Effects of uremic serum of different molecular weight groups ongene and protein expression of connective tissue growth factor gene and proteinexpression in human renal tubular epithelial cells

AIM:To investigate the effects of uremic serum of different molecular weight groups on gene and protein expression of connective tissue growth factor (CTGF) in human renal tubular epithelial cells.METHODS:The serum from 40 chronic renal failure patients and 20 healthy volunteers were collected and uremic serum was segregated to three groups: >10 000 D,5 000-10 000 D,<5 000 D by 10 000 D and 5 000 D molecular weight Centricon Plus 20 Centrifugal Filter Devices.The protein expression of CTGF was examined by Western blotting.The mRNA expression of CTGF was detected by RT-PCR.RESULTS:CTGF gene expression were increased in 2.5%-20% uremic serum groups compared with that in normal control group,and it was the highest in 10% uremic serum groups.CTGF gene expression was increased significantly in molecular weight >10 000 D and 5 000-10 000 D groups,and the highest was in >10 000 D group,but it was no significant difference in <5 000 D group compared with that in normal control group.CTGF protein was increased in different molecular weight uremic serum groups compared with that in normal control group,and gradually increased following the increasing of uremic serum concentration and it was the highest in molecular weight >10 000 D group.CONCLUSION:Human renal tubulointerstitial fibrosis was accelerated significantly by uremic toxin,especially molecular weight >10 000 D uremic toxin through promoting the gene and protein expression of CTGF in renal tubular epithelial cells in patients with chronic renal failure.     

Clinical efficacy and safety of recombinant canine erythropoietin in dogs with anemia of chronic renal failure and dogs with recombinant human erythropoietin-induced red cell aplasia

The efficacy and safety of recombinant canine erythropoietin (rcEPO) therapy was evaluated in 19 dogs with anemia of chronic renal failure (group 1) and 6 dogs with chronic renal failure and recombinant human erythropoietin (rhEPO)-induced red cell aplasia (group 2). Hematocrit (Hct) and absolute reticulocyte count (ARC) were monitored weekly for the first 8 weeks, CBC (including ARC) and serum iron profiles were evaluated monthly, and serum biochemical analyses were performed every 2 months for 6 (group 2) to 12 (group 1) months. For group 1 dogs, median Hct and ARC increased significantly during the 1st week of rcEPO treatment, and median Hct was sustained at >35% after week 5. In contrast, median Hct and ARC for group 2 did not change significantly with rcEPO treatment, even with doses greater than those used in group 1. Nevertheless, 2 (33%) of the 6 dogs in group 2 developed erythroid hyperplasia, reticulocytosis, and increases in Hct with rcEPO treatment. Although median systolic blood pressure did not change significantly in either group, 5 dogs developed systolic blood pressures > or = 180 mm Hg during the study. Appetite and energy level improved in most group 1 dogs with increases in Hct. Recombinant cEPO stimulated erythrocyte production in dogs with nonregenerative anemia secondary to chronic renal failure without causing the profound erythroid hypoplasia that can occur in rhEPO-treated dogs. Unfortunately, rcEPO was not as effective in restoring erythrocyte production in dogs that had previously developed rhEPO-induced red cell aplasia.     

Quantitative assessment of urea generation and elimination in healthy dogs and in dogs with chronic kidney disease

Background: Kinetic assessment of urea, the main end product of protein metabolism, could serve to assess protein catabolism in dogs with chronic kidney disease (CKD). Protein malnutrition and catabolism are poorly documented in CKD and they often are neglected clinically because of a lack of appropriate evaluation tools. Hypothesis: Generation and excretion of urea are altered in dogs with CKD. Animals: Nine dogs with spontaneous CKD (IRIS stages 2–4) and 5 healthy research dogs. Methods: Endogenous renal clearance (Cl_renal) of urea and creatinine was measured first. Exogenous plasma clearance (Cl_plasma, total body clearance) of the 2 markers then was determined by an IV infusion of urea (250–1,000 mg/kg over 20 minutes) and an IV bolus of creatinine (40 mg/kg). Extrarenal clearance (Cl_extra) was defined as the difference between Cl_plasma and Cl_renal. Endogenous urea generation was computed assuming steady‐state conditions. Results: Median Cl_renal and Cl_extra of urea were 2.17 and 0.21 mL/min/kg in healthy dogs and 0.37 and 0.28 mL/min/kg in CKD dogs. The proportion of urea cleared by extrarenal route was markedly higher in dogs with glomerular filtration rate <1 mL/kg/min than in normal dogs, reaching up to 85% of the total clearance. A comparable pattern was observed for creatinine excretion, except in 1 dog, Cl_extra remained <20% of Cl_plasma. Conclusion: Extrarenal pathways of urea excretion are predominant in dogs with advanced CKD and justify exploring adjunctive therapies based on enteric nitrogen excretion in dogs. A trend toward increased urea generation may indicate increased catabolism in advanced CKD.     

