| Kidney injury molecule-1(Kim-1) also known as T cell Ig and mucin1(Tim-1), or hepatitis A virus cellular receptor-1(Havcr-1), is a type1transmembrane protein belonging to the immunoglobulin gene superfamily. Kim-1is undetectable in normal kidney but after acute injury it is induced abundantly in the proximal tubule, where it localizes to the apical surface of epithelial cells. Acting as a non-myeloid phosphatidylserine (PS) receptor, the Kim-1ectodomain binds and internalizes oxidized lipid as well as phosphatidylserine exposed on the outer surface of luminal apoptotic cells. In this way, Kim-1clears the tubule lumen of debris following kidney injury, aiding in nephron repair and tissue remodeling. However, Kim-1is also upregulated in a variety of animal models of CKD, including protein over-load nephropathy, adriamycin-induced nephropathy, angiotensin â…¡-induced renal damage and murine polycystic kidney disease, where it colocalizes with areas of fibrosis and inflammation. It is upregulated in human CKD and its expression correlates directly with interstitial fibrosis in human allografts. The epidemiologic association of AKI with future CKD and colocalization of Kim-1with area of inflammation and fibrosis suggests that it might play a pathogenic role in linking AKI to CKD and renal fibrosis. Transgenic mice with nephron-specific overexpression of Kim-1develop tubular damage, reduced nephron number, fibrosis, high mortality and cardiac hypertrophy. Kim-1rapidly, sensitive, specific injury and recovery process reflects a variety of kidney disease can be a reliable early detection of kidney injury biomarker has broad application prospects in clinical. However, the physiopathology role of KIM-1in the development of kidney disease is still unclear.Zebrafish (Danio rerio), a small tropical freshwater fish, has become an excellent vertebrate model for studying human disease, genetics and development19-20. Approximately70%of all human disease genes have structure and functional homologues in zebrafish21. Because of the anatomical simplicity of zebrafish pronephros (in larvae) and mesonephros (in adult fish), zebrafish have become a popular model for studying acute kidney injury (AKI), polycystic kidney disease (PKD), nephronophthisis and a range of ciliopathies. Rapid breeding and development, ease of genetic manipulation and optical transparency make zebrafish an ideal model organism to explore mechanisms of Kim-1mediated acute and chronic changes in the kidney.In this study, by using phylogenetic and genomic analysis, we characterized the zebrafish Kim gene family to consist of zKim-1, zKim-3and zKim-4. zKim-1shares a high degree of structural and functional homologies with mammalian Kim-1. By creating constitutive and conditional Kim-1transgenic fish, where Kim-1was specifically expressed in tubular epithelial cells, we demonstrate the pathological effects of prolonged overexpression of Kim-1in kidney tubular cells in pronephros as well as mesonephros.Chapter I Comparison of zKim family gene sequence identification of zKim-1splice variantsObjective To explore the distribution of Kim family genes in the zebrafish genome, lay the molecular foundation for the establishmen of Kim-1transgenic zebrafish.Methods Search Kim family gene sequences in a variety of public and cDNA databases, such as Ensembl human genome browser (Ensembl Genome Browser, http://useast.ensembl.org/Dani o_rerio/Info/Index); GenBank, the National Biotechnology information Center (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm. nih.gov/genbank) and the UCSC genome Browser (UCSC Genome Browser, http://genome.ucsc.edu) and so on. Using different program for thecomparsion of zebrafish genome humans and mammalian KIM family genes to identify the zebrafish Kim family geneswith higher degree of similarity score.Determination of the zebrafish Kim family genesby analyzing cDNA gene sequence and protein coding sequence analyze, and examination the structure and function of this proteins. By using rapid amplification of the target gene cDNA ends to identify the zKim-1splice variants.Results Located and confirmed three Kim family genes in zebrafishchromosome21, which are the zKim-1(EMBL accession number ENSDARG00000091692), zKim-3(ENSDARG00000077257), and zKim-4(ENSDARG00000040178) by using NCBI/BLAST, EMBL, Genbank and UCSC data projects. Homology analysis showed that the sequence and structure ofzKim-1, zKim-3, zKim-4shared the highly similarity with hKIM-1, hKIM-3, hKIM-4, respectively. Rapid Amplification of cDNA ends zKim-1showed the presence of three splice variants, named long chain zKim-1(zKim-l-L), short chain zKim-1(zKim-1-S) and immunoglobulinzKim-1(zKim-1-Ig).Conclusion1. Itwas first discovered and confirmed the distribution of KIM family genes in zebrafish genome.2. ZebrafishKIM familygenes sequence are highly homologous to human KIM family genes.3. Zebrafish KIM family genesshared the similar protein structure with human KIM family genes, including extracellular signal peptide,immunoglobulin-like