Correlation analysis between expression of histone deacetylase 6 and clinical parameters in IgA nephropathy patients.

BACKGROUND: It has been demonstrated that histone deacetylase 6 (HDAC6) is involved in various kidney diseases in experimental study. However, correlation between HDAC6 and clinical parameters in IgA nephropathy (IgAN) patients is still unknown. METHODS: A total of 46 human kidney biopsy specimens with IgAN were selected as observation group, specimens of normal renal cortex tissue that was not affected by the tumor from patients with renal carcinoma (n = 7) served as control. We investigated the relationship between HDAC6 and clinical parameters in IgAN. RESULTS: HDAC6 was highly expressed in human kidney biopsy specimens with IgAN compared with control group, while the number of acetyl histone H3 positive cells were significantly decreased. There was a statistical difference in the indexes of albumin, estimated glomerular filtration rate (eGFR), serum urea, serum creatinine, serum uric acid, ß2-microglobulin, cystatin C, cholesterol, high-density lipoprotein, low-density lipoprotein, and HDAC6 positive area among the different Oxford Classification (p < 0.05). The expression of HDAC6 was different in various eGFR levels, the expression of HDAC6 increased with the decreasing of eGFR level, the expression of acetyl histone H3 decreased with the decreasing of eGFR level. In addition, the expression of HDAC6 positively correlated with Masson trichrome positive area, serum urea, serum creatinine, ß2 macroglobulin, and cystatin C, while negatively correlated with eGFR and acetyl histone H3. Multivariate linear regression analysis demonstrated that eGFR and cystatin C were independently associated with HDAC6, respectively (p < 0.05). CONCLUSIONS: These results suggested that high level of HDAC6 expression in IgAN is correlated with renal dysfunction.

DC-SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis.

Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC-SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC-SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC-SIGN was co-expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC-SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti-DC-SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC-SIGN, major histocompatibility complex (MHC) class II and CD80 was up-regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)-γ/interleukin (IL)-4 ratio increased. However, anti-DC-SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC-like function through their expression of DC-SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC-SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects.

[The role of Foxo1 and ubiquitin-proteasome system markers in muscle atrophy caused by chronic kidney disease].

OBJECTIVE: To identity whether there is muscle atrophy phenomenon in end-stage kidney disease patients and to detect the level of transcription factor Foxo1 and the activity of ubiquitin-proteasome system. METHODS: Twenty-two patients in chronic kidney disease (CKD) stage 5 were selected and their mean muscle cross sectional area was measured. mRNA and protein levels of Foxo1, Atrogin-1, MuRF1 in rectus abdominis biopsies obtained from consecutive patients were detected. Control biopsies were obtained from 8 healthy subjects during elective surgery for abdominal wall hernias and 6 subjects during elective surgery for adenomyosis. RESULTS: Compared with the control group, cross sectional area of muscle fibers decreased and the transcription and protein levels of Foxo1, Atrogin-1, MuRF1 were upregulated in CKD group (P < 0.05). Protein level of p-Foxo1 decreased in CKD group (P < 0.05). CONCLUSION: There exist muscle atrophy phenomenon in CKD patients, which may associate with the upregulation of Foxo1 and activation of ubiquitin-proteasome system.

[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.

[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.

Parathyroid hormone-potentiated connective tissue growth factor expression in human renal proximal tubular cells through activating the MAPK and NF-kappaB signalling pathways.

