1H-NMR and 13C-NMR lipid profiles of human renal tissues

Lipids from human renal tissues are studied by means of (1)H- and (13)C-NMR spectroscopy. The total lipid fractions obtained from healthy kidneys, malignant renal cell carcinomas, and benign oncocytomas are characterized and analyzed to elucidate the main differences between the functional and neoplastic tissues. In all cases the lipid components are well identified. The healthy kidney is characterized by high amounts of triglycerides and the presence of cholesterol in its free form. On the contrary, renal cell carcinomas contain high amounts of cholesterol that are almost completely esterified as oleate, suggesting an intracellular localization of the cholesteryl esters synthesis. Cholesteryl esters are considered markers of renal cell carcinomas, thus supporting recent theories that these compounds play a leading role in cell proliferation. Oncocytomas are particularly rich in phosphatidylcholine and, analogous to the healthy kidney, are completely lacking in cholesteryl esters. Healthy kidneys and oncocytomas appear to have other similarities if compared with renal cell carcinomas: a very high fatty acyl/cholesterol ratio, the presence of dolichols, and a higher grade of unsaturation. The (13)C data suggest a new method for the direct evaluation of the saturated/unsaturated fatty acyl ratio.     

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity in cortical and medullary human renal tissue.

Platelet-activating factor (PAF) is one of the most potent inflammatory mediators. It is biosynthesized by either the de novo biosynthesis of glyceryl ether lipids or by remodeling of membrane phospholipids. PAF is synthesized and catabolized by various renal cells and tissues and exerts a wide range of biological activities on renal tissue suggesting a potential role during renal injury. The aim of this study was to identify whether cortex and medulla of human kidney contain the acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity which catalyses the last step of the remodeling biosynthetic route of PAF and is activated in inflammatory conditions. Cortex and medulla were obtained from nephrectomized patients with adenocarcinoma and the enzymatic activity was determined by a trichloroacetic acid precipitation method. Lyso-PAF AT activity was detected in both cortex and medulla and distributed among the membrane subcellular fractions. No statistical differences between the specific activity of cortical and medullary lyso-PAF AT was found. Both cortical and medullary microsomal lyso-PAF ATs share similar biochemical properties indicating common cellular sources.     

The transfer of bovine J blood group activity to erythrocytes: chemical nature of transferable and of non-transferable J1

The bovine J blood group substance exists as a glycosphingolipid (ceramide deca-hexoside as well as ceramide dodecahexoside) and as a glycoprotein. The lipidic form occurs in erythrocyte membranes, both forms are found in serum. The lipidic J substances were isolated from erythrocytes and from serum, and identified by thin-layer chromatography with lipidic J substances isolated from spleen. The glycoprotein nature of the non-lipidic J of serum was evident by pronase-catalysed hydrolysis yielding J-active glycopeptides of lower molecular weights. The lipidic J was completely extracted from lyophilized stroma with chloroform/methanol. From lyophilized serum, however. it was completely extracted only in the presence of water, indicating different binding partners in serum and in erythrocyte membranes. The J lipid was incorporated as intact molecule into the erythrocyte membrane by a simple incubation technique. The incorporation was inhibited by various glyc-erophospholipids (called blockers). The J glycoprotein could not be transferred to the erythrocyte membrane. Three methods are descrjbed which are suitable for the preparation of a blocker-free fraction enriched with J lipids from J-positive serum.     

Changes in Metabolic Profiles during Acute Kidney Injury and Recovery following Ischemia/Reperfusion