Effect of miR-22 secreted by macrophage exosomes on cardiomyocyte autophagy under uremic toxin stimulation

AIM To investigate the effect of microRNA-22 (miR-22) secreted by macrophage exosomes on the autophagy of H9c2 cardiomyocytes under uremic toxin stimulation. METHODS The macrophage-derived exosomes stimulated by indoxyl sulfate (IS) were collected and co-cultured with H9c2 cells. The levels of miR-22 in the macrophages, macrophage-derived exosomes and H9c2 cells were detected by RT-qPCR. The viability of H9c2 cells was measured by CCK-8 assay. The expression of exosome surface marker protein CD63 and autophagy-related proteins LC3 and P62 was determined by Western blot. RESULTS Under IS stimulation, the expression of exosome surface marker protein CD63 in the macrophages was significantly higher than that in control group (P<0.05), and the levels of miR-22 in the macrophages and macrophage-derived exosomes were significantly increased (P<0.01). With the increase in macrophage exosome concentration, the viability of H9c2 cells was decreased gradually (P<0.05), and the stimulation of macrophage exosomes reduced P62 expression and promoted the conversion of LC3-I to LC3-II in a dose-dependent manner (P<0.05). Macrophage-derived exosomes increased the ratio of LC3-II to LC3-I but decreased P62 protein expression in the H9c2 cells transfected with miR-22 mimic compared with the cells transfected with corresponding negative control miRNAs (P<0.05). However, miR-22 inhibitor yielded contrasting results. CONCLUSION IS-stimulated macrophages increase expression of miR-22 in cardiomyocytes through exosomes, and promote autophagy of the cardiomyocytes.     

Relationship between Th17/Treg cells and microinflammatory state in uremia patients

AIM: To investigate the changes of T-helper 17(Th17) and regulatory T (Treg) cells in uremic patients by determining the mRNA expression of retinoid-related orphan receptor γt (RORγt)and forkhead box P3 protein (Foxp3), and to observe the correlation with the status of microinflammation. METHODS: Thirty uremia patients enrolled in our blood purification center during July 2010 to July 2011 were selected in the study and divided into maintaining hemodialysis (MHD) group (n=20) and chronic kidney disease (CKD) group (n=10). Twenty matched healthy volunteers served as controls. Serum samples were collected from all the patients in the morning. The mRNA expression of RORγt and Foxp3,plasma interleukin (IL)-6, IL-10 and IL-17 in these patients were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The content of high-sensitivity C-reactive protein (hs-CRP) was detected by immunoturbidimetric assay (ITA). The relationship between the ratio of Treg/Th17 cells and the status of microinflammation was also analyzed. RESULTS: RORγt mRNA, IL-6 and IL-17 were significantly higher in uremia groups than those in control group (P<0.05), while mRNA expression of Foxp3 and the level of plasma IL-10 were significantly lower in uremia group than those in control group (P<0.05). The ratio of RORγt/Foxp3 mRMA was higher in uremia group than that in control group (P<0.05), and no significant difference between MHD group and CKD group was observed. The concentration of hs-CRP, which was higher than 3 mg/L, was significantly higher in uremia group than that in control group (P<0.05). The level of hs-CRP had positive correlation with RORγt, and negative correlation with Foxp3. CONCLUSION: The ratio of Th17 Treg is abnormal in uremia patients. The disequilibrium of Th17 and Treg cells has a close relationship with the status of microinflammation.     

Effect of pioglitazone on balance of regulatory and effector T cells of uremic apolipoprotein E knockout mice in vitro

AIM: To investigate the effects of pioglitazone on the quantity and function-related factors of regulatory and effector T cells (Treg and Teff ) of uremic apolipoprotein E knockout mice in vitro with or without the stimulation of atherosclerotic plaque-specific antigen oxidized low-density lipoprotein (oxLDL). METHODS: Uremic apolipoprotein E knockout mouse model was established by 2-step surgical procedure. After intervention with different concentrations (2 μmol/L and 20 μmol/L) of pioglitazone and PPARγ antagonist GW9662 (5 μmol/L) on splenocytes of uremic mice for 12 h in the presence or absence of oxLDL (2 mg/L), the levels of CD4⁺ CD25⁺ Foxp3⁺ Treg and IFNγ⁺ CD4⁺ Teff were determined by flow cytometry. The mRNA expressions of Foxp3 and IFNγ was detected by real-time fluorescent quantitative PCR. RESULTS: In vitro, oxLDL induced a Treg/Teff imbalance in splenocytes from the uremic mice. Pioglitazone upregulated the level of Treg and mRNA expression of Foxp3 in the presence of oxLDL, which was not antagonized by GW9662. Meanwhile, pioglitazone downregulated the level of Teff and mRNA expression of IFNγ in the presence or absence of oxLDL, which was reversed by GW9662. CONCLUSION: oxLDL induces a Treg/Teff imbalance in uremic apolipoprotein E knockout mice. Pioglitazone modulates the Treg/Teff imbalance probably through PPARγ-independent and-dependent mechanisms.