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The aim of this study was to assess the fatty acid (FA) percentage distribution in complex lipids of breast milk from mothers on a low docosahexaenoic acid (DHA) diet. We performed a descriptive, cross-sectional study of milk samples (n = 14) collected 90 days after delivery and analyzed them using gas chromatography, thin-layer chromatography, and the Fiske-Subbarow method. Complex lipid distribution was 40.70 ± 5.11% sphingomyelin (SM), 26.03 ± 5.98% phosphatidylethanolamine (PE), 21.12 ± 2.32% phosphatidylcholine, 7.94 ± 1.96% phosphatidylserine, and 4.22 ± 1.25% phosphatidylinositol. Median DHA and arachidonic acid values were 0.13% (0.12; 0.18) and 0.42% (0.33; 0.53), respectively. Mean FA percentage in SM and PE was as follows: palmitic acid, 34.45 ± 1.94% and 5.38 ± 0.94%; oleic acid, 16.50 ± 4.07% and 9.43 ± 4.05%; linoleic acid, 5.91 ± 4.69% and 9.05 ± 4.5%. DHA was not detectable in SM, but it was found in PE (55.33 ± 14.46). In conclusion, breast milk of mothers on a low DHA diet contained 55% DHA in PE, but no DHA in SM., Competing Interests: The authors report no conflicts of interests., (Copyright © 2023 by European Society for European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Epithelial renal cells have the ability to adopt different cellular phenotypes through epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). These processes are increasingly recognized as important repair factors following acute renal tubular injury. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with impact on proliferation, growth, migration, and differentiation which has significant implication in various diseases including cancer and kidney fibrosis. Here we demonstrated that S1P can exert by activating S1P receptor 2 (S1PR2) different functions depending on the stage of cell differentiation. We observed that the differences in the migratory profile of Madin-Darby canine kidney (MDCK) cells depend both on their stage of cell differentiation and the activity of S1PR2, a receptor that can either promote or inhibit the migratory process. Meanwhile in non-differentiated cells S1PR2 activation avoids migration, it is essential on fully differentiated cells. This is the first time that an antagonist effect of S1PR2 was reported for the same cell type. Moreover, in fully differentiated cells, S1PR2 activation is crucial for the progression of EMT - characterized by adherent junctions disassembly, β-catenin and SNAI2 nuclear translocation and vimentin expression- and depends on ERK 1/2 activation and nuclear translocation. These findings provide a new perspective about the different S1PR2 functions depending on the stage of cell differentiation that can be critical to the modulation of renal epithelial cell plasticity, potentially paving the way for innovative research with pathophysiologic relevance., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2022 Wiley Periodicals LLC.)

Collecting duct cells are physiologically subject to the hypertonic environment of the kidney. This condition is necessary for kidney maturation and function but represents a stress condition that requires active strategies to ensure epithelial integrity. Madin-Darby Canine Kidney (MDCK) cells develop the differentiated phenotype of collecting duct cells when subject to hypertonicity, serving as a model to study epithelial preservation and homeostasis in this particular environment. The integrity of epithelia is essential to achieve the required functional barrier. One of the mechanisms that ensure integrity is cell extrusion, a process initiated by sphingosine-1-phosphate (S1P) to remove dying or surplus cells while maintaining the epithelium barrier. Both types start with the activation of S1P receptor type 2, located in neighboring cells. In this work, we studied the effect of cell differentiation induced by hypertonicity on cell extrusion in MDCK cells, and we provide new insights into the associated molecular mechanism. We found that the different stages of differentiation influence the rate of apoptotic cell extrusion. Besides, we used a novel methodology to demonstrate that S1P increase in extruding cells of differentiated monolayers. These results show for first time that cell extrusion is triggered by the single-cell synthesis of S1P by sphingosine kinase 2 (SphK2), but not SphK1, of the extruding cell itself. Moreover, the inhibition or knockdown of SphK2 prevents cell extrusion and cell-cell junction protein degradation, but not apoptotic nuclear fragmentation. Thus, we propose SphK2 as the biochemical key to ensure the preservation of the epithelial barrier under hypertonic stress., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2019 Wiley Periodicals, Inc.)

