1 Department of Pharmacology, University of Copenhagen, DK-2200 Copenhagen N; 2 The Water and Salt Research Center, Institute of Anatomy, and 4 University Institute of Pathology, Aarhus Kommune Hospital, University of Aarhus, DK-8000 Aarhus C; and 3 Department of Physiology and Pharmacology, University of Southern Denmark, DK-5000 Odense C, Denmark
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ABSTRACT |
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This study was designed to examine the effect of bilateral renal denervation (DNX) on thick ascending limb of Henle's loop (TAL) function in rats with liver cirrhosis induced by common bile duct ligation (CBL). The CBL rats had, as previously shown, sodium retention associated with hypertrophy of the inner stripe of the outer medulla (ISOM) and increased natriuretic effect of furosemide in vivo, and semiquantitative immunoblotting showed increased expression of the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) in ISOM from CBL rats. DNX significantly attenuated the sodium retention in the CBL rats, which was associated with normalization of the natriuretic effect of furosemide, as well as a significant reduction in the expression of NKCC2 in the ISOM. However, the marked hypertrophy of the ISOM found in CBL rats was not reversed by DNX. Together, these data indicate that increased renal sympathetic nerve activity known to be present in CBL rats plays a significant role in the formation of sodium retention by stimulating sodium reabsorption in the TAL via increased renal abundance of NKCC2.
common bile duct ligation; furosemide-sensitive sodium-potassium-2chloride cotransporter type 2; thick ascending limb; furosemide; sodium balance; inner stripe of outer medulla
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INTRODUCTION |
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IT IS WELL DESCRIBED THAT rats with liver cirrhosis induced by common bile duct ligation (CBL) develop edema and ascites. Intact renal innervation seems to play a key role in homeostatic regulatory responses to sodium depletion and sodium loading, and it has been suggested that ~30-40% of the renal sodium retention during edema-forming conditions such as liver cirrhosis (12), congestive heart failure (12), and nephrotic syndrome (18) is dependent on intact renal sympathetic innervation. Autoradiographic studies have shown intense norepinephrine labeling throughout the renal tubules (4-7), and renal sympathetic nerve (RSN) stimulation, which does not affect renal blood flow or glomerular filtration rate (GFR), causes a reversible decrease in urinary sodium excretion in rats (39). Moreover, free-flow micropuncture and tubular microperfusion studies have shown that this stimulatory effect of RSN on tubular sodium reabsorption occurs throughout the tubule (11) and the magnitude of the stimulatory effect of RSN seems to be proportional to the density of the renal tubular innervation, being greatest in the thick ascending limb of Henle's loop (TAL) and least in the collecting duct (4). Together, these findings indicate that RSNs directly stimulate renal tubular sodium reabsorption.
We recently investigated renal function in rats with CBL-induced liver cirrhosis (20, 22, 24) and found that the rats had increased natriuretic response to furosemide together with marked hypertrophy of the TAL epithelium in the inner stripe of the outer medulla (ISOM). Moreover, the capacity to increase the medullary interstitial sodium concentration in response to thirsting was enhanced in the CBL rats (23). Together, these observations indicate that increased sodium reabsorption in the TAL plays a significant role in the sodium retention, which eventually will result in the formation of edema and ascites. Interestingly, recent studies from our own as well as other laboratories show that the furosemide-sensitive Na-K-2Cl cotransporter type 2 (NKCC2) exclusively expressed in the TAL and macula densa is significantly increased in other conditions with sodium retention, such as congestive heart failure and sepsis (21, 32, 35).
The present study was therefore designed to examine the effect of bilateral renal denervation on sodium retention as well as renal function and structure, including the expression of NKCC2 and other TAL transporters, the luminal electroneutral sodium-proton exchanger (NHE3) (9), and the basolateral Na-K-ATPase in rats with CBL-induced liver cirrhosis. Renal function was examined in chronically instrumented rats during control conditions and during acute administration of furosemide. To prevent furosemide-induced sodium and water depletion, we used a computerized servo-controlled sodium- and water-replacement system, where losses of sodium and water were replaced momentarily (19).
