Chronic intrarenal hyperinsulinemia does not cause hypertension JOHN E. HALL, MICHAEL W. BRANDS, H. LELAND CARLO A. GAILLARD, AND DREW A. HILDEBRANDT Department of Physiology and Biophysics, Jackson, Mississippi 39216-4505
University
HALL, JOHN E., MICHAEL W. BRANDS, H. LELAND MIZELLE, CARLO A. GAILLARD, AND DREW A. HILDEBRANDT. Chronic intrarenal hyperinsulinemia does not cause hypertension. Am.
J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F663F669, 1991.-Hyperinsulinemia has been postulated to link obesity and hypertension via the antinatriuretic actions of insulin. The main goal of this study was to quantitate the importance of the direct intrarenal actions of insulin, independent of systemiceffects, in altering blood pressureand renal function. This wasaccomplishedby determining the responses to chronic intrarenal insulin infusion in uninephrectomized, chronically instrumented consciousdogs maintained on a 74 meq/day sodiumintake. Insulin wasinfused at rates calculated to raiseintrarenal, but not systemic,insulin to levels similar to those observedin obesehypertensive dogs. Intrarenal insulin infusion (0.6 mU kg-‘. min-‘) for 7 days causedtransient decreasesin sodiumexcretion but no significant changesin potassiumexcretion. Mean arterial pressuredid not changeduring 7 days of insulin infusion, averaging 93 & 4 mmHg during control and 93 t 3 mmHg during insulin infusion. Intrarenal insulin caused small increases in GFR but no significant changesin effective renal plasmaflow or renal vascular resistance. These results demonstrate that insulin causestransient decreasesin sodiumexcretion, but chronic intrarenal hyperinsulinemia does not elevate blood pressure in normal dogs. Additional factors other than the direct sodium-retaining effects of insulin may be important in raising blood pressurein obesity-associatedhypertension. sodiumexcretion; obesity; glomerular filtration rate; kidney
epidemiologicaland experimental evidence that excessive weight gain elevates blood pressure (3, 7, 10, 20, 26, 27, 28), the mechanisms responsible for obesity-associated hypertension are still unclear. Recently, there has been interest in the possibility that hyperinsulinemia in obese individuals may mediate hypertension via an antinatriuretic effect on the kidney (7, 23, 27). Acute studies have demonstrated that insulin reduces sodium excretion in experimental animals and in humans (1, 7, 18, 21), as well as in completely isolated kidneys (22), indicating a direct action of insulin on renal sodium handling. Although the exact mechanisms of insulin-mediated antinatriuresis have not been fully elucidated, an increase in sodium reabsorption with no change in glomerular filtration rate (GFR) has been noted during acute hyperinsulinemia (7). Recent micropuncture studies suggest that insulin ALTHOUGHTHEREISCONSIDERABLE
0363-6127/91$1.50
Copyright
MIZELLE,
of Mississippi
Medical
Center,
may increase sodium reabsorption mainly at a site beyond the proximal convoluted tubule but before the distal convoluted tubule, suggesting enhanced reabsorption in the loop of Henle (18). However, insulin may also increase reabsorption in isolated perfused proximal tubules (2) I’f insulin exerted a sustained antinatriuretic effect this could increase extracellular fluid volume and initiate a sequence of events leading to chronic hypertension (7). As blood pressure increased, the antinatriuretic effects of insulin would be overridden by pressure natriuresis, similar to the escape from sodium retention observed in other models of hypertension caused by antinatriuretic hormones such as aldosterone (15). Although the antinatriuretic actions of insulin offer a plausible explanation for the association between obesity and hypertension, there have been no previous studies, to our knowledge, that have determined whether insulin, at physiological or pathophysiological levels, is capable of causing a sustained antinatriuretic action sufficient to elevate blood pressure. We have recently demonstrated that in conscious dogs chronic intravenous insulin infusion, at rates calculated to increase plasma insulin to levels comparable to those found in obese hypertensive dogs, did not elevate blood pressure (14). However, one important difference between normal dogs and obese hypertensive humans is that dogs are sensitive to the effects of insulin on tissue glucose uptake, whereas obese hypertensive dogs are resistant to insulin’s metabolic actions. Therefore, systemic administration of insulin in dogs would be expected to increase tissue glucose utilization and metabolic rate, causing peripheral vasodilation and possibly a tendency to reduce blood pressure. Indeed, our previous studies in dogs indicate that chronic hyperinsulinemia is associated with marked reductions in total peripheral vascular resistance, increased cardiac output, and reduced blood pressure (5). However, this hypotensive action of insulin would not be expected in insulin-resistant individuals. Therefore, the failure of systemically administered insulin to raise blood pressure could be due, in part, to offsetting effects of peripheral vasodilation, which tend to reduce blood pressure, and the. antinatriuretic actions of insulin, which tend to elevate blood pressure. The goal of the present study was to determine whether the direct renal actions of insulin are capable of causing sustained increases in arterial pressure in conscious dogs.
0 1991 the American
Physiological
Society
F663
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F664
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HYPERINSULINEMIA
Insulin was infused intrarenally at rates calculated to increase renal arterial, but not systemic, insulin concentrations to values comparable to those found in obese hypertensive dogs. Presumably, if the direct renal actions of insulin can cause chronic hypertension, then raising intrarenal insulin levels should elevate blood pressure in the absence of marked increases in systemic insulin concentrations. In addition, our studies were also designed to quantitate the long-term direct effects of insulin on control of renal hemodynamics and electrolyte excretion. METHODS
Experiments were conducted in conditioned conscious mongrel dogs weighing 20.9-27.3 kg (average 22.6 t 0.8 kg). All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and were carried out according to the “Guide for the Care and Use of Laboratory Animals” from the National Institutes of Health and according to the guidelines of the Animal Welfare Act. Surgical procedures were performed under pentobarbital sodium anesthesia and aseptic conditions. Tygon (Norton Plastics, Akron, OH) catheters were implanted in the femoral arteries and veins for measurements of arterial pressure and blood sampling. A small Tygon catheter was placed in the renal artery of one kidney using a modification of the technique developed by Herd and Barger (17), and the contralateral kidney was removed through a retroperitoneal flank incision. All catheters were tunneled subcutaneously, exteriorized in the scapular region for protection, and filled with a heparin solution (1,000 USP U/ml). The dogs were permitted to recover from surgery, antibiotics were administered daily, and rectal temperatures were monitored to ensure that the dogs were afebrile throughout the studies. After a l- to 2-wk recovery period, the dogs were placed in individual metabolic cages in a quiet air-conditioned room with a 12-h light-dark cycle and fitted with harnesses containing a pressure transducer (Statham Medical Instruments, Hato Rey, PR) at heart level. The arterial pressure signals from the polygraph recorder (model 7D; Grass Instruments, Quincy, MA) were sent to an analog-digital converter and analyzed with a digital computer (Turbo X-T; PCs Limited, Austin, TX) using software developed in our laboratory. Analog signals from the polygraph were sampled in bursts of 12 s, l/min, 24 h/day, and the digitized data were processed on the computer to determine systolic, diastolic, and mean arterial pressure, as well as heart rate. The average blood pressures and heart rates for each day were then calculated from values recorded over an 18-h period between 1400 and 0800 h. All routine care of the dogs, including feeding and cleaning of cages, as well as studies of renal function and blood sampling, were done between the hours of 0800 and 1400. To infuse the various solutions continuously, one of the femoral venous catheters was connected to a roller infusion pump (model 375A; Sage Instruments, Cambridge, MA) that delivered -435 ml/day of sterile isoto-
