3-MA was purchased from Selleckchem (Houston, TX, USA). Antibodies to Beclin-1 (#3738), Atg7 (#2631), and p53 (#2527) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to GAPDH (sc-32233), Collagen I (sc-28654), and Caspase-11 (sc-374615) were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). Antibodies to NLRP3 (ab214185), and Caspase-1 (ab179515) were purchased from Abcam (Cambridge, MA, USA). Antibodies to Collagen III (GB11023), and IL-1β (GB11113) were purchased from Servicebio (Wuhan, China). Antibodies to IL-18 (A16737) and GSDMD (A20197) were purchased from ABclonal Biotech (Shanghai, China). Antibody to cathepsin B (12216-1-AP) was purchased from Proteintech Group (Chicago, IL, USA). Anti-mouse secondary antibody (A0216), and anti-rabbit secondary antibody (A0208) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Beclin-1 siRNA and GP-transfect-Mate (G04009) were purchased from GenePharma (Shanghai, China). Antibody to α-SMA (A2547), DMSO and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The HN model was established in male Sprague-Dawley rats (6-8 weeks old, Shanghai Super-B&K Laboratory Animal Corp. Ltd, Shanghai, China) that weighed 200-220g. The animals were housed at the Experimental Animal Center of Tongji University under a 12 h light-dark cycle with food and water supplied ad libitum. The HN model was established as described in our previous study (8). Rats were injected intraperitoneally with 3-MA 15mg/kg in warmed saline daily in order to explore the effect of 3-MA on HN. Rats were randomly divided into four groups, each group including six rats: (1) The rats in control group were given an equivalent amount of saline by gavage and injected with an equivalent amount of saline intraperitoneally; (2) The rats in sham+3-MA group were given an equivalent amount of saline by gavage and injected with an equivalent amount of 3-MA intraperitoneally; (3) The rats in HN group were given a mixture of adenine (0.1 g/kg) and potassium oxonate (1.5 g/kg) by gavage and injected with an equivalent amount of saline intraperitoneally,; (4) The rats in HN+3-MA group were given a mixture of adenine (0.1 g/kg) and potassium oxonate (1.5 g/kg) by gavage and injected with an equivalent amount of 3-MA intraperitoneally. At the end of the experimental period, all animals were killed by exsanguination under anesthesia with inhaled 5% isoflurane in room air and the kidney was collected for the following experiments. All the animal experiments were conducted with approval from the Institutional Animal Care and Use Committee at Tongji University.

Human tubular epithelial cells (HK-2) were attained from ATCC (Manassas, VA). Cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and F-12 containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin in an atmosphere of 5% CO₂ and 95% air at 37°C. Before starting the formal experiments, we passed the primary cells for three generations in order to obtain a stable phenotype. To investigate the effect of 3-MA in uric acid-induced tubular cell injury, subconfluent HK-2 cells were starved for 24 hours in DMEM medium containing 0.5% FBS and then exposed to uric acid (UA, 800 μM) in the presence of 3-MA (0, 1, 5 and 10 mM) for 36 hours. After stimulation for 36 hours, cells were harvested for further analysis. All of the in vitro experiments were repeated no less than three times.

The small interfering (si) RNA oligonucleotides targeted specially for p53, Beclin-1, CTSB, and negative control (NC) siRNA which chemically synthesized by GenePharma (Shanghai, China) were used in this study. The sequence of p53 siRNA is 5′-GACUCCAGUGGUAAUCUACTT-3′ (sense strand) and 5′-GUAGAUUACCACUGGAGUCTT-3′ (antisense strand). The sequence of Beclin-1 siRNA is 5′-CAGUUUGGCACAAUCAAUATT-3′ (sense strand) and 5′-UAUUGAUUGUGCCAAACUGTT-3′ (antisense strand). The sequence of CTSB siRNA is 5′-ACAAGCACUACGGAUACAUTT-3′ (sense strand) and 5′-AUGUAUCCGUAGUGCUUGUTT-3′ (antisense strand). The sequence of NC siRNA is 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense strand) and 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense strand). Briefly, HK-2 cells were grown in 6-well plates and transiently transfected with siRNA at 60-80% confluence at a final concentration of 5 nM using GP-transfect-Mate (GenePharma, Shanghai, China) according to the manufacturer’s instructions. After 6 hours transfection, the cells were incubated with fresh medium alone or administrated with uric acid (800 μM) for an additional 36 hours before being harvested for the further experiments.

