Lecture: New light on the role of claudins in the kidney

The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria and nephrolithiasis. This review describes recent findings on the physiological function of claudins underlying paracellular transport mechanisms with a focus on renal Ca(2+) handling. We have uncovered a molecular mechanism underlying paracellular Ca(2+) transport in the thick ascending limb of Henle (TAL) that involves the functional interplay of three important claudin genes: claudin-14, -16 and -19, all of which are associated with human kidney diseases with hypercalciuria, nephrolithiasis and bone mineral loss. The Ca(2+) sensing receptor (CaSR) signaling in the kidney has long been a mystery. By analyzing small non-coding RNA molecules in the kidney, we have uncovered a novel microRNA based signaling pathway downstream of CaSR that directly regulates claudin-14 gene expression and establishes the claudin-14 molecule as a key regulator for renal Ca(2+) homeostasis. The molecular cascade of CaSR-microRNAs-claudins forms a regulatory loop to maintain proper Ca(2+) homeostasis in the kidney

The kidney tight junction

The tight junction is an important subcellular organelle which plays a vital role in epithelial barrier function. Claudin, as the integral membrane component of tight junctions, creates a paracellular transport pathway for various ions to be reabsorbed by the kidneys. This review summarizes advances in claudin structure, function and pathophysiology in kidney diseases. Different claudin species confer selective paracellular permeability to each of three major renal tubular segments: the proximal tubule, the thick ascending limb of Henle’s loop and the distal nephron. Defects in claudin function can cause a wide spectrum of kidney diseases, such as hypomagnesemia, hypercalciuria, kidney stones and hypertension. Studies using transgenic mouse models with claudin mutations have recapitulated several of these renal disease phenotypes and have elucidated the underlying biological mechanisms. Modern recording approaches based upon scanning ion conductance microscopy may resolve the biophysical nature of claudin transport function and provide novel insight into tight junction architecture

A Search for Spectral Galaxy Pairs of Overlapping Galaxies based on Fuzzy Recognition

The Spectral Galaxy Pairs (SGPs) are defined as the composite galaxy spectra which contain two independent redshift systems. These spectra are useful for studying dust properties of the foreground galaxies. In this paper, a total of 165 spectra of SGPs are mined out from Sloan Digital Sky Survey (SDSS) Data Release 9 (DR9) using the concept of membership degree from the fuzzy set theory particularly defined to be suitable for fuzzily identifying emission lines. The spectra and images of this sample are classified according to the membership degree and their image features, respectively. Many of these 2nd redshift systems are too small or too dim to select from the SDSS images alone, making the sample a potentially unique source of information on dust effects in low-luminosity or low-surface-brightness galaxies that are underrepresented in morphological pair samples. The dust extinction of the objects with high membership degree is also estimated by Balmer decrement. Additionally, analyses for a series of spectroscopic observations of one SGP from 165 systems indicate that a newly star-forming region of our Milky Way might occur.Comment: 16pages, 6figure

Biochemical and biophysical analyses of tight junction permeability made of claudin-16 and claudin-19 dimerization

The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions—two claudins that play key polygenic roles in fatal human renal disease, FHHNC—we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision

Encephalitic alphaviruses exploit caveola-mediated transcytosis at the blood-brain barrier for central nervous system entry

Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB

Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

新型冠状病毒SARS-CoV-2在全球蔓延，给全球公共卫生带来严重威胁。快速研制疫苗、抗体和治疗药物成为科学界面临的重大挑战。由于SARS-CoV-2的高度传染性，采用病毒感染模型进行中和抗体及小分子抑制剂的药效评估需要在高等级生物安全实验室中进行，且常需要数天时间才能完成检测，限制了抗体和药物筛选的效率。发展快速、可视、不依赖于活病毒的新冠病毒入胞检测探针和细胞模型，对于加速新冠病毒抗体和药物的研究有重要意义。夏宁邵教授团队通过CHO真核表达系统高效表达制备出C端融合抗酸荧光蛋白Gamillus的重组新冠病毒spike蛋白STG。STG经SEC分子筛和冷冻电镜确认呈现与天然病毒刺突高度相似的三聚体结构，且与ACE2有很高的亲和力（18.2nM）。STG具备良好的细胞相容性和荧光性质，研究者进一步开发了可定量测定感染恢复期血清、疫苗免疫血清中和抗体（入胞阻断抗体）水平的CSBT检测方法。除了抗体检测评估方面的应用外，该研究发展的探针和模型还可用于筛选分析抑制新冠病毒入胞及胞内转运的小分子化合物。 我校博士后张雅丽，博士生王邵娟、巫洋涛，博士后侯汪衡、袁伦志和深圳市第三人民医院沈晨光博士为共同第一作者。厦门大学夏宁邵教授、袁权教授、程通教授为该论文共同通讯作者。The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system,a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed.In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.This study was supported by National Natural Science Foundation of China (81993149041 for N.X.; 81902057 for Y.Z.; 81871316 and U1905205 for Q.Y.), the National Science and Technology Major Project of Infectious Diseases (No. 2017ZX10304402‐002‐003 for T.C. and No. 2017ZX10202203‐009 for Q.Y.), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003‐005‐003 for T.C.), the Science and Technology Major Project of Fujian (2020YZ014001), the Science and Technology Major Project of Xiamen (3502Z2020YJ01), and the Guangdong Basic and Applied Basic Research Foundation (2020A1515010368 for C.S.). 该研究得到了国家自然科学基金、传染病防治国家科技重大专项、福建省应急科技攻关项目和厦门应急科技攻关项目的支持