María Gabriela Márquez

Cell proliferation and morphometric changes in the rat kidney during postnatal development.

We have investigated the temporal maturation of the rat kidney during the postnatal developmental period. As a result, we observed the following: an active process of cortical cell proliferation and differentiation occurs as late as day 20. The medulla is the most immature zone at birth and displays the greatest morphological changes during this period. At birth, no distinction exists between inner and outer medulla, and the outer and inner strip of the outer medulla can be distinguished as late as day 30. Remodeling of the ECM surrounding collecting ducts occurs in the medulla twice, stopping at day 11 and it occurs in the papilla three times, stopping at day 20. The increase of kidney size is temporally different for each kidney zone. The cortex and the papilla acquire the morphological appearance of the adult kidney before the medulla does. Consequently, the medulla remains at the highest degree of immaturation among the kidney zones for a relatively long postnatal period.

The Effect of Testosterone on the Immune Response 1.-Mechanism of Action on Antibody-Forming Cells

Sex hormones are known to be implicated in humoral and cellular immune responses. In this study we report the effects of orchidectomy and testosterone replacement on the immune response using T-dependent and T-independent antigens. It was found that the response was dependent on the nature of the antigen employed and on the presence of testosterone. The absence of testosterone receptors in spleen lymphocytes was also found. An hypothesis that testosterone regulates the immune system through the enhancement of suppressive activity is advanced.

Developmental study of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT) from Wistar rats

The aim of the present report was to study in growing Wistar rats the development of immunocompetent cells in the bronchus-associated lymphoid tissue (BALT). We found at day 4 postpartum, a high number of TCRgamma/delta+ T cells and very few CD8alpha+, CD8beta+, CD5+, TCRalpha/beta+ T cells in BALT. The latter cells and CD4+ T cells increase with age. Even though T cells expressing TCRgamma/delta outnumber those expressing TCRalpha/beta early in development, until 45 days of age, alpha/beta+ predominate over gamma/delta+ T cells only in adult rats (60 days of age). Moreover, a predominance of suppressor/cytotoxic T cells over T-helper cells was found in 60 days old rats. Surprisingly, more CD8alpha+ than CD8beta+ T cells in BALT are observed. The number of IgA+ B and CD4+ T cells found in the BALT increases with age. The early appearance - 4 days of age - of all T-cell phenotypes in BALT especially of gamma/delta+ T cells may imply a benefit to respond to inhaled antigen soon after birth.

Compartmentalisation between gut and lung mucosae in a model of secondary immunodeficiency: effect of thymomodulin.

Compartmentalisation of mucosal immune response seems to be the result mainly of the preferential migration of activated cells back to their inductive sites. The aim of this report was to demonstrate, in a model of secondary immunodeficiency in Wistar rats (severely protein deprived at weaning and refed with casein 20 %; group R21), that the oral administration of Thymomodulin (group: R21TmB) has different effects on gut and BALT (Bronchus-associated lymphoid tissue). Tissue sections (5μ) were studied by immunohistochemistry 1). The oral administration of Thymomodulin restores only in gut Lamina propria (LP) the IgA B and CD4 T cell populations to control levels. The CD8a and CD25 subpopulations do not vary in gut as they return to control levels when refed with 20% casein diet. All the populations mentioned above remained decreased even after receiving Thymomodulin by the oral route. However, the same behaviour was observed for the TCRδ T cells that were decreased and return to normal levels in both mucosae by the effect of the immunomodulator; 2) when studying the iIEL (intestinal intraepithelial lymphocytes) CD8α, CD25 and TCRγδ T cells, that were increased in R21, return to control levels in R21TmB. In BALT intraepithelium CD8α and CD25 T cells remained decreased, while only TCRγδ T cells (increased in R21) return to control values. Conclusions: 1) there exists a compartmentalisation between both mucosae, as T CD4+ and IgA B+ cells are restored by TmB only in gut; 2) only those iIEL involved in inflammation (CD8α+/CD25+ and TCRγδ+/CD25+) are normalised by means of the Thymomodulin 3) however, in BALT, only TCRγδ+ T cells are restored 4) the oral administration of the present immunomodulator may be useful as a therapeutic agent, although the preferential survival in the tissue of initial stimulation is the major factor in the preferential distribution of activated cells.

Changes in membrane lipid composition cause alterations in epithelial cell-cell adhesion structures in renal papillary collecting duct cells.

In epithelial tissues, adherens junctions (AJ) mediate cell-cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell-cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell-cell adhesion structures in renal papillary collecting duct cells.

