Clinical Science and Molecular Medicine (1977) 53, 149-1 54.
Failure of loading with sodium bicarbonate to protect against acute renal failure induced by mercuric chloride in the rat J. E. BEAUMONT, T. A. KOTCHEN, J. H. GALLA
AND
R. G. LU K E
University of Kentucky Medical Center, Department of Medicine, Lexington, Kentucky, U.S.A.
(Received 10 February 1977; accepted 30 March 1977)
Key words: acute renal failure, intrarenal renin, plasma renin activity, sodium bicarbonate loading.
SY 1. To investigate the mechanism by which sodium loading protects against acute renal failure we compared the effects of prior chronic loading with NaCl, or with NaHC03, on renal function after injection of HgC1,. 2. Twenty-four male Sprague-Dawley rats were divided into three groups of eight rats. One group drank isotonic NaCl solution, a second drank isotonic NaHCOJ solution and the third control group drank deionized water. Acute renal failure was induced by HgCl, on day 9, and the rats were killed 48 h after injection. 3. Net sodium balances and plasma volumes were similar in both groups of sodium-loaded rats. After HgCl, serum creatinine was significantly less and urinary volume was greater in NaC1-loaded than in both NaHCOJoaded and water-drinking animals. 4. Plasma renin activity of both NaCl- and NaHC03-loaded animals was less than that of control rats. However, renal renin content was suppressed by NaCl but not by NaHC03 loading. 5. Loading with NaCl afforded greater protection against HgC1,-induced acute renal failure than NaHC03. Since this difference was not related to changes in sodium balance or plasma volume before HgCl,, or plasma renin activity after HgCL, the results support the hypothesis that intrarenal renin plays a role in the pathogenesis of HgClz-induced acute renal failure in the rat.
Introduction
The renin-angiotensin system may be involved in the pathogenesis of HgClJnduced acute renal failure in the rat (McDonald, Thiel, Wilson, DiBona & Oken, 1969; DiBona, McDonald, Flamenbaum, Dammin & Oken, 1971). Thus plasma renin activity is acutely elevated after HgCl, (DiBona & Sawin, 1971). Chronic saline loading prevents the elevation of plasma renin activity and protects against renal insufficiency (DiBona et al., 1971). However, neither active renin immunization nor acute renin inhibition by administration of deoxycorticosterone and NaCl solution prevents acute renal failure after HgCL (Flamenbaum,Kotchen & Oken, 1972; Flamenbaum, McNeil, Kotchen, Lowenthal & NagIe, 1973a). Although peripheral renin is either blocked by antirenin antibody or suppressed by these two experimental manoeuvres, the content of renin within the kidney is not decreased. Consequently, the relevant factor may be intrarenal rather than circulating renin. Alternatively, renin may not be involved and the protective effect of chronic NaCl loading may reflect plasma volume expansion rather than renin suppression (Matthews, Morgan & Johnson, 1974). W e have recently demonstrated that NaHC03, unlike NaCl, fails to suppress both plasma renin activity and renal renin content in the rat, despite comparable positive sodium balance after loading with both sodium salts
Correspondence: Dr R. G. Luke, University of Kentucky Medical Center, Department of Medicine, Lexington, Kentucky 40506, U.S.A.
149
150
J . E. Beaumont et al.
