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Changes in renal function in the rat during bromosulpho phthalein (BSP) infusion
CliniCa ChimiCa Acta, 43 (1973) 243-247 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
243
CGA 5285
CHANGES IN RENAL FUNCTION IN THE RAT DURING BROMOSULPHO
A. R. NOBLE,
PHTHALEIN
M. A. WINCH
Department of Physiology (Received
July 5.
(BSP) INFUSION
AND K. A. MUNDAY
and Biochemistry,
The University, Southampton So9 gNH, (U.K.)
1972)
SUMMARY
Bromosulphophthalein (BSP) is widely used in liver function tests and in the determination of hepatic blood flow. Some side effects of this compound on kidney function are reported in this paper. When BSP was given in a continuous infusion into rats, using a comparable dose to that used experimentally and clinically for function tests, it caused a 45% reduction in urine flow and a 60% reduction in sodium excretion rate. Potassium excretion rate was unaltered. Calculation of the y. filtered sodium excreted, from measurements of glomerular filtration rate and plasma sodium concentration, indicated that BSP was affecting tubular reabsorption of sodium. This could be either a direct or indirect effect on the tubule. No substantial changes in effective renal blood flow (determined by p-amino hippuric acid clearance) could be detected.
INTRODUCTION
Since the introduction of the bromosulphophthalein (BSP) retention test for liver function in 1925l, it has become widely used in both clinica12v3and experimental studies*+, despite many reservations concerning the validity of the data obtained617 The ability of the liver to extract BSP from blood plasma after a single injection of the dye is used as an index to hepatic function I,*. Clearance by the liver of BSP given in continuous infusion can be used as a crude estimation of hepatic blood flows~lo. The metabolism of BSPS and its excretion in the bileI and urinei have been extensively studied. However, the possible adverse effects of the dye on other organs are less well known. Several reports have been made of anaphylactoid responses to BSP13p1*and Grozman et al.l6 have shown that BSP infusion decreased bile flow in an isolated perfused liver preparation. During experiments on dogs in which BSP was used to measure hepatic blood flow, we noted some side effects of the dye on kidney function. These unpublished observations have now been more fully investigated in the rat, and these results are presented in this paper.
NOBLE
244
et al.
MATERIALS AND METHODS
Each group of animals consisted of eight female Wistar albino rats in the weight range 175-230 g. They were anaesthetised with sodium pentobarbitone (60 mg/kg body wt.). Blood samples were taken from a cartoid artery cannula. The bladder was catheterised and urine collected every 30 min. Tyrode’s solution was infused at the rate of 8.25 ml/kg body wt./h via a jugular vein cannula. Deep body temperature was monitored using an oesophageal thermistor, and was maintained at 37.0-38.0”. After completion of surgery the infusion was started and the animals were left for an equilibrium period of I I/Z-Z h before starting sample collection. When the effects of BSP (Sulphobromophthalein; Koch-Light) were investigated, this was added to the infusate to a concentration of 1.33 mg/ml. This dose is similar to that used both clinicallylo and experimentally18~17 b y other workers for hepatic blood flow estimations Sodium and potassium were estimated using a flame photometer. Effective renal plasma flow (ERPF) was calculated from the clearance, in 3o-min periods of PAH (Para-amino Hippuric Acid; British Drug Houses) by the kidney. This was added to the infusates at a concentration of 8.0 mg/ml. Glomerular filtration rate (GFR) was assessed by creatinine (Koch-Light) clearance, from a concentration in the infusates of 3.0 mg/ml. Blood samples (0.6 ml) were centrifuged for 30 min at IOOOg, the haematocrit measured and plasma samples retained for analysis. Student’s t-test was used for the statistical analysis of the results. RESULTS AND DISCUSSION
The initial experiments were designed to investigate the effect of BSP on the excretion of water, sodium and potassium by the kidney. Two groups of rats were each infused with Tyrode’s solution. BSP was added to the infusate of one group. Urine samples were collected every 30 min for 2-3 h after the equilibration period. The average of the 4-6 estimations of urine volume and urine electrolytes in each individual animal were used in the calculation of the mean for each group of animals. TABLE THE
I
EFFECT
(1.33 Ins/ml)
OF ON
A CONTINUOUS WATER
AND
INFUSION
OF
ELECTROLYTE
TYRODE’S
SOLUTION
HANDLING
BY
THE
WITHOUT
BSP
ADDED
t
P
-45.0%
6.44
244.7 z!z 8.9 178.3 & 14.8
-28.0%
3.84
<0.005
74.8 zt 7.8 I20.7 * 9.9
+62.0%
3.64
<0.005
-60.0%
7.65
0.23
N.S.
