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Uranium, the heaviest of the naturally occurring elements is widely present as environmental contaminant from natural deposits, industrial emissions and most importantly from modern weapons. Histopathological examinations revealed that uranyl nitrate (UN) exposure caused severe damage to pars recta of renal proximal tubule. However, biochemical events involved in cellular response to renal injury are not completely elucidated. We hypothesized that UN exposure would severely damage kidney tissues and alter their metabolic functions. Rats were treated with a single nephrotoxic dose of UN (0.5 mg/kg body weight) i.p. After 5 d, effect of UN was studied on the activities of various enzymes of carbohydrate metabolism, brush border membrane (BBM) and oxidative stress in different kidney tissues. Activity of lactate dehydrogenase increased whereas activities of isocitrate, succinate and malate dehydrogenases, glucose-6-phosphatase and fructose-1,6-bisphosphatase significantly decreased by UN exposure. Activity of glucose-6-phosphate dehydrogenase decreased whereas that of NADP-malic enzyme increased. The activities of BBM enzymes were significantly lowered and after dissociation from BBM excreted in urine. Lipid peroxidation and the activities of superoxide dismutase and glutathione peroxidase increased whereas catalase activity decreased by UN. UN treatment caused specific alterations in the activities of metabolic and membrane enzymes and perturbed antioxidant defenses.

The effect of 1-, 3-, and 5-day fasting on enzymes of brush border membrane and carbohydrate metabolism in the rat small intestine has been studied. Fasting led to significant decrease in the specific activities of brush border marker enzymes alkaline phosphatase and sucrase, as compared to levels in unfasted controls, both in the homogenates and in brush border membrane preparations. Kinetic studies revealed that these changes were due to changes in the maximal velocity (Vmax) only of alkaline phosphatase, but both the Michaelis constant (K m ) and V max were altered for sucrase. Fasting decreased the activities of lactate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase but increased the activities of glucose-6-phosphatase and fructose 1,6-bisphosphatase in mucosal homogenates. The activities of glucose-6-phosphate dehydrogenase and malic enzyme were also decreased by fasting. Thus fasting caused changes in the activities of various enzymes involved in the metabolism of glucose. The results of these enzyme studies suggest that the degradation of glucose is decreased but its production by gluconeogenesis is enhanced upon fasting.

Although dietary fish oil supplementation has been used to prevent the progression of kidney disease in patients with IgA nephropathy, relatively few studies provide a mechanistic rationale for its use. Using an antithymocyte (ATS) model of mesangial proliferative glomerulonephritis, we recently demonstrated that fish oil inhibits mesangial cell (MC) activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. We therefore sought to define potential mechanisms underlying the antiproliferative effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the predominant aomega;-3 polyunsaturated fatty acids found in fish oil, in cultured MC. DHA and EPA were administered to MC as bovine serum albumin fatty-acid complexes. Low-dose (10-50 amu;mol/L) DHA, but not EPA, inhibited basal and epidermal growth factor (EGF)andash;stimulated [3H]-thymidine incorporation in MCs. At higher doses (100 amu;mol/L), EPA and DHA were equally effective in suppressing basal and EGF-stimulated MC mitogenesis. Low-dose DHA, but not EPA, decreased ERK activation by 30% (P alt; .01), as assessed with Western-blot analysis using phosphospecific antibodies. JNK activity was increased by low-dose DHA but not by EPA. p38 activity was not significantly altered by DHA or EPA. Cyclin E activity, as assessed with a histone H1 kinase assay, was inhibited by low-dose DHA but not by EPA. DHA increased expression of the cell cycle inhibitor p21 but not p27; EPA had no effect on p21 or p27. We propose that the differential effect of low-dose DHA vs EPA in suppressing MC mitogenesis is related to down-regulation of ERK and cyclin E activity and to induction of p21.

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 γ-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 + P i cotransport had higher V max and lower K m 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 γ-glutamyltransferase and leucine aminopeptidase. Conversely, BBM from juxtamedullary cortex separated into a major peak with very high activities of γ-glutamyltransferase and leucine aminopeptidase, and lesser activity of alkaline phosphatase. These distinct BBMV fractions showed diverse Na + P i 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.