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nic saline. The renal arterial catheter was connected to a syringe infusion pump (model 944; Harvard Instruments, South Natick, MA) that delivered 45 ml of sterile water per day. All solutions were pumped through a disposable Millipore filter (0.22 pm Cathivex; Millipore, Bedford, MA) to prevent air bubbles, contaminants, or bacteria from passing into the infusion catheters. The infusion tubing and cables from the pressure transducers were protected by ,a flexible vacuum hose attached to a harness that permitted the dogs to move freely in the cage. The dogs were fed two cans (447 g/can) per day of a sodium-deficient diet (H/D Hills Pet Products, Topeka, KS) that provided ~7 meq sodium and 65 meq potassium per day and were given 5 ml of a vitamin syrup (V. A. L. syrup; Fort Dodge Labs, Fort Dodge, IA). Total sodium intake, including the food and the intravenous infusion of sodium chloride, was held constant at -74 meq/day. Experimental protocol. After the dogs were placed in metabolic cages and the intravenous and intrarenal infusions were started, lo-14 days were allowed for the dogs to achieve sodium balance. During that time, the dogs were trained to lie quietly while blood samples were obtained from the arterial catheters and studies of renal function were performed. After a week of control measurements, an intrarenal infusion of insulin (SquibbNOVO, Princeton, NJ) was started at a rate of 0.6 mU+ kg-’ min-’ for 7 days in the sterile water vehicle. Insulin solutions were made fresh daily and changed between 1130 and 1230 h, after measurements of renal clearance and blood and urine samples were obtained. The rate of insulin infusion was calculated to increase renal arterial concentrations by -65 pU/ml. This calculation is based on measurements of effective renal plasma flow (see below) and the assumption that the extraction of [ 1311]iodohippurate, the indicator used for measurement of effective renal plasma flow, averaged ~0.65. Previous studies in our laboratory have indicated that the renal extraction of [1311]iodohippurate is -0.65 in normal dogs (unpublished observations). After 7 days of insulin infusion, postcontrol measurements were made for an additional 7 days. During the control, experimental, and postcontrol periods, blood pressure and heart rate were measured for 18 h each day and 24-h urine excretion of sodium, potassium, and water, as well as 24-h intake of water were measured daily. Glomerular filtration rate, effective renal plasma flow, plasma electrolytes, plasma glucose concentration, and various hormone measurements were made on days 1 and 4 before starting insulin infusions, on days 1, 3, and 6 of insulin infusion, and on days 1, 3, and 6 after stopping insulin infusion. In two dogs, insulin was infused intrarenally at a lower rate of 0.3 mU kg-‘. min-’ for 7 days while measurements of arterial pressure, water and electrolyte balances, and renal function were performed as described above. Analytical methods. Glomerular filtration rate and effective renal plasma flow were estimated from the total clearances of [ ‘251] Iothalamate (Glofil; Isotex Diagnostics, Friendwood, TX) and [ 1311]iodohippurate (Hippuran; Squibb, Princeton, NJ), respectively, as previl
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ously described (16). Renal vascular resistance was calculated as mean arterial pressure/effective renal blood flow, where effective renal blood flow is effective renal plasma flow/(l.O - hematocrit). Plasma and urine sodium and potassium concentrations were determined by flame photometry (IL443; Instrumentation Labs, Lexington, MA). Plasma protein concentration was measured by refractometry (American Optical, Buffalo, NY), and plasma glucose concentration was determined by the hexokinase method (Sigma Diagnostics, St. Louis, MO). Plasma renin activity was measured by radioimmunoassay using ‘““I-angiotensin I from New England Nuclear (Boston, MA) and antibody from Chemicon (El Segundo, CA). Aldosterone was extracted from plasma with 7 vol of dichloromethane, and the dried extract was reconstituted with phosphate gelatin buffer and measured by radioimmunoassay using ““I-aldosterone from Amersham (Arlington Heights, IL) and liquid phase antibody (Diagnostic Products, Los Angeles, CA). Plasma insulin concentration was measured by radioimmunoassay (Cambridge Medical Diagnostics, Billerica, MA). Statistical analyses. Experimental data were compared with control data by analysis of variance and, when appropriate, with Dunnett’s t test for multiple comparisons (9, 19). Statistical significance was considered to be P < 0.05. All data are expressed as means +- SE unless otherwise indicated.
AND
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TUT 60
URINARY SODIUM EXCRETION WWdaY)
60
40
60
URINARY POTASSIUM EXCRETION
6o -
(mEq’day)
40-
‘I
1000
URINE VOLUME (ml/day) 5w
-4
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6
IO
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RESULTS
Figure 1 shows the effects of 7 days of intrarenal insulin infusion at a rate of 0.6 mu. kg-‘. min-’ on mean arterial pressure in uninephrectomized dogs. Mean arterial pressure did not change significantly, averaging 93 + 4 mmHg during control and 93 & 3 mmHg during 7 days of renal arterial insulin infusion. Urinary sodium excretion decreased significantly on the 2nd and 4th day of insulin infusion, averaging 49.1 + 5.5 and 42.2 Ilt 4.9 meq/day compared with an average control value of 64.5 + 3.9 meq/day (Fig. 2). No significant changes in sodium excretion were noted on the other days of insulin infusion, although there was -4O- to 47-meq increase in cumulative sodium balance after 4-6 days of insulin infusion. When the insulin infusion was stopped, there was a transient increase in sodium excretion on the first postcontrol day. Urine volume also decreased significantly on the 1st and 4th day of insulin infusion, averaging 735 + 67 and 858 + 64 ml/day compared with a
MEAN ARTERIAL PRESSURE CmmHg)
F665
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90
.4
-2
FIG. 1. Effects of intrarenal for 7 days on mean arterial (n = 8).
2TIME;days6)
10
12
14
insulin infusion (0.6 mu. kg-‘. pressure in uninephrectomized
min-‘) dogs
FIG. 2. Effect of intrarenal insulin for 7 days on sodium and potassium uninephrectomized dogs (n = 8).
infusion excretion
(0.6 mU.kg-’ .min-‘) and urine volume in
control value of 1,019 -C76 ml/day. After 7 days of insulin infusion, cumulative water balance increased by 824 + 210 ml. Urinary potassium excretion, however, did not change significantly during intrarenal insulin infusion, averaging 63.8 + 4.2 meq/day during control and 61.5 + 4.0 meq/day during insulin infusion. Glomerular filtration rate (Fig. 3) increased by -lO12% during intrarenal insulin infusion, although the changes were statistically significant only on the 6th day of infusion. There were no significant changes in effective renal plasma flow or renal vascular resistance during 7 days of intrarenal insulin infusion. Intrarenal insulin infusion caused no significant changes in plasma glucose concentration (Table 1). There were small increases in plasma insulin concentration, indicating a slight spillover from the renal arterial infusion into the systemic circulation. However, intrarenal insulin infusion caused no significant changes in heart rate, plasma electrolytes, plasma protein concentration, plasma renin activity, or plasma aldosterone concentration. Table 2 shows the effects of intrarenal insulin infusion at a lower rate of 0.3 mu. kg-‘. min-’ in two dogs. Insulin infusion caused no changes in arterial pressure in either dog; for both dogs, arterial pressure averaged 89 mmHg during control and 89 mmHg during 7 days of insulin infusion. Insulin infusion intrarenally also caused no changes in heart rate, plasma electrolytes, or plasma insulin concentration in either dog. Intrarenal insulin
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1
GLOMERULAR FILTRATION RATE (% Control)
,w 90
EFFECTIVE RENAL PLASMA FLOW fi Control)
I I
II
I
110 100 90
T
RENAL VASCULAR RESISTANCE (% Control)
T
100
~ 3
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I
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TIME (days)
FIG. 3. Effect of intrarenal insulin infusion (0.6 mu. kg-‘.min-‘) for 7 days on glomerular filtration rate, effective renal plasma flow, and renal vascular resistance in uninephrectomized dogs (n = 8).
infusion caused -40 meq net retention of sodium after 7 days in one dog, while causing no change in sodium balance in the second dog. Average urinary sodium excretion did not change markedly during intrarenal infusion of insulin at 0.3 mU - kg-‘. min-‘, although there was a small transient decrease in sodium excretion in one dog. DISCUSSION
The most significant finding of the study is that 7 days of intrarenal insulin infusion, at rates calculated to increase renal arterial concentrations to levels comparable TABLE
1. Effects of intrarenal
insulin
p .
