缺血再灌注对大鼠肾脏足细胞podocin的表达影响及意义

目的:观察SD大鼠缺血再灌注肾损伤足细胞表面相关蛋白(podocin)表达的变化,探讨podocin分子在足细胞损伤中的作用.方法:60只雄性SD大鼠随机分为假手术组(S组)30只和模型组(M组)30只,各组又分为lh、3h、6h、12h、24h、48h六个亚组,每个亚组各5只.采用Westemblot蛋白印迹法检测肾皮质足细胞podocin蛋白表达.并观察各组血肌酐、尿素氮的变化.结果:与S组相比,肾缺血再灌注损伤后,M组尿素氮于6h开始升高,且48h达高峰(P＜0.05);血肌酐48 h才升高(P＜0.05).Podocin 蛋白在3h表达开始下降,以后随缺血再灌注时间的延长podocin蛋白的表达逐渐下降,且48h组下降最明显(P＜0.01).结论:(1)缺血再灌注肾损伤时大鼠肾功明显发生改变,尿素氮早期开始升高,血肌酐晚期升高明显,说明造模成功.(2)大鼠肾脏足细胞podocin的蛋白表达随着缺血再灌注时间的延长而改变,早期下降不明显,3h组开始逐渐下降,48h组下降最明显.此结果表明podocin蛋白在肾脏缺血再灌注肾损伤表达减少.我们可以推想在缺血再灌注肾损伤时,podocin随缺血时间延长而表达逐渐减少的同时,也破坏了与足细胞相关蛋白之间的联系,这种关系的破坏最终导致足细胞受损.通过对足细胞表面蛋白podocin的研究,对将来肾脏足细胞损伤的防治提供新的思路.     

Podocin蛋白对肾小球足细胞的影响

目的：探讨肾小球足细胞裂隙膜蛋白podocin对足细胞形态及蛋白尿的影响.方法：32只体重160g～220g的雄性SD大鼠按阿霉素给药剂量随机分成小剂量组（3.0mg/Kg）、肾病剂量组（7.5mg/Kg）、超剂量组（10.0mg/Kg）、正常对照组.于给药第三周末处死大鼠,用氯化苄乙氧铵比浊法检测大鼠24h尿蛋白量,用免疫胶体金电镜检测大鼠肾小球蛋白podocin的表达和足细胞形态.结果：阿霉素组大鼠24h尿蛋白排泄量明显高于正常对照组,尤以肾病组最明显（P＜0.05）;正常对照组大鼠podocin分布在靠近肾小球基底膜（GBM）的足突基底部,主要定位于裂隙隔膜的胞质面,部分金颗粒也可发现于GBM稍远的足突细胞表面.肾病组肾小球足突广泛融合,免疫胶体金颗粒几乎见不到;超剂量组和小剂量组podocin分布在靠近肾小球基底膜（GBM）的足突基底部,免疫胶体金颗粒数明显少于正常对照组,有部分足突退缩.结论：（1）podocin表达减少或消失可能是导致肾小球足突细胞融合的关键因素（2）蛋白尿的发生与肾小球足细胞裂隙膜蛋白podocin的减少或缺失有关.     

C-terminal oligomerization of podocin mediates interallelic interactions

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3′ mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283–382): principally through the first C-terminal helical region (H1, 283–313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332–348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r²?=?0.68, P?=?9.2?×?10^?32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.     

Nephrin expression is increased in anti-Thy1.1-induced glomerulonephritis in rats

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3-7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.     

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin^P118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2^P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin^P118L and MEC-2^P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.     

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte β1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin β1 (podocin-Cre β1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre β1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin β1 activity in epithelial cells. To further explore whether integrin β1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte β1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.     

Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C‐terminal phosphorylation as a determinant of slit diaphragm complex architecture

Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross‐species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross‐species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph‐1 (Kirrel). Moreover, cross‐species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 ( http://proteomecentral.proteomexchange.org/dataset/PXD001005 ).     

Structural features and oligomeric nature of human podocin domain

Podocytes are crucial cells of the glomerular filtration unit and plays a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and charge-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101–125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126–350). We show that the podocin domain majorly homo-oligomerizes into a 16-mer. Circular dichroism and fluorescence spectroscopy suggest that the 16-mer oligomer has considerable secondary structure and moderate tertiary packing.