NLRP3 Deficiency Attenuates Renal Fibrosis and Ameliorates Mitochondrial Dysfunction in a Mouse Unilateral Ureteral Obstruction Model of Chronic Kidney Disease

Abstract

Background and Aims. The nucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) inflammasome has been implicated in the pathogenesis of chronic kidney disease (CKD); however, its exact role in glomerular injury and tubulointerstitial fibrosis is still undefined. The present study was performed to identify the function of NLRP3 in modulating renal injury and fibrosis and the potential involvement of mitochondrial dysfunction in the murine unilateral ureteral obstruction (UUO) model of CKD. Methods. Employing wild-type (WT) and NLRP3^-/- mice with or without UUO, we evaluated renal structure, tissue injury, and mitochondrial ultrastructure, as well as expression of some vital molecules involved in the progression of fibrosis, apoptosis, inflammation, and mitochondrial dysfunction. Results. The severe glomerular injury and tubulointerstitial fibrosis induced in WT mice by UUO was markedly attenuated in NLRP3^-/- mice as evidenced by blockade of extracellular matrix deposition, decreased cell apoptosis, and phenotypic alterations. Moreover, NLRP3 deletion reversed UUO-induced impairment of mitochondrial morphology and function. Conclusions. NLRP3 deletion ameliorates mitochondrial dysfunction and alleviates renal fibrosis in a murine UUO model of CKD.

Figure 1

Demonstration of kidney histopathology at day 14 UUO. Representative photomicrographs of periodic acid-Schiff-stained kidney sections ((a), magnification, ×400) and Masson trichrome-stained kidney sections ((b), magnification, ×400).

Figure 2

Immunohistochemical staining for deposition of collagen I ((a) magnification, ×400) and fibronection ((b), magnification, ×400) in kidney sections from different groups at day 14 after UUO.

Figure 3

NLRP3 deletion attenuates UUO-induced glomerular injury and tubulointerstitial fibrosis as well as deposition of collagen I and fibronectin. Glomerular injury score (a) and tubulointerstitial fibrosis index (b) are evaluated according to PAS staining and Masson staining. Semiquantitative analysis of collagen I (c) and fibronectin (d) positive areas is performed according to immunohistochemical staining. Data represent the mean ± SEM (n = 6). ^∗P < 0.05, WT/Sham group versus WT/UUO group; ^#P < 0.05, NLRP3^−/−/UUO group versus WT/UUO group.

Figure 4

NLRP3 deletion decreases UUO-induced phenotypic alterations in UUO mice. Expression of α-SMA and E-cadherin protein detected in kidney samples from different groups (a). Semiquantitative analysis of α-SMA expression (b) and E-cadherin expression (c) normalized against β-actin. Data represent the mean ± SEM (n = 6). ^∗P < 0.05 compared with WT/Sham group, ^#P < 0.05, NLRP3^−/−/U14 group versus WT/U14 group. U1, U3, U7, and U14 indicate that mice were sacrificed on days 1, 3, 7, and 14 after UUO.

Figure 5

NLRP3 deletion ameliorates UUO-induced cell apoptosis in UUO mice. Representative images of TUNEL staining are shown ((a), magnification, ×200). Bar graph indicates the mean number of TUNEL-positive tubular cells per field (b). Expression of caspase 3 protein was detected in kidney samples (c). Semiquantitative analysis of cleaved caspase 3 normalized against β-actin (d). Data represent the mean ± SEM (n = 6). ^∗P < 0.05, compared with WT/Sham group, ^#P < 0.05, NLRP3^−/−/U14 group versus WT/U14 group. U1, U3, U7, and U14 indicate that mice were sacrificed on days 1, 3, 7, and 14 after UUO.

Figure 6

NLRP3 deletion decreases UUO-upregulated expression of NLRP3 inflammasome-regulated cytokines. Expression of caspase 1, IL-1β, and IL-18 in kidney samples was detected (a). Semiquantitative analysis of caspase 1, IL-1β, and IL-18 normalized against β-actin (b). Data represent the mean ± SEM (n = 6). ^∗P < 0.05, compared with WT/Sham group, ^#P < 0.05, NLRP3^−/−/U14 group versus WT/U14 group. U1, U3, U7, and U14 indicate that mice were sacrificed on days 1, 3, 7, and 14 after UUO.

Figure 7

NLRP3 deletion reverses UUO-induced mitochondrial dysfunction. Representative electron microscopy photomicrographs of ultrastructural morphology of mitochondria (a), (magnification ×12,000). Arrow indicates swollen mitochondria. Expression of PGC-1a in kidney sample was detected, and semiquantitative analysis of PGC-1a normalized against β-actin (b). Semiquantitative analysis of mtDNA expression normalized against 18S performed by real-time PCR (c). Data represent the mean ± SEM (n = 6). ^∗P < 0.05, compared with WT/Sham group, ^#P < 0.05, NLRP3^−/−/U14 group versus WT/U14 group. U1, U3, U7, and U14 indicate that mice were sacrificed on days 1, 3, 7, and 14 after UUO.

Figure 8

NLRP3 deletion attenuates UUO-induced mitochondrial dysfunction. Semiquantitative analysis of ATP synthase (a) and ND1 (b) normalized against 18S performed by real-time PCR. Data represent the mean ± SEM (n = 6). ^∗P < 0.05, compared with WT/Sham group, ^#P < 0.05, NLRP3^−/−/U14 group versus WT/U14 group. U1, U3, U7, and U14 indicate that mice were sacrificed on days 1, 3, 7, and 14 after UUO.