514
Effect of Insulin on Renal Sodium Handling
in Hypertensive Rats
David Finch, Gary Davis, John Bower, and Kent Kirchner
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Spontaneously hypertensive rats have reduced peripheral insulin sensitivity. To determine
whether hypertensive rats demonstrate reduced response to the antinatriuretic effect of insulin,
urinary sodium excretion was determined in hypertensive and normotensive rats (n=7 per
group) before and during euglycemic insulin administration at two infusion rates (21
milliunits/kg load and 4 milliunits/kg/min or 85 milliunits/kg load and 8 milliunits/kg/min).
Hypertensive and normotensive time controls received the vehicle for insulin administration.
Mean arterial pressure was greater (/?<0.05) and inulin clearance was less (/?<0.05) in
hypertensive than normotensive rats before insulin infusion. Baseline fractional sodium
excretion was not different between groups. Low dose insulin infusion reduced (p<0.05)
fractional sodium excretion from 0.81 ±0.43% to 0.31 ±0.07% in hypertensive rats and from
1.05±0.37% to 0.47±0.18% in normotensive rats. High dose insulin infusion reduced (/><0.05)
fractional sodium excretion from 0.67±0.22% to 0.21 ±0.08% in hypertensive rats and from
0.81 ±0.15% to 0.30±0.09% in normotensive rats. Sodium excretion was unchanged in time
controls. The reduction in sodium excretion was similar in both rat groups during low dose and
high dose insulin infusions. Mean arterial pressure and inulin clearance were unchanged from
baseline values during insulin infusion in all rat groups. Glucose requirement to maintain
euglycemia was greater (p<0.05) in normotensive than hypertensive rats at both insulin
infusion rates. Thus, while hypertensive rats have reduced sensitivity to the hypoglycemic
effects of insulin, the antinatriuretic response to insulin is not different from that of
normotensive rats. Furthermore, the presence of hypertension does not modulate the antinatriuretic activity of insulin. (Hypertension 1990;15:514-518)
E
levated plasma insulin levels and resistance to
the hypoglycemic effect of insulin have been
associated with hypertension in human and
animal models of hypertension.1-4 This observation
• has led to speculation that insulin may play a role in
the development of increased blood pressure.5 Such
a hypothesis is strengthened by studies demonstrating that normotensive rats fed a fructose-enriched
diet developed insulin resistance, hyperinsulinemia,
and hypertension.3 Inasmuch as insulin reduces urinary sodium excretion,6"8 an attractive hypothesis is
that sodium-dependent hypertension develops as a
result of persistent insulin antinatriuresis. However,
no data are available that examine the effect of
From the Department of Medicine, University of Mississippi
Medical Center, Jackson, Mississippi.
Portions of the data were presented at the annual meeting of the
Southern Section of the American Federation for Clinical
Research in New Orleans, Louisiana, February 1989, and was
published in abstract form in Clinical Research 1989;37:16A.
Address for reprints: Kent A. Kirchner, MD, Department of
Medicine, University of Mississippi Medical Center, 2500 North
State Street, Jackson, MS 39216-4505.
Received July 17, 1989; accepted in revised form December 19,
1989.
insulin on renal sodium reabsorption under conditions of elevated blood pressure. This information is
fundamental for evaluation of any hypothesis implicating a role for insulin in development or maintenance of hypertension. The current study was performed to examine the effect of insulin on urinary
sodium excretion in glucose-clamped, volumeexpanded spontaneously hypertensive rats (SHR).
Effect of insulin on urinary sodium excretion in
normotensive Wistar-Kyoto (WKY) rats was also
performed for comparison.
