Objective To detect the proteins structure encoded by COL4A4 gene with different missense mutations of thin basement membrane nephropathy(TBMN) and to analyze the effect of gene mutation on the secondary structure of α4(Ⅳ) chain and its association with phenotype． Methods A COL4A4-1inked TBMN patient with FSGS by a missense mutation (g. 1214G>A resulting in p. G405E) diagnosed by clinical manifestations, family history and renal biopsy examination, as well as two controls (one healthy, one pure TBMN carrying a g. 1550G>A mutation resulting in p. G448S) were enrolled in this study. The fragments of cDNA with the two mutations and that of corresponding cDNA from the healthy control were expressed in E. coli. The secondary structures of recombinant polypeptides were analyzed by circular dichroism (CD) spectroscopy. Results CD spectra of healthy control exhibited a negative peak near 208 nm whereas that of TBMN patient with FSGS exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of this patient decreased as compared with that of healthy control. CD spectra of pure TBMN control was slightly changed with the negative peak remaining near 208 nm and the magnitude slightly decreased as compared with that of healthy control. In addition, the secondary structure of polypeptide from healthy control was composed of about 1/4 α-helix and 1/4 β-sheet, whereas that form the patient presented about 1/3 α-helix without any β-sheet. The secondary structure of polypeptide from pure TBMN control was almost the same as the healthy control, except a slight reduction of α-helix and a slight increase of β-sheet. Conclusions Although the glycine substitutions exists in the nearby domain of α4(Ⅳ)chain, the TBMN patient complicating FSGS with severe phenotype and g. 1214G>A mutation and the pure TBMN control with the mild phenotype and g. 1550G>A mutation are revealed with different secondary structures of α4(IV)chain. Moreover, the secondary structure change of α4(IV) chain is consistent with their corresponding phenotype severity.

Objective To investigate the prevalence of chronic kidney disease (CKD) and associated factors in Changsha county of Hunan province. Methods Using a stratified, multistage sampling, 1950 residents (older than 20 years old) from 3 towns of Changsha county were randomly selected to be interviewed and tested for the kidney damage indicators and the associated factors with CKD. Results Eligible data of 1727 subjects were enrolled in the study. After the adjustment of age and gender component, the prevalence of albuminuria was 8.5%, hematuria 5.1%, and reduced eGFR 1.5%. Approximately 14.6% subjects had at least one indicator of kidney damage, and the awareness rate was 16.5%. Age, hypercholesteremia, hypertriglyceridemia, hypertension and diabetes were independently correlated with albuminuria. Female, age, hypertriglyceridemia and hyperuricemia were independently correlated with reduced renal function. Female was independently correlated with hematuria. Conclusions The prevalence of chronic kidney disease is 14.6% and the awareness rate is 16.5% in suburban adult population of the central south area of China. The spectrum and correlated factors of CKD in this county undergoing fast economic development are close to those of Guangzhou and developed countries.

Objective To investigate the expression of inflammatory mediators in renal tubular epithelial cells in patients with diabetic nephropathy (DN) and to explore the possible clinicopathological significance. Methods Twenty-three patients with DN diagnosed by renal biopsy and 10 patients with renal cell carcinoma undergone nephrectomy were allocated into DN group and control group, respectively. The renal expression of NF-κB p50, NF-κB p65, NF-κB p65 mRNA, MCP-1, OPN, α-SMA, and FN was detected by immunohistochemical or in situ hybridization assay. Serum creatinine, urinary N-acetylglucosaminedase (NAG), urinary albumin and 24-hour urinary protein were detected. The correlation between these inflammatory markers and clinicopathological data were analyzed. Results (1)Among all the 23 DN patients, granular degeneration of the renal tubular epithelium, focal tubular atrophy, infiltration of inflammatory cells and interstitial fibrosis were apparent, and none of these were found in control group. (2)Immunohistochemical and in situ hybridization assay showed that, compared with control group, expression of these factors increased significantly in renal tubular cells or interstitium in DN patients, and expression of α-SMA or FN was not found in tubular epithelial cells. (3)Statistics assay showed the tubular NF-κB p65 protein expression was correlated with all of the following factors: NF-κB p50 protein(r=0.792) and NF-κB p65 mRNA (r=0.763), tubular MCP-1 (r=0.825) and OPN (r=0.869) expression, interstitial α-SMA (r=0.327) and FN (r=0.432) expression, proteinuria(r=0.710), estimated glomerular filtration rate (eGFR) (r=-0.728), and urinary NAG (r=0.930), P<0.01 respectively. Conclusion Tubular inflammation may play a role in the pathogenesis and progression of DN.

