MHC class II in renal tubules plays an essential role in renal fibrosis

Abstract

Immunomodulation is considered a potential therapeutic approach for chronic kidney disease (CKD). Although it has been previously reported that CD4⁺ T cells contribute to the development of renal fibrosis, the role of MHC class II (MHCII) in the development of renal fibrosis remains largely unknown. The present study reports that the expression of MHCII molecules in renal cortical tubules is upregulated in mouse renal fibrosis models generated by unilateral ureter obstruction (UUO) and folic acid (FA). Proximal tubule epithelial cells (PTECs) are functional antigen-presenting cells that promote the proliferation of CD4⁺ T cells in an MHCII-dependent manner. PTECs from mice with renal fibrosis had a stronger ability to induce T cell proliferation and cytokine production than control cells. Global or renal tubule-specific ablation of H2-Ab1 significantly alleviated renal fibrosis following UUO or FA treatment. Renal expression of profibrotic genes showed a consistent reduction in H2-Ab1 gene-deficient mouse lines. Moreover, there was a marked increase in renal tissue CD4⁺ T cells after UUO or FA treatment and a significant decrease following renal tubule-specific ablation of H2-Ab1. Furthermore, renal tubule-specific H2-Ab1 gene knockout mice exhibited higher proportions of regulatory T cells (Tregs) and lower proportions of Th2 cells in the UUO- or FA-treated kidneys. Finally, Immunohistochemistry (IHC) studies showed increased renal expression of MHCII and the profibrotic gene α smooth muscle actin (α-SMA) in CKD patients. Together, our human and mouse data demonstrate that renal tubular MHCII plays an important role in the pathogenesis of renal fibrosis.

Keywords: Chronic kidney disease; Folic acid; MHCII; Renal fibrosis; Unilateral ureteral obstruction.

Fig. 1

Renal expression of H2-Ab1 is induced in the UUO and FA models. A Real-time PCR assays showing H2-Ab1 mRNA levels primarily present in the renal cortex, with lower levels in the outer medulla (OM) and inner medulla (IM). Data are presented as the mean ± SEM. **P < 0.01 vs. cortex, n = 4–5. B Western blot assays showing H2-Ab1 protein levels primarily present in the renal cortex, with lower levels in the OM and IM. Representative Western blots are presented. C Intrarenal localization of the H2-Ab1 protein in the renal cortex and medulla as assessed by immunohistochemistry. H2-Ab1 immunoreactivity was observed in the renal cortex and medulla. H2-Ab1 signals (arrowheads) were mainly located in the cortical tubules. Representative immunostaining for H2-Ab1: (1) Cortex-anti-H2-Ab1; (2) Medulla-anti-H2-Ab1; (3) Cortex-isotype-IgG; (4) Medulla-isotype-IgG (bar = 100 μm). D Real-time PCR assays showing that H2-Ab1 mRNA expression was increased in the obstructed kidney after 7 days of UUO. **P < 0.01 vs. the control, n = 5–6. E Western blotting assay showing that renal H2-Ab1 protein expression was upregulated in the obstructed kidney. Representative Western blots are presented. F Representative immunohistochemical staining for increased H2-Ab1 expression in the obstructed kidney (bar = 50 μm). **P < 0.01 vs. the control, n = 5–6. G Real-time PCR assay showing that H2-Ab1 mRNA expression was increased in the fibrotic kidney after 28 days of FA treatment. **P < 0.01 vs. the control, n = 5–6. H Western blotting assay showing that renal H2-Ab1 protein expression was upregulated in the fibrotic kidney. Representative Western blots are presented. I Representative immunohistochemical staining for increased H2-Ab1 expression in the fibrotic kidney (bar = 50 μm). Data are the mean ± SEM. **P < 0.01 vs. the control, n = 5–6

Fig. 2

Renal proximal tubule epithelial cells (PTECs) trigger MHCII-restricted antigen-dependent CD4⁺ T cell proliferation and cytokine production. PTECs from the kidneys of the mice with UUO in the presence (anti-MHCII) or absence (IgG) of MHCII-neutralizing antibodies were cultured and grown in 96-well U bottom plates for 3 days. Adherent cells were pulsed with OVA peptide (OVA_323-339) for 2 h before CFSE-labeled CD4⁺ T cells from OT-II mice were added. After 3 days, CFSE dilution was examined by flow cytometry to quantify proliferating CD4⁺ T cells in cocultures. A Representative histograms demonstrate antigen-induced proliferation of CFSE-labeled OT-II CD4⁺ T cells in cocultures containing OVA peptide-pulsed PTECs from the kidneys of the mice with UUO in the presence or absence of MHCII-neutralizing antibodies. B Quantitation of OT-II CD4⁺ T cell proliferation in cocultures. C IL-2 production in cocultures containing OVA peptide-stimulated renal PTECs from the UUO-induced mice with or without MHCII-neutralizing antibodies. Data are the mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs.^. the control; ^#P < 0.05, ^##P < 0.01 vs. the UUO group, n = 3–4

