Acute Na+,K+-ATPase Inhibition With Bufalin Impairs Pressure Natriuresis in the Rat
Abstract Although it has been reported that Na+,K+-ATPase inhibition with bufalin induces acute and chronic hypertension in the rat, the mechanisms mediating this response are unclear. To examine the role of the kidney in this process, glomerular filtration rate, renal blood flow, and pressure natriuresis were determined in rats treated with bufalin or vehicle during changes in renal perfusion pressure. Mean arterial pressure increased from 123±4 to 149±3 mm Hg (P<.05) after 40 minutes of intravenous bufalin and remained at this level. In control rats, glomerular filtration rate was well autoregulated. In bufalin-treated rats, glomerular filtration rate fell with decreasing renal perfusion pressure. Glomerular filtration rate autoregulatory index was greater in bufalin-treated than control rats (P<.05). Renal blood flow showed a similar pattern. Urine flow and sodium excretion were less in bufalin-treated than control rats at equivalent renal perfusion pressures. The slope of the line describing the relation between urine flow and renal perfusion pressure was greater (P<.05) in control than bufalin-treated rats. Similarly, the slope of the line relating sodium excretion to renal perfusion pressure was greater (P<.05) in control than bufalin-treated rats. Thus, acute increases in blood pressure during Na+,K+-ATPase inhibition are associated with impaired renal autoregulation and pressure natriuresis. This effect may be important in chronic hypertension associated with Na+,K+-ATPase inhibition in the rat.
Inhibition of Na+,K+-ATPase with either bufalin or ouabain has been demonstrated to induce hypertension in the dog and rat.1 2 3 4 The effect of these compounds on blood pressure regulation has been of considerable interest because of experimental data suggesting that endogenous inhibitors of the sodium-potassium pump may play a role in the sustained rise in blood pressure in human essential hypertension.5 6 7 Although bufalin and ouabain have been shown to induce vasoconstriction, increase cardiac contractility, and increase the responsiveness to vasoactive compounds, their effects on kidney function have not been systematically examined.1 2 3 8 9 10 The data that are available are conflicting. Bufalin, the agent most frequently examined because it induces both acute and sustained increases in blood pressure, has been reported to acutely reduce RBF in dogs.10 11 Effects on urine flow and sodium excretion are reported to be unchanged or increased in the rat and unchanged in the dog during infusion.2 3 10 11 GFR has only been reported in sheep and is unchanged during intrarenal bufalin infusion.12 These observations seem to conflict with a central hypothesis of long-term blood pressure control that states that hypertension induced by increases in cardiac output or total peripheral resistance cannot be sustained without impairment in renal excretory capacity. As clearly elucidated by Guyton et al13 and Hall et al,14 15 increases in arterial pressure normally elevate renal sodium excretion above intake until arterial pressure returns to its original set point. Thus, increases in blood pressure that are not associated with reductions in renal excretory capacity should not lead to chronic hypertension. The present study was designed to test the hypothesis that the increase in blood pressure induced by the acute inhibition of the sodium-potassium pump with bufalin is associated with a rightward shift in the pressure-natriuresis relation.