Methods
SHR and WKY rats were obtained from Taconic
Laboratories, Germantown, New York, and maintained on a 20 mmol sodium intake/g diet for 14-21
days before study. On the day of study, rats were
anesthetized with intraperitoneal injections of 5 secbutyl-5-ethyl-2-thiobarbituric acid (Inactin, Promonto,
Hamburg, FRG) in a dose of 80 mg/kg/body wt and
placed on a heated animal table. Rectal temperatures
were maintained between 37° and 38° C with a servoactivated controller (Yellow Springs Instr. Co., Yellow
Springs, Ohio). After tracheostomy, PE-50 catheters
Finch et al
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were introduced into the right jugular vein for inulin
and insulin infusions and into the left jugular vein for
infusion of glucose and saline. A PE-50 catheter was
placed in the femoral artery for blood sampling and
continuous blood pressure monitoring. This catheter
was connected to a transducer (model 4-327-1, TransAmerica Delavel, Pasadena, California), and arterial
pressure was continuously recorded on a polygraph
(Lafayette Instrs., Lafayette, Indiana). A flanged PE50 catheter was placed into the bladder through a
suprapubic incision for urine collection from the right
kidney. From the start of surgery, isotonic Ringer's
solution composed of the following (meq/1): Na+ 140,
Cl" 115, HCO3 30, K+ 5, and containing 5% polyfructosan (Inutest, Laevosan Gesellschaft, Linz, Austria)
was infused at a rate of 3 ml/hr for 20 minutes and at
1.5 ml/hr thereafter. The left kidney was exposed
through a subcostal incision and gently separated from
the adrenal gland and perirenal fat. The kidney was
placed in a Lucite cup, and the upper ureteral segment
was cannulated with PE-50 tubing. The kidney was
bathed continuously with mineral oil. After the surgical
procedure, a volume of 0.15 M NaCl, equivalent to 3%
of the body weight, was infused over a 60-minute
interval. The rate of the 0.15 M NaCl infusion was then
adjusted to match urinary output. Urine was collected
under oil in preweighed vials from the left kidney over
a 45-minute interval for determination of baseline flow
rate, inulin, and sodium concentrations. Blood was
obtained at 15-minute intervals for glucose determination. A larger plasma sample was determined at the
beginning and end of this period for measurement of
inulin and sodium concentrations. After the baseline
interval, SHR or WKY rats were subdivided into three
groups. The first group (rc=7) received an insulin load
of 21 milliunits/kg and a continuous insulin infusion of
4 milliunits/kg/min. This insulin infusion rate produced
plasma insulin concentrations determined by radioimmunoassay of 46 ±8 microunits/ml in SHR and 43 ±8
microunits/ml in WKY rats. The second group (n=7)
received an insulin load of 85 milliunits/kg and a
continuous insulin infusion of 8 milliunits/kg/min. This
insulin infusion rate produced plasma insulin concentrations determined by radioimmunoassay of 198±79
microunits/ml in SHR and 165 ±85 microunits/ml in
WKY rats. One minute after initiation of the insulin
infusion a 25% glucose infusion was begun at a rate of
30 )u,l/min. A 40-minute stabilization period was then
begun during which time blood glucose levels were
determined at 5-minute intervals, and the rate of
glucose infusion was adjusted to maintain the blood
glucose concentration constant at the mean value
measured during that animal's baseline period. After
this interval, a second 45-minute experimental period
was begun. Urine and blood samples were obtained
as described above. The glucose infusion was
adjusted to maintain blood sugars constant. A second
group of SHR and WKY rats («=8 per group) served
as time controls. These rats received the vehicle for
insulin infusion and 0.15 M NaCl in place of the 25%
Insulin Antinatriuresis in Rats
515
glucose infusion. Urine and blood were collected as
described above.
Analytical Techniques
Sodium concentrations in serum and urine were
measured by flame photometry (model 143, Instrumentation Labs., Lexington, Massachusetts). Inulin
concentration in urine and plasma was determined by
the diphenylamine method of Walser et al.9 Blood
glucose concentration was determined by the glucose
oxidase method (Accu-Check II, Boehringer Mannheim, Indianapolis, Indiana). This technique has
been demonstrated to correlate well (r=0.953) with
measurements of serum glucose.10
Analysis of Data
Determination of the concentration of inulin,
sodium, and chloride in blood and urine and urinary
flow rate permitted calculation of whole-kidney glomerularfiltrationrate and urinary excretion rate of sodium
according to standard expressions. Glucose concentrations for each period were the mean value of the
measurements determined for that period. In insulininfused rats, the rate of glucose use by the body was
determined by weighing the glucose infusion syringe
before and after the euglycemic insulin clamp and
determining the rate of glucose administration. The
Student's t test for paired data was used to determine
statistical significance within each group. Analysis of
variance was used to determine statistical significance
among the groups. If analysis of variance indicated that
a statistical significance existed, then Bonferroni's modification of the t test was used to determine statistical
significance among the groups. Statistical significance
was set at the/?<0.05 level.