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METHODS |
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Experimental Animals
Female Wistar rats (230-250 g) from Charles River (Sulzfeld, Germany) were used for the experiments. The animals were housed in a temperature (22-24°C)- and moisture (40-70%)-controlled room with a 12:12-h light-dark cycle (light on from 6:00 AM to 6:00 PM). Animals were given free access to tap water and a diet containing ~140 mmol/kg of sodium, ~275 mmol/kg potassium, and 23% protein. All animal procedures followed the guidelines for the care and handling of laboratory animals established by the Danish government.Animal Preparation
Cirrhosis was induced by CBL as described by Kountouras and co-workers (28). Briefly, biliary obstruction induces portal inflammation and bile duct proliferation, which eventually will result in the formation of cirrhosis. Control rats were subjected to sham-CBL.Bilateral renal denervation (DNX) was performed through flank incisions. The adventitia of the renal vein and artery were carefully dissected under a microscope. All visible nerves were cut, and the vessels were coated with 10% phenol in 95% ethanol. With this procedure, renal norepinephrine content is reduced to <5% of control levels (37).
Three weeks after CBL/sham-CBL and renal denervation/sham denervation, permanent medical-grade Tygon catheters were implanted in the femoral artery and vein and a permanent suprapubic bladder catheter was implanted in the bladder as described previously (22, 38). After instrumentation, the animals were housed individually.
Experimental Groups
The experimental groups were as follows: sham (sham-CBL rats with sham-DNX); sham-DNX (sham-CBL rats with bilateral DNX); CBL (CBL rats with sham-DNX); and CBL-DNX (CBL rats with bilateral DNX).The experiments were performed in animals in two series: series 1 (n = 6-8/group), in which sodium balance studies were performed, plasma samples for measurement of renin and aldosterone were collected, and kidneys were perfusion fixed; and series 2 (n = 7-8/group), in which renal functions studies were performed, and kidneys were used for immunoblotting.
Series 1
Sodium balance studies. Four weeks after CBL or sham operation, the rats were placed in metabolic cages for accurate determination of daily food and water intake. After 2 days of adaptation, sodium balance was measured daily for 3 consecutive days, and the average of the three values was used. Sodium intake was calculated from the amount of diet ingested per 24 h, and sodium loss was estimated from the amount of sodium excreted in the urine within the same 24 h. Sodium balance was then calculated as the difference between sodium intake and sodium excretion.
Measurement of plasma renin and aldosterone.
Five weeks after CBL or sham operation (i.e., 2 days after the
termination of the sodium balance studies), the rats were placed in
restraining cages, arterial blood samples (total volume 1.0 ml) were
drawn from a permanent arterial catheter, and the plasma was stored at
20°C for later measurement of renin and aldosterone. Plasma renin
concentration was measured by ultramicroradioimmunoassay of generated
ANG I with the "antibody-trapping" technique of Lykkegaard and
Poulsen (30). Aliquots of plasma were diluted 20- to
80-fold with Tris buffer containing human albumin, and 5-µl portions
of these samples were incubated for 24 h at 37°C with 20 ml of a reaction mixture that contained purified rat renin substrate (~1,200 ng ANG I equivalents/ml). This incubation was followed by
radioimmunoassay of generated ANG I. Plasma renin concentration was
measured in reference to renin standards obtained from the National
Institute for Biological Standards and Control (Potters Bar, Herts, UK; 1 milli-Goldblatt unit = 160 pg ANG
I · ml
1 · h
1).
Plasma aldosterone concentration was measured by radioimmunoassay using
a commercial kit (Coat-A-Count Aldosterone, DPC, Los Angeles, CA).