(Kotchen, Galla & Luke, 1976). To determine whether protection against HgCIz-induced acute renal failure is related to positive sodium balance and volume expansion rather than to a specific effect on intrarenal renin, the effects of NaCl or NaHC03 loading on renal function after HgClz were compared in the rat. Methods Twenty-four Sprague-Dawley rats were divided into three groups of eight rats. All rats were placed in individual metabolic cages and allowed a 3 days period of adjustment before start of the study. All animals were given ground Purina rat chow, containing 182 pmol of sodium, 307 pmol of potassium and 190 pmol of chloridelg of diet, determined by analysis after nitric acid extraction. In group A, deionized drinking water was replaced with NaCl solution. Group B was given isotonic NaHCO, solution to drink; group C received deionized water. Animals were pair-fed; i.e. the fluid and dietary intakes of group B and group C were determined by the previous daily intake of group A. Urine was collected under mineral oil and preserved by thymol. The study period for groups A, B and C extended from day 1 to day 11. Rats were weighed on day 1 and tail blood was obtained for measurement of serum urea; packed cell volume was measured on days 1 and 9, before HgClz was given. The rats were injected with HgCL (4.5 mg/kg body weight) subcutaneously on day 9 to induce acute renal failure, and 48 h later, on the morning of day 12, they were reweighed and killed by decapitation. Blood was collected in chilled tubes with EDTA for the measurement of plasma renin activity; serum was obtained for urea and creatinine concentrations. Immediately thereafter a single kidney was removed from each animal and frozen for measurement of renal renin content. Plasma volume was measured in comparable groups of animals (n = 8lgroup) on similar regimens, for 7 days, with 1251-labelledalbumin (Belcher & Harris, 1957). On day 7, the rats were anaesthetized by an intraperitoneal injection of sodium ethyl 1-methylpropylmalonylthiourea (Inactin, Promonta, Hamburg) (100 mg/kg body weight). An external jugular vein was cannulated with PESO polyethylene tubing for injection of 1251-labelledalbumin. After an
equilibration period (10 min), blood was obtained for radioactivity counting and measurement of packed cell volume by direct aortic puncture. In the calculation of plasma volume, adjustment was made for plasma trapping. Urinary sodium and potassium concentrations were determined with an IL flame photometer. Plasma creatinine concentration was measured by the method of Kennedy, Hilton & Berliner (1952), and serum urea by the method of Crocker (1967). Urinary chloride was measured with a Buchler chloridometer (Buchler Instrument Division, Nuclear-Chicago Corp., Fort Lee, NJ, U.S.A.). Plasma renin activity was measured in quadruplicate by the radioimmunoassay method of Haber, Koerner, Page, Kliman & Purnode (1969). To maintain constant pH during the incubation (3 h), phosphate buffer, pH 7.4, was added (100 pl/ml) to each sample. To determine if HgCI2 affects the measurement of plasma renin activity, the latter was measured in portions of plasma from each of six rats after addition of HgCl2 (70 pglml). This concentration of HgClz was calculated on the assumption that the total dose of subcutaneous HgClz administered to induce renal failure was present in extracellular fluid. Mean plasma renin activity in aliquots of plasma to which HgClz was added (4.4 pmol h- ml- l , SEM 0.6) did not differ from that in portions not containing HgClz (4.5 pmol h-' d-l,SEM 0.6) and it was concluded that measurement of plasma renin activity is not altered by HgClZin the concentrations used. Renal renin was extracted by ammonium sulphate precipitation and measured as previously described (Flamenbaum et al., 1972). Briefly, a sample of the kidney extract was incubated with excess of sheep renin substrate, and the concentration of angiotensin I generated after incubation for 15 min at 37°C and pH 7.4 was measured by radioimmunoassay. One unit of renal renin is arbitrarily defined as that concentration of renin that will generate 100 ng of angiotensin I during the incubation. When results were compared for the three groups of animals, and when the variances for the three groups were similar, the significance of group differences was computed with analysis of variance (Snedecor & Cochran, 1967). If group variances were dissimilar, then statistical significance for three group comparisons was