Results
Table 1 and Figures 1 and 2 summarize the results
of blood values and urine electrolyte excretion data
during baseline and experimental periods in SHR
and WKY rats. Baseline blood sugars and plasma
sodium concentrations did not differ between any rat
group. Baseline mean arterial pressures were higher
(/?<0.05) and inulin clearances lower (p<0.05) in
SHR compared with WKY rats. Baseline mean arterial pressures and inulin clearances, however, were
not different between subsequent time control and
insulin-infused subgroups of either SHR or WKY
rats. Baseline urine flow rates and absolute urinary
sodium excretion rates were slightly but not statistically lower in SHR than WKY rats. Baseline urine
flow rates and absolute urinary sodium excretion
rates, however, were not different between SHR or
WKY rat time controls and their respective insulintreated counterparts.
Values in SHR or WKY time control rat groups
were not different between baseline and second
collection periods for any parameter measured.
Blood glucose concentrations were not different
between baseline and experimental periods in any
insulin-treated SHR or insulin-treated WKY rat
516
Hypertension
TABLE 1.
Vol 15, No 5, May 1990
Effect of Insulin on Blood Glucose, Blood Pressure and Renal Function
Blood glucose
(mg/dl)
Rat groups
B
WKY time control
(«=8)
WKY low insulin
(n=7)
WKY high insulin
(n=7)
SHR time control
(n=8)
SHR low insulin
(n=7)
SHR high insulin
(n=7)
EXP
Mean arterial
pressure
(mm Hg)
B
EXP
126+12 119+14 112+3 112+3
NS
NS
120+7 116+7
116+2 117±2
NS
NS
110±8 116+7
112+5 114+4
NS
NS
121 + 11 121 + 7 161*±5 165*±5
NS
NS
116+7 119+9 173*±4 172*±4
NS
NS
126+11 120+13 168*±6 155*±8
NS
NS
V
(jtl/min)
(jtl/min)
B
1,299±73
EXP
1,297+84
NS
l,282±109 1,284±84
NS
1,336±86 1,211+90
NS
914*±64 853*+67
NS
l,001*±49 950*±96
NS
995*+62 l,009±116
NS
B
(neq/min)
EXP
11.1+3.6 9.4+3.0
NS
13.2±4.9 8.2+2.3
NS
8.5 + 1.4
5.3+0.9
p<0.05
7.7±0.9 8.2+1.0
NS
6.7±0.8 5.1+0.8
NS
6.8±1.3 3.3+0.4
p<0.05
B
(meq/1)
EXP
2,047±l,066 l,685±907
NS
2,101 ±794
908±348
p<0.05
1,616+389
449±109
p<0.05
l,204±157 1,264±216
NS
1,138+177
448±136
p<0.05
l,027±346
369+157
p<0.05
B
EXP
148+3 150±2
NS
147+1 148+1
NS
149+2 147+1
NS
147+1 149+1
NS
146±2 146±2
NS
148±2 148±2
NS
Values are mean+SEM. B, baseline period; EXP, experimental period; CIN, inulin clearance; V, urine flow rate; UNa V, absolute urinary
sodium excretion rate; PNa, plasma sodium concentration; WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats.
*p<0.05 vs. WKY time control.
Discussion
The current study demonstrates that administration of insulin at rates that produce plasma insulin
80
1?
H WKY
levels in the pathophysiological or pharmacological
range significantly reduce absolute and fractional
urinary sodium excretion rates in both hypertensive
SHR and in normotensive WKY rats. In both rat
groups these effects occur without alterations in
mean arterial pressure or glomerular filtration rate.