m$
AND BLOOD
PRESSURE
to those found in obese hypertensive dogs, failed to elevate arterial pressure. However, insulin infusion did cause transient retention of sodium and water. The antinatriuretic and antidiuretic actions of insulin did not persist during chronic intrarenal hyperinsulinemia even in the absence of increased blood pressure and offsetting pressure natriuresis. Chronic renal actions of insulin. The mechanisms by which insulin reduces sodium excretion during chronic intrarenal infusion are not entirely clear but appear to be related mainly to increased sodium reabsorption. In the present study, we found no reductions in GFR associated with intrarenal insulin infusion. In fact, there were small but consistent increases in GFR during insulin infusion indicating that the sodium retention was due to increased tubular reabsorption. This observation is consistent with previous acute experiments which have demonstrated that insulin elevates sodium reabsorption (7, 18), although there have been no previous reports on the chronic direct effects of insulin on renal function. Although it was not possible in the present studies to determine the tubular site at which insulin increased renal sodium reabsorption, previous acute studies have suggested that insulin may act at multiple parts of the renal tubule. Baum (2) demonstrated that insulin, when added to the basolateral side of isolated proximal tubules, stimulated fluid reabsorption and caused a more negative transepithelial potential difference. However, an increase in fluid reabsorption occurred after the addition of physiological or pharmacological levels of insulin to an insulin-deficient bath. Therefore, these studies did not differentiate whether minimal levels of insulin are required for normal fluid reabsorption or whether hyperinsulinemia actually stimulates fluid reabsorption above normal in proximal tubules. Recent micropuncture studies by Kirchner (18) demonstrated that euglycemic hyperinsulinemia increased chloride reabsorption in the loop of Henle but did not alter proximal tubular fluid or chloride reabsorption in superficial nephrons. However, these studies did not rule out the possibility that insulin may also increase sodium chloride reabsorption in the pars recta portion of the proximal tubule or in the proximal tubule in juxtamedullary nephrons inaccessible to micropuncture. Thus acute administration of insulin increases sodium reabsorption in the loop of Henle and possibly in the proximal tubule. However, further studies are
infusion (0.6 mU- kg-l. min-l) PK, ma
P pro,ern, g/100 ml
ngANG
PRA, I.&-‘.h-’
Aldo, ng/lOO ml
P ‘“su’r”’ pU/ml
P p,ucore, mg/lOO ml
HR beats/min
Control
(average of days 1 and 4) Insulin Day I 3 6
142.1kO.5
4.2kO.05
140.9kO.7 142.9kO.5
4.04a0.16 4.19+0.10
143.9kO.3
4.34kO.07
6.75rt0.18
0.42+0.11
6.68kO.21
0.79+0.21 0.67+0.19
6.62?0.26 6.89-cO.32
0.40+0.07
3.9kO.7
3.7kO.9 2.6kO.4 3.2kO.8
7.8kO.5
9425
86+3
20.5+2.1* 22.6&2.2* l&7+2.6*
8926 86klO &3+3
8754 89+3 93t4
Postcontrol 6.9kkO.21 0.4&0.22 4.3kO.9 12.5t2.6 9&9 90t5 (average of days 1, 3, 141.7eo.4 4.33kO.14 and 6) plasma protein; PRA, plasma renin activity; Aldo, Values are means + SE; n = 8 dogs. P Nn,plasma sodium; Pk, plasma potassium; P PlOtPin, plasma aldosterone; Pinsuiin,plasma insulin; P piurore,plasma glucose; HR, heart rate. * P < 0.05 compared with control. Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.
INTRARENAL TABLE
HYPERINSULINEMIA
AND BLOOD
F667
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2. Effects of in&arena1 insulin infusion (0.3 mUb kg-‘. min-‘) MAP, mmHg
Control (average, 5 days) Insulin Day
HR beats/min
medday
UN&x
v, ml/day
Pinsulin,
d/ml
ng ANG
PRA, I ml-’ l
89-+10
85Ik4
69.6t5.7
1,270*270
9.9t6.6
0.4420.21
89k9 86k7 86&6 88t6 92t8 92-c9 943-10
86t8 86t7 82t8 81k6 82k6 81t6 83k5
78.9t22.3 61.025.0 52.1k4.0 72.4t13.1 72.5k1.2 60.9t1.9 83.7t14.1
1,230+470 1,140f420 970t290 1,205+335 1,190*270 1,260f380 1,310t450
9.4k2.2
0.35t0.21
10.9t6.4
0.3620.23
9.6t0.7
0.4320.38
- h-’
Postcontrol (average, 6 days) 90533 81,t6 72.7HO.3 1,215+373 9.5H.4 0.33kO.08 Values are means t SE; n = 2. MAP, mean arterial pressure; H R, heart rate; U Na V, urinary sodium excretion; V, urine volume; Pimulin,plasma insulin concentration; PRA, plasma renin activity.
needed to determine the exact tubular sites at which chronic hyperinsulinemia influences sodium reabsorption. The mechanisms responsible for the rise in GFR during hyperinsulinemia are not clear. If insulin stimulated sodium reabsorption in the proximal tubule or in the loop of Henle, there would be a reduction in sodium chloride delivery to the macula densa which, in turn, could initiate a secondary vasdilation of afferent arterioles and a rise in GFR (30). Although a macula densa feedback offers a plausible explanation for the increased GFR observed during insulin infusion, insulin may also have a direct renal vasodilator action. Cohen et al. (6) reported that insulin caused vasodilation in isolated kidneys perfused with hyperoncotic albumin to prevent glomerular filtration, indicating that insulin can cause renal vasodilation via a mechanism that is independent of filtration. Therefore, insulin may have multiple actions that tend to increase GFR. Intrarenal infusion of insulin in the present study caused no significant changes in potassium excretion. However, previous studies in our laboratory have demonstrated that intravenous insulin infusion caused marked reductions in potassium excretion lasting for several days. Thus the reduced potassium excretion observed during intravenous insulin infusion is probably due to systemic rather than direct intrarenal effects of insulin. One important effect of systemic hyperinsulinemia is increased cellular uptake of potassium (8, 29), which would tend to cause hypokalemia and a decrease in potassium secretion by the renal tubules. This explanation fits with the observation that intravenous infusion of insulin caused significant hypokalemia (13), whereas intrarenal insulin infusion in the present study caused no significant changes in plasma potassium concentration. Although we observed no major changes in renal potassium handling during renal arterial infusion of insulin, we cannot rule out the possibility that insulin could have transient direct effects on potassium excretion that might not be detected in the 24-h measurement. Insulin could also exert opposite effects on potassium handling in different nephron segments. For example, reduced sodium chloride delivery to the distal tubule, caused by
insulin-mediated effects on proximal or loop sodium reabsorption, would tend to decrease potassium excretion, whereas a stimulatory effect of insulin on sodiumpotassium exchange in the collecting tubule would tend to increase potassium excretion. The net effect could be little or no change in overall potassium excretion. The results in the present study do not allow us to determine the contributions of the multiple effects of insulin on renal potassium handling. However, our results do indicate that the direct actions of insulin on the kidney cause little or no change in overall potassium excretion. Intrarenal actions of insulin and long-term blood pressure regulation. Hyperinsulinemia has been suggested to
link obesity and hypertension, in part, via an antinatriuretic action on the kidney (7). As discussed above, insulin causes sodium and water retention via direct renal actions, and blood pressure is correlated with plasma insulin concentration in obese subjects. If the antinatriuretic action of insulin was sustained, this could lead to chronic hypertension by expansion of extracellular fluid volume. According to this concept, an initial retention of sodium would raise extracellular fluid volume and cardiac output, leading to a rise in blood pressure (7). The antinatriuretic effect of insulin would eventually be offset by pressure natriuresis, and sodium balance would be reestablished. However, the maintenance of sodium balance would occur at the expense of elevated blood pressure. Although the hypothesis that the antinatriuretic actions of insulin may contribute to the hypertension associated with obesity has received considerable attention, most of the supporting evidence is still indirect. Only the acute direct renal effects of insulin have been previously reported, and it is not certain whether the sodium-retaining action of insulin is powerful enough, or can be sustained long enough, to cause chronic hypertension. Insulin infusion has been observed to cause sodium retention in obese subjects, despite the presence of resistance to the effects of insulin on carbohydrate metabolism (27). Furthermore, the sodium retention was comparable to that found in nonobese insulin-sensitive subjects (27). These studies indicate that obese subjects retain renal sensitivity to the antinatriureti c actions of insulin and are consistent with the concept that hyper-
