guanylate-binding protein 2 (GBP2) was significantly upregulated in L-Exo, and NLRP3 inflammasomes was the direct target of GBP2. Further experimentation showed that GBP2 inhibition in vitro and in vivo reserves L-Exo effects, while GBP2 overexpression in vitro and in vivo promotes L-Exo effects. These results demonstrated that L-Exo contains excessive GBP2 and promotes inflammation through targeting NLRP3 inflammasomes, which induced alveolar epithelial cells dysfunction and pyroptosis. These findings demonstrate that L-Exo exerted a deleterious effect on ALI by regulating the GBP2/NLRP3 axis, which might provide new insight on ALI prevention and treatment.

SIRT1 prevents cigarette smoking-induced lung fibroblasts activation by regulating mitochondrial oxidative stress and lipid metabolism.

BACKGROUND: Cigarette smoking (CS) is a strong risk factor for idiopathic pulmonary fibrosis (IPF). It can activate lung fibroblasts (LF) by inducing redox imbalance. We previously showed that clearing mitochondrial reactive oxygen species (mtROS) protects against CS-induced pulmonary fibrosis. However, the precise mechanisms of mtROS in LF need further investigation. Here we focused on mtROS to elucidate how it was regulated by CS in LF and how it contributed to LF activation. METHODS: We treated cells with 1% cigarette smoking extract (CSE) and examined mtROS level by MitoSOX™ indicator. And the effect of CSE on expression of SIRT1, SOD2, mitochondrial NOX4 (mtNOX4), fatty acid oxidation (FAO)-related protein PPARα and CPT1a and LF activation marker Collagen I and α-SMA were detected. Nile Red staining was performed to show cellular lipid content. Then, lipid droplets, autophagosome and lysosome were marked by Bodipy 493/503, LC3 and LAMP1, respectively. And lipophagy was evaluated by the colocalization of lipid droplets with LC3 and LAMP1. The role of autophagy on lipid metabolism and LF activation were explored. Additionally, the effect of mitochondria-targeted ROS scavenger mitoquinone and SIRT1 activator SRT1720 on mitochondrial oxidative stress, autophagy flux, lipid metabolism and LF activation were investigated in vitro and in vivo. RESULTS: We found that CS promoted mtROS production by increasing mtNOX4 and decreasing SOD2. Next, we proved mtROS inhibited the expression of PPARα and CPT1a. It also reduced lipophagy and upregulated cellular lipid content, suggesting lipid metabolism was disturbed by CS. In addition, we showed both insufficient FAO and lipophagy resulted from blocked autophagy flux caused by mtROS. Moreover, we uncovered decreased SIRT1 was responsible for mitochondrial redox imbalance. Furthermore, we proved that both SRT1720 and mitoquinone counteracted the effect of CS on NOX4, SOD2, PPARα and CPT1a in vivo. CONCLUSIONS: We demonstrated that CS decreased SIRT1 to activate LF through dysregulating lipid metabolism, which was due to increased mtROS and impaired autophagy flux. These events may serve as therapeutic targets for IPF patients.

Author Correction: Mir-21 Mediates the Inhibitory Effect of Ang (1-7) on AngII-induced NLRP3 Inflammasome Activation by Targeting Spry1 in lung fibroblasts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Autophagy Attenuates Angiotensin II-Induced Pulmonary Fibrosis by Inhibiting Redox Imbalance-Mediated NOD-Like Receptor Family Pyrin Domain Containing 3 Inflammasome Activation.

AIMS: The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which is activated by reactive oxygen species (ROS) and repressed by autophagy, has been identified as a novel agent of pulmonary fibrosis. Angiotensin II (AngII), the bioactive pro-oxidant in the renin-angiotensin system, aggravates lung fibrosis. However, the effect of AngII on NLRP3 inflammasome and autophagy in lung fibrosis remains unknown. This study investigates the potential link between AngII-induced autophagy in the regulation of NLRP3 inflammasome/IL-1ß axis in lung fibrosis. RESULTS: In vivo, autophagy and the NLRP3 inflammasome were activated in fibrotic patients and positively correlated with oxidation. Treatment with rapamycin promoted autophagy but inhibited oxidation, NLRP3 inflammasome, and lung fibrosis after bleomycin (BLM) infusion. The autophagy inhibitor 3-methyladenine reduced BLM-induced lung fibrosis and concurrently facilitated NLRP3 inflammasome activation and oxidation in fibroblasts. In vitro, AngII promoted intercellular ROS, hydrogen peroxide, and NADPH oxidase 4 (NOX4) protein levels and reduced the glutathione concentration, thereby leading to NLRP3 inflammasome activation and consequent collagen synthesis. AngII induced autophagy, while VAS2870, NOX4, small-interfering RNA (siRNA), and compound C eliminated AngII-induced LC3B augmentation. Moreover, blocking autophagy with bafilomycin A1 or LC3B siRNA resulted in oxidant accumulation, NLRP3 inflammasome hyperactivation, and collagen deposition. Finally, AngII induced P62/SQSTM1, targeting ubiquitinated apoptosis-associated speck-like protein containing a CARD for degradation, thereby contributing to NLRP3 inflammasome inactivation. Innovation and Conclusion: Autophagy attenuates pulmonary fibrosis by regulating NLRP3 inflammasome activation induced by AngII-mediated ROS via redox balance modulation.

