Journal of Ethnopharmacoiogy, 36 ( 1992) 2 13-2
213
17
Elsevier Scientific Publishers Ireland Ltd.
Stevioside effect on renal function of normal and hypertensive rats MS. Melis Departamento de Biologia, Setor de Fisiologia. Faculdade de Filosofa, Ckncias e Letras de Riheirifo Preto Uziversidade de Scio Paula. RibeirZo Preto: CEP 14049 Siio Puulo (Brusilj
(Received May 11, 1991; revision received December 20, 1991; accepted March 24, 1992)
Physiological and pharmacological experiments have suggested that stevioside from the leaves of Stevia rebaudiuna acts as a typical systemic vasodiiator. The effect of stevioside on renal function in both normal and with experimental renal hypertension rats (GII) was evaluated using clearance techniques. Stevioside provoked hypotension, diuresis and natriuresis in both the normal and hypertensive rats. Normal rats presented an increase in renal plasma flow (RPF) and giomeruiar filtration rate (GFR) constant following stevioside administration. The last effect is in part due to vasodilation of both the afferent and efferent arterioles. Moreover, stevioside infusion in hypertensive rats caused an increase in RPF and GFR. These data are consistent with impairment of a renal autoregulation mechanism in this experimental hypertensive model. Key words: stevioside; vasodiiator; experimental Goldbiatt two-kidney (GII) hypertension; fractional sodium and potassium excretion
Introduction Stevioside is a glycoside which occurs in large amounts in the leaves of Stevia rebaudiana, an herbaceous member of the Compositae from Paraguay and Brazil. It is considered sweeter than sucrose and is composed of steviol, a diterpenic carboxylic alcohol and three glucose molecules (Fig. 1) (Mosettig and Nes, 1955; Wood et al., 1955). Stevioside is used commercially as a sweetening agent in both Japan (Kato, 1975) and Brazil (Sakaguchi and Kan, 1982). The effects of stevioside and other natural products of S. rebaudiana on renal function have been the subject of relatively few investigations. Well demonstrated are the effects of stevioside and extracts prepared from the leaves of Stevia on cardiovascular parameters. The first experiments were done by Humboldt and Boeck (1977) and Boeck and Humboldt (1981). They reported that both the stevioside and aqueous extracts of Stevia induces a pronounced decrease in blood pressure and heart rate as well as diuresis. From the results of these investigations it may be concluded that both the stevioside and aqueous extracts of Stevia Correspondence too:Dr. MS. Meiis, Departamento de Bioiogia, Setor de Fisioiogia, Facuidade de Fiiosoiia, Ciencias e Letras de Ribeirzo Preto, Universidade de SZo Pauio, Ribeira?, Preto; CEP 14049 SazI Pauio, Brazil.
may be useful hypotasive agents. More recently, Melis and Sainati (1991) confirmed the earlier observation of Humboldt and Boeck (1977) and added new data about the action of stevioside on renal function. Our results showed that stevioside is a vasoactive drug and produces hypotension, diuresis, natriuresis and kaliuresis per ml of glomerular filtration rate. These effects seem to be analogous to that of verapamil, a specific inhibitor of calcium action in cardiac and vascular musctes. Arterial hypertension is a pathological state resulting from an inappropriate relationship between vascular capacity and blood volume. Experimental Goldblatt two-kidney/one-clip hypertension or GII (Goldblatt et al., 1934) is associated
Fig. I. Structural formula of stevioside (C,sH@O,s).
0378-8741/92/$05.00 0 1992 Eisevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
214
with the increased amounts of renin released by the ischemic kidney and therefore depends on vasoconstriction (Brunner et al., 1971). There is evidence, however, pointing to sodium retention during the early phase, which later depends on the course of hypertension (Gravas et al., 1975). Consequently, both homeostatic factors may be altered under these experimental conditions. The therapeutic agents used for the treatment of hypertension try to compensate for the increased secretion of renin or volume expansion. Among these, stevioside acts as a vasodilator that inhibits Ca2+ influx into smooth and cardiac muscle (Melis and Sainati, 1991). In addition, stevioside inhibits Na+ and fluid reabsorption when injected into the jugular vein (16 mg/kg per h). The purpose of the present study was to determine the effect of stevioside, a vasodilator, on renal function in both the GII hypertension and normal rats. These data may provide important information about the potential clinical treatment with stevioside of Steviu of hypertension rebaudiana.