Cells were washed three times with ice-cold PBS and harvested in RIPA buffer mixed with PMSF and phosphatase inhibitor on ice. The supernatants were collected after centrifugation at 12,000 g for 15 min at 4°C. In addition, the kidney tissue samples were homogenized with cell lysis buffer and with PMSF and phosphatase inhibitor. Proteins were separated by SDS-PAGE and transferred to 0.2 μm nitrocellulose membranes. After incubation with 5% nonfat milk for 1 hour at room temperature, the membranes were incubated with primary antibodies overnight at 4°C and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Bound antibodies were visualized by chemiluminescence detection. Densitometry analysis of immunoblot results was conducted by using Image J software (National Institutes of Health, Bethesda, MD).

Formalin-fixed kidneys were imbedded in paraffin and prepared in 3-μm-thick sections. Sections were de-paraffinized and rehydrated, immersed in citrate buffer and heated in a microwave for retrieval of antigens and quenched with 3% H₂O₂. Sections were incubated with primary antibodies overnight at 4°C and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. For quantifications, 10 random visual fields were analyzed per kidney section. The positive area was calculated with Image J software (National Institutes of Health, Bethesda, MD).

Formalin-Fixed Paraffin-Embedded sections (3 μm) were rehydrated and incubated with primary antibodies against p53, GSDMD, IL-1β, and IL-18 and then Texas Red- or FITC-labeled secondary antibodies (Invitrogen).

After treatments, HK-2 cells were plated on coverslips and then fixed in 4% paraformaldehyde for 10 minutes. Cells were permeabilized by 0.25% Triton X-100 in PBS for 10 minutes. After blocking with 10% normal goat serum, cells were incubated with primary antibodies against NLRP3, Caspase-11, GSDMD, IL-1β, IL-18, or CTSB followed by secondary antibodies. Finally, cells were stained with DAPI and mounted. Images were acquired using Fluorescence Microscope (Leica, DM3000).

After HK-2 cells were treated in accordance with the aforementioned cell culture and treatments and reached confluence, cells were collected from each group for standard transmission electron microscope (TEM) processing to observe the morphology of autophagosome. Multiple autophagic structures such as phagophore, autophagosome, and autolysosome in HK-2 cells were observed at high magnification from each cell and digital images with scale bars were taken.

Data depicted in graphs expressed as means ± SEM for each group. The comparisons between two groups were analyzed by Student’s t-test and one-way analysis of variance was used for comparisons of multiple groups. Statistically significant differences between mean values were marked in each graph. P<0.05 was considered statistically significant.

Our previous study has revealed that the increased expression of LC3II/I and Beclin-1 were observed in the kidney of hyperuricemic injury rats, and numerous autophagic vacuoles appeared in proximal tubular cells in vivo (8). It suggested that hyperuricemia can induce the activation of autophagy. In addition, kidneys with HN displayed severely structural damage as characterized by glomerulosclerosis, tubular dilation, epithelial atrophy, interstitial expansion, and collagen accumulation. Inhibition of autophagy with 3-MA decreased the deposition of extracellular matrix components. However, whether uric acid induced the activation of autophagy in vitro is still unknown, and how autophagy effects on renal tubular cells have not been elucidated. Therefore, we performed in vitro experiments on HK-2 cells with the stimulation of uric acid. As shown in Figure 1A , images from TEM indicated multiple of autophagy-related vacuoles such as autophagosome and autolysosome were observed in uric acid-stimulated HK-2 cells compared to control cells. Treatment with 3-MA remarkably inhibited the autophagic activity ( Figures 1A, B ). Uric acid also induced a significant upregulation of Beclin-1 and Atg7, two autophagy related proteins, 3-MA dose-dependently suppressed these responses ( Figures 1C–E ). Reduction of Beclin-1 expression by its specific siRNA also decreased uric acid-stimulated expression of Atg7 ( Figures 1J–L ). Since epithelial-to-mesenchymal transition (EMT) is involved in the progression of renal fibrosis (24), we found that compared with serum-starved HK-2 cells, uric acid-exposed HK-2 cells expressed higher protein levels of α-SMA, Collagen I, and Collagen III, three mesenchymal markers. Inhibition of autophagy with 3-MA significantly reduced the expression of α-SMA, Collagen I, and Collagen III ( Figures 1F–I ). Consistent with this result, knocking down of Beclin-1 also blocked the expression of α-SMA and Collagen I ( Figures 1M–O ). Taken together, these results indicated that autophagy was activated in the uric acid-stimulated HK-2 cells, which is essential for the occurred of EMT in tubular epithelial cells.

