CTLA4 polymorphisms and de novo malignancy risk after renal transplantation in Chinese recipients.

Genetic polymorphisms in cytotoxic T lymphocyte-associated antigen 4 (CTLA4) play an influential role in graft rejection and the long-term clinical outcome of organ transplantation. We investigated the association of five CTLA4 single-nucleotide polymorphisms (SNPs) (rs733618 C/T, rs4553808 A/G, rs5742909 C/T, rs231775 A/G, and rs3087243 G/A) with de novo malignancy in 1463 Chinese renal transplantation (RT) recipients who underwent a 192-month follow-up. Multivariate analyses revealed that recipient rs231775 genotype is significantly associated with tumorigenesis (P = 0.012). Multiplicative interaction between rs231775 AA and possible risk factors of malignancy revealed two significant results: rs231775 AA × primary diseases and rs231775 AA × number of HLA-mismatch. The frequency of haplotype TACAG was significantly higher in the tumor group (17.07%) than that in the nontumor group (1.53%). In addition, aristolochic acid nephropathy (P = 0.003) and the time of discovery of tumor (P = 0.000) also were independently associated with tumorigenesis. Our data show that the CTLA4 genotype rs231775 AA may be one of risk factors for the development of malignancy and haplotype TACAG was susceptible haplotype in Chinese kidney transplant recipients.

[Effect of Notch1 signal on hepatitis B virus X-mediated abnormal immune activity in renal tubular epithelial cells].

OBJECTIVE: To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with hepatitis B virus X(HBx) gene and its role in immunologic activity. METHODS: The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells. And the shRNA technique was used to silence Notch1. HK-2 cells were divided into 7 groups, including normal culture, pcDNA3.1/myc, HBx, HBx +pcDNA3.1/myc, HBx+Notch1, HBx+ shRNA and HBx+ Notch1 shRNA. Real-time PCR and Western blotting were used to detect the expression of Notch1. The expressions of MHC-IIand CD40 were examined by flow cytometry. And the supernatant contents of IL-4 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of real-time PCR and Western blotting verified that HBx and Notch1 were successfully expressed in HK-2 cells after transfection. The transfection efficiency of shRNA was 70%. Compared with normal culture and pcDNA3.1/myc groups, the expression of Notch1 increased. The expressions of MHC-II and CD40 also significantly increased in the HBx+Notch1 group (9.69% ± 0.52% vs 4.90% ± 0.32%, 21.56% ± 0.71% vs 15.74% ± 0.20%, both P < 0.05) . The supernatant level of IFN-γ was lower in HBx+Notch1 group (11.9 ± 1.7 vs 18.8 ± 0.8, P < 0.05) while the level of IL-4 was higher than control groups (50.2 ± 0.6 vs 28.1 ± 1.2, P < 0.05). And the HBx+Notch1 shRNA group had the opposite results. CONCLUSIONS: An over-expression of HBx gene may up-regulate the expression of Notch1. And Notch1 promotes the expression of immune molecules of renal tubular epithelial cell and regulates the secretion of cytokines so as to cause injury of cells and dysfunction of immune microenvironment.

[Role of Notch1 receptor on apoptosis of renal tubular epithelial cells transfected with HBx gene].

OBJECTIVE: To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with HBx gene and elucidate its role in cell apoptosis. METHODS: The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells and shRNA technique employed for silencing Notch1. HK-2 cells were divided into 7 groups of normal culture, pcDNA3.1/myc, HBx, HBx + pcDNA3.1/myc, HBx + Notch1 gene, HBx + shRNA and HBx + Notch1 shRNA. Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of HBx and detect the expression of Notch1 receptor. Methyl thiazolyl tetrazolium (MTT) assay was used to observe the proliferation rate of HK-2 cells and flow cytometry to detect the apoptosis of HK-2 cells. RESULTS: HBx and Notch1 receptor were successfully expressed in HK-2 cells post-transfection. The transfection efficiency of shRNA was 70%. The expression of Notch1 receptor in pcDNA3.1/myc-HBx group was higher than that in the normal culture and pcDNA3.1/myc groups. And the apoptotic ratio was also higher than that of normal culture and pcDNA3.1/myc group. The difference was significant (14.94% ± 0.32% vs 13.86% ± 0.18%, 13.67% ± 0.54%, both P < 0.05). The apoptotic ratio in the HBx + Notch1 gene group was lower than that in control group (10.10% ± 0.26% vs 14.79% ± 0.02%, P < 0.05). And the growth inhibition ratio of cells was also lower. But the apoptotic ratio and growth inhibition ratio were both higher in HBx + Notch1 shRNA group than those in HBx + shRNA group (both P < 0.05). CONCLUSIONS: HBx gene is successfully transfected into HK-2 cells. And its over-expression may induce apoptosis of HK-2 cells and growth inhibition by regulating Notch1 signal.

