A total of 15 human renal tissue samples and normal tissue (children who underwent resection of Wilms tumor and the normal renal tissue 3 cm outside the lesion was taken as control) were obtained from Shandong Provincial Hospital (Table I). The present study was approved and conducted under the guidance of the ethics committee of the Shandong Provincial Hospital. All samples were obtained with the written informed consent from the patients (or signed by the child's guardian) with PNS. The present study was approved by the clinical research ethics committee of Shandong Provincial Hospital (approval no. SWYX: NO.2020-152).

Conditionally immortalized human podocytes (HPCs) were a kind gift from Professor Fan Yi (Shandong University) (22,23) and maintained in modified RPMI-1640 medium (MilliporeSigma) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin/streptomycin (100 µg/ml, Nanjing KeyGen Biotech Co., Ltd.) at 33°C with 5% CO₂ for 2–3 days. In order to induce podocytes to undergo differentiation, the culture temperature was changed from 33°C to 37°C for 2 weeks; and the other conditions remained unaltered.

The lentiviral transferring plasmid, pCDH-CMV-Puro-CXCL16 (termed pCDH-CXCL16) was purchased from Vigene Biosciences, Inc. The sequences of the CXCL16 shRNA and ERK1/2 shRNA were amplified and inserted into pLKO.1-TRC control (pLKO.1) to generate recombinant plasmid for lentiviral vector production. The construction of recombinant plasmid was as previously described (24). Briefly, the sequences of shCXCL16 and shERK1/2 were amplified and inserted into pLKO.1 to generate recombinant pLKO.1-shCXCL16 and pLKO.1-ERK1/2 for lentivirus production. All shRNA primer sequences are presented in Table II. Using synthetic shCXC16 and shERK1/2 sequences as templates for PCR. PCR amplifications were performed with Phanta Max Super-Fidelity DNA Polymerase (cat. no. DC505; Vazyme Biotech Co., Ltd.), using the following thermocycling conditions: 35 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 15 sec; extension at 72°C for 1 min; and maintained at 4°C. The restriction enzymes were used to perform a double digest of the plasmid and the PCR gel purification products. The products were separated by 1% agarose gel electrophoresis, purified and incubated with T4 ligase overnight at 4°C. The products were transformed into Escherichia coli, and a single colony was randomly selected. The plasmid was extracted using a plasmid extraction kit (cat. no. DC201; Vazyme Biotech Co., Ltd.). The plasmids were subsequently sent to GenScript Biotech for sequencing.

293T cells (Conservation Genetics CAS Kunming Cell Bank) were cultured in a 10 cm cell culture dish at 37°C with 5% CO₂ overnight. At 80% confluence, 293T cells were co-transfected with lentiviral plasmids (5.94 µg lentivirus-CXCL16, 5.94 µg lentivirus-CXCL16 shRNA1, 2, 5.94 µg lentivirus-ERK2 shRNA, 4.86 µg helper plasmid psPAX2 and 9.0 µg pMD2.G using 40 µl Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to produce the recombinant lentivirus. The 4.86 µg empty vectors pCDH and pLKO-1 were also transfected as control. At 10 h post-transfection, the cell culture medium was replaced with DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Following transfection for 48 h, the medium was collected. The lentiviral titer was approximately 1×10⁷ to 1×10⁸ transduction units (TU)/ml.

Different groups of HPCs (5×10³ cells/well) were cultured in 96-well plates. After the HPCs were allowed to adhere to the plate (12 h after cell plating), cell viability was detected at 0, 1, 3 and 5 days. Briefly, the procedure used for the Cell Counting Kit-8 (CCK-8) reagent kit (cat. no. C0038, Beyotime, Institute of Biotechnology) involved the addition of 10% reconstituted CCK-8 reagent in 100 µl of culture medium volume and returning to an incubator at 37°C for 1 h. The optical density (OD) value determined using a microplate reader (BioTek Instruments, Inc.) at 450 nm.

Briefly, 200 µl HPC (2×10⁵ cells) suspension was seeded into the culture insert, while 500 µl RPMI-1640 medium containing 10% FBS were added to the lower chamber. The Transwell chamber was incubated at 37°C for 12 h. The inner surface of the basement membrane was cleaned. The membrane was then dried, inverted. Images were captured from six random of fields (magnification, ×200) using an light microscope (BX54; Olympus Corporation).

