European Journal of Pharmacology, 180 (1990) 69-76 Elsevier
69
EJP 51269
Stereospecificity of the effects of ozolinone on renal hemodynamics and on segmental tubular sodium reabsorption in conscious rats Michael Shalmi, J ~ r g e n S. Petersen a n d Sten C h r i s t e n s e n Department of Pharmacology, Unioersityof Copenhagen, Juliane Maries Vej 20, DK-2100 Copenhagen O, Denmark
Received 16 January 1990, accepted 6 February 1990
The study was performed to elucidate the effects of the two stereoisomers of ozolinone (d,I) on renal hemodynamics and proximal tubular Na reabsorption. Clearance experiments were performed in conscious water-loaded female Wistar rats. The clearances of [3H]inulin, [14C]tetraethylammonium and lithium were used as estimates for glomerular filtration rate, renal plasma flow and delivery of fluid from the proximal tubules, respectively. When the baseline parameters had stabilized, d- or l-ozolinone was injected i.v. in doses of 4, 20 and 100 mg/kg, l-Ozolinone caused a transient and dose-dependent diuretic-natriuretic response with no evidence of a ceiling. At peak natriuresis, 2.5-5 rain after 100 mg/kg of l-ozolinone, the fractional Na excretion was increased from 0.5 to 25%; this was associated with an increased fractional excretion of lithium from 27 to 60%, and small transient decreases of renal hemodynamic parameters, d-Ozolinone had no significant effects except for a small natriuresis after 100 mg/kg. It is concluded that in water-loaded conscious rats l-ozolinone is a powerful diuretic which, in contrast to d-ozolinone, increases the delivery of fluid from the proximal tubule as judged from changes in lithium clearance. Ozolinone; Stereospecificity; Loop diuretic; Lithium clearance; Renal hemodynamics; Sodium reabsorption (proximal, distal)
I. Introduction Ozolinone is a non-sulphonamide loop-diuretic which exists in two stereoisomer forms. A racemic mixture of the stereoisomers, d,l-ozolinone, is formed in vivo by enzymatic hydrolysis of an inactive prodrug, etozoline (Elkapin~), which is registered as a diuretic in Italy and West Germany (Satzinger, 1979; Satzinger and He~-rmann, 1983). The pharmacological properties of etozoline and ozolinone have been examined in detail by Greven, Heidenreich and coworkers (Greven and Heidenreich, 1977; 1978; Greven et al., 1978; 1980a,b; 1984) in experiments with anesthetized rats and
Correspondence to: Sten Christensen, Department of Pharmacology, University of Copenhagen, Juliane Marie.s Vej 20, DK-2100 Copenhagen ~, Denmark.
dogs. When administered i.v., racemic d,lozolinone is more potent and has a faster start of natriuretic action than d,l-etozoline (Greven and Heidenreich, 1977; 1978). Qualitatively, racemic ozolinone has renal effects in anesthetized animals similar to those of furosemide: It increases the excretion of Na, K and C1, lowers urine pH and osmolality, increases RBF (renal blood flow) and proximal free flow pressure, and decreases PAH (para-aminohippurate) transport in the proximal tubules (Greven and Heidenreich, 1978). Micropuncture studies evidenced no change in end proximal T F / P (tubular fluid/plasma) ratios for inulin, Na, K or CI, whereas the early distal tubular T F / P ratios for Na and K were increased, indicating inhibition of electrolyte transport in the loop of Henle (Greven et al., 1978). Like furosemide, ozolinone decreases the intrinsic reabsorptive capacity of the proximal tubular epithelium as
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
70 measured with the shrinking drop technique (Greven et al., 1978). Examination of the individual effects of the two stereoisomers, d- and l-ozolinone showed that both compounds increased RBF and inhibited renal PAH transport, whereas only the levorotatory isomer had diuretic activity and inhibited electrolyte reabsorption in the loop of Henle (Greven et al., 1980b). In addition, it was shown that the dextrorotatory isomer antagonized the natriuretic effect of furosemide and 1-ozolinone in a probenecid-like manner, i.e. by blocking the secretion of these diuretics into the lumen of the proximal tubule. The effects of diuretics on renal hemodynamics and tubular function can be markedly influenced by the anesthesia necessary for performing micropuncture experiments (Elmer et al., 1972). Also, when the site of action of diuretics is under study, micropuncture methods are restricted to the superficial nephron population. For these reasons we have developed an unanesthetized rat model, based on the use of specific clearance markers to evaluate the integrated function of the entire kidney. By using lithium (C Li) clearance as a marker for the delivery of fluid from the proximal tubule, we demonstrated an effect of i.v. furosemide on proximal tubular reabsorption, a site for which micropuncture studies have given conflicting results (Christensen et al., 1986; 1987; 1988; Christensen and Petersen, 1988). In the present study we examined the renal effects of two isomers, d- and I-ozolinone, with particular attention to renal hemodynamics and proximal tubular Na reabsorption. The results indicate that neither of the isomers increases renal plasma flow in conscious rats and that l-ozolinone specifically inhibits Na reabsorption in the proximal tubule, in addition to its effect on the distal nephron.
2. Materials and methods
2.1. Animals Female albino rats (Pan-Wistar strain from the Panum Institute, Copenhagen), weighing 167-210
g, were used. The animals were kept in a temperature- (22 ° C) and moisture- (60%) controlled room with a 12 h light-dark cycle (lights on from 06:00 to 18:00) for at least 1 week prior to the experiments. The rats were given commercial rat pellets (Altromin No. 1324, Chr. Petersen A / S , Ringsted, Denmark) containing approx. 100 mmol Na, 250 mmol K and 180 g protein per kg and tap water ad libitum. The rats were fasted overnight before the experiments.
2.2. Clearance protocol The experiments were performed between 08:00 and 13:00 h. The rats were given an oral test dose of 10 ml 100 m M LiC1/kg (1 m m o l / k g ) 20 min before surgery. This procedure resulted in a relatively stable plasma Li concentration between 0.5 and 0.3 mM during the experiments. The rats were thereafter anesthetized with 1-3% halothane in N20 + 02 (2 : 1) for implantation of catheters in v. jugularis and the urinary bladder, and were given 10 m l / k g isotonic saline i.v. postoperatively over 2 rain. After surgery, lasting approx. 20 rain, the rats were fixed in a restraining cage and allowed to awaken. Water diuresis was induced by the i.v. infusion of 120 m M glucose + 10 mM NaC1 at a rate of 24 m l / h for the first 15 min followed by 6 m l / h throughout the experiment. [3H]Inulin (Amersham, UK; specific activity 3.9 ( C i / m m o l ) and [14C]tetraethylammonium bromide (New England Nuclear, Boston, MA; specific activity 4.8 m C i / m m o l ) were added to the infusion in amounts of 4 and 2 ~ C i / h , respectively. After a 2-h equilibration period the determination of clearance was started with three control periods of 10 min each. Thereafter d- or lozolinone (4, 20 and 100 m g / k g ; N = 6 in each group) was given as an i.v. bolus injection over 2 rain. The urine collecting periods were reduced simultaneously to 2.5 min for the next four periods followed by four periods of 5 min. Blood samples of 150/~l were collected from the tail tip at times 0, 30 and 60 min into heparinized glass capillary tubes and centrifuged immediately. The rats were killed after the experiments by an overdose of pentobarbital.