[Toll-like receptor 4 deposition and its significance in hepatitis B virus associated nephropathy].

OBJECTIVE: To investigate the expression and distribution of Toll-like receptor 4 (TLR4) in renal tissue of HBV associated nephropathy (HBV-GN) and its role in the pathogenesis and clinical manifestations of HBV-GN. METHODS: Renal tissues were sampled from 48 HBV-GN patients confirmed by renal biopsy and 154 non-HBV-GN patients. The distribution of TLR4 in renal tissue and the relationship between the distribution of TLR4 and HBsAg were detected by immunohistochemistry. Integrating case record, correlations between the expression of TLR4 with clinical parameters including pathology, glomeruli, kidney tubules lesions, renal interstitial inflammatory infiltration and blood serum HBV were analyzed. RESULTS: TLR4 mainly distributed in the renal tubular epithelial cells and interstitial areas as brownish red and granular, which was in consistent with HBsAg distribution. The TLR4 positive rate and score in HBV-GN group were higher than those in non-HBV-GN group (P < 0.05). TLR4 positive score was slightly higher in mesangial proliferative glomerulonephritis group and focal segmental glomerulosclerosis group, which had no significant difference (P > 0.05). Kidney tubules lesions were strongly associated with TLR4 expression (r = 0.748, P < 0.001) which increased with aggravation of renal interstitial fibrosis (r = 0.569, P < 0.001), tubular atrophy (r = 0.577, P < 0.001) and inflammatory cell infiltration (r = 0.684, P < 0.001). No obvious correlation with glomeruli lesions was observed (r = 0.293, P = 0.053). Negative correlation could be seen between TLR4 and the renal function (R(2) = 0.784), systolic blood pressure (R(2) = 0.869), high sensitivity C-reactive protein (R(2) = 0.979) and urinary protein (R(2) = 0.615) by regression analysis. Other clinical parameters had no statistical significances. CONCLUSIONS: The expression of TLR4 is abnormal in the renal tissue of HBV-GN patients, mainly in renal tubular epithelial cells and interstitial, which is consistent with the distribution of HBsAg. Its intensity is closely related with renal interstitial lesions, renal function changes and inflammatory cell infiltration. A speculation, that HBV can promote abnormal expression of TLR4 in renal tissues of HBV-GN which may be involved in the lesion progress of HBV-GN, is made upon our study.

Intrarenal activation of renin angiotensin system in the development of cyclosporine A induced chronic nephrotoxicity.

BACKGROUND: The relationship between cyclosporine-induced chronic nephrotoxicity (CAN) and renin-angiotensin II in humans is still contradictory. This study was conducted to detect the levels of renin and angiotensin II (ANGII) both in renal tissue and plasma from kidney transplantation patients suffering from CAN. METHODS: Twenty-six patients with allograft biopsy-proven CsA-related chronic nephrotoxicity (CAN group) and chronic rejection (control group) were enrolled in this study. Renal tissues were subjected to immunohistochemical staining with renin and ANGII antibodies. Renin and ANGII plasma levels were measured when the biopsy was performed. The relationship between expression of renin or ANGII and clinicopathological manifestations were also investigated. The cyclosporine plasma level was obtained 2 hours after morning dose (C2). In vitro, human umbilical vein endothelial cells (HUVEC) and rat mesangial cells (MC) were incubated with different concentrations of CsA (0, 250, 500, 1000 microg/L) for 24 hours. Secretion and expression of renin and ANGII was measured by radioimmunoassay or immunohistochemical staining. RESULTS: Renal pathological scores for renin and ANGII expression were significantly higher in specimens of CAN than in controls (P < 0.05). The plasma levels of renin, ANGII and C(2) in the CAN group were higher than the control group, but no significant difference was found ((0.37 +/- 0.12) ng x ml(-1)x h(-1) vs (0.20 +/- 0.10) ng x ml(-1) x h(-1), P = 0.076; (122.69 +/- 26.73) pg/ml vs (121.88 +/- 36.35) pg/ml, P = 0.977; (719.04 +/- 55.89) ng/ml vs (658.80 +/- 90.78) ng/ml, P = 0.196, respectively). In vitro, renin as well as ANGII expression increased significantly in both HUVEC and MC after the cells were incubated with CsA for 24 hours (P < 0.05). CsA also stimulated the secretion of ANGII in HUVEC and MC in a dose-dependent manner. CONCLUSIONS: Renal allograft biopsy is important to differentiate chronic CsA-related nephropathy from chronic rejection. The intrarenal renin angiotensin system plays an important role in CsA-related chronic nephropathy. The histological lesions of CsA nephrotoxicity fail to correspond spontaneously to either the change of C2 level or the change of renin and ANGII plasma level. CsA stimulates the secretion of ANGII and the expression of renin and ANGII in HUVEC and MC. Blockage of RAS may be helpful for therapeutic intervention in the progression of CsA-related chronic nephropathy.