Fish/flaxseed oil protect against nitric oxide-induced hepatotoxicity and cell death in the rat liver.

Sodium nitroprusside (SNP) is an antihypertensive drug with proven toxic effects attributed mainly to the production of nitric oxide (NO). Polyunsaturated fatty acids (PUFAs) are widely regarded as functional foods and have been shown to ameliorate the harmful effects of many toxicants. This study examined whether feeding of fish oil (FO)/flaxseed oil (FXO) would have any protective effect against SNP-induced hepatotoxicity and cell death. Male Wistar rats were fed either on normal diet or with 15% FO/FXO for 15 days, following which SNP (1.5 mg/kg body weight) was administered intraperitoneally for 7 days. Animals were killed after treatment, and livers were collected for further analysis. We observed that SNP significantly elevated tissue nitrite levels and lipid peroxidation (LPO) with concomitant perturbation in antioxidant defense systems accompanied with dysregulated glucose metabolism and pronounced cellular death. FO/FXO supplementation to SNP-treated rats caused reversal of tissue injury/cell death and markedly decreased LPO and improved antioxidant defense systems. FO/FXO appear to protect against SNP-induced hepatotoxicity by improving energy metabolism and antioxidant defense mechanism.

Protective effect of ω-3 polyunsaturated fatty acids (PUFAs) on sodium nitroprusside-induced nephrotoxicity and oxidative damage in rat kidney.

Sodium nitroprusside (SNP) a nitric oxide (NO) donor has proven toxic effects. Dietary ω-3 polyunsaturated fatty acid (PUFA) has been shown to reduce the severity of numerous ailments. Present study examined whether intake of fish oil (FO)/flaxseed oil (FXO, Omega Nutrition, St Vancouver, Canada) would have protective effect against SNP-induced toxicity. Male Wistar rats (150 ± 10 g) were used in this study. Initially animals were divided into two groups: one fed on normal diet and the other on 15% FO/FXO for 15 days. On the 16th day, SNP (1.5 mg/kg body weight) was administered intraperitoneally for 7 days daily. After 7 days animals were killed, kidneys were harvested for further analysis. SNP induced nephrotoxicity by increasing serum creatinine and blood urea nitrogen, SNP significantly decreased malate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and malic enzyme but increased lactate dehydrogenase and glucose-6-phosphate dehydrogenase. Brush border membrane enzymes such as alkaline phosphatase, γ-glutamyl transpeptidase and leucine amino peptidase were also decreased. The activity of catalase and glutathione peroxidase decreased concomitantly with increased lipid peroxidation, indicating that the significant kidney damage has been inflicted by SNP. Feeding of FO and FXO with SNP ameliorated the changes in various parameters caused by SNP. The results of the present study suggest that ω-3 PUFA-enriched FO and FXO from seafoods and plant sources, respectively, are similarly effective in reducing SNP-induced nephrotoxicity and oxidative damage. Thus, vegetarians who cannot consume FO can have similar health benefits from plant-derived ω-3 PUFA.

Protective effect of ω-3 polyunsaturated fatty acids on L-arginine-induced nephrotoxicity and oxidative damage in rat kidney.

L-Arginine (ARG), an essential amino acid, is the endogenous source of the deleterious nitric oxide. Dietary ω-3 polyunsaturated fatty acid (PUFA)-enriched fish oil (FO) has been shown to reduce the severity of certain types of cancers, cardiovascular disease, and renal disease. Present study examined whether feeding of FO/flaxseed oil (FXO) would have protective effect against ARG-induced nephrotoxicity. ARG-induced nephrotoxicity was recorded by increased serum creatinine and blood urea nitrogen. ARG significantly altered the activities of metabolic and brush border membrane (BBM) enzymes. ARG caused significant imbalances in the antioxidant system. These alterations were associated with increased lipid peroxidation (LPO) and altered antioxidant enzyme activities. Feeding of FO and FXO with ARG ameliorated the changes in various parameters caused by ARG. Nephrotoxicity parameters lowered and enzyme activities of carbohydrate metabolism, BBM and inorganic phosphate (32Pi) transport were improved to near control values. ARG-induced LPO declined and antioxidant defense mechanism was strengthened by both FO and FXO alike. The results of the present study suggest that ω-3 PUFA-enriched FO and FXO from seafoods and plant sources, respectively, are similarly effective in reducing ARG-induced nephrotoxicity and oxidative damage. Thus, vegetarians who cannot consume FO can have similar health benefits from plant-derived ω-3 PUFA.