Mean f S.E.M.
Control + BSP
115.5
Urine sodium concentration (mequiv/l)
Control + BSP
Urine potassium concentration (mequiv/l)
Control t- BSP
Sodium excretion rate (,uequiv/min/kg b. wt.)
Control + BSP
28.1 f 1.39 11.3 & 1.65
Potassium excretion rate (pequiv/min/kg b. wt.)
Control + BSP
8.5 f 0.81 7.7 & 0.63
b. wt.)
AND
KIDNEY
Change BSP
Infusate
Urine flow ($/minjkg
WITH RAT
+
63.7 i
6.1 5.2
-lo’o%
with
BROMOSULPHOPHTHALEIN
AND KIDNEY FUNCTION
245
The results shown in Table I demonstrate a 45% ($ < 0.001) decrease in urine flow in the presence of BSP. This, together with a 28% (p < 0.005) reduction in urine sodium concentration, resulted in a sodium excretion rate 60% (p < 0.001) lower than in the control animals. Potassium excretion was unaltered as the decreased urine flow was accompanied by a 62.5% (p < 0.001) increase in urine potassium concentration. The functional basis of the antidiuretic and antinatriuretic responses reported above was investigated in a second experiment. ERPF and GFR were measured as well as urine and plasma sodium and potassium concentrations. From this data the filtration fraction (the ratio of GFR to ERPF), and the percentage of filtered sodium excreted were calculated. Results obtained are shown in Table II. The renal handling of sodium, potassium and water among the animals in this experiment followed the same pattern as the results reported in Table I. Although a small increase in the GFR was observed in the BSP-infused animals (Table II), this was probably a reflection of the small, but not statistically significant, TABLE
II
MEASUREMENTS INFUSATES
OF RENAL
OF CONTROL
FUNCTION
AND
BY THE INCLUSION
BSP-INFUSED -
OF
PAH
AND
CREATININE
IN THE
RATS
Infus&?
MelWL f S.E.M.
Change with BSP
t
P
Plasma sodium concentration (mequiv/l)
Control +BSP
146.3 f 139.7 f
-4.5%
2.79
PAH clearance (ml/min/kg b. wt.)
Control + BSP
+ 15.0%
1.45
N.S.
Creatinine clearance (ml/min/kg b. wt.)
Control + BSP
7.8 * 0.36 9.7 i 0.55
+22.0y0
2.88
(0.02
Filtration (%)
fraction
Control +BSP
31.2 * 1.04 31.9 + 1.44
+1.4%
-
N.S.
% Filtered sodium excreted
Control +BSP
2.76 & 0.08 0.98 Jr 0.24
-64.5%
7.51
(0.001
Haematocrit
Control +BSP
35.7 f. 1.02 36.1 & 0.90
+ 1.0%
-
N.S
2.0 1.2
25.3 + i 2.3 1.4 29.2
The results obtained for electrolyte and water handling by the kidney in this experiment are not shown here. They are exactly comparable with those shown in Table I. These parameters were not therefore affected by the inclusion of PAH and creatinine in the infusate.
increase in the ERPF. This is supported by the fact that the filtration fraction was constant. Haemodynamic and glomerular permeability changes did not therefore appear to be of major importance in the BSP-induced antidiuresis and antinatriuresis, although distribution of blood flow within the kidney was not investigated. An effect of BSP on the renal tubules was indicated from the calculations of percentage of filtered sodium excreted. A 64.5% decrease in this parameter in the BSP-infused animals showed that there was increased sodium reabsorption in the kidney. This effect was specific for sodium as the overall handling of potassium by the kidney was not affected by the presence of BSP. Despite the increased retention of sodium by the kidney, there was a small but significant decrease in plasma sodium concentration in the BSP-infused animals
NOBLE
246
et d.