It is known that bradykinin (BK) B2 receptor (B2R) is expressed in the collecting duct (CD) cells of the newborn rat kidney, but little is known about its role during early postnatal life. Therefore, we hypothesize that BK could participate in the mechanisms that mediate CD formation during the postnatal renal development. Performing primary cultures, combined with biochemical, immunocytochemical, and time-lapse analysis, we studied the role of BK in CD cell behavior isolated from renal papilla of neonatal rats. A reverse relationship was observed between B2R expression and the degree of CD epithelial cell sheet maturation. BK stimulation induced CD cell association upon B2R activation. The lack of B2R expression in cells showing mature adherens junctions suggested that BK is mostly involved in early adhesive events, thus favoring the initial formation of CD during development. Time-lapse analysis revealed that BK induced a high protrusive activity of CD cells, denoted by ruffle formation and lamellipodia extension. PI3K was involved in the BK-induced CD cell-cell association and the acquisition of the migratory phenotype since, when inhibited, membrane ruffles, and filopodia between cells diminished. Results indicate that the actions of BK mediated by PI3K activation were due to the downstream Akt and Rac pathways. This study, performed with CD cells that were not genetically manipulated, provides new experimental evidence supporting a novel role of BK in rat renal CD organization. As B2R blockade results in abnormal tubular differentiation, our results contribute to better understanding the etiology of human congenital renal malformation and diseases., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ceramides (Cers) and complex sphingolipids with defined acyl chain lengths play important roles in numerous cell processes. Six Cer synthase (CerS) isoenzymes (CerS1-6) are the key enzymes responsible for the production of the diversity of molecular species. In this study, we investigated the changes in sphingolipid metabolism during the differentiation of Madin-Darby canine kidney (MDCK) cells. By MALDI TOF TOF MS, we analyzed the molecular species of Cer, glucosylceramide (GlcCer), lactosylceramide (LacCer), and SM in nondifferentiated and differentiated cells (cultured under hypertonicity). The molecular species detected were the same, but cells subjected to hypertonicity presented higher levels of C24:1 Cer, C24:1 GlcCer, C24:1 SM, and C16:0 LacCer. Consistently with the molecular species, MDCK cells expressed CerS2, CerS4, and CerS6, but with no differences during cell differentiation. We next evaluated the different synthesis pathways with sphingolipid inhibitors and found that cells subjected to hypertonicity in the presence of amitriptyline, an inhibitor of acid sphingomyelinase, showed decreased radiolabeled incorporation in LacCer and cells did not develop a mature apical membrane. These results suggest that hypertonicity induces the endolysosomal degradation of SM, generating the Cer used as substrate for the synthesis of specific molecular species of glycosphingolipids that are essential for MDCK cell differentiation., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location., (Copyright © 2016. Published by Elsevier B.V.)

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 β- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression., (Copyright © 2010 S. Karger AG, Basel.)

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.

Background/aims: Diabetes mellitus may impact on the regulation of renal Na+-glucose cotransporter type 2 (SGLT2), however, previous studies have yielded conflicting results on the effects of streptozotocin (STZ)-induced diabetes on SGLT-mediated glucose transport., Methods: Diabetes was induced in male Wistar rats. The studies were performed at 3 (D3), 7 (D7) and 14 (D14) days after a single i.p. injection of STZ. SGLT2 activity was measured using alpha-14C-methyl glucose uptake in brush-border vesicles (BBV) from renal cortex, and SGLT2 expression was assessed by immunoblotting. Phospholipids were quantified by a modification of Fiske-Subarow's method after being separated by thin-layer chromatography., Results: Glucose uptake was reduced in all groups of diabetic rats. SGLT2 expression decreased in D3 and D7. There was a decrease in sphingomyelin (SM) content and an increase in phosphatidylcholine (PC) content in BBV from D14 versus control, without differences in phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylethanolamine (PE)., Conclusion: The downregulation of SGLT2 activity during STZ-induced diabetes may be a protective mechanism to control the excess of circulating glucose and could be a consequence of a decrease in SGLT2 expression in D3 and D7, whereas altered activity of SGLT2 in D14 could be a consequence of changes in membrane lipid composition. However, we cannot discard the possibility that the decrease in SGLT2 activity could be due to a covalent modification of the active site of the protein., (Copyright 2009 S. Karger AG, Basel.)