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insulinemia may contribute to sodium retention and hypertension in these individuals (7,27). Additional support of the concept that insulin may be a significant factor in obesity-associated hypertension is the observation that there is a correlation between body weight and blood pressure, as well as between insulin and blood pressure in obese subjects (3, 10, 20, 26). Furthermore, when obese individuals are placed on a low-calorie diet, plasma insulin levels fall in parallel with blood pressure, although the fall in blood pressure may occur with or without a negative sodium balance (25). Thus a direct cause and effect relationship between the antinatriuretic actions of insulin and blood pressure in obese subjects has not been demonstrated. In previous studies, we demonstrated that chronic intravenous infusion of insulin, for as long as 28 days at rates that raised plasma insulin to values comparable to that found in obese hypertensive dogs, did not elevate blood pressure in normal dogs, dogs with increased adrenergic activity due to chronic infusion of norepinephrine, angiotensin II-hypertensive dogs, dogs maintained on a high-sodium intake, and dogs in which renal excretory capability was reduced by surgically removing 70% of the kidney mass (13, 14). In fact, chronic insulin administration in some of these studies caused small but significant reductions in blood pressure (13, 14). Recent studies in our laboratory demonstrated that the fall in blood pressure associated with 7 days of insulin infusion was due to decreased total peripheral vascular resistance, since cardiac output was markedly increased by insulin infusion (5). One explanation for the peripheral vasodilation is that systemic administration of insulin increased tissue glucose utilization and metabolic rate, which would tend to cause vasodilation. In normal insulin-sensitive dogs, peripheral vasodilation could, over a period of several days, tend to offset a hypertensive effect of insulin caused by antinatriuresis. However, in obese subjects that are resistant to the metabolic effects of insulin, peripheral vasodilation would not be expected. For this reason, the present studies were designed to examine the long-term direct actions of insulin on the kidney, while preventing large increases in systemic concentrations of insulin that could cause peripheral vasodilation. Yet, even under these conditions, we failed to observe an increase in arterial pressure during chronic insulin infusion at rates that were calculated to raise renal arterial concentrations to levels at least as high as those measured in obese hypertensive dogs. Although insulin did cause mild sodium retention, this effect did not appear to be powerful enough or sustained long enough to cause hypertension. In fact, after 24-48 h of insulin administration there was only mild sodium retention. In previous studies we found much more marked sodium retention during intravenous insulin infusion (5, 13, 14). However, part of the sodium retention observed in our previous studies with intravenous infusion may have been secondary to the fall in blood pressure caused by peripheral vasodilation. The results from the present study indicate that the direct renal actions of insulin, at physiological or pathophysiological levels, are capable of causing only mild and transient sodium
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retention that is insufficient to elevate blood pressure in normal dogs. Although the results in the present study provide no support for the hypothesis that the direct renal actions of insulin cause hypertension, our results do not completely rule out the possibility that hyperinsulinemia might exacerbate the renal actions of other hypertensive stimuli. In previous studies, we found no evidence that hyperinsulinemia potentiated the renal sodium-retaining actions of norepinephrine or angiotensin II, or that hyperinsulinemia elevated blood pressure when adrenergic activity and angiotensin II levels were chronically elevated (13,14). However, insulin has been shown in acute studies to amplify the action of aldosterone on sodium and potassium transport in cultured kidney cells (11). Unfortunately, the relevance of this observation to the long-term antinatriuretic actions of insulin in vivo is still unclear. Also, insulin may have other actions that alter renal function in a manner that could elevate blood pressure. For example, insulin has been shown to stimulate vascular smooth muscle growth (10). If this effect was maintained and occurred in the renal arterioles, this could lead to a slow insidious development of hypertension, as occurs with other disturbances that gradually raise preglomerular vascular resistance (12). In addition, insulin-mediated abnormalities of lipid metabolism (24) could also, over a long period of time, have a damaging effect on the kidney that might lead to hypertension. We can only speculate on the possible importance of these renal actions of insulin. However, the results of the present study indicate that the functional effects of insulin to cause sodium and water retention do not appear to be capable of causing hypertension over a period of 7 days. Our results in the dog do not rule out the possibility that insulin’s direct renal actions might cause hypertension in other species. We have recently reported in preliminary studies that 7 days of euglycemic hyperinsulinemia in rats caused mild hypertension (4). However, because insulin was infused intravenously, we could not determine if the hypertension was due to the direct renal actions of insulin or to some systemic effect which, in turn, influenced kidney function and blood pressure. Also, it is not clear which animal species most closely mimics the human response to hyperinsulinemia. Although the acute effects of insulin have been widely studied, there have been few reports on the blood pressure or renal changes caused by chronic hyperinsulinemia. Recently, Tsutsu et al. (31) measured blood pressure and fasting insulin and glucose levels in patients with insulinoma who had symptoms for an average of 19 mo. No evidence of hypertension was found in these patients despite very high levels of insulin, and no change in blood pressure was observed after resection of the insulinoma. These observations suggest that hyperinsulinemia per se may not cause hypertension in humans, similar to our findings in dogs. However, additional studies are needed to examine potential interactions between insulin and other hypertensive stimuli in the long-term regulation of blood pressure and renal function.
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INTRARENAL
HYPERINSULINEMIA
We thank Nancy Kimmel and Ivadelle Heidke for excellent secretarial assistance and William Dixon and Beth Miller for expert technical assistance. We are also indebted to Dr. Manis J. Smith, Jr., for the radioimmunoassays and to John Fleming, Robert Seaton, and Dr. Jean-Pierre Montani for computer software used in these experiments. This work was supported by National Heart, Lung, and Blood Institute Grants HL-39399, HL-11678, and HL-23502. M. W. Brands and D. A. Hildebrandt were supported by National Institutes of Health Research Service Awards HL-08171 and HL-08931. C, A. Gaillard was supported by a postdoctoral fellowship from the American Heart Association, Mississippi Affiliate. Address for reprint requests: J. E. Hall, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505. Received 30 July 1990; accepted in final form 4 January 1991. REFERENCES 1. ATCHLEY, S. W., R. F. LOEB, D. W. RICHARDS, E. M. BENEDICT, AND M. E. DRISCOLL. On diabetic acidosis. J. Clin. Invest. 12: 297326,1933. 2. BAUM, M. Insulin stimulates transport in rabbit proximal convoluted tubule. J. C&n. Invest. 79: 1104-1109, 1987. 3. BERGLUND, G., S. LJUNGMAN, M. HARTFORD, L. WILHELMSEN, AND P. BJORNTORP. Type of obesity and blood pressure. Hypertension Dallas 4: 692-696,1982. 4. BRANDS, M. W., J. E. HALL, D. A. HJLDEBRANDT, AND H. L. MIZELLE. Hypertension during chronic hyperinsulinemia in conscious rats (Abstract). FASEB J. 4: A817, 1990. 5. BRANDS, M. W., H. L. MIZELLE, C. A. GAILLARD, D. A. HILDEBRANDT, AND J. E. HALL. The hemodynamic response to chronic hyperinsulinemia (Abstract). Am. J. Hypertens. 3: 19A, 1990. 6. COHEN, A. J., D. M. MCCARTHY, AND J. S. STOFF. Direct hemodynamic effect of insulin in the isolated perfused kidney. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F580-F585,1989. 7. DEFRONZO, R. A. The effects of insulin on renal sodium metabolism. Diabetologia 21: 165-171, 1981. 8. DEFRONZO, R., P. FELIG, E. FERRANNINI, AND J. WAHREN. Effect
of graded doses of insulin on splanchnic and peripheral potassium metabolism in man. Am. J. Physiol. 238 (Endocrinol. Metab. 1): E421-E427,1980. 9. DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics 20: 482-491, 1964. 10. FERRARI, P., AND P. WEIDMANN. Insulin, insulin sensitivity and hypertension. J. Hypertens. 8: 491-500, 1990. 11. FIDELMAN, M. L., AND C. 0. WATLINGTON. Insulin and aldoster-
one interaction in Na’ and K+ transport in cultured kidney cells (AG). Endocrinology 115: 1171-1178, 1989. 12. GUYTON, A. C. Arterial Pressure and Hypertension. Philadelphia, PA: Saunders, 1980. 13. HALL, J. E., M. W. BRANDS, S. D. KIVLIGHN, H. L. MIZELLE, D. A. HILDEBRANDT, AND C. A. GAILLARD. Chronic hyperinsulinemia and blood pressure; interaction with catecholamines? Hypertension Dallas
15: 519-527,199O.
PRESSURE
F669
J. E., T. G. COLEMAN, JR. Chronic hyperinsulinemia
H. L. MIZELLE, AND M. J. SMITH, and blood pressure regulation. Am. Electrolyte Physiol. 27): F722-F731,
AND BLOOD 14. HALL,
J. Physiol.
258 (Renal
Fluid
1990. J. E., J. P. GRANGER, R. L. HESTER, AND J.-P. MONTANI. Mechanisms of sodium balance in hypertension: role of pressure natriuresis. J. Hypertens. 4, SuppZ. 4: S57-S65, 1986. 16. HALL, J. E., A. C. GUYTON, AND B. M. FARR. A single-injection technique for measuring glomerular filtration rate. Am. J. Physiol.
15. HALL,
232 (Renal 17. HERD, J.
Fluid
Electrolyte
Physiol.
1): F72-F76,
1977.
