Phosphorus balance calculator: an individualized tool for treatment of hyperphosphatemia in hemodialysis patients.

BACKGROUND AND HYPOTHESIS: Lack of evaluations of the dietary phosphorus and dialysis phosphorus removal in daily clinical practice are the common obstacle to assess phosphorus balance and control phosphorus in hemodialysis patients. We aimed to investigate whether the individualized therapy using phosphorus balance calculator improves phosphorus control. METHODS: A randomized, open-label, multicenter, 4-week clinical trial was conducted. 119 maintenance hemodialysis patients aged 18 to 85 years old and with serum phosphorus level higher than 1.45mmol/l from 3 university teaching hospitals in Shanghai were enrolled. Patients were randomized in a 1:1 ratio to individualized therapy (n=60), or conventional therapy (n=59). The primary outcome was the serum phosphorus concentration after 4-week treatment. Secondary outcomes included the serum calcium and parathyroid hormone (PTH) concentrations, changes in serum phosphorus, calcium and PTH concentrations, and the proportion of patients achieving target ranges of serum phosphorus, calcium and PTH after 4-week treatment. RESULTS: Among 119 randomized participants (mean age, 62 years; 68 male[57%]), 116 completed the trial. By using the phosphorus balance calculator, the individualized group achieved a better phosphorus balance state, significantly reduced the serum phosphorus (1.62±0.45mmol/l versus 1.85±0.45 mmol/l, P=0.006), increased the proportions of patients achieving target serum phosphorus range (41% versus 18%, P=0.006), and had greater adjusted mean difference in change in serum phosphorus over the 4 weeks (-0.47 versus -0.23mmol/l, P=0.010) when compared to conventional therapy. No significant changes were observed in serum calcium and PTH levels, the proportion of patients achieving target serum calcium or PTH levels, and adjusted mean difference of serum calcium and PTH levels over the treatment period. CONCLUSION: Phosphorus balance calculator was proved to improve serum phosphorus control in patients undergoing maintenance hemodialysis, offering a new tool for managing hyperphosphatemia.

Low-Dose Mycophenolate Mofetil for Treatment of Neuromyelitis Optica Spectrum Disorders: A Prospective Multicenter Study in South China.

Objective: To evaluate the efficacy and safety of low-dose mycophenolate mofetil (MMF, 1,000 mg/day) treatment of neuromyelitis optica spectrum disorders (NMOSDs). Methods: This study was a multicenter, open, prospective, follow-up clinical trial. The data include retrospective clinical data from the pretreatment phase and prospective data from the post-treatment phase. From September 2014 to February 2017, NMOSD patients seropositive for aquaporin 4-IgG (AQP4-IgG) were treated with low-dose MMF. Results: Ninety NMOSD patients were treated with MMF for a median duration of 18 months (range 6-40 months). The median annual recurrence rate (ARR) decreased from 1.02 before treatment to 0 (P < 0.0001) after treatment, and the Expanded Disability Status Scale (EDSS) score decreased from 4 to 3 (P < 0.0001). The EDSS score was significantly lower (P = 0.038) after the first 90 days of treatment. The serum AQP4-IgG titer decreased in 50 cases (63%). The median Simple McGill pain score (SF-MPQ) was reduced in 65 patients (88%) with myelitis from 17 (range 0-35) to 11 (range 0-34) after treatment (P < 0.0001). The median Hauser walking index (Hauser Walk Rating Scale) was reduced from 2 (range 1-9) before treatment to 1 (range 0-7) after treatment (P < 0.0001). Adverse events were documented in 43% of the patients, and eight patients discontinued MMF due to intolerable adverse events. Fourteen (16%) of the total patients discontinued MMF after our last follow-up for various reasons and switched to azathioprine or rituximab. Conclusion: Low-dose MMF reduced clinical relapse and disability in NMOSD patients in South China. However, some patients still suffered from adverse events at this dosage. CLINICAL TRIAL REGISTRATION: www.ClinicalTrials.gov, identifier : NCT02809079.

Effects of Nogo-A Silencing on TNF-α and IL-6 Secretion and TH Downregulation in Lipopolysaccharide-Stimulated PC12 Cells.

