Objective To elucidate whether focal segmental glomerulosclerosis (FSGS) is a secondary development of the COL4-linked thin basement membrane nephropathy (TBMN) or the primary FSGS produces thin glomerular basement membrane(GBM). Methods The family members presented microscopic hematuria, increasing proteinuria with the years and a dual pathological diagnosis of FSGS and TBMN was made in the proband. DNA linkage analysis at locus 2q36-37 that contains the COL4A3/COL4A4 genes was performed with polymorphic microsatellite markers D2S434, D2S279, D2S1370, D2S256 and D2S427. Haplotypes were constructed at the COL4A3/COL4A4 loci for affected and unaffected family members. All exons of COL4A3 and COL4A4 genes were screened for mutations in the proband. Mutation screening was also performed for NPHS1, NPHS2, CD2AP, WT1, TRPC6 and ACTN4 to exclude familial FSGS. Mutation or polymorphism found in the family were examined in 50 healthy controls. Results In this family hematuria segregated with the 55224 haplotype at the COL4A3/COL4A4 locus. G to A substitution at nucleotide 1214 resulting in an glycine being replaced by glutamate (G405E) was demonstrated for the first time in exon 20 of COL4A4 gene. G405E was present in all four members of the family with hematuria but not in the seven unaffected family members nor in 50 healthy controls. A novel polymorphism segregating with hematuria (IVS1-4C>T in exon 2 of COL4A3) was also found which was only present in all four affected family members but not in the seven unaffected family members. No mutations were demonstrated in FSGS associated genes, however, a novel SNP (R268Q), which distributed with the disease incompletely, was described in the NPHS1 gene coding nephrin, the podocyte slit diaphragm protein. Conclusions In this family, FSGS occurres on the basis of TBMN. Maybe the particular COL4A3/COL4A4 mutation and polymorphism work together to develop proteinuria and eventually leading to FSGS. But whether the mutation and the polymorphism segregating with the disease predispose to develop FSGS in TBMN patients is required further study.

Objective To investigate the effects of CD4+CD25high regulatory T cells (Treg) and the imbalance of helper T lymphocyte subsets(Th1/Th2) on the immunological mechanism of IgA nephropathy (IgAN) patients. Methods The percentage of Treg and helper T cells subpopulation (Th1/Th2) in the peripheral blood of IgAN patients and healthy controls was examined by flow cytometry. The FOXP3 expression was detected through intracellular staining. The correlation of Treg or Th1/Th2 with clinical parameters of IgAN was analyzed by Spearman or Pearson rank correlation test. Results The percentages of Treg and Th2 cells were significantly higher in peripheral blood of IgAN patients compared to that of healthy controls[Treg(2.14±0.82）％ vs（1.59±0.53)%, Th2（2.57±0.72）％ vs（1.81±1.10）％, all P＜0.05]. Th1/Th2 ratio was significantly reduced in IgAN patients （5.75±1.89 vs 12.73±9.79， P＜0.05）. The percentage of circulating Treg cells was positively correlated with serum IgA concentration (r=0.397, P＜0.05), and was negatively correlated with eGFR（r=－0.376, P＜0.05）. The percentage of circulating Th2 cells was positively correlated with serum IgA（r=0.468, P＜0.05）. Conclusions There is a disorder of T lymphocyte population in the peripheral blood of IgAN patients. The increased Treg and Th2 cells may play an important role in the pathogenesis of IgAN.

Objective To assess the value of blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) in diagnosis and prediction of early acute renal transplant rejection. Methods BOLD-MRI was performed in a cohort of 103 patients undergoing cadaver renal transplantation between Dec 2005 and March 2007. Among them, 82 recipients had normal renal function, 21 had biopsy-proved acute rejection. R2* (1/s) measurements were obtained in the medulla and cortex of grafted kidneys. Results R2* values of the medulla were significantly lower in the acute rejection group [R2*=(14.02±2.68)/s] than that in the normally functioning transplants group [R2*=(16.66±2.82)/s], the difference between these two groups was significant (P<0.01); ROC curve analyses suggested that medullary MR2* values could accurately identify acute rejection in the early post-transplantation period. In the normal functioning transplant group, those with lower medullary R2* values (MR2*<14.9/s, n=23) had higher acute rejection rates than those with higher medullary R2* values (MR2*>14.9/s, n=59) in the first 6 months following transplantation, but the difference between these two groups was not significant (17.39％ vs 8.47％, P=0.259). Conclusions Mean R2* values in the medullary regions of grafted kidneys with BOLD-MRI may be a non-invasive diadynamic criteria with good sensitivity and specificity, and may be a valuable predictor of early acute renal transplant rejection.

