Hypertonic induction of COX2 expression requires TonEBP/NFAT5 in renal epithelial cells.

TonEBP/NFAT5 transcription factor is a master regulator of genes involved in osmoprotection. Cyclooxygenase 2 (COX2) has been reported to be a cytoprotective molecule in the inner renal medulla, where cells are physiologically exposed to the highest osmolality of the body. Our aim was to study whether COX2 expression requires TonEBP/NFAT5. Incubation of MDCK cells in hypertonic NaCl medium (500 mOsm/kg H2O) caused fully translocation of TonEBP/NFAT5 from cytoplasm to nucleoplasm and significantly increased COX2 mRNA, protein and activity levels. TonEBP/NFAT5-siRNA prevented hypertonic induction of COX2 mRNA and protein, leading to a depressed-prostaglandin synthesis and to a decreased cell survival. By using COX2-siRNA and COX2 specific inhibitor NS398, we found that cell survival does not depend on endogenous COX2-induced prostaglandin synthesis, but that cytoprotection strongly correlates with COX2 protein levels. These results demonstrate a new function for TonEBP/NFAT5, i.e., to mediate hypertonic-induced COX2 expression, and suggest that osmoprotection strongly depends on COX2 protein levels.

Lipid-protein interactions in rat renal subcellular membranes: a biophysical and biochemical study.

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.

Heme metabolism and lipid peroxidation in rat kidney hexachlorobenzene-induced porphyria: A compartmentalized study of biochemical pathogenic mechanisms.

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.

Endogenous prostaglandins regulate rat renal phospholipid 'de novo' synthesis.

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.

Ontogenic development of membrane lipids in the chick optic lobe.

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.

Compartmental study of rat renal prostaglandin synthesis during development.

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.

Short- and long-term treatment with indometacin causes renal phospholipid alteration: a possible explanation for indomethacin nephrotoxicity.

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.