Objective To elucidate the features of clinicopathology and mutation in Fabry disease complicated with thin basement membrane nephropathy (TBMN), and to investigate the kindred. Methods Data of clinicopathology and gene mutation of a female patient of Fabry disease complicated with TBMN admitted to the Department of Nephrology in our hospital were analyzed. Members of her kindred were investigated simultaneously. Results Proband was a 41-year-old Chinese woman who presented syndrome of Fabry disease and TBMN including angiokeratomas, chronic pain, tinnitus, vertigo, left ventricular hypertrophy and nephropathy as proteinuria, microscopic hematuria and hypertension. A percutaneous renal biopsy was performed on the proband, which was consistent with FSGS and vaculization of podocytes by light microscopy. Electron microscopy showed concentric lamellated inclusions in some podocytes. Diffuse thinning of glomerular basement membrane (GBM) with a mean thickness of (216±31) nm was found. The diagnosis of TBMN with suspected Fabry disease was identified. Family screening showed that her daughter had microscopic hematuria, tinnitus and neuropathic pain. One of her sisters only had microscopic hematuria. The activity of α-galacsidase A（α-Gal A）enzyme in the proband and her daughter was 33 units and 75 units respectively (the normal range is 100 to 500 units). They all carried the novel GLA mutation 1208 ins 21 bp and COL4A3 SNP c: 3627G>A(p:M1209I). While her sister who only had microscopic hematuria just carried the variant of COL4A3 gene—c: 3627G>A (p:M1209I), and had the normal activity of α-Gal A with no mutation of GLA. Conclusion TBMN should be considered in the patients of Fabry disease with the condition of benign familial hematuria.

Objective To investigate whether the combination of maintenance hemodialysis (MHD) with hemoperfusion(HP) can improve the clearance rate of middle- and large-molecule uremic toxins so as to improve the quality of life and reduce the mortality in MHD patients. Methods A prospective, randomized and controlled clinical trial was carried out. One hundred MHD patients were selected and then randomly divided into two groups after four weeks of run-in period. HD+HP group received MHD alone 2 times a week and the combined treatment of HD with HP (HD+HP) once a week, whereas HD group received MHD alone 3 times a week. The follow up lasted for mean 2 years. The primary outcome was the death of patients. Secondary end points included clinical data, leptin, high sensitive C-reactive protein (hsCRP), interleukin 6 (IL-6), β2 microglobulin (β2-MG), parathyroid hormone (PTH), tumor necrosis factor α(TNF-α) and the indexes of dimensions of Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36 Chinese Edition ). Results At the end of the two-year observation, the serum concentration of leptin, hsCRP, PTH, IL-6, β2-MG and TNF-α, systolic blood pressure(SBP), diastolic blood pressure(DBP), heart rate(HR), cardiothoracic ratio, left ventricular mass index (LVMI), EPO dose and the types of antihypertensive drugs used were lower in HD+HP group as compared to HD group (all P<0.05). HD+HP group had higher hemoglobin(Hb), ejection fraction (EF) and body mass index (BMI) (all P<0.05). No significant differences between two groups were found in terms of serum albumin (Alb), serum iron (SI), total iron binding capacity (TIBC), cardiac output (CO), Kt/V, early/atrial mitral inflow velocities (E/A) (all P>0.05). Besides, the SF-36 indicated that the total score of overall dimensions in HD+HP group was higher (P<0.05) and the quality of life of HD+HP group was evidently better as compared to HD group. The Kaplan-Meier survival curves for the 2-year observation period showed that patients in HD+HP group had obvious survival advantage, while Log-rank test results showed P<0.05. No serious adverse incidents occurred during the HD+HP treatment. Conclusion HD+HP is superior to HD in eliminating regularly middle- and large-molecules uremic toxins and has a potential role in improving the quality of life and survival rate of MHD patients.

