Influence of sphingomyelin metabolism during epithelial-mesenchymal transition associated with aging in the renal papilla.

The renal collecting ducts (CD) are formed by a fully differentiated epithelium, and their tissue organization and function require the presence of mature cell adhesion structures. In certain circumstances, the cells can undergo de-differentiation by a process called epithelial-mesenchymal transition (EMT), in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell-cell adhesion. We have previously shown that in renal papillary CD cells, cell adhesion structures are located in sphingomyelin (SM)-enriched plasma membrane microdomains and the inhibition of SM synthase 1 activity induced CD cells to undergo an EMT process. In the present study, we evaluated the influence of SM metabolism during the EMT of the cells that form the CD of the renal papilla during aging. To this end, primary cultures of renal papillary CD cells from young, middle-, and aged-rats were performed. By combining biochemical and immunofluorescence studies, we found experimental evidence that CD cells undergo an increase in spontaneous and reversible EMT during aging and that at least one of the reasons for this phenomenon is the decrease in SM content due to the combination of decreased SM synthase activity and an increase in SM degradation mediated by neutral sphingomyelinase. Age is a risk factor for many diseases, among which renal fibrosis is included. Our findings highlight the importance of sphingolipids and particularly SM as a modulator of the fate of CD cells and probably contribute to the development of treatments to avoid or reverse renal fibrosis during aging.

Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.

BACKGROUND: Chronic kidney disease (CKD) patients are at high-risk for severe coronavirus disease 2019 (COVID-19). The multicentric, observational and prospective SENCOVAC study aims to describe the humoral response and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in CKD patients. Safety and immediate humoral response results are reported here. METHODS: Four cohorts of patients were included: kidney transplant (KT) recipients, and haemodialysis (HD), peritoneal dialysis (PD) and non-dialysis CKD patients from 50 Spanish centres. Adverse events after vaccine doses were recorded. At baseline and on Day 28 after the last vaccine dose, anti-Spike antibodies were measured and compared between cohorts. Factors associated with development of anti-Spike antibodies were analysed. RESULTS: A total of 1746 participants were recruited: 1116 HD, 171 PD, 176 non-dialysis CKD patients and 283 KT recipients. Most patients (98%) received mRNA vaccines. At least one vaccine reaction developed after the first dose in 763 (53.5%) and after the second dose in 741 (54.5%) of patients. Anti-Spike antibodies were measured in the first 301 patients. At 28 days, 95% of patients had developed antibodies: 79% of KT, 98% of HD, 99% of PD and 100% of non-dialysis CKD patients (P < 0.001). In a multivariate adjusted analysis, absence of an antibody response was independently associated with KT (odds ratio 20.56, P = 0.001) and with BNT162b2 vaccine (odds ratio 6.03, P = 0.023). CONCLUSION: The rate of anti-Spike antibody development after vaccination in KT patients was low but in other CKD patients it approached 100%, suggesting that KT patients require persistent isolation measures and booster doses of a COVID-19 vaccine. Potential differences between COVID-19 vaccines should be explored in prospective controlled studies.

Efectos del ASPO por COVID-19 en factores de riesgo y factores protectores de suicidalidad adolescente en Calingasta, San Juan / Effects of ASPO by COVID-19 on risk factors and protective factors for adolescent suicidality in Calingasta, San Juan

El suicidio representa un grave problema creciente de la salud pública en todo el planeta, y constituye la segunda causa de muerte en jóvenes y adolescente. Este estudio indaga el comportamiento de aspectos psicosociales de la suicidalidad en contexto de pandemia, en un conjunto de poblaciones rurales andinas. El objetivo general consiste en explorar los efectos del ASPO por COVID-19 en factores de riesgo y factores protectores de la suicidalidad adolescente del Departamento Calingassta (Provincia de San Juan (durante el año 2020. Mediante un diseño cuali-cuantitativo flexible, que considera emergentes, se administraron técnicas remotas de producción de datos -formularios digitales y entrevvistas semiestructuradas telefónicas- a referentes de instituciones comunitarias; y se accedió a registros oficiales de casos, así como a información originada en dispositivos virtuaes implementados en pandemia. El análisis incorporó elementos de la teoría fundamentada para elaborar categorías y comparó cifras oficiales, a fin de producir conocimientos situados. Los principales resutados obtenidos indican una disminución de casos de intentos de suicidio y suicidio consumado, aunque no es posible dimensionar con precisión la suicidalidad calingastina durante el ASPO debido a que los registros de ideación suicida y autolesiones no se encuentran sistematizados; si bien se advierten mejores condiciones de formación para su pesquisa y abordaje en agentes de la salud mental de salud pública. Se pusieron en visibilidad carencias de recursos de larga data, que conducen a centrarse en las demandas más urgentes en detrimento del desarrollo de actividades comunitarias de prevención y promoción de la saluld; y se evidenciaron necesidades de capacitación ante el desconocimiento de la Ley Nacional 27.130, y de refuerzo en redes entre dispositivos comunitarios departamentales, formales e informales.