Parkinson's disease (PD) is a common degenerative disease that lacks efficient treatment. Myelin-associated neurite outgrowth inhibitor A (Nogo-A) is relevant with inhibition of nerve regeneration and may play vital role in pathogenesis of PD. The study aimed to establish the shRNA expression plasmids of Nogo-A gene and explore the regulatory effects of Nogo-A silencing on the expression of inflammation factor tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) as well as tyrosine hydroxylase (TH) in lipopolysaccharide- (LPS-) stimulated rat PC12 cells. The results showed that both mRNA and protein levels of Nogo-A in pGenesil-nogoA-shRNA group were downregulated. The viabilities of PC12 cells decreased with increase of LPS concentrations. LPS significantly increased the supernatant TNF-alpha and IL-6 concentrations and reduced TH protein expression in PC12 cells, while silencing Nogo-A could block these effects. These results suggested that LPS can activate PC12 cells to secrete inflammatory cytokines and lower the TH expression, which can be regulated by Nogo-A gene silencing. Nogo-A silencing might provide new ideas for PD treatment in the future.

Crosstalk between peroxisome proliferator-activated receptor-γ and mineralcorticoid receptor in TNF-α activated renal tubular cell.

INTRODUCTION: In our previous study, we observed the crosstalk between peroxisome proliferator-activated receptor-γ (PPAR-γ) and angiotensin II in activated renal tubular cells. The present study is aimed to further explore the crosstalk between PPAR-γ and mineralocorticoid receptor (MR) in tumor necrosis factor (TNF)-α activated renal tubular cells. METHODS: Human proximal renal tubular epithelial cells HK-2 were cultured with the pre-treatment of PPAR-γ agonist, pioglitazone (5 µM), MR antagonist, eplerenone (5 µM), or their combined treatment, followed by activation with TNF-α (20 ng/ml). In the parallel experiment, PPAR-γ inhibitor GW9662 (25 µM) was used to study the independence of PPAR-γ. Gene expression and protein synthesis of intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), MR and PPAR-γ were measured by RT-PCR, ELISA and Western blot, respectively; nuclear factor κB (NF-κB) nuclear translocation activity in the nucleus was examined by EMSA assay. RESULTS: TNF-α effectively activated HK-2 cells by up-regulating gene expression and protein synthesis of ICAM-1, IL-6 and MR and down-regulating PPAR-γ in a dose-dependent manner. TNF-α also significantly induced NF-κB nuclear translocation in HK-2 cells. Dual treatment of pioglitazone and eplerenone demonstrated synergistic effect on reducing ICAM-1 and IL-6 expression and alleviating NF-κB activation when compared with their monotherapies in TNF-α activated renal tubular cells. PPAR-γ antagonist, GW9662, significantly attenuated protective effect on ICAM-1, IL-6 and PPAR-γ expression by pioglitazone, eplerenone and their combined treatment. CONCLUSIONS: Our data suggest that pioglitazone, in a PPAR-γ-dependent manner, trans-represses MR signaling by suppressing NF-κB activation. MR antagonist also restored PPAR-γ expression. Dual treatment of pioglitazone and eplerenone present better efficacy in attenuating excessive inflammatory response in activated renal tubular cells under stimulation of TNF-α than single treatment.

Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice.

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor κB, degradation of inhibitor of κB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor κB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage.

Recombinant GnRH-p53 protein sensitizes breast cancer cells to 5-fluorouracil-induced apoptosis in vitro and in vivo.

An ideal approach to treat cancers with dysfunctional p53 tumor suppressor gene is to reinstate p53 functionality by directly using p53 protein as a therapeutic agent. However, this has not been possible because the cells cannot readily internalize the protein. We constructed a fusion protein consisting of gonadotropin-releasing hormone (GnRH-p53) and p53 moieties. The recombinant protein was directly used to treat human breast cancer cells and athymic nude mice bearing breast cancer xenografts, with or without DNA synthesis-arresting agent 5-fluorouracil (5-FU). Treatments of cells from breast cancer cell-lines MDA-MB-231, T47D, or SKBR-3 with GnRH-p53 in combination with 5-FU significantly enhanced p53-activated apoptosis signals, including PUMA expression, BAX translocation to mitochondria, and activated caspase-3. Intratumoral injection of the GnRH-p53 protein inhibited MDA-MB-231 xenograft growth and induced p53-mediated apoptosis in the tumors. Systemic treatment of the tumor-bearing mice via tail vein injection of GnRH-p53 markedly augmented the anticancer efficacy of 5-FU. Substitution of GnRH-p53 with wild type p53 protein had no effect. Recombinant GnRH-p53 is able to function as a surrogate of p53 with regard to its apoptosis-inducing activity. Combination of GnRH-p53 with DNA-damaging drugs may be of important therapeutic value for cancer treatment.

