Objective To explore the effect of sulodexide on renal injury and podocalyxin expression of podocytes in STZ diabetic desoxycorticosterone acetate(DOCA)-hypertensive rats. Methods Wistar rats were subjected to subcutaneous injection of streptozotocin(STZ), followed by uninephrectomy and subcutaneous administration of DOCA. Diabetic and hypertensive rats were randomly allocated to treatment with sulodexide or a combination of sulodexide and telmisartan for 8 weeks. Blood pressure (BP), 24-hour urinary albumin were measured every 2 weeks. Blood and urinary samples were collected to detect biochemical indexes of plasma and urinary β-acetyl-β-D-glucosaminidase(NAG) at the end of the study. Immunohistochemistry (IHC), RT-PCR and Western blot were performed to examine the expression and distribution of podocalyxin. Results STZ+DOCA-treated rats progressively developed hypertension, albuminuria and hyperglycemia. Hyperlipidemia and hypoinsulinemia were found in diabetic and hypertensive rats compared with controls. Albuminuia was significantly reduced in sulodexide group at week 8 and sulodexide plus telmisartan group at week 6 and week 8. Blood pressure decreased in sulodexide plus telmisartan group. No significant effects on lipid and glucose metabolism were observed in all treated groups. Histopathological index increased in STZ+DOCA-treated rats, but was significantly lower in sulodexide group as well as sulodexide plus telmisartan group. The number of podocytes on glomerular cross-section of the four groups were comparable. Segmental loss and down-regulation of podocalyxin were detected in STZ+DOCA-treated rats, which were greatly attenuated by sulodexinde, meanwhile, combination treatment preserved more podocalyxin expression in glomeruli than sulodexide monotherapy. Conclusion Sulodexide effectively reduces albuminuria, prevents loss of podocyte podocalyxin and alleviates renal damage in STZ diabetic DOCA-hypertensive rats.

objective To assess the effect of aldosterone on the production of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9）and collagen Ⅳ in culture supernatants of podocytes and the possible molecular mechanisms involved in the influence of aldosterone on the synthesis and degradation of extracellular matrix produced by podocytes． Methods Podocytes were treated with aldosterone at the concentration of 10-11, 10-9, 10-7 mol/L respectively. Cultured podocytes were examined at 24, 48 and 72 hours respectively. Spironolactone, a receptor antagonist of aldosterone, was added to observe the blocking effect on aldosterone. An inhibitor of TGF-β1 receptor was used to determine whether the effect of aldosterone on podocytes were mediated through TGF-β1 system. The enzymatic activities of MMP-2 and MMP-9 were assayed by gelatin zymography. Collagen Ⅳ α5 chain and TGF-β1 proteins released into culture supernatants were assessed by Western blot and ELISA analysis. The adhesion rate of podocytes was monitored by flow cytometry. Results Aldosterone increased the activities of MMP-2 and MMP-9 in a dose- and time-dependent manner(P<0.05). Aldosterone decreased the level of collagen Ⅳ α5 chain protein in culture supernatants(P<0.05). Meanwhile, the expression of TGF-β1 was also increased (P<0.05). Spironolactone completely abolished the above-mentioned changes(P<0.05). Blockage of TGF-β1 signaling with SB431542 prevented the aldosterone-induced upregulation of MMP-2 and MMP-9 as well as the downregulation of the collagen IV α5 chain protein and the adhesion rate of podocytes (P<0.05). Conclusions Aldosterone increases the activities of MMP-2 and MMP-9 but decreases the expression of collagen IV α5 chain and the adhension rate of podocytes possibly via TGF-β1 signaling pathway. Such alterations may contribute to glomerular podocyte injury associated with the GBM abnormality caused by the imbalance between matrix synthesis and degradation.

Objective To observe the effects of puromycin aminonucleoside (PAN) and dexamethasone (DEX) on the expression and distribution of podocin in vitro, and to explore the possible mechanism of DEX in improving proteinuria. Methods Mouse podocyte cells (MPCs) in control group were cultured with RPMI-1640 plus 0.02% DMSO, and were subjected to PAN treatment alone (PAN group) or PAN plus DEX (DEX group) for 8, 24,48 hours respectively. The podocyte morphology was observed by phase-contrast microscope, and was analyzed by Image J. The distribution, mRNA and protein expression of podocin were detected by indirect immunocytofluorescence, semi-quantitative RT-PCR and Western blot, respectively. Results The well-developed arborization and interconnection of podocytes were found in control group. PAN treatment led to significant shrinkage of podocytes with decreased distribution at 43% of control group at 8 h, 10% at 24 h and 5.7% at 48 h (P<0.01), respectively, together with podocyte foot process retraction as well as effacement and loss of cell contact. RT-PCR revealed podocin mRNA expression prone to decrease. Western blot showed podocin protein expression was significantly decreased and immunocytochemistry revealed podocin expression was disappeared in the cellular membrane after PAN treatment. DEX significantly prevented the shrinkage of podcytes, with decreased area at 43.9% of control at 8 h, 26.2% at 24 h and 29.6% at 48 h (P<0.05), respectively, and up-regulated the mRNA and protein expression of podocin at 48 h (P<0.05). The abnormal distribution of podocin was also alleviated by DEX. Conclusion DEX exerts a direct action on podocyte via stabilizing mRNA, protein expression and distribution of podocin, which may be associated with the improvement of proteinuria.