A., AND A. C. BARGER. Simplified technique for chronic catheterization of blood vessels. J. Appl. Physiol. 19: 791-794,1965. 18. KIRCHNER, K. A. Insulin increases loop segment chloride reabsorption in the euglycemic rat. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F1206-F1213, 1988. Design: Procedures for the Behavioral 19. KIRK, R. E. Experimental Sciences. Belmont, CA: Wadsworth, 1968, p. 69-98. 20. LUCAS, C. P., J. A. ESTIGARRIBIA, L. L. DARGA, AND G. M. REAVEN. Insulin and blood pressure. Hypertension Dallas 7: 702706,1985. 21. MILLER, J. H., AND M. D. BODGONOFF. Antidiuresis associated with administration of insulin. J. Appl. Physiol. 6: 509-512, 1954. 22. NIZET, A., P. LEFEBORE, AND J. CRABBE. Control by insulin of sodium, potassium, and water by the isolated dog kidney. Pfluegers Arch. 323: 11-20, 1971. 23. REAVEN, G. M. Role of insulin resistance in human disease. Diabetes 37: 1595-1607, 1988. 24. REAVAN, G. M., AND B. B. HOFFMAN. Hypertension as a disease of carbohydrate and lipoprotein metabolism. Am. J. Med. 87, Suppl. 6A: 2%6S, 1989. 25. REISEN, E., R. ABEL, M. MODAN, D. S. SILVERBERG, H. E. ELIAHOU, AND B. MODAN. Effect of weight loss without salt restriction
on the reduction of blood pressure in overweight hypertensive patients. N. Engl. J. Med. 298: l-6 1978. 26. ROBINSON, S. C., M. BRUCER, AND J. Moss. Hypertension and obesity. J. Lab. Clin. Med. 25: 807-822, 1939. 27. ROCCHINI, A. P., U. KATCH, D. KVESELIS, C. MOOREHEAD, M. MARTIN, R. LAMPMAN, AND M. GREGORY. Insulin and renal sodium retention in obese adolescents. Hypertension Dallas 14: 367-374, 1989. 28. ROCCHINI, A. P., C. P. MOOREHEAD, S. DEREMER, AND D. BONDIE. Pathogenesis of weight-related changes in blood pressure in dogs. Hypertension Dallas 13: 922-928, 1989. 29. ROSSETTJ, L., G. KLEIN-R• BBENHAAR, G. GIEBISCH, D. SMITH, AND R. DEFRONZO. Effect of insulin on potassium metabolism. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F60-F64, 1987. 30. SCHNERMANN, J., AND J. BRIGGS. Function of the juxtaglomerular
apparatus: local control of glomerular dynamics. In: The Kidney: Physiology and Pathophysiology, edited by D. W. Seldin and G. Giebisch. New York: Raven, 1985. p. 669-697. 31. TSUTU, N., K. NUNOI, T. KODAMA, R. NOMIYAMA, M. IWASE, AND M. FUJISHIMA. Lack of association between blood pressure and insulin in patients with insulinemia. J. Hypertens. 8: 479-482, I*WV”. wn
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.
University
HALL, JOHN E., MICHAEL W. BRANDS, H. LELAND MIZELLE, CARLO A. GAILLARD, AND DREW A. HILDEBRANDT. Chronic intrarenal hyperinsulinemia does not cause hypertension. Am.
J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F663F669, 1991.-Hyperinsulinemia has been postulated to link obesity and hypertension via the antinatriuretic actions of insulin. The main goal of this study was to quantitate the importance of the direct intrarenal actions of insulin, independent of systemiceffects, in altering blood pressureand renal function. This wasaccomplishedby determining the responses to chronic intrarenal insulin infusion in uninephrectomized, chronically instrumented consciousdogs maintained on a 74 meq/day sodiumintake. Insulin wasinfused at rates calculated to raiseintrarenal, but not systemic,insulin to levels similar to those observedin obesehypertensive dogs. Intrarenal insulin infusion (0.6 mU kg-‘. min-‘) for 7 days causedtransient decreasesin sodiumexcretion but no significant changesin potassiumexcretion. Mean arterial pressuredid not changeduring 7 days of insulin infusion, averaging 93 & 4 mmHg during control and 93 t 3 mmHg during insulin infusion. Intrarenal insulin caused small increases in GFR but no significant changesin effective renal plasmaflow or renal vascular resistance. These results demonstrate that insulin causestransient decreasesin sodiumexcretion, but chronic intrarenal hyperinsulinemia does not elevate blood pressure in normal dogs. Additional factors other than the direct sodium-retaining effects of insulin may be important in raising blood pressurein obesity-associatedhypertension. sodiumexcretion; obesity; glomerular filtration rate; kidney
epidemiologicaland experimental evidence that excessive weight gain elevates blood pressure (3, 7, 10, 20, 26, 27, 28), the mechanisms responsible for obesity-associated hypertension are still unclear. Recently, there has been interest in the possibility that hyperinsulinemia in obese individuals may mediate hypertension via an antinatriuretic effect on the kidney (7, 23, 27). Acute studies have demonstrated that insulin reduces sodium excretion in experimental animals and in humans (1, 7, 18, 21), as well as in completely isolated kidneys (22), indicating a direct action of insulin on renal sodium handling. Although the exact mechanisms of insulin-mediated antinatriuresis have not been fully elucidated, an increase in sodium reabsorption with no change in glomerular filtration rate (GFR) has been noted during acute hyperinsulinemia (7). Recent micropuncture studies suggest that insulin ALTHOUGHTHEREISCONSIDERABLE
0363-6127/91$1.50
Copyright
MIZELLE,
of Mississippi
Medical
Center,
may increase sodium reabsorption mainly at a site beyond the proximal convoluted tubule but before the distal convoluted tubule, suggesting enhanced reabsorption in the loop of Henle (18). However, insulin may also increase reabsorption in isolated perfused proximal tubules (2) I’f insulin exerted a sustained antinatriuretic effect this could increase extracellular fluid volume and initiate a sequence of events leading to chronic hypertension (7). As blood pressure increased, the antinatriuretic effects of insulin would be overridden by pressure natriuresis, similar to the escape from sodium retention observed in other models of hypertension caused by antinatriuretic hormones such as aldosterone (15). Although the antinatriuretic actions of insulin offer a plausible explanation for the association between obesity and hypertension, there have been no previous studies, to our knowledge, that have determined whether insulin, at physiological or pathophysiological levels, is capable of causing a sustained antinatriuretic action sufficient to elevate blood pressure. We have recently demonstrated that in conscious dogs chronic intravenous insulin infusion, at rates calculated to increase plasma insulin to levels comparable to those found in obese hypertensive dogs, did not elevate blood pressure (14). However, one important difference between normal dogs and obese hypertensive humans is that dogs are sensitive to the effects of insulin on tissue glucose uptake, whereas obese hypertensive dogs are resistant to insulin’s metabolic actions. Therefore, systemic administration of insulin in dogs would be expected to increase tissue glucose utilization and metabolic rate, causing peripheral vasodilation and possibly a tendency to reduce blood pressure. Indeed, our previous studies in dogs indicate that chronic hyperinsulinemia is associated with marked reductions in total peripheral vascular resistance, increased cardiac output, and reduced blood pressure (5). However, this hypotensive action of insulin would not be expected in insulin-resistant individuals. Therefore, the failure of systemically administered insulin to raise blood pressure could be due, in part, to offsetting effects of peripheral vasodilation, which tend to reduce blood pressure, and the. antinatriuretic actions of insulin, which tend to elevate blood pressure. The goal of the present study was to determine whether the direct renal actions of insulin are capable of causing sustained increases in arterial pressure in conscious dogs.
0 1991 the American
Physiological
Society
F663
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.
