Vanadate causes hypokalemic E. DAFNIS, Department
M. SPOHN,
of Internal
B. LONIS,
Medicine,
distal renal tubular N. A. KURTZMAN,
Texas Tech University
Dafnis, E., M. Spohn, B. Lonis, N. A. Kurtzman, and S. Sabatini. Vanadate causes hypokalemic distal renal tubular acidosis. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F449-F453, 1992.-Considerable evidence supports the presence of an H+-K+-ATPase along the mammalian nephron. Inhibition of this enzyme might be expected to reduce acid excretion while increasing potassium excretion, thus causing hypokalemic distal renal tubular acidosis (RTA). In this study we administered vanadate at a dose of 5 mg/kg ip for 16 days to rats. These animals developed hypokalemic distal RTA with a blood pH of 7.22 t 0.01, a plasma bicarbonate of 15.2 t 0.6 meq/l, and a plasma potassium of 3.28 t 0.06 meq/l. The vanadate-treated animals had a urine pH of 6.70 t 0.09, a value significantly higher than NH&l-treated animals with the same degree of acidemia (urine pH = 5.25 & 0.04). When cortical collecting tubules (CCT) from these animals were microdissected and H+-K+-ATPase was measured, it was decreased by -75% (P < 0.001); but H+-ATPase was no different from control. In medullary collecting tubule, H+-K’-ATPase was also decreased but less than in CCT. Muscle potassium concentration in the vanadate-treated animals was significantly lower than in controls. These results demonstrate that vanadate causes hypokalemic distal RTA in association with inhibition of collecting tubule H+-K+-ATPase activity. proton-potassium-adenosinetriphosphatase; proton-adenosinetriphosphatase; sodium-potassium-adenosinetriphosphatase; tissue vanadium; rat nephron microdissection
Health
AND
acidosis
S. SABATINI
Sciences Center, Lubbock,
Texas 79430
several hundred patients, but may affect as many as people (16; and S. Nimmannit, personal communication). In the present study vanadate was administered on a chronic basis to rats, and after 10 days measurements of renal function were made. CCT and MCT were microdissected, and activity of the H+-K+-ATPase was measured radiochemically. Na+-K+-ATPase and H+-ATPase activities were also measured. The results show that H+K+-ATPase activity was decreased in both the CCT and MCT. Not surprisingly, Na+-K+-ATPase activities in these same segments were also decreased. In contrast to these results, H+-ATPase activity in the two collecting tubule segments dissected from the vanadate-treated animals was no different from controls. NH&l-treated animals did not exhibit these same changes. These results show that chronic vanadate administration results in distal RTA associated with hypokalemia and a decrease in collecting tubule H+-K+-ATPase activity. These data suggest that H+-K+-ATPase activity is important in distal urinary acidification. 450,000
METHODS
Vanadate (5 mg/kg ip) was administered to male SpragueDawley rats for 10 days. The animals were placed in metabolic IN THE LAST DECADE considerable evidence has been cages for the last 24 h, and on the morning of the 11th day they were anesthetized with Nembutal (50 rng- kg-l. body wt-l ip), published concerning the role of a nonelectrogenic proton after which blood and urine samples were collected for measadenosinetriphosphatase (ATPase) in gastric mucosa. urement of pH, Pco~, Na+, K+, Cl-, and renal function as This proton pump, an H+-K+-ATPase, results in gastric previously described (24). Urine anion gap was calculated as acid secretion and is inhibitable by vanadate, omepradescribed by Batlle et al. (4). Samples of blood, urine, liver, zole, and Sch 28080 (see Ref. 25 for review). Recently, muscle, and kidney cortex and medulla were analyzed for this enzyme has been found in the mammalian kidney, vanadium concentration using electrothermal atomic absorpwith the highest concentrations noted in the cortical tion spectroscopy with the aid of a model 30/40 Varian speccollecting tubule (CCT) (9,11,12,19). The renal enzyme trometer (13). Muscle potassium concentration was measured by atomic absorption (flame). A group of sham-injected controls is stimulated by dietary potassium deprivation of -l-wk duration and is inhibited by hyperkalemia (9-12). In was similarly studied. An additional group of rats were placed on 1.5% NH&l by studies of the perfused outer medullary collecting tubule (MCT) from the potassium-depleted rabbit, Wingo (29) gavage (1 ml. 100 g body wt-’ *day-l followed by ad libitum intake) for 3-4 days as we have previously described (23). In demonstrated a decrease in acidification after the in vitro administration of omeprazole. Parallel with this decrease these animals blood and urine samples were collected for measurement of pH, Pco~, Na’, K+, Cl-, and creatinine as well as was a fall in potassium