Urinary PGE2 in rats with chronic partial unilateral ureteral obstruction.

Urinary prostaglandin E2 (PGE2) was measured in Munich-Wistar rats with surgically created chronic partial unilateral ureteral obstruction (UUO). Mean values of bladder urine PGE2 were higher in sham than in UUO (24.5 +/- 14.4 vs 12.9 +/- 8.2 ng/mg creatinine, respectively, P less than 0.05). Following diuresis, both ureters were cannulated and urine was collected. PGE2 excretion was increased in sham (66.5 +/- 34.4 and 70.1 +/- 44.5 ng/mg creatinine, left and right, respectively). But in UUO, the obstructed kidney excreted less PGE2 than the contralateral kidney (32.1 +/- 6.0 vs 62.3 +/- 40.4 ng/mg creatinine, obstructed vs contralateral, respectively, P = 0.08). PGE2 synthesis was then determined in separated renal medullary and cortical slices. Renal medullary slices from kidneys with severe obstruction synthesized less PGE2 than the contralateral unobstructed side (3.30 +/- 1.22 vs 10.52 +/- 3.23 ng/mg wet wt-30 min, respectively, P less than 0.05) and failed to respond to arachidonic acid stimulation with any significant increase in PGE2 synthesis (3.30 +/- 1.22 vs 4.47 +/- 1.04 ng/mg wet wt-30 min, baseline vs stimulated). In contrast, contralateral unobstructed kidney slices responded with a significant increase in PGE2 synthesis (10.52 +/- 3.23 vs 21.10 +/- 2.50 ng/mg wet wt-30 min, baseline vs stimulated, P less than 0.05). We conclude that chronic partial UUO in the Munich-Wistar rats resulted in significantly less PGE2 elaboration.

Metabolism and disposition of benzidine in the dog.

The dog is an animal model for assessing aromatic amine-induced bladder cancer. For this reason, metabolism and disposition of benzidine in dog was assessed. Dogs were administered a 1 mg/kg i.v. dose of [3H]benzidine (16.4 mCi/mmol). The plasma t1/2 of the radiolabeled material (benzidine plus metabolites) was significantly longer (approximately 3 h) than authentic benzidine (less than 30 min). During the 5 h experiment, the majority of radiolabel was associated with bile, urine and carcass. Bladder transitional epithelium exhibited a consistently higher concentration of bound radioactivity than bladder muscle. A significant amount of binding was observed in DNA from liver, kidney and bladder. DNA from bladder transitional epithelium exhibited the highest concentration of radioactivity. Approximately 30% of the radioactivity recovered following HPLC of urine or bile was identified as unmetabolized benzidine. 3-Hydroxybenzidine was a major metabolite identified in bile (8%) but not urine. Urine samples treated with acid, base or sulfatase yielded 3-hydroxybenzidine (6%) as a major hydrolysis product. Similar treatment of bile samples did not result in increased amounts of 3-hydroxybenzidine. Neither N-acetylated nor N-methylated metabolites of benzidine were observed in urine or bile. Thus, considerable metabolism of benzidine occurs in dogs by pathways that are yet to be determined.

Renal metabolism of formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]-hydrazide.

Formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl]-hydrazide (FNT) is a potent renal carcinogen in the rat. This study assessed the metabolism of FNT by the isolated perfused rat kidney and whole rat. The glomerular filtration rate and the fractional excretion of sodium for the isolated perfused kidney indicated that under the conditions of these experiments FNT did not alter these renal parameters. The half-life (t1/2) for FNT in the isolated perfused kidney was 67 +/- 8 min. Using HPLC, a metabolite of FNT was observed in urine from the isolated perfused kidney. This metabolite had absorbance at 385 nm but not 254 nm and could not be detected electrochemically at +500 mV. While the excretion of FNT decreased with time of perfusion, the metabolite excretion increased. Whole animal studies demonstrated that FNT is rapidly cleared from blood within the first 5 min of administration. The FNT metabolite was excreted at approximately the same rate from 0-30 and 30-60 min after FNT administration. The metabolite was not observed in media from FNT perfused kidneys or plasma from animals administered FNT. Analysis of purified metabolite by liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) determined the structure to be 5-nitro-2-furonitrile. This structure assignment was verified by chemical synthesis. Results demonstrate target organ metabolism of carcinogen.