Bicarbonate in acute renal failure determined by the Kruskal-Wallis Rank test (Bradley, 1968). Comparisons between two groups were made with an unpaired t-test. P 0.7) in
TABLE 1. Weight, net weight gain during the 12 days of study, andpacked cell volume andserum urea before HgC12 in NaCI-loaded, NaHC03-loaded and water-drinking control rats Mean valuesf SEM are shown. There are no significant differences between groups A, B and C. n = 8 for each group. Packed cell volume Group
Weight (g) (Day 1)
Net weight gain (9)
330f7 33524 325+4
21+12
A (NaCl) B (NaHC03) C (water)
9+6
12+4
Day 1
Day 9
46k0.9 48k1.1 46k0.9
48f0.7 49+05
Urea (mmol/l) (Day 1) 14k0.6 16k0.8 18+ 1-7
5020.6
TAELE 2. Total electrolyte excretion and urinary volumes in NaCI-loaded, NaHC03-loaded and water-drinking rats Mean values+seM are shown. n = 8 for each group. Urinary electrolytes (mmol)
Group Na+
(1) Days 1-9 (before HgC12) 62+ 4* A (NaCl) B (NaHC03) 61 f 3* C (water) 21j-1
K+ 30+ 1 29+ 1 28+2
Urinary volume (ml)
c1235+23* 206f 12 164+ 9
75f 5t 23+ I 25k2
(2) Days 10 and 11 (after HgC12) A (NaCl) B (NaHC03) C (water)
14+ 32 6+ 1 1.5k0.1
5+ 0.7t
3f0.4 2+0*1
14f3T 0.8+0.1 0.7k0.1
* Different from group C (P 0.6). During the 48 h period after injection of HgCI, (Table 2, part 2), sodium excretion in 2 days of group A and group B remained significantly more than that of group C. Chloride excretion was significantly more in group A than in group B and group C. Potassium excretion was similar in group A and group B, but in group A it was significantly greater than in group C. During the first 24 h after HgCI2, urinary volume of all three groups did not differ. However, between 24 and 48 h, urinary volumes
of NaHC03-loaded and water-drinking control rats were less than those of NaC1-loaded animals (Table 2, part 2). This accounts for the greater potassium excretion in the latter group. Low urinary volumes in NaHCOt-loaded animals occurred despite a significantly greater fluid intake than control animals (31 ml, SEM 4, versus 12 ml, SEM 2; P
Failure of loading with sodium bicarbonate to protect against acute renal failure induced by mercuric chloride in the rat J. E. BEAUMONT, T. A. KOTCHEN, J. H. GALLA
AND
R. G. LU K E
University of Kentucky Medical Center, Department of Medicine, Lexington, Kentucky, U.S.A.
(Received 10 February 1977; accepted 30 March 1977)
Key words: acute renal failure, intrarenal renin, plasma renin activity, sodium bicarbonate loading.
SY 1. To investigate the mechanism by which sodium loading protects against acute renal failure we compared the effects of prior chronic loading with NaCl, or with NaHC03, on renal function after injection of HgC1,. 2. Twenty-four male Sprague-Dawley rats were divided into three groups of eight rats. One group drank isotonic NaCl solution, a second drank isotonic NaHCOJ solution and the third control group drank deionized water. Acute renal failure was induced by HgCl, on day 9, and the rats were killed 48 h after injection. 3. Net sodium balances and plasma volumes were similar in both groups of sodium-loaded rats. After HgCl, serum creatinine was significantly less and urinary volume was greater in NaC1-loaded than in both NaHCOJoaded and water-drinking animals. 4. Plasma renin activity of both NaCl- and NaHC03-loaded animals was less than that of control rats. However, renal renin content was suppressed by NaCl but not by NaHC03 loading. 5. Loading with NaCl afforded greater protection against HgC1,-induced acute renal failure than NaHC03. Since this difference was not related to changes in sodium balance or plasma volume before HgCl,, or plasma renin activity after HgCL, the results support the hypothesis that intrarenal renin plays a role in the pathogenesis of HgClz-induced acute renal failure in the rat.