Material and Methods Glycoside source Stevioside was extracted and purified from dried S. rebaudiana leaves (identified by Dr. Antonio Barioni Gusman, University of Sao Paulo) by the method of Alvarez et al. (1981). The method produces a stevioside preparation with greater than 90% purity. The primary impurities are various rebaudiosides, mucilage and leaf pigments. The positive identification of stevioside was made by comparison to UV, optical rotation and IR. Experimental methods Studies were performed on male Wistar rats weighing 250-320 g. All animals were fed a standard pellet diet (Purina Nutrimentos Ltda) and had free access to water until the day of the experiment. Two groups of clearance studies were performed: group 1 (n = lo), normal rats utilized as control (N); group 2 (n = lo), hypertensive rats in chronic phase (GII) (H). Details about the surgical preparation of hypertensive animals have been described previously by Goldblatt et al. (1934) and adapted to small animals by Schaffenburg (1959). Sodium pentobarbital (30 mg/kg) was administered intraperitoneally to induce anesthesia. A tracheostomy was performed, both right and left jugular veins were cannulated for infusions and
the urinary bladder was catheterized for timed urine collections. The left carotid artery was cannulated for blood collection and for recording mean arterial blood pressure measurement using a Statham strain-gauge transducer connected to a Physiograph Six-B (Narco Bio-Systems). After giving a priming dose of inulin and paminohippuric acid (PAH), (30 mg/lOO g and 2 mgrat, respectively) the infusion of a sustaining dose (0.05 mg/min per 100 g inulin and 40 mg/lO ml infusion PAH) in isotonic saline with 3% mannitol was initiated at a rate of 0.05 ml/min with a Harvard infusion pump. After a stabilization period of 60 min, a control period was started (C). After this period, a priming dose of stevioside (16 mg/kg i.v.) in isotonic saline was given and an in sequence intravenous infusion of stevioside was begun at a rate of 16 mg/kg per h. A 30-min collection corresponding to the stevioside period was performed (S). A recovery period (R) of 30 min was obtained after the termination of the stevioside infusion. Blood samples were drawn from the carotid artery at the midpoint of each clearance period. Urine was collected quantitatively from the bladder in preweighed containers and urine volume determined gravimetrically. Inulin concentration in plasma and urine was determined by the anthrone method (Furhr et al., 1955). Plasma and urinary PAH concentrations were measured by the method of Smith et al. (1945). Sodium and potassium concentrations in urine and plasma were determined with a Klina flame photometer (Beckman Instruments). The Tukey test was used for statistical analysis of the data, and the results are presented as the means + S.E.M., with the critical level of significance set at P < 0.05.
Results Paramaters of renal function of both normal (N) and hypertensive (H) rats did not differ in the control phase of experiments; only mean arterial blood pressure (MAP) was significantly lower in N than in H rats (P < 0.01) (Table 1). The effect of stevioside treatment in N and H animals on MAP, glomerular filtration rate (GFR) and PAH clearance (C PAH), an indicator of renal plasma flow (RPF) are shown in Fig. 2. Both stevioside-treated N and H rats showed a marked hypotension. MAP in N rats was 110.0 + 6.50 at C and decreased to 72.3 f 4.79 mmHg at S (P < 0.01) and in H from 152.0 f 7.31 at C to 110.0 f 6.50 mmHg at S (P < 0.01). There was
TABLE
1
CHARACTERIZATION OF THE RENAL AND MEAN ARTERIAL PRESSURE IN MOTENSIVE AND HYPERTENSIVE TROL PERIOD Parameters
(ml/min
per kg)
C,, (mhmin V/GFR (%) F, NaC (%) F, K+ (%)
per kg)
RATS IN THE CON-
Normotensive
Hypertensive
110
152 7.48 19.30 1.62 0.68 27.8
PA (mHg) GFR
FUNCTION BOTH NOR-
* 6.25 6.30 f 0.57
16.70 1.38 0.56 21.40
f f f f
3.76 0.11 0.04 2.48
l
f f f f f
C
7.31** 1.30 3.50 0.77 0.10 3.02
a 5 + Y
rots rots
C-Control period S - Stevioslde period R-Recovery period
C
S
R
Fig. 2. Mean arterial pressure (MAP), poru-aminohippurate clearance (f&B) and glomerular filtration rate (GFR) of both normal and hypertensive rats submitted to stevioside. The symbols for each period are given on the abscissa. C, control period; S, intravenous infusion of stevioside (16 mg/kg per h); R, recovery period (each period lasted 30 min). Results are the means * S.E.M. **P co.01 compared with the control period; n= 10.
S
0
Normol
69
Hypertenswe
R
rots rots
C -Control period S - Stewoslde period R-Recovery period
60
30
C
S
R
Fig. 3. Urine flow as percentage of glomerular filtration rate (V/GFR), fractional urinary sodium excretion (F, Na+) and fractional potassium excretion (F, K+) of both normal and hypertensive rats treated with stevioside. Results are the means + S.E.M. **P < 0.01 compared with period; n = 10. Other abbreviations as in Fig. 2.
_
Normal
C
I?
no significant change in GFR at any time during the experiment in N animals, however a significant increase in GFR was observed when stevioside was administered in H rats (7.48 f 1.31 vs. 13.67 f 1.20 ml/min per kg at C and S periods, respectively (P < 0.01). Both N and H animals
q Hypertenswe
R
90
Results are the means f S.E.M. Abbreviations: MAP, mean arterial pressure; GFR, glomerular filtration rate; CPAH, para-amino-hyppurate clearance; V/GFR, PAH urine flow as percentage of glomerular filtration rate; F, Na+, fractional urinary sodium excretion; F, K+, fractional urinary potassium excretion. Significance relates to normotensive values: **P < 0.01, n = 20.