Male Sprague-Dawley rats weighing 230 to 350 g were maintained on tap water and standard rodent chow after arrival in our animal facility. All animals were housed according to institutional guidelines, and the studies were approved by the Jackson Veterans Affairs Medical Center Animal Care and Use Committee. On the day of study, rats were anesthetized with injections of 5, sec-butyl-5-ethyl-2-thiobarbituric acid (Inactin, Promonta) in a dose of 80 mg/kg IP. Animals were placed on a thermostatically controlled animal table, and body temperatures were maintained at 37°C with a servo-activated controller (Vestavia Scientific Co). After tracheostomy, polyethylene catheters (PE-50) were placed in the right carotid and femoral arteries to allow for continuous measurement of arterial pressure (Micro-Med Inc) above and below the renal arteries. Catheters were placed in the right and left jugular vein for intravenous infusions. A flanged PE-50 polyethylene catheter was placed in the bladder for urine collection. The aorta was exposed through a midline abdominal incision, and ultramicro clamps (Vestavia Scientific Co) were placed above and below the renal arteries. Ligatures were then loosely placed around the superior mesenteric and celiac arteries. To compensate for the reduction in plasma volume attendant on abdominal surgery, rats received a 1.2 mL/100 g body wt infusion of 5% albumin during the surgical procedure. Additionally, all rats received an infusion of 154 mmol/L sodium chloride containing 1% bovine serum albumin at a rate of 100 μL/min through one of the jugular venous catheters. An infusion of [125I]iothalamate (Glofil, Isotex Inc) in 154 mmol/L sodium chloride was infused at a rate of 1.2 mL/h for measurement of GFR. Two groups of rats were examined (n=8 per group). The first group received an infusion of 10% ethanol, 90% 154 mmol/L sodium chloride containing 200 μg/mL bufalin (Sigma Chemical Co) at a rate of 50 μL/min (10 μg/min) IV for 40 minutes. This was then reduced to a rate of 12.5 μL/min (2.5 μg/min) and continued throughout the experiment. This protocol resulted in an increase in systemic blood pressure to approximately 149 mm Hg, which was sustained throughout the study without evidence of cardiac arrhythmia. The control group received the vehicle for bufalin (10% ethanol in 90% 154 mmol/L sodium chloride) throughout the experiment.
Forty minutes after completion of surgery, RPP was adjusted to approximately 100 mm Hg by tightening of the aortic clamp above the renal arteries. After a 15-minute equilibration period, urine was collected over a 40-minute experimental period. A blood sample (400 μL) was obtained at the midpoint of the collection period. The plasma was removed and the red cells were suspended in a small amount (400 μL) of 154 mmol/L sodium chloride and returned to the animal. In the control group, the aortic clamp was then released, allowing RPP to return to its baseline values (approximately 125 mm Hg). After a 15-minute equilibration period, urine and blood samples were collected over a 40-minute experimental period as described above. RPP was then increased approximately 25 mm Hg above baseline value by tightening of the aortic clamp below the renal arteries and the ligatures on the celiac and superior mesenteric arteries. Urine and blood were again collected as previously described. The clamps were then released and a final ambient blood pressure was determined. The bufalin-infused group was studied in a similar manner at RPPs of 100, 125, 150, and 180 mm Hg. To ensure that the order of collection did not influence the findings, the order of pressure adjustment was varied in some rats from each group.
After the final blood pressure determination, the animal was exsanguinated while still under anesthesia and the kidneys were removed and weighed.
In two additional sets of rats (n=6 per group), RBF was determined with 131I-hippuran (Isotex Inc) with the use of an identical protocol.
Urine flow rate was determined by change in weight of preweighed vials. Plasma and urine samples were evaluated for [125I]iothalamate and 131I-hippuran activity on a gamma counter (Packard Instrument Inc). Sodium concentration in urine and plasma samples was determined with the use of a flame photometer (model 943, Instrumentation Laboratories).
Analysis of Data
Determination of the activity of [125I]iothalamate, activity of 131I-hippuran, sodium concentration in plasma and urine, and urine flow rate permitted calculation of GFR, RBF, and urinary sodium excretion rate according to standard expressions.16 RBF was calculated from hippuran clearance, renal hippuran extraction, and hematocrit as described previously. Urinary excretion data, RBF, and GFR were all expressed per gram kidney weight.
To examine the effects of bufalin on the autoregulation of GFR and RBF, the autoregulatory index was calculated according to the method of Sample and de Wardener as previously described from our laboratory.16 17
All values are expressed as mean±SE. Statistical significance between values at different RPPs within a single group of animals was determined with the use of ANOVA for repeated measures followed by Tukey test. Statistical significance between groups was determined with the use of the unpaired Student’s t test.
