Inflammasome-Independent Role of NLRP3 Mediates Mitochondrial Regulation in Renal Injury

Abstract

The NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome has been implicated in renal inflammation and fibrosis. However, the biological function of inflammasome-independent NLRP3 in non-immune cells is still unclear. We evaluated the role of inflammasome-independent NLRP3 in renal tubular cells and assessed the value of NLRP3 as a therapeutic target for acute kidney injury (AKI). Various renal tubular cell lines and primary cultured tubular cells from NLRP3 knockout (KO) mice were used for in vitro studies. We also tested the role of tubular NLRP3 in AKI with a unilateral ureter obstruction model (UUO). Hypoxia induced significant increase of NLRP3 independent of ASC, caspase-1, and IL-1β. NLRP3 in renal tubular cells relocalized from the cytosol to the mitochondria during hypoxia and bound to mitochondrial antiviral signal protein (MAVS). The deletion of NLRP3 or MAVS in renal tubular cells attenuated mitochondrial reactive oxygen species (ROS) production and depolarization of the mitochondrial membrane potentials under hypoxia. In response to UUO, NLRP3 KO mice showed less fibrosis, apoptosis, and ROS injury than wild type (WT) mice. Compared with WT kidney, mitophagy was up-regulated in NLRP3 KO kidney relative to the baseline and it was protective against AKI. Our results indicate that inflammasome-independent NLRP3 in renal tubular cells plays important role in mitochondrial ROS production and injury by binding to MAVS after hypoxic injury. This mitochondrial regulation in the absence of NLRP3 increases autophagy and attenuates apoptosis after UUO. We suggest that inflammasome-independent NLRP3 could be a therapeutic target of AKI to prevent the progression of chronic kidney disease.

Keywords: NLRP3; acute kidney injury (AKI); apoptosis; mitochondrial ROS; mitophagy.

Figure 1

NLRP3 is increased by hypoxia in renal tubular epithelial cells without releasing IL-1 β. (A) HK-2, HKC-8, TERT and HTE cells were subjected to 6 h of hypoxia. IL-1β expression was analyzed by immunoblotting. (B,C) Expression of NLRP3, ASC, caspase-1, and HIF-1α proteins was detected in HK-2 cells after 0, 1, 3, 6, 9, 12, or 24 h of hypoxia exposure. ^*p < 0.05 vs. Control. (D,E) Expression of NLRP3, ASC, and HIF-1α proteins was detected in PTEC after 3 and 6 h of hypoxia exposure. ^*p < 0.05 vs. WT control.

Figure 2

NLRP3 relocalizes into the mitochondria during hypoxia in HK-2 cells. (A–C) HK-2 cells were subjected to 6 h of hypoxia and analyzed by confocal microscopy for NLRP3, ASC and MitoTracker staining. (D) Expression of NLRP3, ASC and Caspase-1 was analyzed by immunoblotting in hypoxia-exposed cells. (E) HK-2 cells were transfected with siASC and siCaspase-1 and subjected to 6 h of hypoxia. NLRP3 expression was analyzed by immunoblotting. Cell lysates were fractionated into cytosolic or mitochondrial fraction. Prohibitin and GAPDH were used to confirm that the mitochondrial and cytosolic fractions were well separated.

Figure 3

NLRP3 is required for mitochondrial dysfunction in HK-2 cells. (A) HK-2 cells were transfected with siNLRP3 and NLRP3 pCMV6 vector and subjected to 6 h of hypoxia. NLRP3 expression was analyzed by immunoblotting. (B–D) siNLRP3 or NLRP3 pCMV6 transfected HK-2 cells were stained with MitoSOX and analyzed by flow cytometry and confocal microscopy.(E–G) siNLRP3 or NLRP3 pCMV6 transfected HK-2 cells were stained with JC-1 and analyzed by flow cytometry and confocal microscopy. Representative histograms and quantified levels are shown. ^*p < 0.05 vs. Control, #p < 0.05 vs. Hypoxia. (H) HK-2 cells were transfected with siNLRP3 and subjected to 6 h of hypoxia. Cell lysates were fractionated into cytosolic or mitochondrial fraction and then immunoblotted for AIF, cytochrome c, and Bax. (I) HK-2 cells were transfected with siNLRP3 and NLRP3 pCMV6 vector; and subjected to 6 h of hypoxia. Genomic DNA and cytosolic mtDNA were isolated from cells and mtDNA in cytosolic fraction was analyzed by real-time PCR. ^*p < 0.05 vs. Control, #p < 0.05 vs. Hypoxia.