Changes of metabolism have been implicated in renal ischemia/reperfusion injury (IRI). However, a global analysis of the metabolic changes in renal IRI is lacking and the association of the changes with ischemic kidney injury and subsequent recovery are unclear. In this study, mice were subjected to 25 minutes of bilateral renal IRI followed by 2 hours to 7 days of reperfusion. Kidney injury and subsequent recovery was verified by serum creatinine and blood urea nitrogen measurements. The metabolome of plasma, kidney cortex, and medulla were profiled by the newly developed global metabolomics analysis. Renal IRI induced overall changes of the metabolome in plasma and kidney tissues. The changes started in renal cortex, followed by medulla and plasma. In addition, we identified specific metabolites that may contribute to early renal injury response, perturbed energy metabolism, impaired purine metabolism, impacted osmotic regulation and the induction of inflammation. Some metabolites, such as 3-indoxyl sulfate, were induced at the earliest time point of renal IRI, suggesting the potential of being used as diagnostic biomarkers. There was a notable switch of energy source from glucose to lipids, implicating the importance of appropriate nutrition supply during treatment. In addition, we detected the depressed polyols for osmotic regulation which may contribute to the loss of kidney function. Several pathways involved in inflammation regulation were also induced. Finally, there was a late induction of prostaglandins, suggesting their possible involvement in kidney recovery. In conclusion, this study demonstrates significant changes of metabolome kidney tissues and plasma in renal IRI. The changes in specific metabolites are associated with and may contribute to early injury, shift of energy source, inflammation, and late phase kidney recovery.     

Enrichment of rat tissue lipids with fatty acids that are prostaglandin precursors.

The effects of supplementation of a complete diet with ethyl arachidonate and with ethyl dihomo-gamma-linolenate (20 : 3Omega6) on the fatty acid composition of plasma and tissue lipid classes were studied in normal rats. 2. These prostaglandin precursors were incorporated in varying degrees into all lipid classes of the tissues that were investigated. The largest elevations were seen in plasma and tissue triacylglycerols. Significant increases were also observed in phospholipids, cholesteryl esters and the free fatty acid fraction. 3. Following the feeding of the ester of 20 : 3Omega6, arachiodonate levels also rose in the lipids of some tissues. In others, such as the renal medulla and platelets, and increase in 20 : 3Omega6 content occurred without a rise in 20 : 4. 4. Platelet aggregation is known to be stimulated by 20 : 4 (via active metabolites), but not by 20 : 3Omega6. The ability to modify 20 : 3Omega6 levels selectively in certain tissues is of interest in light of such pharmacologic differences from 20 : 4.     

Quantitative real-time PCR for the measurement of 11beta-HSD1 and 11beta-HSD2 mRNA levels in tissues of healthy dogs.

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) exists in two isoforms, 11beta-HSD1 and 11beta-HSD2. 11beta-HSD1 generates active cortisol from cortisone and appears to be involved in insulin resistant states. 11beta-HSD2 protects the mineralocorticoid receptor from inappropriate activation by glucocorticoids and is important to prevent sodium retention and hypertension. The purposes of the present study were to develop two real-time PCR assays to assess 11beta-HSD1 and 11beta-HSD2 mRNA expression and to evaluate the tissue distribution of the two isoforms in dogs. Thirteen different tissues of 10 healthy dogs were evaluated. Both real-time PCR assays were highly specific, sensitive and reproducible. Highest 11beta-HSD1 mRNA expression was seen in liver, lung, and renal medulla; highest 11beta-HSD2 mRNA expression in renal cortex, adrenal gland, and renal medulla. Higher 11beta-HSD1 than 11beta-HSD2 mRNA levels were found in all tissues except adrenal gland, colon, and rectum. Our results demonstrate that the basic tissue distribution of 11beta-HSD1 and 11beta-HSD2 in dogs corresponds to that in humans and rodents. In a next step 11beta-HSD1 and 11beta-HSD2 expression should be assessed in diseases like obesity, hypercortisolism, and hypertension to improve our knowledge about 11beta-HSD activity, to evaluate the dog as a model for humans and to potentially find new therapeutic options.     

Mevalonate metabolism by renal tissue in vitro

Previous studies from this laboratory have demonstrated that the kidneys rather than the liver play the major role in the in vivo metabolism of circulating mevalonic acid. Kidneys, however, convert mevalonic acid primarily to the precursors of cholesterol, squalene and lanosterol, rather than to cholesterol. This study was designed to define the specific tissue site within the kidney responsible for mevalonic acid metabolism. Tissue slices from rat and dog renal cortex and medulla and glomeruli and tubules were isolated, and the incorporation of (14)C-labeled mevalonic acid into the nonsaponifiable lipids squalene, lanosterol, and cholesterol was determined in these tissues. The results demonstrate that the renal cortex is the primary site of mevalonic acid metabolism within the kidney and that the glomerulus is responsible for 95% of the mevalonic acid metabolized by the renal cortex. As was the case for the whole kidney, the major metabolites of mevalonate in the glomeruli are squalene and lanosterol.     