To investigate the lesion of podocyte and the expression of renal integrin-linked kinase (ILK) in a diet-induced hyperlipidemic model of rats. Methods Thirty-six 6-8 week-old female Wistar rats were randomly assigned into three groups, high-fat diet group, simvastatin group (fed with high-fat diet plus 10 mg&#8226;kg－1&#8226;d－1 simvastatin), and control group. Six rats in each group were sacrificed at the 4th and 10th week respectively. The levels of serum cholesterol and triglyceride were determined by enzymic method. The morphology of podocyte was observed and photographed with electron microscope. The expression of ILK mRNA was determined by RT-PCR. The expressions of ILK and desmin protein were determined by Western blot analysis. The distribution of ILK in renal tissue was examined by immunohistochemical staining. Results Levels of serum cholesterol and triglyceride, the expression of desmin, renal ILK mRNA and protein, as well as the foot process effacement were significantly up-regulated in both high-fat diet group and simvastatin group as compared with control group. However, all of the above parameters were ameliorated in simvastatin group as compared with high-fat diet group (P<0.01). ILK was mainly expressed in glomerular podocytes and renal tubular cells by immunohistochemical staining, and its change was similar to the results detected by Western blot analysis in each group. A positive correlation was found between ILK protein expression and desmin expression in renal tissue (r=0.93107, R2=0.8669, P<0.01). Conclusions Podocyte lesion can be induced by high-fat diet, which is correlated with over-expression of renal ILK. Simvastatin may play an important role in protecting against podocyte injury induced by hyperlipoidemia, properly through down-regulating ILK expression in renal tissue.

Objective To examine mutations in the WT1 and PLCE1 gene in three Chinese families with autosomal recessive steroid-resistant nephrotic syndrome (SRNS) once mutations in NPHS2 had been excluded. Methods Peripheral blood samples were collected for genetic analysis from three probands of three Chinese families and their parents, and two probands’ siblings, and 50 adult volunteers with normal urinalysis. Genomic DNA was isolated from peripheral blood leucocytes. Ten exons and exon-intron boundaries of WT1, and 31 exons and exon-intron boundaries of PLCE1 were amplified by polymerase chain reaction (PCR). Mutational analysis was performed by DNA sequencing directly and RFLP (restriction fragment length polymorphism) and/or PCR. Results No mutation in both WT1 and PLCE1 was identified in three probands from three Chinese families with autosomal recessive SRNS. However, three variants of WT1, 126C>T, IVS5-64A>G and 903A>G, and 13 variants of PLCE1, -134A>G, 810T>C, 960G>A, IVS11-28C>G, IVS15+26A>C, 4724G>C, IVS20+40C>T, IVS21+64G>A, IVS22-26T>A, 5320C>T, 5780A>G, IVS27+24A>G and IVS31+48_49insT, were detected in three probands and some controls, indicating that all these variants were gene polymorphisms. WT1 polymorphism IVS5-64A>G, and PLCE1 polymorphism IVS22-26T>A were novel. Conclusion All the encoding exons and exon-intron boundaries of both WT1 and PLCE1 in three probands are examined, and no causative mutations in WT1 and PLCE1 are found, suggesting that mutation in WT1 and PLCE1 genes is not a major cause of the Chinese families with autosomal recessive SRNS.

Objective To investigate the clinical features and the effect of gender differences on phenotype of Gitelman syndrome (GS) patients. Methods Clinical features and biochemical parameters were compared and analyzed to look for correlation between male and female GS patients. Results More male patients suffered from nocturia than female patients （P < 0.05）, and there were no statistical differences in other clinical features between males and females. The level of serum creatinine was higher in male patients than that in female ones [（82.7±43.3） μmol/L vs （58.7±12.7） μmol/L], but estimated glomerular filtration rate was similar [（143.0±48.4） ml&#8226;min-1&#8226;（1.73 m2）-1 vs (138.0±38.9) ml&#8226;min-1&#8226;（1.73 m2）-1] between male patients and female patients. The urinary potassium and chloride excretion fraction were higher in male group than those in female group (33.0%±22.9% vs 17.0%±4.7%; 2.30%±1.59% vs 1.23%±0.39%, P< 0.05, respectively). Statistical differences were not observed in other laboratory parameters. Three patients with impaired renal function were all male. Conclusions More male patients suffer from nocturia than female patients. Male patients seem to be prone to impaired renal function. It is speculated that different density of sodium-chloride cotransporter in renal tubule may account for gender differences.