Furthermore, when the reduction in urinary sodium
excretion rate produced by insulin administration
was expressed as a percent of baseline values, the
magnitude of the antinatriuretic response of insulin
was similar in both SHR and WKY rats. This
occurred despite the fact that the amount of glucose
required to maintain euglycemia in SHR was less
than half that required to maintain euglycemia in
WKY rats. Thus, while SHR and WKY rats demonstrate differences in insulin-stimulated glucose disposal, the antinatriuretic effect of insulin is qualitatively and quantitatively similar in both strains.
Whether the antinatriuretic effect of insulin is
preserved during the insulin resistance associated
with hypertension has been uncertain. Based on
studies in isolated perfused kidneys, Rostand and
associates11 suggested that variations in circulating
60
2.0
1.8
1.6
50
1.4
70 "
uctlon In absoluti
sodium iMcretion
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groups. Mean arterial pressure was unchanged between
baseline and experimental periods in SHR or WKY
insulin-treated rats. Absolute and fractional urinary
sodium excretion rates were markedly reduced during
both high dose and low dose insulin administration in
both SHR and WKY rat groups (Table 1, Figure 2).
The percent reduction in absolute urinary sodium
excretion produced by either high or low dose insulin
infusion was not different between SHR and WKY rats
(Figure 1). Furthermore, the percent reduction in
sodium excretion was not different between rats receiving high dose and low dose insulin infusion. The
glucose infusion rate required to maintain euglycemia
during low dose insulin administration was 7.5 ±1.2
mg/kg/min in WKY rats and 2.3±1.1 mg/kg/min in
SHR (p<0.05). The glucose infusion rate required to
maintain euglycemia during high dose insulin administration was 16.5 ±2.6 mg/kg/min in WKY rats and
6.0±2.6 mg/kg/min in SHR (p<0.05).
40
30
20
10
0
|SHR
i
Time Control
Low Dose Insulin
High Dose Insulin
£ 12
o 1.0
S. 0.8
0.6
0.4
0.2
I
|H
II
•
Low Dose Insulin
High Dose Insulin
FIGURE 1. Bar graph showing reduction in absolute urinary
sodium excretion rate during insulin infusion expressed as a
percent of the baseline value in Wistar-Kyoto (WKY) rats
(hatched bar) and spontaneously hypertensive rats (SHR)
(solid bar) during low dose and high dose insulin infusion.
0.0
WKY
SHR
WKY
SHR
WKY
SHR
•FIGURE 2. Bar graph showing fractional urinary sodium excre-
tion rate (FeNa) in baseline (hatched bar) and insulin infusion
(solid bar) periods in time control and low dose and high dose
insulin-treated Wistar-Kyoto (WKY) rats and spontaneously
hypertensive rats (SHR). *p<0.05 vs. baseline period.
Finch et al Insulin Antinatriuresis in Rats
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insulin concentrations reduce the availability of insulin receptor sites in the kidney and significantly
attenuate the antinatriuretic effect of subsequent
insulin administration. Consequently, insulin may be
less antinatriuretic in the setting of the insulin resistance associated with hypertension. On the other
hand, Ferrannini and associates1 suggested that insulin resistance associated with essential hypertension
in humans is limited to the nonoxidative pathways of
intracellular glucose disposal. In that study the ability
of insulin to promote cellular potassium uptake and
to inhibit lipolysis remained unimpaired. In our
current study, the observation that insulin produces
similar reductions in urinary sodium excretion in
both SHR and WKY rats demonstrates that the
antinatriuretic response to insulin is also unimpaired
in the resistance that accompanies hypertension. The
observation that doubling the insulin infusion rate
produced insignificant additional reductions in urinary sodium excretion further suggests that insulin
antinatriuresis is maximal in both hypertensive and
normotensive rats at plasma insulin concentrations
within the pathophysiological range. Thus, resistance
to the action of insulin may involve some biological
effects but not others. A similar situation has recently
been demonstrated in obesity. In that condition,
equivalent decreases in urinary sodium excretion
have been reported in insulin-resistant obese adolescents and normal subjects during euglycemic insulin
administration at rates producing plasma insulin
concentrations similar to those observed in our high
dose insulin rat groups.12
Most investigators have suggested that insulin
reduces urinary sodium excretion through a direct
tubular effect.6-8 Although the mechanism responsible for the antinatriuretic response to insulin is not
discernible from the present study, the observation
that insulin reduced urinary sodium excretion without altering glomerular nitration rate, filtered sodium
load, or mean arterial pressure in either SHR or
WKY rats is consistent with a primary tubular rather
than a hemodynamic mechanism. The observation
that the antinatriuretic response was not different
between SHR and WKY rat groups despite the large
difference in mean arterial pressure and, presumably,
renal perfusion pressure would also be consistent
with a tubular mechanism for the effect of insulin.