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.

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.

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.

Lamin A/C is the most studied nucleoskeletal constituent. Lamin A/C expression indicates cell differentiation and is also a structural component of nuclear speckles, which are involved in gene expression regulation. Hypertonicity has been reported to induce renal epithelial cell differentiation and expression of TonEBP (NFAT5), a transcriptional activator of hypertonicity-induced gene transcription. In this paper, we investigate the effect of hypertonicity on lamin A/C expression in MDCK cells and the involvement of TonEBP. Hypertonicity increased lamin A/C expression and its distribution to nucleoplasm with speckled pattern. Microscopy showed codistribution of TonEBP and lamin A/C in nucleoplasmic speckles, and immunoprecipitation demonstrated their interaction. TonEBP silencing caused lamin A/C redistribution from nucleoplasmic speckles to the nuclear rim, followed by lamin decrease, thus showing that hypertonicity induces lamin A/C speckles through a TonEBP-dependent mechanism. We suggest that lamin A/C speckles could serve TonEBP as scaffold thus favoring its role in hypertonicity.

Background/aims: The renal sodium glucose cotransporter (SGLT2) and the water channel aquaporin-2 (AQP2) play a critical role in tubular sodium and water reabsorption and in the regulation of extracellular fluid volume both in physiologic and pathophysiologic conditions. However, there is little information about SGLT2 and AQP2 expression and/or activity in hypertension and there are no reports during hypertension induced by chronic nitric oxide synthase (NOS) inhibition., Methods: Hypertension was induced in rats by oral administration of N(G)-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg/24 h) for 6 (H6) or 12 (H12) weeks. SGLT2 activity was measured using alpha-(14)C-methylglucose active uptake. The expression level of transporters was assessed by immunohistochemistry and/or immunoblotting., Results: SGLT2 activity was reduced in both H6 and H12; this was due neither to a decrease in SGLT2 expression nor to a change in membrane phospholipid composition. In H6, AQP2 expression diminished only in the inner medulla (IM), while in H12 it diminished in both outer (OM) and IM. This reduced expression of AQP2 may partially account for the increased urinary volume and decreased urinary osmolality in H12, since we obtained a strong correlation between AQP2 expression and these urinary parameters in both OM and IM., Conclusion: We propose that in rats in which hypertension is induced by NOS inhibition, SGLT2 activity and AQP2 expression are modified to compensate for the elevated arterial pressure. However, we cannot discount the possibility that the observed changes are due to the decrease in NO production itself.

Phosphatidylcholine (PC) is the most abundant phospholipid in mammalian cell membranes. Several lines of evidence support that PC homeostasis is preserved by the equilibrium between PC biosynthetic enzymes and phospholipases catabolic activities. We have previously shown that papillary synthesis of PC depends on prostaglandins (PGs) that modulate biosynthetic enzymes. In papillary tissue, under bradikynin stimulus, arachidonic acid (AA) mobilization (the substrate for PG synthesis) requires a previous phospholipase C (PLC) activation. Thus, in the present work, we study the possible involvement of PLC in PC biosynthesis and its relationship with PG biosynthetic pathway on the maintenance of phospholipid renewal in papillary membranes; we also evaluated the relevance of CDP-choline pathway enzymes compartmentalization. To this end, neomycin, U-73122 and dibutiryl cyclic AMP, reported as PLC inhibitors, were used to study PC synthesis in rat renal papilla. All the PLC inhibitors assayed impaired PC synthesis. PG synthesis was also blocked by PLC inhibitors without affecting cyclooxygenase activity, indicating a metabolic connection between both pathways. However, we found that PC biosynthesis decrease in the presence of PLC inhibitors was not a consequence of PG decreased synthesis, suggesting that basal PLC activity and PGs exert their effect on different targets of PC biosynthetic pathway. The study of PC biosynthetic enzymes showed that PLC inhibitors affect CTP:phosphocholine cytidylyltransferase (CCT) activity while PGD(2) operates on CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), both activities associated to papillary enriched-nuclei fraction. The present results suggest that renal papillary PC synthesis is a highly regulated process under basal conditions. Such regulation might occur at least at two different levels of the CDP-choline pathway: on the one hand, PLC operates on CCT activity; on the other, while PGs regulate CPT activity.