Cell apoptosis was measured using an Apoptosis Detection kit [cat. no. 40302ES20; Yeasen Biotechnology (Shanghai) Co., Ltd.]. The different groups of HPCs were detached with 0.25% trypsin (cat. no. T1320, Beijing Solarbio Science & Technology Co., Ltd.), centrifuged at 200 × g for 5 min at room temperature and washed three times with cold 4°C PBS. Subsequently, 200 µl binding buffer were used for re-suspension, followed by the addition of 5 µl PI reagent and 5 µl Annexin-V-FITC. The mixture was reacted in an incubator at 25°C for 15 min in the dark and 400 µl binding buffer were then added. Finally, the cells were detected using a FACScan flow cytometer (BD Biosciences) within 2 h at wavelengths of 488 and 615 nm. Stained cells were analyzed using the FACScan flow cytometry system (BD Biosciences). The apoptotic rate was calculated as the percentage of early + late apoptotic cells.

mRNA was extracted from cells (1×10⁷/ml) and tissues (20 mg) using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and was reverse transcribed into cDNA using the PrimeScript Reverse Transcription kit (Vazyme Biotech Co. Ltd), the temperature protocol used for reverse transcription was as follows: 50°C for 20 min and 85°C for 2 min. qPCR was performed using the ABI-7300 System (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a total volume of 20 µl according to the manufacturer's protocols. The primers sequences used were as follows: CXCL16 forward, 5′-AAACCACCATTCACACTGCG-3′ and reverse, 5′-GAGTCAGGTGCCACAGGTAT-3′; ERK2, forward, 5′-GATCTGTGACTTTGGCCTGG-3′; and reverse, 5′-TGTGGTTCAGCTGGTCAAGA-3′; β-actin, forward, 5′-GGCATCCTCACCCTGAAGTA-3′; and reverse, 5′-GGGGTGTTGAAGGTCTCAAA-3′. The following thermocycling conditions were used for qPCR: 95°C for 5 min; 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Relative gene expression was determined using the 2^−ΔΔCq method (25) using β-actin as an internal control.

The HPCs in the different groups were harvested and extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc) supplemented with protease and phosphatase inhibitors(Thermo Fisher Scientific, Inc). Total protein was quantified using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Total protein (equal, 30 µg/lane) was separated on 10% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were then incubated with 5% non-fat milk (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 h. Finally, the membranes were incubated with the following primary antibodies at 4°C overnight: CXCL16 (1:1,000; cat. no. ab101404, Abcam), ERK1/2 (1:1,000; cat. no. ab17942, Abcam), ZO-1 (1:1,000; cat. no. ab96587, Abcam), P-cadherin (1:1,000; cat. no. ab2420604, Abcam), vimentin (1:1,000; cat. no. ab137321, Abcam) N-cadherin (1:1,000; cat. no. ab76011, Abcam) and β-actin (1:1,000; cat. no. ab8226, Abcam). The following day, the membranes were washed three times and then incubated with goat anti-rabbit IgG (1:10,000; cat. no. ab205718, Abcam) or anti-mouse IgG (1:10,000; cat. no. ab6789, Abcam) at room temperature for 2 h. The membranes were detected using ECL reagent (Thermo Fisher Scientific, Inc.) and images of each protein band were obtained using an enhanced chemiluminescence detection system (4600; Tanon Science and Technology Co., Ltd.).

The clinical tissues were provided by Shandong Provincial Hospital for IHC and were then fixed in 10% formaldehyde for 24 h, immersed in 75% ethanol for 2 h followed by tissue dehydration: 80% ethanol 20 min, 90% ethanol 20 min, 95% ethanol 20 min, 100% ethanol I 10 min, 100% ethanol II 10 min, xylene I 10 min, xylene II 10 min, xylene III 10 min, paraffin I 40 min, paraffin wax II 1 h, Paraffin III 1 h and embedded in paraffin. The tissue was sectioned at 5 µm and attached to slides. After blocking endogenous peroxides and non-specific proteins, the tissues sections were incubated with primary antibodies, CXCL16 (cat. no. ab119350, Abcam) and ERK1/2 (cat. no. ab17942, Abcam) at 4°C overnight. The following day, the sections were washed three times and were incubated with HRP-goat-anti-rabbit antibody (1:10,000; cat. no. ab205718, Abcam) at 37°C for 30 min. The tissues sections were then stained with diaminobenzidine for 3 min and the nuclei were counterstained with hematoxylin for 2 min at room temperature. The CXCL16 and ERK1/2 antibodies were used at a dilution of 1:200. IHC images of each tissues were randomly obtained using a light microscope (BX53M; Olympus Corporation), then were analyzed using the Image-Pro Plus 6.0 image analysis system (Media Cybernetics, Inc.).

