Uric acid regulates NLRP3/IL-1β signaling pathway and further induces vascular endothelial cells injury in early CKD through ROS activation and K⁺ efflux

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 [16]. 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.

Total RNA was extracted through TRIzol reagent (Invitrogen Life Technologies, USA) and then was reverse-transcribed into cDNA using the Primer Script RT reagent kit (Takara Bio, China). qPCR was performed with SYBR Premix Ex Taq™ II kit (Takara Bio, China). The primers used for IL-1β, caspase 1, NLRP3, and ASC were listed in Table 1. GAPDH was used as a control. The mRNA levels were calculated relative to internal control using 2^-ΔΔCt method.

The animal model was established as described [17]. 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.

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.

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).

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).

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.

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 (Fig. 1). UA significantly increased the protein expression of IL-1β in cells culture supernatant and the mRNA level of IL-1β in cells, and the influence presented by concentration and time dependent manner (Fig. 1a, b). However, the expression of IL-1β precursor (pro-IL-1β) showed no obvious change. We also found that with the increase of UA concentration and incubation time, the level of ICAM-1 protein increased consequently (Fig. 1c). Therefore, UA could induce the expression of ICAM-1 and IL-1β in HUVECs.

In order to investigate the effects of UA on NLRP3 inflammasome, the expression of NLRP3, ASC, and pro-caspase 1 was measured by western blotting and qPCR after the treatment by different concentrations or different incubation time of UA (Fig. 2). We found that high concentrations (10 and 20 mg/dL) of UA or longer incubation time (12 and 24 h) of UA at 20 mg/dL significantly increased the expression of ASC and NLRP3 in cells and the protein level of caspase 1 in medium supernatant. The influence presented concentration and time dependence (Fig. 2a). Accordingly, UA significantly promoted the mRNA expression of NLRP3 and caspase 1 in HUVECs with the concentration and time dependent manner (Fig. 2b).

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.

To investigate the regulation of UA on downstream cytokines expression by activating NLRP3, the NLRP3 inflammasome components and downstream cytokines expression were measured after NLRP3 siRNA or caspase 1 inhibitor treatment, respectively. We found that UA could increase the expression of caspase 1, NLRP3, IL-1β, and ICAM-1 in HUVECs, but the trends were significantly suppressed by siRNA NLRP3 treatment. However, no remarkable changes were observed in the expression of pro-IL-1β and pro-caspase 1 after siRNA NLRP3 treatment (Fig. 3a). Caspase 1 activated by the combination of NLRP3 complexes is an important regulator of the cleavage of pro-IL-1β into mature IL-1β. After the treatment of caspase 1 inhibitor (Ac-YVAD-CHO), the expression of caspase 1, IL-1β, and ICAM-1 markedly declined, but no significant difference was found in the expression of pro-IL-1β and pro-caspase 1 (Fig. 3b). Therefore, UA might regulate the level of downstream cytokines by activating NLRP3.

To unfold whether UA regulate the activation of NLRP3 inflammasome via ROS activation or K⁺ efflux, we blocked K⁺ efflux using extracellular high concentration K⁺, and treated cells with ROS inhibitor (Apocynin, APO), ROS scavenger (N-acetylcysteine, NAC), 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 (Fig. 4a). Then we found that UA could induce the activation of caspase 1 and NLRP3, and increase the expression of IL-1β and ICAM-1, but pretreatment with APO or NAC significantly inhibited these trends (Fig. 4b). Mito-TEMPO pretreatment or high concentration of K⁺ in extracellular significantly inhibited the formation of NLRP3 inflammasome, activation of caspase 1, release of IL-1β and expression of ICAM-1 induced by UA (Fig. 4c, d). These findings suggested that UA could regulate activation of NLRP3 inflammasome and expression of inflammatory factors by activating ROS or regulating K⁺ efflux.

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).

During the experiment, no dead rat, and no glomerular, tubule, and interstitial lesions in all groups (Fig. 5a). The serum creatinine in four groups were also no changes, but the content of serum UA in group C was significant higher than group A and group B, and allopurinol presented inhibition function on the increase of UA (Table 2). In the group C accompanied with increase of serum UA, the expressions of IL-1β, TNF-α, and ICAM-1 in rat serum were significantly higher than group A, group B, and group D (Table 3). The mRNA and protein levels of NLRP3, caspase 1, ASC, and IL-1β in group C were remarkably higher than group A and group B, and significantly decreased in group D (Fig. 5b, c). For the histopathology in the group A, we found that the structure of aorta was clear, the endothelial cells arranged closely, and there was no inflammatory cells aggregation in the vessel wall. Meanwhile, slight infiltration of inflammatory cells could be seen on the vessel wall in group B. However, edematous endothelial cells appeared foam like change, a little inflammatory cells aggregation in the wall, smooth muscle cells proliferation and structural disorder were also found in group C. Compared with group C, the lesions in group D were lighter. The proliferation of smooth muscle cells was not obvious, and the structure of cells arranged orderly in group D (Fig. 5d). We investigated the histopathology changes of five mice for each group. The histopathology changes of mice vascular were similar in the same group, and representative pictures were shown in the study. These results demonstrated that UA might induce early vascular endothelial injury by activating NLRP3/IL-1β signaling pathway.