Proteomics uncovers ICAM2 (CD102) as a novel serum biomarker of proliferative lupus nephritis

Abstract

Objectives: This study aimed to identify novel, non-invasive biomarkers for lupus nephritis (LN) through serum proteomics.

Methods: Serum proteins were detected in patients with LN and healthy control (HC) groups through liquid chromatography-tandem mass spectrometry. The key networks associated with LN were screened out using Cytoscape software, followed by pathway enrichment analysis. The best candidate biomarkers were selected by machine learning models, further validated in a larger independent cohort. Finally, the expression of these candidate markers was verified in kidney tissue samples, and the mechanism was explored by knocking down the expression of intercellular adhesion molecule 2 (ICAM2) through in vitro cell transfection with siRNA.

Results: Following the serum proteomic screening of LN, a key network of 20 proteins was identified. Machine learning models were used to select ICAM2 (CD102), metalloproteinase inhibitor 1 (TIMP1) and thrombospondin 1 (THSB1) for validation in independent cohorts. ICAM2 exhibited the highest area under the curve (AUC) value in distinguishing LN from HC (AUC=0.92) and was significantly correlated with activity index, proteinuria, albumin and anti-dsDNA antibody levels. Particularly, ICAM2 was significantly elevated in proliferative LN and was associated with specific pathological attributes, outperforming conventional parameters in distinguishing proliferative LN from non-proliferative LN. ICAM2 expression was also elevated in renal tissue samples from patients with proliferative LN. In vitro, knockdown of ICAM2 expression can inhibit the activation of the PI3K/Akt pathway and alleviate the injury of glomerular endothelial cells.

Conclusion: ICAM2 (CD102) may serve as a potential serum biomarker for proliferative LN that reflects renal pathology activity, potentially contributing to the progression of LN through the PI3K/Akt pathway.

Keywords: Autoimmune Diseases; Autoimmunity; Lupus Nephritis.

Figure 1. Differentially expressed protein screening for lupus nephritis (LN) and healthy controls (HC). (A) Principal coordinate analysis (PCoA) plots were derived using 40 patients with LN and 40 HCs for proteomics. (B) Volcano plots highlighting significant differences in protein expression between LN and HCs (|fold change|>2 and p<0.05 by Student’s t-test). Red indicates expression upregulation in LN group, blue indicates expression upregulation in LN. (C) Selection of 25 hub proteins from protein-protein interaction (PPI) network through maximal clique centrality (MCC) algorithm using the plugin CytoHubba. The red nodes represent proteins with high MCC values, while the yellow nodes represent proteins with low MCC values. (D) Selection of the most significant module from PPI network using the plugin MCODE. (E) The pathway enrichment analysis enriched by Metascape. (F) Top 20 proteins prioritised by random forest analysis ranked by MeanDecreaseAccuracy and MeanDecreaseGini.

Figure 2. ELISA validation of three serum biomarkers using an independent cohort and their correlation with clinical indices. (A–C) A cohort of 80 serum samples: 30 healthy controls (HC, green) and 50 patients with lupus nephritis (LN, red) were tested by ELISA for the levels of ICAM2, TIMP1 and THBS1. (D) The discriminatory abilities of the three serum markers were assessed in distinguishing patients with LN from HCs using receiver operating characteristic (ROC) curve analysis. (E–K) The correlation of ELISA-assayed serum ICAM2 levels with clinical indices in LN. r denotes Spearman’s correlation coefficient. ****p<0.0001. AUC, area under the curve.

Figure 3. Serum ICAM2 levels in lupus nephritis (LN) classes and their association with renal histology. (A) Serum ICAM2 was significantly elevated in patients with LN III (±V) and LN IV (±V) when compared with healthy controls (HC) or LN V. (B) Serum ICAM2 outperformed C3, C4 or anti-dsDNA antibody in discriminating LN V from LN III (±V) or IV (±V). Values in the plot indicate areas under the curve. (C–H) The correlation of serum ICAM2 levels with particular pathological attributes of the activity index. r denotes Spearman’s correlation coefficient. *p<0.05; **p<0.01; ****p<0.0001. AUC, area under the curve; ROC, receiver operating characteristic.

Figure 4. Expression of ICAM2 within the renal tissues in lupus nephritis (LN). (A) Immunohistochemical analysis of ICAM2 protein expression was performed in kidney sections. The representative images were shown in paracancer controls and LN class III, IV and V patients. (B, C) ICAM2 expression in LN glomeruli and tubulointerstitium from Nephroseq database. *p<0.05; ****p<0.0001. HC, healthy control; MLN, membranous lupus nephritis (class V); PLN, proliferative lupus nephritis (class III (±V) or IV (±V)).

Figure 5. Downregulation of ICAM2 inhibited the activation of PI3K/Akt pathway and reduced the injury of human renal glomerular endothelial cells (HRGECs) induced by immunoglobulin G (IgG) extracted from lupus nephritis (LN) serum. (A) Representative immunofluorescence images of the costaining of ICAM2 (green) and CD31 (red) in control and patients with LN. Scar bar=10 µm. (B, C) Western blotting and quantitative analysis of ICAM2 and VCAM1 in HRGECs (n=3). (D–F) Western blotting and quantitative analysis of ICAM2 and VCAM1 in HRGECs after ICAM2 knockdown (n=3). (G–K) Western blotting and quantitative analysis of Akt, p-Akt, PI3K and p-PI3K in HRGECs after ICAM2 knockdown (n=3). *p<0.05; **p<0.01; ***p<0.001.