Objective To investigate the clinical features of pneumocystis pneumonia (PCP) in patients with chronic kidney disease. Methods Clinial data of 21 cases of the primary and secondary kidney diseases complicated with PCP, excluding renal transplantation, were analyzed retrospectively. Results Twenty-one cases consisted of 6 cases of primary renal diseases and 15 cases of secondary renal diseases. Twenty patients（95.2%） were receiving immunosuppressive therapy at the PCP onset. Main manifestations were fever, progressive dyspnea, cough with no or seldom sputum. Twenty patients presented obvious hypoxemia and 12 of them were type I respiratory failure. X-ray and CT imaging of 20 patients revealed diffuse pulmonary interstitial shadows or ground glass opacities in both lungs. All the patients were treated with trimethoprim-sulfamethoxazole. Eleven patients died accounting for 52.3%. Compared with the survivors, elder age (60.91±15.08 vs 44.50±14.83, P<0.05), lower blood oxygen pressure at onset [(48.11±19.05)mm Hg vs (65.91±13.13)mm Hg, P<0.01], higher percentage of respirator application and other secondary lung infection were found in dead patients. No PCP relapsed after average 16-month follow-up in the survival patients. Conclusions PCP is a severe complication with high mortality during immunosuppressive therapy in patients with chronic renal disease. Early diagnosis and proper treatment are important to improve prognosis.

Objective To explore the mechanism of protecting cells from ischemic reperfusion injury by constructing specific small interference RNA(siRNA) to inhibit Na+-H+ exchanger-1 (NHE-1) expression in human renal tubular epithelial cell (HKC). Methods The siRNA was designed and synthesized based on human NHE-1 complete sequence, and was transfected into HKC. The irrespective siRNA transfected group was used as control. The cells were treated with 10 μmol/L antimycin A to induce ischemia and anoxyaemia environment. NHE-1 expression was examined by RT-PCR and Western blot. The intracellular pH (pHi), Ca2+ or Na+ concentrations were detected by BCECF/AM, Fluo-3/AM and SBFI-AM, respectively, combining with laser confocal assay system. Nucleic morphology was determined by Hoechst 33342. Cellular apoptosis was examined by Annexin V/PI staining and flow cytometry. Fluorescent probe JC-1 was used to detect the change of mitochondrial transmembrane potential. Results The specific siRNA could efficiently inhibit NHE-1 expression in HKC. Compared with the irrespective siRNA transfected group, the mRNA and protein expression of NHE-1 was significantly down-regulated in NHE-1 siRNA transfected group (all P<0.05). After treatment with antimycin A, the mRNA and protein expression of NHE-1 was significantly up-regulated in both groups, however, it was less than that in irrespective siRNA transfected group. At the same time, the ratio of apoptosis decreased （8.9%±2.9% vs 18.8%±3.2%, 17.4%±3.6％, P<0.05） and mitochondrial transmembrane potential rose significantly in NHE-1 siRNA transfected group as compared to irrespective siRNA transfected group and antimycin A group. The intracellular Na+, H+ and Ca2+ concentrations increased in NHE-1 siRNA transfected group treated with antimycin A, but their levels were lower than those in irrespective siRNA transfected group with the same treatment (P<0.05). Conclusions The synthesized siRNA can inhibit the expression of NHE-1 and can protect HKC from ischemia reperfusion injury induced by antimycin A. The mechanism might be via suppressing the expression of NHE-1 to delay intracelluar Na+ accumulation, attenuate intracellular Ca2+ overloading, and inhibit the decrease of mitochondrion transmembrane potential and reduce cellular apoptosis.