71
2.3. Drugs and vehicles Ozolinone exists as two stereoisomers, d- and I-ozolinone, which were supplied by G~Sdeke A G (Freiburg, FRG). The stereoisomers are labile and racemization occurs rapidly when they are exposed to light or kept at temperatures higher than - 1 0 ° C . The ozolinone isomers were therefore dissolved immediately before use in 1 M N H 3 and diluted appropriately in redistiiled water to a concentration giving the dosage required (4, 20 and 100 m g / k g ) in a 1-ml solution. The p H of the infusate was between 8 and 9. According to the manufacturer, solutions of the isomers should be stable at least for 1 h when this method is used.
mean extraction fraction of 0.90 determined previously (Petersen et al., 1987): G F R = C~n; R P F = C-rEA/0.90. The filtration fraction was calculated as FF = G F R / R P F . TEA was used as R P F marker instead of P A H for two reasons. It has been shown that lithium at levels higher than those used as a test dose in this study does not influence the uptake of TEA in rat kidney cortical slices ( G e m b a et al., 1984; Sughihara et al., 1985) whereas both tissue accumulation and renal clearance of P A H were decreased by the ion. Furthermore, glucose infusion decreases the extraction fraction of PAH, but does not alter that of TEA (Rasmussen et al., in press).
2.6. Statistics 2.4. Analyses The diuresis was determined by weighing to the nearest milligram. The concentrations of Li and N a in plasma and urine were determined by atomic absorption spectrophotometry as previously described (Christensen et al., 1987). [3H]Inulin and [laC]TEA ([14C]tetraethylammonium) in plasma and urine were determined by double-label scintillation counting of 30 /~1 samples + 270 /tl water mixed with 2.5 ml Optiflour ® in a LKB betacounter model 1217.
All results are mean values + S.E.M. The relationship between individual values for Cei/C~n and FF was evaluated by linear regression. Oneway analysis of variance followed by Duncan's test for multiple comparison was used to evaluate differences between means in fig. 5. Differences between means were considered statistically significant when the 0.05 probability level was achieved.
3. Results
2.5. Calculations 3.1. Time-control experiments" Renal clearances (C) and fractional excretions (FE) were calculated by the standard formula and expressed a s / t l / m i n per 100 g b.w. and %, respectively. Li clearance was used to estimate the delivery of Na out of the proximal tubules (Thornsen, 1984). We have recently provided circumstantial evidence that the increase of CL~ observed after administration of another loop-diuretic, furosemide, reflects an increased delivery of Na ions from the proximal tubules (Christensen et al., 1988). The fractional proximal N a excretion was accordingly calculated as FELl = FENa prox = CI.i/C~n and the fractional distal Na excretion as FENa dist = C N a / C L i " CIn was used as a measure for G F R (glomerular filtration rate) and CTV.Awas used as a measure for R P F after division by the
Figure 1 indicates the time course of the various renal parameters in 6 rats receiving an infusion of hypotonic glucose-saline solution only. After a 2-h equilibration (not shown in fig. 1), urine flow and the clearance of marker substances became reasonably constant, except for a small but steady increase in N a excretion which, however, could not obscure the relatively fast changes induced by the diuretics. The mean values for CTEA, C i , and CLi indicated a normal renal function and were similar to the values determined previously in rats without water diuresis (Christensen et al., 1986; Petersen and Christensen, 1987). A diuresis of about 80 ~tl/min per 100 g was maintained by infusion of a hypotonic glucose-saline solution. The fractional
72 l
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~.
1200, CIn (GFR)
-~
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mediate doses and a tendency to an initial increase, followed by a more sustained decrease after the highest (100 m g / k g ) dose. Almost identical changes were observed for C i, used as a marker for G F R . With 1-ozolinone the fall in C1, was somewhat greater than the fall in CTEA, SO that the filtration fraction decreased momentarily for this isomer, while it remained largely unchanged for the d-form (fig. 2, lower panel).
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3.3. Effects of d- and l-ozolinone on total and fractional segmental Na excretion
1.5. UNaV 1.O i 0.5-
l-Ozolinone (4-100 m g / k g ) caused a short-term and dose-dependent increase in Na excretion with peak effects 2.5-5 min after bolus injections (fig.
1.5. FENa (CNa/CIn) 1.00.5- _ _ z - %
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Fig. 4. Effects of 1- and d-ozolinone on indices of the fractional escape of Na ions from the proximal tubules (CLi//CIn) and the distal ncphron (C~a/CLi). The curves indicate mean values for five to six rats for each drug and dose level. Diuretics were injected at the times indicated by arrows. The predrug level indicated by a horizontal line is the mean for all rats.
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cretion of N a ions delivered to the distal nephron, from 3 up to 43%; d-ozolinone had no effect on the distal rejection of N a ions, apart from a modest increase after 100 m g / k g . The effects of the two isomers on absolute and fractional segmental N a excretion are summarized in fig. 5, where the average peak responses are plotted against the dose of diuretics. 1-Ozolinone is seen to be a highly effective natriuretic substance which inhibits up to 25% of tubular N a reabsorption without any evidence of having reached its maximal effect within the dose range investigated. The increase of FE ~ (C NJ C la) can be attributed to an increased fractional output from the proximal tubule (CLi/CIn) as well as from the distal nephron (C ~ a / C Li)" However, t h e dose-response curves were quite different for the two parts of the nephron: the effect on FE[. i reached its maximum at doses of 20 m g / k g , while
74
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DOSE OF OZOLINONE ( m g / k g ) Fig. 5. Dose-response curves indicating the effects of 1- (e) and d- ( o ) ozolinone on total and fractional Na excretion as well as on indices for the fraction',d delivery of Na ions from the proximal tubules ( C l . i / C L n ) and the distal n e p h r o n (CN~/CLi). Each point indicates the m e a n + S . E . M , of the individual peak values shown in figs. 2-4.
there was no detectable maximum for the inhibition of distal Na reabsorption, d-Ozolinone showed no significant natriuretic effect at low or intermediate doses, and only a weak effect after 100 m g / k g .