Activation of hypoxia-inducible factor-1 ameliorates postischemic renal injury via inducible nitric oxide synthase.

Hypoxia-inducible factor-1 (HIF-1) could ameliorate renal ischemia reperfusion injury (IRI), but the underlying mechanism remains elusive. In the current study, we aim to investigate the possible role of prolyl hydroxylases inhibitor dimethyloxalylglycine (DMOG) in inducing delayed preconditioning-like effects against IRI. Mice were divided into four groups (n = 6): sham group; IRI group; DMOG group: pretreated with DMOG 24 h before IRI; and GW274150 + DMOG group: pretreated with DMOG followed by iNOS inhibitor GW274150 treatment 24 h before IRI. The results showed that the protein level of HIF-1a and the expression of its targets inducible nitric oxide synthase (iNOS), erythropoietin, and heme oxygenase-1 were obviously increased after administration of DMOG. Histological analysis of renal function showed improvement in tubulointerstitial injury due to ischemia by delayed preconditioning with DMOG. GW274150 antagonized the delayed renal protection afforded by DMOG as reflected by deteriorated renal dysfunction, aggravated histological injury, increased renal cell apoptosis, and increased vimentin expression in the kidney. In conclusion, our data demonstrate that DMOG pretreatment induces delayed renal protection against IRI in mice and the beneficial effects are mitigated by pharmacological inhibition of iNOS, suggesting that the protective effects derived from HIF-1 activation via DMOG in the kidney are partially mediated by iNOS.

Human rhomboid family-1 gene RHBDF1 participates in GPCR-mediated transactivation of EGFR growth signals in head and neck squamous cancer cells.

Epidermal growth factor receptor (EGFR) is an activated oncogene in many cancers. It can be transactivated by ligands of G protein-coupled receptors (GPCRs). We show here that a novel gene, human rhomboid family-1 (RHBDF1), which was recently reported to have a pivotal role in epithelial cancer cell growth in culture and in xenograft tumors, participates in the modulation of GPCR-mediated EGFR transactivation. The RHBDF1 protein localizes mainly in the endoplasmic reticulum. Silencing the RHBDF1 gene in head and neck squamous cancer cell line 1483 cells with siRNA causes an inhibition of gastrin-releasing peptide (GRP) -induced phosphorylation of EGFR and EGFR-dependent signaling proteins p44/42 MAPK and AKT, accompanied by an inhibition of GRP-induced survival, proliferation, and invasion of the cells. The EGFR signaling pathway itself remains intact, however, as the cells remain responsive to exogenous EGF. In addition, RHBDF1 gene silencing disrupts GRP-stimulated secretion of EGFR ligand TGF-alpha, but not the production of latent TGF-alpha, whereas engineered overexpression of RHBDF1 markedly accelerates the secretion of TGF-alpha. These findings are consistent with the view that RHBDF1 is critically involved in a GPCR ligand-stimulated process leading to the activation of latent EGFR ligands.

Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth.

The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers.

[Effect of the mutation of promoter region in Wilson disease ATP7B gene on the expression of reporter gene].

OBJECTIVE: To find out the relationship between mutation of ATP7B gene promoter region and pathogenesis of Wilson disease(WD). METHODS: Two of 48 WD patients presented C-->T base substitution mutations at the position -183. DNA sequences of the promoter region from normal and mutant samples were separated. The fragments containing the promoter region were cloned upstream of the luciferase. Luciferase activity was analyzed. RESULTS: The luciferase activity of reporter gene containing normal sequence of ATP7B gene promoter region did not show significant difference as compared with that of reporter gene containing mutant promoter(n=3, P > 0.05). CONCLUSION: No influence of C-->T base substitution mutations on the activity of promoter was observed in study. The results suggest that WD pathogenesis relates little to the mutations of the promoter region in Chinese.