Angiotensin-(1-7) Attenuated Cigarette Smoking-related Pulmonary Fibrosis via Improving the Impaired Autophagy Caused by Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4-Dependent Reactive Oxygen Species.

Cigarette smoking is acknowledged as the major risk factor of pulmonary fibrosis. Angiotensin (Ang) II has been reported to aggravate smoking-induced lung fibrosis, whereas the effect of Ang-(1-7) on smoking-related lung fibrosis remains unknown. The autophagy, being activated by reactive oxygen species (ROS), is identified as a novel mechanism of pulmonary fibrosis. However, whether autophagy is involved in regulation of smoking-induced lung fibrosis still needs investigation. Here, we aim to investigate the effect of Ang-(1-7) on smoking-related lung fibrosis by the regulation of autophagy and ROS. In vivo, Ang-(1-7) was constantly infused into passive smoking rats for 8 weeks. In vitro, primary lung fibroblasts were pretreated with antioxidant, nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) 4 siRNA, or light chain (LC) 3B siRNA before exposure to cigarette smoke extract (CSE). GFP-mCherry red fluorescent protein-LC3 advenovirus was introduced to evaluate the autophagic flux in cells. We found that Ang-(1-7) reduced hydrogen peroxide (H2O2) concentration, protein levels of NOX4, and autophagy impairment, as well as improving lung fibrosis induced by smoking stimulation in vivo. In vitro, CSE treatment elevated NOX4 protein expression and ROS production, resulting in the accumulation of impaired autophagosomes in fibroblasts. LC3B depletion enhanced CSE-induced collagen synthesis. Treatment with antioxidants or NOX4 siRNA inhibited CSE-induced insufficient autophagic flux and collagen production. In contrast, the action of Ang-(1-7) opposed the effects of CSE. In conclusion, Ang-(1-7) improves smoking-induced pulmonary fibrosis via attenuating the impaired autophagy caused by NOX4-dependent ROS in vivo and in vitro.

Mir-21 Mediates the Inhibitory Effect of Ang (1-7) on AngII-induced NLRP3 Inflammasome Activation by Targeting Spry1 in lung fibroblasts.

MicroRNA-21 (mir-21) induced by angiotensin II (AngII) plays a vital role in the development of pulmonary fibrosis, and the NLRP3 inflammasome is known to be involved in fibrogenesis. However, whether there is a link between mir-21 and the NLRP3 inflammasome in pulmonary fibrosis is unknown. Angiotensin-converting enzyme 2/angiotensin(1-7) [ACE2/Ang(1-7)] has been shown to attenuate AngII-induced pulmonary fibrosis, but it is not clear whether ACE2/Ang(1-7) protects against pulmonary fibrosis by inhibiting AngII-induced mir-21 expression. This study's aim was to investigate whether mir-21 activates the NLRP3 inflammasome and mediates the different effects of AngII and ACE2/Ang(1-7) on lung fibroblast apoptosis and collagen synthesis. In vivo, AngII exacerbated bleomycin (BLM)-induced lung fibrosis in rats, and elevated mir-21 and the NLRP3 inflammasome. In contrast, ACE2/Ang(1-7) attenuated BLM-induced lung fibrosis, and decreased mir-21 and the NLRP3 inflammasome. In vitro, AngII activated the NLRP3 inflammasome by up-regulating mir-21, and ACE2/Ang(1-7) inhibited NLRP3 inflammasome activation by down-regulating AngII-induced mir-21. Over-expression of mir-21 activated the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1, resulting in apoptosis resistance and collagen synthesis in lung fibroblasts. These results indicate that mir-21 mediates the inhibitory effect of ACE2/Ang(1-7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts.

MicroRNA-21 Mediates Angiotensin II-Induced Liver Fibrosis by Activating NLRP3 Inflammasome/IL-1ß Axis via Targeting Smad7 and Spry1.

AIMS: Angiotensin II (AngII), a vasoconstrictive peptide of the renin-angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1-7) [Ang-(1-7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1ß axis in liver fibrosis. RESULTS: In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1-7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1-7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects. Innovation and Conclusions: Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1-7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1-20.

Angiotensin-(1-7) Improves Liver Fibrosis by Regulating the NLRP3 Inflammasome via Redox Balance Modulation.

