Objective To study the mutations of apolipoprotein E(apoE)gene in 4 Chinese lipoprotein glomerulopathy(LPG)patients and their family members，and to investigate the pathogenesis of LPG. Methods Urinalysis was performed on the family members of two patients， and they were screened for the level of serum creatinine，serum lipid and serum lipoprotein. The mutation of apoE gene was detected by direct sequencing after PCR of apoE exons. PCR RFLP was used to identify the mutations in family members. Results Two kinds of mutation of apoE gene were identified in these 4 patients. A 9 bp deletion in exon 4， resulting in a 3-amino acid deletion (residues 142-144-0) was confirmed in 2 patients and a point mutation in exon 3 resulting in Arg25Cys was found in another 2 patients. Above mutations were found in the patients′ relatives who had normal urinalysis results. Conclusions The 3-amino acid deletion (142-144-0) of apoE gene is the common mutation to cause LPG in Chinese patients. Both mutations could be found in their relatives without LPG.

Objective To investigate whether the risk factors of cardiovascular disease exist in early stage of ADPKD patients with normal renal function. Methods Morphologic，mechanical and functional sonographic parameters of arteries were examined by high-frequency ultrasonography in 32 hypertensive and 28 normotensive ADPKD patients with preserved renal function， 25 patients with essential hypertension， and 30 healthy subjects were included in the study. Brachial artery endothelial dependent vasodilation (EDV)， carotid intima-media thickness (IMT) and distensibility were measured by high-resolution vascular ultrasound. Results EDV was more significant in hypertensive patients with ADPKD as compared to patients with essential hypertension and normotensive patients with ADPKD， and EDV is also more significant in normotensive ADPKD patients as compared to healthy subjects. Carotid IMT was significant greater in both hypertensive and normotensive patients with ADPKD compared with healthy subjects. Moreover， carotid cross-sectional distensibility (CD) and incremental elastic modulus (Einc) differed significantly between both hypertensive and normotensive ADPKD patients and healthy subjects. Conclusion Both hypertensive and normotensive patients with ADPKD showed significant endothelial dysfunction， increased IMT and decreased distensibility， which are predictors of atherosclerosis.

Objective To evaluate the feacibility of microalbumin(MA)，alpha-1-microglobulin(α1-MG)，N-acetyl-beta-D-glucosaminidase(NAG)，retinol-binding protein(RBP)as predictors of outcome in critically ill patients. Methods A prospective study was underwent in 30 critically ill patients.Urinary samples were collected at ICU admission and on the third，seventh ICU day for MA，α1-MG，NAG，RBP measurement.The severity of illness was assessed by APACHEⅡ score calculated on the first ICU day，and the degree of organ dysfunction was assessed using SOFA score calculated on the first，third，seventh ICU day. Results MA，α1-MG，the durations of ICU stay and mechanical ventilation，APACHEⅡscore， SOFA score were relative factors of MODS and death in ICU. There was significant association of APACHEⅡ with MA(r=0.397)， α1-MG(r=0.448)， and RBP(r=0.465)， respectively. The area under ROC curve of APACHEⅡ score，SOFA score，MA，α1-MG，RBP，NAG to predicate the death in ICU were 0.875﹙P < 0.05﹚，0.825﹙P < 0.05﹚， 0.820﹙P < 0.05﹚，0.730，0.530，0.620. Conclusion MA，α1-MG and RBP are valuable predictors of outcome in critically ill patients.

