Microarray-based analysis of renal complement components reveals a therapeutic target for lupus nephritis

Abstract

Background: Screening abnormal pathways and complement components in the kidneys of patients with lupus nephritis (LN) and NZB/W mice may help to identify complement-related therapeutic targets for LN.

Methods: KEGG and GO enrichment assays were used to analyze kidney microarray data of LN patients and NZB/W mice. Immunohistochemistry and immunofluorescence assays were used to measure renal expression of complement-related proteins and TGFβ1. Cytokines were measured using RT-qPCR and ELISA.

Results: We screened the renal pathogenic pathways present in LN patients and NZB/W mice and selected the complement activation pathway for further study. The results indicated greater renal expression of C1qa, C1qb, C3, C3aR1, and C5aR1 at the mRNA and protein levels. C3 appeared to be a key factor in LN and the renal signaling downstream of C1 was inhibited. There were significant correlations between the expression of TGFβ1 and C3. Analysis of primary cell cultures indicated that TGFβ1 promoted the expression of C3 and that a TGFβ1 antagonist decreased the levels of C3 and C3aR. TGFβ1 inhibition significantly inhibited the deposition of complement-related factors in the kidneys of NZB/W mice.

Conclusions: At the onset of LN, there are significant increases in the renal levels of C3 and other complement pathway-related factors in patients with LN and NZB/W mice. C3 may lead to albuminuria and participate in the pathogenesis of LN. TGFβ1 promotes C3 synthesis, and TGFβ1 inhibition may block the progression of LN by inhibiting the synthesis of C3 and other complement components.

Keywords: C3aR1; Complement C3; Lupus nephritis; SB431542; TGFβ1.

Fig. 1

Renal pathways and complement genes in LN patients and healthy controls based on microarray results. A Enrichment and p-values of some upregulated KEGG pathways in the kidneys of LN patients and NZB/W F1 mice. B Cluster analysis of the expression of C1qa, C1qb, C3, C3aR1, and C5aR1 in the glomeruli (GLO) and renal tubules (TUB) in each group. C mRNA levels of C1qa, C1qb, C3, C3aR1, and C5aR1 in the renal tubules of patients with LN (N = 32) and healthy controls (N = 15). * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 2

Immunohistochemical analysis of complement components in the glomeruli and renal tubules of LN patients and healthy controls. A Expression of C1q, C3, C5, C3aR, C5aR1, and CR3 in glomeruli (GLO) and renal tubules (TUB). B Immunohistochemistry -paraffin section (IHC-P) scores of C1q (N = 3, p < 0.01). C IHC-P scores of C3 (N = 6, p < 0.001). D IHC-P scores of C3aR (N = 5, p < 0.01). E IHC-P scores of C5 (N = 6, p < 0.001). F IHC-P scores of C5aR1 (N = 6, p < 0.001). G The IHC-P scores of CR3 (N = 3, p < 0.01). All IHC-P scores were determined using ImageJ software

Fig. 3

Expression of renal complement components in NZB/W mice during the progression of LN. A Cluster analysis showing the expression of C1qa, C1qb, C1qc, C3aR1, C3, C5aR1, CR3, and CR4 in the control group (16 weeks, N = 8), early-stage group (23 weeks, N = 6), and late-stage group (36 weeks, N = 10). B Renal mRNA levels of complement components in each group (* p < 0.05, ** p < 0.01, *** p < 0.001, ▲ significant difference between late-stage and early-stage). C Immunohistochemical analysis of C3 and C3aR. D Complement activation pathway analysis based on kidney microarray results of LN patients and NZB/W mice, red boxes show significantly upregulated genes

Fig. 4

GO enrichment analysis and the levels of complement C3 in LN patients. A Directed acyclic graph of GO enrichment, showing upregulated genes in biological process in LN patients and NZB/W mice. Squares: complement-related GO terms; red squares or red circles: significant enrichment; # significant enrichment only in LN patients; ## significant enrichment only in NZB/W mice; the unmarked terms: have significant enrichment in LN patients and NZB/W mice. B Levels of urinary C3 in healthy controls (N = 11), SLE patients without LN (N = 14) and patients with LN (N = 39). *** p < 0.001. C Levels of plasma C3 in SLE patients without LN (N = 66) and patients with LN (N = 76). *** p < 0.001. D Correlation between the urinary levels of C3 and 24 h urine protein (N = 37)

Fig. 5

Relationships between the levels of TGFβ1 and C3 in LN patients. A Plasma levels of TGFβ1 in SLE patients without LN (N = 26) and patients with LN (N = 25). * p < 0.05. B Correlation of the protein levels of TGFβ1 and C3 in plasma of LN patients (N = 38). C Correlation of the mRNA levels of TGF β1 and C3 in PBMCs of LN patients (N = 18). D Urine levels of TGFβ1 in SLE patients without LN (N = 14) and patients with LN (N = 26). *** p < 0.001. E Correlation between the levels of TGFβ1 and 24 h urine protein in patients with LN (N = 26). F Correlation between the urine levels of TGFβ1 and C3 in LN patients (N = 26). G Immunohistochemical analysis of TGFβ1 and C3 in the glomeruli (GLO) and renal tubules (TUB) of patients with different classes of LN

Fig. 6

Effects of TGFβ1 on the production of C3 and other complement components. A mRNA levels of C3 (N = 12), C3AR1 (N = 10), and C1qa (N = 8) in the presence of TGFβ1 or SB431542 in bone marrow cell-cultures of NZB/W mice. B mRNA levels of C3 (N = 14), C3aR1 (N = 15), and C1qa (N = 14) in the presence of TGFβ1 or SB431542 in kidney primary cell-cultures of NZB/W mice. C mRNA levels of C3 (N = 11), C3AR1 (N = 5), and C1qa (N = 7) in the presence of TGFβ1 or SB431542 in PBMC-cultures of LN patients. * p < 0.05, ** p < 0.01, *** p < 0.001. D Immunohistochemical analysis of C1q, C3, C5, and their receptors (C3aR, C5aR1) in the kidneys of NZB/W mice treated with SB431542 or saline as control. E Immunofluorescence analysis of C3 in the kidney of NZB/W mice: 32-week-old mice treated with SB431542, 32-week-old mice treated with saline, and 8-week-old mice without treatment. Red fluorescence indicates C3 and blue fluorescence indicates nuclei