Introduction
The renin-angiotensin system may be involved in the pathogenesis of HgClJnduced acute renal failure in the rat (McDonald, Thiel, Wilson, DiBona & Oken, 1969; DiBona, McDonald, Flamenbaum, Dammin & Oken, 1971). Thus plasma renin activity is acutely elevated after HgCl, (DiBona & Sawin, 1971). Chronic saline loading prevents the elevation of plasma renin activity and protects against renal insufficiency (DiBona et al., 1971). However, neither active renin immunization nor acute renin inhibition by administration of deoxycorticosterone and NaCl solution prevents acute renal failure after HgCL (Flamenbaum,Kotchen & Oken, 1972; Flamenbaum, McNeil, Kotchen, Lowenthal & NagIe, 1973a). Although peripheral renin is either blocked by antirenin antibody or suppressed by these two experimental manoeuvres, the content of renin within the kidney is not decreased. Consequently, the relevant factor may be intrarenal rather than circulating renin. Alternatively, renin may not be involved and the protective effect of chronic NaCl loading may reflect plasma volume expansion rather than renin suppression (Matthews, Morgan & Johnson, 1974). W e have recently demonstrated that NaHC03, unlike NaCl, fails to suppress both plasma renin activity and renal renin content in the rat, despite comparable positive sodium balance after loading with both sodium salts
Correspondence: Dr R. G. Luke, University of Kentucky Medical Center, Department of Medicine, Lexington, Kentucky 40506, U.S.A.
149
150
J . E. Beaumont et al.
(Kotchen, Galla & Luke, 1976). To determine whether protection against HgCIz-induced acute renal failure is related to positive sodium balance and volume expansion rather than to a specific effect on intrarenal renin, the effects of NaCl or NaHC03 loading on renal function after HgClz were compared in the rat. Methods Twenty-four Sprague-Dawley rats were divided into three groups of eight rats. All rats were placed in individual metabolic cages and allowed a 3 days period of adjustment before start of the study. All animals were given ground Purina rat chow, containing 182 pmol of sodium, 307 pmol of potassium and 190 pmol of chloridelg of diet, determined by analysis after nitric acid extraction. In group A, deionized drinking water was replaced with NaCl solution. Group B was given isotonic NaHCO, solution to drink; group C received deionized water. Animals were pair-fed; i.e. the fluid and dietary intakes of group B and group C were determined by the previous daily intake of group A. Urine was collected under mineral oil and preserved by thymol. The study period for groups A, B and C extended from day 1 to day 11. Rats were weighed on day 1 and tail blood was obtained for measurement of serum urea; packed cell volume was measured on days 1 and 9, before HgClz was given. The rats were injected with HgCL (4.5 mg/kg body weight) subcutaneously on day 9 to induce acute renal failure, and 48 h later, on the morning of day 12, they were reweighed and killed by decapitation. Blood was collected in chilled tubes with EDTA for the measurement of plasma renin activity; serum was obtained for urea and creatinine concentrations. Immediately thereafter a single kidney was removed from each animal and frozen for measurement of renal renin content. Plasma volume was measured in comparable groups of animals (n = 8lgroup) on similar regimens, for 7 days, with 1251-labelledalbumin (Belcher & Harris, 1957). On day 7, the rats were anaesthetized by an intraperitoneal injection of sodium ethyl 1-methylpropylmalonylthiourea (Inactin, Promonta, Hamburg) (100 mg/kg body weight). An external jugular vein was cannulated with PESO polyethylene tubing for injection of 1251-labelledalbumin. After an