Renal handling of 5-nitrofuran nephrotoxins in the rat.

Formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl)-hydrazide (FNT) and 3-hydroxymethyl-1-([3-(5-nitro-2-furyl)-allydidene]amino)hydantoin (HMN) were investigated to determine whether differences in the renal handling of these two chemicals might provide evidence to explain their different patterns of toxicity and carcinogenicity. The isolated perfused rat kidney and whole animal were used. In the isolated perfused rat kidney, both FNT and HMN had similar half-lives (t1/2) but the urinary excretion and renal clearance of HMN (2.1 +/- 0.4 greater than those of FNT (0.2 +/- 0.1 nmol/min and 0.06 +/- 0.01 ml/min, respectively). Probenecid increased the t1/2 and decreased the metabolic clearance of HMN but did not have any effect on FNT t1/2 or clearance. These differences in excretion of FNT and HMN could not be explained on the basis of protein binding. The total clearances of FNT and HMN were similar and significantly higher than that of the 5-nitrofuran bladder carcinogen ANFT. In the whole animal, the urinary excretion of HMN was about 10-fold greater than that of FNT. The t1/2 of both FNT and HMN was less than 5 min in the whole animal. Probenecid decreased the urinary excretion of HMN from 9.7 +/- 1.4% to 4.4 +/- 1.0% (p less than 0.05). Compared with HMN, FNT has less urinary excretion but a similar elimination t1/2, suggesting a greater nonrenal clearance. HMN but not FNT has tubular excretion. Thus, alterations in substituents of 5-nitrofurans markedly alter their renal handling and may partially explain their diverse toxic effects.

Renal metabolic/excretory coupling.

Renal metabolic/excretory coupling is the enhancement of urinary excretion dependent upon renal metabolism. The nitrofurothiazoles, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), are model compounds used to study metabolic/excretory coupling. FANFT is deformylated to ANFT by renal deformylase enhancing ANFT excretion. In the rat, ANFT excretion after oral FANFT administration was 100-fold greater than ANFT excretion when ANFT was administered. FANFT and ANFT uptake into purified proximal tubules achieved equilibrium within 60 s and was demonstrated in nonviable tubules. FANFT partitioned into oil better than ANFT. Albumin inhibited FANFT and ANFT uptake into oil and decreased tubular uptake by 65%. Tubular FANFT uptake was threefold or greater than that of ANFT uptake with or without albumin. Renal deformylase was predominantly cytosolic and yielded apparent Km and Vmax of 6.7 microM and 6.1 nmol ANFT.min-1.mg protein-1, respectively. Deformylase activity was abolished by boiling, was specific for N-formylated compound, and was not altered by dinitrophenol treatment. Renal metabolic/excretory coupling for FANFT/ANFT combines energy-independent uptake with metabolism (deformylation), resulting in enhanced urinary ANFT excretion.

Effect of peroxidase inhibitors on an in vivo metabolite of the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in rats.

Peroxidase metabolism of 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was evaluated in vitro and in vivo. In vitro metabolism of ANFT was characteristic of the hydroperoxidase activity of prostaglandin H synthase. The peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole, significantly reduced ANFT binding to trichloroacetic acid precipitable material and glutathione conjugate formation. Isolated perfused kidneys rapidly converted the glutathione conjugate to its corresponding mercapturic acid (ANFT-MA). With both radiochemical and electrochemical techniques, ANFT-MA was identified in the urine of rats given N-[14C]-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, the carcinogenic N-formyl analogue of ANFT. ANFT was the major urinary metabolite with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide not detected. A 30-min pretreatment with 6-n-propyl-2-thiouracil and methimazole significantly reduced urinary excretion of ANFT-MA in rats given N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (150 mg/kg) from 14.8 +/- 2.1 (SE) to 7.9 +/- 0.8 and 6.2 +/- 1.1 nmol/18 h, respectively. Peroxidase inhibitor pretreatment did not alter the excretion of ANFT or prostaglandin E2. These results provide further in vitro and in vivo support for the involvement of peroxidases, i.e., the hydroperoxidase activity of prostaglandin H synthase, in ANFT metabolism.

