An immortalized PTEC line HK-2 was purchased from American Type Culture Collection. HK-2 cells were cultured in DMEM/F12 supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin (all Gibco; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (FBS; Australian origin; Biological Industries USA, Inc.) at 37°C in an atmosphere containing 95% air and 5% CO₂. In order to avoid the interference of Ig in FBS, the medium was replaced with serum-free medium 24–48 h prior to cell harvest. HK-2 cells were treated with different concentrations (2, 5 and 10 ng/ml) of TGF-β1 (Sigma-Aldrich; Merck KGaA).

A human kidney sample of macroscopically normal cortical tissue was obtained from a patient (male, 31 years old) undergoing nephrectomy as a result of renal carcinoma without obvious renal dysfunction. A single-cell suspension was prepared by digesting renal cortex with 1 mg/ml collagenase I (Sigma-Aldrich; Merck KGaA) at 37°C for 20 min. PTECs were sorted using phycoerythrin (PE)-conjugated anti-CD10 (cat. no. 312203) and allophycocyanin (APC)-conjugated anti-CD13 (cat. no. 301705; both BioLegend, Inc.) by fluorescence-activated cell sorting (BD FACSAria II Special Order System) as previously described (20). The corresponding isotype control antibodies (cat. nos. 400111 and 400119; both BioLegend, Inc.) were used to exclude non-specific staining. Double positively labeled living cells were isolated as PTECs. A single PTEC was manually selected under an inverted light microscope using a capillary pipette and was then transferred to a 0.2-ml thin-wall PCR tube containing lysis buffer (21). Single PTEC RNA extraction and cDNA synthesis were carried out according to previously described methods (21). A total of five single PTECs were used to detect Ig gene transcription and rearrangement.

Total RNA was extracted from HK-2 cells, peripheral blood mononuclear cells [PBMCs, isolated from a 31-year-old female healthy donor using Ficoll (cat. no. 7111011; Dakewe Biotech., Ltd.)] and kidney cortex (from the same patient used in single PTEC isolation) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the RNA concentration was assessed using a NanoDrop spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.). Subsequently, 2 µg total RNA was reverse-transcribed to cDNA using the RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.). PCR was performed using primers targeting the constant regions of Igγ, Igκ, Igλ and activation-induced cytidine deaminase (AID). Nested PCR was performed to amplify the variable region of Igγ, low-density lipoprotein receptor-related protein 2 (LRP2) and recombination activating gene (RAG)1 and RAG2. The PCR products were separated by electrophoresis on a 1.0% agarose gel and was visualized using GelRed (cat. no. 41003; Biotium, Inc.). The primers of AID, RAG1/2 and the constant regions of Igγ, Igκ, Igλ used in this study refer to primers used by Jing et al (19). The primers of Igγ variable region refer to primers used by van Dongen et al (22). The other primers used for PCR are listed in Table SI. The thermocycling conditions are listed in Table SII.

PCR products of the Igγ variable region obtained from single PTECs, HK-2 cells and PBMCs were respectively cloned into a pGEM-T Easy Vector system I (cat. no. A1360; Promega Corporation), which was transformed into TOP10 Competent cells (CB104; Tiangen Biotech Co., Ltd.). Briefly, 5 µl ligation products were added to 30 µl TOP10 competent cells, incubated on ice for 30 min, heat shocked at 42°C for 90 sec and incubated on ice for 5 min. Then, 500 µl LB was added and left to stand at 37°C for 40 min before inoculating part of the bacterial liquid on Petri dishes coated with 0.1 mmol/l IPTG and 20 µg/ml X-Gal. Dishes were inverted at 37°C overnight. In all, 5–16 white colonies per sample were chosen randomly, and sequenced using an ABI 3730XL Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). The rearranged V(D)J sequences were compared with those in the basic local alignment search tool (https://www.ncbi.nlm.nih.gov/igblast/) to identify the best matching germline gene segments and junctions following primer trimming.

HK-2 cells were lysed in TSD lysis buffer [1% SDS, 50 mM Tris-HCl (pH 7.5), 50 mM DTT] containing a protease inhibitor cocktail (Applygen Technologies Inc.), sonicated at ice-water for 1 min (working 5 sec and resting 15 sec; 3 times) and lysed for 30 min at room temperature. Following centrifugation at 12,000 × g for 10 min at 4°C, the protein concentration of the cell lysate was determined using a BCA kit (Applygen Technologies Inc.). Subsequently, 5X reducing loading buffer was added to the lysate, boiled at 100°C for 10 min, and the samples were immediately used for western blot analysis. Serum, used as a positive control for Ig, was isolated from a healthy donor (the same donor as used in PBMCs) by centrifugation at 2,103 × g for 10 min at room temperature.