absorption. More recently, Cheval for calculation of the urine anion gap at the time of renal artery et al. (7) showed Sch 28080-inhibitable potassium reab- cannulation. sorption (as measured by rubidium uptake) in collecting The left renal artery was then cannulated, and the kidney tubule segments (CCT and MCT) from normal rats. was perfused for 15 min in situ at a rate of 0.7 ml/min with a These data suggest that the renal H+-K+-ATPase en- balanced salt solution containing 400 U/ml collagenase at 4OC, pH 7.4 (23). The kidneys were then cut along the corticopapilzyme may be important in overall acid-base and potascontainsium regulation in both collecting tubule segments; thus lary axis and incubated in 3 ml of collagenase-albumin ing Hanks’ solution at 35°C for 12 min. The tissues were its inhibition should result in hypokalemic metabolic acidosis. The acidification defect would resemble “clas- continuously bubbled with compressed air (3 psi). After incumicrosic” hypokalemic distal renal tubule acidosis (RTA). An bation, the pyramids were rinsed and immediately dissected as previously described (23). The tubule segments outbreak of hypokalemic distal RTA in northeastern were identified (CCT and MCT), and tubule length was measThailand, which has been tentatively ascribed to natuured. To remove most of the extracellular potassium, nephron rally occurring contamination of water, possibly with segments were incubated for 15 min at 37°C in potassium-free vanadate, underscores the potential importance of the buffer as described by Doucet and Marsy (9). They were then H+-K+-ATPase (16). This disorder has been studied in subjected to a two-step hypotonic-hypothermic shock, and 0363-6127/92 $2.00 Copyright 0 1992 the American Physiological Society F449 Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (136.186.001.082) on November 26, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.
F450
VANADATE
AND DISTAL
ATPase activities were determinedusing [y-32P]ATP (37”C, 15 min, pH 7.4). Na+-K+-ATPase and H+-ATPase activities were measuredaspreviously described(23). H+-K+-ATPase activity was measuredfirst in a sodium-freebuffer containing (in mM) 150 tris(hydroxymethyl)aminomethane hydrochloride, 10 MgCl*, 1 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’tetraacetic acid, 2 ouabain, 2 N-ethylmaleimide (NEM), 2 NaN3, and 4 KCl, aswell as20 pg/ml oligomycin. High specific activity [T-~~P]ATP (2-10 Ci/mmol) was added in tracer amounts (10 nCi/pl) to vanadium-free ATP (final concentration, 12mM). For determination of the H’-K+-ATPase, activity wasthen measuredin a solution containing zero potassiumand 200PM Sch 28080,a concentration greater than two- to threefold that neededto inhibit the enzyme under the above conditions. H+-K+-ATPase activity is defined as the difference in activity found in the presenceand absenceof potassium and Sch 28080. Enzyme activity is expressedas picomoles per millimeter tubule length per hour of ATP hydrolyzed. All sampleswere run in triplicate or quadruplicate, and appropriate corrections were madefor blanks and the spontaneoushydrolysis of ATP (23) Sodium orthovanadate was obtained from Aldrich Chemical (Milwaukee, WI) and was diluted as previously describedjust before administration (3). Milli-Q water was obtained from Millipore (San Francisco, CA) and was usedin the preparation of all the solutions.RadiolabeledATP was obtained from New England Nuclear (Boston, MA). All other chemicals and reagents were purchasedfrom Sigma Chemical (St. Louis, MO) and were of highest purity. Statistical significance was assessed using the Student’s t test or analysisof covariance, where appropriate, with P < 0.05 being significant. RESULTS
The effect of 10 days of chronic vanadate administration on blood and urine parameters in rats is shown in Table 1. Also included in Table 1 are data on some blood and urine parameters in a separate group of animals given 1.5% NH&l per OS for 3-4 days (by gavage). Both experimental groups of animals developed hyperchloremic metabolic acidosis with blood bicarbonate concen-
RENAL
TUBULE
trations in the range of 15-16 meq/l. The vanadatetreated group developed hypokalemia with a plasma potassium of 3.28 t 0.06 meq/l, a value significantly lower than either the sham-injected controls (4.43 t 0.06 meq/ 1) or the NH&l-treated animals (4.81 t 0.23 meq/l). Glomerular filtration rate and serum creatinine in the vanadate-treated group were no different from control. Urine pH in the vanadate-treated animals was slightly higher than controls, but was inappropriately high for animals with metabolic acidosis. As has been reported in the literature and as shown in the NH&l-treated animals (Table l), rats with this degree of metabolic acidosis that have intact distal acidification elaborate a urine pH of
