Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.     

Morphogenesis and ultrastructure of the peripolar cells in the mouse kidney.

Peripolar cells are located in the outer layer of the Bowman's capsule. They surround the vascular pole of the renal corpuscle and project into the urinary space. Morphologically they are characterized by the presence of secretory granules within their cytoplasm. In order to study their embryological development, we used 60 C57bl mice embryos (15th to 19th gestational day), 10 newborn mice (2 hours to 6 days old), 10 preadult mice (8-30 days old) and 4 adults (4 months old). Some granular cells, dispersed at the outer and inner layer of the Bowman's capsule, appear on the 17th gestational day. Later, these cells are found around the vascular pole of the renal corpuscle, located exclusively at the outer layer of the Bowman's capsule. Their granules are spherical and variously dense, they are surrounded by a membrane and their number increases progressively with time and reaches a maximum on the 4th postnatal day. Following that, there is a diminution and then their population stabilizes. By the end of the first month, there are only a few such cells (mean number 1 to 2). They become smaller and they always project into the urinary space.     

Gene expression profiling of the developing mouse kidney and embryo

The metanephros is formed from the reciprocal inductive interaction of two precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB). The UB induces MM to condense and differentiate forming the glomerulus and renal tubules, whilst the MM induces the UB to differentiate into the collecting tubules of the mature nephron. Uninduced MM is considered the progenitor cell population of the developing metanephros because of its potential to differentiate into more renal cell types than the UB. Previous studies have identified the phenotype of renal precursor cells; however, expression of candidate marker genes have not been analysed in other tissues of the murine embryo. We have assayed up to 19 candidate genes in eight embryonic tissues at five gestation stages of the mouse embryo to identify markers definitively expressed by renal cells during metanephric induction and markers developmentally regulated during kidney maturation. We then analysed their expression in other developing tissues. Results show Dcn, Hoxc9, Mest, Wt1 and Ywhaq were expressed at moderate to high levels during the window of metanephric specification and early differentiation (E10.5-E12.5 dpc), and Hoxc9, Ren1 and Wt1 expression was characteristic of mature renal cells. We demonstrated Cd24a, Cdh11, Mest, Scd2 and Sim2 were regulated during brain development, and Scd2, Cd24a and Sip1 expression was enriched in developing liver. These markers may be useful negative markers of kidney development. Use of a combination of highly expressed and negative markers may aid in the identification and removal of non-renal cells from heterogeneous populations of differentiating stem cells.     

The significance of perfusion fixation in the lysosomal histochemistry of the mouse and rabbit kidney

Summary In rabbit kidneys fixed by immersion into formol the Gomori-Barka's histochemical method for acid phosphatase was practically negative, while in perfusion fixed material a strong positive reaction was obtained. A similar observation was made in connection with the -glucuronidase activity of the mouse kidney.     

Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney.

Branching morphogenesis of epithelium is a common and important feature of organogenesis; it is, for example, responsible for development of renal collecting ducts, lung airways, milk ducts of mammary glands and seminal ducts of the prostate. In each case, epithelial development is controlled by a variety of mesenchyme-derived molecules, both soluble (e.g. growth factors) and insoluble (e.g. extracellular matrix). Little is known about how these varied influences are integrated to produce a coherent morphogenetic response, but integration is likely to be achieved at least partly by cytoplasmic signal transduction networks. Work in other systems (Drosophila tracheae, MDCK models) suggests that the mitogen-activated protein (MAP) kinase pathway might be important to epithelial branching. We have investigated the role of the MAP kinase pathway in one of the best characterised mammalian examples of branching morphogenesis, the ureteric bud of the metanephric kidney. We find that Erk MAP kinase is normally active in ureteric bud, and that inhibiting Erk activation with the MAP kinase kinase inhibitor, PD98059, reversibly inhibits branching in a dose-dependent manner, while allowing tubule elongation to continue. When Erk activation is inhibited, ureteric bud tips show less cell proliferation than controls and they also produce fewer laminin-rich processes penetrating the mesenchyme and fail to show the strong concentration of apical actin filaments typical of controls; apoptosis and expression of Ret and Ros, are, however, normal. The activity of the Erk MAP kinase pathway is dependent on at least two known regulators of ureteric bud branching; the GDNF-Ret signalling system and sulphated glycosaminoglycans. MAP kinase is therefore essential for normal branching morphogenesis of the ureteric bud, and lies downstream of significant extracellular regulators of ureteric bud development.     