F664
INTRARENAL
HYPERINSULINEMIA
Insulin was infused intrarenally at rates calculated to increase renal arterial, but not systemic, insulin concentrations to values comparable to those found in obese hypertensive dogs. Presumably, if the direct renal actions of insulin can cause chronic hypertension, then raising intrarenal insulin levels should elevate blood pressure in the absence of marked increases in systemic insulin concentrations. In addition, our studies were also designed to quantitate the long-term direct effects of insulin on control of renal hemodynamics and electrolyte excretion. METHODS
Experiments were conducted in conditioned conscious mongrel dogs weighing 20.9-27.3 kg (average 22.6 t 0.8 kg). All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and were carried out according to the “Guide for the Care and Use of Laboratory Animals” from the National Institutes of Health and according to the guidelines of the Animal Welfare Act. Surgical procedures were performed under pentobarbital sodium anesthesia and aseptic conditions. Tygon (Norton Plastics, Akron, OH) catheters were implanted in the femoral arteries and veins for measurements of arterial pressure and blood sampling. A small Tygon catheter was placed in the renal artery of one kidney using a modification of the technique developed by Herd and Barger (17), and the contralateral kidney was removed through a retroperitoneal flank incision. All catheters were tunneled subcutaneously, exteriorized in the scapular region for protection, and filled with a heparin solution (1,000 USP U/ml). The dogs were permitted to recover from surgery, antibiotics were administered daily, and rectal temperatures were monitored to ensure that the dogs were afebrile throughout the studies. After a l- to 2-wk recovery period, the dogs were placed in individual metabolic cages in a quiet air-conditioned room with a 12-h light-dark cycle and fitted with harnesses containing a pressure transducer (Statham Medical Instruments, Hato Rey, PR) at heart level. The arterial pressure signals from the polygraph recorder (model 7D; Grass Instruments, Quincy, MA) were sent to an analog-digital converter and analyzed with a digital computer (Turbo X-T; PCs Limited, Austin, TX) using software developed in our laboratory. Analog signals from the polygraph were sampled in bursts of 12 s, l/min, 24 h/day, and the digitized data were processed on the computer to determine systolic, diastolic, and mean arterial pressure, as well as heart rate. The average blood pressures and heart rates for each day were then calculated from values recorded over an 18-h period between 1400 and 0800 h. All routine care of the dogs, including feeding and cleaning of cages, as well as studies of renal function and blood sampling, were done between the hours of 0800 and 1400. To infuse the various solutions continuously, one of the femoral venous catheters was connected to a roller infusion pump (model 375A; Sage Instruments, Cambridge, MA) that delivered -435 ml/day of sterile isoto-
AND
BLOOD
PRESSURE
nic saline. The renal arterial catheter was connected to a syringe infusion pump (model 944; Harvard Instruments, South Natick, MA) that delivered 45 ml of sterile water per day. All solutions were pumped through a disposable Millipore filter (0.22 pm Cathivex; Millipore, Bedford, MA) to prevent air bubbles, contaminants, or bacteria from passing into the infusion catheters. The infusion tubing and cables from the pressure transducers were protected by ,a flexible vacuum hose attached to a harness that permitted the dogs to move freely in the cage. The dogs were fed two cans (447 g/can) per day of a sodium-deficient diet (H/D Hills Pet Products, Topeka, KS) that provided ~7 meq sodium and 65 meq potassium per day and were given 5 ml of a vitamin syrup (V. A. L. syrup; Fort Dodge Labs, Fort Dodge, IA). Total sodium intake, including the food and the intravenous infusion of sodium chloride, was held constant at -74 meq/day. Experimental protocol. After the dogs were placed in metabolic cages and the intravenous and intrarenal infusions were started, lo-14 days were allowed for the dogs to achieve sodium balance. During that time, the dogs were trained to lie quietly while blood samples were obtained from the arterial catheters and studies of renal function were performed. After a week of control measurements, an intrarenal infusion of insulin (SquibbNOVO, Princeton, NJ) was started at a rate of 0.6 mU+ kg-’ min-’ for 7 days in the sterile water vehicle. Insulin solutions were made fresh daily and changed between 1130 and 1230 h, after measurements of renal clearance and blood and urine samples were obtained. The rate of insulin infusion was calculated to increase renal arterial concentrations by -65 pU/ml. This calculation is based on measurements of effective renal plasma flow (see below) and the assumption that the extraction of [ 1311]iodohippurate, the indicator used for measurement of effective renal plasma flow, averaged ~0.65. Previous studies in our laboratory have indicated that the renal extraction of [1311]iodohippurate is -0.65 in normal dogs (unpublished observations). After 7 days of insulin infusion, postcontrol measurements were made for an additional 7 days. During the control, experimental, and postcontrol periods, blood pressure and heart rate were measured for 18 h each day and 24-h urine excretion of sodium, potassium, and water, as well as 24-h intake of water were measured daily. Glomerular filtration rate, effective renal plasma flow, plasma electrolytes, plasma glucose concentration, and various hormone measurements were made on days 1 and 4 before starting insulin infusions, on days 1, 3, and 6 of insulin infusion, and on days 1, 3, and 6 after stopping insulin infusion. In two dogs, insulin was infused intrarenally at a lower rate of 0.3 mU kg-‘. min-’ for 7 days while measurements of arterial pressure, water and electrolyte balances, and renal function were performed as described above. Analytical methods. Glomerular filtration rate and effective renal plasma flow were estimated from the total clearances of [ ‘251] Iothalamate (Glofil; Isotex Diagnostics, Friendwood, TX) and [ 1311]iodohippurate (Hippuran; Squibb, Princeton, NJ), respectively, as previl
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INTRARENAL
HYPERINSULINEMIA
ously described (16). Renal vascular resistance was calculated as mean arterial pressure/effective renal blood flow, where effective renal blood flow is effective renal plasma flow/(l.O - hematocrit). Plasma and urine sodium and potassium concentrations were determined by flame photometry (IL443; Instrumentation Labs, Lexington, MA). Plasma protein concentration was measured by refractometry (American Optical, Buffalo, NY), and plasma glucose concentration was determined by the hexokinase method (Sigma Diagnostics, St. Louis, MO). Plasma renin activity was measured by radioimmunoassay using ‘““I-angiotensin I from New England Nuclear (Boston, MA) and antibody from Chemicon (El Segundo, CA). Aldosterone was extracted from plasma with 7 vol of dichloromethane, and the dried extract was reconstituted with phosphate gelatin buffer and measured by radioimmunoassay using ““I-aldosterone from Amersham (Arlington Heights, IL) and liquid phase antibody (Diagnostic Products, Los Angeles, CA). Plasma insulin concentration was measured by radioimmunoassay (Cambridge Medical Diagnostics, Billerica, MA). Statistical analyses. Experimental data were compared with control data by analysis of variance and, when appropriate, with Dunnett’s t test for multiple comparisons (9, 19). Statistical significance was considered to be P < 0.05. All data are expressed as means +- SE unless otherwise indicated.
AND
BLOOD
r
TUT 60
URINARY SODIUM EXCRETION WWdaY)
60
40
60
URINARY POTASSIUM EXCRETION
6o -
(mEq’day)
40-
‘I
1000
URINE VOLUME (ml/day) 5w
-4
-2
0
2
4
6
6
IO
12
14
TIME (days)
RESULTS
Figure 1 shows the effects of 7 days of intrarenal insulin infusion at a rate of 0.6 mu. kg-‘. min-’ on mean arterial pressure in uninephrectomized dogs. Mean arterial pressure did not change significantly, averaging 93 + 4 mmHg during control and 93 & 3 mmHg during 7 days of renal arterial insulin infusion. Urinary sodium excretion decreased significantly on the 2nd and 4th day of insulin infusion, averaging 49.1 + 5.5 and 42.2 Ilt 4.9 meq/day compared with an average control value of 64.5 + 3.9 meq/day (Fig. 2). No significant changes in sodium excretion were noted on the other days of insulin infusion, although there was -4O- to 47-meq increase in cumulative sodium balance after 4-6 days of insulin infusion. When the insulin infusion was stopped, there was a transient increase in sodium excretion on the first postcontrol day. Urine volume also decreased significantly on the 1st and 4th day of insulin infusion, averaging 735 + 67 and 858 + 64 ml/day compared with a
MEAN ARTERIAL PRESSURE CmmHg)
F665
PRESSURE
90
.4
-2
FIG. 1. Effects of intrarenal for 7 days on mean arterial (n = 8).
2TIME;days6)
10
12
14
insulin infusion (0.6 mu. kg-‘. pressure in uninephrectomized
min-‘) dogs
FIG. 2. Effect of intrarenal insulin for 7 days on sodium and potassium uninephrectomized dogs (n = 8).
infusion excretion
(0.6 mU.kg-’ .min-‘) and urine volume in
control value of 1,019 -C76 ml/day. After 7 days of insulin infusion, cumulative water balance increased by 824 + 210 ml. Urinary potassium excretion, however, did not change significantly during intrarenal insulin infusion, averaging 63.8 + 4.2 meq/day during control and 61.5 + 4.0 meq/day during insulin infusion. Glomerular filtration rate (Fig. 3) increased by -lO12% during intrarenal insulin infusion, although the changes were statistically significant only on the 6th day of infusion. There were no significant changes in effective renal plasma flow or renal vascular resistance during 7 days of intrarenal insulin infusion. Intrarenal insulin infusion caused no significant changes in plasma glucose concentration (Table 1). There were small increases in plasma insulin concentration, indicating a slight spillover from the renal arterial infusion into the systemic circulation. However, intrarenal insulin infusion caused no significant changes in heart rate, plasma electrolytes, plasma protein concentration, plasma renin activity, or plasma aldosterone concentration. Table 2 shows the effects of intrarenal insulin infusion at a lower rate of 0.3 mu. kg-‘. min-’ in two dogs. Insulin infusion caused no changes in arterial pressure in either dog; for both dogs, arterial pressure averaged 89 mmHg during control and 89 mmHg during 7 days of insulin infusion. Insulin infusion intrarenally also caused no changes in heart rate, plasma electrolytes, or plasma insulin concentration in either dog. Intrarenal insulin
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.
