糖尿病肾病肾小球nephrin表达的变化

目的： 探讨糖尿病大鼠肾小球滤过屏障外层裂孔膜关键蛋白nephrin mRNA和蛋白表达的改变及其意义。 方法：建立糖尿病动物模型，测定血糖（BG）、总胆固醇（TC）、甘油三酯(TG)、尿白蛋白排泄率（UAER）水平，并用RT-PCR和免疫组织化学方法检测肾小球nephrin mRNA和蛋白的表达。 结果：糖尿病组（D组）BG、TC显著高于正常对照组（N组），肾小球nephrin mRNA和蛋白表达明显低于N组，UAER显著高于N组（均P<0.05）。 结论：持续糖脂代谢紊乱显著抑制糖尿病大鼠肾小球nephrin mRNA和蛋白的表达，破坏肾小球滤过屏障结构完整性，增加UAER，这可能是糖尿病肾病发生发展的机制之一。     

Podocalyxin与糖尿病肾病的关系

足细胞标记蛋白(podocalyxin)作为足突顶端质膜的主要构成部分,是足突顶膜区主要的带负电荷的唾液酸蛋白,参与维持足细胞的正常结构和滤过屏障。糖尿病肾病早期以肾脏肥大和肾小球高滤过为特征。在肾小球足细胞的检测中,podocalyxin作为最常用的标记蛋白之一,对监测肾小球疾病的发生和发展起到极其重要的作用。深入研究podocalyxin对糖尿病肾小球病变的早期诊断及治疗有着重要的指导意义。     

儿童激素耐药肾病综合征相关致病基因及其临床检测策略

肾病综合征（NS）是因肾小球滤过屏障受损而引起的一绀临床综合征。肾小球滤过屏障由内皮细胞、基底膜和足细胞构成。多项研究表明,足细胞在肾小球滤过屏障中起关键作用,特别是足细胞相邻足突构成的裂隙膜（slitcliaphragm,SD）[1-4]。依据对激素治疗的反虚可将Ns分为激素敏感型（SSNS）和激素耐药型（SRNS）[5]。     

足突裂孔隔膜相关蛋白分子研究进展

肾小球足细胞的足突裂孔隔膜是肾小球滤过屏障的重要结构 ,其特异的结构及功能蛋白分子是维持肾小球滤过屏障功能的基础 ,已获得的裂孔隔膜相关蛋白的信息资料增进了对足细胞及其足突裂孔隔膜生物学作用的认识 ,有利于进一步揭示蛋白尿的发生机制 ,为肾脏病基础与临床研究开辟了新的广阔的前景。     

糖尿病肾病血浆瘦素和血清胱抑素C、白细胞介素-18水平检测的临床意义

糖尿病是我国的常见病、多发病,其中相当一部分患者由于治疗不及时或其它原因出现微血管病变,特别是肾病的发生。对于糖尿病肾病的发生,早诊疗、早治疗是关键。肾小球滤过率（GFR）是对肾小球滤过功能评价的客观指标。菊粉清除率虽然可作为GFR评价的金标准（参考方法）,但是由于复杂费时,偎难作为常规法用于临床。     

血、尿β2-m及尿mAlb联检在DN早期诊断中的临床价值

糖尿病肾病（DN）是糖尿病最严重的并发症之一,是糖尿病致残、致死的重要原因。糖尿病对肾小球,肾小管的损伤一直是糖尿病肾病研究的重点。约30％～40％的1型糖尿病和5％～20％的2型糖尿病患者在病程高达（10—15）年间发生DN,因此DN的早期诊断是防止DN的关键。血、尿β2-m及尿mAIb能准确、简单、快捷地对肾小球滤过功能和肾小管重吸收功能进行早期评价,为此我们对2型糖尿病患者三项指标联检来探讨三者对糖尿病肾病早期诊断的临床价值。     

血清CysC在早期DN中的诊断价值

Grubb于1985年首先报道了血清胱氨酸蛋白酶抑制剂C（serum cystatin C，Cys C）和视黄醇结合蛋白（retinol—binding protein RBP）与肾小球滤过功能和肾血流量相关密切，可以作为评价肾小球滤过功能的指标。为评价上述检测项目在早期糖尿病肾病（DN）诊断中的价值，本文分别对100例糖尿病患者和100例健康体检者的血Cys C、RBP、Sur和Scr进行检测分析，目的在于评价Cys C在早期DN诊断中的临床价值。     

