A total of 57 patients with LN were recruited from the Affiliated Drum Tower Hospital of Nanjing University Medical School, and all patients fulfilled the American College of Rheumatology (ACR) revised criteria (1997) [8]. A total of 27 sex- and age-matched volunteers were recruited as healthy controls (HCs). LN inclusion criteria consisted of 18 years of age or older, The Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K) ≥ 8 combined with LN, and fulfilled the British Isles Lupus Assessment Group (BILAG) A 1 item or BILAG B grade 2 item [9]. The exclusion criteria consisted of patients with other connective tissue diseases complicated with an infection or tumor who were pregnant or breastfeeding.

The patients with LN were divided into two groups. SLEDAI-2 K was used to judge the level of disease activity [10]. SLEDAI-2 K scores ≤ 4 represented inactive disease, and SLEDAI-2 K scores ≥ 8 represented active disease. In this study, 16 patients with RA who fulfilled the 2010 ACR Rheumatoid Arthritis Classification Criteria [11] and 16 patients with Sjogren’s syndrome (SS) who fulfilled the 2002 American-European Consensus Group (AECG) Classification Criteria [12] were included as control groups. All patients exhibited active disease. The simple disease activity index (SDAI) of patients with RA was > 3.3 points, and patients with SS had damage to at least one system.

Venous blood and urine samples were harvested from all subjects. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation methods according to the manufacturer’s instructions (STEMCELL Technologies, Vancouver, CA). RNA was extracted from the PBMCs using TRIzol in accordance with the manufacturer’s instructions (Invitrogen, Carlsbad, USA).

The proteomic profiling of nine patients with active LN using TMT-LC–MS/MS was analyzed on a Thermo Orbitrap Elite mass spectrometer (Thermo Finnigan, San Jose, CA). Differentially expressed (DE) proteins were identified and quantified. Total RNA was extracted from PBMCs and sequenced using Illumina HiSeq 2500 (Illumina, USA). Mass spectrometry and RNA-seq services were carried out by Shanghai Shengzi Biological Technology Co., Ltd.

The following selection criteria for the DE genes were used: FDR ≤ 0.05 and FC ≥ 2. DE genes were analyzed by DAVID 6.8 (https://david.ncifcrf.gov/) using the GO and Kyoto Encyclopedia of Genes and Genome (KEGG) databases. The protein sequence and human protein data from Ensembl were compared using BLASTP. A similarity of 85% was used as the cutoff to associate the protein with the transcriptome.

C57BL6/J (B6) female mice (Animal Center of Nanjing Medical University, China) and MRL/lpr female mice (Shanghai SLAC Laboratory Animals Co., Ltd, Shanghai, China) were purchased at 4 weeks of age and housed in the Animal Center of Nanjing Drum Tower Hospital under specified pathogen-free (SPF) conditions. Sixteen-week-old MRL/lpr mice were randomly divided into three groups. The mice were intraperitoneally injected with PBS, 10 µg mouse IGFBP2 antibody (cat: MAB797, R&D), and rat IgG2A ISOtype control antibody (cat: MAB006, R&D) each week for 4 weeks.

For real-time PCR, RNA was extracted from cells and tissues using TRIzol according to the manufacturer’s instructions (Invitrogen, Carlsbad, USA). cDNA was synthesized from 1 μg of total RNA using HiScript II Q RT SuperMix (Vazyme Biotech). RT–qPCR was performed in accordance with the manufacturer’s instructions for the SYBR Green Master MIX (Low ROX Premixed) and Applied Biosystems QuantStudio 6 Flex Real-Time PCR System. The primer sequences (Genscript Biotech, Nanjing, China) are listed in supplementary Table 1.

The concentrations of IGFBP2 in the plasma and supernatant of homogenized tissues were measured using an IGFBP2 R&D Systems ELISA kit (Minneapolis, MN; cat no: DBG200 for humans and cat no: DY797 for mice). All samples were diluted to 1:10 for plasma and 1:50 for the supernatants. The absorbance was read by spectrophotometry at 450 nm. A standard curve was constructed to calculate the concentration of IGFBP2. All measurements were repeated at different dilutions to confirm the validity of the analyses.

The following reagents were used for flow cytometric analysis: mouse antibodies: CD3 BUV395, CD4 FITC, CD8 BV510, CD25 APC, CD69 Percp Cy5.5, Ki67 PE Cy7, Foxp3 PE, IFN-γ BV786, IL-4 PE-CF594, IL-17 BV421, IL-2 APC-R700, and live dye APC Cy7 (780). All antibodies were purchased from eBioscience (San Diego, CA). To detect intracellular factors, the cells were incubated with 0.1 μg/mL phorbol 12-myristate 13-acetate (PMA), 5 μg/mL ionomycin, and 25 μg/mL BFA at 37 °C for 6 h. All analytical flow cytometry was performed on a BD FACSCalibur™ Flow Cytometer (BD Immunocytometry Systems, San Diego, CA) using FlowJo software (TreeStar, Ashland, OR) for data analysis.

SDS–PAGE and immunoblotting were used to detect the level of protein expression. Briefly, protein samples (20 µg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824). Then, the membranes were detected with HRP-linked secondary antibodies. GAPDH (cell signaling) was used as an internal reference. The protein bands were visualized using a Tanon-5200 Imaging System (Shanghai Tanon Science & Technology).

LN was pathologically classified according to the International Society of Nephrology/Renal Pathology Society [13]. Kidney biopsy samples from 15 LN patients, 3 primary nephrotic syndrome patients, and normal renal tissue samples adjacent to renal cancer were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson’s trichrome stain. The cells were counted under high-power magnification to calculate the histological score for kidney disease [14, 15]. Antibodies and dyes used for IHC and IF were listed as follows: anti-IGFBP2 (ab188200, Abcam), Alexa 488-conjugated goat anti-rabbit IgG, Alexa Fluor 594-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific), and Hoechst 33258. The sections were observed under an inverted fluorescence microscope (Olympus, Tokyo Japan). ImageJ software was used for image acquisition and processing. The level of mouse urine protein was determined using a Bradford assay kit (KGI, Nanjing, China).

Statistical analyses were performed using GraphPad Prism version 7.0 for Windows (GraphPad Software, La Jolla, CA). All values were expressed as the mean ± SD. DE proteins were identified by the fold-change threshold (fold ≥ 1.5) and an independent t test. The data at the time points before and after treatment were tested using a paired t test, and comparisons between the other two groups were assessed using an unpaired t test. Three groups or more than three groups were compared using one-way analysis of variance. A value of P < 0.05 was considered to be statistically significant.