Uric acid regulates NLRP3/IL-1β signaling pathway and further induces vascular endothelial cells injury in early CKD through ROS activation and K+ efflux
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.
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. 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.
UA could regulate NLRP3/IL-1β signaling pathway through ROS activation and K+ efflux and further induce vascular endothelial cells injury in early stages of CKD.
KeywordsUric acid NLRP3/IL-1β Early CKD ROS activation K+ efflux
Apoptosisassociated specklike protein
Chronic kidney disease
Human umbilical vein endothelial cells
Intercellular adhesion molecule-1
Mitochondrial ROS inhibitor
NLR family pyrin domain-containing 3
Quantitative real-time PCR
Tumor necrosis factor-alpha
Vascular adhesion molecule-1
In recent years, chronic kidney disease (CKD) has become one of the most recognized public health problems worldwide [1, 2]. Hyperuricemia is prevalent in early CKD patients for the reason that the function of kidney excreting UA has been impaired, and UA increases continuously as the further deterioration of renal function [3, 4]. Meanwhile, several studies have demonstrated that hyperuricemia is an independent risk factor of cardiovascular diseases [5, 6, 7]. It was reported that CKD is highly consistent with cardiovascular diseases, and the risk of cardiovascular diseases increase as the decline of renal function . In addition, cardiovascular diseases have become the most common complication of CKD and the leading cause of death in CKD patients . Endothelial dysfunction is considered to be one of the major causes of cardiovascular diseases . The increase of UA could significantly damage cardiovascular endothelial cells, and endothelial dysfunction is positively correlated with serum UA level . Moreover, UA may damage vascular endothelial cells via oxidative stress, inflammation and abnormal lipid metabolism [11, 12]. However, the specific mechanism remains unclear.
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 increase of multiple fractions of NLRP3 has been identified in several diseases such as acute sterile inflammation, CKD and atherosclerosis . UA is reported to be an important signal of NLRP3 inflammasome activation . And activation of NLRP3 inflammasome in vascular endothelial cells eventually leads to endothelial dysfunction and contributes to the development of cardiovascular diseases . Interleukin-1 beta (IL-1β), a member of interleukin-1 (IL-1) cytokine superfamily, can be activated by caspase 1, so activation of NLRP3 inflammasome is very important for the maturation and secretion of IL-1β. Meanwhile, the activation of reactive oxygen species (ROS) and potassium ions (K+) efflux also participate in the activation of NLRP3 inflammasome . Therefore, NLRP3/IL-1β pathway may be closely related to vascular endothelial cells injury. However, in early-stage CKD patients whether UA activates NLRP3/IL-1β and further induces vascular endothelial cells injury through ROS activation and K+ efflux are still not well known.
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 K+ efflux in vitro, and further validated them in vivo. The aim of this study is to provide a new perspective for clinical drug therapy.
Cells culture and treatment
HUVECs were isolated by collagenase A perfusion from umbilical cords (obtained from the Department of Obstetrics, Hunan Provincial People’s Hospital) and cultured as described . The cells were incubated in 1640 medium at 37 °C with 5% CO2. 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 (0, 5, 10, 20 mg/dL) 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 (0, 6, 12, 24 h, respectively) on the expression of IL-1β and ICAM-1 by western blotting and qPCR.
Total 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 (Millipore, USA). After blocking with TBST (5% milk), the membranes were incubated with primary antibody obtained from Abcam (1:1000, Hong Kong, China) 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 (Millipore, USA) was used for immunodetection, and the density of the bands was detected by Image J software.
