Objective To make sure the effect of dialysate composition on HPMCs.Methods Cell strains were subculturing.There are six groups in this experiment: Group 1(control);group 2(4.25% Glucose);group 3(1.75mmol/L Ca~(2+));group 4(1.25 mmol/L Ca~(2+));group 5(4.25% Glucose+1.75mmol/L Ca~(2+));group 6(4.25% Glucose+1.25mmol/L Ca~(2+)).The capacity of proliferation of HPMCs was assessed by MTT assay. Results Proliferation of HPMCs was inhibited in 4.25% glucose group in time dependence.Ca~(2+) induce proliferation of HPMCs,however,no effect on proliferation of HPMCs exists in different Ca~(2+) group.Conclusion High glucose can inhibit cell proliferation;Ca~(2+)(1.75mmol/L,1.25mmol/L) can promote the proliferation and 1.25mmol/L is better.

Objective To investigate the roles of TLR3 pathway activiated by polyI:C in proliferation and apoptosis of multiple myeloma (MM) RPMI8226 cell line.Methods RPMI8226 cells were cultured in RPMI 1640 with different dose of polyl:C.Cells were collected in different time.Proliferation and apoptosis were detected by CCK-8 kit and flow cytometry,separately.Results The proliferation of RPM18226 was inhibited by polyI:C,and it was dose and time dependent,24 h:12.30％ ±2.04％,22.50％±2.20％,37.90％ ±1.30％ ; 48 h:17.80％ ±1.52％,29.60％ ±0.85％,45.80％ ±1.68％ ;72 h:25.10％±1.01％,34.60％±1.27％,60.50％±2.08％,P＜0.05.RPMI8226 cells were incubated with 50 μg/ml,100 μg/ml and 200 μg/ml polyI:C for 48 h.Apoptotic rate were 5.60％ ±1.06％,8.71％ ±1.06％ and 13.93％ ±1.17％,P＜0.05.TLR3 and TRIF mRNA expression increased obviously and dose dependent,TLR3:1.41±0.10,2.24±0.16,4.08±0.13; TRIF:1.07±0.16,1.97±0.13,3.56±0.19,P＜0.05.Conclusion The proliferation of MM cells were inhibited by TLR3 pathway obviously,and apoptosis was induced by polyI:C.

Objective To examine the expression of IgA1 and B1a positive cells in palatine tonsils of IgA nephropathy (IgAN) patients, and to analyze the association between B1a cells and clinicopathological changes. Methods Eight patients diagnosed as IgAN by renal biopsy and 8 chronic tonsillitis patients without nephritis as control were enrolled in the study.Immunofluorescence and laser scanning confocal microscope (LSCM) were applied to observe the localization and quantitative calculation of Bla and IgA1 positive cells. Statistic analysis of the association of B1a cells with proteinuria and pathological Lee's grading was performed. Results Bla cells were mainly localized in germinal center of tonsil, and IgA1 positive cells were mainly localized in subepithelium of tonsil. Compared to control group, the percent of B1a cells and IgA1 positive cells was significantly higher in IgAN (P＜0.01). There was a positive correlation between Bla cells and IgA1 cells (P＜0.05). In IgAN, the percent of B1a cells in patients with hematuria and proteinuria was obviously higher than that of patients with hematuria only (P＜0.05). The number of Bla cells in IgAN patients with≥Lee's grade Ⅲ was significantly higher than that of those ＜ grade Ⅲ (P＜0.05). Conclusions IgA1 may be secreted by Bla cells in the tonsil of IgAN patients. The number of B1a cells is correlated with exacerbation of proteinuria and pathological severity, which may play an important role in pathogenesis of IgAN.

