Demonstration of human kidney differentiation antigens with monoclonal antibodies

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.     

Developmental modifications of sheep kidney antigens

We have used monoclonal antibodies to study the changes in the expression of four kidney antigens during organogenesis in the sheep. Two of these antibodies, EE24.6 and EJ30.1, label intensely only the adult kidney, whereas the other two, EK17.1 and EJ15.1, bind to the extracellular matrix of the embryonic kidney. For EJ15.1, the staining of the extracellular matrix decreases temporarily during the second half of intrauterine life, a period during which a light staining appears in the mesangium. For the other, EK17.1, the extracellular matrix staining in the stroma gradually decreases as the embryo grows, while staining of the mesangium and the arterial intima becomes evident. With EK17.1, fibronectin is identified in the extracellular matrix of the embryonic kidney and intracellularly in the mesangial cells after these cells have colonized the glomerulus. The mesonephros staining seems to be the same as that of the metanephros.In the adult, extraglomerular vascular endothelial cells bind EK17.1, whereas intraglomerular endothelial cells do not express fibronectin, which suggests a functional difference between endothelial cells in these two localizations.     

Antigenic study of the differentiation of the human kidney using monoclonal antibodies

The production of monoclonal antibodies against human embryonic renal cells allowed to display on the adult human kidney some antigens typical of certain structures or tissues: the proximal convoluted tubule for EG 9-11 and EG 19-6 monoclonal antibodies, the glomerular basement membrane for EG 14-1, the urothelium for EE 24-6, the connective tissue for EK 8-1 and EK 17-1 and probably the capsular and tubular basement membranes for EK 8-1. Simultaneously, we could follow the spatial and temporal repartition of the antigens during the renal development. One of them (EI 16-1) seemed to disappear in the adult and might correspond to a foetal type-antigen.     

Changes in the protein spectrum of the chick embryo mesonephros and metanephros in the course of development

The protein spectra of two fractions (the soluble and the membrane fraction) of chick embryo kidney homogenates were isolated by electrophoresis on polyacrylamide gels with the aim of detecting the kidney differentiation process at the molecular level and, at the same time, of evaluating similarities in the construction of the mesonephros and metanephros at this level. Corresponding stages of the above two types of kidney were chosen for studying changes in protein structure during differentiation--i.e. the outset of differentiation (the 6-day mesonephros, the 11-day metanephros) and the stage of full maturity (the 14-day mesonephros, the 20-day metanephros). A total of 36 proteins was distinguished. The analysis of the protein spectra showed that the number of proteins changes but slightly during differentiation; the protein composition of the two types of kidney during differentiation altered by 20-35% of the total number of proteins; the similarity of the protein composition of the corresponding stages of mesonephros and metanephros, expressed as the proportion of the number of identical proteins, was greater than the mutual similarity of different developmental stages of the same type of kidney. The percentage of different proteins at corresponding stages of the kidneys varied from 5% to 23% of the total number of proteins detected.     

Peroxisomes in permanent and provisional kidneys

Peroxisomes in three forms of vertebrate kidney (pronephros, mesonephros, and metanephros), as permanent or provisional kidney, are summarized concerning their ultrastructure and developmental changes. Because the peroxisome is known to be diverse in mammalian metanephros, and species difference is its distinctive feature among cell organelles, information should be obtained on each kidney of each species. The ultrastructural and biochemical features of peroxisomes have at least been partly delineated in the metanephros and mesonephros, but nothing is known about the pronephros. Ultrastructural studies of the metanephric peroxisomes are present in mammals, birds, and reptiles, but information on their development is restricted to mammals and birds. As for the mesonephric peroxisomes, both ultrastructural and developmental data have been accumulating on mammals and amphibians, and ultrastructural information is present on fishes, but not on birds and reptiles. At present, studies on peroxisomes of provisional kidney have been restricted to mammalian mesonephros. The common features of renal peroxisomes previously examined are that they are spherical cell organelles with a single limiting membrane in ultrastructure, and are positive for catalase. Information on the ultrastructure and enzymes is not sufficient at present for comparing the ontogenesis of renal peroxisomes with their phylogenesis. Part of this study appeared as an original paper (11), and was presented as a poster at the International Symposium/CREST Research Conference, Peroxisome: Biogenesis, Function and Disease.     

