Remdesivir Alleviates Acute Kidney Injury by Inhibiting the Activation of NLRP3 Inflammasome

Abstract

Acute kidney injury (AKI) is a frequent clinical complication in critically ill patients, and it rapidly develops into renal failure with high morbidity and mortality. However, other than dialysis, no effective therapeutic interventions can offer reliable treatment to limit renal injury and improve survival. Here, we firstly reported that remdesivir (RDV, GS-5734), a broad-spectrum antiviral nucleotide prodrug, alleviated AKI by specifically inhibiting NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in macrophages. Mechanically, RDV effectively suppressed the activities of nuclear transcription factor (NF)-κB, mitogen-activated protein kinase (MAPK), which further led to the reduction of the inflammasome genes of NLRP3 transcription, limiting the activation of NLRP3 inflammasome in vivo and in vitro. RDV also inhibited other pro-inflammatory genes including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12, IL-1β, and interferon-β (IFN-β), leading to the reduction of inflammatory factors release. Thus, RDV can ameliorate AKI via modulating macrophage inflammasome activation and inflammatory immune responses and may have a therapeutic potential for patients with AKI in clinical application.

Keywords: LRR-; NF-κB; NLRP3 inflammasome or NOD-; acute kidney injury; macrophage; pyrin domain-containing protein 3 (NLRP3)inflammasome; remdesivir.

Figure 1

RDV inhibits the activation of NLRP3 inflammasome. (A) RT-PCR analysis of NLRP3 mRNA expression in mouse peritoneal macrophages stimulated with LPS, RDV, and LPS + RDV for 12 h. (B) Western blot analysis of NLRP3, NLRC4 and AIM2 in mouse peritoneal macrophages under LPS, RDV and LPS+RDV stimulation. (C) Relative intensity analysis of NLRP3, NLRC4 and AIM2. (D) Western blot analysis of NLRP3, ASC, NLRC4, AIM2 in mouse peritoneal macrophages under LPS and LPS+RDV stimulation at different time points. (E) Relative intensity analysis of NLRP3, ASC, NLRC4 and AIM2. (F) Western blot analysis of pro-Capase-1, Caspase-1, pro-IL-1β, IL-1β, Caspase-3, Cleaved-Capase-3, GSDMD, GSDMD-N. (G) Relative intensity analysis of Caspase-1/pro-Capase-1, pro-IL-1β/GAPDH, IL-1β/GAPDH, Cleaved-Capase-3/Caspase-3, GSDMD/GAPDH, GSDMD-N/GAPDH. (H) ASC specks formation assay of the effect of LPS and LPS + RDV stimulation for 12 h and treatment with ATP in the last 40 min. Percentages of ASC speck formation marked on the images. Data are representative as mean values ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

RDV suppresses the activation of NF-κB and MAPK signaling pathway. (A) Western blot analysis of p-p65, p65, p-p38, p38, p-ERK, ERK in mouse peritoneal macrophages under of LPS, RDV and LPS+RDV stimulation at the indicated time point. (B) Relative intensity analysis of p-p65/p65, p-p38/p38, p-ERK/ERK, p-IκBα/IκBα. (C) Macrophages were stimulated with LPS, LPS + RDV and nuclear translocation of p65 (Red) was determined. (D) Quantitative analysis of p65-positive nuclei. Data are presented as mean values ± SD from three independent experiments. *P < 0.05**; P < 0.01.

Figure 3

RDV inhibits the expression of pro-inflammatory cytokines. (A) RT-PCR analysis of TNF-α, IL-6, IL-1β, IL-12, iNOS, and IFN-β mRNA expressions in mouse peritoneal macrophages stimulated with LPS, RDV, LPS + RDV for 12 h. (B) ELISA analysis of TNF-α, IL-12, IL-6, and IL-1β in cell culture supernatant stimulated with LPS and LPS+RDV for 12 h and 24 h and treatment with ATP in the last 40 min. (C) Western blot analysis of iNOS in mouse peritoneal macrophages under LPS and LPS+RDV stimulation at different time points and relative intensity analysis of iNOS. (D) Western blot analysis of iNOS in the kidney and relative intensity analysis of iNOS. Data are representative as mean values ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

RDV alleviates LPS-induced AKI. (A) HE and PAS staining of kidney tissue sections. (B) Right panel showing the renal inflammation score based on HE staining. (C) SCr, BUN and urinary albumin levels detected by ELISA. Data are representative as mean values ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Effects of RDV on inflammatory injury in LPS-induced AKI mice. (A) Western blot analysis of NLRP3, NLRC4 and AIM2 in kidneys. (B, D, F) Relative intensity analysis of NLRP3, NLRC4, AIM2, p-ERK/ERK, p-IκBα/IκBα, cleaved-caspse-3/caspase-3, GSDMD/GAPDH, GSDMD-N/GAPDH. (C) Key points of apoptosis and pyroptosis pathways in kidneys were detected by Western blot. (E) Western analysis of p-p65/p65, p-p38/p38, p-IκBα/IκBα. (G) ELISA analysis of serum level of IL-1β, IL-6, TNF-α, IL-12. Data are representative as mean values ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Work model for RDV alleviates AKI by inhibiting NLRP3 inflammasome activation. RDV inhibits NF-kB and MAPK signaling activation, which reduces the transcription of NLRP3, IL-1β, TNF-α, IL-6, IL-12, GSDMD inhibiting the NLRP3 inflammasome formation, apoptosis, pyroptosis and pro-inflammatory cytokine release and contributing to the alleviation of AKI.