Major histocompatibility complexes are up-regulated in glomerular endothelial cells via activation of c-Jun N-terminal kinase in 5/6 nephrectomy mice

Abstract

Background and purpose: This study aims to explore the mechanism underlying the up-regulation of major histocompatibility complex (MHC) proteins in glomerular endothelial cells in 5/6 nephrectomy mice.

Experimental approach: C57/BL6 mice were randomly allocated to sham-operated (2K) and 5/6 nephrectomy (5/6Nx) groups. Mouse splenic lymphocytes, from either syngeneic or allogeneic background, were injected into 5/6Nx mice after total body irradiation. Human glomerular endothelial cells (HGECs) were cultured for experiments in vitro. Western blots, PCR, immunohistochemical and fluorescent staining were used, along with assays of tissue cytokines, lymphocyte migration and renal function.

Key results: Four weeks after nephrectomy, expression of both mRNA and protein of MHC II, CD80, and CD86 were increased in 5/6Nx glomerular endothelial cells. After total body irradiation, 5/6Nx mice injected with lymphocytes from Balb/c mice, but not those from C57/BL6 mice, exhibited increased creatinine levels, indicating that allograft lymphocyte transfer impaired renal function. In HGECs, the protein levels of MHC and MHC Class II transactivator (CIITA) were increased by stimulation with TNF-α or IFN-γ, which promoted human lymphocytes movement. These increases were reduced by JNK inhibitors. In the 5/6Nx mice, JNK inhibition down-regulated MHC II protein in glomerular endothelial cells, suggesting that JNK signalling participates in the regulation of MHC II protein.

Conclusion and implications: Chronic inflammation in mice subjected to nephrectomy induces the up-regulation of MHC molecules in glomerular endothelial cells. This up-regulation is reduced by inhibition of JNK signalling.

Keywords: IFN-γ; JNK; MHC Class II transactivator; TNF-α; glomerular endothelial cell; major histocompatibility complex; nephrectomy.

FIGURE 1

Mice subjected to 5/6 nephrectomy exhibit chronic inflammation in the cortex of remnant kidneys and increase MHC protein in glomeruli, 4 weeks after the surgery. (a) Isolated glomeruli under microscopy (left, low resolution; right, high resolution). (b) Glomeruli count in the kidney of 2K and 5/6Nx mice. n = 9; ^* P < 0.05, significantly different from the control 2K mice. (c) Levels of inflammatory cytokines in the kidney of 2K and 5/6Nx mice. n = 5; ^* P < 0.05, significantly different from the control 2K mice. (d–i) Presence of MHC molecule complex in glomeruli of 2K and 5/6Nx. Presence of MHC I (d), MICA (f), and MHC II (h) by immunohistochemistry staining. Presence of MHC I (e), MICA (g), MHC II (i), and CD31⁺ endothelial cells by immunofluorescent examination

FIGURE 2

Allograft lymphocyte transfer deteriorates renal function in 5/6Nx mice. (a) Protocol for the total body irradiation and lymphocyte transfer in mice subjected to 5/6 nephrectomy. (b) Serum levels of creatinine. (c) CD3 T lymphocytes and subgroups in the kidney after total body irradiation and lymphocyte transfer. (d) Morphological changes in the kidney after lymphocyte transfer. n = 5–6; ^* P < 0.05, significantly different from control 2K mice; ^# P < 0.05, significantly different from 5/6Nx mice; ^$ P < 0.05, significantly different from 5/6Nx mice subjected to irradiation

FIGURE 3

The up‐regulated MHC II protein is mainly expressed in CD31⁺ endothelial cells. (a) The presence of CD31⁺ endothelial cells in the kidney after lymphocyte transfer (upper: absolute value; lower: percentage value when normalized to the total examined cells measured by FACS). (b) Quantification of MHC II⁺ (upper), CD80⁺ (middle), and CD86⁺ (lower) signals in the CD31⁺ group. (c) Quantification of MHC II⁺ (upper), CD80⁺ (middle), and CD86⁺ (lower) signals in the CD31⁻ group. (d) The presences of MHC II⁺, CD80⁺, and CD86⁺ signals in the CD31⁺ group measured by FACS. (e) The presences of MHC II⁺, CD80⁺, and CD86⁺ signals in the CD31⁻ group measured by FACS. n = 5–6; ^* P < 0.05, significantly different from control 2K mice