M. SPOHN,
of Internal
B. LONIS,
Medicine,
distal renal tubular N. A. KURTZMAN,
Texas Tech University
Dafnis, E., M. Spohn, B. Lonis, N. A. Kurtzman, and S. Sabatini. Vanadate causes hypokalemic distal renal tubular acidosis. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F449-F453, 1992.-Considerable evidence supports the presence of an H+-K+-ATPase along the mammalian nephron. Inhibition of this enzyme might be expected to reduce acid excretion while increasing potassium excretion, thus causing hypokalemic distal renal tubular acidosis (RTA). In this study we administered vanadate at a dose of 5 mg/kg ip for 16 days to rats. These animals developed hypokalemic distal RTA with a blood pH of 7.22 t 0.01, a plasma bicarbonate of 15.2 t 0.6 meq/l, and a plasma potassium of 3.28 t 0.06 meq/l. The vanadate-treated animals had a urine pH of 6.70 t 0.09, a value significantly higher than NH&l-treated animals with the same degree of acidemia (urine pH = 5.25 & 0.04). When cortical collecting tubules (CCT) from these animals were microdissected and H+-K+-ATPase was measured, it was decreased by -75% (P < 0.001); but H+-ATPase was no different from control. In medullary collecting tubule, H+-K’-ATPase was also decreased but less than in CCT. Muscle potassium concentration in the vanadate-treated animals was significantly lower than in controls. These results demonstrate that vanadate causes hypokalemic distal RTA in association with inhibition of collecting tubule H+-K+-ATPase activity. proton-potassium-adenosinetriphosphatase; proton-adenosinetriphosphatase; sodium-potassium-adenosinetriphosphatase; tissue vanadium; rat nephron microdissection
Health
AND
acidosis
S. SABATINI
Sciences Center, Lubbock,
Texas 79430
several hundred patients, but may affect as many as people (16; and S. Nimmannit, personal communication). In the present study vanadate was administered on a chronic basis to rats, and after 10 days measurements of renal function were made. CCT and MCT were microdissected, and activity of the H+-K+-ATPase was measured radiochemically. Na+-K+-ATPase and H+-ATPase activities were also measured. The results show that H+K+-ATPase activity was decreased in both the CCT and MCT. Not surprisingly, Na+-K+-ATPase activities in these same segments were also decreased. In contrast to these results, H+-ATPase activity in the two collecting tubule segments dissected from the vanadate-treated animals was no different from controls. NH&l-treated animals did not exhibit these same changes. These results show that chronic vanadate administration results in distal RTA associated with hypokalemia and a decrease in collecting tubule H+-K+-ATPase activity. These data suggest that H+-K+-ATPase activity is important in distal urinary acidification. 450,000
METHODS
Vanadate (5 mg/kg ip) was administered to male SpragueDawley rats for 10 days. The animals were placed in metabolic IN THE LAST DECADE considerable evidence has been cages for the last 24 h, and on the morning of the 11th day they were anesthetized with Nembutal (50 rng- kg-l. body wt-l ip), published concerning the role of a nonelectrogenic proton after which blood and urine samples were collected for measadenosinetriphosphatase (ATPase) in gastric mucosa. urement of pH, Pco~, Na+, K+, Cl-, and renal function as This proton pump, an H+-K+-ATPase, results in gastric previously described (24). Urine anion gap was calculated as acid secretion and is inhibitable by vanadate, omepradescribed by Batlle et al. (4). Samples of blood, urine, liver, zole, and Sch 28080 (see Ref. 25 for review). Recently, muscle, and kidney cortex and medulla were analyzed for this enzyme has been found in the mammalian kidney, vanadium concentration using electrothermal atomic absorpwith the highest concentrations noted in the cortical tion spectroscopy with the aid of a model 30/40 Varian speccollecting tubule (CCT) (9,11,12,19). The renal enzyme trometer (13). Muscle potassium concentration was measured by atomic absorption (flame). A group of sham-injected controls is stimulated by dietary potassium deprivation of -l-wk duration and is inhibited by hyperkalemia (9-12). In was similarly studied. An additional group of rats were placed on 1.5% NH&l by studies of the perfused outer medullary collecting tubule (MCT) from the potassium-depleted rabbit, Wingo (29) gavage (1 ml. 100 g body wt-’ *day-l followed by ad libitum intake) for 3-4 days as we have previously described (23). In demonstrated a decrease in acidification after the in vitro administration of omeprazole. Parallel with this decrease these animals blood and urine samples were collected for measurement of pH, Pco~, Na’, K+, Cl-, and creatinine as well as was a fall in potassium absorption. More recently, Cheval