Fig. 3

H2-Ab1 gene deficiency attenuates UUO-induced renal fibrosis in mice. A Masson trichrome staining demonstrating intensive fibrosis in tubulointerstitial areas (arrowheads) in mice with both genotypes after UUO. However, the H2-Ab1^−/− mice exhibited much less renal fibrosis than the wild-type (WT) mice. Representative images: (1) WT; (2) WT + UUO; (3) H2-Ab1^−/−; (4) H2-Ab1^−/−+UUO (bar = 100 μm). B Semiquantitative analysis of (A) showing significantly increased collagen staining in the kidneys of the WT mice with UUO, which was markedly attenuated in the H2-Ab1^−/− mice. C Real-time PCR assay indicating that the mRNA expression of collagen I, collagen IV, fibronectin, Actα and TGFβ was markedly increased in the kidneys of the UUO-treated WT mice and was significantly attenuated in the H2-Ab1^−/− mice. D Western blotting assay showing that H2-Ab1^−/− deficiency resulted in a marked decrease in UUO-induced protein expression of fibronectin, α-SMA and collagen IV. E Representative immunostaining for α-SMA by immunohistochemistry: (1) WT; (2) WT + UUO; (3) H2-Ab1^−/−; (4) H2-Ab1^−/−+UUO (bar = 50 μm). F Semiquantification of α-SMA protein immunoreactivity. α-SMA protein expression was significantly lower in the H2-Ab1^−/− mice than in the WT mice after UUO. Data are the mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. WT; ^#P < 0.05 vs. WT + UUO, n = 6–8

Fig. 4

Renal tubule-specific H2-Ab1 gene deficiency attenuates UUO-induced renal fibrosis in mice. A Masson trichrome staining indicates substantial fibrosis (arrowheads) in the tubulointerstitial areas of the wild-type (WT) mice after UUO. However, renal histological changes were markedly attenuated in the kH2-Ab1^−/− mice following UUO. Representative images: (1) WT; (2) WT + UUO; (3) kH2-Ab1^−/−; (4) kH2-Ab1^−/−+UUO (bar = 100 μm). B Semiquantitative analysis of (A) showing significantly increased collagen staining in the kidneys of the WT mice with UUO, which was markedly reduced in the kH2-Ab1^−/− mice. C Real-time PCR assay demonstrating that the mRNA expression of collagen I, collagen IV, fibronectin, Actα and TGFβ was markedly increased in the kidneys of the UUO-treated WT mice and was significantly attenuated in the kH2-Ab1^−/− mice. D Western blotting assays showed that the kH2-Ab1^−/− mice had markedly decreased UUO-induced protein expression of fibronectin, a-SMA, and collagen IV. E Representative immunostaining for α-SMA by immunohistochemistry: (1) WT; (2) WT + UUO; (3) kH2-Ab1^−/−; (4) kH2-Ab1^−/−+UUO (bar = 50 μm). F Semiquantification of α-SMA protein immunoreactivity. α-SMA protein expression was markedly lower in the kH2-Ab1^−/− mice than in the WT mice after UUO. Data are the mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. WT; ^#P < 0.05 vs. WT + UUO, n = 6–8

Fig. 5

Analysis of CD4⁺ T cells and their subtypes in the kidneys of the WT and kH2-Ab1⁻/⁻ mice after UUO. A FACS analysis of changes in CD4⁺ T cells in the kidneys of the WT and kH2-Ab1^−/− mice after UUO. The percentages of renal CD4⁺ T cells in the WT mice were markedly increased following UUO and were significantly decreased in the kH2-Ab1^−/− mice. B The percentages of Tregs, FOXP3-positive CD4⁺ T cells, were markedly upregulated in the UUO-treated WT mouse kidneys and were further elevated in the kH2-Ab1^−/− mice. C The percentages of renal Th1 cells and IFN-r-positive CD4⁺ T cells were increased in the WT mice with UUO; however, there were no obvious changes in the kH2-Ab1^−/− mice with UUO. D The percentages of renal Th2 cells and IL-4-positive CD4⁺ T cells in the WT mice were markedly increased following UUO and were significantly decreased in the kH2-Ab1^−/− mice. E The percentages of renal Th17 cells and IL-17A-positive CD4⁺ T cells were increased in the WT mice with UUO; however, no obvious changes were observed in the kH2-Ab1^−/− mice with UUO. F Representative immunostaining for CD4 by immunohistochemistry: (1) WT; (2) WT + UUO; (3) kH2-Ab1^−/−; (4) kH2-Ab1^−/−+UUO (bar = 50 μm). A marked increase in CD4 expression in the kidneys of the mice after UUO treatment was significantly decreased with renal tubule-specific ablation of H2-Ab1. G Real-time PCR assay demonstrating that the mRNA expression of FOXP3, T-bet, Gata-3, and RORγt was markedly increased in the kidneys of the UUO-treated WT mice. The mRNA expression of FOXP3 was further upregulated in the kH2-Ab1^−/− mice after UUO, but the mRNA expression of Gata-3 was significantly decreased in the kH2-Ab1^−/− mice after UUO. Data are the mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. WT; ^#P < 0.05 vs. WT + UUO, n = 6–8

Fig. 6

Increased renal expression of MHCII and the profibrotic gene α-SMA in CKD patients. A Masson trichrome staining demonstrating intensive fibrosis and immunohistochemistry staining showing marked increases in MHCII and α-SMA immunoreactivities in tubulointerstitial areas (arrowheads) in the CKD patients. Representative images: (1) Masson staining in healthy kidneys; (2) Masson staining in CKD patient kidneys; (3) MHCII staining in healthy kidneys; (4) MHCII staining in CKD patient kidneys; (5) α-SMA staining in healthy kidneys; (6) α-SMA staining in CKD patient kidneys (bar = 100 μm). B Semiquantitative analysis of (A) showing significantly increased extracellular matrix protein deposition in the kidneys of the CKD patients. C Semiquantification of MHCII protein immunoreactivity. MHCII protein expression was significantly elevated in the kidneys of the CKD patients. D Semiquantification of α-SMA protein immunoreactivity. α-SMA protein expression was markedly increased in the kidneys of the CKD patients. Data are the mean ± SEM. ^**P < 0.01 vs. the controls, N = 6–8