Hydrogen sulfide attenuates renal I/R‑induced activation of the inflammatory response and apoptosis via regulating Nrf2‑mediated NLRP3 signaling pathway inhibition

Abstract

Renal ischemia/reperfusion (I/R) injury can lead to acute renal failure, delayed graft function and graft rejection. Nucleotide‑binding oligomerization domain NOD‑like receptor containing pyrin domain 3 (NLRP3)‑mediated inflammation participates in the development of renal injury. Nrf2 accelerates NLRP3 signaling pathway activation and further regulates the inflammatory response. In addition, hydrogen sulfide serves a protective role in renal injury; however, the detailed underlying mechanism remains poorly understood. The present study investigated whether Nrf2 and NLRP3 pathway participate in hydrogen sulfide‑regulated renal I/R‑induced activation of the inflammatory response and apoptosis. Wild‑type and Nrf2‑knockout (KO) mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. A total of 20 mg/kg MCC950 (an NLRP3 inhibitor) was injected intraperitoneally daily for 14 days prior to surgery. Renal tissue and blood were collected from the I/R model mice to analyze NLRP3 and Nrf2 mRNA expression levels, NLRP3, PYD and CARD domain containing, caspase‑1, IL‑1β, Nrf2 and heme oxygenase 1 protein expression levels, cell apoptosis, the secretion of tumor necrosis factor‑α, IL‑1β and IL‑6 cytokines and renal histopathology and function. Renal I/R activated the NLRP3 and Nrf2 signaling pathways. Conversely, MCC950 treatment inhibited activation of the NLRP3 signaling pathway, and prevented I/R‑induced renal injury, release of cytokines and apoptosis in renal I/R model mice. Sodium hydrosulfide (NaHS) not only alleviated upregulation of NLRP3 protein expression levels, but also relieved renal injury, release of cytokines and cell apoptosis induced by renal I/R in wild‑type mice, but not in Nrf2‑KO mice. NaHS alleviated NLRP3 inflammasome activation, renal injury, the inflammatory response and cell apoptosis via the Nrf2 signaling pathway in renal I/R model mice.

Keywords: NLR family pyrin domain containing 3; Nrf2; apoptosis; hydrogen sulfide; renal ischemia/reperfusion injury.

Figure 1.

Renal I/R upregulates NLRP3 expression levels. Mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. Following 24 h reperfusion, renal tissue was collected to analyze NLRP3 protein and mRNA expression levels by (A and B) western blotting, (C) reverse transcription-quantitative PCR and (D and E) immunohistochemistry (scale bar, 100 µm). Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con. I/R, ischemia/reperfusion; NLRP3, NLR family pyrin domain containing 3; Con, control.

Figure 2.

MCC950 downregulates expression levels of NLRP3 signaling pathway-associated proteins following renal I/R. Mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. Mice were injected with 20 mg/kg MCC950 intraperitoneally daily for 14 days prior to surgery. Following 24 h reperfusion, renal tissue was collected for (A) western blot analysis of (B) NLRP3, (C) ASC, (D) caspase-1 and (E) IL-1β expression levels. Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con; ^#P<0.05 vs. I/R. I/R, ischemia/reperfusion; NLRP3, NLR family pyrin domain containing 3; ASC, PYD and CARD domain containing; Con, control.

Figure 3.

MCC950 alleviates renal function and injury and decreases cytokine levels and apoptosis following renal I/R. Mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. Mice were injected with 20 mg/kg MCC950 intraperitoneally daily for 14 days prior to surgery. Following 24 h reperfusion, renal tissue and blood were collected to analyze (A) Cr, (B) BUN and (C) KIM-1 levels. (D) Histopathological changes were assessed and (E) scored. Scale bar, 20 µm. (F) TNF-α, (G) IL-1β and (H) IL-6 expression levels and (I and J) apoptosis were assessed. Scale bar, 100 µm. Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con; ^#P<0.05 vs. I/R. I/R, ischemia/reperfusion; Cr, creatinine; BUN, blood urea nitrogen; KIM-1, kidney injury molecule-1; Con, control.

Figure 4.

Nrf2 expression levels are upregulated following renal I/R. Mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. At 24 h post-reperfusion, renal tissue was collected for (A) western blot analysis of (B) nucleic, (C) total protein and mRNA expression levels of Nrf2, (D) reverse transcription-quantitative PCR and (E and F) immunohistochemistry. Scale bar, 100 µm. Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con. I/R, ischemia/reperfusion; Con, control.

Figure 5.

Nrf2 regulates the inhibition of NLRP3 inflammasome activity. Wild-type and Nrf2-KO mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. (A) At 24 h post-reperfusion, renal tissue was collected for western blot analysis of (B) Nrf2, (C) HO-1, (D) NLRP3, (E) ASC, (F) caspase-1 and (G) IL-1β protein expression levels. Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con wild-type mice; ^#P<0.05 vs. I/R wild-type mice. NLRP3, NLR family pyrin domain containing 3; ASC, PYD and CARD domain containing; HO-1, heme oxygenase 1; KO, knockout; Con, control; I/R, ischemia/reperfusion.

Figure 6.

NaHS downregulates I/R-induced upregulation of NLRP3 protein expression levels via the Nrf2 signaling pathway. Wild-type and Nrf2-KO mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. Mice were treated with 50 µmol/kg intraperitoneal NaHS prior to renal ischemia. (A) At 24 h post-reperfusion, renal tissue was collected for western blot analysis of (B) Nrf2, (C) NLRP3, (D) caspase-1 and (E) IL-1β protein expression levels. Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con wild-type mice; ^#P<0.05 vs. I/R wild-type mice; ^&P<0.05 vs. I/R+NaHS wild-type mice. NaHS, sodium hydrosulfide; I/R, ischemia/reperfusion; NLRP3, NLR family pyrin domain containing 3; KO, knockout; Con, control.

Figure 7.

NaHS alleviates renal dysfunction, excessive release of cytokines and cell apoptosis induced by renal I/R via the Nrf2 signaling pathway. Wild-type and Nrf2-KO mice underwent surgery to induce renal I/R via clamping of the bilateral renal pedicles. Mice were treated with 50 µmol/kg intraperitoneal NaHS prior to renal ischemia. (A) At 24 h post-reperfusion, blood and renal tissue were collected to measure (B) histopathological changes and (C) Cr, (D) BUN and (E) KIM-1 levels. Scale bar, 20 µm. (F and G) Apoptosis and expression levels of (H) TNF-α, (I) IL-1β and (J) IL-6 were assessed. Scale bar, 100 µm. Data are presented as the mean ± SD (n=3). *P<0.05 vs. Con wild-type mice; ^#P<0.05 vs. I/R wild-type mice; ^&P<0.05 vs. I/R+NaHS wild-type mice; ^$P<0.05 vs. Con KO mice. NaHS, sodium hydrosulfide; I/R, ischemia/reperfusion; KO, knockout; BUN, blood urea nitrogen; KIM-1, kidney injury molecule-1; Con, control; Cr, creatinine.