Western blotting was carried out according to standard procedures. Briefly, 30 µg proteins were separated by SDS-PAGE on 10% gels and were transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked in 5% skimmed milk at room temperature for 1 h and was incubated with primary antibodies at 4°C overnight, including rabbit anti-human Igγ (cat. no. ab109489; 1:1,000), rabbit anti-human Igγ4 (cat. no. ab109493; 1:1,000), anti-Igκ (cat. no. ab124727; 1:10,000), anti-Igλ (cat. no. ab124719; 1:20,000), rabbit anti-human β-actin (cat. no. ab8227; 1:2,000) (all from Abcam), and RP215 monoclonal antibody (mAb) (donated by Professor Xiaoyan Qiu, Peking University, Beijing, China; 1:1,000), which specifically identified a carbohydrate-associated epitope on non-B-Igγ. The membrane was then incubated with goat anti-rabbit (cat. no. 926-32211) or anti-mouse (cat. no. 926-32210) IgG-IRDyeTM680CW secondary antibodies (both 1:10,000; both LI-COR Biosciences) at room temperature for 1 h. The signal was detected using the Odyssey Imaging system and Odyssey V3.0 software (both LI-COR Biosciences). ImageJ software (version 1.8.0; National Institutes of Health) was used for semi-quantification.

After HK-2 cells had been cultured in DMEM/F12 without FBS for 48 h, the culture supernatant was collected after centrifugation at 2,103 × g for 10 min at 4°C. The cell supernatant was purified by affinity chromatography using protein G Sepharose, according to the manufacturer's instructions (cat. no. 17-0618-02; Thermo Fisher Scientific, Inc.). The eluent was ultra-filtered to replace the elution buffer (0.1 M Glycine; pH 2.4) with PBS. The purified proteins were separated by SDS-PAGE on 10% gels, detected by western blotting, and further analyzed by mass spectrometry, performed by Beijing Protein Innovation Co., Ltd.

HK-2 cells were cultured on cover-slips, which were fixed in cold undiluted acetone for 5 min at room temperature. Subsequently, the slides were washed twice in PBS and blocked with 5% FBS/PBS at room temperature for 20 min, after which they were incubated with primary antibodies at 4°C overnight. The antibodies were the same as those used in western blotting: Rabbit anti-human Igγ (1:150), anti-human Igκ (1:250), anti-human Igλ (1:250) and RP215 mAb (1:200); PBS was used as a negative control. After washing in PBS, the slides were incubated with fluorescein isothiocyanate-labeled goat anti-rabbit (cat. no. A11008) or goat anti-mouse (cat. no. A11001) IgG antibodies (1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Nuclei were stained with DAPI. Images were captured under a Leica DFC300 FX fluorescence microscope (Leica Microsystems GmbH).

Paracancerous renal cortices were collected from four male patients (age, 38–49 years) with renal carcinoma. Normal paracancerous renal cortices following nephrectomy were fixed with 10% formalin for 48 h at room temperature, and then embedded in paraffin. Paraffin-embedded human kidney samples were cut into 3-µm sections, and deparaffinized and rehydrated through a series of graded ethanol concentrations. Antigen retrieval was performed by boiling in 0.05 M Tris-EDTA (pH 9.0) in a pressure cooker for 3 min. The sections were then incubated with 3% H₂O₂ solution for 10 min at room temperature to eliminate endogenous peroxidase and incubated with normal goat serum (cat. no. ZLI-9022, ZSGB-BIO, China) for 30 min at room temperature to block nonspecific antibody binding sites at room temperature. Subsequently, indirect immunohistochemical staining was performed with primary antibodies at 4°C overnight, including RP215 mAb (1:200; 5 µg/ml), rabbit anti-human Igγ (1:2,000), anti-human Igκ (1:1,000), anti-human Igλ (1:1,000) (the same antibodies as used in western blotting). Sections without primary antibodies were used as negative controls. The slides were then incubated with undiluted horseradish peroxidase-labelled secondary antibodies (cat. nos. PV-6001 and PV-6002; both OriGene Technologies, Inc.) at room temperature for 30 min. Bound antibodies were detected using diaminobenzidine. Finally, the slides were counterstained with hematoxylin. Images were captured using a light microscope (×200 magnification).