Expression of lymphatic endothelium-specific hyaluronan receptor LYVE-1 in the developing mouse kidney

Our knowledge of the embryonic development of the lymphatic vessels within the kidney is limited. The aim of this study was to establish the time of appearance and the distribution of intra-renal lymphatic vessels in the developing mouse kidney by using the lymphatic marker, LYVE-1. Kidneys from embryonic day 12 (E12) to E18, from neonates at post-natal day 1 (P1) to P21, and from adults were studied. In the adult mouse kidney, LYVE-1 was expressed mainly in the lymphatic endothelial cells (LECs) and in a subset of endothelial cells in the glomerular capillaries. However, in the developing mouse kidney, LYVE-1 was also expressed transiently in F4/80⁺/CD11b^– immature macrophages/dendritic cells and in the developing renal vein. LYVE-1⁺ lymphatic vessels connected with extra-renal lymphatics were detected in the kidney at E13. F4/80⁺/CD11b^–/LYVE-1⁺ immature macrophages/dendritic cells appeared prior to the appearance of LYVE-1⁺ renal lymphatic vessels and were closely intermingled or even formed part of the lymphatic vascular wall. Prox1 was expressed only in the LYVE-1⁺ LECs from fetus to adult-hood, but not in LYVE-1⁺ endothelial cells of the developing renal vein and macrophages/dendritic cells. Thus, lymphatic vessels of the kidney might originate by extension of extra-renal lymphatics through an active branching process possibly associated with F4/80⁺/CD11b^–/LYVE-1⁺ macrophages/dendritic cells.     

Effect of hormones on hydrolase activities and DNA synthesis in kidney of the developing mouse

The postnatal development of brush border enzyme activities, namely maltase, trehalase, alkaline phosphatase, gamma-glutamyltranspeptidase, and leucylnaphthylamidase, as well as the ontogenic profile of DNA synthesis has been determined in the mouse kidney. In addition, these parameters were evaluated following daily administration of hormones during 3 days to 8-day-old mice. Insulin or epidermal growth factor induced a 34% increase of maltase activity over that of 11-day-old controls. Trehalase activity was precociously and significantly augmented by cortisone alone or combined with thyroxine (p less than 0.05), although thyroxine alone had no influence. Only epidermal growth factor had a significant effect on alkaline phosphatase activity. gamma-Glutamyltranspeptidase activity was significantly decreased when insulin and thyroxine were given simultaneously, but was not modified by any of the hormones injected separately. The level of leucylnaphthylamidase activity was enhanced by 70% after cortisone injection, but it was significantly reduced by thyroxine injected in combination with insulin or cortisone. The incorporation of [3H]thymidine into DNA was increased by 107% after epidermal growth factor administration, but it was decreased by 33% after the cortisone treatment. In spite of this precocious reduction, the level of incorporation was still 2 times higher than that in adult mice. These results show that hormones act separately or in cooperation to accelerate or retard the maturation of the suckling mouse kidney.     

Morphogenesis of ductal networks in the mouse prostate

Postnatal growth and development of the glandular architecture of the ventral and dorsolateral lobes of the mouse prostate (VP and DLP) were investigated by microdissection techniques that permitted precise quantification of the numbers of primary ducts emerging from the urethra, the terminal ductal tips, and ductal branch-points. At birth both the right and left lobe of the VP consisted of 1-3 main ducts that had already undergone secondary and tertiary branching. In contrast, at birth the more complex DLP had 9-12 unbranched main ducts per lobe on both the right and left sides. During the first 15 days after birth, 80.7% of tips and 76.4% of branch-points of the adult gland formed in the VP, and 70.4% of tips and 53.6% of the branch-points formed in the DLP. Ductal branching was completed by 60 to 90 days. The DLP developed in three stages: first, formation of unbranched main ducts (first 10 days); second, distal branching of each main duct resulting in 3-5 terminal branches per main duct (10-15 days after birth); third, elaboration of intraductal mucosal infolding in distal ducts after 30 days of age. Ducts of the lateral prostate (LP), a ventrolateral subdivision of the DLP, initiated branching morphogenesis between 1 to 5 days after birth. The LP grew into and became embedded within the capsule of the VP, which may explain why the ductal architecture of these two lobes are similar. These heretofore unrecognized differences in the organogenesis and morphology of the mouse VP and DLP and the striking morphological heterogeneity both between and within the lobes of the mouse prostate may be morphological manifestations of functional heterogeneities within the prostate.     