Histological examinations. Then, the rats were anesthetized with halothane-nitrous oxide, and the left kidney was perfused in vivo for 3 min with 1.5% glutharaldehyde in Tyrode's solution with added 2.25% dextran T-40 (perfusion pressure: 150 mmHg) and postfixed in perfusion fluid for later stereological examination. The kidneys were sliced at a 90° angle on the longitudinal axis of the kidney. The 2-mm-thick slices were embedded in paraffin, and 3- to 4-µm-thick sections were cut and stained with hematoxylin-eosin. From this, the volume fractions of the different renal zones were measured stereologically (17). All sections were investigated by light microscopy point counting (using a stage motor), in systematic order with random starting points. The number of points hitting within each zone was estimated. The total number of hitting points within each kidney slice was 200-300, and each field of vision included a grid with 4 points. Data from all 2-mm-thick slices were included, which means that approximately five to seven sections from each kidney were examined. When the volume fractions of the different zones are known, the absolute volumes of the zones can be calculated by multiplying the volume fractions with the volume of the kidney (equal to the kidney weight, assuming that the specific gravity of the kidney is 1 g/cm3) (22).
Series 2
Renal clearance studies.
Renal hemodynamic and tubular responses to furosemide were examined by
clearance techniques in conscious, chronically instrumented rats 4 wk
after a CBL/sham-CBL operation. Before the clearance experiments, all
rats were adapted to the restraining cage used for these experiments by
training them for two periods of 2 h each on consecutive days.
Clearance experiments were started at 8:00 AM. Clearance of
[14C]tetraethylammonium bromide was used as a marker for
effective renal plasma flow, clearance of [3H]inulin as a
marker for GFR, and clearance of lithium (CLi) as a marker
for distal delivery (42). In addition to minor amounts of
lithium in the infusion solutions, lithium was added to the diet (12 mmol/kg diet) for 3 days before the experiments to avoid acute effects
of lithium on renal function (29). The clearance experiments were performed as follows. Clearance markers in 150 mM
glucose, 13 mM NaCl, and 3 mM LiCl were infused at a constant rate of
2.5 ml/h throughout the experiments. After a 90-min
equilibration period where steady-state levels of the tracer substances
were reached, urine was collected in 2 × 30-min control periods
to characterize baseline values of systemic and renal hemodynamics and
tubular function. Then, infusion of furosemide was started at a
constant rate of 0.50 mg/h, and urine was collected in 8 × 30-min
periods during furosemide infusion. To avoid sodium and water
depletion, all furosemide-induced water and sodium losses were
immediately replaced using a computerized servo-controlled water- and
sodium-replacement system (19, 28), which originally was
developed by Andersen and Bie (2) for sodium and water replacement in dogs. The servo system consists of a sodium-sensitive electrode (Radiometer) that continuously measures sodium concentration in the urine, a balance (Sartorius model MC 1, Göttingen,
Germany) that registers urine production (integration period: 1 min),
and two infusion pumps (model 200-7103, Harvard Apparatus) that
infuse 50 mM glucose and 300 mM NaCl, respectively. The input from the electrode and the balance is analyzed on-line in Lab-View, and the
infusion rate of the two infusion pumps is continuously altered to
replace the urinary water and sodium losses. The accuracy of this
system lies within the micromolar range for urinary sodium concentration between 10 and 150 mM. Arterial blood samples (300 µl
each) were collected into ammonium-heparinized capillary tubes at the
end of the equilibration period, at the end of the control period, and
once every hour during furosemide infusion. All blood samples were
immediately replaced with heparinized blood from a donor rat. Mean
arterial pressure (MAP) was measured throughout. Electrolytes in
samples of plasma and urine were determined by atomic absorption
spectrometry, using a PerkinElmer series 2380 and a PerkinElmer Analyst
300 atomic absorption spectrometer. [14C]tetraethylammonium and [3H]inulin were
determined by double-label scintillation counting in a Packard 2250 CA
liquid scintillation counter. Renal clearances (C) and fractional
excretions (FE) were calculated by the standard formula
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Semiquantitative immunoblotting.