FIGURE 4

TNF‐α and IFN‐γ, but not TGF‐β1, increase MHC proteins in HGECs, but not in HAECs. (a) Representative western blots of MHC I, MICA, and MHC II proteins in HAECs (left) and HGECs (right), after 72‐h stimulation with TNF‐α (3 ng·ml⁻¹), TGF‐β1 (5 ng·ml⁻¹), or IFN‐γ (10 ng·ml⁻¹). Densitometric quantification of MHC I (b), MICA (c), and MHC II (d) protein in HGECs (n = 5). ^* P < 0.05, significantly different from cells under control condition

FIGURE 5

IFN‐γ (a) and TNF‐α (b) regulate MHC protein differently. (a) (left) Representative western blots of MHC I, MICA, MHC II, phosphorylated p65 of NF‐κB, total p65 NF‐κB protein, phosphorylated MAPK–JNK, total MAPK–JNK, and GAPDH protein in HGECs when stimulated with IFN‐γ (10 ng·ml⁻¹). (a) (right) Densitometric quantification of MHC I, MICA, MHC II, phosphorylated p65 of NF‐κB, total p65 NF‐κB protein, phosphorylated MAPK–JNK, and total MAPK–JNK in HGEC when stimulated with IFN‐γ. n = 5; ^* P < 0.05, significantly different from cells under control condition. (b) (left) Representative western blots of MHC I, MICA, MHC II, phosphorylated p65 of NF‐κB, total p65 NF‐κB protein, phosphorylated MAPK–JNK, total MAPK–JNK, and GAPDH protein in HGECs when stimulated with TNF‐α (3 ng·ml⁻¹). (b) (right) Densitometric quantification of MHC I, MICA, MHC II, phosphorylated p65 of NF‐κB, total p65 NF‐κB protein, phosphorylated MAPK–JNK, and total MAPK–JNK in HGEC when stimulated with TNF‐α. n = 5; ^* P < 0.05, significantly different from cells under control conditions

FIGURE 6

(a) Protocol of lymphocyte migration in HGECs stimulated with TNF‐α (3 ng·ml⁻¹) or IFN‐γ (10 ng·ml⁻¹). (b) Lymphocyte count, in the absence of PHA, in the lower chamber containing freshly changed medium. (c) Lymphocyte count, in the presence of PHA, in the lower chamber containing freshly changed medium. (d) Lymphocyte count, in the absence of PHA, in the lower chamber containing 3‐day experimental medium. (e) Lymphocyte count, in the presence of PHA, in the lower chamber containing 3‐day experimental medium. (f) Lymphocyte count in the lower chamber after IFN‐γ stimulation during 72 h. (g) Lymphocyte count in the lower chamber after TNF‐α stimulation during 72 h. n = 5; ^* P < 0.05, significantly different from with cells under control conditions; ^# P < 0.05, significantly different from cells under the same condition, either with IFN‐γ or TNF‐α, respectively

FIGURE 7

Inhibition of MAPK–JNK partly restores renal function in 5/6Nx mice. (a) Serum levels of creatinine. (b) Quantification of MHC II⁺ (upper), CD80⁺ (middle), and CD86⁺ (lower) signals in the CD31⁺ group in 5/6Nx mice treated with JNK inhibitors. (c) Quantification of MHC II⁺ (upper), CD80⁺ (middle), and CD86⁺ (lower) signals in the CD31⁻ group in 5/6Nx mice treated with JNK inhibitors. (d) The presence of MHC II⁺, CD80⁺, and CD86⁺ signals in the CD31⁺ group measured by FACS in 5/6Nx mice treated with JNK inhibitors. (e) The presence of MHC II⁺, CD80⁺, and CD86⁺ signals in the CD31⁻ group by FACS in 5/6Nx mice treated with JNK inhibitors. n = 9–10; ^* P < 0.05, significantly different from control mice (2K); ^# P < 0.05, significantly different from 5/6Nx mice. (f) Quantification of TNF‐α, IFN‐γ, IL‐5, IL‐13, and IL‐17 in CD3⁺ lymphocytes. n = 5; ^* P < 0.05, significantly different from control mice (2K); ^# P < 0.05, significantly different from 5/6Nx mice