Values were presented as the mean ± standard deviation. Differences between two groups were analyzed using a Student's t-test. One-way analysis of variance and Tukey or LSD post hoc test were used for comparisons between multiple groups. The correlation between ERK1/2 and CXCL16 was measured by Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The present study first evaluated CXCL16 expression levels in renal tissues from patients from PNS and healthy individuals. As illustrated in Fig. 1A, CXCL16 expression in podocyte of the renal tissues of the 15 patients with PNS was significantly upregulated compared with that in the paired normal tissues (P<0.001). Accordingly, five tissues from each group were randomly selected for western blot analysis. As illustrated in Fig. 1B, CXCL16 protein expression from the podocyte in PNS renal tissues was significantly higher than that in normal tissues (P<0.001). Furthermore, IHC was conducted. As illustrated in Fig. 1C, CXCL16 expression in podocyte of the renal tissue of patients with PNS was significantly higher than that of the normal control (P<0.001).

Previous studies have indicated that CXCL16 promotes mouse podocyte migration and regulates the progression of PNS; however, there are still areas of uncertainty. To clarify this, the present study examined the effects of CXCL16 after overexpression or knockdown on human podocytes. HPCs were transfected with lentivirus to overexpress or knockdown CXCL16. Western blot analysis confirmed the overexpression or knockdown of CXCL16 (P<0.01, Fig. 2A and B). The overexpression of CXCL16 suppressed the growth of human podocytes and the knockdown of CXCL16 promoted the proliferation of podocytes (P<0.01, P<0.001; Fig. 2C and D). Consistent with this finding, CXCL16 overexpression promoted HPC apoptosis and the knockdown of CXCL16 suppressed the apoptosis of HPCs (P<0.05; Fig. 2E and F). In addition, Transwell assays were performed on HPCs stably transfected with CXCL16 or CXCL16 shRNA. The results indicated that CXCL16 overexpression promoted HPC migration (P<0.001; Fig. 2G), whereas CXCL16 knockdown significantly suppressed the migration of HPCs (P<0.01; Fig. 2H). Overall, these findings demonstrated that CXCL16 regulated the progress of PNS by inhibiting the growth and promoting the apoptosis of podocytes, which caused podocytes to lose the characteristics of normal cells and obtain a higher migration and movement ability.

In order to reveal the potential downstream targets of CXCL16, a literature search was performed. The results demonstrated that ERK1/2 may be the potential downstream target of CXCL16 and may be regulated by CXCL16 (26,27). To verify the association between CXCL16 and ERK1/2, the expression of ERK1/2 in PNS was verified. The ERK2 mRNA and ERK1/2 protein levels were evaluated in the same renal tissues from patients with PNS using qPCR (P<0.001, Fig. 3A), western blot analysis (P<0.01, Fig. 3B) and IHC (P<0.001, Fig. 3C). Notably, compared with normal tissues, the expression of ERK1/2 in PNS tissues was also upregulated and there was a positive association between ERK2 and CXCL16 expression (Fig. 3D). The expression of CXCL16 and ERK1/2 was then verified in HPCs in which was stably overexpressed or knocked down. As shown in Fig. 3E and F, ERK1/2 expression increased with the overexpression of CXCL16 expression and decreased with the knockdown of CXCL16 (P<0.01). The aforementioned results suggested that ERK1/2 may be a potential downstream target of CXCL16.

To define the role of ERK1/2 upregulation in PNS, the recombinant lentivirus plasmid of ERK1/2 shRNA was first constructed and the expression of ERK2 was knocked down in HPCs overexpressing CXCL16 (P<0.001, Fig. 4A). The effects of this knockdown were then evaluated on the ability of cell proliferation and apoptosis. As indicated in Fig. 4B and C, the knockdown of ERK1/2 expression reversed the inhibitory effects of CXCL16 on HPC proliferation and reduced the apoptosis of HPCs which was induced by CXCL16 (P<0.5, P<0.01 and P<0.001). Subsequently, the effects of ERK2 knockdown on podocyte migration were evaluated and the results were consistent with the expectations. The knockdown of ERK2 inhibited the increased migratory ability of HPCs induced by CXCL16 (P<0.01 and P<0.001; Fig. 4D). EMT of HPCs is the main pathway leading to HPC dysfunction, proteinuria and glomerulosclerosis (28,29). Therefore, the present study detected the expression of N-cadherin, P-cadherin, vimentin and zonula occludens-1 (ZO-1) proteins related to EMT. Of note, it was found that the knockdown of ERK1/2 expression in CXCL16-overexpressing HPCs reversed the CXCL16-induced expression of protein molecules associated with EMT (P<0.5 and P<0.01; Fig. 5A and B).