Decreased Renal Excretion of Uric Acid Following Diuretic Administration in Rats

Kazumi IWAKI and Yukio YONETANI

Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan

Accepted December 19, 1983

Abstract-In order to evaluate the cause of diuretic-induced hyperuricemia which has been well documented in clinical studies, clearance experiments were performed in rats using furosemide and trichlormethiazide. The net flux in the tubular transport of uric acid was reabsorptive, and the fractional excretion of uric acid responded sensitively to the transtubular transport inhibitors, sodium probenecid and pyrazinoic acid. When the hemoconcentration was induced by highly potent doses of test diuretics, the inulin clearance and the fractional excretion of uric acid clearly de creased. The contraction of body fluid produced by intraperitoneal administration of polyethylene glycol resulted in marked decrease of inulin clearance and fractional excretion of uric acid. The decrease of uric acid excretory capacity under the treatment with trichlormethiazide was completely corrected by saline loading. Moreover, no significant change was found in the pyrazinoic acid-suppressible fractional excretion of uric acid between the diuretic-treated rats and the control animals. These studies suggest that furosemide and trichlormethiazide-induced changes in the renal handling of uric acid are mediated by the fluid volume con traction and that the decrease in fractional excretion of uric acid by test diuretics is the result of reabsorptive enhancement of uric acid.

Hyperuricemia incident to the treatment the decrease of inulin clearance and the with diuretics is well recognized (1-6), and increase of plasma uric acid level. These obstruction in the renal transport system of results corresponded well to the reports on uric acid has been discussed as the cause clinically observed hyperuricemia induced by (2-6). the diuretics (2-6). Accordingly, the present The transport of uric acid in the mammalian communication contributes to the under kidney involves glomerular filtration, tubular standing of the hyperuricemia-inducing effect reabsorption and tubular secretion, though of diuretics in pharmacological studies with the relative importance of uric acid movement experimental animals. in each direction differs among species (7, 8). The rat is a useful animal for evaluating Materials and Methods drug effects on uric acid excretion because Animals and drug treatments: The drugs the net flux in the tubular excretory transport used were sodium probenecid, pyrazinoic acid of uric acid is reabsorptive, although the (Nakarai Chem.), furosemide, trichlorme fractional excretion of uric acid is obviously thiazide (Shionogi) and polyethylene glycol higher than that in man (9-13). Thus, we 4000 (average molecular weight 3000, Wako utilized rats as the animal for evaluating the Chem.). Sodium probenecid and furosemide action of diuretics on the uric acid transport were prepared in our laboratory from com system. mercially avaiable probenecid (Sigma) and The animals treated with furosemide and furosemide solution for injection (Lasix, trichlormethiazide clearly decreased the Hoechst). Sodium probenecid was dissolved fractional excretion of uric acid, together with in saline. Pyrazinoic acid was dissolved in 0.2 N sodium hydroxide solution, then plasma concentrations of inulin and uric neutralized with hydrochloric acid. Fur acid, the resultant plasma was mixed with osemide and trichlormethiazide were sus 10 volumes of 0.007 N acetic acid, heated for pended in 1 % gum arabic solution. These 2 min in boiling water and then centrifuged. agents were administered at 2 ml/kg of body Inulin concentrations in deproteinized plasma weight. Polyethylene glycol was dissolved at and diluted urine were measured by a a concentration of 0.2 g/ml in water and was fluorometric method adapted from the pro administered at 3 ml/rat. The animals of the cedure described by Vurek and Pegram (14). control group were given saline or 1 % gum Uric acid concentrations in the above arabic solution instead of the test agent. specimens were measured by the fluorometric Experiments were performed on male method described previously (15). Wistar strain rats weighing 270-300 g. Hematocrits were measured in heparinized They were permitted free access to food and microhematocrit tubes. Sodium and potas water until the time of the experiment. The sium in plasma were determined by atomic animal room was maintained at 22-23°C and absorption spectrophotometry, and other illuminated at intervals of 12 hr from 8:00 a.m. components in plasma were determined by a to 8:00 p.m. standard autoanalyzer technique (Technicon In experiments on the effects of diuretics, Autoanalyzer, SMA-MICRO system) in which the animals were housed in metabolism cages chloride, creatinine, urea-nitrogen and protein to measure the change in urine volume and were estimated by the method of Zall et al. deprived of food but not water. Furosemide (16), Chasson et al. (17), Marsh et al. (18) (30 mg/kg, i.p.) and trichlormethiazide (10 and Skeggs and Hochstrasser (19), re mg/kg, p.o.) were administered twice at spectively. 10:00 a.m. and 6:00 p.m. A 24-hr urine was The abbreviations used in this paper are collected after the first administration, and C,n, inulin clearance; PCa, plasma uric acid; then the rats were sacrificed by decapitation UUaV, urine-excreted amount of uric acid; to determine blood components or used for Cu, uric acid clearance; and FELa, fractional clearance studies. The procedures for the excretion of uric acid (C„a/Cin). All data are treatments with sodium probenecid, given as means±S.E. and Student's t-test was pyrazinoic acid and polyethylene glycol are used to assess statistical significance. described in the legend of each figure or table. Results Clearance studies: The rats were anes Effects of transtubular transport inhibitors thetized with sodium pentobarbital (50 mg/ on renal handling of uric acid: First, studies kg, i.p.). Polyethylene catheters were inserted were undertaken to confirm whether the into the left femoral vein for infusion of fluid, inhibitor of renal tubular reabsorption and into the right femoral artery for collection of renal tubular secretion of uric acid in man are blood, and into the urinary bladder for also effective in rats used in the present collection of urine. Next, the animals were clearance experiments. Sodium probenecid placed on a thermostatically controlled heated (100 mg/kg, i.v.) and pyrazinoic acid (50 table to maintain the body temperature at mg/kg, i.v.) were administered just after the 37-38°C and were infused with 0.25% first urine collection. Sodium probenecid sodium chloride solution containing 4% produced marked elevations in the urine mannitol, 1.5% inulin and 0.26% sodium excreted amount, the clearance and the pentobarbital at a flow rate of 1.8 ml/hr. fractional excretion of uric acid compared to After a 60-min equilibration period, con the pre-treatment period. The inulin clearance tinuous 20-min urine collections were taken. and the plasma uric acid level did not change A 0.2 ml arterial blood sample was obtained in the treatment with sodium probenecid at the midpoint of each urine collection period. (Fig. 1A). On the other hand, pyrazinoic acid Analysis: The sampled blood was im produced decreases in the urine-excreted mediately cooled in ice water, then cen amount, the clearance and the fractional trifuged as soon as possible. To determine the excretion of uric acid compared to the pre Fig. 1. Effects of sodium probenecid and pyrazinoic acid on renal handling of uric acid in rats. Sodium probenecid (100 mg/kg) and pyrazinoic acid (50 mg/kg) were administered at 80 min from the tail vein. "P<0 .01 compared with the value during the pre-treatment period (60-80 min).