(Table II). Haematocrit was constant indicating that the blood volume had not altered markedly. The possibility of effects of BSP on sodium movement into the extra-cellular space, or into other tissues, was therefore investigated in a third experiment. The experimental protocol used was similar to the previous experiments except that the kidneys were removed just before starting the saline infusion. No significant differences were observed in the haematocrit, sodium and potassium concentrations in the plasma or blood pressure between the controls and the BSP infused rats (Table III). These results indicate that the effects of BSP on sodium balance are mediated by a change in sodium reabsorption in the kidney tubule. The reason for the small decrease in plasma sodium levels is not known. TABLE
III
THE EFFECT OF A CONTINUOUS INFUSION OFTYRODE'SSOLUTION, ON pLASMASODIUMANDPOTASSIUMLE"ELS,HAEMATOCRITANDTHE NEPHRECTOMISED RATS Infusate
Mean
&
S.E.M.
WITH ANDWITHOUTADDED BSP, BLOODPRESSUREOFACUTELY
Change
with
t
P
-1.4%
0.41
N.S.
+1.5%
0.98
N.S.
0.07
N.S.
BSP Haematocrit
Control + BSP
Plasma sodium concentration (mequiv/l)
Control + BSP
Plasma potassium (mequiv/l)
Control + BSP
4.76 f 0.22 4.80 c 0.23
- 1.0%
Control + BSP
80-100 mm Hg 80-IOO mm Hg
-
Blood pressure
concentration
36.7 * 0.99 36.2 & 0.58 138.3 & 140.7 f
1.6 1,s
No previous reports have been made of possible side effects of BSP on kidney function. It is possible that the responses to the BSP infusion which we have observed could adversely affect patients or experimental animals in situations where this compound is used for hepatic function tests. We are at present unable to speculate on this. There is no obvious relationship to the anaphylactoid reactions to BSP reported in the literature13p1*. REFERENCES I 2 3 4 5 6 7 8 g IO
II IZ 13 14
S. M. ROSENTHAL AND E. C. WHITE, J. Amer. Med. Assoc., 84 (1925) 112. J. VERSIECK AND F. BARBIER, Acta Gastroenterol. Be&., 28 (1965) 651. A. H. SERBY AND L. BLOCH, Amer. J. Med. Sci., 176 (1928) 367. A. M. MILLS AND C. A. DRAGSTEDT, Proc. Sot. Exp. Biol. Med., 34 (1936) 228. I. URUSHIHATA, K. MAEDA, J. NAKAGAWA, K. ITO AND T. MAYAKE, Gastvoenterol. /up., (1967) 128. J. ROSENGARTEN AND T. HELENA, Polski Tygod. Lekar. Wiadomosii Lekav., 21 (1965) 356. J. F. MONAGHAN, Penna. Med.J., 48 (1945) 1036. M.MIKULECKY, CZin. Chim. Acta, 21 (1968) 43. A. D. BENDER AND S. M. HORVATH, Proc. Penna. Acad. Sci., 37 (1964) 77. A. FISCHER, in CH. ROUILLER (Ed)., The Liver, Vol. I, Academic Press, New York, 1964, p. 331. K. MURATA, K. NAGAI AND M. MIZUTA, Gastroenterol. Jap., 2 (1967) 129. K. WINKLER, Stand. J. Clin. Lab. Invest., 22 (1968) 41. S. LANDAU, J. Amer. Med. Assoc., 202 (1967) 238. M. J. PHILLIPS, Brit. Med. J., (1965) 595.
2
BROMOSULPHOPHTHALEIN
AND KIDNEY
FUNCTION
15 R. J. GROZMAN, B. KOTELANSKI, J. KENDLER AND H. J. ZIMMERMAN, Proc. Sot. Med., 132 (1969) 712. 16 T. H. Woo AND S. K. KONG, Amer. J. Physiol., 204 (1963) 778. 17 H. S. LEW. E. LEE, K. LEE AND S. K. HONG, Amer. J. Physiol., 203 (1~62) 644.