Characteristics of the compensatory renal growth of the remnant embryonic chick kidneys

Growth of the remnant embryonic kidney (the mesonephros), as expressed by wet weight, was more rapid in the chick embryos with experimentally induced unilateral renal agenesis compared to controls. The difference was significant between embryonic days 8-12, when the doubled weights of remnant kidneys were increased compared with the weights of paired control kidneys. The excessive growth of the mesonephros ceased on day 14, when the normal physiological regression of the embryonic kidney begins. In the definitive kidney, the metanephros, no significant differences in weights of the control vs. remnant metanephros were found on days 10-14. The characteristics of increased mesonephric growth were evaluated by determination of DNA/protein ratios in homogenates of the kidneys. Significant cellular hypertrophy was found in both the mesonephros and metanephros of the embryos with URA on day 10. Additionally, a non-significant cellular hyperplasia was also revealed in the remnant mesonephros on day 8. This gives evidence that the growth stimuli to the mesonephroi were probably strongest between days 8-10 and that they manifested in the remnant mesonephros only.     

Polysialic acid and N-CAM localisation in embryonic rat kidney: mesenchymal and epithelial elements show different patterns of expression

The expression of polysialic acid (PSA) and the neural cell adhesion molecule (N-CAM) during the embryonic development of rat kidney was investigated using immunocytochemistry and immunoblotting. A monoclonal antibody (mAb 735), which recognised only long chain PSA, and polyclonal antibodies specific for N-CAM were employed. At the earliest stages of metanephros formation, PSA and N-CAM immunostaining was found in both embryonic anlagen, namely the uretic bud and the metanephrogenic mesenchyme. Reactivity in uretic bud derivatives declined during embryonic development and was generally absent in the collecting duct system by embryonic day 18 (E18). Uninduced metanephrogenic mesenchyme was immunostained throughout development while induced mesenchymal cells showed greatly increased PSA and N-CAM immunoreactivity during their transformation into epithelium. This staining declined rapidly as nephrons differentiated. These processes were preceded by sorting of PSA and N-CAM to the basolateral plasma membrane. Similar N-CAM and PSA patterns were observed in mesonephros development. In adult kidney parenchyma both PSA and N-CAM were undetectable. Immunoblotting of samples of embryonic kidney with mAb 735 revealed a broad band ranging from 140 to greater than 200 x 10(3) Mr. N-CAM antibodies revealed reactivity in a band of 140 x 10(3) Mr after removal of PSA by endoneuraminidase treatment. Expression of N-CAM and PSA in both embryonic anlagen indicates that neither molecular species acts primarily as an inductive signal. These molecules were localised in areas where changes in cell adhesion during organogenesis might be important and thus may be involved in the grouping of developing cells.     

Kidney development conserved over species: essential roles of Sall1

The kidney develops in three stages: pronephros, mesonephros, and metanephros. Molecular mechanisms underlying these three steps are similar, and we can thus examine genetic cascades occurring during development. The induction system for pronephros in vitro has been established in Xenopus. Using this system, we isolated Sall1 that is essential for the initial step in metanephros formation. The potential mechanisms involved and future directions regarding kidney development are discussed.     

Comparative immunohistochemical analysis of the expression of cytokeratins, vimentin and alpha-smooth muscle actin in human foetal mesonephros and metanephros

The human mesonephros is currently regarded as a simplified version of the foetal metanephros, primarily due to the close morphological resemblance between these two structures. The aim of the present study was to define whether human mesonephric and foetal metanephric nephrons share immunophenotypical traits in their corresponding structures (glomeruli, proximal and distal tubules). For this purpose we first investigated immunohistochemically the overall expression and topographical distribution of cytokeratins 7, 8, 18, 19, and 20, vimentin and -smooth muscle actin in mature mesonephric nephrons and compared the results with those obtained in maturing-stage foetal metanephric nephrons. No expression of cytokeratins 7 and 20 was found. Cytokeratins 8, 18, and 19 and vimentin showed a restricted and basically coincident expression along the different components of both mesonephric and metanephric nephrons. These findings indicate that the intermediate filament protein profile of human mature mesonephric nephrons closely recapitulates that observed in developing metanephros and thereby strengthens the concept that human mesonephros, a transient ontogenic structure, is largely similar to the foetal metanephros.The sole difference between human mesonephros and foetal metanephros was the divergent expression of -smooth muscle actin. This protein exhibited an increasingly accentuated mesangial expression paralleling the morphological maturation of metanephric glomerulus, whereas it was absent from the mesonephric one. This would suggest that the mesangial cells in these two renal structures have a different function during the foetal life.     