Based on the aforementioned role of uric acid in the activation of autophagy, we further investigated the mechanism involved. It is illustrated that p53 triggered the activation of autophagy in response to DNA damage (25), we thus examined whether uric acid upregulated autophagy through p53 signaling pathway. We established a rat model of HN induced by adenine and potassium oxonate for 3 weeks. As shown in Figure 2A , compared to the sham rats, HN rats displayed increased expression of p53 in the kidney. To further verify the role of p53 on the activation of autophagy, we examined the effect of p53 knockdown in HK-2 cells exposure to uric acid by using siRNA specifically targeting p53. As demonstrated in Figures 2B–E , reduction of p53 expression by its specific siRNA decreased uric acid stimulated expression of Beclin-1 and Atg7. These results demonstrated that uric acid triggered autophagy may be mediated by p53 signaling pathway.

Hyperuricemia can trigger inflammatory responses in the kidney, and the inflammasome family has several members and behaves a magnificent effect on inflammatory responses. Particularly, NLRP3 inflammasome is considered to be a pivotal component of inflammation (26). Recently, emerging studies have demonstrated that there is an interaction between autophagy and NLRP3 inflammasome, more importantly, the interaction plays a crucial role in metabolic diseases (27). However, whether autophagy can activate the NLRP3 inflammasome in HN is still unknown. Thus, we get down to examine the effect of autophagy inhibition on the activation of NLRP3 inflammasome. As shown in Figures 3A, B , NLRP3 was significantly increased in HN rats, while administration with 3-MA resulted in the reduction of NLRP3. Immunohistochemistry staining also demonstrated that the expression level of NLRP3 was increased in the kidney of HN rats, which is contrary to that in sham rats. Treatment with 3-MA largely inhibited the expression as indicated by reduced staining positive areas ( Figures 3C, D ).

To further investigate the interplay between autophagy and NLRP3 inflammasome, we used both 3-MA and Beclin-1 siRNA to inhibit autophagy for in vitro studies. Exposure of HK-2 cells to uric acid resulted in increased expression of NLRP3, treatment with 3-MA inhibited the upregulation of NLRP3, which occurred in a dose-dependent manner ( Figures 3E, F ). The immunofluorescence staining was used to evaluate the expression and localization of NLRP3 in response to uric acid. We found that the cells were positively stained for NLRP3 in the cytoplasm stimulated by uric acid, treatment with 10 mM 3-MA significantly decreased the expression of NLRP3 ( Figures 3G, H ). In consistent with this result, immunoblotting analysis indicated that inhibition of autophagy with Beclin-1 siRNA was also impede the NLRP3 overexpression ( Figures 3I, J ). Taken together, these results suggest that inhibition of autophagy prevents the activation of NLRP3 inflammasome both in vivo and in vitro.

Shao et al. demonstrated that NLRP3 inflammasome leads to the activation of pyroptosis (28). In addition, a large number of studies indicate that a close relationship between autophagy and pyroptosis (29, 30). To investigate the role of autophagy in the NLRP3 inflammasome-mediated pyroptosis in HN, we adopted autophagy inhibitor 3-MA to verify the relationship among them. As shown in Figures 4A–D , proteins level of caspase-1, caspase-11, and GSDMD markedly increased in HN rats, pretreatment with 3-MA dramatically reduced the elevated expression of caspase-1, caspase-11, and GSDMD in the HN kidney. Immunohistochemistry of caspase-11 demonstrated that caspase-11 was predominantly localized in tubular epithelial cells in injured kidneys associated with HN, and 3-MA reduced the number of caspase-11-positive cells ( Figures 4E, F ). Moreover, immunofluorescence staining of GSDMD further revealed that GSDMD was primarily located in renal tubular epithelial cells and highly expressed in injured kidneys ( Figures 4G, H ). Thus, our results implied hyperuricemic induced autophagy and activated the NLRP3 inflammasome-mediated pyroptosis, and treatment with 3-MA was effective in ameliorating this responses.