4. Discussion
The present study confirmed the stereospecificity of the optical isomers of ozolinone, since only the l-form caused significant natriuresis in unanesthetized, water-loaded rats. Greven et al. (1980a, b) reported that doses of d-ozolinone as high as 100 m g / k g had no natriuretic effect in saline-loaded inactin-anesthetized rats, while in our study the same dose produced a small but consistent increase in Na excretion (fig. 5). This minor discrepancy between the two studies may be explained by differences in rat preparations and in time schedules used. We used 2.5-min urine collections, while Greven et al. (1980b) used 30rain collections, and it is evident from fig. 2 that
the natriuretic response to 100 m g / k g d-ozolinone had already ceased after 10 min. However, it cannot be decided on the basis of the present results whether the small natriuresis observed after 100 m g / k g d-ozolinone was due to a lack of absolute selectivity or to partial racemization of d-ozolinone in the freshly prepared infusate despite all efforts to avoid this (see Methods). The effects of the ozolinone isomers on renal hemodynamics and G F R deserve particular consideration, since they are not consistent with the results of Greven and co-workers (Greven and Heidenreich, 1978; Greven et al., 1978; 1980a,b). We used the clearance of tetraethylammonium (TEA) as a marker for R P F because both ozolinone isomers inhibit the tubular secretion of p-aminohippurate (Greven et al., 1980b). Using this marker we found small and rather inconsistent changes in C.rE A after 4 and 20 m g / k g 1-ozolinone and a 20% decrease after 100 m g / k g of both isomers (fig. 2). In sharp contrast with these results, Greven and co-workers reported that the racemic mixture and each of the optical isomers of ozolinone increased RBF measured electromagneticaily in anesthetized dogs (Greven and Heidenreich, 1978) and rats (Greven et al., 1978; 1980b). The reason for this apparent discrepancy may be the use of barbiturate anesthesia in the studies of Greven and co-workers. With reference to another loop-diuretic, furosemide, it is well known that only studies in anesthetized animals evidenced a significant increase in RBF, while we found that furosemide decreased CTEA in the conscious rat model, even when fluid losses were instantly replaced (Christensen and Petersen, !988). Ludens et al. (1970) showed that the increase in RBF after i.v. furosemide administration to pentobarbital-anesthetized dogs was dependent on the pre-existing renal vascular resistance and RBF. The higher the resistance - due to renal vasoconstriction caused by anesthesia - the higher the increase of RBF. In unanesthetized dogs, having low renal vascular resistance, 1 m g / k g furosemide caused a decrease of RBF followed by a small increase (Ludens and Williamson, 1970). It appears from these studies that anesthetized animals are not a suitable model for studying the effects of diuretics on renal hemodynamics. Our studies have
75 indicated that, in the unanesthetized rat preparation, the renal hemodynamics response to two different loop-diuretics, furosemide and ozolinone, is one of no change or decrease rather than an increase of RBF. The overall interpretation of these studies may be that loop-diuretics only act as renal vasodilators when the renal vessels are constricted. Both furosemide (Burke and Duchin, 1979) and d,l-ozolinone (Greven et al.l, 1978) have been reported to increase proximal intratubular pressure and would thus be expected to decrease the GFR, unless a concomitant dilatation of the preglomerular arterioles would tend to maintain the effective filtration pressure. As also demonstrated for furosemide (Christensen and Petersen, 1988), both dand 1-ozolinone caused a decrease of Cin (fig. 2). However, the fall in Cin relative to the fall in CTEA was greater for 1-ozolinone than for dozolinone, resulting in an initial dose-independent drop in filtration fraction for the l-form (fig. 2, bottom). This difference between the two isomers could be due to the difference in effects on tubular fluid reabsorption, since only the diuretically active, 1-ozolinone, would be expected to increase proximal intratubular pressure. However, this possibility is argued against by the observation that 100 m g / k g of d-ozolinone, which had a greater natriuretic effect than 4 m g / k g l-ozolinone (fig. 3), did not cause a fall in the filtration fraction (fig. 2). l-Ozolinone proved to be a very effective natriuretic in conscious rats, since, during peak natriuresis, the 100 m g / k g dose induced a Na excretion of 33 g m o l / m i n per 100 g b.w. or 25% of the filtered amount (fig. 5). In comparison, 120 m g / k g furosemide caused a peak natriuresis of 22 # m o l / m i n per 100 g b.w. or (since the G F R dropped simultaneously) 24% of the filtered amount (Christensen and Petersen, 1988). Furthermore, inspection of the dose-response curve for Na excretion or FEN, (fig. 5) reveals that the slopes had not even started to decrease in the dose interval investigated, which means that the theoretical maximal response may be significantly higher than a FENa = 25%. Since the rat is relatively insensitive to loop-diuretics (Olsen, 1977), the dose which can be administered i.v. is limited
by the water solubility of the drugs, so that it may not be possible to give maximally effective doses. However, it is hypothesized from the shape of the dose-response curve that the maximal efficacy of l-ozolinone in rats is not less than F E N , - - 3 0 % , which suggests inhibition of Na reabsorption in the proximal tubules (Goldberg, 1973; Brater, 1983). When fluid losses were replaced instantly, 120 m g / k g furosemide was shown to increase F E ~ , to 37%, a percentage indicating a proximal effect of this diuretic (Christensen and Petersen, 1988). Assuming that CLi reflects the output of sodium and water from the proximal tubules it is possible to dissociate the effects of ozolinone on the proximal tubule and the 'distal' nephron, which includes the loop of Henle, the distal convoluted tubules and the collecting ducts. After l-ozolinone administration, FEL~ was increased from 27% to a maximum of 60% after 20-100 m g / k g (fig. 5). These data are quantitatively similar to those obtained with furosemide (Christensen and Petersen, 1988) and suggest that maximal inhibition of proximal Na reabsorption is achieved with submaximal diuretic doses. Despite the fall in FF in the first periods after administration of l-ozolinone, there was no significant correlation between the peak values for F F and FELi. For example, an increase in FELi occurred after 100 m g / k g d-ozolinone without any change in FF. This suggests that a fall in FF, leading to a decrease in peritubular oncotic pressure or protein concentration, cannot be responsible for the change in proximal reabsorption rate, as previously suggested for loop diuretics in general (Goldberg, 1973). It is therefore suggested that 1-ozolinone, like furosemide (Christensen and Petersen, 1988), has a direct effect on the reabsorption rate in the proximal tubule which is independent of hemodynamic factors. The molecular basis of this effect, which can now be included among the stereospecific actions of the ozolinone molecule, remains unknown. The effects of 1-ozolinone on the 'distal' nephron, in this context most probably representing mainly the loop of Henle, showed no evidence of reaching a maximum (fig. 5). This observation is similar to our previous findings with furosemide
76 ( C h r i s t e n s e n a n d P e t e r s e n , 1988) b u t in c o n t r a d i c tion to the effects found with bumetanide (Shalmi et al., 1989). T h e
fractional distal reabsorption
r a t e w a s r e d u c e d f r o m 97% in t h e c o n t r o l s t a t e to 57% a f t e r 100 m g / k g
l-ozolinone. The very effec-
tive n a t r i u r e s i s i n d u c e d b y 1 - o z o l i n o n e m a y t h u s be
attributed
in g e n e r a l
to i m p a i r m e n t
of Na
r e a b s o r p t i o n in t h e l o o p o f H e n l e , a n i m p a i r m e n t w h i c h is a c c e n t u a t e d b y a n i n c r e a s e d d e l i v e r y o f N a ions f r o m the p r o x i m a l tubules. However, the further natriuresis obtained when the dose was i n c r e a s e d f r o m 20 t o 100 m g / k g is d u e to i n h i b i tion of the distal N a r e a b s o r p t i o n only.
