Selenium nanoparticles alleviate ischemia reperfusion injury-induced acute kidney injury by modulating GPx-1/NLRP3/Caspase-1 pathway

Abstract

Rationale: Acute kidney injury (AKI) is a common critical illness in the clinic and currently lacks effective treatment options. Ischemia reperfusion injury (IRI) is a major pathogenic factor for AKI. Due to the deficiency of selenium (Se) in AKI patients, we intended to treat IRI-induced AKI using a Se rebalancing strategy in the present study. Methods: Sodium selenate, ascorbic acid, and bovine serum albumin (BSA) were employed to prepare nanomaterials termed Se@BSA nanoparticles (NPs) using a simple method. Experiments with human renal tubular epithelial HK-2 cells exposed to hypoxia/reoxygenation (H/R) and IRI-AKI mice were used to evaluate the therapeutic efficiency of Se@BSA NPs. Transcriptome sequencing, further molecular biology experiments, and pathologic analysis were performed to investigate the underlying mechanisms. Results: Se@BSA NPs accumulated in mouse kidneys and could be endocytosed by renal tubular epithelial cells after intravenous administration. In vitro studies showed that Se@BSA NP treatment markedly increased the levels of glutathione peroxidase (GPx)-1 and suppressed NLRP3 inflammasome activation in H/R cells, which resulted in reductions in the proteolytic cleavage of pro-Caspase-1 into active Caspase-1 and the maturation of inflammatory factors. Mouse experiments confirmed these findings and demonstrated an inspiring mitigative effect of Se@BSA NPs on IRI-induced AKI. Owing to modulation of the GPx-1/NLRP3/Caspase-1 pathway, Se@BSA NPs dramatically inhibited fibrosis formation after AKI. Conclusion: This study provides an effective therapeutic option by applying easy-to-produce Se-containing nanomaterials to remedy Se imbalance and impede inflammatory responses in the kidney, which is a promising candidate for AKI treatment.

Keywords: acute kidney injury; glutathione peroxidase-1; inflammasome; ischemia reperfusion injury; selenium nanoparticles.

Figure 1

(A) Diagram depicting the preparation of Se@BSA NPs. (B) TEM image of Se@BSA NPs. The scale bar indicates 200 nm. The region of interest is magnified in the inset graph, in which the scale bare indicates 20 nm. (C) EDS images of Se@BSA NPs. The yellow and blue signals indicate Se and sulfur, respectively. The scale is 20 nm. (D) XPS spectrum of Se@BSA NPs. (E) FT-IR spectra of Se@BSA NPs. (F) Zeta potential determination. The results are presented as the mean ± SD. (G) DLS detection of the hydrodynamic diameters of Se@BSA NPs at different times.

Figure 2

(A) Survival of HK-2 cells exposed to Se@BSA NPs. The results are shown as the mean ± SD. (B) Hemolysis of Se@BSA NPs. The results are shown as the mean ± SD. (C) Determination of CRE and BUN (D) contents in mice. The results are shown as the mean ± SD. n.s., not significantly. (E) HE staining of the organs of mice treated with sterile PBS and Se@BSA NPs. The scale bar indicates 200 µm. (F) In vitro imaging of FITC-labeled Se@BSA NPs in mouse hearts, livers, spleens, lungs, and kidneys at 0, 3, 6, 12 and 24 h post intravenous administration. (G) ICP-MS determining the content of Se element in mouse organs at different times after Se@BSA NP treatment. (H) Confocal images of HK-2 cells exposed to FITC-labeled Se@BSA NPs for 6 h. The scale bar indicates 20 µm. (I) Immunofluorescence microscopy revealing the colocalization of AQP1 (red) and FITC-labeled Se@BSA NPs (green) in mouse kidneys. The scale bar indicates 20 µm.