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney.

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in omega-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5mg/kg body weight) intraperitoneally. After 5d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism.

Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in kidney and other rat tissues.

Trichloroethylene (TCE), an industrial solvent, is a major environmental contaminant. Histopathological examinations revealed that TCE caused liver and kidney toxicity and carcinogenicity. However, biochemical mechanism and tissue response to toxic insult are not completely elucidated. We hypothesized that TCE induces oxidative stress to various rat tissues and alters their metabolic functions. Male Wistar rats were given TCE (1000 mg/kg/day) in corn oil orally for 25 d. Blood and tissues were collected and analyzed for various biochemical and enzymatic parameters. TCE administration increased blood urea nitrogen, serum creatinine, cholesterol and alkaline phosphatase but decreased serum glucose, inorganic phosphate and phospholipids indicating kidney and liver toxicity. Activity of hexokinase, lactate dehydrogenase increased in the intestine and liver whereas decreased in renal tissues. Malate dehydrogenase and glucose-6-phosphatase and fructose-1, 6-bisphosphatase decreased in all tissues whereas increased in medulla. Glucose-6-phosphate dehydrogenase increased but NADP-malic enzyme decreased in all tissues except in medulla. The activity of BBM enzymes decreased but renal Na/Pi transport increased. Superoxide dismutase and catalase activities variably declined whereas lipid peroxidation significantly enhanced in all tissues. The present results indicate that TCE caused severe damage to kidney, intestine, liver and brain; altered carbohydrate metabolism and suppressed antioxidant defense system.

Studies on the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney.

Gentamicin (GM)-induced nephrotoxicity limits its long-term clinical use. Several agents/strategies were attempted to prevent GM nephrotoxicity but were not found suitable for clinical practice. Dietary fish oil (FO) retard the progression of certain types of cancers, cardiovascular and renal disorders. We aimed to evaluate protective effect of FO on GM-induced renal proximal tubular damage. The rats were pre-fed experimental diets for 10 days and then received GM (80 mg/kg body weight/day) treatment for 10 days while still on diet. Serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport in rat kidney were analyzed. GM nephrotoxicity was recorded by increased serum creatinine and blood urea nitrogen. GM increased the activities of lactate and glucose-6-phosphate dehydrogenases whereas decreased malate, isocitrate dehydrogenases; glucose-6 and fructose-1,6-bisphosphatases; superoxide dismutase, catalase, glutathione peroxidase and BBM enzymes. In contrast, FO alone increased enzyme activities of carbohydrate metabolism, BBM and oxidative stress. FO feeding to GM treated rats markedly enhanced resistance to GM elicited deleterious effects and prevented GM-induced decrease in 32Pi uptake across BBM. Dietary FO supplementation ameliorated GM-induced specific metabolic alterations and oxidative damage due to its intrinsic biochemical/antioxidant properties.

Time dependent effect of gentamicin on enzymes of carbohydrate metabolism and terminal digestion in rat intestine.

Gentamicin (GM) is an aminoglycoside antibiotic commonly used against life threatening gram negative bacterial infections, however, nephrotoxicity remains the major concern for its long term use. Although its effects on kidney are well characterized but there have been no studies regarding its effects on intestine. We hypothesize that GM causes adaptive coordinated effect on enzymes of carbohydrate metabolism and terminal digestion/ absorption in rat intestine. Rats were administered a nephrotoxic dose of GM (80 mg /kg body weight) daily for 15 days and a time dependent effect was observed on various enzyme activities. Activities of lactate (LDH), malate (MDH) and isocitrate (ICDH) dehydrogenases, significantly increased and peaked at different time intervals of GM treatment. Whereas LDH activity remained higher, MDH and ICDH activity slowly declined from their peak values. Activities of fructose-1,6-bisphosphatase, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase increased but malic enzyme decreased in a time dependent manner. Activity of alkaline phosphatase and sucrase significantly increased but gamma-glutamyl transpeptidase activity decreased. GM administration increased lipid peroxidation, glutathione peroxidase but decreased superoxide dismutase and catalase activities. The results indicate that GM treatment selectively upregulated certain enzymes of carbohydrate metabolism and terminal digestion/absorption and perturbed antioxidant defenses.

Effect of potassium dichromate on renal brush border membrane enzymes and phosphate transport in rats.