The effects of a high glucose concentration (HGC) on renal phosphatidylcholine (PtdCho) biosynthesis were studied. In control rats, HGC increased papillary PtdCho biosynthesis. In chronic diabetic rats, an increase above that induced by diabetes was observed. Such glucose-responsive phospholipid pools were shown to be transient in adult control rats, while in acute diabetic and aged control and chronic diabetic rats they seem to be of slow breakdown or permanent. Deoxyglucose evokes the HGC effect only in the presence of 5 mM glucose. Neomycin, which blocks phospholipase C action, corrected the HGC effect in control and chronic diabetic rats, but not the increase due to diabetes. CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity was increased by both in vivo and simulated diabetes. Therefore, transient high extracellular glucose levels promote a reversible increase in papillary (32)P-PtdCho, while diabetes causes an irreversible increase resulting in PtdCho accumulation, possibly related to papillary necrosis., (Copyright 2001 S. Karger AG, Basel)

In the present study, the effects of hexachlorobenzene (HCB) on lipid peroxidation and heme metabolism in the different constitutive suborgans of the kidney were determined. For this purpose, conjugated diene and malondialdehyde levels, as lipid peroxidation parameters, and porphyrin accumulation, uroporphyrinogen decarboxylase activity, and its inhibitor formation, as measures of heme metabolism, were determined in renal cortex, medulla, and papilla. Adult Wistar rats were treated with HCB during 1, 2, 3, or 4 weeks. A significant increase in cortical conjugated dienes was observed from the 1st week of treatment. The malondialdehyde levels rose by 47, 34, and 28% after 2, 3, and 4 weeks of intoxication, respectively. The porphyrin content showed a tenfold increase after 4 weeks of treatment, and the uroporphyrinogen decarboxylase activity was reduced by 26 and 58% with respect to control values after 3 and 4 weeks of treatment, respectively. The results demonstrate a direct correlation between the oxidative environment and the effect elicited by the drug on heme metabolism in the renal cortex. In contrast, in papilla and medulla, where the antioxidant systems were higher, HCB showed no porphyrinogenic effect.

During maturation, rat renal papillary microsomes suffer a rearrangement in their fatty acid phospholipid composition. The most significant changes in total phospholipids are the increase in their content of the 20:4 and a decrease in the levels of 14:0, 16:0, 18:1, 22:6 and 20:3 fatty acids. The changes in total phospholipid fatty acid content are a reflection of the variations in the individual phospholipid composition. During this period, microsomal cholesterol, phospholipid, and protein concentrations present no variations. Steady state fluorescence anisotropy obtained by using TMA-DPH (see text) as a fluorescence probe denoted higher values for 70- versus 10-day-old microsomes. Using DPH as a probe, steady state fluorescence anisotropy was determined in whole microsomes, as well as in total lipid and phospholipid vesicles, from both 10- and 70-day-old papillary cells. No differences were detected in phospholipid and total lipid vesicles between days 10 and 70. On the other hand, 10-day-old microsomes appeared to be less fluid than adult microsomes. The results indicate that these structural changes in kidney membranes during development might affect protein-lipid interaction and, therefore, the activity of many membrane enzymes.

Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [32P]orthophosphate ([32P]Pi) or [3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns "de novo" synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [32P]Pi into PPIs. Of the "de novo" synthesized [3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [32P]Pi labeling, indicating that PPI formation mainly involves a no "de novo" synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [32P]Pi Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occurred in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [gamma-32P]-adenosine triphosphate (ATP) or [32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [32P]from [32P]Pi was incorporated more efficiently into PPIs than that from [gamma-32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI synthesis. These data suggest that PPI formation is not necessarily a futile cycle in PMNs.