Acknowledgements This study was supported by a grant from the Danish Medical Research Council and by Gi~leke AG (Freiburg, FRG) who also supplied thc ozolinone isomers. The authors thank Pia EIsman for technical assistance and Karen Friis for editorial assistance.
References Brater, D.C., 1983, Pharmacodynamic considerations in the use of diuretics, Ann. Rev. Pharmacol. Toxicol. 23, 45. Burke, T.J. and K.L. Duchin, 1979, Glomerular filtration during furosemide diuresis in the dog, Kidney Int. 16, 672. Christensen, S., J.S. Petersen, E. Steiness and F. Andreasen, 1987, Dose dependence of proximal and distal tubular effects of furosemide in conscious rats, J. Pharmacol. Exp. Ther. 241,987. Christensen, S. and J.S. Petersen, 1988, Effects of furosemide on renal haemodynamics and proximal tubular sodium reabsorption in conscious rats, Br. J. Pharmacol. 95, 353. Christensen, S., M. Shalmi and J.S. Petersen, 1988, Lithium clearance as an indicator of proximal tubular sodium handling during furosemide diuresis, J. Pharmacol. Exp. Ther. 246, 753. Christensen, S., E. Steiness and H. Christensen, 1986, Tubular sites of furosemide natriuresis in volume-replaced and volume-depleted conscious rats, J. Pharmacol. Exp. Ther. 239, 211. Elmer, M., P.C. I£~kildsen, L.~. Kristensen and P.P. Leyssac, 1972, A comparison of renal function in rats anesthetized with inactin and sodium amytal, Acta Physiol. Scand. 86, 41. Gemba, M., N. Mikuriya, A. Tachibana and M. Nakajima, 1984, Effect of lithium on organic ion transport i.n rat kidney cortical slices, Jap. J. Pharmacol. 34, 128. Goldberg, M., 1973, The renal physiology of diuretics, in: Handbook of Physiology, eds. J. Orloff, R.W. Berlinger and S.R. Geiger (Physiological Society, Washington, D.C.) p. 1003.
Greven, J., M. Beckers, W. Defrain, K. Meywald and O. Heidenreich, 1980a, Studies with the optically active isomers of the new diuretic drug ozolinone. I1. Inhibition by d-ozolinone of furosemide-induced diuresis, Pfliigers Arch. 384, 61. Greven, J., W. Defrain, K. Glaser, K. Meywald and O. Heidenreich, 1980b, Studies with the optically active isomers of the new diuretic drug ozolinone. I. Differences in stereoselectivity of the renal target structures of ozolinone, Pfliigers Arch. 384, 57. Greven, J., K. Glaser, B. K~511ing and O. Heidenreich, 1984, Attenuation by d-ozolinone of I-ozolinone-induced diuresis in rats, European J. Pharmacol. 98, 331. Greven, J. and O. Heidenreich, 1977, Effect of etozolin on whole kidney function and fluid and electrolyte reabsorption in rat proximal convoluted tubules and loops of Henle, Arzneim. Forsch. 27, 1755. Greven, J. and O. Heidenreich, 1978, Effects of ozolinone, a diuretic active metabolite of etozoline, on renal function. I. Clearance studies in dogs, Naunyn-Schmiedeb. Arch. Pharmacol. 304, 283. Greven, J., H. Klein and O. Heidenreich, 1978, Effects of ozolinone, a diuretic active metabolite of etozolinc, on renal function. II. Localization of tubular site of diuretic action by micropuncture in the rat, Naunyn-Schmiedeb. Arch. Pharmacol. 3134, 289. Ludens. J.H., D.C. Heitz, M.J. Brody and H.E. Williamson, 1970, Differential effect of furosemide on renal and limb blood flows in the conscious dog, J. Pharmacol. Exp. Ther. 171,300. Ludens, J.H. and H.E. WillJamson, 1970, Effect of furosemide on renal blood flow in the conscious dog, Proc. Soc. Exp. Biol. Med. 133, 513. Olsen, U.B., 1977, The pharmacology of bumetanide, Acta pharmacol. Toxicol. 41, Suppl. Ill, 5. Petersen, J.S. and S. Christensen, 1987, Superiority of tetraethylammonium to p-aminohippurate as a marker for renal plasma flow during furosemide diuresis, Renal Physiol. 10, 102. Rasmussen, S.N., M. Shalmi, M. Hansen and S. Christensen, 1990, Tetraethylammonium and p-aminohippurate as clearance markers for renal plasma flow in the rat during saline and glucose infusion, Renal Physiol. Biochem. (in press). Satzmger, G., 1979, Neuere Entwicklungen in der Chemie der Diuretika, Deutsche Apoteker Z. 119, 1773. Satzirtger, G. and W. Herrmann, 1983, European Patent No. 00 16 635. Shalmi, M., J.S. Petersen and S. Christensen, 1989, Effects of intravenous bumetanide administration on renal haemodynamics and proximal and distal tubular sodium reabsorpfion in conscious rats, Pharmacol. Toxicol. 65, 313. Sugihara, K., A. Tachibana and M. Gemba, 1985. Evidence for in vivo effect of lithium on p-aminohippurate transport in rat kidny, preliminary study, Jap. J. Pharmacot. 38, 127. Thomsen, K., 1984, Lithium clearance: A new method for determining proximal and distal tubular reabsorption of sodium and water, Nephron 37, 217.