0
S
the
control
following stevioside administration displayed significantly increased mean values of CPA” when compared to control period values; CPAH in N rats was 16.7 f 3.76 at C and increased to 34.3 f 2.55 ml/min per kg at S (P < 0.01) and in H rats 19.3 f 3.5 at C to 35.1 f 2.4 ml/min per kg at S (P < 0.01). One interesting finding was the incomplete normalization of MAP, GFR and CPAH in both N and H rats during the recovery period, i.e., they did not return back to the control conditions. Another was the GFR of N rats remained unaltered in all experiments, in contrast with high GFR in both S and R periods seen in the H animals. Figure 3 summarizes the effect of stevioside on urinary flow as a percentage of GFR (V/GFR) and fractional excretion of sodium (F, Na+) and potassium (F, K+) in both N and H animals. The data show that this infusion induced a marked diuretic effect at S when compared with the control experiments in the two groups of rats studied (1.38% f 0.11% vs. 2.36% f 0.11%; P < 0.01 and 1.62% f 0.77%~~. 3.55% f 1.56% P < 0.01 in N and H rats, respectively). Moreover, both groups of stevioside-treated rats showed natriuresis and kaliuresis. F, Na+ in the N rats was 0.56% f 0.04% at C and increase to 1.47% f 0.12% at S (P c 0.01); and in the H rats F, Na+ was 0.68% f O.lO”/o at C and increase to
216
2.49% f 2.04%at S (P < 0.01). F, K+ in the N aminals was 21.4% f 2.48% at C and 69.3% f 8.05% at S (P < 0.01) and in H 27.8% f 3.05% at C and 63.4% f 6.08% (P < 0.01). Thus, stevioside promoted diuresis, natriuresis and kaliuresis in normal and hypertensive rats and these effects remained after cessation of the stevioside infusion.
The dose of stevioside used in the present study is similar to that employed in cardiovascular experiments (Boeck and Humboldt, 1981), but is higher than the dose used for sweetening purposes. The average amount of stevioside per day used to sweeten foods and beverages in humans is 300 mg (Sakaguchi and Kan, 1982). The evaluation of renal function in Goldblatt 2-kidney/l-clip hypertension (GII) in the control phase of the experiment indicates values fully comparable to the results obtained for normal rats (Table 1). Under this chronic experimental hypertensive condition, the renal function of the ischemic kidney is very different from the normal kidney (Kramer and Ochwadt, 1972; Schwietezer and Gertz, 1979). On the other hand, when this function is evaluated as the sum of both the normal and the ischemic kidney, the result is analogous to that of a normal animal. In the present study the stevioside infusion in both groups of animals studied caused a significant decrease in MAP (Fig. 2). This finding is consistent with the idea that stevioside is an important hypotensor agent, a fact confirmed in other studies (Beech and Humboldt, 1977; Boeck and Humboldt, 198 1; Melis and Sainati, 199 1). Our previous studies (Melis and Sainati, 1991) suggested that the hypotensive response of stevioside would more likely be through a calcium antagonist mechanism, as is the case for verapamil. This mechanism inhibits calcium influx by blocking excitation-coupling in smooth muscle and promoting vasodilation. In our experiments, the infusion of stevioside in N and H rats was associated with an increase in CPA”, an indicator of renal plasma flow (RPF), probably by lowering renal vascular resistance (Fig. 2). In spite of the increase in RPF, GFR did not change in N rats, a finding consistent with other studies, in which verapamil was infused i.v. to rats (MacLaughlin et al., 1979; Melis and Maciel, 1986). It appears stevioside promotes
vasodilation of both afferent and efferent arterioles through its vasoactive influence. A similar conclusion was obtained by Baylis et al. (1976) infusing prostaglandin E,, acetylcholine and bradykinin into the renal artery of rats. In addition, they suggested that the relative constancy of GFR could be the result of opposite variations in RPF and glomerular capillary ultrafiltration coefficient. Despite the increase of CPAH in normal rats after stevioside, some renal auto-regulatory capacity may exist since GFR did not change. In contrast, stevioside infusion in hypertensive rats showed increase of both CPAH and GFR (Fig. 2). These data may be interpreted as implying that stevioside preferentially promotes dilation in the afferent arteriole of renal vasculature thus increasing GFR during increased renal blood flow. However, this explanation is not valid for normal rats, since in these animals the resistance of both afferent and efferent vessels is probably affected. There is no readily apparent explanation for the conflicting findings. However, it must be considered that the inference of segmental resistance changes from filtration fraction alone should be done with caution, as changes in other parameters, like glomerular capillary pressure, ultrafiltration coefticient, peritubular capillary pressure and proximal tubular pressure, are unknown. In addition, redistribution of blood flow or filtration rate among different nephron populations may be involved. If these populations differ sharply in their hemodynamic characteristics, estimates of pre- or postglomerular resistance changes from wholekidney measurements of renal function may be misleading. It is concluded that in hypertensive rats stevioside mediates an impairment of renal hemodynamics. Normal rats following stevioside administration presented significant increases in sodium and water excretion (Fig. 3) revealing reduction in tubular reabsorption of sodium and water. This finding is in agreement with data obtained by Earley and Friedler (1966) in which intrarenal and intravenous infusion of a vasodilator, under normal conditions, is normally associated with an increase in urinary sodium excretion. Since GFR did not change, the natriuresis and diuresis provoked by stevioside in the normal rats is probably due to changes in Starling forces around the proximal convolution. On the other hand, in the hypertensive rats (Fig. 3), the modifications in renal excretory function after stevioside are not dissociated from changes in the GFR or in sodium and potassium filtered load. Several factors can ac-