equilibration period (10 min), blood was obtained for radioactivity counting and measurement of packed cell volume by direct aortic puncture. In the calculation of plasma volume, adjustment was made for plasma trapping. Urinary sodium and potassium concentrations were determined with an IL flame photometer. Plasma creatinine concentration was measured by the method of Kennedy, Hilton & Berliner (1952), and serum urea by the method of Crocker (1967). Urinary chloride was measured with a Buchler chloridometer (Buchler Instrument Division, Nuclear-Chicago Corp., Fort Lee, NJ, U.S.A.). Plasma renin activity was measured in quadruplicate by the radioimmunoassay method of Haber, Koerner, Page, Kliman & Purnode (1969). To maintain constant pH during the incubation (3 h), phosphate buffer, pH 7.4, was added (100 pl/ml) to each sample. To determine if HgCI2 affects the measurement of plasma renin activity, the latter was measured in portions of plasma from each of six rats after addition of HgCl2 (70 pglml). This concentration of HgClz was calculated on the assumption that the total dose of subcutaneous HgClz administered to induce renal failure was present in extracellular fluid. Mean plasma renin activity in aliquots of plasma to which HgClz was added (4.4 pmol h- ml- l , SEM 0.6) did not differ from that in portions not containing HgClz (4.5 pmol h-' d-l,SEM 0.6) and it was concluded that measurement of plasma renin activity is not altered by HgClZin the concentrations used. Renal renin was extracted by ammonium sulphate precipitation and measured as previously described (Flamenbaum et al., 1972). Briefly, a sample of the kidney extract was incubated with excess of sheep renin substrate, and the concentration of angiotensin I generated after incubation for 15 min at 37°C and pH 7.4 was measured by radioimmunoassay. One unit of renal renin is arbitrarily defined as that concentration of renin that will generate 100 ng of angiotensin I during the incubation. When results were compared for the three groups of animals, and when the variances for the three groups were similar, the significance of group differences was computed with analysis of variance (Snedecor & Cochran, 1967). If group variances were dissimilar, then statistical significance for three group comparisons was
Bicarbonate in acute renal failure determined by the Kruskal-Wallis Rank test (Bradley, 1968). Comparisons between two groups were made with an unpaired t-test. P 0.7) in
TABLE 1. Weight, net weight gain during the 12 days of study, andpacked cell volume andserum urea before HgC12 in NaCI-loaded, NaHC03-loaded and water-drinking control rats Mean valuesf SEM are shown. There are no significant differences between groups A, B and C. n = 8 for each group. Packed cell volume Group
Weight (g) (Day 1)
Net weight gain (9)
330f7 33524 325+4
21+12
A (NaCl) B (NaHC03) C (water)
9+6
12+4
Day 1
Day 9
46k0.9 48k1.1 46k0.9
48f0.7 49+05
Urea (mmol/l) (Day 1) 14k0.6 16k0.8 18+ 1-7
5020.6
TAELE 2. Total electrolyte excretion and urinary volumes in NaCI-loaded, NaHC03-loaded and water-drinking rats Mean values+seM are shown. n = 8 for each group. Urinary electrolytes (mmol)
Group Na+
(1) Days 1-9 (before HgC12) 62+ 4* A (NaCl) B (NaHC03) 61 f 3* C (water) 21j-1
K+ 30+ 1 29+ 1 28+2
Urinary volume (ml)
c1235+23* 206f 12 164+ 9
75f 5t 23+ I 25k2
(2) Days 10 and 11 (after HgC12) A (NaCl) B (NaHC03) C (water)
14+ 32 6+ 1 1.5k0.1
5+ 0.7t
3f0.4 2+0*1
14f3T 0.8+0.1 0.7k0.1
* Different from group C (P 0.6). During the 48 h period after injection of HgCI, (Table 2, part 2), sodium excretion in 2 days of group A and group B remained significantly more than that of group C. Chloride excretion was significantly more in group A than in group B and group C. Potassium excretion was similar in group A and group B, but in group A it was significantly greater than in group C. During the first 24 h after HgCI2, urinary volume of all three groups did not differ. However, between 24 and 48 h, urinary volumes
of NaHC03-loaded and water-drinking control rats were less than those of NaC1-loaded animals (Table 2, part 2). This accounts for the greater potassium excretion in the latter group. Low urinary volumes in NaHCOt-loaded animals occurred despite a significantly greater fluid intake than control animals (31 ml, SEM 4, versus 12 ml, SEM 2; P