69
EJP 51269
Stereospecificity of the effects of ozolinone on renal hemodynamics and on segmental tubular sodium reabsorption in conscious rats Michael Shalmi, J ~ r g e n S. Petersen a n d Sten C h r i s t e n s e n Department of Pharmacology, Unioersityof Copenhagen, Juliane Maries Vej 20, DK-2100 Copenhagen O, Denmark
Received 16 January 1990, accepted 6 February 1990
The study was performed to elucidate the effects of the two stereoisomers of ozolinone (d,I) on renal hemodynamics and proximal tubular Na reabsorption. Clearance experiments were performed in conscious water-loaded female Wistar rats. The clearances of [3H]inulin, [14C]tetraethylammonium and lithium were used as estimates for glomerular filtration rate, renal plasma flow and delivery of fluid from the proximal tubules, respectively. When the baseline parameters had stabilized, d- or l-ozolinone was injected i.v. in doses of 4, 20 and 100 mg/kg, l-Ozolinone caused a transient and dose-dependent diuretic-natriuretic response with no evidence of a ceiling. At peak natriuresis, 2.5-5 rain after 100 mg/kg of l-ozolinone, the fractional Na excretion was increased from 0.5 to 25%; this was associated with an increased fractional excretion of lithium from 27 to 60%, and small transient decreases of renal hemodynamic parameters, d-Ozolinone had no significant effects except for a small natriuresis after 100 mg/kg. It is concluded that in water-loaded conscious rats l-ozolinone is a powerful diuretic which, in contrast to d-ozolinone, increases the delivery of fluid from the proximal tubule as judged from changes in lithium clearance. Ozolinone; Stereospecificity; Loop diuretic; Lithium clearance; Renal hemodynamics; Sodium reabsorption (proximal, distal)
I. Introduction Ozolinone is a non-sulphonamide loop-diuretic which exists in two stereoisomer forms. A racemic mixture of the stereoisomers, d,l-ozolinone, is formed in vivo by enzymatic hydrolysis of an inactive prodrug, etozoline (Elkapin~), which is registered as a diuretic in Italy and West Germany (Satzinger, 1979; Satzinger and He~-rmann, 1983). The pharmacological properties of etozoline and ozolinone have been examined in detail by Greven, Heidenreich and coworkers (Greven and Heidenreich, 1977; 1978; Greven et al., 1978; 1980a,b; 1984) in experiments with anesthetized rats and
Correspondence to: Sten Christensen, Department of Pharmacology, University of Copenhagen, Juliane Marie.s Vej 20, DK-2100 Copenhagen ~, Denmark.
dogs. When administered i.v., racemic d,lozolinone is more potent and has a faster start of natriuretic action than d,l-etozoline (Greven and Heidenreich, 1977; 1978). Qualitatively, racemic ozolinone has renal effects in anesthetized animals similar to those of furosemide: It increases the excretion of Na, K and C1, lowers urine pH and osmolality, increases RBF (renal blood flow) and proximal free flow pressure, and decreases PAH (para-aminohippurate) transport in the proximal tubules (Greven and Heidenreich, 1978). Micropuncture studies evidenced no change in end proximal T F / P (tubular fluid/plasma) ratios for inulin, Na, K or CI, whereas the early distal tubular T F / P ratios for Na and K were increased, indicating inhibition of electrolyte transport in the loop of Henle (Greven et al., 1978). Like furosemide, ozolinone decreases the intrinsic reabsorptive capacity of the proximal tubular epithelium as
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
70 measured with the shrinking drop technique (Greven et al., 1978). Examination of the individual effects of the two stereoisomers, d- and l-ozolinone showed that both compounds increased RBF and inhibited renal PAH transport, whereas only the levorotatory isomer had diuretic activity and inhibited electrolyte reabsorption in the loop of Henle (Greven et al., 1980b). In addition, it was shown that the dextrorotatory isomer antagonized the natriuretic effect of furosemide and 1-ozolinone in a probenecid-like manner, i.e. by blocking the secretion of these diuretics into the lumen of the proximal tubule. The effects of diuretics on renal hemodynamics and tubular function can be markedly influenced by the anesthesia necessary for performing micropuncture experiments (Elmer et al., 1972). Also, when the site of action of diuretics is under study, micropuncture methods are restricted to the superficial nephron population. For these reasons we have developed an unanesthetized rat model, based on the use of specific clearance markers to evaluate the integrated function of the entire kidney. By using lithium (C Li) clearance as a marker for the delivery of fluid from the proximal tubule, we demonstrated an effect of i.v. furosemide on proximal tubular reabsorption, a site for which micropuncture studies have given conflicting results (Christensen et al., 1986; 1987; 1988; Christensen and Petersen, 1988). In the present study we examined the renal effects of two isomers, d- and I-ozolinone, with particular attention to renal hemodynamics and proximal tubular Na reabsorption. The results indicate that neither of the isomers increases renal plasma flow in conscious rats and that l-ozolinone specifically inhibits Na reabsorption in the proximal tubule, in addition to its effect on the distal nephron.
2. Materials and methods
2.1. Animals Female albino rats (Pan-Wistar strain from the Panum Institute, Copenhagen), weighing 167-210
g, were used. The animals were kept in a temperature- (22 ° C) and moisture- (60%) controlled room with a 12 h light-dark cycle (lights on from 06:00 to 18:00) for at least 1 week prior to the experiments. The rats were given commercial rat pellets (Altromin No. 1324, Chr. Petersen A / S , Ringsted, Denmark) containing approx. 100 mmol Na, 250 mmol K and 180 g protein per kg and tap water ad libitum. The rats were fasted overnight before the experiments.
2.2. Clearance protocol The experiments were performed between 08:00 and 13:00 h. The rats were given an oral test dose of 10 ml 100 m M LiC1/kg (1 m m o l / k g ) 20 min before surgery. This procedure resulted in a relatively stable plasma Li concentration between 0.5 and 0.3 mM during the experiments. The rats were thereafter anesthetized with 1-3% halothane in N20 + 02 (2 : 1) for implantation of catheters in v. jugularis and the urinary bladder, and were given 10 m l / k g isotonic saline i.v. postoperatively over 2 rain. After surgery, lasting approx. 20 rain, the rats were fixed in a restraining cage and allowed to awaken. Water diuresis was induced by the i.v. infusion of 120 m M glucose + 10 mM NaC1 at a rate of 24 m l / h for the first 15 min followed by 6 m l / h throughout the experiment. [3H]Inulin (Amersham, UK; specific activity 3.9 ( C i / m m o l ) and [14C]tetraethylammonium bromide (New England Nuclear, Boston, MA; specific activity 4.8 m C i / m m o l ) were added to the infusion in amounts of 4 and 2 ~ C i / h , respectively. After a 2-h equilibration period the determination of clearance was started with three control periods of 10 min each. Thereafter d- or lozolinone (4, 20 and 100 m g / k g ; N = 6 in each group) was given as an i.v. bolus injection over 2 rain. The urine collecting periods were reduced simultaneously to 2.5 min for the next four periods followed by four periods of 5 min. Blood samples of 150/~l were collected from the tail tip at times 0, 30 and 60 min into heparinized glass capillary tubes and centrifuged immediately. The rats were killed after the experiments by an overdose of pentobarbital.