Objective To determine the morphology, bacteria and endotoxin content of bio-films on the inner surface of PVC tubes in hemodialysis water treatment system. Methods We dissolved biofilms of segments before and after reverse osmosis machine for bacterial count and identification. We studied biofilm structure of segments before and after reverse osmosis machine with eyes and scanning electron microscope. Biofilms of all 7 segments were dissolved for qualitative and quantitative assay of endotoxin. Results The inner surface of segment before reverse osmosis machine was homogeneously distributed with activated carbon powder deposition. The segment after reverse osmosis machine was normal. With scanning electron microscope, biofilm with successive surface and sandwich was found on the inner surface of segment before reverse osmosis machine, formed by clustering bacillus, activated carbon powder and some coccus. Bacteria of the same shape and length were found on segment after reverse osmosis machine, but fewer and looser. Bacterial culture and identification showed the former was mostly gram-negative bacillus, the latter was only a few micrococcus. Endotox-in of biofilm was between 2. 0 EU/mL and 4. 0 EU/mL. Quantitative assay showed: segment after softener (2.821 ±0. 807) EU/mL; segment after active charcoal canister(3. 635 ±0. 427) EU/ mL; segment before reverse osmosis machine (3.687 ±0.271) EU/mL; segment after reverse osmosis machine (2. 041 ±0. 295) EU/mL; exit of power pump (1. 983 ±0. 390) EU/mL; the 1st dead space (2. 373 ± 0. 535) EU/mL; and the 2nd dead space (2. 858 ± 0. 690) EU/mL. Conclusion Biofilms are found on the inner surface of segment before and after reverse osmosis machine . Endotoxin level from high to low is as follows: segment before reverse osmosis machine, segment after active charcoal canister, the 2 nd dead space, segment after softener, the 1 st dead space, segment after reverse osmosis machine, exit of power pump. The character of the bacteria and endotoxin of the biofilm can help us find better ways to control them.

Objective To investigate the role of mitochondrial respiratory chain in the hyperpermeability of human peritoneal mesothelial cells (HPMCs) induced by high glucose peritoneal glucose PDS was also added. Transmesothelial electrical resistance (TER) measurement was examined for detection of permeability damage in HPMCs. Immunostaining and Western blotting analysis were used to detect claudin-1 expression. Mitochondrial superoxide (MitoSOX) Red staining and respiratory chain complexes activities were determined for detection of mitochondrial reactive oxygen species (ROS) production and mitochondrial complexes activities. Results TER was decreased in a time- and concentration-dependent manner after culture with high glucose PDS for was also down-regulated significantly by high glucose PDS (P＜0.01). Complex Ⅲ activity was inhibited (10.8% of control, P＜0.01) accompanied with increased mitochondrial ROS generation.These changes were partially prevented by glutathione. Conclusion Mitochondrial respiratory complex Ⅲ pathway has crucial importance in maintaining TER of HPMCs, which may reveal a valuable target for novel therapies to fight hyperpermeability of peritoneum during the prolonged PD treatment.

Objective To investigate the effects of connective tissue growth factor (CTGF) siRNA delivered by pRetro-Super (PRS) retrovirus vector on extracellular matrix and VEGF expression in human peritoneal mesothelial cells (HPMC). Methods Four pairs of oligonucleotides including 64 bp DNA were designed and synthesized in vitro according to siRNA target sequence and PRS retrovirus desire.PRS-CTGF-siRNA1-4 recombinant retrovirus vectors were constructed.The recombinant retrovirus vectors containing CTGF-siRNA were transferred into PT67 packaging cell lines with lipefectamine 2000,then infected HPMC.mRNA expression was determined by semi-quantitative RT-PCR and protein expression was determined by Western blot.Results Both mRNA and protein expressions of CTGF,FN,Col I,laminin (LN) and VEGF were significantly increased in HPMC with 5 μg/L TGF-β1 stimulation (P＜0.01,respectively).CTGF,FN,Col I,LN mRNA and protein and VEGF mRNA expression stimulated by TGF-β1 were significantly decreased in HPMC infected with PRS-CTGF-siRNA1～4 retrovirus vectors (P＜0.01,respectively).The inhibitory rates on CTGF were 69.3%,22.2%,27.4% and 38.8%,respectively (P＜0.01).At the same time,there was also a significant reduction of VEGF protein expression in HPMC infected with PRS-CTGF-siRNA1 vector (P＜0.01).There was no significant difference in HPMC infected with PRS void vector. Conclusion CTGF siRNA delivered by PRS retrovirus vector can effectively inhibit the enhancement of extracellular matrix and VEGF expression stimulated by TGF-β1 in HPMC.