217
count for the diuresis and natriuresis observed in this hypertensive experimental condition. First, reduction in the water and sodium reabsorption in the proximal tubule by modification in physical peritubular factors through the stevioside provoked renal vasodilation. Second, elevation of GFR by impairment of an autoregulatory renal mechanism, thus increasing the sodium filtered load. Third, redistribution of blood flow among different nephron populations involved in this hypertensive condition (Stumpe et al., 1969). Moreover, stevioside may act directly on the mechanism of tubular sodium reabsorption in the two groups of animals studied. Therefore, the experimental model used in the present study is not suitable for discriminating between vascular and tubular effects. The luminal sodium concentrations and luminal fluid flow rate are factors capable of regulating potassium secretion by the distal tubule of the rat (Malnic et al., 1964; Engbretson and Stoner, 1987). If stevioside affects proximal sodium reabsorption in all groups analysed, it increases, therefore, the rate at which sodium and fluid are delivered to the distal tubule, supporting a sodium-potassium exchange at the level of the peritubular membrane. Both normal and hypertensive animals showed incomplete normalization of all parameters studied in the recovery period, i.e., did not return to the control conditions, indicating that the stevioside may not be totally eliminated from the animals at this time. In summary, our results suggest stevioside is a hypotensive agent and may be considered for the clinical treatment of hypertension. References Alvarez, M., Bracht, A. and Ishii, E.L. (1981) Hydrolysis of Steviu rebaudiuna glycosides with the gastric juice of megalobulimus-paranaguensis. Arquivos de Biologiu e Tecnologia 24, 179-183. Baylis, C., Deen, W.M., Mayers, B. and Brenner, B.M. (1976) Effect of some vasodilator drugs on transcapillary fluid exchange in the renal cortex. American Journal of Physiology 230, 1148-I 158. Beech, E.M.A. and Humboldt, G. (1981) Cardio-circulatory effects of total water extract in normal persons and of stevioside in rats. Cienciu e Cultura 32, 208-210. Brunner, H.S., Kirshman, J.D., Sealey, J.E. and Laragh, J.H. (1971) Hypertension of renal origin, evidence for two different mechanism. Science 174, 1344- 1346.
Earley, L.E. and Friedler, R.M. (1966) The effects of combined vasodilation and pressor agents on renal hemodynamics and the tubular reabsorption of sodium. Journal of Clinical Investigation 45, 542-551. Furhr, J., Kaczmarczyk, J. and Krukgen, C.D. (1955) Eine einfache calorimetrische method zur Inulinbestimmung fuer Nieren clearance Untersuchungen bei Stoffwechselgesunden und Diabetiken. Klinische Wochenschrtft 33, 729-730. Gravas, J., Brunner, H.R., Thurston, H. and Laragh, J.H. (1975) Reciprocation of renins dependency with sodium volume dependency in renal hypertension. Science 188, 1316-1317. Goldblatt, H., Linch, B., Hanzal, R.F. and Summerville, W.W. (1943) Studies on experimental hypertension. The production of persistent elevation of systolic blood pressure by means of renal ischemia. Journal of Experimental Medicine 59, 347-379. Humboldt, G. and Beech, E.M.A. (1977) Efeito do edulcorante natural, (stevioside) e sintetico (sacarina) sobre o ritmo cardisco em ratos. Arquivos Brasileiros de Cardiologia 30 275-277. Kato, I. (1975) Some views on the utilization and security of stevioside. Shokuhin Kogyo Tokyo 18, 44-49 Kramer, P. and Ochwadt, B. (1972) Sodium excretion in Goldblatt hypertension. Pfluegers Archives 332, 332-345. MacLaughlin, M., Mello Aires, M. and Malnic, G. (1979) Efeito do verapamil em diferentes pa&metros da fun&o renal. Arquivos Brasileiros de Cardiologia 32, 335-342. Malnic, G., Klose, R.M. and Giebisch, G. (1964) Micropuncture study of renal potassium excretion in the rat. American Journal of Physiology 206, 674-686. Melis, M.S. and Maciel, R.E. (1986) Participacao das prostaglandinas no mecanismo de a&o renal do verapamil. Cit%cia e Cultura 38, 154-159. Melis, M.S. verapamil stevioside. Mosettig, E.
and Sainati, A.R. (1991) Effect of calcium and on renal function of rats during treatment with Journal of Ethnopharmacology 33, 257-262. and Nes, W.R. (1955) Stevioside. II: The structure
of the aglucon. Journal of Organic Chemistry 20, 884-888. Sakaguchi, M. and Kan, T. (1982) As pesquisas Japonesas corn Steviu rebaudiana (Bert.) Bertoni e o esteviosideo. Cienciu e Cultura 34, 235-248. Schaffenburg, C.A. (1959) Device to control constriction of main renal artery for production of hypertension in small animal. Proceedings of the Society for Experimental Biology and Medicine 101, 676. Schwiltzer, G. and Gertz, K.H. (1979) Changes of hemodynamics and glomerular ultrafiltration in renal hypertension of rats. Kidney International 15, 134-143. Smith, H.W., Finkelstein, N., Alminosa, L., Crawford, B. and Graber, M. (1945) The renal clearances of substitute hippuric acid derivatives and other aromatic acids in dog and man. Journal of Clinical Investigations 24, 338-340. Stumpe, K.O., Lowitz, H.D. and Ochwadt, B. (1969) Function of juxtamedullary nephrons in normotensive and chronically hypertensive rats. Pfluegers Archives 313, 43-52. Woods, H.B. Jr., Allerton, R., Diehi, H.W. and Fletcher, H.G., Jr. (1955) Stevioside. I: The structure of the glucose moieties. Journal of Organic Chemistry 20, 875-879.