71
2.3. Drugs and vehicles Ozolinone exists as two stereoisomers, d- and I-ozolinone, which were supplied by G~Sdeke A G (Freiburg, FRG). The stereoisomers are labile and racemization occurs rapidly when they are exposed to light or kept at temperatures higher than - 1 0 ° C . The ozolinone isomers were therefore dissolved immediately before use in 1 M N H 3 and diluted appropriately in redistiiled water to a concentration giving the dosage required (4, 20 and 100 m g / k g ) in a 1-ml solution. The p H of the infusate was between 8 and 9. According to the manufacturer, solutions of the isomers should be stable at least for 1 h when this method is used.
mean extraction fraction of 0.90 determined previously (Petersen et al., 1987): G F R = C~n; R P F = C-rEA/0.90. The filtration fraction was calculated as FF = G F R / R P F . TEA was used as R P F marker instead of P A H for two reasons. It has been shown that lithium at levels higher than those used as a test dose in this study does not influence the uptake of TEA in rat kidney cortical slices ( G e m b a et al., 1984; Sughihara et al., 1985) whereas both tissue accumulation and renal clearance of P A H were decreased by the ion. Furthermore, glucose infusion decreases the extraction fraction of PAH, but does not alter that of TEA (Rasmussen et al., in press).
2.6. Statistics 2.4. Analyses The diuresis was determined by weighing to the nearest milligram. The concentrations of Li and N a in plasma and urine were determined by atomic absorption spectrophotometry as previously described (Christensen et al., 1987). [3H]Inulin and [laC]TEA ([14C]tetraethylammonium) in plasma and urine were determined by double-label scintillation counting of 30 /~1 samples + 270 /tl water mixed with 2.5 ml Optiflour ® in a LKB betacounter model 1217.
All results are mean values + S.E.M. The relationship between individual values for Cei/C~n and FF was evaluated by linear regression. Oneway analysis of variance followed by Duncan's test for multiple comparison was used to evaluate differences between means in fig. 5. Differences between means were considered statistically significant when the 0.05 probability level was achieved.
3. Results
2.5. Calculations 3.1. Time-control experiments" Renal clearances (C) and fractional excretions (FE) were calculated by the standard formula and expressed a s / t l / m i n per 100 g b.w. and %, respectively. Li clearance was used to estimate the delivery of Na out of the proximal tubules (Thornsen, 1984). We have recently provided circumstantial evidence that the increase of CL~ observed after administration of another loop-diuretic, furosemide, reflects an increased delivery of Na ions from the proximal tubules (Christensen et al., 1988). The fractional proximal N a excretion was accordingly calculated as FELl = FENa prox = CI.i/C~n and the fractional distal Na excretion as FENa dist = C N a / C L i " CIn was used as a measure for G F R (glomerular filtration rate) and CTV.Awas used as a measure for R P F after division by the
Figure 1 indicates the time course of the various renal parameters in 6 rats receiving an infusion of hypotonic glucose-saline solution only. After a 2-h equilibration (not shown in fig. 1), urine flow and the clearance of marker substances became reasonably constant, except for a small but steady increase in N a excretion which, however, could not obscure the relatively fast changes induced by the diuretics. The mean values for CTEA, C i , and CLi indicated a normal renal function and were similar to the values determined previously in rats without water diuresis (Christensen et al., 1986; Petersen and Christensen, 1987). A diuresis of about 80 ~tl/min per 100 g was maintained by infusion of a hypotonic glucose-saline solution. The fractional
72 l
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mediate doses and a tendency to an initial increase, followed by a more sustained decrease after the highest (100 m g / k g ) dose. Almost identical changes were observed for C i, used as a marker for G F R . With 1-ozolinone the fall in C1, was somewhat greater than the fall in CTEA, SO that the filtration fraction decreased momentarily for this isomer, while it remained largely unchanged for the d-form (fig. 2, lower panel).
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3.3. Effects of d- and l-ozolinone on total and fractional segmental Na excretion
1.5. UNaV 1.O i 0.5-
l-Ozolinone (4-100 m g / k g ) caused a short-term and dose-dependent increase in Na excretion with peak effects 2.5-5 min after bolus injections (fig.
1.5. FENa (CNa/CIn) 1.00.5- _ _ z - %
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L- OZOLINONE
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Fig. 4. Effects of 1- and d-ozolinone on indices of the fractional escape of Na ions from the proximal tubules (CLi//CIn) and the distal ncphron (C~a/CLi). The curves indicate mean values for five to six rats for each drug and dose level. Diuretics were injected at the times indicated by arrows. The predrug level indicated by a horizontal line is the mean for all rats.
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cretion of N a ions delivered to the distal nephron, from 3 up to 43%; d-ozolinone had no effect on the distal rejection of N a ions, apart from a modest increase after 100 m g / k g . The effects of the two isomers on absolute and fractional segmental N a excretion are summarized in fig. 5, where the average peak responses are plotted against the dose of diuretics. 1-Ozolinone is seen to be a highly effective natriuretic substance which inhibits up to 25% of tubular N a reabsorption without any evidence of having reached its maximal effect within the dose range investigated. The increase of FE ~ (C NJ C la) can be attributed to an increased fractional output from the proximal tubule (CLi/CIn) as well as from the distal nephron (C ~ a / C Li)" However, t h e dose-response curves were quite different for the two parts of the nephron: the effect on FE[. i reached its maximum at doses of 20 m g / k g , while
74
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DOSE OF OZOLINONE ( m g / k g ) Fig. 5. Dose-response curves indicating the effects of 1- (e) and d- ( o ) ozolinone on total and fractional Na excretion as well as on indices for the fraction',d delivery of Na ions from the proximal tubules ( C l . i / C L n ) and the distal n e p h r o n (CN~/CLi). Each point indicates the m e a n + S . E . M , of the individual peak values shown in figs. 2-4.
there was no detectable maximum for the inhibition of distal Na reabsorption, d-Ozolinone showed no significant natriuretic effect at low or intermediate doses, and only a weak effect after 100 m g / k g .