RNA isolation and qPCR
qPCR primer sequences used in this study (h: human, r: rat)
Establishment of early-stage CKD animal model
The animal model was established as described . Briefly, 20 male Sprague-Dawley rats (220~250 g, 6~8 weeks old) were obtained from Hunan Slac Jingda Experimental Animal Co. Ltd. (Changsha, China) and kept in the animal experimental center of the First Affiliated Hospital of Hunan Normal University (Changsha, China). The rats were raised in standard cages, water and food were freely available. After different treatments, animals were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital (45 mg/kg). The region for surgery was shaved and then cleaned with 75% alcohol. The right kidney was exposed through a longitudinal incision under the right costal arch. The renal pedicle was clamped and ligated after separation of renal capsule and perirenal fat. The right kidney was resected with scissors. Tissues and skins were sutured, respectively. Chlortetracycline was then applied to the incision. The entire procedure mentioned above was conducted in Group A (sham operation group, n = 5), but nephrectomy was not applied. Animals in Group C (hyperuricemia group, n = 5) and D (allopurinol and hyperuricemia group, n = 5) were fed with potassium oxonate (OXO) or/and allopurinol. Animals in Group A and B (operation group, n = 5) were gavaged with drinking water with the same amount as in Group C and D.
Kidney histopathological examination
After 10 weeks of gavage treatment, animals were euthanized with prolonged exposure to isofluorane inhalation (Respirator mode: frequency 60–70 bpm, breathing ratio 1:1, tidal volume 2–3 mL/100 g; The induction concentration of isoflurane was 4% and the maintenance concentration was 2%.) 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.
Detection of serum creatinine and UA
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 (TBA-C16000, Toshiba, Japan).
Influence of UA on the production of ROS
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 (PerkinElmer, US).
Measurements of IL-1β, TNF-α, and ICAM-1 by ELISA
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 (eBioscience, San Diego, CA, USA).
Data were shown as the mean ± SD, and analyzed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA). The unpaired two-tailed 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.
UA induced the expression of IL-1β and ICAM-1 in HUVECs
UA induced the expression of NLRP3 complexes and activation of NLRP3 inflammasome in HUVECs
Furthermore, immunoprecipitation and Western blotting were used to detect the effects of UA treatment on the binding of components in NLRP3 inflammasome. The cells lysates were extracted by A/G immunomagnetic beaded with NLRP3 antibody, then the protein expression of ASC, NLRP3, and pro-caspase 1 was measured. We found that the levels of pro-caspase 1 and ASC increased with elevating of UA concentration, and the expression of NLRP3 was same in different groups (Fig. 2c). On the condition that cells lysates were extracted by A/G immunomagnetic beaded with ASC antibody, the level of NLRP3 and pro-caspase 1 elevated with increasing of UA concentration with the same expression of ASC in different groups (Fig. 2d). Therefore, we concluded that UA could induce the expression of NLRP3 complexes and activation of NLRP3 inflammasome in HUVECs.
NLRP3 siRNA or inhibitor of caspase 1 affected the components of NLRP3 inflammasome and the expression of downstream cytokines
UA regulated the activation of NLRP3 inflammasome by activation of ROS and K+ efflux
UA induced the vascular endothelial injury of early stage CKD by activating NLRP3/ IL-1β pathway
To investigate the molecular mechanism of UA-induced vascular endothelial injury in early CKD, the results in cells level were further validated through animal experiments. Rats were separated randomly into four groups: Group A served as the control; Group B, in which animals only received right nephrectomy; Group C, in which animals received right nephrectomy and then gavaged with OXO (800 mg/kg, twice a day); Group D, in which animals received right nephrectomy and then gavaged with OXO (800 mg/kg, twice a day) and allopurinol (25 mg/kg, once a day).