Objective To identify the outcome of pregnancy and the alteration of renal function in women with nephrotic syndrome. Methods From 2003 to 2007, 59 pregnant women with nephrotic syndrome in our hospital were enrolled in the study. Their clinical data were retrospectively analyzed, including the time of kidney disease onset, 24-hour proteinuria, serum albumin, serum creatinine, blood uric acid, blood pressure, fetal survival, fetal mortality, rate of premature delivery, birth weight of the newborn, and proteinuria, renal function, blood pressure of the patients during their postpartum follow-up. Logistic regression analysis was used to identify the risk factors influencing the outcome of the patients and the newborns. Results The average gestational week was (20.35±9.40) weeks when proteinuria was detected in these pregnant women. The 24-hour proteinuria ranged from 3.5 to 15 g/24 h (median 5.1 g/24 h). The serum albumin was between 10 and 28 g/L(median 22.5 g/L). The serum creatinine was between 32 and 825 μmol/L (median 84 μmol/L) and the serum uric acid ranged from 196 to 793 μmol/L (median 385.5 μmol/L). Pregnancy-induced hypertension syndrome occurred in 75% of the patients, among whom 55.5% suffered from preeclampsia. Forty-three(72.9%) newborns survived , among whom 76.7% (33/43) were premature births and 62.8% (27/43) were low birth weight infants. 50% of the pregnant women still had nephrotic syndrome after delivery. 75% of 24 patients with pre-existing chronic glomerulonephritis had increased proteinuria during pregnancy. Among the 38 patients with renal insufficiency, 36.8% had poorer renal function after delivery. 23.7% of the patients progressed into end stage renal failure after delivery, 80% of whom had serum creatinine≥265 μmol/L. 89% of the patients had persistent hypertension after childbirth. The Logistic regression analysis indicated hyperuricemia during pregnancy (P=0.018, OR=1.012) and the increase of serum creatinine (P=0.039, OR=1.005) were risk factors of renal failure in pregnant women after delivery. Hyperuricemia(P=0.012, OR=1.006）was the risk factor of fetal death. Conclusions Pregnancy with nephrotic syndrome leads to a low fetal survival. Hyperuricemia is the most important risk factor of the poor outcome of pregnant women and newborn.

Objective To elucidate the clinical features of nutcracker syndrome complicated with IgA nephropathy (IgAN) and to increase its level of diagnosis and treatment. Methods Clinical data of 14 cases of nutcracker syndrome complicated with IgA nephropathy (patient group) and 36 cases of nutcracker syndrome (control group) were analyzed retrospectively. Nutcracker syndrome was diagnosed by ultrasonography and magnetic resonance angiography (MRA) and IgAN by renal biopsy. Differences of clinical data and images in two groups were analyzed. Results Gender, age and blood pressure of two groups were not significantly different. Higher Scr level [(81.2±21.3) μmol/L vs (61.2±11.8) μmol/L, P<0.01], more severe proteinuria [(1.1±0.6) g/d vs (0.3±0.2) g/d, P<0.01] and hematuria (2.3±0.9 vs 1.5±1.3, P<0.05) in patient group were found. Differences of ultrasonography and MRA in two groups were not significant. Conclusion Renal biopsy should be considered in cases of nutcracker syndrome with persistence of proteinuria, hematuria or abnormal morphology of urinary red blood cell.