4. Discussion
The present study confirmed the stereospecificity of the optical isomers of ozolinone, since only the l-form caused significant natriuresis in unanesthetized, water-loaded rats. Greven et al. (1980a, b) reported that doses of d-ozolinone as high as 100 m g / k g had no natriuretic effect in saline-loaded inactin-anesthetized rats, while in our study the same dose produced a small but consistent increase in Na excretion (fig. 5). This minor discrepancy between the two studies may be explained by differences in rat preparations and in time schedules used. We used 2.5-min urine collections, while Greven et al. (1980b) used 30rain collections, and it is evident from fig. 2 that
the natriuretic response to 100 m g / k g d-ozolinone had already ceased after 10 min. However, it cannot be decided on the basis of the present results whether the small natriuresis observed after 100 m g / k g d-ozolinone was due to a lack of absolute selectivity or to partial racemization of d-ozolinone in the freshly prepared infusate despite all efforts to avoid this (see Methods). The effects of the ozolinone isomers on renal hemodynamics and G F R deserve particular consideration, since they are not consistent with the results of Greven and co-workers (Greven and Heidenreich, 1978; Greven et al., 1978; 1980a,b). We used the clearance of tetraethylammonium (TEA) as a marker for R P F because both ozolinone isomers inhibit the tubular secretion of p-aminohippurate (Greven et al., 1980b). Using this marker we found small and rather inconsistent changes in C.rE A after 4 and 20 m g / k g 1-ozolinone and a 20% decrease after 100 m g / k g of both isomers (fig. 2). In sharp contrast with these results, Greven and co-workers reported that the racemic mixture and each of the optical isomers of ozolinone increased RBF measured electromagneticaily in anesthetized dogs (Greven and Heidenreich, 1978) and rats (Greven et al., 1978; 1980b). The reason for this apparent discrepancy may be the use of barbiturate anesthesia in the studies of Greven and co-workers. With reference to another loop-diuretic, furosemide, it is well known that only studies in anesthetized animals evidenced a significant increase in RBF, while we found that furosemide decreased CTEA in the conscious rat model, even when fluid losses were instantly replaced (Christensen and Petersen, !988). Ludens et al. (1970) showed that the increase in RBF after i.v. furosemide administration to pentobarbital-anesthetized dogs was dependent on the pre-existing renal vascular resistance and RBF. The higher the resistance - due to renal vasoconstriction caused by anesthesia - the higher the increase of RBF. In unanesthetized dogs, having low renal vascular resistance, 1 m g / k g furosemide caused a decrease of RBF followed by a small increase (Ludens and Williamson, 1970). It appears from these studies that anesthetized animals are not a suitable model for studying the effects of diuretics on renal hemodynamics. Our studies have
75 indicated that, in the unanesthetized rat preparation, the renal hemodynamics response to two different loop-diuretics, furosemide and ozolinone, is one of no change or decrease rather than an increase of RBF. The overall interpretation of these studies may be that loop-diuretics only act as renal vasodilators when the renal vessels are constricted. Both furosemide (Burke and Duchin, 1979) and d,l-ozolinone (Greven et al.l, 1978) have been reported to increase proximal intratubular pressure and would thus be expected to decrease the GFR, unless a concomitant dilatation of the preglomerular arterioles would tend to maintain the effective filtration pressure. As also demonstrated for furosemide (Christensen and Petersen, 1988), both dand 1-ozolinone caused a decrease of Cin (fig. 2). However, the fall in Cin relative to the fall in CTEA was greater for 1-ozolinone than for dozolinone, resulting in an initial dose-independent drop in filtration fraction for the l-form (fig. 2, bottom). This difference between the two isomers could be due to the difference in effects on tubular fluid reabsorption, since only the diuretically active, 1-ozolinone, would be expected to increase proximal intratubular pressure. However, this possibility is argued against by the observation that 100 m g / k g of d-ozolinone, which had a greater natriuretic effect than 4 m g / k g l-ozolinone (fig. 3), did not cause a fall in the filtration fraction (fig. 2). l-Ozolinone proved to be a very effective natriuretic in conscious rats, since, during peak natriuresis, the 100 m g / k g dose induced a Na excretion of 33 g m o l / m i n per 100 g b.w. or 25% of the filtered amount (fig. 5). In comparison, 120 m g / k g furosemide caused a peak natriuresis of 22 # m o l / m i n per 100 g b.w. or (since the G F R dropped simultaneously) 24% of the filtered amount (Christensen and Petersen, 1988). Furthermore, inspection of the dose-response curve for Na excretion or FEN, (fig. 5) reveals that the slopes had not even started to decrease in the dose interval investigated, which means that the theoretical maximal response may be significantly higher than a FENa = 25%. Since the rat is relatively insensitive to loop-diuretics (Olsen, 1977), the dose which can be administered i.v. is limited
by the water solubility of the drugs, so that it may not be possible to give maximally effective doses. However, it is hypothesized from the shape of the dose-response curve that the maximal efficacy of l-ozolinone in rats is not less than F E N , - - 3 0 % , which suggests inhibition of Na reabsorption in the proximal tubules (Goldberg, 1973; Brater, 1983). When fluid losses were replaced instantly, 120 m g / k g furosemide was shown to increase F E ~ , to 37%, a percentage indicating a proximal effect of this diuretic (Christensen and Petersen, 1988). Assuming that CLi reflects the output of sodium and water from the proximal tubules it is possible to dissociate the effects of ozolinone on the proximal tubule and the 'distal' nephron, which includes the loop of Henle, the distal convoluted tubules and the collecting ducts. After l-ozolinone administration, FEL~ was increased from 27% to a maximum of 60% after 20-100 m g / k g (fig. 5). These data are quantitatively similar to those obtained with furosemide (Christensen and Petersen, 1988) and suggest that maximal inhibition of proximal Na reabsorption is achieved with submaximal diuretic doses. Despite the fall in FF in the first periods after administration of l-ozolinone, there was no significant correlation between the peak values for F F and FELi. For example, an increase in FELi occurred after 100 m g / k g d-ozolinone without any change in FF. This suggests that a fall in FF, leading to a decrease in peritubular oncotic pressure or protein concentration, cannot be responsible for the change in proximal reabsorption rate, as previously suggested for loop diuretics in general (Goldberg, 1973). It is therefore suggested that 1-ozolinone, like furosemide (Christensen and Petersen, 1988), has a direct effect on the reabsorption rate in the proximal tubule which is independent of hemodynamic factors. The molecular basis of this effect, which can now be included among the stereospecific actions of the ozolinone molecule, remains unknown. The effects of 1-ozolinone on the 'distal' nephron, in this context most probably representing mainly the loop of Henle, showed no evidence of reaching a maximum (fig. 5). This observation is similar to our previous findings with furosemide
76 ( C h r i s t e n s e n a n d P e t e r s e n , 1988) b u t in c o n t r a d i c tion to the effects found with bumetanide (Shalmi et al., 1989). T h e
fractional distal reabsorption
r a t e w a s r e d u c e d f r o m 97% in t h e c o n t r o l s t a t e to 57% a f t e r 100 m g / k g
l-ozolinone. The very effec-
tive n a t r i u r e s i s i n d u c e d b y 1 - o z o l i n o n e m a y t h u s be
attributed
in g e n e r a l
to i m p a i r m e n t
of Na
r e a b s o r p t i o n in t h e l o o p o f H e n l e , a n i m p a i r m e n t w h i c h is a c c e n t u a t e d b y a n i n c r e a s e d d e l i v e r y o f N a ions f r o m the p r o x i m a l tubules. However, the further natriuresis obtained when the dose was i n c r e a s e d f r o m 20 t o 100 m g / k g is d u e to i n h i b i tion of the distal N a r e a b s o r p t i o n only.
