The expression of NOD2, NLRP3 and NLRC5 and renal injury in anti-neutrophil cytoplasmic antibody-associated vasculitis

Abstract

Background: Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are intracellular sensors of pathogens and molecules from damaged cells to regulate the inflammatory response in the innate immune system. Emerging evidences suggested a potential role of NLRs in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study aimed to investigate the expression of nucleotide-binding oligomerization domain containing protein 2 (NOD2), NOD-like receptor family pyrin domain containing 3 (NLRP3) and NOD-like receptor family CARD domain containing 5 (NLRC5) in kidneys of AAV patients, and further explored their associations with clinical and pathological parameters.

Methods: Thirty-four AAV patients in active stage were recruited. Their renal specimens were processed with immunohistochemistry to assess the expression of three NLRs, and with double immunofluorescence to detect NLRs on intrinsic and infiltrating cells. Analysis of gene expression was also adopted in cultured human podocytes. The associations between expression of NLRs and clinicopathological parameters were analyzed.

Results: The expression of NOD2, NLRP3 and NLRC5 was significantly higher in kidneys from AAV patients than those from normal controls, minimal change disease or class IV lupus nephritis. These NLRs co-localized with podocytes and infiltrating inflammatory cells. The mean optical density of NOD2 in glomeruli was significantly higher in crescentic class than non-crescentic class, and correlated with levels of proteinuria and serum creatinine at renal biopsy. The mean optical density of NLRC5 in glomeruli was significantly higher in crescentic class than non-crescentic class, and correlated with proteinuria level, Birmingham Vasculitis Activity Score and the proportion of crescents in the renal specimen.

Conclusions: The expression of three NLRs was upregulated in kidneys of AAV patients. The expression of NOD2 and NLRC5 was associated with the severity of renal lesions in AAV.

Keywords: ANCA; NOD-like receptors; Vasculitis.

Fig. 1

Immunohistochemistry staining for NOD2, NLRP3, NLRC5 in glomeruli and tubulointerstitium of renal specimens. a Immunohistochemical staining of NLRs in glomeruli of normal controls. b Immunohistochemical staining of NLRs in glomeruli of AAV patients. c Immunohistochemical staining of NLRs in tubulointerstitium of normal controls. d Immunohistochemical staining of NLRs in tubulointerstitium of AAV patients. Scale bar = 50 μm in the bottom right

Fig. 2

Different expression of NOD2, NLRP3, NLRC5. a The mean optical density of NOD2 in different conditions. b The mean optical density of NLRP3 in different conditions. c The mean optical density of NLRC5 in different conditions. NC: normal control. MCD: minimal change disease. LN lupus nephritis, AAV anti-neutrophil cytoplasmic antibody-associated vasculitis. Data were shown as mean ± standard error. ns no significance. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Double immunofluorescence staining of NOD2 and glomerular intrinsic cells in AAV patients. a Co-localization of NOD2 (red) and CD31 (green). b Co-localization of NOD2 (red) and integrin-α (green). c Co-localization of NOD2 (red) and synaptopodin (green). Scale bar = 50 μm in the bottom right

Fig. 4

Double immunofluorescence staining of NLRP3 and glomerular intrinsic cells in AAV patients. a Co-localization of NLRP3 (red) and CD31 (green). b Co-localization of NLRP3 (red) and integrin-α (green). c Co-localization of NLRP3 (red) and synaptopodin (green). Scale bar = 50 μm in the bottom right

Fig. 5

Double immunofluorescence staining of NLRC5 and glomerular intrinsic cells in AAV patients. a Co-localization of NLRC5 (red) and CD31 (green). b Co-localization of NLRC5 (red) and integrin-α (green). c Co-localization of NLRC5 (red) and synaptopodin (green). Scale bar = 50 μm in the bottom right

Fig. 6

Double immunofluorescence staining of NLRs and infiltrating cells in AAV patients. a Co-localization of NOD2 (red) and CD68 (green). b Co-localization of NLRP3 (red) and CD68 (green). c Co-localization of NLRC5 (red) and CD68 (green). Scale bar = 50 μm in the bottom right

Fig. 7

The mRNA expression of three NLRs in human podocytes. The mRNA expression of NOD2, NLRP3 and NLRC5 in cultured human podocytes was elevated upon TNF-α stimulation. Bars represent mean ± standard error of repeated measurements of four independent experiments. ns no significance. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle treatment

Fig. 8

Correlations of NLRs and clinicopathological data in AAV patients. a, b The mean optical density of NOD2 in glomeruli correlated with proteinuria and serum creatinine. c–e The mean optical density of NLRC5 in glomeruli correlated with proteinuria, BVAS and proportion of crescents. f Glomerular expression of three NLRs in different categories of Berden classification. Data were shown as mean ± standard error. ns no significance. *P < 0.05