Expression And Effect Of HMGB1/TLR/NF-κB Signal Pathway In Murine Lupus Nephritis

Objective:Systemic lupus erythematosus (SLE) is a systemic autoimmune disease affecting multiple organs including the kidney, and lupus nephritis (LN) is a capital cause of death and disability. Although the pathogenesis of SLE has not yet entirely clear, immune dysfunction induced by lymphocyte dysfunction and unbalanced cytokine secretion is the core of the pathogenesis. As an important late inflammatory mediator, HMGB1 involved in the occurrence and development of rheumatoid arthritis, SLE, inflammatory diseases and other autoimmune diseases [2-3]. Our early experiments revealed that the level of HMGB1 protein in the peripheral blood of patients with lupus nephritis increased and HMGB1 promoted on the proliferation of human mesangial cell, which meaned that HMGB1 is one of the important inflammatory cytokines in the pathogenesis of lupus nephritis, and related to the mesangial cell proliferation [8].However, the expression of HMGB1 in renal tissue and the possible mechanism in lupus nephritis lack in-depth study. Therefore , in order to explore the possible effect of HMGB1/TLR/NF-κB signal pathway in the pathogenesis of lupus nephritis , BXSB mouse model was used to detect the expression of HMGB1, Toll-like receptor (TLR) and NF-κB in renal tissue changes and analyzing the correlation among them.Methods : Sixteen BXSB mice (age: 11 weeks; weight: 18–22 g) were purchased from the Department of Medical Immunology of the Peking University Health Science Center. These mice will herein be referred to as the LN group. The animals were housed in a room with an alternating cycle of 12 h light/12 h dark at 22±3?C and were fed with a standard diet. Fourteen C57BL/6 mice were randomly chosen as normal controls. All animals were allowed to acclimatize for 1 week before the experiment. Normal control mice and BXSB mice were sacrificed after 4 weeks. The serum , obtained from the angular vein ,was analyzed using ELISA to assess renal function and the level of HMGB1 protein. Urine was collected to detect the concentration of micro-albumin. The renal cortex was removed, cleaned and washed, and portions were immediately fixed in buffered neutral formalin for histopathological sections or in 2.5% glutaraldehyde for transmission electron microscope evaluation. Another portion of the renal cortex was stored in liquid nitrogen for extraction of proteins and RNA.PAS stain and TEM was used to observe the morphological changes of renal tissue; RT-PCR and immunohistochemical staining was used to test the expression of HMGB1, TLR2, TLR4mRNA and protein; RT-PCR, Werstern Blot and immunohistochemical staining was used to detect the expression and translocation of NF-κBmRNA and P-NF-κB protein; immunohistochemical staining was used to check the expression of PCNA protein.Results :1. Comparison of renal function in different groups of miceCompared with normal control group, the level of BUN and Upro in serum of LN group were significantly higher (P <0.01 respectively ); however, no significant difference was found in the endogenous creatinine clearance (SCr) between C57BL/6 and BXSB mice (P>0.05).2. The level of serum HMGB1 protein in different groupsThe level of HMGB1 protein in serum of LN group was (0.57±0.03) and significantly higher than that in normal control group (0.54±0.00) (P <0.01).3. Morphological changes of renal tissue in different groupsElectron microscopy showed the thickening of the glomerular basement membrane (GBM), a partial fusion of foot processes of epithelial cells and subepithelial electron-dense deposits in BXSB mice . Staining for PAS also showed the thickening of the GBM and the extracellular matrix (ECM) deposits; however, no obvious ECM accumulation was found in the kidneys of normal control mice.4. The expression of PCNA protein in different groupsCompared with normal control group, the expression of PCNA protein was significantly increased, mainly located in the nucleus of glomerular intrinsic cells.5. The expression of HMGB1mRNA and protein in different groupsThe IOD ratio of HMGB1 mRNA expression in the BXSB mice, normalized by 18SrRNA as an internal control, was 1.40 times higher than that in the normal control group (1.13±0.08 vs. 0.81±0.08).The HMGB1 protein was located in nuclear, cytoplasmic or extracellular milieu. There was little expression of HMGB1 found in the glomeruli of C57BL/6 mice, and expression was mostly located in the nuclear of the tubule. In BXSB mice, however, the expression of HMGB1 was markedly higher and was primarily confined to the cytoplasm or the extracellular milieu of the glomeruli, especially in proliferative glomeruli6. The expression of TLR2, 4mRNA and protein in different groupsCompared with normal control group, the expression of TLR2/4mRNA and protein were significantly higher, mainly located in the membrane of glomerular intrinsic cells. The TLR2 and TLR4 proteins were found at the plasma membrane, and there was no marked expression of TLR2 or TLR4 in C57BL/6 mice. Conversely, expression of both TLR2 and TLR4 was significantly increased in BXSB mice, and these proteins were primarily located at the plasma membrane of inherent cells in the glomeruli, with little expression located in renal proximal tubule epithelial cells.7. The expression and translocation of NF-κBp65mRNA and protein in different groupsCompared with normal control group, the expression of p-NF-κBp65mRNA and protein expression were significantly higher, mainly located in the nuclei of intrinsic glomerular cells and renal tubular epithelial cells.8. Correlation AnalysisIn LN group, there were positive correlation between the expression of HMGB1 and TLR2/4, PCNA protein respectively (r = 0.911, P = 0.012; r = 0.854, P = 0.000; r = 0.909, P = 0.000 respectively), as well as between P-NF-κBp65 and TLR2/4, PCNA protein(r = 0.984, P = 0.000; r = 0.935, P = 0.006; r = 0.944, P = 0.005respectively) .Conclusions:1 HMGB1 could induce the form of proliferative glomerulonephritis by promoting the proliferation of inherent cell of glomeruli ;2 HMGB1 could activate P-NF-κBp65 through combining with its cell-surface receptor–TLR2, TLR4, which may contribute to the pathogenesis of lupus nephritis.