Objective To investigate the effects and mechanism of Megsin gene transfection on mesangial cell proliferation and type Ⅳ collagen excretion. Methods Rat Megsin cDNA eukaryotic expressing vector was constructed and transfected to cultured rat mesangial cells. Cell proliferation was measured by determining [3H]-thymidine (3H-TdR) incorporation. The mRNA expression of PDGF-BB， TGF-β1， IL-1β， IL-6， IL-10 and TNF-α in mesangial cells was detected by RT-PCR. Excretion of PDGF-BB， TGF-β1 and type Ⅳ collagen in supernatant of cultured mesangial cells was measured by ELISA. Impacts of PDGF-BB neutralizing antibody in cell proliferation and expression of TGF-β1 mRNA was also detected. Results A time-dependent up-regulation of mRNA expression of PDGF-BB and TGF-β1 was detected in cultured mesangial cells when transfected with Megsin gene. The concentration of PDGF-BB， TGF-β1 and type Ⅳ collagen in the cell supernatant rose accordingly. PDGF-BB neutralizing antibody suppressed the incorporation of 3H-TdR and the mRNA expression of TGF-β1 in rat mesangial cells transfected with Megsin gene significantly.Conclusion Over expression of Megsin gene enhances the mRNA expression， extracellular excretion of PDGF-BB and TGF-β1 in mesangial cells， which further leads to mesangial cell proliferation and type Ⅳ collagen excretion.

Objective To evaluate the effect of ALD on podocyte apoptosis and the possible roles of Akt in ALD-induced apoptosis. Methods The cultured rat podocytes were incubated with increasing concentrations of ALD (10-9~10-5 mol/L) for variable time periods. Apoptosis was evaluated by cell nucleus staining and flow cytometry. RT-PCR was used to examine the expression of mineralocorticoid receptor (MR)and 11 Beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2) mRNA in podocyte. Activation of Akt/PKB was evaluated by performing Akt kinase assay. Results ALD induced podocyte apoptosis in a dose- and time-dependent manner. The proapoptotic effect was attenuated by the presence of spironolactone (10-7mol/L). The expression of MR and 11β-HSD2 mRNA was demonstrated in the podocytes by RT-PCR. ALD also inhibited the activity of Akt in a dose-dependent manner， but the inhibitory effect was significantly ameliorated by the presence of spironolactone. The activity of Akt was negatively correlated with podocyte apoptosis. Conclusion ALD induces apoptosis in rat podocytes through the signaling mechanism by which Akt is inhibited.

Objective To study whether Hirsutella sinensis (HS) can antagonize aristolochic acid (AA) induced fibrogenesis on human proximal tubular epithelial cells (HKC). Methods HKC were incubated with medium alone， medium with HS 10 mg/L， medium with AA-Na 40 mg/L or medium with AA-Na 40 mg/L and HS 10 mg/L， respectively. After 12 h (for mRNA) or 36 h (for protein)，cells were lysed，and the mRNA and protein expression level of transforming growth factor-β1 (TGF-β1)， connective tissue growth factor (CTGF)， tissue inhibitor of metalloproteinase-1 (TIMP-1)and plasminogen activator inhibitor-1 (PAI-1) of HKC were measured by RT-PCR， ELISA (for TGF-β1， TIMP-1 and PAI-1) and Western blotting (for CTGF)， respectively. Results The mRNA and protein expression of TGF-β1，PAI-1，CTGF and TIMP-1 were significantly up-regulated by AA-Na 40 mg/L. Compared with the control group， the mRNA expression of TGF-β1，CTGF，TIMP-1 and PAI-1 was up-regulated to 1.24，1.31，1.27 and 1.36 times，respectively(P﹤0.05). Their protein expression was up-regulated to 2.50，1.75，2.13 and 1.46 times， respectively(P﹤0.05). HS significantly inhibited the up-regulation of TGF-β1，CTGF，and TIMP-1 expression induced by AA. Compared with the AA-Na stimulation group， the inhibition rates of TGF-β1，CTGF and TIMP-1 mRNA expression in the HS group were 12%，20% and 17%， respectively(P﹤0.05)， and the inhibition rates of their protein expression were 25%，20%， and 37%， respectively (P﹤0.05).HS did not inhibit the up-regulation of PAI-1 expression induced by AA. Conclusion The AA- induced fibrogenic effects on HKC can be antagonized by HS， which may be through down-regulating the factors promoting extra cellular matrix (ECM) synthesis (TGF-β1 and CTGF) and inhibiting ECM degradation (TIMP-1).