Objective To investigate the effect of in vitro high glucose stimulation on the expression of adiponectin receptor (adipoR) in human kidney proximal tubular cells.Methods The HK-2 cells were cultured in the low glucose DMEM culture medium containing 10% fetal bovine serum until the cells were adherent and 80% confluence. After cultured in the serum-free DMEM for 24 hours, these cells were stimulated with glucose-containing 1mg/ml, 2mg/ml, 4mg / ml, 6mg/ml, 8mg/ml serum-free DMEM for 48 hours. Then RT-PCR and western blot were used to analyze adipoR ( R1, R2) expression levels. The HK-2 cells were cultured respectively in high glucose (4mg/ml) , low glucose (1mg/ml) DMEM culture medium containing 10% fetal bovine serum to cultivate 0h, 12h, 24h, 48h, 72h, 96h, then RT - PCR was applied to analyze adipoR (R1, R2) mRNA expression levels semi-quantitatively. Results Two kinds of adiponectin receptor gene were both expressed in HK-2 cells, and the quantity of gene expression of adipoR1 (0. 63 ±0. 12) was 3. 9 times to adipoR2 (0. 16 ±0.03) , the difference was statistically significant ( P<0. 01). The different concentrations of glucose and different time of high glucose on HK-2 cells had no significant effect ( P>0. 05 ) on adipoR gene expression. Expression of adipoR 1 protein in HK-2 cells was detected by western blot, and it was not affected by glucose concentration ( P>0. 05).Conclusion adi-poR1 and adipoR2 gene were both expressed in HK-2 cells, and the adipoR1 was the major one, which suggested that adipoR1 played a more significant role in kidney disease. The expression of adipoRl/R2 of HK-2 cells was not affected by high glucose concentration.

Objective To investigate the correlation of infiltration of mast cells in kidney with renal interstitial fibrosis, expression of TGF-β1 and stem eel] factor (SCF) in rat models withprotein-overload nephropathy. Methods Sixty uninephrectomized SD rats were randomly divided into model group [intraperitoneal injections of bovine serum albumin (BSA)] and control group (intraperitoneal injections of equal volume of saline). Ten rats from both groups were sacrificed respectively at week 3, 7 and 11 after injection. 24 h urinary protein and serum biochemistry of these SD rats at the time of sacrifice were measured. The intensity of mast cell infiltration was examined by toluidine blue (TB) staining and immunohistochemistry using a monoclonal anti-MC chymase antibody. The expression of TGF-β1 and SCF was detected byimmunohistochemistry, using a monoclonal mouse anti-rat TGF-β1 antibody and a polyclonal rabbstanti-rat SCF antibody. Results Severe proteinuria was induced in the rats by BSA injectionpeaked at week 7 [(199.1±98.4) mg/d] after the BSA injection and gradually decreased until week11 [(133.7±67.8) mg/d]. Renal injury was accompanied with chymase-postitive and TB-postitive mast cell infiltration, in close proximity to areas of interstitial fibrosis. With aggravation oflesions degree, the number of mast cells increased,the difference between the modal rats and control rats was significant (P<0.05). Immunostainahle expression of SCIF and TGF-β1 was detected in tubular as well as interstitial cells, and increased with the BSA injection. The difference between the model rats and control rats was significant (P<0.05). Mast cells were positively correlated with interstitial fibrosis (r=0.772, P<0.01), expression of TGF-β1 (r=0.521, P<0.01) and SCF(r=0.916,P<0.01). Conclusions Increased infiltration of mast cells is involved in interstitial fibrosis of rats with protein-overload nephropathy. Proteinuria may attract mast cells to kidney by chemot actions of SCF,and mast cells may contribute to the development of renal fibrosis by secreting chymase and increasing expression of TGF-β1.