Chromium is widely used in industry but exposure to chromium compounds in the workplace can result in nephrotoxicity. Various nephrotoxicants affect the brush border membrane (BBM) lining the epithelial cells of the proximal tubule, but there have been no studies regarding the effect of potassium dichromate (K2Cr2O7), a hexavalent chromium compound, on renal BBM. In the present work, the effect of administering a single intraperitoneal dose (15 mg/kg body weight) of K2Cr2O7 on rat renal BBM enzymes and inorganic phosphate (Pi) transport was studied. The animals were administered normal saline (control) or K2Cr2O7 and sacrificed 1, 2, 4 and 8 days after treatment. K2Cr2O7 induced reversible damage to the rat kidney function as indicated by serum creatinine (Scr) and urea nitrogen levels. The activities of BBM marker enzymes were significantly decreased in isolated BBM vesicles (BBMV) and homogenates of cortex and medulla on 1, 2 and 4 days after administration of K2Cr2O7 with complete recovery to control values after 8 days. The decrease in the activities of the enzymes was mainly due to changes in maximum velocity (V(max)) values, while the Michaelis constant (Km) remained unchanged. The sodium dependent Pi transport across BBMV was reduced by 50% after treatment with K2Cr2O7. Thus, the administration of a single dose of K2Cr2O7 leads to impairment in the functions of renal BBM. These results suggest that the nephrotoxicity of K2Cr2O7 may be mediated, at least in part, by its effect on renal BBM.

cADP-ribose/ryanodine channel/Ca2+-release signal transduction pathway in mesangial cells.

Signaling via release of Ca2+ from intracellular stores is mediated by several systems, including the inositol 1,4,5-trisphosphate (IP3) and cADP-ribose (cADPR) pathway. We recently discovered a high capacity for cADPR synthesis in rat glomeruli and cultured mesangial cells (MC). We sought to determine whether 1) cADPR synthesis in MC is regulated by cytokines and hormones, 2) ryanodine receptors (RyRs) are expressed in MC, and 3) Ca2+ is released through RyRs in response to cADPR. We found that ADP-ribosyl cyclase, a CD38-like enzyme that catalyzes cADPR synthesis, is upregulated in MC by tumor necrosis factor-alpha, interleukin-1beta, and all-trans retinoic acid (atRA). [3H]ryanodine binds to microsomal fractions from MC with high affinity in a Ca2+-dependent manner; binding is enhanced by specific RyR agonists and blocked by ruthenium red and cADPR. Western blot analysis confirmed the presence of RyR in MC. Release of 45Ca2+ from MC microsomes was stimulated by cADPR; release was blocked by ruthenium red and 8-bromo-cADPR. ADPR (non-cyclic) was without effect. In MC, TNF-alpha and atRA amplified the increment of cytoplasmic Ca2+ elicited by vasopressin. We conclude that MC possess elements of a novel ADP-ribosyl cyclase-->cADPR-->RyR-->Ca2+-release signaling pathway subject to regulation by proinflammatory cytokines and steroid superfamily hormones.

Nephron distribution of total low Km cyclic AMP phosphodiesterase in mouse, rat and rabbit kidney.