Objective To investigate the effects of repeated low dose intravenous infusion of low molecular weight iron dextran and iron sucrose on oxidative stress in chronic renal failure(CRF) rats. Methods CRF model was established by 5/6 subtotal nephrectomy (5/6 Nx). Four weeks after removing the right kidney, successful rats were randomly divided into low molecular weight iron dextran group, sucrose iron group and CRF control group. The sham group was established simultaneously. The dose of iron administrated in each rat was similar in iron dextran group and sucrose iron group. There were 6 rats in each group. Animals were observed for 6 weeks，then the blood, urine and renal tissue samples were collected, and indexes of renal function, anemia, iron status and oxidative stress were investigated. Results The hemoglobulin (Hb) level in iron groups was significantly higher as compared to control group (P<0.05) but was not significantly different between two iron groups. The levels of serum iron, ferritin and saturation rate of transferring (TS) were obviously lower in control group as compared to sham group (P<0.05). Levels of above 3 indexes were significantly higher in two iron groups as compared to control group (P<0.05), but were not significantly different between two iron groups. Concentration of plasma advanced oxidation protein products (AOPP) was obviously higher in two iron groups than that in control group [(127.84±21.19) μmol/L, (134.21±29.38) μmol/L vs (81.83±19.93) μmol/L, P<0.05]. Plasma malonaldehyde (MDA) was significantly higher in iron sucrose group than that in iron dextran group [(6.06±0.73) nmol/L vs (4.99±0.80) nmol/L, P<0.05]. Serum levels of superoxide dismutase (SOD) and total anti-oxidant capacity (TAOC) had no significant differences among three CRF groups. Concentration of plasma glutathione peroxidase (GSH-Px) was significantly decreased in three CRF groups as compared to sham group (P<0.05), while plasma GSH-Px was significantly lower in sucrose iron group than that in iron dextran group and control group [(2123.11±74.78) nmol&#8226;ml-1&#8226;min-1 vs (2352.84±163.90) nmol&#8226;ml-1&#8226;min-1, (2310.23±125.99) nmol&#8226;ml-1&#8226;min-1, P<0.05]. Conclusions Injection of intravenous iron can partially improve the anemia and the iron status indexes in 5/6 Nx CRF rats. Repeated low dose intravenous infusion of iron dextran and iron sucrose can aggravate the oxidative stress state in CRF rats, and the iron sucrose is worst.

Objective To observe the formation of asymmetric dimethylarginine(ADMA) and the expression of dimethylarginine dimethylaminohydrolase 2 (DDAH-2) of human umbilical vein endothelial cells(HUVECs) stimulated by uric acid (UA), and to explore the role of ADMA-DDAH axis in the vascular endothelial dysfunction induced by uric acid. Methods HUVECs were cultured in M199 medium supplemented with 10% FBS. Cells were exposed to different concentrations of UA (0, 60, 120 mg/L) for 6 h and 24 h. Under different concentrations and times, the level of ADMA in cell suspension was detected by high performance liquid chromatography (HPLC) technique; the gene and protein expressions of DDAH-2 were detected by RT-PCR and Western blotting; the fluorescence intensity of intracellular 2’,7’-dichlorofluorescein (DCF) which represented the productions of ROS was detected by the flow cytometry (FCM). The activity of DDAH-2 in HUVCEs which were exposed to different concentrations of UA (0, 60, 120 mg/L) or UA (120 mg/L) +NAC (10 mmol/L) for 24 h was estimated by directly measuring the amount of ADMA metabolized by the enzyme and the role of NAC in the activity was studied. Results The expression of ADMA induced by urid acid was dose-depent and higher at 24 h than that at 6 h in the same dosage(all P<0.05). The dosage and stimulation time of UA did not have any influence on the expression of intracellular DDAH-2 (all P>0.05). When HUVECs exposed to UA(120 mg/L) for 24 h, the production of intracellular ROS was significantly increased while the activity of DDAH-2 was decreasesd (all P<0.05) as compared to 60 mg/L stimulation. This effect could be inhibited by the intervention of anti-oxidant NAC. Conclusions The high UA stimulation on HUVECs can increase the expression of intracellular ROS and inhibit the activity of DDAH-2 which increases the concentration of ADMA by decreasing the degradation of ADMA as well as the formation of NO. DDAH-ADMA axis may participate in the vascular endothelial dysfunction induced by UA.