domain, mucin domain and an intracellular tyrosine phosphorylation sites.4. ZKim-1present in three splice variants, which are the long-chain zKim-1(zKim-1-L), short chain zKim-1(zKim-1-S) and immunoglobulin zKim-1(zKim-1-Ig). Chapter â…¡ The tissues and organs distribution of zKim family genes expressing in zebrafish and the regulation of zKim-1expression and functional analysisObjective To analyze the distribution ofKIM family genesand zKim-1splice variants(long chain zKim-1, short chain zKim-1, immunoglobulin zKim-1) in different zebrafish tissues and organs. Base on the mechanism that KIM-1upregulated dramtically in proximal tubular epithelial cells after kidney injury, to investigate whether zKim-1share the same regulatory mechanism with human KIM-1expression. In vitro, to examine whether zKim-1share the similar protein function, acting as a non-myeloid phosphatidylserine (PS) receptor and engulfing apoptotic cells debris and oxidized lipid.Methods Real-time quantitative PCR analysis the distribution ofKim family genes and zKim-1splice variants (long chain zKim-1, short chain zKim-1, immunoglobulin zKim-1) in different tissues and organs. Base on the mechanism that KIM-1significantly upregulated in proximal tubular epithelial cells after kidney injury, determinate the zKim-1splice variants expression pattern in gentamicin induced acute kidney injury zebrafish model by using real-time quantitative PCR, immunofluorescence staining and immunohistochemical staining. In vitro, examine the phagocytosis function of zKim-1by coincubation of apoptosis cell debris and oxidation lipidwith zKim-1expressing cells and human KIM-1expressing cells,respectively.Results A relative small amount of zebrafish Kim family genesexpressing in various normal tissues and organs, where zKim-1expressing a little bit in the fin and a slightly expression of zKim-3in muscle. The expression of zKim-1was significantly increased after kidney tubular injury, while there are no significant changes on the expression of zKim-3and zKim-4in kidney (P<0.05). zKim-lalso can endocytosis of apoptotic cells debris and oxidation lipid.Conclusion1. In healthy and normal tissue, the expression of Kim family genesis undetectable or trackable.2. zKim-1shared the same expression regulatory mechanism as human KIM-1that zKim-1was significantly upregulaed in the proximal renal epithelial cells after kidney injury.3. In vitro, similar to human KIM-1,zKim-1can phagocytosis of oxidized lipid and clean the apoptotic cells debris.Chapter â…¢ Establishment of tissue-specific expression of Kim-1transgenic zebrafish model and phenotype identificationObjective To create a Kim-1special expressing in kidney tubular epithelial cells zebrafish model and investigate the effect of Kim-1expressing in zebrafish larvaepronephrons and mesonephrosin adult zebrafish.Methods Firstly, creating the expression plasmid vector where zKim-1are specific expressing in kidney tubular epithelial cells. Cloning zKim-1and fusing with red fluorescent protein (zKim-1-RFP) and subcloning to kidney tubular epithelial cells-specific expression vector plasmids by using some specific restriction sites.We using cadherin17(Cadherinl7, cdhl7), asakidney tubular epithelial cells-specific marker in zebrafish to drive zKim-1expressing in in zebrafish larvaepronephrons and mesonephrosin adult zebrafish. The kidney tubular epithelial cells specific zKim-1expressing vector plasmids was injected into one cell stage of zebrafish embryos by using nano injection under microscope.Real-time observation of zKim-1fusing red fluorescent protein expressing by using the red channel of fluorescence microscope. For the control groups, the cadherin17drive red fluorescent protein expressing vector plasmids was injected into one cell stage of zebrafish embryos where red fluorescent were directly expressing in kidney tubular epithelial cells without the expression of zKim-1. Identify and verify the Kim-1transgenic zebrafish model and control groupsby Western blot. The various physiological and pathological indicators were measured in Kim-1transgenic zebrafish model, including the response activity, pericardial and whole body edemain zebrafish larvae. Detect the kidney function by the measurement of the delay clearance of dextran which were injected in Kim-1transgenic zebrafish larvae and control groups. For the pathological changes, hematoxylin-eosin staining and immunofluorescence staining examine the expression of Kim-1and some correlated proteins in Kim-1transgenic zebrafish and control groups. Further, the microscopic pathology changes were detected by electron microscope in Kim-1transgenic zebrafish and control group.Results Compared with control groups, Kim-1transgenic zebrafish shows the less response activities. Kim-1transgenic zebrafish had significant pericardial and body edema, which is positively correlated to the severity expression of zKim-1in the kidney tubular cells. The kidney function was affected in Kim-1transgenic zebrafish, where the clearance of dextran was