血清β2-MG、基质金属蛋白酶与经颅多普勒超声在糖尿病肾病患者诊断中的相关性探讨

糖尿病肾病(DN)是糖尿病(DM)最严重和最常见的慢性并发症之一,其早期诊断和早期治疗对提高患者生存质量尤为重要。β2-微球蛋白(β2-MG)是一种低分子量蛋白质,容易透过肾小球滤膜,但几乎全部由近曲小管摄取,并在局部降解。血清β2-MG升高表明肾小球滤过率降低,可以反映肾小球和/或肾小管功能的损害情况[1]。DN发生时,细胞外基质     

肾脏足细胞裂孔隔膜的分子组成

足细胞裂孔隔膜结构的改变是导致肾小球疾病产生蛋白尿的主要原因 ,已陆续发现包括nephrin在内的与裂孔隔膜相关的多个蛋白分子 ,如ZO 1,CD2AP ,P cadherin ,podocin ,NEPH1,FAT及Filtrin等 ,它们的存在及相互作用对于保证裂孔隔膜的完整性和滤过功能有着直接或间接的作用 ,这些蛋白分子的发现有利于进一步阐明蛋白尿的发病机制 ,同时也为临床监测蛋白尿进展及制定治疗方案提供新的思路     

C反应蛋白及尿微量白蛋白与糖尿病肾病的关系探讨

在糖尿病肾病的发病过程中,炎症因子可能起重要作用.C反应蛋白(CRP)是一种敏感的非特异性的炎症标志物[1-2],它反映炎症的严重程度.尿微量白蛋白(MA)是中分子量的肾小球蛋白,在糖尿病肾病早期,肾小球滤过增多,且肾小管不易对其吸收,排除增加.RP、MA的升高与糖尿病肾损伤的程度存在一定的关系.本文对糖尿病患者血清CRP、MA进行检测,以探讨CRP和MA与糖尿病肾病的关系.     

Changes in the expression of nephrin gene and protein in experimental diabetic nephropathy

Diabetic nephropathy is a major complication of diabetes leading to thickening of the glomerular basement membrane, glomerular hypertrophy, mesangial expansion, and overt renal disease. The pathophysiologic mechanisms of diabetic nephropathy remain poorly understood. Nephrin is a recently found podocyte protein crucial for the interpodocyte slit membrane structure and maintenance of an intact filtration barrier. Here we have assessed the role of nephrin in two widely used animal models of diabetes, the streptozotocin model of the rat and the nonobese diabetic mouse. In both models, the expression levels of nephrin-specific mRNA as determined by real-time quantitative polymerase chain reaction increased up to two-fold during several weeks of follow-up. Immunohistochemical stainings revealed nephrin also more centrally within the glomerular tuft along with its preferential site in podocytes. Interestingly, as detected by immunoblotting, nephrin protein was also found in the urine of streptozotocin-induced rats. We conclude that nephrin is connected to the early changes of diabetic nephropathy and thus may contribute to the loss of glomerular filtration function.     

Increased dynamin expression precedes proteinuria in glomerular disease

Dynamin plays an essential role in maintaining the structure and function of the glomerular filtration barrier. Specifically, dynamin regulates the actin cytoskeleton and the turnover of nephrin in podocytes, and knocking down dynamin expression causes proteinuria. Moreover, promoting dynamin oligomerization with Bis-T-23 restores podocyte function and reduces proteinuria in several animal models of chronic kidney disease. Thus, dynamin is a promising therapeutic target for treating chronic kidney disease. Here, we investigated the pathophysiological role of dynamin under proteinuric circumstances in a rat model and in humans. We found that glomerular Dnm2 and Dnm1 mRNA levels are increased prior to the onset of proteinuria in a rat model of spontaneous proteinuria. Also, in zebrafish embryos, we confirm that knocking down dynamin translation results in proteinuria. Finally, we show that the glomerular expression of dynamin and cathepsin L protein is increased in several human proteinuric kidney diseases. We propose that the increased expression of glomerular dynamin reflects an exhausted attempt to maintain and/or restore integrity of the glomerular filtration barrier. These results confirm that dynamin plays an important role in maintaining the glomerular filtration barrier, and they support the notion that dynamin is a promising therapeutic target in proteinuric kidney disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Nephrin Localizes at the Podocyte Filtration Slit Area and Is Characteristically Spliced in the Human Kidney

Defects in the newly reported gene NPHS1 in chromosome 19 cause the massive proteinuria of Finnish type congenital nephrotic syndrome (CNF). Together with its gene product, nephrin, NPHS1 is providing new understanding of the pathophysiological mechanisms of glomerular filtration. Here we show the characteristic splicing of NPHS1 mRNA in the normal and CNF kidneys and localize nephrin exclusively in the glomerulus and to the filtration slit area by light and immunoelectron microscopy. These results indicate that nephrin is a new protein of the interpodocyte filtration slit area with a profound role in the pathophysiology of the filtration barrier.     

Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction

Semaphorin-3A (Sema3a), a guidance protein secreted by podocytes, is essential for normal kidney patterning and glomerular filtration barrier development. Here, we report that podocyte-specific Sema3a gain-of-function in adult mice leads to proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Reversibility of the glomerular phenotype upon removal of the transgene induction provided proof-of-principle of the cause-and-effect relationship between podocyte Sema3a excess and glomerular disease. Mechanistically, excess Sema3a induces dysregulation of nephrin, matrix metalloproteinase 9, and αvβ3 integrin in vivo. Sema3a cell-autonomously disrupts podocyte shape. We identified a novel direct interaction between the Sema3a signaling receptor plexinA₁ and nephrin, linking extracellular Sema3a signals to the slit-diaphragm signaling complex. We conclude that Sema3a functions as an extracellular negative regulator of the structure and function of the glomerular filtration barrier in the adult kidney. Our findings demonstrate a crosstalk between Sema3a and nephrin signaling pathways that is functionally relevant both in vivo and in vitro.The glomerular filter is a size-selective barrier composed of three layers: fenestrated endothelium, glomerular basement membrane (GBM), and podocyte foot processes.¹ Disruption of any of these components of the glomerular filtration barrier causes loss of permselectivity, proteinuria, and glomerular disease.¹ Podocyte foot processes are linked by slit diaphragms, which are modified adherens junctions composed of extracellular domains of nephrin molecules associated to a multiprotein complex.^2,3 Gene mutations in slit-diaphragm proteins and their actin-associated proteins cause familial nephrotic syndrome.^4–7 The GBM is a complex of type IV collagen (α3, α4, and α5) and laminin 521 (α5β2γ1) chains, perlecan, syndecan, entactin, and agrin. Imbalance of collagen and laminin chain expression results in abnormalities of GBM ultrastructure and proteinuria.^8,9 Loss of glomerular endothelial fenestration due to inhibition of vascular endothelial growth factor (VEGF-A) signaling or to excess soluble Flt-1 causes proteinuria and preeclampsia.^10,11Semaphorin-3A (Sema3a) is a secreted guidance protein involved in axon pathfinding and in cardiovascular, lung, and kidney patterning.^12,13 In the normal kidney, Sema3a is expressed in podocytes and collecting ducts.⁷ Loss-of-function studies during kidney development showed that Sema3a inhibits endothelial cell migration into glomeruli and limits ureteric bud branching.^14,15 Sema3a gain-of-function during development resulted in glomerular hypoplasia, delayed podocyte differentiation, and absent slit diaphragms.¹⁵ Exposure of cultured podocytes to recombinant Sema3a induced down-regulation of podocin and decreased the interactions among nephrin, podocin, and CD2AP.¹⁶ Systemic administration of Sema3a to adult mice induced transient, reversible foot-process effacement and proteinuria similar to that induced by protamine sulfate.^17,18 We observed increased podocyte Sema3a protein and mRNA expression in mice with diabetic nephropathy.^13,19 Taken together, our previous studies suggested that excess Sema3a might disrupt the glomerular filtration barrier in the mature kidney, particularly in the setting of diabetes.The goal of the present study was to define whether excess podocyte Sema3a per se causes glomerular disease in adult mice, and to examine the mechanism involved. Here, we report that induction of podocyte-specific Sema3a overexpression in adult mice causes a proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Mechanistically, we show that excess Sema3a induces dysregulation of nephrin, MMP-9, and αvβ3 integrin in vivo, and we identify a novel interaction between the Sema3a signaling receptor plexinA₁ and nephrin that links Sema3a signals to the slit-diaphragm signaling complex. Collectively, these findings establish that Sema3a functions as an extracellular negative regulator of the integrity and function of the glomerular filtration barrier.     