Methylamines and polyols in kidney, urinary bladder, urine, liver, brain, and plasma. An analysis using 1H nuclear magnetic resonance spectroscopy

Methylamines and polyols are known to behave as organic osmolytes in the adaptation of many cells to hyperosmolar conditions. Using 1H nuclear magnetic resonance spectroscopy to analyze perchloric acid extracts we have examined several tissues in the rat for the presence of these compounds. Methylamines such as glycerophosphorylcholine, choline and betaine were observed in the renal inner medulla, urinary bladder, urine, liver, brain, and plasma. Myoinositol was relatively abundant in the renal inner medulla and brain whereas sorbitol was detected only in the inner medulla. A variety of unidentified compounds was also detected in each tissue. Although these methylamines and polyols are known to respond to osmotic changes in the renal inner medulla, their responses in other tissues remain to be investigated.     

On lipid droplets in renal interstitial cells

Summary As the result of histochemical studies, it has been shown that the lipid droplets in the interstitial cells of the renal medulla of the rat contain simple saturated and unsaturated lipids. A possible correlation is suggested between the lipid droplets and the biologically active substances of a lipid character (vasodepressor lipid, medullin, and prostaglandin) which have been isolated from the renal medulla during recent years.This work was supported by a grant from the Danish State Research Foundation.     

Cutaneous lipid excretion

In this research we have studied the lipids excretion of skin by comparison of three groups of subjects at first age, young age and old people. We evidence the amount of the lipidic fractions and the differences between the ages in the lipids of skin and their components. We have also underlined the great significance of the skin as an important factor in the homeostasis.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Alterations of renal cortex and medullary glycosaminoglycans in aging dog kidney

Age-related changes in renal function have been attributed to alterations in the chemical composition of the kidney tissues. Hence, the glycosaminoglycan composition of the renal cortex and medulla at varying age intervals was investigated. Glycosaminoglycans were isolated from the tissues by means of digestion with collagenase and pronase and purified by ethanol precipitation. Subsequent separation of various polyanions was accomplished by ion exchange chromatography on a Dowex 1-X2 column, using sodium chloride buffers of increasing ionic strengths. The glycosaminoglycans in each fraction were identified and quantitated by digestion with specific enzymes, including hyaluronidase, chondroitinase AC and ABC. The enzyme resistant material was separated and further digested with nitrous acid to quantitate the proportion of heparon sulfate. The results indicate that the glycosaminoglycan content of the renal medulla was much higher than the cortex at all the age intervals studied, and age-induced reduction was mainly cortical. There was a significant reduction in the heparan sulfate content of the cortex in aging. Interestingly, the major glycosaminoglycan content of the medulla was hyaluronic acid, which showed a sharp increase during aging, whereas heparan sulfate declined. Chondroitin sulfate was not altered due to age in either tissue. The molecular weight of hyaluronic acid was determined by column chromatography. Results indicate that the size of hyaluronate in the cortex was small and did not vary with age. In the medulla of the younger age group, a considerable amount of large size hyaluronate was observed. As age increased, the size decreased. The results strongly suggest that alteration in the renal glycosaminoglycans may be partly responsible for the age related protinuria and ionic imbalance.     

Properties and subcellular distribution of guanylate cyclase activity in rat renal medulla: correlation with tissue content of guanosine 3',5'-monophosphate.