Five days after the renal clearance studies, the rats were anesthetized
with halothane/N2O, and the right kidney was removed, immediately frozen in liquid nitrogen, and stored at 80°C until processing for membrane fractionation. The outer medulla was isolated and homogenized using a tissue homogenizer (Ultra-Turrax T8, Ika, Staufen, Germany) in 10 ml of ice-cold homogenizing buffer containing 300 mM sucrose, 25 mM imidazol, 1 mM EDTA-disodium salt, and the following protease inhibitors: Pefabloc (0.1 mg/ml buffer) and leupeptin (4 µg/ml buffer); and phosphatase inhibitors sodium orthovanadate (184 µg/ml), sodium fluoride (1.05 mg/ml), and okadeic acid 82 (ng/ml), pH adjusted to 7.2 with 0.1 M HCl. Protein
concentration in the homogenate was measured by use of a commercial kit
(Pierce BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). All
samples were then diluted to a final protein concentration of 4 µg/µl with the additon of sample buffer (in the final solution: 486 mM Tris · HCl, pH 6.8, 7% glycerol, 104 mM SDS,
0.0875 mM bromphenol blue), dithiothreitol (25 mM in the
final solution), and homogenizing buffer. The samples were then
solubilized at 60°C for 10 min.
Statistics
Data are presented as means ± SE. To evaluate the effect of furosemide, the average values during the two 30-min control periods were compared with the average values during the last three 30-min periods of the furosemide-induced diuresis. Comparisons were performed by two-way analysis of variance followed by Fisher's least significant difference test. Differences were considered significant at the 0.05 level. ![]() |
RESULTS |
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Daily sodium intake was similar in all groups, but daily sodium
excretion significantly decreased in the CBL rats, which indicated the
presence of sodium retention relative to the sham-operated control
animals (Table 1). DNX had no effect on
sodium handling in the sham-operated control rats, but sodium retention
was significantly attenuated in CBL-DNX rats (Table
2). Twenty-four-hour urine production and
urine osmolality were similar in the sham and CBL rats. DNX had no
effect on urine production or urine osomolality in the sham-operated
rats, but in CBL rats DNX significantly increased the production of
solute-free urine. Plasma levels of renin, aldosterone, sodium, and
potassium as well as plasma osmolality were similar in all four groups
(Table 2).
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Renal Function Studies
Baseline values.
MAP and effective renal plasma flow (ERPF) were similar in all four
groups (Table 3). GFR was decreased
compared with controls, and the effective filtration fraction was
therefore, as previously shown (22, 23, 25), significantly
decreased in the CBL rats. DNX had no significant effects on effective
filtration fraction or GFR in either CBL or sham-CBL rats. There were
no significant differences in CLi between the CBL and
sham-CBL groups, whereas DNX increased CLi in both the CBL
and the sham-CBL rats. Baseline values for V and urinary sodium
excretion rate (UNaV) were similar in all
groups1 (data not shown).
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Effect of furosemide on renal sodium handling.
Constant furosemide infusion under conditions in which water and sodium
depletion was prevented by use of a computerized servo-controlled system induced a prolonged and sustained diuretic and natriuretic response in all four groups. Within all the groups, diuresis and natriuresis reached a steady state after an ~150-min infusion (Fig.
1). The furosemide-induced natriuresis
was as previously shown (20, 22-24) to be
significantly increased in the CBL rats [UNaV (CBL:
22.9 ± 3.1 vs. sham: 13.4 ± 2.0 µmol · min
1 · 100 g body wt
1, P < 0.05);
FENa (CBL: 20.2 ± 2.6 vs. sham: 10.2 ± 1.7%, P < 0.01)]. Similarly, the change in
fractional distal sodium excretion (CNa/CLi)
was significantly increased in the CBL rats
[
CNa/CLi (CBL: 30.8 ± 3.9 vs. sham:
19.8 ± 2.4%, P < 0.01)]. DNX normalized the
natriuretic response to furosemide in CBL rats [
UNaV
(CBL-DNX: 14.6 ± 2.1 vs. CBL: 22.9 ± 3.1 µmol · min
1 · 100 g body wt
1, P < 0.05);
FENa (CBL-DNX: 11.7 ± 2.6 vs. CBL: 20.2 ± 2.6%, P < 0.01);
CNa/CLi
(CBL-DNX: 19.3 ± 2.9 vs CBL: 30.8 ± 3.9%, P < 0.01)]. DNX had no significant effect on the
furosemide-induced natriuretic response in the sham-CBL rats.
