Solution structure analysis of cytoplasmic domain of podocyte protein Neph1 using small/wide angle x-ray scattering (SWAXS)

Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 Å, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD·ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys-761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into the molecular structure and function of Neph1-CD.     

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.     

肾内科进修生临床带教体会

进修教育是继续医学教育的重要组成部分,是基层医院医生深造的一条重要途径,是基层培养业务技术骨干的重要手段,也是大型综合性医院临床工作的重要组成部分。总结我科进修医生临床教学中的经验包括:分析构成,因材施教;理论知识与临床技能双管齐下;注重医德医风、职业道德的培养,强化法制观念,从多方面着手,培养高素质的肾内科进修医生,收到了满意的效果。     

PBL教学法在肾科临床见习中的应用

目的：探讨以问题为基础的教学法（Problem Based learning,PBL）在肾科临床见习教学中的应用价值。方法：首都医科大学宣武医院63名参加内科学肾脏病临床见习的七年制学生随机分成对照组和观察组,对照组采用传统的基于授课的教学法进行教学,观察组用PBL教学法进行教学,比较2组学习成绩的差异;向学生发放问卷调查,调查学生对2种教学方法的认可程度。结果：2组学生的填空题、选择题及简答题成绩比较无显著性差异（P〉0.05）;观察组病例分析题成绩好于对照组（P=0.001）;培养临床思维、激发学习兴趣方面和自主学习能力方面PBL教学法优于传统教学法（P〈0.01）。结论：PBL教学法有助于学生临床思维和临床技能的培养,从而使教学质量得到提高。     

Human monocyte chemiluminescence triggered by IgG aggregates. Requirement of phospholipase activation and modulation by Fc receptor ligands

Human mononuclear cells stimulated with soluble IgG aggregates generated chemiluminescence, a response attributable to monocytes. Some requirements of this reaction were examined by preincubation of the cells with a variety of inhibitors. The protease antagonists TPCK and TLCK, the phospholipase inhibitors quinacrine and BPB, and the calcium channel blocker verapamil were all inhibitory at micromolar concentrations. The oxygen metabolite scavengers SOD and catalase were less inhibitory. These findings are consistent with a major role for arachidonic acid metabolites in the generation of light. Modulation of monocyte Fc-mediated chemiluminescence also occurred by preincubation of the cells with Fc ligands. While IgG aggregates and monomeric IgG blocked Fc-dependent chemiluminescence, IgG Fc fragments were stimulatory of this response.     

Increased SHP-1 Protein Expression by High Glucose Levels Reduces Nephrin Phosphorylation in Podocytes

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr¹¹²⁷ and Tyr¹¹⁵² are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2^+/C96Y) compared with control littermate mice (Ins2^+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.     

Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase,Nox4, in Mesangial Cells

Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.     

An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Audoradiography demonstrated that the inhibitor shifted the electrophoretic position of ¹²⁵I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated ¹²⁵I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.     

Urinary excretion of prostaglandin E2 and prostaglandin F2α in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE₂ than female animals.