dramtically delayed. The survival of Kim-1transgenic zebrafish was significantly lower than the control groups,which is closely related to theseverity expression of zKim-1in kidney tubular cells.The Kim-1transgenic zebrafish pathology showed the atrophy tuble with lumendisappearance, swellen tubular epithelial cells with accumulation of vacuolar. Immunofluorescence staining showed the colocalization of zKim-1expression and active caspase3in Kim-1transgenic zebrafish. Electron microscopy showed the brush border loss in the tubular epithelial cells and accompanied by a large part of phagosome in the cytoplasm and karyopyknosis in Kim-1transgenic zebrafish.Conclusion1. Successfullycreated Kim-1transgenic zebrafish model for the first time.This models can be a potential biological pharmaceutical research tools that provides a new target for the treatment of human acute and chronic kidney disease.2. Kim-1transgenic zebrafish showed kidney injury in short and long term, which has important significance for the study of the effect of Kim-1in acute and chronic kidney diseasedevelopment.3. The kidney tubular epithelial cells-specific expression of Kim-1induced kidney injury may be associated with Kim-1act as PS receptor, which mediated phagocytosis of potentially toxic or non-toxic proteins from tubular lumen and caused cell stress.Chapter IV Creation of conditional Kim-1transgenic zebrafish model and pathophysiological role of sustainability Kim-1expressing inthe kidney injuryObjective Tocreate conditional Kim-1expressing in zebrafish kidney tubular epithelial cells and investigate the effect of Kim-1expression in zebrafish larvae pronephrons and mesonephros in adult zebrafish.Methods Establishd the conditional Kim-1transgenic zebrafish model by appling the principle of Cre mediated Loxp sites recombinase. First, cloning zKim-1cDNA and fusing with red fluorescent protein, subcloning into ubiquitin (ubiquitin, ubi) drive the special expression of Loxp sites, where the green fluorescent protein can switc to the red fluorescent protein expression by the Cre dependent recombinase activity. Here, we replaced the red fluorescent protein by zKim-1fusing red fluorescent protein to create the Kim-1with loxp sites expressing vector plasmids. Second, cloning Cre cDNA and subcloning into the cadherin17 drive tubular epithelial cell-specific expressing vector plasmids. The expression plasmids were injected into one cell stage of zebrafish embryos by microscopy nano-injection. Three months later, crossing these two transgenic fish lines to create the double transgenic zebrafsih, which the Cre mediated recombination of zKim-1overexpressing in kidney tubular epithelial cells. The physiology and pathology indicators were examined in this conditional Kim-1trangenic zebrafish, including the response activity, pericardial and whole body edema in zebrafish larvae. For the pathological changes, hematoxylin-eosin staining and immunofluorescence staining examine the expression of Kim-1and some correlated proteins in Kim-1transgenic zebrafish and control groups. Further, the microscopic pathologychanges were detected by electron microscope in Kim-1transgenic zebrafish and control groupResults Compared with control groups conditional Kim-1transgenic zebrafish had less response activity. These transgenic zebrafish developed pericardiak and body edema after the addition of the severity ofzKim-1expressing in the tubular epithelial cells. The survival analysis showed that conditional Kim-1transgenic zebrafish had lower survival rate than control groups. For the kidney pathology examination, it showed the similar pathologic changes to Kim-1transgenic zebrafish. The conditional Kim-1transgenic zebrafish also had irregular tuble with lumendisappearance, swellen tubular epithelial cells accompanied with vacuolization. Theterminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test showed that there were more TUNEL positive cell in conditional Kim-1transgenic zebrafish kidney. Electron microscopy furher showed the brush border loss in the tubular epithelial cells and accompanied by a large parts of phagosome in the cytoplasm and karyopyknosis in conditionalKim-1transgenic zebrafish.Conclusion1.Successfully established conditional Kim-1 transgenic zebrafish model, determinated the affect of Kim-1expressing in tubular epithelial cells without the development affects by expressing Kim-1in early stage of pronephrons.2. As a potential and powerful animal models, conditionalKim-1transgenic zebrafish can be used to study the the pathophysiology role of Kim-1under different background of kidney disease, such as diabetes and chronic proteinuria.3.It further comfirmed that persistent expression of Kim-1in tubular epithelial cells cause kidney injury, which is similar to result of overexpression KIM-1in mouse cause kidney injury and fibrosis. These conditional Kim-1transgenic zebrafish can be useful animal model for drug screening,applying to clinical targeted therapy. |
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