213
17
Elsevier Scientific Publishers Ireland Ltd.
Stevioside effect on renal function of normal and hypertensive rats MS. Melis Departamento de Biologia, Setor de Fisiologia. Faculdade de Filosofa, Ckncias e Letras de Riheirifo Preto Uziversidade de Scio Paula. RibeirZo Preto: CEP 14049 Siio Puulo (Brusilj
(Received May 11, 1991; revision received December 20, 1991; accepted March 24, 1992)
Physiological and pharmacological experiments have suggested that stevioside from the leaves of Stevia rebaudiuna acts as a typical systemic vasodiiator. The effect of stevioside on renal function in both normal and with experimental renal hypertension rats (GII) was evaluated using clearance techniques. Stevioside provoked hypotension, diuresis and natriuresis in both the normal and hypertensive rats. Normal rats presented an increase in renal plasma flow (RPF) and giomeruiar filtration rate (GFR) constant following stevioside administration. The last effect is in part due to vasodilation of both the afferent and efferent arterioles. Moreover, stevioside infusion in hypertensive rats caused an increase in RPF and GFR. These data are consistent with impairment of a renal autoregulation mechanism in this experimental hypertensive model. Key words: stevioside; vasodiiator; experimental Goldbiatt two-kidney (GII) hypertension; fractional sodium and potassium excretion
Introduction Stevioside is a glycoside which occurs in large amounts in the leaves of Stevia rebaudiana, an herbaceous member of the Compositae from Paraguay and Brazil. It is considered sweeter than sucrose and is composed of steviol, a diterpenic carboxylic alcohol and three glucose molecules (Fig. 1) (Mosettig and Nes, 1955; Wood et al., 1955). Stevioside is used commercially as a sweetening agent in both Japan (Kato, 1975) and Brazil (Sakaguchi and Kan, 1982). The effects of stevioside and other natural products of S. rebaudiana on renal function have been the subject of relatively few investigations. Well demonstrated are the effects of stevioside and extracts prepared from the leaves of Stevia on cardiovascular parameters. The first experiments were done by Humboldt and Boeck (1977) and Boeck and Humboldt (1981). They reported that both the stevioside and aqueous extracts of Stevia induces a pronounced decrease in blood pressure and heart rate as well as diuresis. From the results of these investigations it may be concluded that both the stevioside and aqueous extracts of Stevia Correspondence too:Dr. MS. Meiis, Departamento de Bioiogia, Setor de Fisioiogia, Facuidade de Fiiosoiia, Ciencias e Letras de Ribeirzo Preto, Universidade de SZo Pauio, Ribeira?, Preto; CEP 14049 SazI Pauio, Brazil.
may be useful hypotasive agents. More recently, Melis and Sainati (1991) confirmed the earlier observation of Humboldt and Boeck (1977) and added new data about the action of stevioside on renal function. Our results showed that stevioside is a vasoactive drug and produces hypotension, diuresis, natriuresis and kaliuresis per ml of glomerular filtration rate. These effects seem to be analogous to that of verapamil, a specific inhibitor of calcium action in cardiac and vascular musctes. Arterial hypertension is a pathological state resulting from an inappropriate relationship between vascular capacity and blood volume. Experimental Goldblatt two-kidney/one-clip hypertension or GII (Goldblatt et al., 1934) is associated
Fig. I. Structural formula of stevioside (C,sH@O,s).
0378-8741/92/$05.00 0 1992 Eisevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
214
with the increased amounts of renin released by the ischemic kidney and therefore depends on vasoconstriction (Brunner et al., 1971). There is evidence, however, pointing to sodium retention during the early phase, which later depends on the course of hypertension (Gravas et al., 1975). Consequently, both homeostatic factors may be altered under these experimental conditions. The therapeutic agents used for the treatment of hypertension try to compensate for the increased secretion of renin or volume expansion. Among these, stevioside acts as a vasodilator that inhibits Ca2+ influx into smooth and cardiac muscle (Melis and Sainati, 1991). In addition, stevioside inhibits Na+ and fluid reabsorption when injected into the jugular vein (16 mg/kg per h). The purpose of the present study was to determine the effect of stevioside, a vasodilator, on renal function in both the GII hypertension and normal rats. These data may provide important information about the potential clinical treatment with stevioside of Steviu of hypertension rebaudiana.
