IGFBP2 function as a novel biomarker for active lupus nephritis

Figures

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  Fig. 1. TMT detection of differential protein expression between the active LN and HC groups. A Statistical histograms of GO ontology biological processes, cellular components, and molecular functions. B Bar graph displaying the top 10 enriched GO biological process functions. C Network analysis diagram. D Scatter plot of DE proteins and corresponding transcripts analyzed by the proteome and transcriptome. The x-axis is the transcript log 2 (fold-change), and the y-axis is the protein log 2 (fold-change). R = 0.0244 was the Pearson correlation coefficient of the two sets of data. The transcript was differentially expressed according to whether it was expressed in different colors. E Heatmap of proteins that were differentially expressed at the proteome and transcriptome levels and were consistently up- or downregulated. F Real-time PCR validation tests of IGFBP2 gene expression were performed in peripheral blood mononuclear cells (PBMCs) of the control (n = 10) and LN (n = 24) groups. ELISA validation tests were performed to verify IGFBP2 expression in the plasma of the controls (n = 18), inactive LN (n = 18), and active LN patients (n = 23). G Validation of IGFBP2 expression in the…

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  Fig. 2. The level of plasma IGFBP2 expression in LNs and correlation analysis with clinical indicators. A Level of IGFBP2 expression in the plasma of RA (n = 16), SS (n = 16), active LN (n = 17), and control (n = 18) patients. B The level of urine IGFBP2 expression in LN patients (n = 12) was higheer than that in HC (n = 12) patients (P < 0.01). C There was no significant difference between urinary IGFBP2 in patients with inactive (n = 19) and active SLE (n = 23). D There was no correlation between plasma and urinary IGFBP2 levels in LN patients (n = 26). E Correlation analysis between plasma IGFBP2 levels and SLE activity-related indicators. F Correlation analysis between the plasma IGFBP2 and SLEDAI score at 3 months after treatment and change in SLEDAI before and after treatment. G The ROC curve revealed that IGFBP2 showed an AUC value of 0.992, with a sensitivity of 0.909 and specificity of 1.0 (95% CI 0.974–1.0, P < 0.001) at the cutoff of 512 ng/mL between inactive and active LN. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not statistically significant

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  Fig. 3. The level of IGFBP2 expression in the kidneys of LN. A IGFBP2 expression in normal kidney issues and LNs by IF staining (n = 3). B IHC staining of IGFBP2 in HC (n = 3), MN (n = 3), and II–V type LN kidneys (n = 3). Original magnification, × 400. The positive area is quantitatively displayed as a histogram on the right. *P < 0.05; **P < 0.01

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  Fig. 4. The distribution of IGFBP2 in the organs of mice. A The level of IGFBP2 expression in the blood of MRL mice (16 weeks) (n = 4) was higher than that of C57BL/6 mice (n = 3). B ELISA detection of the IGFBP2 content in the various organs of C57BL/6 and MRL/lpr mice revealed that in most organs of MRL/lpr mice, the protein content of IGFBP2 was higher than that of the C57BL/6 mice. C WB showed the expression of IGFBP2 in the lymph nodes of MRL/lpr and C57BL/6 mice (n = 3). D IHC showed that the level of IGFBP2 in the lymph nodes of MRL/lpr was significantly higher than that in the lymph nodes of C57BL/6 mice (intracellular tan deposition) (n = 3). *P < 0.05

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  Fig. 5. The IGFBP2 blocking effect in mice following treatment. A MRL/lpr mice were divided into the PBS group (n = 4), ISO group (n = 4), and anti-IGFBP2 group (n = 4). The ELISA results showed that compared with PBS and ISO groups, the level of plasma IGFBP2 in the anti-IGFBP2 group was decreased. B PCR and C WB showed the level of renal IGFBP2 protein expression in each of the three groups. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not statistically significant

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  Fig. 6. Improvement of LN after a IGFBP2 blockade. A MRL/lpr mice were divided into the PBS group (n = 4), ISO group (n = 4), and anti-IGFBP2 group (n = 4). The skin lesions in the anti-IGFBP2 group were significantly improved. B Compared with the PBS and ISO groups, the lymph/body weight ratio was decreased in the anti-IGFBP2 group. C Trend in the reduction of urine protein following treatment. D HE, PAS, and Masson staining (original magnification, × 400), quantified histogram of glomerulus, tubulointerstitium and perivascular pathology. *P < 0.05; **P < 0.01

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  Fig. 7. IGFBP2 blockade affects T cells and AKT/mTOR pathway. A Effect on peripheral blood T cell immunity by flow cytometry in PBS group (n = 4), ISO group (n = 4), and anti-IGFBP2 group (n = 4). B Protein expression and phosphorylation levels of AKT, mTOR, RPS6KB1, and 4E-BPI in anti-IGFBP2 group (n = 4) and ISO group (n = 3) were detected by Western blotting. The quantitative analysis of proteins was performed using ImageJ software. *P < 0.05; **P < 0.01; ns, not statistically significant