247 Exp. Biol.
243
CGA 5285
CHANGES IN RENAL FUNCTION IN THE RAT DURING BROMOSULPHO
A. R. NOBLE,
PHTHALEIN
M. A. WINCH
Department of Physiology (Received
July 5.
(BSP) INFUSION
AND K. A. MUNDAY
and Biochemistry,
The University, Southampton So9 gNH, (U.K.)
1972)
SUMMARY
Bromosulphophthalein (BSP) is widely used in liver function tests and in the determination of hepatic blood flow. Some side effects of this compound on kidney function are reported in this paper. When BSP was given in a continuous infusion into rats, using a comparable dose to that used experimentally and clinically for function tests, it caused a 45% reduction in urine flow and a 60% reduction in sodium excretion rate. Potassium excretion rate was unaltered. Calculation of the y. filtered sodium excreted, from measurements of glomerular filtration rate and plasma sodium concentration, indicated that BSP was affecting tubular reabsorption of sodium. This could be either a direct or indirect effect on the tubule. No substantial changes in effective renal blood flow (determined by p-amino hippuric acid clearance) could be detected.
INTRODUCTION
Since the introduction of the bromosulphophthalein (BSP) retention test for liver function in 1925l, it has become widely used in both clinica12v3and experimental studies*+, despite many reservations concerning the validity of the data obtained617 The ability of the liver to extract BSP from blood plasma after a single injection of the dye is used as an index to hepatic function I,*. Clearance by the liver of BSP given in continuous infusion can be used as a crude estimation of hepatic blood flows~lo. The metabolism of BSPS and its excretion in the bileI and urinei have been extensively studied. However, the possible adverse effects of the dye on other organs are less well known. Several reports have been made of anaphylactoid responses to BSP13p1*and Grozman et al.l6 have shown that BSP infusion decreased bile flow in an isolated perfused liver preparation. During experiments on dogs in which BSP was used to measure hepatic blood flow, we noted some side effects of the dye on kidney function. These unpublished observations have now been more fully investigated in the rat, and these results are presented in this paper.
NOBLE
244
et al.
MATERIALS AND METHODS
Each group of animals consisted of eight female Wistar albino rats in the weight range 175-230 g. They were anaesthetised with sodium pentobarbitone (60 mg/kg body wt.). Blood samples were taken from a cartoid artery cannula. The bladder was catheterised and urine collected every 30 min. Tyrode’s solution was infused at the rate of 8.25 ml/kg body wt./h via a jugular vein cannula. Deep body temperature was monitored using an oesophageal thermistor, and was maintained at 37.0-38.0”. After completion of surgery the infusion was started and the animals were left for an equilibrium period of I I/Z-Z h before starting sample collection. When the effects of BSP (Sulphobromophthalein; Koch-Light) were investigated, this was added to the infusate to a concentration of 1.33 mg/ml. This dose is similar to that used both clinicallylo and experimentally18~17 b y other workers for hepatic blood flow estimations Sodium and potassium were estimated using a flame photometer. Effective renal plasma flow (ERPF) was calculated from the clearance, in 3o-min periods of PAH (Para-amino Hippuric Acid; British Drug Houses) by the kidney. This was added to the infusates at a concentration of 8.0 mg/ml. Glomerular filtration rate (GFR) was assessed by creatinine (Koch-Light) clearance, from a concentration in the infusates of 3.0 mg/ml. Blood samples (0.6 ml) were centrifuged for 30 min at IOOOg, the haematocrit measured and plasma samples retained for analysis. Student’s t-test was used for the statistical analysis of the results. RESULTS AND DISCUSSION
The initial experiments were designed to investigate the effect of BSP on the excretion of water, sodium and potassium by the kidney. Two groups of rats were each infused with Tyrode’s solution. BSP was added to the infusate of one group. Urine samples were collected every 30 min for 2-3 h after the equilibration period. The average of the 4-6 estimations of urine volume and urine electrolytes in each individual animal were used in the calculation of the mean for each group of animals. TABLE THE
I
EFFECT
(1.33 Ins/ml)
OF ON
A CONTINUOUS WATER
AND
INFUSION
OF
ELECTROLYTE
TYRODE’S
SOLUTION
HANDLING
BY
THE
WITHOUT
BSP
ADDED
t
P
-45.0%
6.44
244.7 z!z 8.9 178.3 & 14.8
-28.0%
3.84
<0.005
74.8 zt 7.8 I20.7 * 9.9
+62.0%
3.64
<0.005
-60.0%
7.65
0.23
N.S.