Objective To investigate the influence of uremic serum on the monocyte chemoattractant protein 1(MCP-1) expression of human umbilical vascular endothelial cells (HUVECs) in vitro and the effect of up-regulation of heme oxygenase 1 (HO-1) on the synthesis of MCP-1 of HUVECs in uremic milieu. Methods HUVECs were incubated to confluence and then preincubated with hemin and/or protoporphyrin zinc IX（ZnPP）for 6 hours. The cultures were subsequently incubated with M199 cell medium containing 10% serum of healthy people or with medium containing 10% serum of maintenance hemodialysis (MHD) patients. HO-1 protein and mRNA expression was detected by immunohistochemistry and semi-quantitative RT-PCR. MCP-1 mRNA expression was measured by semi-quantitative RT-PCR, and MCP-1 protein was quantified by ELISA. Results Up-regulated expression of MCP-1 mRNA and protein was detected in HUVECs incubated with medium containing 10% serum of MHD patients. The protein synthesis was 2.95 folds of the control. Hemin induced expression of HO-1 mRNA and protein, and concurrently inhibited the up-regulated MCP-1 expression induced by uremic serum. Such effects of hemin could be blocked by ZnPP. Conclusions Uremic serum induces the expression of MCP-1 in HUVECs. Up-regulated expresson of endothelial HO-1 induced by hemin inhibits the enhancement of MCP-1 synthesis. HO-1 may be beneficial to the alleviation of endothelial cell injury in uremic milieu.

Objective To investigate the relationship between high-glucose-induced fibronectin(FN) expression and up-regulation of integrin-linked kinase(ILK) in human kidney tubular epithelial cells(HKC) and kidney of CD-1 mice. Methods Cultured human kidney tubular epithelial cells and streptozotocin (STZ)-induced diabetic model of CD-1 mice were enrolled in this study. Western blot was used to detect the expression of FN and ILK. The kinase dead ILK plasmid (pCMV-kdILK) were transferred to HKC. Results Four weeks after injection of STZ, CD-1 mice had higher blood glucose level as compared to the control [(20.3±2.7) mmol/L vs (6.1±1.4) mmol/L, P<0.01]. Meanwhile, expression of FN and ILK was significantly increased in diabetic mice as compared to the control(P<0.01). There was positive correlation between the expression of FN and ILK(r=0.899, P<0.01). High-glucose could up-regulate FN and ILK expression in cultured HKC in a time- and dose-dependent manner. Blockage of ILK activation by pCMV-kdILK abrogated high-glucose-incuced FN expression in HKC. Conclusions High-glucose can induce FN expression through up-regulating ILK expression. Blockage of ILK activation abrogates this effect.

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 lipofectamine 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 effects of total anthraquinone in rheum on aquaporin 2 and aquaporin 4 expression in rat kidney and explore its diuresis mechanism. Methods Thirty-two SD rats were randomly divided into control group, low dose group, medium dose group and high dose group. Total anthraquinone in rheum was administered to rats at different doses. Urinary volume of 24 h, Na+ concentration and osmolality were detected. Rats were sacrificed 5 days later. Blood samples were taken from the abdominal aorta to detect blood biochemical indicators. Kidneys of rats were removed to detect AQP2, AQP4 expression through immunohistochemistry, Western blot, and RT-PCR. Results Compared with control group, there were significantly increased 24 h urine output of rats in medium and high dose group[(16.21±1.96), (18.16±1.8) ml vs（13.85±1.25）ml, P＜0.05]. 24 h urine output in low-dose group did not change significantly. AQP2 protein and mRNA expression were significantly decreased in rats’ kidneys of medium and high dose group(P＜0.01)， The AQP4 protein and mRNA expression was significantly down-regulated in high dose group (P＜0.01). In medium does group, the AQP4 protein expression was down-regulated(P＜0.01), without significant decrease in the mRNA expression. Protein and mRNA expression of AQP2 and AQP4 did not significantly change in low dose group. Conclusion Total anthraquinone in rheum can reduce the expression level of AQP2 and AQP4 in rat kidney, which is probably one mechanism of diuresis caused by rheum.