Studies on esterase histochemistry of amphibian kidney

Summary In the hope that the histochemical picture of the kidney may help to understand its role in excretion and osmoregulation, an effort is here made to study the distribution of esterases in amphibian kidney.The kidneys of adults and tadpoles of the frog, Rana tigrina and the toad, Bufo melanostictus were used for this study. Some of these animals were subjected to dehydration for 3–4 days and to the effect of 150 mM NaCl for 8–12 days before their kidneys were used. The esterases were visualised using tweens, naphthol esters and 5-bromoindoxyl acetate as substrates. These were accompanied by activator/inhibitor studies.Very interesting results were obtained in the distribution of the esterases. Tween esterase and -naphthyl acetate esterase were found in the proximal tubules of the adult frog kidney only while 5-bromoindoxyl acetate esterase was found to be present in all the animals tested. On the other hand, naphthol AS acetate esterase was absent in the tadpole stages of the frog and toad. Further 5-bromoindoxyl acetate esterase and naphthol AS acetate esterase were demonstrated in the glomeruli of frogs and toads subjected to NaCl solution. Activator/ inhibitor studies helped in characteristically differentiating these different esterases.There seems to be a relationship between the distribution of the different esterases and the excretory and osmoregulatory adaptations of these animals which differ in the adult and tadpole stages and in the experimental conditions mentioned. The possible implications of the esterase distribution is discussed in considerable details.U.G.C. Research Scholar.     

Enzyme histochemistry and hormones of the developing gastrointestinal tract of the chick embryo

Summary The oxidative enzymes are important in most metabolic processes. Their localisations in embryonic tissues may indicate the onset of important metabolic processes essential for both histodifferentiation and chemodifferentiation. The diphosphate nucleotide and the triphosphate nucleotide diaphorases have been demonstrated as early as the 8th day.Isocitric dehydrogenase, glucose-6-phosphate dehydrogenase and lactic dehydrogenase have been demonstrated histochemically in the epithelial cells and in muscle cells of both stomach-complex and intestine as early as the 6th day. The intensity of these histochemical reactions appears to increase with the age of the embryo reaching a maximum before hatching and indicating the onset of secretory and absorptive functions.     

Enzyme histochemistry and hormones of the developing gastrointestinal tract of the chick embryo

Summary The presence of 5-Hydroxytryptamine, entero-glucagon and insulin in the developing chick duodenum is demonstrated. High levels of enteroglucagon and insulin were found during early embryonic life, enteroglucagon and insulin were found to persist at low levels after hatching.     

Enzyme histochemistry and hormones of the developing gastrointestinal tract of the chick embryo

Summary Glucose-6-phosphate is an important metabolite which acts as the substrate linking three important metabolic cycles. The glycolytic pathway, the pentose cycle and the tricarboxylic acid cycle. The enzyme glucose-6-phosphatase which metabolises this substrate is therefore of key importance. This enzyme was found to fluctuate and to be highly sensitive to insulin during the differentiation of the gastrointestinal tract. The enzyme has the same localisation in the epithelium of the developing gut as glycogen, uridine diphosphate glucoseglycogen transglucosylase and phosphorylase. It increases in activity just before hatching when the absorptive functions of the gut start and may then assume this new function. The importance of uridine diphosphate glucoseglycogen transglucosylase and phosphorylase in the developing gastrointestinal tract is also discussed.     

Enzyme histochemistry and hormones of the developing gastrointestinal tract of the chick embryo

Summary During the differentiation of the gastrointestinal tract cell death occurs. In the present study the activity of lysosomal enzymes which may be associated with this process are investigated. These enzymes are leucine aminopeptidase, acid phosphatase and beta galactosidase. The action of glucagon on leucine aminopeptidase and acid phosphatase is reported.