MAP at the conclusion of the surgical procedure and before infusion of bufalin or its vehicle was similar in both groups. MAP increased to 149±3 mm Hg (P<.05) after infusion of bufalin for 40 minutes. MAP was sustained at this level throughout the experiment in the bufalin group.
Renal function at each group’s ambient RPP is shown in the Table⇓. There were no differences in GFR, urinary flow rate, and absolute or fractional urinary sodium excretion between vehicle- and bufalin-treated rats at their ambient RPPs. RBF was lower (P<.05) in bufalin-treated rats than control rats when measured at their ambient RPPs.
In the control group, GFR was unaltered during changes in RPP (Fig 1⇓). GFR decreased with decreasing RPP in the bufalin group. The GFR fell to 411±109 μL/min per gram kidney weight at 100 mm Hg, a value significantly lower than that observed at 125 mm Hg (Fig 1⇓). The absolute differences in GFR between the control and bufalin groups reached statistical significance only at an RPP of 100 mm Hg. The GFR autoregulatory index between 150 and 100 mm Hg was significantly greater (P<.05) in bufalin-treated rats than in control rats (Fig 2⇓).
RBF was unaltered during changes in RPP in control rats (Fig 3⇓). RBF decreased during decreases in RPP between 180 and 150 mm Hg and between 125 and 100 mm Hg in bufalin-treated rats. RBF was significantly less in bufalin-treated rats than control rats at 150 and 100 mm Hg. The RBF autoregulatory index between 150 and 100 mm Hg was less (P<.05) in control rats than bufalin-treated rats.
Absolute urinary sodium excretion also increased with increasing RPP in both groups (Fig 4⇓). Absolute urinary sodium excretion was significantly lower (P<.05) in bufalin-treated rats at an RPP of 125 and 150 mm Hg than control rats. The slope of the line relating sodium excretion to RPP over the range of pressures for which comparable data were available (100 to 150 mm Hg) was significantly greater (P<.05) in the control group (252.8±56.6 μmol/min per millimeter of mercury) compared with the bufalin group (72.4±21.8 μmol/min per millimeter of mercury). Fractional urinary sodium excretion demonstrated a similar pattern (data not shown).
Urinary flow rate increased as RPP increased in both groups (data not shown). Urinary flow rate was significantly less (P<.05) in the bufalin-treated group compared with the control group at 100, 125, and 150 mm Hg. The slope of the line relating urinary flow rate to RPP over the range of pressures for which comparable data were available (100 to 150 mm Hg) was significantly greater (P<.05) in the control group (1.26±0.27 μL/min per millimeter of mercury) than in the bufalin-treated group (0.35±0.09 μL/min per millimeter of mercury).
The present study demonstrates that acute inhibition of Na+,K+-ATPase results in significant disruption of renal hemodynamics and impairment in pressure natriuresis.
Bufalin was chosen to inhibit Na+,K+-ATPase in the present study because Na+,K+-ATPase inhibition with this compound induces both acute and chronic hypertension in the rat.3 4 In the rat, in vitro studies have demonstrated that bufalin is a more potent inhibitor of myocardial Na+,K+-ATPase than ouabain.18 19 This may in part account for the ability of bufalin to induce both acute and chronic increases in blood pressure whereas ouabain induces increases in blood pressure only when given chronically. Although studies with ouabain-induced hypertension may be more relevant for examining the potential role of Na+,K+-ATPase inhibition in the pathogenesis of hypertension in humans,5 the renal effects of chronic Na+,K+-ATPase inhibition are difficult to separate from effects due to structural renal damage induced by exposure of the kidney to prolonged elevations in RPP. Thus, acute studies are more appropriate to determine the potential role of the kidney in hypertension induced by inhibition of Na+,K+-ATPase.