Objective To elucidate the mechanism of angiotensin (Ang)Ⅱ in regulation of renin synthesis and secretion in primary cultured juxtaglomerular granular cells (JGCs). Methods Mice JGCs were isolated and cultured as described before. Real-time PCR was used to demonstrate the expression of angiotensin converting enzyme ( CE) and angiotencin receptor (AT1，AT2) in JGCs. Ang Ⅱ was co-cultured with JGCs stimulated by PGE2 and isoproternol or not. Renin activity in supernatant was detected by radioimmunoassay and renin mRNA expression was examined by real-time PCR. Different concentrations of AngⅡ were co-cultured with JGCs with different time (1 h, 4 h and 24 h), cAMP in cell lysate and supernatant was measured by Cayman Cyclic AMP EIA Kit. The cytoplasmic calcium was decreased by BAPTA-AM, and increased by thapsigargin and cyclopiazonic acid, which could be used to observe the cAMP concentration affected by calcium. Adenylyl cyclase type 5, 6 (AC5, AC6) mRNA expression of JGCs co-cultured with AngⅡ was measured by real-time PCR. Results Real-time PCR confirmed the expression of ACE and AT mRNA in JGCs of WT mice. Angiotensin Ⅱ reduced renin secretion in primary cultures of JGCs [(370.6±36.9) vs (299.6±25.7) ng AngI&#8226;ml-1&#8226;h-1, P=0.014]. Angiotensin Ⅱ dose-dependently down-regulated forskolin-stimulated cAMP production in JGCs. Thapsigargin and cyclopiazonic acid decreased cAMP level. BAPTA-AM increased cAMP production obviously [(11.09±0.48) vs (3.55±0.47) nmol/L, P<0.01]. Ang Ⅱ inhibited AC5 mRNA expression by 42.12%, but did not inhibit AC6 mRNA expression. Conclusions Angiotensin Ⅱ can directly inhibit renin synthesis and secrestion maybe through reducing AC-stimulated cAMP levels. Crosstalk between calcium and cAMP system may exist in precise regulation of renin in JGCs.

Objective To observe the effect of Xuebijing injection on the expression of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 protein (HMGB-1) in rat peritoneal mesothelial cells (PMCs) induced by lipopolysaccharide (LPS). Methods PMCs were isolated from rat colic omentum and the 3rd generation cells were used in the experiment. PMCs were incubated with LPS at different concentrations (1,10,100 mg/L); with LPS (10 mg/L) for 2, 6, 12, 18, 21, 24, 36 h; with Xuebijing injection at different concentrations (2,10,20 g/L) after incubation with LPS (10 mg/L) for 2 h. PMCs in the control group were incubated with medium. HMGB-1 mRNA was detected by RT-PCR. TNF-α and HMGB-1 protein in supernatants was detected by ELISA. Results Compared to the control group, the expression of HMGB-1 mRNA and protein was significantly increased in groups stimulated by LPS in a time- and dose-dependent manner (all P<0.05); the expression of TNF-α was increased in the groups stimulated by LPS in a dose-dependent manner(P<0.05). In the groups stimulated by LPS (10 mg/L), the expression of TNF-α appeared double hump within 36 hours. Compared to LPS (10 mg/L) group, Xuebijing injection significantly inhibited the expression of HMGB-1 and TNF-α (all P<0.05） in a dose-dependent manner. Conclusions HMGB-1 as a late mediator of inflammatory responses may play a role in the pathogenesis of peritoneal dialysis related peritonitis. Xuebijing injection can reduce peritoneal inflammatory impairment by inhibiting the up-regulation of TNF-α and HMGB-1 induced by LPS.