Material and Methods Glycoside source Stevioside was extracted and purified from dried S. rebaudiana leaves (identified by Dr. Antonio Barioni Gusman, University of Sao Paulo) by the method of Alvarez et al. (1981). The method produces a stevioside preparation with greater than 90% purity. The primary impurities are various rebaudiosides, mucilage and leaf pigments. The positive identification of stevioside was made by comparison to UV, optical rotation and IR. Experimental methods Studies were performed on male Wistar rats weighing 250-320 g. All animals were fed a standard pellet diet (Purina Nutrimentos Ltda) and had free access to water until the day of the experiment. Two groups of clearance studies were performed: group 1 (n = lo), normal rats utilized as control (N); group 2 (n = lo), hypertensive rats in chronic phase (GII) (H). Details about the surgical preparation of hypertensive animals have been described previously by Goldblatt et al. (1934) and adapted to small animals by Schaffenburg (1959). Sodium pentobarbital (30 mg/kg) was administered intraperitoneally to induce anesthesia. A tracheostomy was performed, both right and left jugular veins were cannulated for infusions and
the urinary bladder was catheterized for timed urine collections. The left carotid artery was cannulated for blood collection and for recording mean arterial blood pressure measurement using a Statham strain-gauge transducer connected to a Physiograph Six-B (Narco Bio-Systems). After giving a priming dose of inulin and paminohippuric acid (PAH), (30 mg/lOO g and 2 mgrat, respectively) the infusion of a sustaining dose (0.05 mg/min per 100 g inulin and 40 mg/lO ml infusion PAH) in isotonic saline with 3% mannitol was initiated at a rate of 0.05 ml/min with a Harvard infusion pump. After a stabilization period of 60 min, a control period was started (C). After this period, a priming dose of stevioside (16 mg/kg i.v.) in isotonic saline was given and an in sequence intravenous infusion of stevioside was begun at a rate of 16 mg/kg per h. A 30-min collection corresponding to the stevioside period was performed (S). A recovery period (R) of 30 min was obtained after the termination of the stevioside infusion. Blood samples were drawn from the carotid artery at the midpoint of each clearance period. Urine was collected quantitatively from the bladder in preweighed containers and urine volume determined gravimetrically. Inulin concentration in plasma and urine was determined by the anthrone method (Furhr et al., 1955). Plasma and urinary PAH concentrations were measured by the method of Smith et al. (1945). Sodium and potassium concentrations in urine and plasma were determined with a Klina flame photometer (Beckman Instruments). The Tukey test was used for statistical analysis of the data, and the results are presented as the means + S.E.M., with the critical level of significance set at P < 0.05.
Results Paramaters of renal function of both normal (N) and hypertensive (H) rats did not differ in the control phase of experiments; only mean arterial blood pressure (MAP) was significantly lower in N than in H rats (P < 0.01) (Table 1). The effect of stevioside treatment in N and H animals on MAP, glomerular filtration rate (GFR) and PAH clearance (C PAH), an indicator of renal plasma flow (RPF) are shown in Fig. 2. Both stevioside-treated N and H rats showed a marked hypotension. MAP in N rats was 110.0 + 6.50 at C and decreased to 72.3 f 4.79 mmHg at S (P < 0.01) and in H from 152.0 f 7.31 at C to 110.0 f 6.50 mmHg at S (P < 0.01). There was
TABLE
1
CHARACTERIZATION OF THE RENAL AND MEAN ARTERIAL PRESSURE IN MOTENSIVE AND HYPERTENSIVE TROL PERIOD Parameters
(ml/min
per kg)
C,, (mhmin V/GFR (%) F, NaC (%) F, K+ (%)
per kg)
RATS IN THE CON-
Normotensive
Hypertensive
110
152 7.48 19.30 1.62 0.68 27.8
PA (mHg) GFR
FUNCTION BOTH NOR-
* 6.25 6.30 f 0.57
16.70 1.38 0.56 21.40
f f f f
3.76 0.11 0.04 2.48
l
f f f f f
C
7.31** 1.30 3.50 0.77 0.10 3.02
a 5 + Y
rots rots
C-Control period S - Stevioslde period R-Recovery period
C
S
R
Fig. 2. Mean arterial pressure (MAP), poru-aminohippurate clearance (f&B) and glomerular filtration rate (GFR) of both normal and hypertensive rats submitted to stevioside. The symbols for each period are given on the abscissa. C, control period; S, intravenous infusion of stevioside (16 mg/kg per h); R, recovery period (each period lasted 30 min). Results are the means * S.E.M. **P co.01 compared with the control period; n= 10.
S
0
Normol
69
Hypertenswe
R
rots rots
C -Control period S - Stewoslde period R-Recovery period
60
30
C
S
R
Fig. 3. Urine flow as percentage of glomerular filtration rate (V/GFR), fractional urinary sodium excretion (F, Na+) and fractional potassium excretion (F, K+) of both normal and hypertensive rats treated with stevioside. Results are the means + S.E.M. **P < 0.01 compared with period; n = 10. Other abbreviations as in Fig. 2.
_
Normal
C
I?
no significant change in GFR at any time during the experiment in N animals, however a significant increase in GFR was observed when stevioside was administered in H rats (7.48 f 1.31 vs. 13.67 f 1.20 ml/min per kg at C and S periods, respectively (P < 0.01). Both N and H animals
q Hypertenswe
R
90
Results are the means f S.E.M. Abbreviations: MAP, mean arterial pressure; GFR, glomerular filtration rate; CPAH, para-amino-hyppurate clearance; V/GFR, PAH urine flow as percentage of glomerular filtration rate; F, Na+, fractional urinary sodium excretion; F, K+, fractional urinary potassium excretion. Significance relates to normotensive values: **P < 0.01, n = 20.
