The NLRP3 inflammasome promotes renal inflammation and contributes to CKD

Abstract

Inflammation significantly contributes to the progression of chronic kidney disease (CKD). Inflammasome-dependent cytokines, such as IL-1β and IL-18, play a role in CKD, but their regulation during renal injury is unknown. Here, we analyzed the processing of caspase-1, IL-1β, and IL-18 after unilateral ureteral obstruction (UUO) in mice, which suggested activation of the Nlrp3 inflammasome during renal injury. Compared with wild-type mice, Nlrp3(-/-) mice had less tubular injury, inflammation, and fibrosis after UUO, associated with a reduction in caspase-1 activation and maturation of IL-1β and IL-18; these data confirm that the Nlrp3 inflammasome upregulates these cytokines in the kidney during injury. Bone marrow chimeras revealed that Nlrp3 mediates the injurious/inflammatory processes in both hematopoietic and nonhematopoietic cellular compartments. In tissue from human renal biopsies, a wide variety of nondiabetic kidney diseases exhibited increased expression of NLRP3 mRNA, which correlated with renal function. Taken together, these results strongly support a role for NLRP3 in renal injury and identify the inflammasome as a possible therapeutic target in the treatment of patients with progressive CKD.

Figure 1.

The inflammasome is activated in mice after UUO. (A) IL-1β, IL-18, and caspase-1 activation in mice after UUO. Immunoblotting of kidney tissue lysates from C57Bl/6 mice at 3, 7, and 14 days after UUO. Contralateral (Ctrl) and sham kidneys are used as controls. The appearance of mature (processed) IL-1β, IL-18, and the p10 subunit of active caspase-1 is observed in the ligated (Lig) kidneys at 7 to 14 days, consistent with inflammasome activation. (B) Nlrp3 mRNA expression in mice at 3, 7, and 14 days after UUO (quantitative real-time PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). Data are mean ± SD Nlrp3 mRNA expression. P < 0.001, UUO versus sham, n = 4. (C) Nlrp3 protein expression in mouse kidney at 3, 7, and 14 days after UUO (immunoblotting).

Figure 2.

Nlrp3 contributes to tubular injury in mice undergoing UUO. (A) Histology (hematoxylin and eosin) of kidney sections from Nlrp3^+/+ and Nlrp3^−/− mice after 14 days of UUO. (B) Tubular injury scoring in Nlrp3^+/+ (n = 6), Nlrp3^+/− (n = 6), and Nlrp3^−/− (n = 10) mice at 14 days after UUO (mean score, Nlrp3^+/+ versus Nlrp3^−/− P < 0.05). (C) Quantification of intact cortical tubular area in Nlrp3^+/+ and Nlrp3^−/− mice at 14 days after UUO (mean ± SD tubular area; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6 to 10). (D) Immunofluorescence TUNEL staining of kidney sections from Nlrp3^+/+ and Nlrp3^−/− mice. Sham Nlrp3^+/+ kidneys are shown as controls. (E) Quantification of TUNEL-positive cells in Nlrp3^+/+ and Nlrp3^−/− kidney sections (mean ± SD cell number/high-power field; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 4).

Figure 3.

Renal inflammation is reduced in Nlrp3^−/− mice after UUO. (A) Immunohistochemistry of kidney tissues with the leukocyte marker CD11b at days 7 and 14 after UUO in Nlrp3^+/+ and Nlrp3^−/− mice. (B) Quantification of CD11b⁺ infiltrate area at 14 days after UUO (mean ± SD % area; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6 Nlrp3^+/+ and 10 Nlrp3^−/−). (C) F4/80 immunofluorescence (macrophages) and esterase staining (granulocytes, examples marked with arrows) in Nlrp3^+/+ and Nlrp3^−/− mouse kidneys at 14 days after UUO. Sham Nlrp3^+/+ kidneys are shown as controls. (D) CCL2 (monocyte chemoattractant protein-1) chemokine levels in mouse kidneys at 3, 7, and 14 days after UUO (immunobead assay, mean ± SD; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6 to 10).

Figure 4.

Renal cytokine maturation is reduced in Nlrp3^−/− mice after UUO. (A) Caspase-1 immunoblotting in Nlrp3^+/+ and Nlrp3^−/− mice at 14 days after UUO. Appearance of caspase-1 p10 subunit indicates activation. Sham and contralateral (Ctrl) kidneys are used as controls. (B) Quantification of processed caspase-1 normalized to pro-caspase-1 (mean ± SD; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6 Nlrp3^+/+ and 10 Nlrp3^−/−). (C) IL-1β immunoblotting in Nlrp3^+/+ and Nlrp3^−/− mice at 7 days after UUO. Appearance of mature 17-kD IL-1β indicates activation. (D) Quantification of processed IL-1β normalized to pro-IL-1β (mean ± SD; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6). (E) IL-18 immunoblotting in Nlrp3^+/+ and Nlrp3^−/− mice at 7 days after UUO. Appearance of mature 18-kD IL-18 indicates activation (F) Quantification of processed IL-18 normalized to pro-IL-18 (mean ± SD; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6).

Figure 5.