Phosphonoformic acid was a potent competitive inhibitor of Na+/Pi cotransport by brush border membrane vesicles isolated from human renal cortex (Ki 31 microM). The Na+/Pi cotransport system in rat renal brush border membrane vesicles was much less sensitive to inhibition (Ki 210 microM). Na+/Pi cotransport by cultured cells derived from human renal cortex also was inhibited by phosphonoformic acid. Both in isolated membranes and cells the inhibition was dose-dependent, competitive, and specific for Pi. The effectiveness of phosphonoformic acid on Na+/Pi cotransport in human kidney suggests that its administration may be a useful maneuver to increase renal Pi excretion.

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.

We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.

The role of N-linked oligosaccharide side chains in the biogenesis and function of Na+-coupled transporters in renal luminal brush-border membrane (BBM) is not known. We examined the question of how in vivo inhibition by alkaloid swainsonine of alpha-mannosidase, a key enzyme in processing of glycoproteins in the Golgi apparatus, affects Na+/H+ antiport and Na+/Pi symport as well as activities of other transporters and enzymes in rat renal BBM. Administration of swainsonine to thyroparathyroidectomized rats, control or treated with 3,5,3'-triiodothyronine, markedly decreased the rate of Na+/H+ antiport, but had no effect on the rate of Na+/Pi symport across renal BBM vesicles (BBMV). Moreover, administration of swainsonine did not change activities of Na+ gradient, ([extravesicular Na+] greater than [intravesicular Na+])-dependent transport of D-glucose, L-proline, or the amiloride-insensitive 22Na+ uptake by BBMV; the activities of the BBM enzymes alkaline phosphatase, gamma-glutamyltransferase, or leucine aminopeptidase in BBMV were also not changed. The in vitro enzymatic deglycosylation of BBM by incubating freshly isolated BBMV with bacterial endoglycosidase F also resulted in a decreased rate of Na+/H+ antiport, but not Na+-coupled symports of Pi, L-proline, and D-glucose, or the activities of the BBM enzymes were not significantly affected. Similar incubation with endoglycosidase H was without effect on any of these parameters. Both the modification of BBMV glycoproteins by administration fo swainsonine in vivo as well as the in vitro incubation of BBMV with endoglycosidase F resulted in a decrease of the apparent Vmax of Na+/H+ antiport, but did not change the apparent Km of this antiporter for extravesicular Na+ and did not increase H+ conductance of BBM. Taken together, our findings suggest that intact N-linked oligosaccharide chains of the biantennary complex type in renal BBM glycoproteins are required, directly or indirectly, for the transport function of the Na+/H+ antiporter inserted into BBM of renal proximal tubules.

Since phosphonoformic acid (PFA) acts as a specific competitive inhibitor of Na+-Pi co-transport across renal brush-border membrane (BBM), we employed the [14C]PFA as a probe to determine the mechanism of its interaction with rat renal BBM. The binding of [14C]PFA to BBM vesicles (BBMV), with Na+ present in extravesicular medium (Na+o), was time- and temperature-dependent. The replacement of Na+o with other monovalent cations reduced the PFA binding by -80%. Cl- was the most effective accompanying monovalent anion as NaCl for maximum PFA binding. The Na+o increased the apparent affinity of BBMV for [14C]PFA binding, but it did not change the maximum binding capacity. The maximum [14C]PFA binding was achieved at Na+o approximately equal to 50 mM. The extent of Na+-dependent [14C]PFA binding correlated (r = 0.98; p less than 0.01) with percent inhibition by an equimolar dose of PFA of the (Na+o greater than Na+i)-dependent BBMV uptake of 32Pi. Intravesicular Na+ (Na+i) decreased [14C]PFA binding, on BBMV, and this inhibition by Na+i was dependent on the presence of Na+o. The increase in Na+i, at constant [Na+]o, decreased the Vmax, but not the Km, for [14C]PFA binding on BBMV. Bound [14C]PFA was displaced from BBMV by phosphonocarboxylic acids proportionally (r = 0.99; p less than 0.05) to their ability to inhibit (Na+o greater than Na+i)-gradient-dependent Pi transport, whereas other monophosphonates, diphosphonates, L-proline, or D-glucose did not influence the [14C]PFA binding. The Na+-dependent binding of [14C]PFA and of [3H]phlorizin by BBMV was 10 times higher than binding of these ligands to renal basolateral membranes and to mitochondria. [14C]PFA probably binds onto the same locus on the luminal surface of BBM, where Pi and Na+ form a ternary complex with the Na+-Pi co-transporter.