Rat renal papilla is the zone of the kidney enjoying the most active phospholipid metabolism and also the highest prostaglandin production. We studied the phospholipid biosynthesis and the relationship between phospholipid de novo synthesis and prostaglandin biosynthesis in rat renal papilla. Indomethacin inhibited the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Exogenous PGF2alpha and PGD2 restored biosynthetic activity in the presence of indomethacin and also increased the activity of the enzymes involved in the Kennedy pathway. The decrease in phospholipid biosynthesis maintained a linear relationship with the decrease in prostaglandin biosynthesis. Moreover, esculetin, which stimulates prostaglandin synthesis, brought about a significant increase in 32P incorporation to the three phospholipids studied. The evidence presented in this paper indicates that renal PGF2alpha and PGD2 modulate phospholipid de novo synthesis in rat renal papilla.

The fatty acid content of rat renal phospholipids was examined during development. An increase in the arachidonic acid content of a particular fraction of phosphatidylcholine (PC2), phosphatidylethanolamine and phosphatidylinositol between 10 and 20 days of age could be observed in the papilla, and these levels were maintained into adulthood, while in the 20:4 content of phosphatidylserine changes were found between 30-day-old rats and adults. In the other fraction of phosphatidylcholine (PC1), saturated fatty acids such as 16:0 and 14:0 decreased, while no changes occurred in the stearic acid (18:0) content. The pattern found for the medulla did not differ significantly from that of the papilla. In contrast, the cortex content of arachidonic acid at 10 days of age was higher than that for papilla and medulla. Levels increased between 10 and 20 days of age, returning to the original values by 30 days with no further variations in the adult. These changes in arachidonic acid content with age in addition to the differences found between the three kidney zones might explain some causes of incomplete renal function in newborns.

The developmental profiles of the lipid composition and their de novo synthesis and remodelling in the optic lobe of the chicken were studied. The 32P incorporation to phospholipids showed an active de novo synthesis mainly of phosphatidylinositol and of a particular fraction of phosphatidylcholine during the early stages of the embryo development, concomitantly with the beginning of synaptogenesis. This de novo synthesis of phospholipids strongly increased at hatching. On the other hand, phosphatidylinositol presented an active lipid exchange (acylation-deacylation) in the early stages of embryogenesis, indicating a strong incorporation of 14C-arachidonic acid during this period, followed by a fast drop in specific activity. Two different fractions of phosphatidylcholine were isolated by high-performance thin-layer chromatography with a different profile of fatty acid composition, disclosing their different physicochemical behavior, metabolic activities and evolution during embryogenesis. 32P incorporation into phosphatidylethanolamine remained very low during the earliest stages of embryogenesis, showing an increase when the process of synaptogenesis began, until hatching, when radioactivity reached a plateau. 14C-arachidonic acid incorporation into phosphatidylethanolamine was minimal. Furthermore, the phosphatidylethanolamine pool was progressively enriched in its ethanolamine plasmalogen throughout the development. Chromatographic analysis of lipid extracts showed the presence of cerebroside traces after 16 days of embryo incubation. At hatching, a remarkable increase in non-hydroxylated cerebrosides was observed concurrently with the appearance of hydroxylated ones. These glycosphingolipids, as well as the sulfatides, were markedly increased in the lipid extracts of optic lobes of adult animals, indicating the progressive development and maturity of the myelin sheath.

The effect of acute and chronic streptozotocin-induced diabetes on phospholipid metabolism in papillary, medullary and cortical slices was studied. No changes were observed in either the phospholipid content or composition in the papilla, medulla and cortex from acute and chronic diabetic rats. With reference to (U-14C)glycerol incorporation in the papilla from chronic diabetic rats, it increased in PtdCho, PtdIns and PtdEtn. In the medulla, the incorporation to PtdCho decreased in the acute diabetic state, while in the chronic diabetic state the incorporation to PtdCho and PtdIns decreased. No changes were observed in cortical phospholipids from diabetic rats. As regards 23P-sodium orthophosphate incorporation to phospholipids, while it increased in PtdCho, PtdIns and PtdEtn in the papilla from acute and chronic diabetic rats, in the medulla from both groups, the incorporation in PtdCho and PtdIns decreased, and in the cortex from acute diabetic rats it decreased only in PtdIns. Even though no changes were detected in phospholipid composition, alterations in phospholipid metabolism were induced by experimental diabetes.