Possible role of autoantibodies against nephrin in an experimental model of chronic graft-versus-host disease

Nephrin, a product of the NPHS1 gene, is a component of the slit diaphragms that are found between glomerular foot processes and is a crucial element for glomerular filtration barrier. Recently, nephrin has been focused in a number of studies of proteinuria development including various types of acquired glomerular diseases including minimal change nephrotic syndrome and membranous nephropathy. However, the precise role of nephrin in such acquired glomerular diseases is still unknown. To analyse the role of nephrin further, two kinds of anti-nephrin antibodies were raised in the rabbits and applied to an experimental mouse model of chronic graft-versus-host disease, in which (C57BL/10 x DBA/2) F1 mice developed clinically apparent severe proteinuria with significant glomerular lesions 7 weeks after parental DBA/2 cell transfer. Antibody-sandwich ELISA detected anti-nephrin antibodies during week 2 to week 6, with the peak at week 2 or week 4. Colocalization of nephrin and IgG on week 4, week 6, and week 8 was revealed by confocal microscopic analysis, suggesting that in situ immune complex formation with nephrin in glomerular lesion. Taken together, it seems to be suggested nephrin and its autoantibody have a certain role in the development of glomerular lesion in our model mice.     

Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors

Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.     

Podocyte-associated molecules in puromycin aminonucleoside nephrosis of the rat

Molecules of central functional significance for the glomerular podocytes are rapidly emerging and have been shown to be distinctly involved in diseases with altered glomerular filtration barrier. Here we used the puromycin aminonucleoside (PA) nephrosis model in the rat to study some key proteins associated with the maintenance of the functional glomerular filtration barrier in vivo. The molecules studied included the filtration slit component nephrin, the hairpin-like membrane protein podocin, the basolateral adhesion molecules beta1 integrin and alpha-dystroglycan, and the cytoskeleton-linking intermediary beta-catenin and the actin-associated alpha-actinin-4. The results showed diminished protein levels of podocin and nephrin in the PA-treated group. beta-catenin showed distinct down-regulation at 3 days of induction, and the control level was reached at 10 days. beta1 integrin was markedly up-regulated during induction. alpha-actinin-4 was not changed at the studied time points. The results show distinct differences in the different domains of podocytes during PA-induced proteinuria.     

Nephrin—signature molecule of the glomerular podocyte?

In recent years there has been an explosion of interest in the glomerular podocyte, which plays a central role in control of glomerular filtration. A host of new molecules have been identified as playing essential roles in the maintenance of podocyte integrity in both humans and mouse models. Of all of these, arguably the most pivotal is nephrin, a transmembrane receptor molecule located at the specialized podocyte cell–cell junction, termed the slit diaphragm. Mutations in this gene cause the most severe form of congenital nephrotic syndrome, and many interacting proteins have now been described to form a large multiprotein complex with complex dynamics. There is little evidence of functional nephrin expression outside the glomerulus, and there are accumulating data that nephrin is essential for the unique properties of podocyte biology. Utilizing a powerful human cell culture model, comparing wild‐type with nephrin‐null podocytes, we can show that several crucial functional properties of podocytes depend on nephrin, including insulin responsiveness and cytoskeletal reorganization. Thus, it is reasoned that nephrin is a signature molecule required to define distinct podocyte characteristics. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genetics of nephrotic syndrome: new insights into molecules acting at the glomerular filtration barrier

Nephrotic syndrome is caused by increased permeability of the glomerular filtration barrier for macromolecules. The identification of mutations of various podocyte-expressed proteins as causes of familial nephrotic syndrome has significantly contributed to shedding light into the molecular pathogenesis of nephrotic proteinuria and into the physiology of the glomerular sieve. More recent findings have changed our conception of the glomerular filtration barrier from a relatively static structure to a highly dynamic one. Both the multiprotein slit diaphragm complex around nephrin and the integrin receptor complex that mediates binding of the podocyte to the glomerular basement membrane, may translate outside–inside signaling and lead to podocyte actin cytoskeleton rearrangement. This may enable the podocyte network to adapt to environmental changes and respond to injury. Disturbance in these processes may not only be involved in the pathogenesis of hereditary nephrotic syndrome but also in that of more common acquired proteinuric diseases. Elucidation of the molecular mechanisms involved will possibly open the way to new therapeutic approaches.