Aging and the kidney.

Numerous anatomic and physiologic alterations occur in the kidney with aging. These changes affect the ability of elderly patient(s) to maintain homeostasis and alter response to medications, stress, illness, or changes in diet, mobility, or environment. Drug-induced illness and drug interactions are major problems in the elderly. Bone disease and fractures are associated with negative calcium balance and decreased production of 1,25-dihydroxycholecalciferol seen with aging. The geriatric patient is not immune to the primary glomerular diseases that occur in younger patients, although the relative incidence of pathologic diagnoses may differ. The high incidence of membranous glomerulonephritis in the elderly, and the well-known association between malignancy and membranous nephropathy strongly favor aggressive evaluation of the nephrotic syndrome in the geriatric age group. Attention must be given to consideration of appropriate end-stage renal disease treatment alternatives for the geriatric population, which now comprises the fastest-growing segment of the end-stage renal disease population.

Role of renal metabolism and excretion in 5-nitrofuran-induced uroepithelial cancer in the rat.

5-Nitrofurans have been used in the study of chemical carcinogenesis. There is substantial evidence that N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) is deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) in the process of FANFT-induced bladder cancer. Paradoxically, ANFT is less potent as a uroepithelial carcinogen than FANFT when fed to rats. Feeding aspirin with FANFT to rats decreases the incidence of bladder cancer. Isolated kidneys were perfused with 5-nitrofurans to determine renal clearances and whether aspirin acts to decrease urinary excretion of the carcinogen. In FANFT-perfused kidneys, FANFT was deformylated to ANFT and excreted (1.06 +/- 0.22 nmol/min) at a rate eightfold higher than excretion of FANFT. In kidneys perfused with equimolar ANFT, excretion of ANFT was 0.25 +/- 0.05 nmol/min, which suggests a coupling of renal deformylation of FANFT to excretion of ANFT in FANFT-perfused kidneys. Neither aspirin nor probenecid altered the urinary excretion or half-life of FANFT or ANFT. In rats fed 0.2% FANFT as part of their diet, coadministration of aspirin (0.5%) increased urinary excretion of ANFT during a 12-wk feeding study, which suggests decreased tissue binding or metabolism of ANFT. Kidney perfusion with acetylated ANFT (NFTA), a much less potent uroepithelial carcinogen, resulted in no ANFT excretion or accumulation, which indicates the specificity of renal deformylase. Renal deformylase activity was found in broken cell preparations of rat and human kidney. These data describe a unique renal metabolic/excretory coupling for these compounds that appears to explain the differential carcinogenic potential of the 5-nitrofurans tested. These results are consistent with the hypothesis that aspirin decreases activation of ANFT by inhibiting prostaglandin H synthase.

Effect of phosphodiesterase inhibitors on bradykinin-mediated prostaglandin E2 and cyclic AMP synthesis in renal papillary collecting tubule cells.