The activity of cAMP degradation enzyme, cAMP phosphodiesterase (cAMP PDE), in renal tubules is a critically important factor in determining cellular cAMP levels, particularly in response to hormones. In this study we examine the nephron distribution of cAMP PDE activity in the mouse, rat and rabbit kidney and important cellular regulators of cAMP PDE, namely calmodulin and adenosine triphosphate (ATP). We assayed total low Km cAMP PDE in microdissected tubule segments, using 10(-6) M (3H) cAMP as a substrate. Activities were expressed in fentomol cAMP hydrolyzed per minute per mm tubular length or per one glomerulus. The content of ATP was measured in outer medullary collecting duct and medullary thick ascending limb of Henle's loop with microbioluminescence assay using firefly luciferase. In mouse kidney, cAMP PDE was significantly higher in all tubular segments compared to glomerulus. Proximal convoluted tubule, proximal straight tubule, medullary thick ascending limb of Henle's loop (mTAL), and outer medullary collecting duct (OMCD) had intermediated activity. Greater cAMP PDE activity was detected in cortical ascending limb of Henle's loop (cTAL), cortical collecting duct and in distal convoluted tubule (DCT). The highest activity was found in connecting tubules. In rat, nephron distribution of cAMP PDE activities was similar to mouse, except that activity in glomeruli was higher than in mouse glomeruli. In rabbit, nephron distribution of cAMP PDE activities was different from those of mouse and rat. There was no single prominent segment with high cAMP PDE activity. DCT and cTAL showed low enzyme activity. Overall, the highest cAMP PDE activities were measured in the mouse and the lowest were measured in the rabbit nephrons, with those of rat nephron showing an intermediate activity. The maximum effective dose of the calmodulin antagonist, trifluoperazine (200 microM), inhibited cAMP PDE in all nephron segments from the rat kidney. However, there is no statistical significance of its inhibition among nephron segments. In OMCD and mTAL of the rat kidney, cAMP PDE activity was inhibited by ATP (5 mM to approximately 10 mM) which is far beyond the physiological concentartion of ATP in normal epithelial cell. Actual determinations of ATP in mTAL and OMCD were 0.1 mM and 0.17 mM, respectively. These observations show that distal segments of tubules have more active catabolism of cAMP than proximal segments. cAMP PDE in each nephron segment appear to be almost equally dependent on trifluoperazine-sensitive pathway that may reflect the Ca2+-calmodulin system. Cellular concentration of ATP might not be involved in the regulation of the total low Km cAMP PDE activity in rat mTAL and OMCD.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells.

Nicotinic acid-adenine dinucleotide phosphate (NAADP), a molecule derived from beta-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not beta-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.

Effect of reversible and irreversible ischemia on marker enzymes of BBM from renal cortical PT subpopulations.

The effect of the reversible and relatively irreversible ischemia induced acute renal failure (ARF) in the activities of alkaline phosphatase (AlkPase) and gamma-glutamyltransferase (GGTase) after early (15-30 min) and prolonged (45-60 min) ischemia in the homogenates, and the brush-border membranes (BBM) from rat renal whole, superficial (SC), and juxtamedullary (JMC) cortices were studied. The enzyme activities declined progressively in proportion to the duration of ischemia. Early blood reflow of 15 min to the ischemic rats caused a further decrease in the enzyme activities. However, prolonged reflow (up to 120 min) resulted in partial reversal of the ischemic effect in the early but not in the prolonged ischemic rats. The decrease in the enzyme activities was due to the loss of membrane-bound enzyme components from the damaged BBM into the supernatant fraction as membrane-free enzymes. The activities of AlkPase and GGTase were significantly more decreased by the ischemia in the brush-border membrane vesicles (BBMV)-JMC than in BBMV-SC. The rate of recovery due to reflow for AlkPase was greater in BBMV-SC than apparently for GGTase in BBMV-JMC in early ischemic (15-30 min) rats.

Differential properties of brush-border membrane vesicles from early and late proximal tubules of rat kidney.

We describe the preparation and properties of BBM vesicles (BBMV) from the superficial and juxtamedullary rat renal cortex using Ca(2+)-precipitation method and/or by density gradient centrifugation. BBMV were characterized by the presence of BBM marker enzymes as distributed along microdissected proximal convoluted tubule and proximal straight tubules from superficial and juxtamedullary cortex. In tubules from both superficial and juxtamedullary cortex, the activities of gamma-glutamyltransferase and leucine aminopeptidase were 5-10 times higher in proximal straight tubules than in proximal convoluted tubule. The alkaline phosphatase was higher in proximal convoluted tubules than in straight tubules from superficial cortex, but it was lower in proximal convoluted than straight tubules from the juxtamedullary cortex. The Na+/Pi cotransport had higher Vmax and lower Km in BBMV from superficial cortex than from BBMV from juxtamedullary tissue. BBMV from superficial cortex separated on Percoll gradient showed a high activity of alkaline phosphatase and low activities of gamma-glutamyltransferase and leucine aminopeptidase. Conversely, BBM from juxtamedullary cortex separated into a major peak with very high activities of gamma-glutamyltransferase and leucine aminopeptidase, and lesser activity of alkaline phosphatase. These distinct BBMV fractions showed diverse Na+/Pi cotransport properties and BBM marker enzyme distributions. Thus, using the outlined methodology it is feasible to prepare BBMV derived predominantly from proximal convoluted tubules or from proximal straight tubules located in either superficial or deep cortical nephrons.

Effect of chronic lithium treatment upon the Na(+)-coupled cotransporters in renal brush border membranes.