OBJECTIVE: To construct the cell model of epithelium to mesenchymal transition of proximal tubule cells induced by high glucose and to determine the expression of Smad anchor for receptor activation (SARA). METHODS: Protein expression of vimentin, Zona occludens-1(ZO-1), and SARA was determined by Western blot, and their mRNA expressions were detected by Real-time PCR. RESULTS: After stimulation by 30 mmol/L D-glucose, the protein and mRNA expression levels of vimentin in HK-2 cells increased in a time-dependent manner while the expression of ZO-1 was reduced significantly, especially at 48 h. Meanwhile, SARA was also decreased in a time-dependent manner. CONCLUSION High glucose can induce renal epithelium to mesenchymal transition, and SARA may be involved in this process as a protector.
Cell Differentiation ; Cells, Cultured ; Epithelial Cells ; cytology ; Glucose ; pharmacology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Kidney Tubules, Proximal ; cytology ; metabolism ; Membrane Proteins ; metabolism ; Mesoderm ; cytology ; Phosphoproteins ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; Signal Transduction ; Transforming Growth Factor beta1 ; pharmacology ; Vimentin ; metabolism ; Zonula Occludens-1 Protein

OBJECTIVE: To investigate the prevalence and distribution of chronic kidney disease(CKD) in Second Xiangya Hospital of Central South University. METHODS: We retrospectively analyzed the medical records of 3547 CKD patients (>or=14 years old) admitted to Second Xiangya Hospital for the 1st time from January 2003 to December 2008. Glomerular filtration rate (GFR) was estimated by using the MDRD equation abbreviated [eGFR=186.3*SCr-1.154*age-0.203*0.742 (for women) mL/min.1.73 m2]. Data of patients' gender, age, admission number, etiologic and functional diagnose, course of disease, blood pressure, hemoglobin, urine, renal function, blood albumin, and kidney ultra-B were collected. RESULTS: (1) The most common causes for CKD were primary glomerulonephritis(55.20%), hypertension (14.55%) and diabetes (11.78%). The composition of the causes during this 6 years was not significantly different (P>0.05). The proportion of chronic glomerulonephritis declined while that of hypertensive nephropathy and diabetic nephropathy increased as time passed. (2) Patients were respectively distributed from stage 3 to stage 5 at the following percentages: 11.59% (stage 3), 23.03% (stage 4), and 65.38% (stage 5). Middle-aged and young patients (41 years60 years) accounted for 36.88% of the CKD patients. Chronic glomerulonephritis was often seen at 21-40 and 41-60 year olds. Patients with lupus nephritis were younger, 48.35% of whom were 21-40 years old. On the contrary, patients with hypertensive nephropathy and diabetic nephropathy were much older. They were mainly seen in older than 40 year groups. (3) Obstructive nephropathy and gout nephropathy occurred mainly in men while it seemed much easier for women to suffer from lupus nephritis. (4) The majority of CKD patients was accompanied by anemia (94.28%) and hypertension (56.91%), and the incidence and degree of anemia and hypertension increased as CKD developed (P<0.005). CONCLUSION The top 3 causes of CKD are chronic glomerulonephritis, hypertensive nephropathy, and diabetic nephropathy. The proportion of hypertensive nephropathy and diabetic nephropathy is on the rise. Elderly people are the high risk group for CKD.
Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; China ; epidemiology ; Chronic Disease ; Diabetic Nephropathies ; epidemiology ; Female ; Glomerulonephritis ; epidemiology ; Humans ; Hypertension ; complications ; Kidney Diseases ; classification ; epidemiology ; etiology ; Male ; Middle Aged ; Prevalence ; Retrospective Studies ; Risk Factors ; Young Adult