0
S
the
control
following stevioside administration displayed significantly increased mean values of CPA” when compared to control period values; CPAH in N rats was 16.7 f 3.76 at C and increased to 34.3 f 2.55 ml/min per kg at S (P < 0.01) and in H rats 19.3 f 3.5 at C to 35.1 f 2.4 ml/min per kg at S (P < 0.01). One interesting finding was the incomplete normalization of MAP, GFR and CPAH in both N and H rats during the recovery period, i.e., they did not return back to the control conditions. Another was the GFR of N rats remained unaltered in all experiments, in contrast with high GFR in both S and R periods seen in the H animals. Figure 3 summarizes the effect of stevioside on urinary flow as a percentage of GFR (V/GFR) and fractional excretion of sodium (F, Na+) and potassium (F, K+) in both N and H animals. The data show that this infusion induced a marked diuretic effect at S when compared with the control experiments in the two groups of rats studied (1.38% f 0.11% vs. 2.36% f 0.11%; P < 0.01 and 1.62% f 0.77%~~. 3.55% f 1.56% P < 0.01 in N and H rats, respectively). Moreover, both groups of stevioside-treated rats showed natriuresis and kaliuresis. F, Na+ in the N rats was 0.56% f 0.04% at C and increase to 1.47% f 0.12% at S (P c 0.01); and in the H rats F, Na+ was 0.68% f O.lO”/o at C and increase to
216
2.49% f 2.04%at S (P < 0.01). F, K+ in the N aminals was 21.4% f 2.48% at C and 69.3% f 8.05% at S (P < 0.01) and in H 27.8% f 3.05% at C and 63.4% f 6.08% (P < 0.01). Thus, stevioside promoted diuresis, natriuresis and kaliuresis in normal and hypertensive rats and these effects remained after cessation of the stevioside infusion.
The dose of stevioside used in the present study is similar to that employed in cardiovascular experiments (Boeck and Humboldt, 1981), but is higher than the dose used for sweetening purposes. The average amount of stevioside per day used to sweeten foods and beverages in humans is 300 mg (Sakaguchi and Kan, 1982). The evaluation of renal function in Goldblatt 2-kidney/l-clip hypertension (GII) in the control phase of the experiment indicates values fully comparable to the results obtained for normal rats (Table 1). Under this chronic experimental hypertensive condition, the renal function of the ischemic kidney is very different from the normal kidney (Kramer and Ochwadt, 1972; Schwietezer and Gertz, 1979). On the other hand, when this function is evaluated as the sum of both the normal and the ischemic kidney, the result is analogous to that of a normal animal. In the present study the stevioside infusion in both groups of animals studied caused a significant decrease in MAP (Fig. 2). This finding is consistent with the idea that stevioside is an important hypotensor agent, a fact confirmed in other studies (Beech and Humboldt, 1977; Boeck and Humboldt, 198 1; Melis and Sainati, 199 1). Our previous studies (Melis and Sainati, 1991) suggested that the hypotensive response of stevioside would more likely be through a calcium antagonist mechanism, as is the case for verapamil. This mechanism inhibits calcium influx by blocking excitation-coupling in smooth muscle and promoting vasodilation. In our experiments, the infusion of stevioside in N and H rats was associated with an increase in CPA”, an indicator of renal plasma flow (RPF), probably by lowering renal vascular resistance (Fig. 2). In spite of the increase in RPF, GFR did not change in N rats, a finding consistent with other studies, in which verapamil was infused i.v. to rats (MacLaughlin et al., 1979; Melis and Maciel, 1986). It appears stevioside promotes
vasodilation of both afferent and efferent arterioles through its vasoactive influence. A similar conclusion was obtained by Baylis et al. (1976) infusing prostaglandin E,, acetylcholine and bradykinin into the renal artery of rats. In addition, they suggested that the relative constancy of GFR could be the result of opposite variations in RPF and glomerular capillary ultrafiltration coefficient. Despite the increase of CPAH in normal rats after stevioside, some renal auto-regulatory capacity may exist since GFR did not change. In contrast, stevioside infusion in hypertensive rats showed increase of both CPAH and GFR (Fig. 2). These data may be interpreted as implying that stevioside preferentially promotes dilation in the afferent arteriole of renal vasculature thus increasing GFR during increased renal blood flow. However, this explanation is not valid for normal rats, since in these animals the resistance of both afferent and efferent vessels is probably affected. There is no readily apparent explanation for the conflicting findings. However, it must be considered that the inference of segmental resistance changes from filtration fraction alone should be done with caution, as changes in other parameters, like glomerular capillary pressure, ultrafiltration coefticient, peritubular capillary pressure and proximal tubular pressure, are unknown. In addition, redistribution of blood flow or filtration rate among different nephron populations may be involved. If these populations differ sharply in their hemodynamic characteristics, estimates of pre- or postglomerular resistance changes from wholekidney measurements of renal function may be misleading. It is concluded that in hypertensive rats stevioside mediates an impairment of renal hemodynamics. Normal rats following stevioside administration presented significant increases in sodium and water excretion (Fig. 3) revealing reduction in tubular reabsorption of sodium and water. This finding is in agreement with data obtained by Earley and Friedler (1966) in which intrarenal and intravenous infusion of a vasodilator, under normal conditions, is normally associated with an increase in urinary sodium excretion. Since GFR did not change, the natriuresis and diuresis provoked by stevioside in the normal rats is probably due to changes in Starling forces around the proximal convolution. On the other hand, in the hypertensive rats (Fig. 3), the modifications in renal excretory function after stevioside are not dissociated from changes in the GFR or in sodium and potassium filtered load. Several factors can ac-