Mean f S.E.M.
Control + BSP
115.5
Urine sodium concentration (mequiv/l)
Control + BSP
Urine potassium concentration (mequiv/l)
Control t- BSP
Sodium excretion rate (,uequiv/min/kg b. wt.)
Control + BSP
28.1 f 1.39 11.3 & 1.65
Potassium excretion rate (pequiv/min/kg b. wt.)
Control + BSP
8.5 f 0.81 7.7 & 0.63
b. wt.)
AND
KIDNEY
Change BSP
Infusate
Urine flow ($/minjkg
WITH RAT
+
63.7 i
6.1 5.2
-lo’o%
with
BROMOSULPHOPHTHALEIN
AND KIDNEY FUNCTION
245
The results shown in Table I demonstrate a 45% ($ < 0.001) decrease in urine flow in the presence of BSP. This, together with a 28% (p < 0.005) reduction in urine sodium concentration, resulted in a sodium excretion rate 60% (p < 0.001) lower than in the control animals. Potassium excretion was unaltered as the decreased urine flow was accompanied by a 62.5% (p < 0.001) increase in urine potassium concentration. The functional basis of the antidiuretic and antinatriuretic responses reported above was investigated in a second experiment. ERPF and GFR were measured as well as urine and plasma sodium and potassium concentrations. From this data the filtration fraction (the ratio of GFR to ERPF), and the percentage of filtered sodium excreted were calculated. Results obtained are shown in Table II. The renal handling of sodium, potassium and water among the animals in this experiment followed the same pattern as the results reported in Table I. Although a small increase in the GFR was observed in the BSP-infused animals (Table II), this was probably a reflection of the small, but not statistically significant, TABLE
II
MEASUREMENTS INFUSATES
OF RENAL
OF CONTROL
FUNCTION
AND
BY THE INCLUSION
BSP-INFUSED -
OF
PAH
AND
CREATININE
IN THE
RATS
Infus&?
MelWL f S.E.M.
Change with BSP
t
P
Plasma sodium concentration (mequiv/l)
Control +BSP
146.3 f 139.7 f
-4.5%
2.79
PAH clearance (ml/min/kg b. wt.)
Control + BSP
+ 15.0%
1.45
N.S.
Creatinine clearance (ml/min/kg b. wt.)
Control + BSP
7.8 * 0.36 9.7 i 0.55
+22.0y0
2.88
(0.02
Filtration (%)
fraction
Control +BSP
31.2 * 1.04 31.9 + 1.44
+1.4%
-
N.S.
% Filtered sodium excreted
Control +BSP
2.76 & 0.08 0.98 Jr 0.24
-64.5%
7.51
(0.001
Haematocrit
Control +BSP
35.7 f. 1.02 36.1 & 0.90
+ 1.0%
-
N.S
2.0 1.2
25.3 + i 2.3 1.4 29.2
The results obtained for electrolyte and water handling by the kidney in this experiment are not shown here. They are exactly comparable with those shown in Table I. These parameters were not therefore affected by the inclusion of PAH and creatinine in the infusate.
increase in the ERPF. This is supported by the fact that the filtration fraction was constant. Haemodynamic and glomerular permeability changes did not therefore appear to be of major importance in the BSP-induced antidiuresis and antinatriuresis, although distribution of blood flow within the kidney was not investigated. An effect of BSP on the renal tubules was indicated from the calculations of percentage of filtered sodium excreted. A 64.5% decrease in this parameter in the BSP-infused animals showed that there was increased sodium reabsorption in the kidney. This effect was specific for sodium as the overall handling of potassium by the kidney was not affected by the presence of BSP. Despite the increased retention of sodium by the kidney, there was a small but significant decrease in plasma sodium concentration in the BSP-infused animals
NOBLE
246
et d.