Lectin activity and distribution of chicken lactose lectin I in the extracellular matrix of the chick developing kidney

A lectin activity inhibitable by thiodigalactose, N-acetyllactosamine, lactulose, lactose and by an antibody raised against CLL I (chicken-lactose lectin I) has been investigated in the chick embryo developing kidney. At post-induction stages this activity was found in both mesonephros and metanephros. In immunofluorescence and immunoelectron microscopy, the extracellular distribution of CLL I was similar in the mesonephros and the metanephros. The lectin was never found intracellularly; cultured kidney cells did not express any endogenous lectin but were rich in lectin-receptor sites, which led to the hyphothesis that CLL I is not produced in situ but could be adsorbed on renal cells. Potential physiological roles for embryonic lectins are discussed.     

Involvement of FGF and BMP family proteins and VEGF in early human kidney development

The spatial and temporal pattern of the appearance of the fibroblast growth factor proteins (FGF-8 and FGF-10), the bone morphogenetic proteins (BMP-2/4 subfamily and BMP-7) and the vascular endothelial growth factor protein (VEGF) was investigated in the human mesonephros and metanephros of the 5-9 week-old conceptuses. In the mesonephros, both FGF's and BMP's were found in all structures and their expression slightly decreased in the early fetal period. VEGF positivity appeared in all mesonephric structures, and increased in the fetal period coincidently with formation of the mesonephric blood vessel network. In the metanephros, FGF-8 first appeared only in the metanephric mesenchyme, but from the 7th week on, its reactivity increased and spread to other metanephric structures. FGF-10 positive cells appeared in all metanephric structures already in the 5th week, and slightly intensified with progression of development. Cell survival and nephrogenesis in the permanent kidney might be associated with the appearance of both growth factors. Both BMP-2/4 and BMP-7 displayed a similar pattern of reactivity in all metanephric structures, and their reactivity intensified with advancing development. Alterations in their pattern of appearance might lead to the formation of small and dysplastic kidneys. Already in the earliest developmental stages, VEGF protein appeared in all metanephric structures. At later stages, VEGF showed more intense reaction in the collecting system than in the differentiating nephrons and interstitium. Due to VEGF involvement in vasculogenesis and angiogenesis, abnormal VEGF appearance might lead to impaired formation of the blood vessel network in the human permanent kidney.     

Ontogeny of calbindin-D28K and calretinin in developing chick kidney

The ontogeny of two calcium-binding proteins (calbindin-D_28k and calretinin) was studied by immunohistochemical techniques in developing chick kidney. This study showed the presence of calbindin on the 5th incubation day and calretinin on the 7th incubation day in mesonephric distal and connecting tubules, and in the medial wall of the Wolffian duct. At later stages, immunostaining for these two proteins, in particular for calretinin, was also demonstrated in some metanephric proximal tubules. Glomeruli and Bowman's capsules were negative both in the mesonephros and metanephros. The presence of calretinin in the developing kidney has thus been demonstrated for the first time. The early expression of calbindin and calretinin in mesonephric distal tubules suggests their role in regulating the final excretion of calcium. The different patterns of immunoreactivity of the walls of the Wolffian duct can be correlated with their different histogenetic and histological features.     