Objective To study the expression and regulation of aquaporins (AQP) in cystic epithelial cells of jck mice with polycystic kidney disease. Methods Localization and regulation of AQP1, AQP2, AQP3 and AQP4 protein were analyzed by using the immunofluorescence and Western blotting. Results Kidneys of jck homozygous mice were 4 folds larger than those of litter matched wild-type mice. There were multiple cysts and fibrosis in the renal tissue of jck mice. The epithelial cells in cysts were flat in shape. Blood urea level in jck mice was (42.6 ± 6.7) mmol/L, which was 5 folds higher than that in wild-type mice [(8.4±1.9) mmol/L] (P<0.01). Immunofluorescence analysis showed that AQP1 was expressed in the apical and basolateral membranes of epithelial cells in proximal tubules, as well as in the thin descending limb of Henle and endothelial cells of descending vasa recta. There was no AQP1 expression in epithelial cells of cysts. AQP2 was expressed in the apical membranes of collecting ducts and renal cysts. AQP3 and AQP4 were expressed in basolateral membranes of collecting duct and renal cystic epithelial cells of jck mice. Western blot analysis showed the same protein sizes of AQP1, AQP2, AQP3 and AQP4 in both jck and wild-type kidneys. However, AQP1 expression was down-regulated in jck kidneys(P<0.01). Conclusion The renal cystic epithelia expresses AQP2, AQP3 and AQP4, which indicates that epithelial cells in renal cysts are derived from renal collecting ducts in jck mice and aquaporins may play an important role in renal cyst development.

Objective To explore the role of NG2 proteoglycan in the pathogenesis of glomerulosclerosis. Methods Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for NG2 mRNA , named as Psilencer-NG2, was constructed. Then, rat mesangial cells (RMC) were transfected with Psilencer-NG2, Psilencer-NC (negative control), pcDNA/NG2 (NG2 over-expressive vector) and empty vector pcDNAⅠrespectively. The expression of endogenous NG2 in RMCs was examined by real-time PCR and Western blotting. Cell proliferation was analyzed by MTT assay and flow cytometry. The expression of laminin was detected by real-time PCR. Results Transfection of pcDNA/NG2 into HBZY-1 cells resulted in over-expression of NG2 mRNA and protein(P<0.05, P<0.05). Transfection of Psilencer-NG2 led to reduced expression of NG2 mRNA and protein(P<0.01, P<0.01). The expression of laminin β1 significantly increased due to overexpression of NG2 and decreased by treating with NG2 siRNA. According to MTT assay, overexpression of NG2 significantly stimulated the proliferation of mesangial cells while NG2 silencing inhibited it. NG2 increased the cell number in S phase and decreased the cell number in G0/G1 phase, while silencing NG2 induced the decrease of cell number in S phase and the increase of cell number in G0/G1 phase. Conclusion NG2 actively participates in the pathogenesis of glomerulosclerosis by stimulating proliferation of RMCs and increasing the deposition of ECM.

Objective To develop a model of type 2 diabetes with early renal injury on spontaneously hypertensive rats (SHR). Methods The 6-week old SHR were fed with the diets enriched with sucrose (20%, W/W), lard (10%, W/W), cholesterol (2.5%, W/W) and chleolate (1%, W/W) to induce insulin resistance. Hyperglycemia was developed by intraperitoneal injection of streptozotocin (STZ, 35 mg/kg). Wistar-Kyoto rats (WKY) were used as normal controls. Rats with plasma glucose (PGL) ≥16.7 mmol/L were diagnosed as diabetes. Eight weeks after the induction of diabetes, plasma triglyceride (TG), cholesterol (CHO), glucose, systolic pressure(SP), 24-h urine protein excretion (Upro) were examined in all the rats, and the homeostasis model assessment of insulin resistance (HOMA-IR) was analyzed. Renal pathological changes were studied by immunohistochemical staining and electron microscope. Results After 2 weeks on the high sucrose and fat diets, the model rats exhibited significant increase in basal PGL, TG and CHO levels as compared to control rats (P＜0.05, respectively). The insulin resistance was developed in model rats demonstrated by the higher HOMA-IR(5.03±0.38 vs 2.61±0.34, P＜0.05). At the end of the experiment, model rats were associated with hypertension. Upro level was significantly increased in model rats compared with that in controls [(57.58±16.54) mg/24 h vs (5.35±1.90) mg/24 h, P＜0.01]. The kidney hypertrophy index (KWI) was significantly increased in the model rats compared to controls (P＜0.05). Moreover, the diabetic model rats showed glomerular hypertrophy, foot process effacement, micro villous transformation, glomerular basement membrane (GBM) thickening. Conclusion A rat model is successfully established, which presents typical features of human type 2 diabetes and can be served as an ideal model to study the diabetic nephropathy.