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development.

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.

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.

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity.

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and ß- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.

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.

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.

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.

Membrane lipid composition plays a central role in the maintenance of epithelial cell adhesion to the extracellular matrix.

Focal contacts (FC) are membrane-associated multi-protein complexes that mediate cell-extracellular matrix (ECM) adhesion. FC complexes are inserted in detergent-resistant membrane microdomains enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2); however, the influence of membrane lipid composition in the preservation of FC structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell adhesion structures. We biochemically characterized the lipid composition of membrane-containing FC complexes. By using cholesterol and PtdIns(4,5)P2)affecting agents, we demonstrated that such agents did not affect any particular type of lipid but induced the formation of new FC-containing domains of completely different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that phospholipid composition plays an essential role in the in vivo maintenance of FC structures involved in cell-ECM adhesion.

Bradykinin modulates focal adhesions and induces stress fiber remodeling in renal papillary collecting duct cells.

Focal adhesions (FAs) are specialized regions of cell attachment to the extracellular matrix. Previous works have suggested that bradykinin (BK) can modulate cell-matrix interaction. In the present study, we used a physiological cellular model to evaluate the potential role of BK in modulating FAs and stress fibers. We performed a quantitative morphometric analysis of FAs in primary cultured rat renal papillary collecting duct cells, which included size, axial ratio (shape), and average length. After 1, 5, or 10 min of incubation with BK, cultured cells were immunostained and analyzed by confocal microscopy. Although the shape of FAs was not altered, BK induced a decrease in the number of vinculin-stained FAs per cell, and a decrease in both their size and their average length, but not in talin-containing FAs, thus suggesting that BK could be inducing a restructuring of FAs. BK also induced a remodeling of the actin filament assemblies rather than their dissipation. Since we have previously demonstrated that BK stimulates activation of PLCbeta in rat renal papillae, we attempted to determine whether BK can modulate FA restructuring by this mechanism, by pretreating cultured cells with the PLCbeta inhibitor U73122. The present study, performed under physiological conditions with cells that were not genetically manipulated, provides new experimental evidence supporting the notion that the intrarenal hormone BK modulates FAs and actin cytoskeleton organization through a mechanism that involves the activation of PLCbeta. We propose this finding as a novel mechanism for BK modulation of tubular collecting duct function.

Is DRM lipid composition relevant in cell-extracellular matrix adhesion structures?

Focal adhesions mediate cell-extracellular matrix adhesion. They are inserted in detergent-resistant membrane microdomains enriched in phosphatidylinositol-4,5-bisphosphate. In spite of the relevance that membrane lipids appear to have on cell adhesion structures, to our knowledge, there are no previous reports on the membrane lipid composition where focal adhesions are located in vivo or on how changes in local membrane composition contribute to focal adhesion maintenance. This may be due to the fact that the explosion of information in the fields of genomics and proteomics has not been matched by a corresponding advancement of knowledge in the field of lipids. The physiological importance of lipids is illustrated by the numerous diseases to which lipid abnormalities contribute. To gain insight into the role of membrane lipid composition in the preservation of epithelial cell adhesion to the substratum, how specific changes in the membrane lipid composition in vivo affect the maintenance of focal adhesions in renal papillae collecting duct cells has been previously studied. It is currently considered that phosphatidylinositol-4,5-bisphosphate plays a crucial role in the maintenance of assembled focal adhesion. However, such pool of polyphosphoinositides has to be part of a domain of a specific lipid composition to serve as a membrane lipid stabilizing the focal adhesion plaque.

The role of transforming growth factor-{beta} in a model of oral tolerance to the diet antigen dextrin.

In a model of immunodeficiency provoked by protein deficiency, cytosol fractions from gut IELS isolated from immunodeficient rats presenting oral tolerance to dextrin showed increased expression of TGF-beta to reduce the effect of higher levels of inflammatory cytokines.