Hyaluronate and hyaluronidase in the developing chick embryo kidney

Changes in hyaluronate concentration and hyaluronidase activity were measured at progressive stages of chick embryo kidney development. Hyaluronate accumulates in the immature metanephros containing undifferentiated mesenchyme and early stages of tubular epithelium formation. Differentiation of the epithelium to mature tubules and renal corpuscles occurs over a period when hyaluronate concentrations are decreasing. The initial decrease in hyaluronate is accompanied by an increase in hyaluronidase activity in the metanephros. A similar peak of hyaluronidase activity was found to occur during mesonephros development. Chondroitin sulfate levels remain almost constant throughout the course of metanephric differentiation.     

Expression of intermediate filaments,EGF and TGF-α in early human kidney development

The spatial and temporal expression patterns of cytokeratins, vimentin, epithelial growth factor (EGF) and transforming growth factor alpha (TGF-α), were investigated in the 5–9-week old human mesonephros and metanephros. Vimentin was found in all mesonephric structures, while cytokeratins were seen only in the mesonephric tubules. EGF and TGF-α were detected early in all mesonephric structures, and immunoreactivity to both factors decreased in later stages. In the 5–6-week metanephros, vimentin immunoreactivity was found in all structures and later increased in the collecting system and interstitium. In the 5th week, cytokeratins 8 and 19 appeared in the ureteric bud and ampullae, and later showed increasing immunoreactivity in the collecting system and nephrons. The coexpression of intermediate filament proteins in metanephric development is a temporary feature and might be associated with mesenchymal to epithelial transformation of developing nephrons. In adult kidneys, such coexpression is associated with fibrosis or carcinomatous changes. At early stages, immunoreactivity to EGF and TGF-α was detected in all metanephric structures and from the 7th week onward, it decreased in differentiating nephrons. EGF and TGF-α patterns of appearance indicate their role in induction, proliferation and growth of metanephric structures. Disturbances in that pattern might cause reduction in kidney growth.     

Expression of aquaporin-1 (AQP1) in the adult and developing sheep kidney

The distributions of aquaporin-1 mRNA and protein were studied by hybridization histochemistry with a homologous riboprobe and immunohistochemistry, in the adult sheep kidney. Heaviest labelling occurred in the thin descending limb (DTL) of the loop of Henle in the inner stripe of the outer medulla, with apparent decreasing expression in the inner medulla, outer stripe of the outer medulla and cortex, but no quantitation was performed. Only proximal tubules (PT) (convoluted and straight) and DTL labelled. The glomerulus showed no labelling, consistent with the pattern in the rat but different to that in the human. During ontogeny, no labelling occurred in the mesonephros at 27 or 41 days of gestation (term = 145–150 days) but other structures did label at 27 days (heart, lung bud, blood vessels surrounding developing spinal cord). Labelling first occurred faintly in the metanephros at 41 days of gestation and increased throughout gestation consistent with morphological development of nephrons.     

Ultrastructural manifestations of the functional plasticity of the renal glomerular endothelium in vertebrates

Studied has been the fine structure of the kidney's glomerulus endotheliocyte of man (age 2-6 years) and fish (year-old Atlantic salmon Salmo salar L.), as organisms occupying extreme place in development of kidney in phylogenetic line of the vertebrates. Revealed has been the uniformity of geometric reconstruction of vascular endothelium of kidney's glomerulus of investigated objects under heterogeneous factors' influence (compensatory hyperfunction of man and environment influence of fish). Formation of star-shaped structures with large open fenestrae in its central part (diameter 100-150 nm) both in the local parts of endothelium and in space between them (diameter 250-350 nm) has been observed. Such construction peculiarity sharply increases permeability of capillaries of kidney's glomerulus and thus appears as a characteristic sign of strenuous functional activity of the filtration apparatus of metanephros of man and mesonephros of fish. Revealed reaction of endothelium is considered as universal and nonspecific.     

环境毒素对未成熟肾脏的影响

肾脏是人体主要的排泄器官,它的发育经过原肾、中肾、后肾3个阶段。外界环境中的化学物质、生物及物理因素的毒性作用可损害未成熟肾脏的发育,并严重影响胚胎和儿童的正常发育和身体健康。本文就影响肾脏血流动力学的药物、有机阴离子和有机阳离子、真菌毒素、环境污染物等具体毒素对未成熟肾脏的影响及其临床表现的研究进展作一综述。