Furosemide produced similar changes in MAP, GFR, ERPF, and
FELi in all four groups (data not shown).
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Renal histopathology.
The stereological analysis performed on the in vivo perfusion-fixed
kidneys showed, in accordance with previous reports from our laboratory
(20, 22), that CBL rats had marked and selective hyperthrophy of the ISOM (absolute volume: 154 ± 17 vs. 227 ± 14 mm3, P < 0.01). DNX had no effect on the hypertrophy of the ISOM in CBL rats
(Fig. 2).
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Expression of sodium transporters in renal outer medulla.
Figure 3A shows an immunoblot
of membrane fractions (40 µg protein/lane) from renal outer medullary
preparations. The affinity-purified anti-NKCC2 antibody recognizes a
broad band at ~165 kDa corresponding to the furosemide-sensitive
type-2 Na-K-2Cl cotransporter exclusively expressed in the TAL and in
the macula densa (14). Densitometry of all samples (Fig.
3B) revealed an increased expression of NKCC2 in the outer
medulla in CBL rats compared with sham-operated controls (sham:
100 ± 14 vs. CBL: 131 ± 4%, P < 0.05).
DNX significantly decreased the expression of NKCC2 in CBL rats, as
shown in Fig. 3, C and E. Results of the
densitometry were CBL: 100 ± 15 vs. CBL-DNX: 57 ± 11%,
P < 0.05, and sham: 100 ± 27 vs. CBL-DNX:
62 ± 21%, not significant. DNX had no effect on NKCC2 expression in the sham-DNX rats (data not shown). Figure
4A shows an immunoblot of
membrane fractions (40 µg protein/lane) from renal outer
medullary preparations. The affinity-purified anti-NHE3
antibody recognizes a band at ~86 kDa corresponding to NHE3 expressed
in the TAL and in the proximal tubules (9). Densitometry
of all samples (Fig. 4B) revealed a decreased expression of
NHE3 in the outer medulla of CBL rats compared with sham-operated
controls (sham: 100 ± 8 vs. CBL: 57 ± 4%,
P < 0.05). Renal denervation had, as shown in Fig. 4,
C-F, no effect on the expression of NHE3 in
CBL rats. Similarly, DNX had no effect on NHE3 expression in the
sham-DNX rats (data not shown).
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Expression of AQP2 in the renal outer medulla.
Finally, we measured the expression level of AQP2 protein in the outer
medulla. AQP2 is expressed in the principal cells of the collecting
ducts, and the affinity-purified anti-AQP2 antibody recognizes the
29-kDa and the 35- to 50-kDa band, corresponding to nonglycosylated and
glycosylated AQP2 protein, respectively. In accordance with previous
reports from our laboratory (23, 25), AQP2 expression was
significantly decreased in CBL rats (Fig. 5, A and
B). As shown in Fig.
5, C-F, renal denervation had no
effect on the expression of AQP2 in CBL rats. Similarly, the expression
of AQP2 was unchanged in the sham-DNX rats (data not shown).
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DISCUSSION |
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The major finding of the present study is that DNX ameliorates the development of renal tubular dysfunction in rats with CBL-induced liver cirrhosis by mechanisms most probably involving inhibition of increased sodium reabsorption in the TAL. The sodium balance studies revealed that DNX normalized 24-h sodium balance, and the clearance studies showed that DNX normalized the increased natriuretic response to furosemide found in CBL rats. Moreover, DNX significantly reduced the expression of NKCC2. However, the marked hypertrophy of the ISOM found in CBL rats was not reversed by DNX.