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.

IgA B and T cells in the intestinal villi of immunodeficient rats orailly treated with thymomodulin

Previous studies on the effect of the oral administration of bacterial immunomodulators (IM-104 and RN-301) during the protein free diet period, have shown an increase on B and T cell gut repopulation, accompanied by IgA antibody production. The usefulness of oral administration of the immunomodulator thymomodulin (TmB) during the protein refeeding period was investigated. TmB allowed the recovery of a normal repopulation of gut lamina propria with IgA B and CD5 T cells and decreases to control values the number of activated intraepithelial lymphocytes (CD25+T cell subset). Therefore, the oral administration of TmB may be useful as a therapeutic agent as it seems to improve the repopulation of intestinal villi with immunocompetent cells. Also, it seems to regulate the immunosurveillance at the epithelium level as it increases the CD5+T cells but decreases the activated ones. (AU)

IgA B and T cells in the intestinal villi of immunodeficient rats orailly treated with thymomodulin

Previous studies on the effect of the oral administration of bacterial immunomodulators (IM-104 and RN-301) during the protein free diet period, have shown an increase on B and T cell gut repopulation, accompanied by IgA antibody production. The usefulness of oral administration of the immunomodulator thymomodulin (TmB) during the protein refeeding period was investigated. TmB allowed the recovery of a normal repopulation of gut lamina propria with IgA B and CD5 T cells and decreases to control values the number of activated intraepithelial lymphocytes (CD25+T cell subset). Therefore, the oral administration of TmB may be useful as a therapeutic agent as it seems to improve the repopulation of intestinal villi with immunocompetent cells. Also, it seems to regulate the immunosurveillance at the epithelium level as it increases the CD5+T cells but decreases the activated ones.

Effect of RN-301 immunomodulator on bronchus-associated lymphoid tissue (Balt) in protein depleted rats at weaning

It has been previously demonstrated in Wistar rats that severe protein deprivation at weaning, even after refeeding with a 20 percent casein diet for 21 days, provokes alterations in IgA+ B cell and T cell populations from gut and GALT (gut associated lymphoid tissue) that are reverted by immunomodulator IM-104. In the present report, we investigated the influence of RN-301 (quite similar to IM-104) given by the oral or subcutaneous route during the protein deprivation period, in the seeding of BALT with IgA+ B and CD5+T cells. The immunomodulator RN-301 contains LPS from E. coli and membrane and ribosomal fractions of P. acne. Tissue sections of the lower respiratory tract were studied by immunohistochemistry. The immunomodulator RN-301 administered by the oral route favours the significant increase in the seeding of the BALT lamina propria with IGA+B and CD5+T cells (p < 0.001). However, the RN-301 given by the subcutaneous route does not favour the repopulation of the BALT lamina propria. The ribosomal fractions from P. acne associated with LPS from E. coli contained in the immunomodulator RN-301 administered by the oral route may rescue the small resting lymphocytes in the gut-associated lymphoid tissue (GALT). This event favours their proliferation and migration to the BALT. (AU)

Effect of RN-301 immunomodulator on bronchus-associated lymphoid tissue (Balt) in protein depleted rats at weaning

It has been previously demonstrated in Wistar rats that severe protein deprivation at weaning, even after refeeding with a 20 percent casein diet for 21 days, provokes alterations in IgA+ B cell and T cell populations from gut and GALT (gut associated lymphoid tissue) that are reverted by immunomodulator IM-104. In the present report, we investigated the influence of RN-301 (quite similar to IM-104) given by the oral or subcutaneous route during the protein deprivation period, in the seeding of BALT with IgA+ B and CD5+T cells. The immunomodulator RN-301 contains LPS from E. coli and membrane and ribosomal fractions of P. acne. Tissue sections of the lower respiratory tract were studied by immunohistochemistry. The immunomodulator RN-301 administered by the oral route favours the significant increase in the seeding of the BALT lamina propria with IGA+B and CD5+T cells (p < 0.001). However, the RN-301 given by the subcutaneous route does not favour the repopulation of the BALT lamina propria. The ribosomal fractions from P. acne associated with LPS from E. coli contained in the immunomodulator RN-301 administered by the oral route may rescue the small resting lymphocytes in the gut-associated lymphoid tissue (GALT). This event favours their proliferation and migration to the BALT.