for calculation of the urine anion gap at the time of renal artery et al. (7) showed Sch 28080-inhibitable potassium reab- cannulation. sorption (as measured by rubidium uptake) in collecting The left renal artery was then cannulated, and the kidney tubule segments (CCT and MCT) from normal rats. was perfused for 15 min in situ at a rate of 0.7 ml/min with a These data suggest that the renal H+-K+-ATPase en- balanced salt solution containing 400 U/ml collagenase at 4OC, pH 7.4 (23). The kidneys were then cut along the corticopapilzyme may be important in overall acid-base and potascontainsium regulation in both collecting tubule segments; thus lary axis and incubated in 3 ml of collagenase-albumin ing Hanks’ solution at 35°C for 12 min. The tissues were its inhibition should result in hypokalemic metabolic acidosis. The acidification defect would resemble “clas- continuously bubbled with compressed air (3 psi). After incumicrosic” hypokalemic distal renal tubule acidosis (RTA). An bation, the pyramids were rinsed and immediately dissected as previously described (23). The tubule segments outbreak of hypokalemic distal RTA in northeastern were identified (CCT and MCT), and tubule length was measThailand, which has been tentatively ascribed to natuured. To remove most of the extracellular potassium, nephron rally occurring contamination of water, possibly with segments were incubated for 15 min at 37°C in potassium-free vanadate, underscores the potential importance of the buffer as described by Doucet and Marsy (9). They were then H+-K+-ATPase (16). This disorder has been studied in subjected to a two-step hypotonic-hypothermic shock, and 0363-6127/92 $2.00 Copyright 0 1992 the American Physiological Society F449 Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (136.186.001.082) on November 26, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.
F450
VANADATE
AND DISTAL
ATPase activities were determinedusing [y-32P]ATP (37”C, 15 min, pH 7.4). Na+-K+-ATPase and H+-ATPase activities were measuredaspreviously described(23). H+-K+-ATPase activity was measuredfirst in a sodium-freebuffer containing (in mM) 150 tris(hydroxymethyl)aminomethane hydrochloride, 10 MgCl*, 1 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’tetraacetic acid, 2 ouabain, 2 N-ethylmaleimide (NEM), 2 NaN3, and 4 KCl, aswell as20 pg/ml oligomycin. High specific activity [T-~~P]ATP (2-10 Ci/mmol) was added in tracer amounts (10 nCi/pl) to vanadium-free ATP (final concentration, 12mM). For determination of the H’-K+-ATPase, activity wasthen measuredin a solution containing zero potassiumand 200PM Sch 28080,a concentration greater than two- to threefold that neededto inhibit the enzyme under the above conditions. H+-K+-ATPase activity is defined as the difference in activity found in the presenceand absenceof potassium and Sch 28080. Enzyme activity is expressedas picomoles per millimeter tubule length per hour of ATP hydrolyzed. All sampleswere run in triplicate or quadruplicate, and appropriate corrections were madefor blanks and the spontaneoushydrolysis of ATP (23) Sodium orthovanadate was obtained from Aldrich Chemical (Milwaukee, WI) and was diluted as previously describedjust before administration (3). Milli-Q water was obtained from Millipore (San Francisco, CA) and was usedin the preparation of all the solutions.RadiolabeledATP was obtained from New England Nuclear (Boston, MA). All other chemicals and reagents were purchasedfrom Sigma Chemical (St. Louis, MO) and were of highest purity. Statistical significance was assessed using the Student’s t test or analysisof covariance, where appropriate, with P < 0.05 being significant. RESULTS
The effect of 10 days of chronic vanadate administration on blood and urine parameters in rats is shown in Table 1. Also included in Table 1 are data on some blood and urine parameters in a separate group of animals given 1.5% NH&l per OS for 3-4 days (by gavage). Both experimental groups of animals developed hyperchloremic metabolic acidosis with blood bicarbonate concen-
RENAL
TUBULE
trations in the range of 15-16 meq/l. The vanadatetreated group developed hypokalemia with a plasma potassium of 3.28 t 0.06 meq/l, a value significantly lower than either the sham-injected controls (4.43 t 0.06 meq/ 1) or the NH&l-treated animals (4.81 t 0.23 meq/l). Glomerular filtration rate and serum creatinine in the vanadate-treated group were no different from control. Urine pH in the vanadate-treated animals was slightly higher than controls, but was inappropriately high for animals with metabolic acidosis. As has been reported in the literature and as shown in the NH&l-treated animals (Table l), rats with this degree of metabolic acidosis that have intact distal acidification elaborate a urine pH of