While the most frequent and prominent side effect of the chronic lithium (Li) administration is dysfunction of collecting ducts and polyuric syndrome, some reports also suggest defects in functions of proximal tubules. We determined the transport properties of brush border membranes (BBM) from kidneys of rats chronically fed for four weeks a chow containing Li (60 mmol/kg; Li-rats) and compared them with BBM from placebo-treated controls. BBM from Li-rats did not differ from controls in Na(+)-gradient-dependent transport of 32Pi, D-[3H]-glucose or L-[3H]-proline, but showed a considerable increase in the transport rates of [14C]-citrate (delta + 53%; P < 0.001) and [14C]-succinate (delta + 48%, P < 0.005), and also enhanced the rate of Na(+)-H+ antiport across BBM (delta + 30%, P < 0.05). In contrast, direct addition of 1 mM Li to BBMV in vitro inhibited Na(+)-dependent transports of both citrate and succinate. The Li-rats had higher plasma level of citrate and decreased (delta - 50%) renal clearance of citrate. Our results show that chronic exposure to oral Li in vivo results in increased Na(+)-gradient-dependent transport of polycarboxylic acids and increased Na(+)-H+ antiport in BBM of proximal tubules; these changes are contrary to effects of Li added to isolated BBM in vitro.

Effect of phosphate deprivation on enzymes of the cyclic adenosine monophosphate metabolism in rat proximal convoluted and proximal straight tubules.

Phosphate deprivation causes a resistance to the phosphaturic effect of parathyroid hormone (PTH). The present study determined whether acute phosphate deprivation alters basal or stimulated activities of key enzymes of the cyclic adenosine monophosphate (cAMP) metabolism in microdissected proximal convoluted and proximal straight tubules, since blunted cAMP levels in these proximal subsegments might account for refractoriness to the effect of PTH on phosphate reabsorption in the proximal convoluted and proximal straight tubule segments. In the proximal convoluted tubules of rats fed a normal-phosphate diet (NPD), PTH increased the adenylate cyclase activity by tenfold. In the proximal convoluted tubule of rats fed a low-phosphate diet (LPD), PTH also increased the adenylate cyclase activity by tenfold. In addition, forskolin increased the adenylate cyclase activity to levels similar to PTH in the proximal convoluted tubule of rats fed NPD or LPD. In the proximal straight tubule of rats fed NPD, PTH resulted in an approximately fivefold increase in adenylate cyclase activity. In the proximal straight tubule of rats fed LPD, PTH resulted in a fourfold increase in adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was markedly decreased (46%) in the proximal straight tubule of phosphate-deprived rats. The cAMP-phosphodiesterase activity in the proximal convoluted tubule was significantly increased by 26% in phosphate-deprived rats. The cAMP-phosphodiesterase activities in the proximal straight tubules from rats fed NPD or LPD were similar. We conclude that distinct differences in key enzymes of cAMP metabolism exist in the proximal convoluted and proximal straight tubule subsegments. Further, phosphate deprivation affects the cAMP-phosphodiesterase and adenylate cyclase activities differently in these nephron subsegments.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural requirement of monophosphates for inhibition of Na+-Pi cotransport in renal brush border membrane.

Using the chemical structural analogs of phosphonoacetic acid (PAA) and related phosphonate compounds, we investigated which structural features are required for competitive inhibition of Na+-Pi cotransport in rat renal cortical brush border membrane (BBM) vesicles (BBMV). The effects of compounds on [Nao+ greater than Nai+]-gradient-dependent 32Pi uptake by BBMV were examined using various inhibitor-to-32Pi concentration ratios in the transport assay medium. The replacement of a phosphono-group with an arsono-group in PAA, or the substitution of a carboxylic group in PAA by an amino or hydroxyl group, totally abolished the inhibitory action on Na+-Pi cotransport. Decreased electronegativity of carboxyl in PAA by coupling with hydrazine or hydroxylamine lowered the inhibitory potenty of PAA. Substitution of H at the alpha-carbon of PAA with ethyl or p-Cl-phenyl groups completely abolished the inhibitory activity, whereas alpha-halogenation with Br greatly increased the inhibitory potency of PAA, close to that of phosphonoformic acid (PFA). The inhibition by all the active tested monophosphates was strictly competitive. The tested compounds displaced [14C]PFA pre-bound onto BBMV in the presence of 100 mM NaCl. The ability of monophosphates to inhibit Na+-Pi cotransport across BBM and the binding of [14C]PFA were closely correlated (r = 0.925; P greater than 0.001). These results show that: (a) strong electronegativity at both ends of the PAA molecule is needed for inhibitory action, (b) an alpha-aliphatic or aromatic substituent at the alpha-carbon probably hinders the access of the inhibitor to the Pi-binding site of the Na+-Pi cotransporter in BBM, whereas (c) an alpha-electrophilic substituent--Br--enhances the inhibitory potency of PAA. The tested compounds inhibited Na+-Pi cotransport by binding, in the presence of Na+, on the same site on the luminal surface of BBM as did PFA and, by extension, Pi.