Figure 3

(A) Diagram depicting the establishment of the IRI-AKI mouse model. (B) Se contents in the kidneys of IRI-AKI mice. The results are shown as the mean ± SD. ^*, P < 0.05; ^***, P < 0.001. (C) CRE and BUN contents in IRI-AKI mice after Se@BSA NP treatment. The results are shown as the mean ± SD. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. (D) Pictures of mouse kidneys. (E) HE staining of the kidneys of IRI-AKI mice treated with Se@BSA NPs. The scale bar indicates 200 µm. (F) Kim-1 mRNA expression relative to β-actin expression in mouse kidneys. The results are shown as the mean ± SD. ^***, P < 0.001. (G) Western blotting detecting the Kim-1 expression in the kidneys. β-actin was used as the reference. Histogram shows the results of gray analysis for the protein bands. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (H) Immunohistochemical staining showing the Kim-1 expression in the kidneys. The scale bar indicates 200 µm.

Figure 4

(A) Top eight enriched pathways in KEGG analysis. (B) Heat map showing the differential expression of NLRP3 inflammasome-related proteins in IRI-AKI mice treated with Se@BSA NPs. (C) NLRP3 mRNA expression relative to β-actin expression in mouse kidneys. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (D) Western blotting detecting the NLRP3 expression in the kidneys. β-actin was used as the reference. Histogram shows the results of gray analysis for the protein bands. The results are shown as the mean ± SD. ^*, P < 0.05; ^***, P < 0.001. (E) Confocal images indicating the expression of NLRP3 (green) and AQP1 (red) in mouse kidneys. The scale bar indicates 20 µm. (F) Western blotting detecting the NLRP3 expression in HK-2 cells. β-actin was used as the reference. Histogram shows the results of gray analysis for the protein bands. The results are shown as the mean ± SD. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. (G) Confocal images indicating the expression of NLRP3 (green) in HK-2 cells. The scale bar indicates 20 µm. (H) Western blotting revealing the comparable inhibition of Se@BSA NPs to CY-09 on NLRP3 expression in HK-2 cells. β-actin was used as the reference. Histogram shows the results of gray analysis for the protein bands. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001.

Figure 5

(A) IL-1β and IL-18 mRNA expression relative to β-actin in mouse kidneys. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (B) Western blotting detecting the IL-1β, IL-18, and Caspase-1 expression in the kidneys. β-actin was used as the reference. Histogram shows the results of gray analysis for the protein bands. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (C) Immunohistochemical staining showing the IL-1β and IL-18 (D) expression in the kidneys. The scale bar indicates 200 µm. (E) Western blotting detecting the IL-1β, IL-18, and Caspase-1 expression in HK-2 cells. β-actin was used as the reference. The results of gray analysis are presented as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001.

Figure 6

(A) Counts of fluorescent cells by flow cytometry. (B) Average fluorescence intensity of HK-2 cells. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (C) GPx-1 mRNA expression relative to β-actin in HK-2 cells. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (D) Western blotting detecting the GPx-1 expression in HK-2 cells. β-actin was used as the reference. The results of gray analysis are presented as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (E) Confocal images indicating the expression of GPx-1 (red) in HK-2 cells. The scale bar indicates 20 µm. (F) Western blotting detecting the GPx-1 expression in mouse kidneys. β-actin was used as the reference. The results of gray analysis are shown as the mean ± SD. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. (G) Confocal images showing the expression of GPx-1 (green) and AQP1 (red) in mouse kidneys. The scale bar indicates 20 µm. (H) Western blotting detecting the NLRP3 and GPx-1 expression in HK-2 cells. The results of gray analysis are shown as the mean ± SD. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001.

Figure 7

(A) HE and Masson staining of the kidneys of IRI-AKI mice in the absence and presence of Se@BSA NP treatment. The scale bar indicates 200 µm. (B) Determination of CRE and BUN contents in IRI-AKI mice at 2 weeks after Se@BSA NP treatment. The results are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001. (C) Confocal images showing the expression of fibronectin (red) and α-Sma (E, red) in mouse kidneys. The scale bar indicates 20 µm. (D) Immunohistochemical staining showing the collagen I expression in the kidneys. The scale bar indicates 200 µm. (F) Western blotting detecting the fibronectin, α-Sma and collagen I expression in mouse kidneys. The results of gray analysis are shown as the mean ± SD. ^**, P < 0.01; ^***, P < 0.001.