The factors responsible for the differences in carbohydrate metabolism observed between hypertensive and normotensive conditions in humans and
between SHR and WKY rats are incompletely elucidated. Reduced rates of insulin removal, higher
plasma glucagon concentrations, and reduced intracellular glucose disposal have all been reported.1-13-15
Abnormalities in the suppression of hepatic glucose
production, although not a significant component of
insulin resistance associated with hypertension in
humans,1 have yet to be excluded in the SHR.
Although the mechanisms responsible for the lower
amount of glucose required to maintain euglycemia
in the hypertensive rats in the current study are
517
uncertain, the observation that insulin-induced
sodium retention is not prevented by the increases in
mean arterial pressure suggests an attractive link
between mechanisms that lead to increases in plasma
insulin concentration and the development or maintenance of hypertension. It should be recognized,
however, that mean arterial pressure was not altered
by the short duration of insulin administration in any
rat group in the current study. Thus, if insulin does
increase mean arterial pressure, a more chronic
exposure to the hormone is required. However,
development of volume-dependent hypertension
through persistent insulin-induced antinatriuresis is
possible only if escape from insulin-mediated sodium
retention does not occur with time. In this regard,
Hall and associates16 noted no increase in mean
arterial pressure after 28 days of insulin administration to two thirds nephrectomized dogs. Clearly,
further studies will need to be performed before the
role of insulin in the development and maintenance
of hypertension can be fully evaluated.
Finally, although the current study demonstrates
that the antinatriuretic response is equivalent and
maximal at pathophysiological insulin levels in both
normotensive and hypertensive rats, whether the
threshold for induction of insulin antinatriuresis and
dose-response curve for this effect are truly identical
remains to be determined.
In summary, the current study demonstrates that
insulin produces equivalent antinatriuresis in hypertensive and normotensive animals. This antinatriuretic response appears to be mediated through
tubular rather than hemodynamic mechanisms.
References
1. Ferrannini E, Buzzigoli G, Bonadonna R, Giorico M, Oleggini
M, Graziadei L, Pedrinelli R, Brandi L, Bevilacqua S: Insulin
resistance in essential hypertension. N Engl J Med 1987;
317:350-357
2. Shen D, Shieh S, Fuh M, Chen Y, Reaven G: Resistance to
insulin stimulated glucose uptake in patients with hypertension. J Clin Endocrinol Metab 1988;66:580-583
3. Hwang I, Ho H, Hoffman B, Reaven G: Fructose-induced
insulin resistance and hypertension in rats. Hypertension 1987;
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4. Mondon C, Reaven G: Evidence of abnormalities of insulin
metabolism in rats with spontaneous hypertension. Metabolism
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5. O'Hare J: The enigma of insulin resistance and hypertension.
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12. Rocchini AP, Katch V, Kveselis D, Moorhead C, Martin M,
Lampman R, Gregory M: Insulin and renal sodium retention
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Metabolism 1988;37:303-305
14. Horl WH, Schaefer RM, Heidland A: Abnormalities of carbohydrate metabolism in spontaneously hypertensive rats. Klin
Wochenschr 1988;66:924-927
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spontaneous hypertension. Kidney Int 1988;33:296
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35:326
KEY WORDS • insulin • sodium excretion
hypertension • spontaneously hypertensive rats
• essential
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Effect of insulin on renal sodium handling in hypertensive rats.
D Finch, G Davis, J Bower and K Kirchner
Hypertension. 1990;15:514-518
doi: 10.1161/01.HYP.15.5.514
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