Table 1. Changes of urine volume, hematocrit and blood components in furosemide and trichlorme thiazide-treated rats

treatment period. The plasma uric acid level duced significant changes in blood compo slightly elevated in the pyrazinoic acid-treated nents as detailed in Table 1. Plasma uric acid rats, but the inulin clearance did not change level increased in both diuretic-treated in the treatment with pyrazinoic acid (Fig. groups. Moreover, creatinine, urea-nitrogen 1 B). Thus, both transtubular transport in and protein levels in plasma were sig hibitors showed reasonable effects, but all nificantly higher in the drug-treated group parameters did not change in the treatment compared to the control. With respect to the with saline. plasma electrolytes, both drugs did not Effects of furosemide and trichlormethizide change the potassium level, but decreased on blood components and renal handling of the chloride level. Sodium level decreased uric acid: Furosemide (30 mg/kg, i.p.) and only with furosemide treatment. trichlormethiazide (10 mg/kg, p.o.) given Next, clearance studies were performed twice daily, sufficient to produce marked in order to understand changes in the renal diuresis, increased the hematocrit and pro transport of uric acid due to the treatments with both diuretics. As indicated in Fig. 2, body fluid on renal transport of uric acid was the two drugs gave similar results in the studied using rats which had received an clearance experiment. The inulin clearance intraperitoneal administration of polyethylene was lower and the plasma uric acid level was glycol. Fluid loss into the peritoneal cavity higher in the diuretic-treated animals com produced by polyethylene glycol resulted in pared to the control. The urine-excreted a marked rise of the hematocrit at 3 hr after amount, the clearance and the fractional the administration, from 47.8±0.5% in the excretion of uric acid were significantly lower nontreated group to 55.4±0.7% (P<0.01). in the drug-treated animals. In the clearance experiment, the inulin Effect of polyethylene glycol-induced clearance and the urine-excreted amount, the volume contraction on renal handling of uric clearance and the fractional excretion of uric acid: The acute effect of contraction of the acid were lower, and the plasma uric acid

Fig. 2. Effects of furosemide and trichlormethiazide on renal handling of uric acid in rats. Treatment with diuretics were performed as described in the text. Blood and urine specimens for three clearance periods were obtained; then the average values were determined. *P<0.05, **P<0.01, compared with the control group.

