Dynamics of differentiated-renal epithelial cell monolayer after calcium oxalate injury: The role of cyclooxygenase-2.

AIMS: Calcium oxalate (Oxa), constituent of most common kidney stones, damages renal tubular epithelial cells leading to kidney disease. Most in vitro studies designed to evaluate how Oxa exerts its harmful effects were performed in proliferative or confluent non-differentiated renal epithelial cultures; none of them considered physiological hyperosmolarity of renal medullary interstitium. Cyclooxygenase 2 (COX2) has been associated to Oxa deleterious actions; however, up to now, it is not clear how COX2 acts. In this work, we proposed an in vitro experimental system resembling renal differentiated-epithelial cells that compose medullary tubular structures which were grown and maintained in a physiological hyperosmolar environment and evaluated whether COX2 â†’ PGE2 axis (COX2 considered a cytoprotective protein for renal cells) induces Oxa damage or epithelial restitution. MAIN METHODS: MDCK cells were differentiated with NaCl hyperosmolar medium for 72 h where cells acquired the typical apical and basolateral membrane domains and a primary cilium. Then, cultures were treated with 1.5 mM Oxa for 24, 48, and 72 h to evaluate epithelial monolayer restitution dynamics and COX2-PGE2 effect. KEY FINDINGS: Oxa completely turned the differentiated phenotype into mesenchymal one (epithelial-mesenchymal transition). Such effect was partially and totally reverted after 48 and 72 h, respectively. Oxa damage was even deeper when COX2 was blocked by NS398. PGE2 addition restituted the differentiated-epithelial phenotype in a time and concentration dependence. SIGNIFICANCE: This work presents an experimental system that approaches in vitro to in vivo renal epithelial studies and, more important, warns about NSAIDS use in patients suffering from kidney stones.

Fatty Acid Percentage Distribution in Complex Lipids of Breast Milk From Mothers on a Low Docosahexaenoic Acid Diet.

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.

Participation of prostaglandin D2 in the mobilization of the nuclear-localized CTP:phosphocholine cytidylyltransferase alpha in renal epithelial cells.

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.

Glycosphingolipid synthesis is essential for MDCK cell differentiation.

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.

The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions.

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.

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells.

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.