Figure 4

The mitochondrial adaptor MAVS mediates NLRP3 mitochondrial localization. (A) Hypoxia-exposed HK-2 cells were analyzed by confocal microscopy for NLRP3 and MAVS expression. (B) HK-2 cells were transfected with siMAVS and subjected to 6 h of hypoxia. Cell lysates were immunoprecipitated using anti-NLRP3 antibody, and the immunoprecipitates were immunoblotted with anti-MAVS antibody. (C,D) HK-2 cells were transfected with siMAVS and subjected to 6 h hypoxia. HK-2 cells were stained MitoSOX and analyzed by flow cytometry. (E,F) HK-2 cells were transfected with siMAVS and subjected to 6 h of hypoxia. HK-2 cells were stained with JC-1 and analyzed by flow cytometry. Representative histograms and quantified levels are shown. ^*p < 0.05 vs. Control, #p < 0.05 vs. Hypoxia.

Figure 5

NLRP3 deficiency in PTEC inhibits hypoxia-induced mitochondrial ROS and apoptosis. (A,B) PTEC were stained with MitoSOX and analyzed by flow cytometry. Representative images and quantitative analysis of mean fluorescence intensities are shown. ^*p < 0.05 vs. WT. (C) Analysis of the cell death phenotype by Annexin-V/PI staining followed by flow cytometry. (D,E) Expression of cytochrome c, Bax, cleaved caspase-3 and PARP proteins was detected in WT and NLRP3 KO PTEC after 3 and 6 h of hypoxia exposure. ^*p < 0.05 vs. WT control, #p < 0.05 vs. WT hypoxia. NLRP3^−/−, NLRP3 KO.

Figure 6

NLRP3 contributes to tubular injury in mice undergoing UUO. (A,B) Masson's trichrome–stained of kidney sections from WT (C57BL/6) mice and NLRP3 KO mice after 3 and 7 days of UUO (Original magnification, x400). Semiquantitative scoring of tubulointerstitial fibrosis in Masson's trichrome–stained sections. (C,D) Representative images of TUNEL staining are shown (Original magnification, ×200). Bar graph indicates the mean number of TUNEL-positive tubular cells per field. (E,F) Expression of caspase-3 and PARP proteins was detected in kidney tissue samples. (G,H) Immunohistochemistry was used to measure the levels of the oxidative stress marker 8-OHdG (Original magnification, ×400). Bar graph indicates the mean number of 8-OHdG-positive cells. ^*p < 0.05 vs. WT sham, #p < 0.05 vs. WT UUO.

Figure 7

NLRP3 depletion is associated with mitophagy, which protects mitochondria against damage. (A,B) Expression of LC3, Parkin, and PINK1 proteins was detected in WT and NLRP3 KO PTEC after 3 and 6 h of hypoxia exposure. ^*p < 0.05 vs. WT control, #p < 0.05 vs. WT hypoxia. (C) PTEC were transfected with LC3-GFP vector and subjected to 6 h of hypoxia. Hypoxia-exposed PTEC were stained with Lysotracker-Red and analyzed by confocal microscopy. (D,E) Expression of NLRP3, LC3, Parkin, PINK1 and HIF-1α proteins was detected in kidney tissue samples. ^*p < 0.05 vs. WT sham, #p < 0.05 vs. WT UUO. (F) Transmission electron microscopy analysis of mitochondrial morphology. Arrows indicate mitophagy. Original magnification × 1,000 nm.