Expression of immunoglobulin G in human proximal tubular epithelial cells

Abstract

Proximal tubular epithelial cells (PTECs) have innate immune characteristics, and produce proinflammatory factors, chemokines and complement components that drive epithelial‑mesenchymal transition (EMT). Our previous studies revealed that human mesangial cells and podocytes were able to synthesize and secrete immunoglobulin (Ig)A and IgG, respectively. The aim of the present study was to evaluate the expression of Igs in PTECs. Firstly, IgG was detected in the cytoplasm, the cell membrane and the lumen of PTECs in the normal renal cortex by immunohistochemistry. Secondly, Igγ gene transcription and V(D)J recombination were detected in single PTECs by nested PCR and Sanger sequencing. Thirdly, Igγ, Igκ and Igλ were clearly detected in an immortalized PTEC line (HK‑2) by immunostaining and western blotting, in which RP215 (an antibody that predominantly binds to non‑B cell‑derived IgG) was used. In addition, Igγ, Igκ and Igλ gene transcripts, conservative V(D)J recombination in the Igγ variable region, recombination activating gene 1/2 and activation‑induced cytidine deaminase were all detected in HK‑2 cells. These data suggested that PTECs may express IgG in a similar manner to B cells. Furthermore, IgG expression was upregulated by TGF‑β1 and may be involved in EMT.

Keywords: proximal tubular epithelial cells; single cell; HK‑2; IgG; epithelial‑mesenchymal transition.

Figure 1.

IgG expression in renal tubular epithelial cells of the kidney cortex. Representative images of Igγ, Igκ and Igλ expression in renal tubular epithelial cells from normal kidney cortex. IgG expression was detected by immunohistochemistry using antibodies against human IgG heavy and light chains. NC-Rabbit and NC-Mouse indicates PBS instead of primary antibody and goat anti-rabbit (NC-rabbit) or goat anti-mouse (NC-mouse) as the secondary antibody. The red arrows indicate proximal tubular epithelial cells and the blue arrows indicate distal convoluted tubular cells (scale bar, 100 µm). NC, negative control; Ig, immunoglobulin.

Figure 2.

Rearranged IgG was detected in sorted single PTECs by nested PCR. (A) PTECs were sorted by FACS using antibodies against CD10-PE and CD13-APC. The corresponding isotype control antibody was used to exclude non-specific staining. FACS analysis revealed that 4.1% of cells were double-positive. PCR analysis of (B) Igγ and the B-cell marker gene CD19, and (C) PTEC marker gene LRP2. PBMCs were used as the positive control for IgG and CD19, and cDNA from kidney cortex was used as a positive control for LRP2. Water instead of cDNA was used as a negative control. Ig, immunoglobulin; PBMCs, peripheral blood mononuclear cells; PTECs, proximal tubular epithelial cells; LRP2, low-density lipoprotein receptor-related protein 2; FACS, fluorescence-activated cell sorting.

Figure 3.

Expression of IgG heavy and light chains in HK-2 cells. (A) Expression and cellular localization of Igγ, Igκ and Igλ in HK-2 cells was assessed by immunofluorescence staining. Green indicates positive staining of Igs and blue indicates nuclear staining by DAPI. Scale bar, 75 µm (upper panel), 25 µm (lower panel). (B) Expression of IgG heavy and light chains in HK-2 lysates was detected by western blotting under reducing conditions. Human serum was used as a positive control. The absence of a band in the medium eliminates the possibility of human Ig heavy chain and light chain expression in the culture medium containing 10% fetal bovine serum, which was considered as a negative control. (C) A 55-kDa band of Igγ was collected from the culture supernatant and purified by protein G. (D and E) Mass spectrometry results of the 55-kDa protein detected in the culture supernatant of HK-2 cells. The bold red sequences refer to the alignment of an amino acid sequence with Igκ chain protein and Ig heavy chain variable region protein in the National Center for Biotechnology Information database. Ig, immunoglobulin.

Figure 4.

Transcription and V(D)J recombination of IgG heavy and light chains in HK-2 cells. (A) Transcription of IgCγ, IgCγ4, IgCκ and IgCλ were amplified by RT-PCR. CD19 was used to eliminate B-cell interference. (B) IgVγ and RAG1/RAG2 mRNA expression was amplified by nested RT-PCR, whereas the AID mRNA was amplified by RT-PCR. PBMCs acted as a positive control. Water acted as a negative control. R acted as a negative control. (C) Variable region sequences and mutations of IGHV4-4/IGHD2-8/IGHJ5. Identity with the homologous germline sequence is indicated by dots. Each nucleotide mutation is indicated. The mutation hotspots in germline genes are underlined. The red letters refer to the junctions. CDR, complementarity determining region; FR, framework region; R, cDNA template replaced by DNase-treated RNA; RT-PCR, reverse transcription-PCR; AID, activation-induced cytidine deaminase; RAG, recombination activating genes; IgC, immunoglobulin constant region; IgV, immunoglobulin variable region.

Figure 5.

Expression of Igγ in HK-2 cells induced by TGF-β1. (A) HK-2 cells were stimulated by TGF-β1 and Igγ expression was assessed by immunofluorescence staining. Green indicates positive staining of Igγ, blue indicates nuclear staining by DAPI. Scale bar, 75 µm (upper panel), 25 µm (lower panel). (B) Representative western blotting images of Igγ in HK-2 cells treated with TGF-β1. Serum acts as the positive control. Cell culture medium containing 10% fetal bovine serum was used as a negative control. (C) Gray value of Igγ relative to β-actin upon treatment with different concentrations of TGF-β1. ****P<0.0001 and ***P<0.001 vs. untreated control group. *P<0.05 vs. 10 ng/ml TGF-β1. Ig, immunoglobulin.