Isolated rabbit renal papillary collecting tubule cells were used to examine the effects of phosphodiesterase inhibitors on intracellular cyclic AMP and prostaglandin synthesis. Experiments performed on confluent primary tissue cultures demonstrated that bradykinin increases intracellular cyclic AMP by a prostaglandin-dependent mechanism. Phosphodiesterase inhibitors induced a dose-dependent decrease in bradykinin-stimulated prostaglandin synthesis. Fifty percent inhibition occurred with approximately 0.7 mM 3-isobutyl-1-methylxanthine (IBMX). Inhibition was found to be reversible. IBMX did not inhibit bradykinin-induced prostaglandin synthesis as a result of increased intracellular cyclic AMP. The nonmethylxanthine phosphodiesterase inhibitor RO 20-1724 also reduced bradykinin-stimulated prostaglandin synthesis. IBMX inhibited calcium-ionophore-A23187-induced prostaglandin synthesis but did not inhibit arachidonic acid stimulation of prostaglandin synthesis. The data demonstrate that bradykinin increased renal papillary collecting tubule cell cyclic AMP in a prostaglandin-dependent manner. Based on the data presented, phosphodiesterase inhibitors act to decrease arachidonic acid availability for prostaglandin synthesis, independent of changes in cellular cyclic AMP content.

Independent effects of bradykinin on adenosine 3',5'-monophosphate and prostaglandin E2 metabolism by rabbit renal medulla.

Bradykinin-stimulated increases in renal prostaglandin (PG) synthesis are thought to result in subsequent increases in cAMP content. This study assesses the relationship between bradykinin-stimulated increases in PGE2 and cAMP syntheses in renal inner medullary slices. Bradykinin-mediated increases in cAMP (2 min) preceded those in PGE2 (5 min) synthesis. Forskolin, an activator of adenylate cyclase, increased cAMP, while 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor, reduced cAMP. However, neither agent altered bradykinin-stimulated PGE2 synthesis. Aspirin decreased basal and abolished bradykinin-stimulated PGE2 production, but did not alter bradykinin-induced increases in cAMP content. Maximal stimulatory concentrations of 1-methyl-3-isobutylxanthine, a cyclic nucleotide phosphodiesterase inhibitor, and bradykinin were additive in their capacity to increase inner medullary cAMP content. These results suggest that 1-methyl-3-isobutylxanthine and bradykinin increase cAMP by separate mechanisms and that bradykinin increases inner medullary cAMP by a direct effect on the production of that cyclic nucleotide. Bradykinin-mediated increases in cAMP and PGE2 syntheses by renal medullary slices are independent effects of this renally acting hormone.

Effects of angiotensin II on phosphatidylinositol and polyphosphoinositide turnover in rat kidney. Mechanism of prostaglandin release.

Prostaglandin (PG) release from rat inner medullary tissue has been shown to be stimulated by angiotensin II, bradykinin, and arginine vasopressin. PG release from inner medullary has also been demonstrated to be a Ca2+-dependent process. We performed the following studies in an attempt to determine the mechanism by which angiotensin II stimulates PG release and to identify the lipid source serving as the donor for arachidonic acid (AA) in the Ca2+-dependent reaction. After 5 min of incubation, slices of inner medulla prelabeled with [14C]AA released 2.0-fold as much radiolabel into the media in the presence of Ca2+ as in the absence of Ca2+. After 30 min of incubation, the neutral lipids lost 6.1% of their [14C]AA label, phosphatidylethanolamine lost 12.5%, phosphatidylcholine 13.3%, and phosphatidylinotisol (P[I) 27%. The divalent ionophore A23187 (5 microM) increased 3.7-fold the formation of immunoassayable prostaglandin E2 at 30 min in the presence of Ca2+. Angiotensin II increased immunoassayable PGE2 formation 1.3-fold at 2 min and 1.5- to 1.8-fold by 30 min. In addition, angiotensin II rapidly increased the incorporation of [32P]orthophosphate to significantly higher values into PI, phosphatidic acid, diphosphoinositol, and triphosphoinositol by 30 s, returning to control values by 2 min of incubation. The data suggest that PI may be a major source of arachidonic acid in the Ca2+-dependent release of PG, that angiotensin II stimulates a smaller or different pool of AA release than the divalent ionophore, and the angiotensin II stimulation of PG is a Ca2+-mediated process associated with increased "phosphatidylinositol-polyphosphoinositide" turnover in the rat inner medulla.