The present study clearly demonstrates that acute inhibition of Na+,K+-ATPase has adverse effects on renal function, which could contribute to an increase in arterial pressure. Bufalin not only impaired autoregulation of both RBF and GFR but also decreased RBF and GFR. The autoregulatory index for GFR and RBF increased from slightly >0 in normal animals to >1 after inhibition of Na+,K+-ATPase with bufalin (P<.05). When autoregulatory indexes are expressed in this manner, a value of 0 reflects perfect autoregulation whereas a level >1 reflects the absence of autoregulation in a system in which vascular resistance is fixed. These observations in vivo are consistent with in vitro findings that inhibition of Na+,K+-ATPase in isolated vessels or vascular rings produces dose-dependent vasoconstriction and inhibition of the responses to vasodilators.2 20 21 Our studies are also in agreement with previous work by Tamaki and associates22 demonstrating that acute intrarenal ouabain administration impairs autoregulation of RBF and GFR in the dog.
Acute inhibition of Na+,K+-ATPase also blunted the relationship between RPP and urinary fluid or sodium excretion. Alterations in pressure natriuresis or diuresis could be due to increases in preglomerular resistance either because of intrinsic changes in afferent arteriolar tone or because of resetting of the macula densa feedback mechanism.14 15 Alternatively, Na+,K+-ATPase inhibition could reduce the glomerular capillary filtration coefficient (Kf) or have direct effects on increasing tubular reabsorption. Which of these factors accounts for the alterations in pressure natriuresis remains to be determined.
Whether the alterations in RBF and GFR and the changes in the pressure-natriuresis relationship are due to direct effects of Na+,K+-ATPase inhibition on the renal vasculature and tubular transport or result from responses to systemic Na+,K+-ATPase inhibition remains to be determined. Inhibition of Na+,K+-ATPase with ouabain has been proposed to increase blood pressure through excitation of the sympathetic nervous system, whereas bufalin has been postulated to potentiate vasoconstrictor responses to norepinephrine.2 10 23 On the other hand, inhibition of Na+,K+-ATPase with bufalin has been shown to cause concentration-dependent contraction of human arterial rings and produces renal vasoconstriction after direct infusion into the renal artery.10 21 Thus, the alterations in renal function observed after Na+,K+-ATPase inhibition in the present study could occur through either direct or indirect mechanisms.
Bufalin-induced hypertension has been examined previously by others. Pamnani and associates3 reported that bolus intravenous administration of bufalin induced dose-dependent increases in blood pressure, heart rate, and cardiac contractility. Eliades and associates2 confirmed these observations during intravenous bufalin infusion and further demonstrated that bufalin infused into the canine forelimb induced vasoconstriction, potentiated the vasoconstrictor responses to norepinephrine, and reduced the vasodilator response to potassium chloride. None of the previous studies rigorously examined renal function. RBF reportedly has been reduced during Na+,K+-ATPase inhibition with bufalin in the dog, which is in agreement with our present observations in the rat. Urinary sodium and water excretion have been noted to be unchanged from control values during bufalin infusion in most previous studies.2 3 10 18 This lack of an increase in urinary fluid or sodium excretion despite a large increase in MAP is suggestive evidence that normal pressure-natriuretic and diuretic responses were impaired during Na+,K+-ATPase inhibition with bufalin in these reports. Thus, the findings from these previous studies would be consistent with our observations as well.
In summary, the present study demonstrates that inhibition of Na+,K+-ATPase with bufalin results in significant increases in renal vascular resistance and impairment of the regulation of RBF and GFR and markedly reduces pressure natriuresis and diuresis in the rat. These events may contribute to the sustained increases in blood pressure observed during chronic Na+,K+-ATPase inhibition in the rat.
Selected Abbreviations and Acronyms
|GFR||=||glomerular filtration rate|
|MAP||=||mean arterial pressure|
|RBF||=||renal blood flow|
|RPP||=||renal perfusion pressure|
This study was supported by research funds from the Department of Veterans Affairs.
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