The concentration of serum creatinine and UA after different kinds of operations
30.4 ± 7.2
54.8 ± 9.4
31.9 ± 4.8
55.9 ± 6.1
CKD + OXO
31.2 ± 5.7
178.5 ± 8.4*
CKD + OXO + ALLO
30.7 ± 7.7
74.9 ± 6.1#
The expression of IL-1β, TNF-α, and ICAM-1 after different kinds of operations
CKD + OXO
CKD + OXO + ALLO
42.38 ± 7.96
45.72 ± 8.13
88.73 ± 5.64*
47.25 ± 6.14#
163.70 ± 10.83
174.20 ± 11.24
293.10 ± 12.31*
183.60 ± 14.28#
284.10 ± 22.64
311.50 ± 27.83
592.18 ± 98.20*
328.70 ± 76.12#
UA 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 [18, 19]. While UA may be an important risk factor for CKD, high serum UA content is closely associated with several adverse pathological and cellular processes, such as inflammation, endothelial dysfunction, oxidative stress, reduction of NO production and decrease of biological activity, which increases the harm of CKD . In this study, we found that UA could significantly increase the level of some inflammation factors including IL-1β and ICAM-1 in HUVECs and serum of early CKD rats. The occurrence of many human diseases are related to the abnormal expression of IL-1β, which is a promising target for clinical intervention of inflammation-related diseases. ICAM-1, an important adhesion molecule and high expression in vascular endothelial cells, can promote adhesion between leukocytes and endothelial cells. The increase of IL-1β and ICAM-1 induced by UA probably account for vascular endothelium damage. Furthermore, we also observed the inflammatory cells aggregation in the wall, smooth muscle cells proliferation and structural disorder in histological level for the rats treated by nephrectomy and OXO, which could cause hyperuricemia. These findings above in vivo and in vitro identified the adverse effect of UA on vascular endothelium.
It was reported that endothelial cells damage induced by inflammatory factors plays a key role in the pathogenesis of vascular diseases [20, 21]. NLRP3 inflammasome, a multiprotein complex, can activate caspase 1 and lead to the secretion of IL-1β. The activation of NLRP3 inflammasome may lead to the development of endothelial dysfunction [22, 23] and further accelerate the injury of vascular. In this study, we have found that UA activated NLRP3 inflammasome and increased the expression of IL-1β, caspase 1, and ICAM-1 in HUVECs. In vivo, edematous endothelial cells appeared foam like change after OXO treatment, which could cause hyperuricemia. Therefore, UA may cause vascular endothelial injury by activating NLRP3 inflammasome and promoting the level of inflammatory factors.
Some reports indicated that cytoplasmic K+ concentration significantly influences the activation of NLRP3 inflammasome [24, 25]. The cytoplasmic K+ concentration of healthy cells is ~ 140–150 mM, which does not induce NLRP3 activation [26, 27]. ATP, a strong activator of NLRP3 inflammasome, could decrease the cytoplasmic K+ concentration by 50% . Therefore, we used extracellular high concentration K+ for blocking K+ efflux, and observed the inhibition of NLRP3 inflammasome activation. Interestingly, the activation of apoptosome also were correlated with the K+ concentration . ROS production leads to activation of NLRP3 inflammasome through release of the ROS-sensitive NLRP3 ligand thioredoxin-interacting protein (TXNIP) . The interplay between ROS production and K+ efflux in the activation of NLRP3 inflammasome remains unclear, but low intracellular K+ concentration induces ROS production and vice versa [28, 29, 30]. Therefore, UA might activate NLRP3 inflammasome by activating ROS or promoting K+ efflux.
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+ 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.
We would like to give our sincere gratitude to the reviewers for their constructive comments.
YW performed experimental studies, acquired data, prepared and edited manuscript. ZQL designed the study and did literature research. OYSX and CY analysed data. GYT performed clinical studies. LYM designed the study and reviewed the manuscript. All authors read and approved the final manuscript.
Funding was supported by RENSHU Funding Project of Hunan Provincial People’s Hospital (No.RS201702). The funding body did not have a role in study design nor collection, analysis, interpretation of the data and did not influence the writing the manuscript.
Ethics approval and consent to participate
The use of laboratory animals, the umbilical cord collection and use procedure were all approved by the Medical Ethical Committee of Hunan Normal University (Approved number: 2017050). All of the donors were given and accepted written informed consent.
Consent for publication
The authors declare that they have no competing interests.
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