F666
INTRARENAL
HYPERINSULINEMIA
1
GLOMERULAR FILTRATION RATE (% Control)
,w 90
EFFECTIVE RENAL PLASMA FLOW fi Control)
I I
II
I
110 100 90
T
RENAL VASCULAR RESISTANCE (% Control)
T
100
~ 3
10
I
12
14
TIME (days)
FIG. 3. Effect of intrarenal insulin infusion (0.6 mu. kg-‘.min-‘) for 7 days on glomerular filtration rate, effective renal plasma flow, and renal vascular resistance in uninephrectomized dogs (n = 8).
infusion caused -40 meq net retention of sodium after 7 days in one dog, while causing no change in sodium balance in the second dog. Average urinary sodium excretion did not change markedly during intrarenal infusion of insulin at 0.3 mU - kg-‘. min-‘, although there was a small transient decrease in sodium excretion in one dog. DISCUSSION
The most significant finding of the study is that 7 days of intrarenal insulin infusion, at rates calculated to increase renal arterial concentrations to levels comparable TABLE
1. Effects of intrarenal
insulin
p .
m$
AND BLOOD
PRESSURE
to those found in obese hypertensive dogs, failed to elevate arterial pressure. However, insulin infusion did cause transient retention of sodium and water. The antinatriuretic and antidiuretic actions of insulin did not persist during chronic intrarenal hyperinsulinemia even in the absence of increased blood pressure and offsetting pressure natriuresis. Chronic renal actions of insulin. The mechanisms by which insulin reduces sodium excretion during chronic intrarenal infusion are not entirely clear but appear to be related mainly to increased sodium reabsorption. In the present study, we found no reductions in GFR associated with intrarenal insulin infusion. In fact, there were small but consistent increases in GFR during insulin infusion indicating that the sodium retention was due to increased tubular reabsorption. This observation is consistent with previous acute experiments which have demonstrated that insulin elevates sodium reabsorption (7, 18), although there have been no previous reports on the chronic direct effects of insulin on renal function. Although it was not possible in the present studies to determine the tubular site at which insulin increased renal sodium reabsorption, previous acute studies have suggested that insulin may act at multiple parts of the renal tubule. Baum (2) demonstrated that insulin, when added to the basolateral side of isolated proximal tubules, stimulated fluid reabsorption and caused a more negative transepithelial potential difference. However, an increase in fluid reabsorption occurred after the addition of physiological or pharmacological levels of insulin to an insulin-deficient bath. Therefore, these studies did not differentiate whether minimal levels of insulin are required for normal fluid reabsorption or whether hyperinsulinemia actually stimulates fluid reabsorption above normal in proximal tubules. Recent micropuncture studies by Kirchner (18) demonstrated that euglycemic hyperinsulinemia increased chloride reabsorption in the loop of Henle but did not alter proximal tubular fluid or chloride reabsorption in superficial nephrons. However, these studies did not rule out the possibility that insulin may also increase sodium chloride reabsorption in the pars recta portion of the proximal tubule or in the proximal tubule in juxtamedullary nephrons inaccessible to micropuncture. Thus acute administration of insulin increases sodium reabsorption in the loop of Henle and possibly in the proximal tubule. However, further studies are
infusion (0.6 mU- kg-l. min-l) PK, ma
P pro,ern, g/100 ml
ngANG
PRA, I.&-‘.h-’
Aldo, ng/lOO ml
P ‘“su’r”’ pU/ml
P p,ucore, mg/lOO ml
HR beats/min
Control
(average of days 1 and 4) Insulin Day I 3 6
142.1kO.5
4.2kO.05
140.9kO.7 142.9kO.5
4.04a0.16 4.19+0.10
143.9kO.3
4.34kO.07
6.75rt0.18
0.42+0.11
6.68kO.21
0.79+0.21 0.67+0.19
6.62?0.26 6.89-cO.32
0.40+0.07
3.9kO.7
3.7kO.9 2.6kO.4 3.2kO.8
7.8kO.5
9425
86+3
20.5+2.1* 22.6&2.2* l&7+2.6*
8926 86klO &3+3
8754 89+3 93t4
Postcontrol 6.9kkO.21 0.4&0.22 4.3kO.9 12.5t2.6 9&9 90t5 (average of days 1, 3, 141.7eo.4 4.33kO.14 and 6) plasma protein; PRA, plasma renin activity; Aldo, Values are means + SE; n = 8 dogs. P Nn,plasma sodium; Pk, plasma potassium; P PlOtPin, plasma aldosterone; Pinsuiin,plasma insulin; P piurore,plasma glucose; HR, heart rate. * P < 0.05 compared with control. Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 11, 2019.
INTRARENAL TABLE
HYPERINSULINEMIA
AND BLOOD
F667
PRESSURE
2. Effects of in&arena1 insulin infusion (0.3 mUb kg-‘. min-‘) MAP, mmHg
Control (average, 5 days) Insulin Day
HR beats/min
medday
UN&x
v, ml/day
Pinsulin,
d/ml
ng ANG
PRA, I ml-’ l
89-+10
85Ik4
69.6t5.7
1,270*270
9.9t6.6
0.4420.21
89k9 86k7 86&6 88t6 92t8 92-c9 943-10
86t8 86t7 82t8 81k6 82k6 81t6 83k5
78.9t22.3 61.025.0 52.1k4.0 72.4t13.1 72.5k1.2 60.9t1.9 83.7t14.1
1,230+470 1,140f420 970t290 1,205+335 1,190*270 1,260f380 1,310t450
9.4k2.2
0.35t0.21
10.9t6.4
0.3620.23
9.6t0.7
0.4320.38
- h-’
Postcontrol (average, 6 days) 90533 81,t6 72.7HO.3 1,215+373 9.5H.4 0.33kO.08 Values are means t SE; n = 2. MAP, mean arterial pressure; H R, heart rate; U Na V, urinary sodium excretion; V, urine volume; Pimulin,plasma insulin concentration; PRA, plasma renin activity.
needed to determine the exact tubular sites at which chronic hyperinsulinemia influences sodium reabsorption. The mechanisms responsible for the rise in GFR during hyperinsulinemia are not clear. If insulin stimulated sodium reabsorption in the proximal tubule or in the loop of Henle, there would be a reduction in sodium chloride delivery to the macula densa which, in turn, could initiate a secondary vasdilation of afferent arterioles and a rise in GFR (30). Although a macula densa feedback offers a plausible explanation for the increased GFR observed during insulin infusion, insulin may also have a direct renal vasodilator action. Cohen et al. (6) reported that insulin caused vasodilation in isolated kidneys perfused with hyperoncotic albumin to prevent glomerular filtration, indicating that insulin can cause renal vasodilation via a mechanism that is independent of filtration. Therefore, insulin may have multiple actions that tend to increase GFR. Intrarenal infusion of insulin in the present study caused no significant changes in potassium excretion. However, previous studies in our laboratory have demonstrated that intravenous insulin infusion caused marked reductions in potassium excretion lasting for several days. Thus the reduced potassium excretion observed during intravenous insulin infusion is probably due to systemic rather than direct intrarenal effects of insulin. One important effect of systemic hyperinsulinemia is increased cellular uptake of potassium (8, 29), which would tend to cause hypokalemia and a decrease in potassium secretion by the renal tubules. This explanation fits with the observation that intravenous infusion of insulin caused significant hypokalemia (13), whereas intrarenal insulin infusion in the present study caused no significant changes in plasma potassium concentration. Although we observed no major changes in renal potassium handling during renal arterial infusion of insulin, we cannot rule out the possibility that insulin could have transient direct effects on potassium excretion that might not be detected in the 24-h measurement. Insulin could also exert opposite effects on potassium handling in different nephron segments. For example, reduced sodium chloride delivery to the distal tubule, caused by
insulin-mediated effects on proximal or loop sodium reabsorption, would tend to decrease potassium excretion, whereas a stimulatory effect of insulin on sodiumpotassium exchange in the collecting tubule would tend to increase potassium excretion. The net effect could be little or no change in overall potassium excretion. The results in the present study do not allow us to determine the contributions of the multiple effects of insulin on renal potassium handling. However, our results do indicate that the direct actions of insulin on the kidney cause little or no change in overall potassium excretion. Intrarenal actions of insulin and long-term blood pressure regulation. Hyperinsulinemia has been suggested to
link obesity and hypertension, in part, via an antinatriuretic action on the kidney (7). As discussed above, insulin causes sodium and water retention via direct renal actions, and blood pressure is correlated with plasma insulin concentration in obese subjects. If the antinatriuretic action of insulin was sustained, this could lead to chronic hypertension by expansion of extracellular fluid volume. According to this concept, an initial retention of sodium would raise extracellular fluid volume and cardiac output, leading to a rise in blood pressure (7). The antinatriuretic effect of insulin would eventually be offset by pressure natriuresis, and sodium balance would be reestablished. However, the maintenance of sodium balance would occur at the expense of elevated blood pressure. Although the hypothesis that the antinatriuretic actions of insulin may contribute to the hypertension associated with obesity has received considerable attention, most of the supporting evidence is still indirect. Only the acute direct renal effects of insulin have been previously reported, and it is not certain whether the sodium-retaining action of insulin is powerful enough, or can be sustained long enough, to cause chronic hypertension. Insulin infusion has been observed to cause sodium retention in obese subjects, despite the presence of resistance to the effects of insulin on carbohydrate metabolism (27). Furthermore, the sodium retention was comparable to that found in nonobese insulin-sensitive subjects (27). These studies indicate that obese subjects retain renal sensitivity to the antinatriureti c actions of insulin and are consistent with the concept that hyper-