217
count for the diuresis and natriuresis observed in this hypertensive experimental condition. First, reduction in the water and sodium reabsorption in the proximal tubule by modification in physical peritubular factors through the stevioside provoked renal vasodilation. Second, elevation of GFR by impairment of an autoregulatory renal mechanism, thus increasing the sodium filtered load. Third, redistribution of blood flow among different nephron populations involved in this hypertensive condition (Stumpe et al., 1969). Moreover, stevioside may act directly on the mechanism of tubular sodium reabsorption in the two groups of animals studied. Therefore, the experimental model used in the present study is not suitable for discriminating between vascular and tubular effects. The luminal sodium concentrations and luminal fluid flow rate are factors capable of regulating potassium secretion by the distal tubule of the rat (Malnic et al., 1964; Engbretson and Stoner, 1987). If stevioside affects proximal sodium reabsorption in all groups analysed, it increases, therefore, the rate at which sodium and fluid are delivered to the distal tubule, supporting a sodium-potassium exchange at the level of the peritubular membrane. Both normal and hypertensive animals showed incomplete normalization of all parameters studied in the recovery period, i.e., did not return to the control conditions, indicating that the stevioside may not be totally eliminated from the animals at this time. In summary, our results suggest stevioside is a hypotensive agent and may be considered for the clinical treatment of hypertension. References Alvarez, M., Bracht, A. and Ishii, E.L. (1981) Hydrolysis of Steviu rebaudiuna glycosides with the gastric juice of megalobulimus-paranaguensis. Arquivos de Biologiu e Tecnologia 24, 179-183. Baylis, C., Deen, W.M., Mayers, B. and Brenner, B.M. (1976) Effect of some vasodilator drugs on transcapillary fluid exchange in the renal cortex. American Journal of Physiology 230, 1148-I 158. Beech, E.M.A. and Humboldt, G. (1981) Cardio-circulatory effects of total water extract in normal persons and of stevioside in rats. Cienciu e Cultura 32, 208-210. Brunner, H.S., Kirshman, J.D., Sealey, J.E. and Laragh, J.H. (1971) Hypertension of renal origin, evidence for two different mechanism. Science 174, 1344- 1346.
Earley, L.E. and Friedler, R.M. (1966) The effects of combined vasodilation and pressor agents on renal hemodynamics and the tubular reabsorption of sodium. Journal of Clinical Investigation 45, 542-551. Furhr, J., Kaczmarczyk, J. and Krukgen, C.D. (1955) Eine einfache calorimetrische method zur Inulinbestimmung fuer Nieren clearance Untersuchungen bei Stoffwechselgesunden und Diabetiken. Klinische Wochenschrtft 33, 729-730. Gravas, J., Brunner, H.R., Thurston, H. and Laragh, J.H. (1975) Reciprocation of renins dependency with sodium volume dependency in renal hypertension. Science 188, 1316-1317. Goldblatt, H., Linch, B., Hanzal, R.F. and Summerville, W.W. (1943) Studies on experimental hypertension. The production of persistent elevation of systolic blood pressure by means of renal ischemia. Journal of Experimental Medicine 59, 347-379. Humboldt, G. and Beech, E.M.A. (1977) Efeito do edulcorante natural, (stevioside) e sintetico (sacarina) sobre o ritmo cardisco em ratos. Arquivos Brasileiros de Cardiologia 30 275-277. Kato, I. (1975) Some views on the utilization and security of stevioside. Shokuhin Kogyo Tokyo 18, 44-49 Kramer, P. and Ochwadt, B. (1972) Sodium excretion in Goldblatt hypertension. Pfluegers Archives 332, 332-345. MacLaughlin, M., Mello Aires, M. and Malnic, G. (1979) Efeito do verapamil em diferentes pa&metros da fun&o renal. Arquivos Brasileiros de Cardiologia 32, 335-342. Malnic, G., Klose, R.M. and Giebisch, G. (1964) Micropuncture study of renal potassium excretion in the rat. American Journal of Physiology 206, 674-686. Melis, M.S. and Maciel, R.E. (1986) Participacao das prostaglandinas no mecanismo de a&o renal do verapamil. Cit%cia e Cultura 38, 154-159. Melis, M.S. verapamil stevioside. Mosettig, E.
and Sainati, A.R. (1991) Effect of calcium and on renal function of rats during treatment with Journal of Ethnopharmacology 33, 257-262. and Nes, W.R. (1955) Stevioside. II: The structure
of the aglucon. Journal of Organic Chemistry 20, 884-888. Sakaguchi, M. and Kan, T. (1982) As pesquisas Japonesas corn Steviu rebaudiana (Bert.) Bertoni e o esteviosideo. Cienciu e Cultura 34, 235-248. Schaffenburg, C.A. (1959) Device to control constriction of main renal artery for production of hypertension in small animal. Proceedings of the Society for Experimental Biology and Medicine 101, 676. Schwiltzer, G. and Gertz, K.H. (1979) Changes of hemodynamics and glomerular ultrafiltration in renal hypertension of rats. Kidney International 15, 134-143. Smith, H.W., Finkelstein, N., Alminosa, L., Crawford, B. and Graber, M. (1945) The renal clearances of substitute hippuric acid derivatives and other aromatic acids in dog and man. Journal of Clinical Investigations 24, 338-340. Stumpe, K.O., Lowitz, H.D. and Ochwadt, B. (1969) Function of juxtamedullary nephrons in normotensive and chronically hypertensive rats. Pfluegers Archives 313, 43-52. Woods, H.B. Jr., Allerton, R., Diehi, H.W. and Fletcher, H.G., Jr. (1955) Stevioside. I: The structure of the glucose moieties. Journal of Organic Chemistry 20, 875-879.