Mercuric chloride (HgCl2) is a well-known renal toxic that causes acute renal failure. The effect of HgCl2 treatment and the protection by thyroxine were studied in rat renal papilla (P), outer medullary inner stripe (OMIS), outer medullary outer stripe (OMOS) and cortical phospholipids (PhLs). HgCl2 brought about an increase in the total phospholipid content in P and OMIS but a drop in OMOS and cortex. Only phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) accounted for such changes. Thyroxine, injected on HgCl2-treated rats, partially reversed the effect of the toxic metal in P and OMIS while completely reversed the PtdCho drop in OMOS and cortex. However, the hormone failed to recover the sphingomyelin increase in P, the PtdEtn shortage in OMIS, OMOS and partially reversed the drop in the cortex. When thyroxine was injected without toxic treatment, no effect was observed in the phospholipid content of any kidney zone. Results obtained by using 32P as a precursor to study the PhL de novo synthesis were consistent with those of the phospholipid content. Thus a radioactivity increase--associated with PtdCho and PtdEtn--was observed in the kidney zones where said endogenous PhLs had risen. But in OMOS and cortex, where PtdCho and PtdEtn had dropped, they were also accompanied by a decrease in radioactivity. The thyroxine-induced recovery phase also paralleled the phospholipid content results with those of the de novo synthesis. We suggest that the decrease in the renal phospholipid de novo synthesis may constitute one biochemical explanation of the selective renal toxic effect exerted by HgCl2 and that the increase observed in the renal phospholipid metabolism--induced by the toxic treatment in OMIS and P--may represent a protective mechanism of these zones against toxic injury. Moreover, recovery promoted by thyroxine treatment in OMOS and cortex was accompanied by the reversion of the corresponding PtdCho decrease induced by HgCl2.

The biosynthesis of prostaglandins (PGs) from the endogenous and exogenous precursor, arachidonic acid (AA), in renal papilla, medulla and cortex from neonatal to adult rats was investigated. Rat renal papilla and medulla incubated in the presence of [1-14C]AA released radioactive PGE2, PGF2 alpha and PGD2 which increased with age. No radioactive prostaglandins were found in the supernatants of renal cortex at any age studied. The amount of total prostaglandins released from the endogenous precursor also increased from 10 to 70 days of age, PGD2 being the prostaglandin that showed the most important rise. In the cortex, only PGE2 release increased with age. Cyclooxygenase (COX) activity was measured in papillary, medullary and cortical homogenates by using [1-14C]AA as substrate. Papillary and medullary COX activity increased after 10 days of age and continued to rise up to day 30 thereafter remaining unaltered until adulthood. Cortical COX activity was very low and decreased with age. These findings indicate the low capacity of the neonatal rat kidney to synthesize PGs.

We have studied microsomal phospholipid, cholesterol and protein concentration in rat renal papilla, medulla and cortex during postnatal development, and the relationship between these membranes biochemical parameters and morphological changes. We also determined DNA concentration in each kidney zone. No changes were observed either in papillary microsomal phospholipids, proteins and cholesterol or in DNA concentration from 10-to 70-day-old rats. Medullary microsomal proteins and cholesterol did not change but a significant increase was observed in the microsomal phospholipid concentration during development; in this case, medullary DNA was significantly lower at 70 than at 10 days. In contrast, all biochemical parameters in renal cortex were significantly higher during development except for DNA concentration which suffered a great decrease. These biochemical findings demonstrate that the developmental pattern is different in each zone of the kidney and confirm the fact that the papilla, in newborn rats, is almost fully developed whereas the renal cortex and medulla are immature.

Phospholipid content was studied in kidneys from rats treated with indometacin. Short-term treatment was performed by using low (1 and 5 mg/kg/day) and high (10 and 50 mg/kg/day) doses of indometacin. Long-term treatment was achieved by using only low doses of indometacin. Short-term treatment at low doses did not result in any change in the phospholipid content. In rats administered higher concentrations, indometacin caused a marked increase in all papillary phospholipid contents, but no effect was observed in the medulla, and an increase in sphingomyelin and phosphatidylethanolamine was observed in the cortex. Long-term treatment with administration of 1 mg/kg/day of indometacin led to an increase in all papillary phospholipids from the 2nd week of treatment. Medullary phospholipids also increased, but no changes were observed in cortical phospholipids. These results show that indometacin causes phospholipid accumulation in rat kidney and that the papilla is the most sensitive renal tissue.