Dexamethasone blocks adaptive increase of Na+-Pi cotransport in renal brush border membrane elicited by thyroid hormone.

Dexamethasone administered to rats blocks and/or reverses adaptive increases in the rate of Na+-Pi cotransport, and also in the Na+-dependent binding of [14C]-phosphonoformic acid (PFA) by renal brush border membrane (BBM) vesicles elicited by thyroid hormone (T3). In contrast, dexamethasone had no effect on Na+-independent binding of [14C]-phosphonoformic acid, on Na+-dependent transport of D-glucose or on Na+-dependent binding of phlorizin by BBMV which indicates that its inhibitory effect is specific for Na+-Pi cotransport system of BBM. These findings suggest that glucocorticoids antagonize T3-elicited adaptive enhancement of Na+-Pi cotransport in renal proximal tubules by blocking the T3-stimulated de novo synthesis of Na+-Pi symporters and/or their insertion into BBM.

Different mechanisms of adaptive increase in Na+-Pi cotransport across renal brush-border membrane.

We explored the biochemical mechanism by which thyroid hormone (T3) and low-phosphate diet (LPD) cause an adaptive increase in Na+-Pi cotransport across renal brush-border membrane (BBM). The rate of Na+-Pi cotransport was determined by 32Pi uptake by BBM vesicles (BBMV), and the number of Na+-Pi symporters was assessed by binding of [14C]phosphonoformic acid (PFA) on BBMV. In BBMV of both T3-treated rats and LPD-fed rats, the Na+ gradient-dependent 32Pi uptake increased (Vmax increased; Km Pi was not changed). The Na+-dependent [14C]PFA binding on BBMV increased (higher Vmax, no change in Km PFA) in response to T3, but it remained unchanged in rats fed LPD. Both the increase of Na+-Pi cotransport and of Na+-dependent [14C]PFA binding in response to T3 were blocked by actinomycin D or cycloheximide. Addition of benzyl alcohol to BBMV in vitro increased Na+-Pi cotransport, but [14C]PFA binding did not change; the [3H]phlorizin binding and cotransports of other solutes decreased or did not change. The exposure of BBMV to cholesterol decreased Na+-Pi cotransport without changing [14C]PFA binding. We suggest that the adaptive increase of Na+-Pi cotransport elicited by T3 is due to an increase in number of Na+-Pi cotransporters in BBM. In contrast, in response to LPD the number of Na+-Pi cotransporters is unchanged, and the increased Na+-Pi cotransport is due to faster translocation of Na+ with Pi due to enhanced fluidity of BBM.

Rat atrial natriuretic factor (ANP-III) inhibits phosphate transport in brush border membrane from superficial and juxtamedullary cortex.

Previous studies have shown that administration of synthetic atrial natriuretic factor (ANF, 101-126) decreases sodium-dependent phosphate transport across renal brush border membrane vesicles (BBMV) in rats fed a normal or low phosphate diet. In the present study, infusion of rat ANF (atriopeptin III (ANP-III), 103-126 rat ANF) to rats fed a normal phosphate diet caused natriuretic and phosphaturic effects similar to those of ANF (101-126), but unlike ANF (101-126) did not increase the glomerular filtration rate. The effect of ANP-III infusion on sodium-dependent transport of phosphate was also determined in BBM vesicles isolated from the superficial cortex (BBMV-SC) and juxtamedullary cortex (BBMV-JM). The results indicate that ANP-III decreases phosphate transport across BBMV-SC and BBMV-JM similarly (20-24%). However, it had no effect on sodium-dependent transport of proline in these vesicles. The infusion of ANP-III to rats fed a normal phosphate diet inhibits phosphate uptake both in BBMV-SC and BBMV-JM and causes phosphaturia without increments in glomerular filtration rate.