Objective To investigate whether aldosterone(Aldo)can activate Na+-H+ exchange isoform 1 and increase its expression in rat mesangial cells(MC)，and this effect on the proliferation of exracellular matrix(ECM) in rat mesangial cells. Methods Rat mesamgial cells were cultured and then divided as followings: control group(Con)， Aldo group (Aldo of the concentration of 10-7mol/L was added)，Aldo+spironolactone group(spironolactone 10-9mol/L and Aldo 10-7mol/L were added)， Aldo+dimethylamiloride(DMA) group(DMA 25 μmol/L and Aldo 10-7 mol/L were added) and spironolactone group ( spironolactone 10-9 mol/L was added). Twenty-four hours later， the mesangial cells， the supernatants and the RNAs of the cells in different groups were collected. The NHE activity was calculated from the initial rate of Na+ dependent pHi recovery after acid load.NHE1 mRNA expression and protein abundance were measured by real-time PCR and flow cytometry analysis. The mRNA expression and protein level of FN were examined by real-time PCR and ELISA respectively. Results After 24 h exposure of MCs to aldosterone(10-7 mmol/L)， NHE1 activity was increased compared to control[Con(4.48±0.25)%， Aldo (5.29±0.11)%， P < 0.05]， the antagonists of mineralocorticoid receptor(MR) spironolactone and the NHE-1 inhibitor DMA could inhibit this effect[Aldo+Spir(4.92±0.35)%， Aldo+DMA (4.07±0.23)%， vs Aldo， P < 0.05]. NHE1 mRNA expression was increased by 1.16 fold of Con(P < 0.05)， and spironolactone could inhibit this effect to 81.9% of Aldo(P < 0.05).NHE1 protein abundance was increased by 2.9 fold of Con(P < 0.01)， and spironolactone could inhibit this effect to only 52.3% of Aldo(P < 0.05). Spironolactone itself had no effect on NHE1 mRNA expression and protein abundance. FN mRNA expression was increased by 1.03 fold of Con(P < 0.05)， spironolactone and DMA could inhibit this effect to 91.21% and 92.30% of Aldo respectively(P < 0.01). ELISA showed the similar result in the concentration of FN(ng/ml) (Con 17.84±3.77， Aldo 51.66±1.40，P < 0.01； Aldo+Spir 29.60±1.99，Aldo+DMA 25.75±4.66， vs Aldo， P < 0.01). Conclusion Aldosterone can activate NHE1 and induce NHE-1-dependent proliferation of ECM in RMCs.

Objective To investigated the inhibitory effect of an IgG-Fc region specific inhibitory peptide on the ANCA-accelerated apoptosis of neutrophils. Methods The peptide was prepared by solid-phase peptide synthesis and its biological activity was identified by rosette formation assay. ANCA was prepared from the sera of active Wegener′s granulomatosis (WG) and microscopic polyangiitis(MPA) patients. Neutrophils isolated from the blood of healthy volunteers were primed with TNF-α(2 ng/ml) then incubated with ANCA. At different intervals(3， 6， 12， 18 hours) the neutrophils were harvested to assess the apoptosis by flow cytometric analysis of JC-1 staining， Sub-G1 population and formation TUNEL technique. Results Tg19320 bound tightly to human IgG dose- dependently and inhibited statistically the rosette formation between SRBC-IgG and U937 cells(20.3% vs 53.2%，P < 0.01).Furthermore，tg19320 blocked ANCA-accelerated apoptosis of neutrophils at different intervals and made the apoptosis have no significant difference with control groups. Conclusion The small peptide can intervene the apoptosis induced by ANCA，which supports the role of FcγR in primary systemic small vasculitis(PSV) pathogenesis and perhaps opens new avenues for the development of drugs to treat PSV.