Objective To investigate the effect of swifch from cyclosporine to FK506 on renal allograft outcome after initial acute rejection. Methods Clinical outcome of patients who experienced first acute rejection episode were retrospectively analyzed. After initial acute rejection, 23 patients were switched to FK506-based immunosuppression, and 63 patients continued CsA-based immunosuppression. Demographic data, lipid, serum creatinine, uric acid, incidence of recurrent acute rejection and graft survival were analyzed and compared. Results During one year after anti-rejection therapy, incidence of biopsy-proved recurrent rejection events was significantly lower with FK506 therapy (1/23, 4.35%) compared with CsA therapy (16/63, 25.40%) (P=0.033). 5-year graft survival rate of FK506-based immunosuppression group was higher than that of CsA-based immunosuppression group(100.0% vs 81.4%). Serum uric acid level of FK506-based immunosuppression group from 24 months to 36 months after initial rejection were significantly lower than that of CsA-based immunosuppression group [(265.5±147.9) μmol/L, (245.8±88.9) μmol/L vs (428.5±119.3) μmol/L, (441.2±125.3) μmol/L, P<0.01, respectively]. Conclusion Conversion to FK506 therapy can significantly reduce recurrent rejection episode, and decreasing serum uric acid level provides long-term benefits to graft survival.

Objective To evaluate the protective effects of mycophenolate mofetil (MMF) on the kidneys of diabetic rats and elucidate the associated mechanisms. Methods Wistar rats were divided into three groups: normal control rats, diabetic rats, and diabetic rats received MMF(15 mg&#8226;kg－1&#8226;d－1). Eight weeks later, 24 h urinary albumin excretion, creatine clearance rate(Ccr), and blood glucose were measured, and kidney pathology was observed. Inmmunohistochemistry and RT-PCR were used to analyze the expression of matrix metalloproteinase-9(MMP-9) and transforming growth factor β1 (TGF-β1 ). Results As compared with normal control rats, the 24 h urinary albumin excretion [(26.80±0.82) mg vs (6.64±1.42) mg], blood glucose[(22.18±3.36) mmol/L vs (6.40±0.87) mmol/L], Ccr[(0.220±0.380) ml/min vs(0.098±0.015) ml/min] of the diabetic rats were rised remarkbably. The 24 h urinary albumin excretion [(16.17±1.15) mg] and Ccr [(0.207±0.377) ml/min] of the diabetic rats received MMF were lower as compared to diabetic rats(P＜0.05). MMP-9 in renal tissue of normal control rats was mainly expressed in glomerular mesangial cells and renal tubular epithelial cells. Such MMP-9 expression was weak in diabetic rats and improved in the diabetic rats received MMF. There were significant differences among 3 groups. The expression of TGF-β1 was on the contrary. Conclusion Mycophenolate mofetil decreases 24 h urinary albumin, Ccr and glomerular volume, which may be associated with the increase of MMP-9, the decrease of TGF-β1 expression and extracellular matrix deposition in renal tissue.

Objective To investigate the role of recombinant human interleukin 6 (rhIL-6) in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells (HUASMC), and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with (treatment groups) or without(control group) rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control cells and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group (treated with rhIL-6 10 μg/L and 50 μg/L, respectively) was determined calorimetrically by the o-cresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase (BAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP2) and osteoprotegerin(OPG) were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretic mobility shift assays(EMSA) was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK(SB203580) and PKC(DHC) after 6 hours stimulation by rhIL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition. Compared with control group, rhIL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time(12 and 24 hours), and of OPN and OPG at later hours(24 and 72 hours). RhIL-6 still induced an increasing of binding activity of Cbfα1, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenic transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical studies. The role of IL-6 in HUASMCs may partially achieved through the PKC cell signal transduction way.

Objective To build an antibody microarray based on direct labeling strategy for microalbuminuria measurement, and evaluate it’s technical potentiality for clinical application. Methods Urine samples of diabetic patients were collected. Antibody microarrays were constructed by preparation of array support, array fabrication, then protein assay and data analysis were performed. Procedure conditions for each step especially the labeling of samples were optimized. The set-ups were evaluated in terms of sensitivity, specificity and reproducibility. Urinary albumin excretion in the samples was detected by fabricated protein array, which was compared to that detected with immunoturbidimetry. Results The signal intensity was best when protein quality ratio of pure albumin or urine sample against NHS-biotin was 2:1. A calibration curve with a correlation coefficient of 0.9995 was established. The lower limit of detection was 0.0617 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs as 6.78%－9.22% and 3.35%－7.59%, respectively. Compared with the immunoturbidimetry, the antibody microarray was able to detect the extremely lower grade albumin in urine samples. The correlation coefficient of the results obtained by the two methods was 0.9199 (P＜0.01). Conclusion An antibody microarray based on direct labeling strategy for microalbuminuria measurement is successfully established, which is comparable to immunoturbidimetry in its accuracy and will have great potential for clinical use with its high throughput, sensitivity, specifity and reproducibility.