UUO activates redundant inflammatory pathways in mouse kidneys. (A) Nlrp1b and Nlrp12 mRNA expression (quantitative real time reverse transcriptase–PCR, normalized to GAPDH) in ligated (Lig) or contralateral (Ctrl) Nlrp3^+/+ mouse kidneys at 14 days after UUO. (B) Hyaluronan content (ELISA) in kidney lysates from Nlrp3^+/+ and Nlrp3^−/− mice at 14 days after UUO (sham versus Nlrp3^+/+, P < 0.01, versus Nlrp3^−/−, P < 0.05; Nlrp3^+/+ versus Nlrp3^−/−, NS; n = 5) (C) Immunoblotting for biglycan and HMGB1 in kidney lysates from Nlrp3^+/+ and Nlrp3^−/− mice at 3, 7, and 14 days after UUO. Fourteen-day sham and contralateral kidneys are used as controls.

Figure 6.

Renal fibrosis is reduced in Nlrp3^−/− mice after UUO. (A) Masson trichrome staining on Nlrp3^+/+ and Nlrp3^−/− kidneys at 14 days after UUO. (B) Quantification of renal fibrosis area at 14 days (mean ± SD area; P < 0.01, Nlrp3^+/+ versus Nlrp3^−/−; n = 6 Nlrp3^+/+ and 10 Nlrp3^−/−). (C) E-cadherin and α-smooth muscle actin (αSMA) expression in Nlrp3^+/+ and Nlrp3^−/− kidneys at 14 days after UUO (immunoblotting). Fourteen-day sham and contralateral (Ctrl) kidneys are used as controls. (D) Quantification of E-cadherin and αSMA expression in Nlrp3^+/+ and Nlrp3^−/− mice at 14 days normalized to β-tubulin (mean ± SD; P < 0.05, Nlrp3^+/+ versus Nlrp3^−/−; n = 6 to 10).

Figure 7.

UUO in bone marrow chimeric mice show that Nlrp3 mediates renal injury from renal cells and infiltrating leukocytes. (A) Histology (hematoxylin and eosin) of kidney sections from Nlrp3 BM chimeric mice after 14 days of UUO. Contralateral kidney is from an Nlrp3^+/+ to Nlrp3^+/+ mouse. (B) Quantification of intact cortical tubular area in Nlrp3 BM chimeric mice at 14 days after UUO (mean ± SD tubular area; Nlrp3^+/+ to Nlrp3^+/+ [n = 5]), Nlrp3^+/+ to Nlrp3^−/− [n = 5] and Nlrp3^−/− to Nlrp3^+/+ [n = 12; NS]; all groups versus Nlrp3^−/− to Nlrp3^−/− [n = 8; P < 0.05]). (C) E-cadherin and IL-18 expression in kidneys of Nlrp3 BM chimeric mice at 14 days after UUO (immunoblotting). Sham and contralateral (Ctrl) kidney samples shown are from Nlrp3^+/+ to Nlrp3^+/+ mice. (D) Quantification of E-cadherin expression in Nlrp3 chimeras at 14 days normalized to β-tubulin (mean ± SD; Nlrp3^+/+ to Nlrp3^+/+, Nlrp3^+/+ to Nlrp3^−/−, and Nlrp3^−/− to Nlrp3^+/+ [NS]; all groups versus Nlrp3^−/− to Nlrp3^−/− [P < 0.05]). Magnification, ×40.

Figure 8.

NLRP3 is expressed in primary renal TECs and contributes to apoptosis. (A) Semiquantitative reverse transcriptase–PCR for human and mouse NLRP3 mRNA in HEK293, primary human proximal tubular cells (HPTCs), THP-1 monocyte/macrophages, mouse B8 cells, primary C57Bl/6 TECs, and primary mouse BM macrophages. (B) Immunoblotting for NLRP3 in PMA-stimulated HPTCs and THP-1 monocyte/macrophages. Immunoblotting for mouse Nlrp3 in LPS-stimulated primary C57Bl/6 TECs and primary mouse BM macrophages. (C) Immunoblotting for poly(ADP-ribose) polymerase-1 (PARP1) cleavage (cPARP1) in Nlrp3^+/+ and Nlrp3^−/− primary TECs induced to undergo apoptosis with cyclohexamide and TNF-α. (D) Quantification of cPARP1 normalized to total PARP1 in Nlrp3^+/+ and Nlrp3^−/− primary TECs induced to undergo apoptosis (mean ± SD; P < 0.01, Nlrp3^+/+ versus Nlrp3^−/−; n = 9). (E) Flow cytometry (propidium iodide) of primary TECs after H₂O₂-induced necrosis (mean ± SD % propidium iodide–positive cells; NS, Nlrp3^+/+ versus Nlrp3^−/−; n = 3).

Figure 9.

NLRP3 mRNA is expressed in human nondiabetic kidney disease. RNA is isolated from renal biopsies of patients with nondiabetic kidney disease. Normal n = 16; IgA nephropathy (IgA) n = 9; minimal change disease (MCD) n = 3; membranous glomerulonephritis (MGN) n = 3; crescentic GN (CrGN) n = 5; lupus nephritis (SLE) n = 3; secondary FSGS n = 9; ATN n = 7; hypertension/vascular nephrosclerosis (HTN) n = 4. (A) NLRP3 mRNA expression is assessed by quantitative real-time PCR. Values are normalized to endogenous 18S mRNA expression levels (mean NLRP3 mRNA expression, normal versus disease, all P < 0.01). (B) Scatter plot of NLRP3 mRNA expression (log) against renal function (creatinine, μmol/L) in patients with nondiabetic kidney disease (r = 0.51, P < 0.01; n = 33).