Data are presented as the means ± standard deviation and were analyzed using SPSS 20.0 for Windows (IBM Corp.). All experiments were repeated 3 times. The differences among multiple groups were analyzed using one-way ANOVA and Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Normal renal cortexes, which were collected from donors undergoing nephrectomy as a result of renal cell carcinoma, were used to determine the IgG expression in renal tubular epithelial cells by immunohistochemistry using antibodies against human Igγ, Igκ, Igλ and RP215 (an antibody that predominantly binds to non-B-IgG). Positive staining of IgG heavy and light chains was not only detected in the PTECs but also in distal convoluted tubule epithelial cells, either in the cytoplasm, cell membrane or tubular lumen (Fig. 1).

To avoid the interference of residual blood, binding and transcytosis of IgG by PTECs in the kidney cortex, and to obtain direct evidence of IgG expression in PTECs, single PTECs were sorted from human kidney cortex using CD10 and CD13 co-labeling by flow cytometry (20). As shown in Fig. 2A, CD10/CD13 double-positive PTECs accounted for 4.1% of viable cells in the sample. The isolated PTECs were further confirmed using the specific marker gene LRP2, and B-cell contamination was eliminated by CD19. Igγ variable region transcripts were amplified in five single PTECs by nested PCR (Fig. 2B and C). Sanger sequencing results revealed that PTECs exhibited functional and conservative VDJ recombination of IgG heavy chain (Table I), indicating that IgG expression may occur in PTECs.

Since it was difficult to detect IgG protein expression in a single PTEC and to obtain enough PTECs for western blotting, the HK-2 cell line, which is comprised of immortalized PTECs, was selected to further confirm IgG protein expression. Immunofluorescence analysis demonstrated positive staining of Igγ, Igκ and Igλ in the cytoplasm, and stronger positive staining of RP215 predominantly in the cytoplasm and cell membrane (Fig. 3A).

Subsequently, the expression of IgG heavy and light chains in HK-2 cells were detected by western blotting under reducing conditions. To eliminate FBS interference in the culture medium, medium containing FBS was blotted with corresponding antibodies and stained negative. A commercial IgG antibody was able to detect serum-derived IgG but not HK-2-derived IgG. By contrast, RP215 could detect HK-2-derived IgG, but not serum IgG. Both commercial IgG antibody and RP215 were able to detect Igγ at 55 kDa. An Igγ4 (36 kDa) band was detected in HK-2 cells, consistent with the predicted molecular weight. Furthermore, Igκ (25 kDa) and Igλ (50 kDa, dimer) expression was observed in the cell lysates (Fig. 3B). Subsequently, IgG in the cell supernatant was purified by protein G, confirmed by western blotting and sequenced by mass spectrometry, which demonstrated that the 55-kDa band contained fragments of the Igκ chain and Ig heavy chain variable region, according to the National Center for Biotechnology Information (NCBI) database (Fig. 3C-E). These data suggested that HK-2 cells produced and secreted IgG protein.

Ig gene transcription and functional V(D)J recombination is a prerequisite for Ig expression. To confirm the expression of IgG in HK-2 cells, Igγ, Igκ and Igλ transcripts were assessed by amplifying the constant and variable regions in HK-2 cells (Fig. 4A and B). Sequencing of the constant PCR products exhibited high homology with the published sequence in the NCBI database. T-A cloning and Sanger sequencing demonstrated that the Igγ in HK-2 cells displayed conservative V(D)J recombination with VH4-4/D2-8/JH5. By contrast, the diversity of V(D)J recombination was observed in PBMCs, which eliminated primer bias (Table II). Similar to B cells, HK-2-derived IgG displayed typical productive V(D)J recombination with the V-D and D-J junctions (Fig. 4C). Moreover, the somatic hypermutations were detected in HK-2-derived Igγ (range, 6.8–8.4%) with 7 out of 15 hotspot motifs (RGYW/WRCY, W=A/T, R=A/G, Y=C/T) presenting with mutations.

In addition, AID transcripts (essential element for somatic hypermutation and class switch recombination in B cells), and RAG1 and RAG2 [necessary for V(D)J recombination] were detected in HK-2 cells (Fig. 4B), indicating that the mechanism underlying Ig synthesis in HK-2 cells may be similar to that in B cells.

HK-2 cells were stimulated with various concentrations of TGF-β1 for 48 h. Immunofluorescence staining revealed that cytoplasmic IgG exhibited enhanced positive staining compared with the control group (Fig. 5A). Western blotting confirmed that IgG was significantly upregulated by TGF-β1 (P<0.05; Fig. 5B and C).