Objective To study the effect of oxidative modification of hydrochlorous acid (HOCl) on human serum albumin (HSA) and the relationship between the AOPPs and HOCl-treated HSA. Methods Purified HSA (60 mg/ml) was treated with HOCl (0， 1， 5， 10， 20， 30， 40， 50 and 60 mmol/L). Size-exclusion chromatography was applied to estimate molecular weights of oxidized products of HSA by HOCl and spectrum scan from 190 nm~400 nm was performed to observe the spectrum characteristics of all variants of HSA. Results Major products of HSA after exposure to HOCl were dimer and hexmer of HSA. The first-order process could be employed to describe the oxidative dynamics of monomer and dimer of HSA oxidized by HOCl. To AOPPs formation mediated by oxidant was identified as pseudo first-order reaction. However， formation hexmer was much in accordance with second-order reaction. Hexmer was also a major contributor to AOPPs in all types of modified HSA. Spectral analysis showed that red shift of absorbance maximum of polymers of HSA occurred， suggesting that a possibility that polymers of HSA were cross linked by tyrosine residues in protein. Conclusions Protein aggregation is primary consequence of HSA after its exposure to HOCl. Hexmer of HSA is the major contributor to AOPPs.

Objective To observe the regulatory mechanism of β-catenin in modulating the expression of E-cadherin in TGF-β1 induced epithelial-mesenchymal transition (EMT). Methods The substrate mimetic peptide and control peptide were constructed. The human tubular cells(HK2 cell line) were cultured with 10 ng/ml TGF-β1 for 72 hours， in the group of the substrate-analogue peptide and control peptide the cells were incubated with 2 μmol/l peptide and 10ng/ml TGF-β1 for 72 hours. The expression of E-cadherin and α-SMA was measured by Western blot. Tyrosine phosphorylation of β-catenin was measured by Western blot as well.The intracellular disposition of β-catenin was observed by confocal microscope. Results In the normal HK-2 cells， E-cadherin was expressed， α-SMA was almost not expressed，and β-catenin in the state of dephosphorylation was mainly expressed on the membrane of the cell.In the model group， E-cadherin was not expressed， the expression of α-SMA was up-regulated，and tyrosine of β-catenin was phosphorylated and located in the cell nucleus.The experimental results of the cells of the substrate mimetic peptide group was similar to those of the normal group， so do the results of the cells of the control peptide group and the model group. Conclusions In the human tubular cells， the up-regulation of tyrosine phosphorylation of β-catenin， followed by β-catenin moving from cell membrane to the cell nucleus leads to the disassociation of the complex of E-cadherin/β-catenin， which is involved in the loss of E-cadherin ，expression of α-SMA and EMT induced by TGF-β1.

Objective To explore the effects of Sinomenine(Sin) on phenotype transdifferentiation in renal tubular epithelial cells. Methods Mice primary tubular epithelial cells and cell line (MCT) were cultured in vitro. Model group cells were stimulated by IL-1β(10 ng/ml). The cells of Sin group were co-incubated with Sin(10，100，500 μmol/L) simultaneously. The cells without IL-1β and Sin treatment were regarded as control group. The protein expression of α-smooth muscle actin(SMA) and Vimentin was assessed by immunohistochemistry and Western blotting. The mRNA expression of α-SMA and Vimentin was examined by RT-PCR. Secretion of matrix metalloproteinases was determined by zymography. Results The mRNA expression and protein level of α-SMA and Vimentin， and the enzyme activity of MMP-2 and MMP-9 in tubular epithelial cells stimulated with IL-1β were significantly higher than those in the controls. Sinomenine significantly reduced the expression levels of protein and mRNA of α-SMA and Vimentin. At the same time， the MMP-2 and MMP-9 activities were reduced markedly. Conclusion Sinomenine can decrease the transdifferentiation in renal tubular epithelial cells stimulated by inflammatory factor IL-1β and prevent renal interstitial fibrosis.