Acknowledgements This study was supported by a grant from the Danish Medical Research Council and by Gi~leke AG (Freiburg, FRG) who also supplied thc ozolinone isomers. The authors thank Pia EIsman for technical assistance and Karen Friis for editorial assistance.
References Brater, D.C., 1983, Pharmacodynamic considerations in the use of diuretics, Ann. Rev. Pharmacol. Toxicol. 23, 45. Burke, T.J. and K.L. Duchin, 1979, Glomerular filtration during furosemide diuresis in the dog, Kidney Int. 16, 672. Christensen, S., J.S. Petersen, E. Steiness and F. Andreasen, 1987, Dose dependence of proximal and distal tubular effects of furosemide in conscious rats, J. Pharmacol. Exp. Ther. 241,987. Christensen, S. and J.S. Petersen, 1988, Effects of furosemide on renal haemodynamics and proximal tubular sodium reabsorption in conscious rats, Br. J. Pharmacol. 95, 353. Christensen, S., M. Shalmi and J.S. Petersen, 1988, Lithium clearance as an indicator of proximal tubular sodium handling during furosemide diuresis, J. Pharmacol. Exp. Ther. 246, 753. Christensen, S., E. Steiness and H. Christensen, 1986, Tubular sites of furosemide natriuresis in volume-replaced and volume-depleted conscious rats, J. Pharmacol. Exp. Ther. 239, 211. Elmer, M., P.C. I£~kildsen, L.~. Kristensen and P.P. Leyssac, 1972, A comparison of renal function in rats anesthetized with inactin and sodium amytal, Acta Physiol. Scand. 86, 41. Gemba, M., N. Mikuriya, A. Tachibana and M. Nakajima, 1984, Effect of lithium on organic ion transport i.n rat kidney cortical slices, Jap. J. Pharmacol. 34, 128. Goldberg, M., 1973, The renal physiology of diuretics, in: Handbook of Physiology, eds. J. Orloff, R.W. Berlinger and S.R. Geiger (Physiological Society, Washington, D.C.) p. 1003.
Greven, J., M. Beckers, W. Defrain, K. Meywald and O. Heidenreich, 1980a, Studies with the optically active isomers of the new diuretic drug ozolinone. I1. Inhibition by d-ozolinone of furosemide-induced diuresis, Pfliigers Arch. 384, 61. Greven, J., W. Defrain, K. Glaser, K. Meywald and O. Heidenreich, 1980b, Studies with the optically active isomers of the new diuretic drug ozolinone. I. Differences in stereoselectivity of the renal target structures of ozolinone, Pfliigers Arch. 384, 57. Greven, J., K. Glaser, B. K~511ing and O. Heidenreich, 1984, Attenuation by d-ozolinone of I-ozolinone-induced diuresis in rats, European J. Pharmacol. 98, 331. Greven, J. and O. Heidenreich, 1977, Effect of etozolin on whole kidney function and fluid and electrolyte reabsorption in rat proximal convoluted tubules and loops of Henle, Arzneim. Forsch. 27, 1755. Greven, J. and O. Heidenreich, 1978, Effects of ozolinone, a diuretic active metabolite of etozoline, on renal function. I. Clearance studies in dogs, Naunyn-Schmiedeb. Arch. Pharmacol. 304, 283. Greven, J., H. Klein and O. Heidenreich, 1978, Effects of ozolinone, a diuretic active metabolite of etozolinc, on renal function. II. Localization of tubular site of diuretic action by micropuncture in the rat, Naunyn-Schmiedeb. Arch. Pharmacol. 3134, 289. Ludens. J.H., D.C. Heitz, M.J. Brody and H.E. Williamson, 1970, Differential effect of furosemide on renal and limb blood flows in the conscious dog, J. Pharmacol. Exp. Ther. 171,300. Ludens, J.H. and H.E. WillJamson, 1970, Effect of furosemide on renal blood flow in the conscious dog, Proc. Soc. Exp. Biol. Med. 133, 513. Olsen, U.B., 1977, The pharmacology of bumetanide, Acta pharmacol. Toxicol. 41, Suppl. Ill, 5. Petersen, J.S. and S. Christensen, 1987, Superiority of tetraethylammonium to p-aminohippurate as a marker for renal plasma flow during furosemide diuresis, Renal Physiol. 10, 102. Rasmussen, S.N., M. Shalmi, M. Hansen and S. Christensen, 1990, Tetraethylammonium and p-aminohippurate as clearance markers for renal plasma flow in the rat during saline and glucose infusion, Renal Physiol. Biochem. (in press). Satzmger, G., 1979, Neuere Entwicklungen in der Chemie der Diuretika, Deutsche Apoteker Z. 119, 1773. Satzirtger, G. and W. Herrmann, 1983, European Patent No. 00 16 635. Shalmi, M., J.S. Petersen and S. Christensen, 1989, Effects of intravenous bumetanide administration on renal haemodynamics and proximal and distal tubular sodium reabsorpfion in conscious rats, Pharmacol. Toxicol. 65, 313. Sugihara, K., A. Tachibana and M. Gemba, 1985. Evidence for in vivo effect of lithium on p-aminohippurate transport in rat kidny, preliminary study, Jap. J. Pharmacot. 38, 127. Thomsen, K., 1984, Lithium clearance: A new method for determining proximal and distal tubular reabsorption of sodium and water, Nephron 37, 217.