BACKGROUND: Secondary hyperparathyroidism is a universal complication of chronic renal diseases. One of the pathological consequences of hyperparathyroidism is impairment of the renal interstitium and tubules. However, the molecular mechanism of renal tubular interstitial impairment induced by parathyroid hormone (PTH) remains unclear. Enhanced and prolonged expression of connective tissue growth factor (CTGF) has been associated with fibrosis and inflammation in the kidney. The purpose of this study was to investigate the effects of PTH on CTGF expression patterns in human proximal tubular cell line-HK-2 cells. METHODS: We treated cells with various concentrations of PTH for the indicated periods of time in the presence or absence of the mitogen-activated protein kinase (MAPK) inhibitor (PD98059) or the NF-κB inhibitor (PDTC). RESULTS: Quantitative real-time RT-PCR analysis revealed that PTH at a concentration of 10(-12)-10(-10) M increased the mRNA levels of CTGF, which was similar to the trends of CTGF protein levels detected by immunoblotting assay. Our data clearly show the ability of human proximal tubular HK-2 cells to produce CTGF after the treatment with PTH. In addition, we showed that PTH induced the phosphorylation of MAPK p42 and p44, and increased NF-κB-binding activities in the PTH-treated cells. Moreover, both PD98059 and PDTC inhibited the effect of PTH on the expression of CTGF, which strongly suggests that these pathways play important roles in the PTH-induced CTGF upregulation in renal tubular cells. CONCLUSIONS: Our results indicated for the first time that PTH may enhance the expression of CTGF in human kidney proximal tubular cells, suggesting that PTH may play an important role in the fibrotic and inflammatory process that is a hallmark for progression of chronic kidney disease.

[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.

Risk factors for heart valve calcification in chronic kidney disease.

Cardiovascular disease (CVD) is a common cause of death in patients with chronic kidney disease (CKD). Aortic and mitral valve calcification (AVC and MVC, respectively) are critical indicators of CVD and all-cause mortality in CKD patients.We conducted a single center retrospective study of Chinese inpatients with CKD to identify risk factors associated with valve calcification (VC).Of 288 enrolled CKD patients, 22.9% had VC, all of which exhibited AVC, while 21.2% exhibited MVC. The VC group were significantly older than the non-VC group (70.42 ±â€Š11.83 vs 56.47 ±â€Š15.00, P < .001), and contained more patients with history of coronary artery disease (12.1% vs 4.5%, P = .025) or stroke (18.2% vs 5.4%, P < .001). Subjective global assessment scoring indicated that more VC patients were mid/severely malnourished. Levels of prealbumin, cholesterol (Ch), triglycerides, low-density lipoprotein (LDL), apolipoprotein E, ejection fraction, and fraction shortening were significantly lower, and blood C reactive protein, IL-6, left ventricular internal end diastole diameter measured in end diastole, and interventricular septum thickness (IVST) levels were significantly higher in the VC group. Bone metabolism did not differ significantly between the 2 groups. Multivariable logistic regression analysis indicated that age, blood Ch, and LDL levels were significantly associated with VC.Advanced age, increased IVST, hypocholesterolemia, and hyper-LDL cholesterolemia were key risk factors for VC in Han patients with CKD.

Effect of CTLA-4 gene polymorphisms on long-term kidney allograft function in Han Chinese recipients.

Single nucleotide polymorphisms (SNPs) of cytotoxic T lymphocyte associated antigen-4 gene (CTLA-4) have been associated with graft rejection and long-term clinical outcome after organ transplantation. Our aim was to examine the association between CTLA-4 SNPs (rs733618, rs4553808, rs5742909, rs231775, rs3087243) and long-term allograft function in Chinese renal transplant recipients. Genotyping of CTLA-4 SNPs was performed in 292 renal transplantation recipients. To assess long-term allograft function, the estimated glomerular filtration rate (eGFR) was determined 1, 3, 6, 12, 24, 36, 48 and 60 months after renal transplantation. CTLA-4 rs733618 and rs3087243 alleles and genotypes as well as the rs5742909 and rs231775 genotypes were significantly associated with long-term allograft function after transplantation (P<0.05). Patients with favorable genotypes had higher allograft function during the 60 months after transplantation. The TACGG, CACAG and CGTAA haplotypes were also associated with long-term kidney function after renal transplantation (P<0.05 or P<0.01). In sum, the favorable CTLA-4 rs5742909TT genotype, CTLA-4 rs733618C and rs3087243A alleles, and CACAG and CGTAA haplotypes, as well as the unfavorable rs733618TT, rs3087243GG and rs231775GG genotypes and TACGG haplotype could potentially serve as effective indicators of long-term allograft function in Chinese renal transplantation recipients.

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.