Objective To explore the role of oxidative stress in the epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells and the protective effect of probucol in rat model with diabetic nephropathy(DN). Methods Thirty SD rats were randomly divided into normal control group, DN group, probucol treatment group (supplemented 1% probucol dietary). Twenty-four hours urinary protein excretion (UTP) was measured at the 3rd, the 8th and the 12th week respectively. The biochemical indicators including blood glucose (BG), lipids [triglyceride (TG), total cholesterol (TC)], low-density lipoprotein (LDL), serum creatinine (Scr), creatinine clearance rate (Ccr),kidney tissue malondialdehyde (MDA) level and glutathione peroxidase (GSH-Px) activity were assessed at the end of the 12th week in all groups. The renal pathological changes were evaluated by hematoxylin & eosin(HE) and Masson staining. The protein expression of specificity protein 1 (Sp1), α-smooth muscle actin (α-SMA) and E-cadherin was also detected and analyzed by immunohistochemistry and Western blotting. Results Compared with the normal control group, the BG, TC, LDL, Scr, 24 h UTP and MDA level of renal tissue increased significantly and the Ccr reduced in the rats of DN group (all P<0.01). The pathological scores and the expression of Sp1 and α-SMA in renal tissue were higher in the DN animals than that in the other animals (all P<0.01), the expression of E-cadherin downregulated significantly in the DN animals (P<0.01). The MDA level of renal tissue was positively correlated to the expression of α-SMA and Sp1 protein in DN group (r＝0.896, P<0.01; r＝0.862, P<0.01, respectively), and negatively correlated to the expression of E-cadherin protein (r＝-0.673, P<0.01). In the diabetic animals treated with probucol, the Scr, 24 h UTP, pathological scores, MDA content,expression of Sp1 and α-SMA in renal tissue were lower than those in the diabetic animals (all P<0.01). The Ccr and the expression of E-cadherin upregulated obviously (all P<0.01). Conclusion Oxidative stress plays an important role in the EMT process of tubular epithelial cells. Probucol can slow down renal disease progression in DN rats through anti-oxidant, downregulating the expression of Sp1 protein and inhibiting the renal tubular EMT.

Objective To investigate the effect of intermedin(IMD) on tubular cells apoptosis induced by renal ischemia/reperfusion(I/R) injury and its associated mechanism. Methods A total of twenty-four male Wistar rats were randomly divided into four groups (control group, I/R group, empty plasmid group and IMD group). One week after the removal of right kidney, ultrasound plasmid was used to transfect empty or IMD plasmid into the left kidney. Renal I/R model was made by clasping the left renal artery for 45 minutes. Tubular cell apoptosis was determined by TUNEL. Expression of Bax, Bcl-2, Fas was detected by semi-quantitative RT-PCR. Activity of caspase-8 and caspase-9 was evaluated with commercially available kits respectively. Protein level of caspase-3 was measured by Western blotting analysis. Results Compared with control group, apoptosis of tubular epithelial cells, expression of Bax and Fas, activities of caspase-8 and caspase-9, as well as protein level of caspase-3 were all significantly increased in I/R group (all P<0.05). IMD pre-treatment significantly inhibited all these effects (all P<0.05). There were no differences of above parameters between empty plasmid group and I/R group. Conclusion IMD pre-treatment protects against renal I/R injury by inhibion of tubular epithelial cell apoptosis.

Objective To investigate the renoprotection of tubular L-FABP in murine IgA nephropathy (IgAN） induced by bone marrow transplantation(BMT). Methods IgAN models were reconstituted by BMT from IgAN-prone mice into mice (Tg) transgenically tubular overexpressing human L-FABP (hL-FABP) and wild type (WT) mice. These recipients were sacrificed at 6 and 12 weeks after BMT and their kidneys were collected. The expressions of hL-FABP, fibronectin (FN) and monocyte chemoattractant protein-1(MCP-1) mRNA were detected by real-time PCR. hL-FABP, FN, type Ⅳ collagen (Col Ⅳ), hemeoxygenase-1(HO-1) and 4-hydroxy-2-nonenal (4-HNE) modified proteins were detected by Western blotting. The distribution of hL-FABP and FN protein in kidney was detected by immunohistochemistry. The level of serum IgA, urinary albumin and urinary hL-FABP was detected by ELISA. Results (1) IgAN was reconstituted in both Tg and WT mice by BMT: mesangial IgA deposition and up-regulation of serum IgA. The levels were not significantly different between two groups (Tg-ddY and WT-ddY). (2) hL-FABP was expressed in proximal tubular cells of normal Tg mice. The mRNA (1.62±0.32 vs 0.46±0.09, P<0.01) and protein expression (1.74±0.76 vs 1.14±0.31, P<0.01) of hL-FABP was up-regulated in Tg-ddY kidney and urinary hL-FABP level (?滋g/g creatinine) was significantly increased (59.87±26.75 vs 31.01±14.86, P<0.05) at the 6th week after BMT. (3) WT-ddY mice showed a significantly higher urinary albumin level (mg/L) (828±656 vs 82±22, P<0.01), severer mesangial matrix expansion (P<0.01)，more glomerular FN and Col Ⅳ deposition at the 12th week. (4) Up-regulation of renal hL-FABP was associated with significant suppression of renal HO-1 expression (P<0.05), accumulation of 4-HNE modified proteins (P<0.05) and MCP-1 mRNA expression (P<0.01) in Tg-ddY mice. Conclusion Tubular L-FABP may lessen the progression of glomerular damage at early stages of IgAN by reducing oxidative stress and inflammatory mediators.