(Table II). Haematocrit was constant indicating that the blood volume had not altered markedly. The possibility of effects of BSP on sodium movement into the extra-cellular space, or into other tissues, was therefore investigated in a third experiment. The experimental protocol used was similar to the previous experiments except that the kidneys were removed just before starting the saline infusion. No significant differences were observed in the haematocrit, sodium and potassium concentrations in the plasma or blood pressure between the controls and the BSP infused rats (Table III). These results indicate that the effects of BSP on sodium balance are mediated by a change in sodium reabsorption in the kidney tubule. The reason for the small decrease in plasma sodium levels is not known. TABLE
III
THE EFFECT OF A CONTINUOUS INFUSION OFTYRODE'SSOLUTION, ON pLASMASODIUMANDPOTASSIUMLE"ELS,HAEMATOCRITANDTHE NEPHRECTOMISED RATS Infusate
Mean
&
S.E.M.
WITH ANDWITHOUTADDED BSP, BLOODPRESSUREOFACUTELY
Change
with
t
P
-1.4%
0.41
N.S.
+1.5%
0.98
N.S.
0.07
N.S.
BSP Haematocrit
Control + BSP
Plasma sodium concentration (mequiv/l)
Control + BSP
Plasma potassium (mequiv/l)
Control + BSP
4.76 f 0.22 4.80 c 0.23
- 1.0%
Control + BSP
80-100 mm Hg 80-IOO mm Hg
-
Blood pressure
concentration
36.7 * 0.99 36.2 & 0.58 138.3 & 140.7 f
1.6 1,s
No previous reports have been made of possible side effects of BSP on kidney function. It is possible that the responses to the BSP infusion which we have observed could adversely affect patients or experimental animals in situations where this compound is used for hepatic function tests. We are at present unable to speculate on this. There is no obvious relationship to the anaphylactoid reactions to BSP reported in the literature13p1*. REFERENCES I 2 3 4 5 6 7 8 g IO
II IZ 13 14
S. M. ROSENTHAL AND E. C. WHITE, J. Amer. Med. Assoc., 84 (1925) 112. J. VERSIECK AND F. BARBIER, Acta Gastroenterol. Be&., 28 (1965) 651. A. H. SERBY AND L. BLOCH, Amer. J. Med. Sci., 176 (1928) 367. A. M. MILLS AND C. A. DRAGSTEDT, Proc. Sot. Exp. Biol. Med., 34 (1936) 228. I. URUSHIHATA, K. MAEDA, J. NAKAGAWA, K. ITO AND T. MAYAKE, Gastvoenterol. /up., (1967) 128. J. ROSENGARTEN AND T. HELENA, Polski Tygod. Lekar. Wiadomosii Lekav., 21 (1965) 356. J. F. MONAGHAN, Penna. Med.J., 48 (1945) 1036. M.MIKULECKY, CZin. Chim. Acta, 21 (1968) 43. A. D. BENDER AND S. M. HORVATH, Proc. Penna. Acad. Sci., 37 (1964) 77. A. FISCHER, in CH. ROUILLER (Ed)., The Liver, Vol. I, Academic Press, New York, 1964, p. 331. K. MURATA, K. NAGAI AND M. MIZUTA, Gastroenterol. Jap., 2 (1967) 129. K. WINKLER, Stand. J. Clin. Lab. Invest., 22 (1968) 41. S. LANDAU, J. Amer. Med. Assoc., 202 (1967) 238. M. J. PHILLIPS, Brit. Med. J., (1965) 595.
2
BROMOSULPHOPHTHALEIN
AND KIDNEY
FUNCTION
15 R. J. GROZMAN, B. KOTELANSKI, J. KENDLER AND H. J. ZIMMERMAN, Proc. Sot. Med., 132 (1969) 712. 16 T. H. Woo AND S. K. KONG, Amer. J. Physiol., 204 (1963) 778. 17 H. S. LEW. E. LEE, K. LEE AND S. K. HONG, Amer. J. Physiol., 203 (1~62) 644.
247 Exp. Biol.