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insulinemia may contribute to sodium retention and hypertension in these individuals (7,27). Additional support of the concept that insulin may be a significant factor in obesity-associated hypertension is the observation that there is a correlation between body weight and blood pressure, as well as between insulin and blood pressure in obese subjects (3, 10, 20, 26). Furthermore, when obese individuals are placed on a low-calorie diet, plasma insulin levels fall in parallel with blood pressure, although the fall in blood pressure may occur with or without a negative sodium balance (25). Thus a direct cause and effect relationship between the antinatriuretic actions of insulin and blood pressure in obese subjects has not been demonstrated. In previous studies, we demonstrated that chronic intravenous infusion of insulin, for as long as 28 days at rates that raised plasma insulin to values comparable to that found in obese hypertensive dogs, did not elevate blood pressure in normal dogs, dogs with increased adrenergic activity due to chronic infusion of norepinephrine, angiotensin II-hypertensive dogs, dogs maintained on a high-sodium intake, and dogs in which renal excretory capability was reduced by surgically removing 70% of the kidney mass (13, 14). In fact, chronic insulin administration in some of these studies caused small but significant reductions in blood pressure (13, 14). Recent studies in our laboratory demonstrated that the fall in blood pressure associated with 7 days of insulin infusion was due to decreased total peripheral vascular resistance, since cardiac output was markedly increased by insulin infusion (5). One explanation for the peripheral vasodilation is that systemic administration of insulin increased tissue glucose utilization and metabolic rate, which would tend to cause vasodilation. In normal insulin-sensitive dogs, peripheral vasodilation could, over a period of several days, tend to offset a hypertensive effect of insulin caused by antinatriuresis. However, in obese subjects that are resistant to the metabolic effects of insulin, peripheral vasodilation would not be expected. For this reason, the present studies were designed to examine the long-term direct actions of insulin on the kidney, while preventing large increases in systemic concentrations of insulin that could cause peripheral vasodilation. Yet, even under these conditions, we failed to observe an increase in arterial pressure during chronic insulin infusion at rates that were calculated to raise renal arterial concentrations to levels at least as high as those measured in obese hypertensive dogs. Although insulin did cause mild sodium retention, this effect did not appear to be powerful enough or sustained long enough to cause hypertension. In fact, after 24-48 h of insulin administration there was only mild sodium retention. In previous studies we found much more marked sodium retention during intravenous insulin infusion (5, 13, 14). However, part of the sodium retention observed in our previous studies with intravenous infusion may have been secondary to the fall in blood pressure caused by peripheral vasodilation. The results from the present study indicate that the direct renal actions of insulin, at physiological or pathophysiological levels, are capable of causing only mild and transient sodium
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retention that is insufficient to elevate blood pressure in normal dogs. Although the results in the present study provide no support for the hypothesis that the direct renal actions of insulin cause hypertension, our results do not completely rule out the possibility that hyperinsulinemia might exacerbate the renal actions of other hypertensive stimuli. In previous studies, we found no evidence that hyperinsulinemia potentiated the renal sodium-retaining actions of norepinephrine or angiotensin II, or that hyperinsulinemia elevated blood pressure when adrenergic activity and angiotensin II levels were chronically elevated (13,14). However, insulin has been shown in acute studies to amplify the action of aldosterone on sodium and potassium transport in cultured kidney cells (11). Unfortunately, the relevance of this observation to the long-term antinatriuretic actions of insulin in vivo is still unclear. Also, insulin may have other actions that alter renal function in a manner that could elevate blood pressure. For example, insulin has been shown to stimulate vascular smooth muscle growth (10). If this effect was maintained and occurred in the renal arterioles, this could lead to a slow insidious development of hypertension, as occurs with other disturbances that gradually raise preglomerular vascular resistance (12). In addition, insulin-mediated abnormalities of lipid metabolism (24) could also, over a long period of time, have a damaging effect on the kidney that might lead to hypertension. We can only speculate on the possible importance of these renal actions of insulin. However, the results of the present study indicate that the functional effects of insulin to cause sodium and water retention do not appear to be capable of causing hypertension over a period of 7 days. Our results in the dog do not rule out the possibility that insulin’s direct renal actions might cause hypertension in other species. We have recently reported in preliminary studies that 7 days of euglycemic hyperinsulinemia in rats caused mild hypertension (4). However, because insulin was infused intravenously, we could not determine if the hypertension was due to the direct renal actions of insulin or to some systemic effect which, in turn, influenced kidney function and blood pressure. Also, it is not clear which animal species most closely mimics the human response to hyperinsulinemia. Although the acute effects of insulin have been widely studied, there have been few reports on the blood pressure or renal changes caused by chronic hyperinsulinemia. Recently, Tsutsu et al. (31) measured blood pressure and fasting insulin and glucose levels in patients with insulinoma who had symptoms for an average of 19 mo. No evidence of hypertension was found in these patients despite very high levels of insulin, and no change in blood pressure was observed after resection of the insulinoma. These observations suggest that hyperinsulinemia per se may not cause hypertension in humans, similar to our findings in dogs. However, additional studies are needed to examine potential interactions between insulin and other hypertensive stimuli in the long-term regulation of blood pressure and renal function.
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We thank Nancy Kimmel and Ivadelle Heidke for excellent secretarial assistance and William Dixon and Beth Miller for expert technical assistance. We are also indebted to Dr. Manis J. Smith, Jr., for the radioimmunoassays and to John Fleming, Robert Seaton, and Dr. Jean-Pierre Montani for computer software used in these experiments. This work was supported by National Heart, Lung, and Blood Institute Grants HL-39399, HL-11678, and HL-23502. M. W. Brands and D. A. Hildebrandt were supported by National Institutes of Health Research Service Awards HL-08171 and HL-08931. C, A. Gaillard was supported by a postdoctoral fellowship from the American Heart Association, Mississippi Affiliate. Address for reprint requests: J. E. Hall, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216-4505. Received 30 July 1990; accepted in final form 4 January 1991. REFERENCES 1. ATCHLEY, S. W., R. F. LOEB, D. W. RICHARDS, E. M. BENEDICT, AND M. E. DRISCOLL. On diabetic acidosis. J. Clin. Invest. 12: 297326,1933. 2. BAUM, M. Insulin stimulates transport in rabbit proximal convoluted tubule. J. C&n. Invest. 79: 1104-1109, 1987. 3. BERGLUND, G., S. LJUNGMAN, M. HARTFORD, L. WILHELMSEN, AND P. BJORNTORP. Type of obesity and blood pressure. Hypertension Dallas 4: 692-696,1982. 4. BRANDS, M. W., J. E. HALL, D. A. HJLDEBRANDT, AND H. L. MIZELLE. Hypertension during chronic hyperinsulinemia in conscious rats (Abstract). FASEB J. 4: A817, 1990. 5. BRANDS, M. W., H. L. MIZELLE, C. A. GAILLARD, D. A. HILDEBRANDT, AND J. E. HALL. The hemodynamic response to chronic hyperinsulinemia (Abstract). Am. J. Hypertens. 3: 19A, 1990. 6. COHEN, A. J., D. M. MCCARTHY, AND J. S. STOFF. Direct hemodynamic effect of insulin in the isolated perfused kidney. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F580-F585,1989. 7. DEFRONZO, R. A. The effects of insulin on renal sodium metabolism. Diabetologia 21: 165-171, 1981. 8. DEFRONZO, R., P. FELIG, E. FERRANNINI, AND J. WAHREN. Effect
of graded doses of insulin on splanchnic and peripheral potassium metabolism in man. Am. J. Physiol. 238 (Endocrinol. Metab. 1): E421-E427,1980. 9. DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics 20: 482-491, 1964. 10. FERRARI, P., AND P. WEIDMANN. Insulin, insulin sensitivity and hypertension. J. Hypertens. 8: 491-500, 1990. 11. FIDELMAN, M. L., AND C. 0. WATLINGTON. Insulin and aldoster-
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