To investigate a possible action of insulin on the rat kidney papilla, the binding of 125I-insulin to papilla microsomes was examined. This binding was specific to insulin in that it was displaced by increasing concentrations of unlabelled porcine insulin and to a lesser extent by porcine proinsulin and IGF-I, but not by IGF-II and bGH. Scatchard plot of the binding data was curvilinear consistent with either two classes of receptors with different affinities or a single class of receptors that showed negative cooperativity. A small fraction of 125I-insulin (maximum 2%) was degraded during incubation, but with a Km two order of magnitude higher than the constant of affinity for binding. Insulin stimulates the incorporation of phosphate to phosphatidylcholine in a dose-dependent manner, reaching a maximum with 10 nM insulin. This data showed both the presence of specific insulin receptors in the kidney papilla and an insulin action through the synthesis of phospholipids by insulin.

The present study provides information regarding the effects of the sow follicular fluid (FF) on the motility of isolated segments of swine and rabbit oviducts. In addition, the concentration of prostaglandins (PGs) F2 alpha, E2 and E1 in the follicular fluid of sow ovaries isolated at different stages of the sex cycle as well as the generation of the same PGs by walls of ovarian follicles in early and late proestrus, in estrus, in metestrus and in diestrus, were explored. The stimulatory contractile effect of proestrous FF in isolated segments of sow fimbria was antagonized by polyphloretin phosphate (PPP), a PG receptor blocker and by indomethacin, an inhibitor of PG synthesis. The positive inotropism evoked by the FF was mimiked by bradykinin and the influences of both interventions were similarly antagonized by PPP. It appears plausible that the inotropic effect of the preovulatory FF on the sow fimbria could be not only by PGs already present in the fluid, but also by the stimulation of the synthesis of tubal PGs by follicular fluid bradykinin. The FF also stimulated the ampullary tubal segments isolated from proestrous sows whereas the same volume of FF depressed significantly the isometric developed tension of rabbit ampulla. The total concentration of the three PGs in the FF from late proestrous follicles was significantly greater than that of the same PGs in the other two stages of the sex cycle (early proestrus and diestrus), whereas the concentration of each PG (PGE2, PGF2 alpha or PGE1), did not differ within any of the stages of the cycle. Furthermore, the total amount of the three PGs produced by the walls of follicles from late proestrous ovaries was also significantly greater than that generated by ovarian follicles from early proestrus, estrus, metestrus and diestrus. In summary the results document that the concentration of each one of the PGs measured (E2, E1 or F2 alpha) attained maximal values at the time of ovulation. The results regarding the effects of FF on the inotropic activity of fimbrial and ampullary segments of sow oviducts also suggest that the fluid might play a physiological role, favouring the capture and transfer of ova into the oviducts at the moment of ovulation.

In view of the fact that mepacrine (Mp) is usually used as an inhibitor of the endogenous phospholipase A2, and since this enzyme produces the release of arachidonic acid (AA) from membrane phospholipids, we studied the effect of different concentrations of Mp on the mobilization of [1-14C]AA in rat renomedullary phospholipids. During the acylation period, 0.1 mM Mp did not produce any significant change in the incorporation of [1-14C]AA into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and only a slight increase in phosphatidylinositol (PI). Higher concentrations of Mp (0.5 to 1.0 mM) produced a decrease of radioactivity in PE and PC with an increase in PI. Using prelabeled slices, a dose-dependent decrease in the 14C-radioactivity in PE and PC was observed, with a parallel increase in PI. This effect of Mp persisted even in the presence of a physiological activator of phospholipase A2, bradykinin (BK). No change in the net amount of phospholipids was observed at any of the Mp concentrations used. The results of this study show that Mp, at concentrations generally used to inhibit phospholipase A2, produced a transfer of arachidonic acid from PE and PC to PI, rather than a blockade in the release of AA from membrane phospholipids.