To further determine the role of autophagy in activating pyroptosis, we used autophagy inhibitor 3-MA as well as knockdown of Beclin-1 targeted by siRNA to inhibit autophagy for in vitro studies. Uric acid was found to induce a significant upregulation of caspase-1, caspase-11, and GSDMD, pretreatment with 3-MA dose-dependently reduced expression level of caspase-1, caspase-11 and GSDMD ( Figures 5A–D ). Immunofluorescence assay further confirmed that the expression of caspase-11 and GSDMD significantly increased in response to uric acid, treatment with 3-MA reduced the expression of caspase-11 and GSDMD in HK-2 cells ( Figures 5E–G ). As expected, knockdown of Beclin-1 reduced expression levels of caspase-1 and GSDMD ( Figures 5H–J ), which performed the same effects with inhibitor. Collectively, these data further conform that 3-MA inhibits the process of pyroptosis in HK-2 cells exposed to uric acid.

After the process of pyroptosis, the freed-out N-terminal domain of GSDMD further regulates Pannexin and Potassium iron efflux, causes osmotic potential disruption, cell swelling and lysis, as well as releases of inflammatory cytokines such as IL-1β and IL-18 (31). To evaluate whether autophagy plays a crucial role in mediating the release of inflammatory cytokines after pyroptosis, we examined the effect of autophagy inhibition on the release of IL-1β and IL-18 in vivo and in vitro. Immunofluorescence staining showed that IL-1β and IL-18 were mainly located in the cytoplasm of renal tubules and highly expressed in the kidney of HN rats. 3-MA treatment inhibited the expression of IL-1β and IL-18 ( Figures 6A–C ). We further verified the role of autophagy in regulating the release of inflammatory cytokines in HK-2 cells exposed to uric acid. We found that after 36 hours uric acid stimulation, the cells were positively stained for IL-1β and IL-18 in the cytoplasm. Pretreatment with 10 mM 3-MA significantly reduced their expression ( Figures 6D–F ). All in all, these results indicate that inhibition of autophagy with 3-MA prevents the release of IL-1β and IL-18 both in vivo and in vitro.

Previous study has indicated that crystal-induced (such as monosodium urate) lysosomal damage plays a crucial role in activating the NLRP3 inflammasome (32). Lysosomes contain several proteolytic enzymes, one of which is the CTSB family. First, we investigated whether CTSB was released into cytoplasm in the kidney of HN rats. Protein level of CTSB significantly increased in HN rats compared to the sham rats, administration with 3-MA reduced its expression ( Figures 7A, B ). Immunohistochemistry staining pointed out that CTSB mainly expressed in the cytoplasm of damaged tubular epithelial cells, and remarkably reduced after 3-MA treatment ( Figures 7C, D ). At the same time, in our in vitro study, we found that CTSB expression in HK-2 cells was significantly increased after 36 hours uric acid exposure compared with the sham group, treatment with 3-MA reduced the protein level of CTSB in a dose-dependent manner ( Figures 7E, F ). Immunofluorescence staining confirmed the effect of 3-MA in decreasing the release of CTSB in cytoplasm ( Figures 7G, H ). Consistent with these results, knockdown of Beclin-1 also reduced protein level of CTSB ( Figures 7I, J ). To further examine whether release of CTSB was involved in the activation of NLRP3 inflammasome and pyroptosis, HK-2 cells were transfected with CTSB siRNA to blockage the activities of CTSB. Transfected with CTSB siRNA caused a significant reduction of CTSB protein levels. Knockdown of CTSB had less expression of NLRP3, caspase-1, and GSDMD ( Figures 8A–E ). Taken together, these results demonstrated that CTSB was critical for uric acid-induced activation of NLRP3 inflammasome and pyroptosis, which is associated with autophagy.

In the present study, we found that chronic exposure to uric acid induces dysregulation of autophagy, which is mediated by p53 pathway. The activation of autophagy further activated NLRP3 inflammasome and pyroptosis. The release of CTSB from autolysosome plays a crucial role in the activation of NLRP3 inflammasome and pyroptosis, which leads to the release of IL-1β and IL-18. Blockade of autophagy inhibits the aforementioned responses, thus prevents the development and progression of hyperuricemic nephropathy ( Figure 8F ).