Fig. 3. Effect of polyethylene glycol-induced volume contraction on renal handling of uric acid in rats. Polyethylene glycol (20 w/v%, 3 ml/rat) was intraperitoneally administered 3 hr before the clearance study. Blood and urine specimens for three clearance periods were obtained; then the average values were determined. **P<0.01 compared with the nontreated group. Fig. 4. Effect of saline loading on trichlormethiazide induced changes in renal handling of uric acid in rats. Treatment with trichlormethiazide was performed as described in the text. Saline (5 ml/rat) was loaded 2 hr before the clearance study. Blood and urine specimens for three clearance periods were obtained; then the average values were determined. **P<0.01 compared with the control group. level was higher in volume-contracted animals compared to the nontreated group (Fla. 3). Effect of saline loading on the decrease of uric acid excretion induced by trichlorme thiazide: The correction of body fluid volume by saline loading was performed using rats with marked diuresis produced by treatment with trichlormethiazide. Volume restoration to the control level at 2 hr after saline loading was indirectly evidenced by the decrease in the hematocrit from 50.5±0.7% in the trichlormethiazide-treated group to 47.6± 0.5% (P<0.01) in the group that received saline loading. The hematocrit of the control group was 47.2±0.4%. In the clearance Fig. 5. Relationship between the effect of pyrazi experiment, the decreases of the inulin noic acid on FEua and its dose in rats. Pyrazinoic clearance, the urine-excreted amount of uric acid (0: 10 mg/kg, n=5; •: 25 mg/kg, n=5; A: acid, the uric acid clearance and the fractional 50 mg/kg, n=5; A: 100 mg/kg, n=5) was adminis excretion of uric acid by trichlormethiazide tered at 80 min from the tail vein. "P<0.05, MMP<0 treatment returned to the control levels in the .01, compared with the value during the animals loaded with saline. The increase of pre-treatment period (60-80 min). the plasma uric acid level by trichlorme thiazide treatment was also prevented by (Fig. 5). Pyrazinoic acid was administered saline loading (Fig. 4). intravenously just after the first urine col Pyrazinoic acid -suppressible fractional lection. The fractional excretion of uric acid excretion of uric acid under treatment with decreased temporarily at the 10 mg/kg dose diuretics: The dose-response relationship and rapidly returned to the pre-treatment between the decrease in fractional excretion level. Doses over 25 mg/kg resulted in a of uric acid induced by pyrazinoic acid and lasting decrease in the fractional excretion of its dose was determined in the control rats uric acid. The maximum response was cosuric in man, respectively. Thus, we concluded that the rat kidneys used in the present clearance study have considerably greater bidirectional fluxes in regard to the transtubular uric acid transport. On the basis of the effectiveness of this experimental design, we clarified the main subject of diuretic-induced decrease of uric acid excretion. Uric acid retention accompanied by diuretic therapy is presumed to result either from diminished secretion or accelerated reabsorption of uric acid in the renal tubules (2-6). This change in renal handling of uric Fig. 6. Pyrazinoic acid-suppressible FEva in acid is known to be closely associated with diuretic-treated rats. Treatment with diuretics (0: diuretic-induced extracellular fluid volume Control, n=8; •: Fursemide, n=8; A: Trichlorme contraction (4-6). A close relationship thiazide, n=7) were performed as described in the between the state of hydration in the ex text. Pyrazinoic acid (50 mg/kg) was administered tracellular fluid volume and the renal at 80 min from the tail vein. ""P<0.01 compared transport of uric acid in rats was also noted by with the control group. Weinman et al. (20). Thus, highly potent doses of furosemide and trichlormethiazide observed at 50 mg/kg. The decrease in the were given to the rat to rapidly change the fractional excretion of uric acid in rats body fluid volume. As seen in Table 1, the treated with 100 mg/kg was similar to that increase of plasma protein level together with in the animals treated with 50 mg/kg. the elevated hematocrit value obviously Figure 6 shows the result of the study to reflects the hemoconcentration, and the assess the pyrazinoic acid-suppressible and changes of creatinine, urea-nitrogen and -nonsuppressible components in the fractional chloride levels in plasma suggest obstructions excretion of uric acid in the diuretic-treated in renal excretory capacity and electrolyte animals. As previously stated, the treatments balance. In the clearance study, inulin with furosemide and trichlormethiazide sig clearance, urine-excreted amount of uric acid nificantly decreased the fractional excretion and uric acid clearance decreased, while the of uric acid. This excretion in diuretic-treated level of plasma uric acid increased in diuretic rats responded well to pyrazinoic acid, and treated rats compared to the control group. the decrease in the excretion was almost Moreover, the fractional excretion of uric parallel to that in control animals. Pyrazinoic acid fell in the experimental animals due to acid-suppressible fractional excretion of uric secretory inhibition and/or reabsorptive acid was 0.25±0.02, 0.21 ±0.03 (P>0.05) enhancement. and 0.1 9±0.02 (P>0.05) in the control, Further experiments were performed to furosemide-treated and trichlormethiazide investigate the mechanism of diuretic-induced treated rats, respectively. changes in renal handling of uric acid. By employing polyethylene glycol, often used to Discussion produce volume-depleted animals (23, 24), While net reabsorption of uric acid in the in the clearance study, we were able to rat nephron has been described by many confirm the importance of the extracellular investigators, the value of fractional excretion fluid volume contraction on both the re of uric acid varies from 0.1 to 0.9 (8-11, 20 duction in the filtration rate and the trans 22). The value in the present study was tubular transport for uric acid. Moreover, the approximately 0.4, and it responded reason restoration of uric acid excretory capacity in ably to sodium probenecid and pyrazinoic the animals loaded with saline after tri acid, known to be uricosuric and antiuri chlormethiazide treatment suggests that the diuretic-induced uric acid retention might be 4 Suki, W.N., Hull, A.R., Rector, F.C. and Seldin, triggered by the volume contraction. 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