Objective To explore whether the change of S phase kinase-associated protein 2 (Skp2) expression could regulate mesangial cell proliferation. Methods Skp2 siRNA and control siRNA, pIRES-GFP-Skp2 plasmid and pIRES-GFP plasmid were designed and synthesized. Cell transfection was performed by Lipofectamine 2000. Skp2 mRNA and protein levels were detected by semiquantitative PCR and Western blotting respectively. Primary culture rat mesangial cells were divided into 6 groups: 0%FCS, 20%FCS, 10%FCS+pIRES-GFP plasmid, 10%FCS+pIRES-GFP-Skp2 plasmid, 20%FCS+control siRNA, 20%FCS+Skp2 siRNA. Cell number was detected by MTT. S phase entry was measured by BrdU labeling. Cell cycle profile was determined by flow cytometric analysis. Results Skp2 mRNA level was significantly down-regulated by Skp2 siRNA compared to control siRNA. Skp2 protein level increased after pIRES-GFP-Skp2 plasmid transfection compared to pIRES-GFP plasmid. MTT, BrdU labeling and cell cycle profile demonstrated that cell number (A: 0.419±0.088 vs 0.305±0.036, P＜0.01) and S-phase cells (BrdU labeling positive cell: 0.21±0.04 vs 0.15±0.03, P＜0.01；S-phase cell number：20.18±0.64 vs 14.33±0.37, P＜0.01) obviously increased after Skp2 plasmid transfection, while decreased after Skp2 siRNA transfection compared to control groups(A: 0.328±0.069 vs 0.482±0.133, P＜0.01; BrdU labeling positive cell: 0.17±0.01 vs 0.24±0.00, P＜0.01; S-phase cell number: 16.52±0.75 vs 23.81±1.25, P＜0.01). Conclusion Over-expression of Skp2 stimulates mesangial cell proliferation while down-regulated expression of Skp2 inhibits mesangial cell proliferation.

Objective To observe the change of Wnt-β-catenin signaling’s location and expression in kidney repair after acute kidney injury induced by ischemla reperfusion (I/R). Methods Ischemia reperfusion injury in BAT-gal reportor mice was made and blood sample was taken from tails on the 1th day after injury. Mice were sacrified on the 2th or 7th day and kidneys and blood were collected. Renal pathological change was observed by PAS stain. The changes of location and expression of Wnt-β-catenin signaling were detected by immunofluorescence costainning X-gal-LTL, X-gal-NKCC2, X-gal-DBA respectively. The protein expressions of the Wnt4 and co-receptor Lrp6 were assessed by Western blotting. Results PAS-stained kidney sections showed desquamative or flattened epithelia, necrotic debris on day 2 and regenerating tubules on day 7. An injury-induced enhancement of the Wnt pathway response (X-gal staining) in kidney cortex and out-medulla. Immunolabelling of kidney sections from injured BAT-gal mice revealed that X-gal staining was detected in kidney epithelial cells (double-labelled with LTL or NKCC2). Western blotting showed the Wnt4 protein was up-regulated and phospho-Lrp6, indicative of active canonical Wnt signaling, was noted in kidney cortex from day 2 after I/R, but in control kidney cortex pLrp6 was not detected. Conclusion Wnt-β-catenin signaling is activited after acute kidney I/R injury and is required for tubular epithelial repair and regeneration following kidney I/R injury.