AIMS: Angiotensin II (Ang II) aggravates hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Angiotensin-(1-7) [Ang-(1-7)], which counter-regulates Ang II, has been evidenced to protect against hepatic fibrosis. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, being activated by reactive oxygen species (ROS), is identified as a novel mechanism of liver fibrosis. However, whether the NLRP3 inflammasome involves in regulation of Ang II-induced hepatic fibrosis remains unclear. This study investigates the different effects of the Ang II and Ang-(1-7) on collagen synthesis by regulating the NLRP3 inflammasome/Smad pathway via redox balance modulation. RESULTS: In vivo, Ang-(1-7) improved bile duct ligation-induced hepatic fibrosis, reduced H2O2 content, protein levels of NOX4, and the NLRP3 inflammasome, whereas it increased glutathione (GSH) and nuclear erythroid 2-related factor 2 (Nrf2) antioxidant response element (ARE). In vitro, Ang II treatment elevated NOX4 protein expression and ROS production in hepatic stellate cells (HSCs), whereas it inhibited GSH and Nrf2-ARE, resulting in the activation of the NLRP3 inflammasome in the mitochondria of HSCs. NLRP3 depletion inhibited Ang II-induced collagen synthesis. Furthermore, Ang II increased NLRP3 and pro-IL-1ß levels by activating the Toll-like receptor 4 (TLR4)/MyD88/NF-κB pathway. Treatment with antioxidants, NOX4 small interference RNA (siRNA), or Nrf2 activator inhibited Ang II-induced NLRP3 inflammasome activation and collagen synthesis. In contrast, the action of Ang-(1-7) opposed the effects of Ang II. INNOVATION AND CONCLUSIONS: Ang-(1-7) improved liver fibrosis by regulating NLRP3 inflammasome activation induced by Ang II-mediated ROS via redox balance modulation. Antioxid. Redox Signal. 24, 795-812.

[Comparison of matrix metalloproteinase-9 between Wistar rat and spontaneously hypertensive rat in pulmonary injury model].

OBJECTIVE: To compare the expression of matrix metalloproteinase-9 (MMP9) and observe the change of cigarette smoke-induced pulmonary injury in Wistar and spontaneously hypertensive (SH) rats. METHODS: A total of 10 male Wistar rats and 10 male SH rats were randomly divided into four groups of Wistar control, Wistar cigarette smoking, SH control and SH cigarette smoking (n=5 each). The control groups were fed routinely while the cigarette smoking groups were placed into a homemade organic glass box and exposed to cigarette smoking for twice daily 6 days a week. After 8-week treatment, general condition, lung function and pathological changes of lung tissues were detected. The expressions of nuclear factor (NF)-κB, IκB-α and MMP9 were determined by Western blot. And real-time polymerase chain reaction (RT-PCR) was employed to detect the mRNA levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Immunohistochemistry was used to detect the expression of MMP9 in lung tissue. RESULTS: Both cigarette smoking groups were marantic along with intermittent cough and polypnea. Resistance index (RI) in Wistar control, Wistar cigarette smoking, SH control and SH cigarette smoking groups was (0.23±0.04), (0.33±0.05), (0.33±0.02) and (0.46±0.08) cmH2O·s·ml(-1) respectively. RI increased in Wistar cigarette smoking group versus Wistar control group. And SH cigarette smoking group was significantly higher than SH control and Wistar cigarette smoking groups (P<0.05). Hematoxylin & eosin staining demonstrated that the lesion of chronic bronchitic inflammation and emphysema existed in two cigarette smoking groups while it was more severe in SH rats than Wistar rats. The mean alveolar number (MAN) was significantly reduced in two cigarette smoking groups compared with that in control groups (P<0.05, respectively). And mean lining interval (MLI), damage index (DI), bronchial smooth muscle index and collagen index significantly increased in Wistar cigarette smoking group compared with Wistar control group, SH cigarette smoking group significantly increased than that in SH control and Wistar cigarette smoking groups (P<0.05, respectively). On Western blot, the protein level of NF-κB in Wistar control, Wistar cigarette smoking, SH control and SH cigarette smoking groups was (0.322±0.014), (0.558±0.044), (0.373±0.029) and (1.156±0.197) respectively. The protein level of MMP9 in these groups was (0.255±0.070), (0.456±0.089), (0.594±0.184) and (0.847±0.138) respectively. The protein levels of NF-κB and MMP9 increased obviously in Wistar cigarette smoking group versus Wistar control group and those in SH cigarette smoking group were significantly higher than SH control and Wistar cigarette smoking groups (P<0.05). Additionally, IκB-α expression in SH cigarette smoking group was markedly decreased in comparison to that in SH control group and Wistar cigarette smoking group (P<0.05). The mRNA level of TNF-α increased obviously in two cigarette smoking groups in comparison to those in controls (P<0.05) and the mRNA level of IL-6 significantly increased in the SH cigarette smoking group compared with SH control group and Wistar cigarette smoking group (P<0.05). As for immunohistochemistry, the MMP9 positive cells were prominently distributed in the lungs of cigarette smoking groups versus the stained cells in the control group. And SH cigarette smoking group was significantly higher than Wistar cigarette smoking group (P<0.05). CONCLUSION: Both Wistar and SH rats induced by cigarette smoking may have an onset of lung injury symptom analogous to chronic obstructive pulmonary disease and SH rat are more severe.