Stratification of Sphingosine Kinase-1 Expression and Activity in Rat Kidney

Sphingosine-1-phosphate, the product of sphingosine kinase (SK) activity, is a sphingolipid metabolite that regulates cell growth, survival and migration. It is also known to affect diuresis, natriuresis and renovascular contraction in rats, although the mechanisms through which it affects these processes are not known. No previous report has addressed the differences among the kidney zones regarding endogenous SK expression and activity. Therefore, we examined SK1 distribution and activity in the various kidney zones: cortex, medulla and papilla. We found that SK1 expression does not correlate with enzyme activity. Study of the expression showed that the enzyme is highly expressed in cortex, followed by medulla and papilla. However, medulla had the highest enzyme activity. In all kidney zones, SK1 expression was mainly cytosolic. Regarding enzyme activity, whereas we found no difference between cytosol, membrane and nucleus in renal medulla, the membrane-bound enzyme presented the highest activity in cortex and papilla. SK1 distribution observed by immunohistochemical staining showed higher expression in cortical proximal convoluted epithelial cells. In medulla, immunostaining was observed as patches of staining, whereas in papilla, positive immunostaining was exclusively restricted to collecting duct cells. We also evaluated the effects of bradykinin and angiotensin II on SK1 activity.

Differential Branching of the Sphingolipid Metabolic Pathways with the Stage of Development

We have studied sphingomyelin metabolism in the papillae of neonatal (10-day-old) and adult (70-day-old) kidneys of male Wistar rats because sphingolipid second messengers generated by sphingomyelin metabolism are involved in cellular processes such as proliferation, differentiation and apoptosis. We showed that sphingomyelin and ceramide concentrations in homogenized rat papilla tissue increase with the time whereas sphingosine-1-phosphate content decreases. This is consistent with the finding of a higher biosynthesis of the latter sphingolipid in neonatal than in adult rat papillae. De novo synthetized ceramide was, however, higher in adult than in neonatal papilla homogenates probably accounting for the high ceramide content of the adult rat papilla. These results suggest an active de novo pathway not ending in sphingomyelin but instead arresting at ceramide in adult rat papilla tissue, partially metabolized into sphingosine-1-phosphate in neonatal rat papillae. The activity and the expression of sphingosine kinase, one of the enzymes involved in ceramide metabolism, was found to be higher in neonatal than adult rat kidney tissue. The intracellular distribution of sphingosine kinase was also different; in neonatal rat tissue the enzyme was predominantly associated with plasma membranes but it was cytosolic in adult rat papilla tissue. These findings seem to indicate that, in rat renal papillae, the developmental regulation of sphingosine kinase expression and activity addresses the sphingolipid metabolism to the formation of the proliferative metabolite sphingosine-1-phosphate in the neonatal period, and ceramide, which is associated with cell arrest and differentiation in the adult tissue. These data are consistent with the proliferative state necessary for tubular elongation during the neonatal period and the maintenance of the differentiated state in the adult tissue.

Physiologically induced restructuring of focal adhesions causes mobilization of vinculin by a vesicular endocytic recycling pathway.

In epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents.

Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2 in renal papillary collecting duct cells

Focal adhesions (FAs) are structures of cell attachment to the extracellular matrix. We previously demonstrated that the intrarenal hormone bradykinin (BK) induces the restructuring of FAs in papillary collecting duct cells by dissipation of vinculin, but not talin, from FAs through a mechanism that involves PLCbeta activation, and that it also induces actin cytoskeleton reorganization. In the present study we investigated the mechanism by which BK induces the dissipation of vinculin-stained FAs in collecting duct cells. We found that BK induces the internalization of vinculin by a noncaveolar and independent pinocytic pathway and that at least a fraction of this protein is delivered to the recycling endosomal compartment, where it colocalizes with the transferrin receptor. Regarding the reassembly of vinculin-stained FAs, we found that BK induces the formation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-enriched vinculin-containing vesicles, which, by following a polarized exocytic route, transport vinculin to the site of FA assembly, an action that depends on actin filaments. The present study, which was carried out with cells that were not genetically manipulated, shows for the first time that BK induces the formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2, which transport vinculin to the site of FA assembly. Therefore, the modulation of the formation of these vesicle-like structures could be a physiological mechanism through which the cell can reuse the BK-induced internalized vinculin to be delivered for newly forming FAs in renal papillary collecting duct cells.

The inhibition of sphingomyelin synthase 1 activity induces collecting duct cells to lose their epithelial phenotype

Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.