The properties of the guanylate cyclase systems of outer and inner medulla of rat kidney were examined and compared with those of the renal cortex. A gradation in steady-state cyclic guanosine 3',5'-monophosphate (cGMP) levels was observed in incubated slices of these tissues (inner medula greater than outer medulla greater than cortex). This correlated with the proportion of total guanyl cyclase activity in the 100 000 g particulate fraction of each tissue, but was discordant with the relative activities of guanylate cyclase (highest in cortex) and of cGMP-phosphodiesterase (lowest in cortex) in whole tissue homogenates. Soluble guanylate cyclase of cortex and inner medulla exhibited typical Michaelis-Menten kinetics with an apparent Km for MnGTP of 0.11 mM, while the particulate enzyme from inner medulla exhibited apparent positive cooperative behavior and a decreased dependence on Mn2+. Thus, the particulate enzyme could play a key role in regulating cGMP levels inthe intact cell where Mn2+ concentrations are low. The soluble and particulate enzymes from inner medulla were further distinguished by their responses to several test agents. The soluble enzyme was activated by Ca2+, NaN3, NaNo2 and phenylhydrazine, whereas particulate activity was inhibited by Ca2+ and was unresponsive to the latter agents. In the presence of NaNo2, Mn2+ requirement of the soluble enzyme was reduced and equivalent to that of the particulate preparation. Moreover, relative responsiveness of the sollble enzyme to NaNO2 was potentiated when Mg2+ replaced Mn2+ as the sole divalent cation. These changes in metal requirements may be involved in the action of NaNO2 to increase cGMP in intact kidney. Soluble guanylate cyclase of cortex was clearly more responsive to stimulation by NaN3, Nano2, and phenylhydrazine that was soluble activity from either medullary tissue. The effectiveness of the agonists on soluble activity from outer and inner medulla cound also be distinguished. Accordingly, regulation and properties of soluble guanylate cyclase, as well as subcellular enzyme distribution, and distinct in the three regions of the kidney.     

Phospholipase C activity in rat kidney. Effect of deoxycholate on phosphatidylinositol turnover

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.     

Renal cytochrome P-450's-electrophoretic and electron paramagnetic resonance studies.

The cytochrome P-450's of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450's on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450's but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.     

Effects of water-deficit on lipids of safflower aerial parts

Three-week-old plants of safflower (Carthamus tinctorius L.) were subjected to a water-deficit stress. The lipid composition of the shoot parts of both control (well-watered) and water stressed plants was analyzed. Experimental data revealed that moderate stress induced an increase in total lipid content within all lipidic classes. However, severe water-deficit induced a sharp decrease in the total lipid content and specially in polar lipids, particularly in phosphatidylethanolamine, phosphatidylcholine, monogalactosyl-diacylglycerol and digalactosyl-diacylglycerol. Also, the content of neutral lipids was increased. Concerning the fatty acid composition, water-deficit induced a decrease in their degree of unsaturation expressed by a reduction in the proportions of linolenic (18:3) and linoleic (18:2) acids and most of lipidic classes.     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla.

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions. Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from which the colelcting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [14C] arachidonate as substrate. Of the prostaglandin in synthetase activity 39% was found in the collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

A histochemical analysis of adrenal lipids in Ox and Sheep

Summary The adrenals of Ox and Sheep were analysed by various histochemical methods available for lipids. The cortex was found to be rich in choline containing phospholipids, unsaturated phospholipids and masked lipids compared with the medulla. Unsaturated lipids and free fatty acids were more abundant in the cortex. There was slightly more cholesterol and plasmalogen in the medulla than the cortex. Formaldehyde fixation apparently increased the relative number of carboxyl groups in the adrenaline storing cells. Osmium tetroxide reacted with catechol amines and differentiated between the adrenaline and noradrenaline storing cells in the ox medulla. The histochemical results are compared with previous biochemical findings on the nature and distribution of adrenal lipids.     

Sophorose lipid production from lipidic precursors: Predictive evaluation of industrial substrates

Summary Sophorose lipids stand out as biosurfactants with a wide potential for industrial application and which can be produced in good yield from glucose and a lipidic cosubstrate.Candida bombicola CBS 6009 (ATCC 22214) was used in the present study. The influence of the lipidic cosubstrate on various aspects of production performance of these glycolipids (final concentration, yield) and on product composition (in particular, the structure of the hydroxy fatty acid vegetable and animal oils, markedly influenced product composition. In terms of production performance, the best substrates were oils or esters rich in C18:0 and C18:1 fatty acids. Optimal overall performance was obtained with esters (340 g L^–1 sophorose lipids with rapeseed esters). Conclusions drawn from the results allow predictive evaluation of lipidic industrial substrates.