An increasing number of studies have evaluated TAL function in conditions with impaired renal sodium handling. The present study confirmed previous findings from our own laboratory (20, 22-24, 26) indicating that sodium reabsorption in the TAL is increased in cirrhotic rats and plays a significant role in the sodium retention that eventually results in the formation of edema and ascites. However, not only liver cirrhosis seems to be associated with altered TAL function. Increased NKCC2 expression has also been found in rats with congestive heart failure (32, 35, 41), and recently we have shown that sepsis-induced acute renal failure is associated with increased NKCC2 expression (26). Moreover, Alvarez-Guerra and Garay (1) have reported increased natriuretic effect of bumetanide associated with increased bumetanide-sensitive rubidium uptake in TAL from Dahl-S hypertensive rats, and Manning and co-workers (31) have recently shown increased NKCC2 expression in rats with prenatally programmed hypertension induced by a maternal low-protein diet during pregnancy. Together, these data seem to support a role of regulation of NKCC2 abundance in a number of pathophysiological conditions with impaired renal sodium handling.
RSNs are important modulators of renal sodium excretion through release
of the neurotransmitter norepinephrine. DiBona and co-workers
(13) have demonstrated that RSN activity is increased during conditions with extracellular volume expansion, including liver
cirrhosis and congestive heart failure (13). Moreover, long-term sodium balance studies have shown that renal denervation significantly attenuates the development of excess sodium accumulation in rats with liver cirrhosis or congestive heart failure
(12). Several segments of the nephron are closely
associated with sympathetic neuronal varicosities
(4-7), and the highest number of neural fibers per
tubule is found in the TAL (7). The TAL possesses both
2 (33)- and
-adrenergic receptors
(16), and the selective
-adrenergic receptor agonist
isoproterenol increases sodium reabsorption in the TAL
(3). Thus renal nerve activity seems to be involved in the
regulation of sodium reabsorption in TAL. In the present study,
bilateral DNX prevented the excess sodium retention in CBL rats. This
normalization of the sodium balance was associated with a significantly
reduced expression of NKCC2 in the outer medulla in CBL rats and with
normalization of the natriuretic response to furosemide. Together,
these findings indicate that increased RSN activity known to be present
in CBL rats plays a significant role in the formation of sodium
retention by stimulating sodium reabsorption in the TAL.
We also examined the expression of NHE3 in the outer medulla. NHE3, which plays a major role in the regulation of urine acidification and might work as an alternative route for TAL sodium reabsorption, was significantly decreased in CBL rats. The mechanism behind this finding is unknown. However, DNX had no effect on the expression of NHE3 in both sham and CBL rats, indicating that changes in the abundance of this transporter are not affected by RSN.
CBL rats had, as was previously shown (20, 22), marked hypertrophy of the ISOM. Similar morphological changes are found in rats chronically treated with vasopressin (8, 10), and we have shown that this hypertrophy is absent in vasopressin-deficient Brattleboro rats with CBL-induced liver cirrhosis (22). Moreover, we have shown that chronic treatment with the somatostatin analog octreotide prevents the development of ISOM hypertrophy in CBL rats by an unknown mechanism (22). Despite the marked effect of DNX on TAL function in CBL rats, DNX did not prevent the formation of ISOM hypertrophy, which strongly indicates that RSN stimulation has functional but not hypertrophic action in the TAL.
Renal denervation increased the formation of solute-free urine in CBL rats. The final regulation of urine production is regulated by vasopressin and depends on 1) expression and membrane targeting of AQP2 in the collecting ducts and 2) the magnitude of the corticomedullary osmotic gradient generated by sodium reabsorption in the TAL. As was previously shown (23, 25), CBL rats had significantly decreased expression of AQP2. DNX had no effect on the abundance of AQP2 in either normal or CBL rats. However, because DNX prevented the formation of increased TAL sodium reabsorption, the formation of an increased corticomedullary osmotic gradient (21) most probably was prevented as well, resulting in the production of an increased amount of solute-free urine.
In summary, the present data indicate that RSN activity plays a significant role in the formation of sodium retention in CBL rats by stimulating sodium reabsorption in the TAL. An increasing number of reports support the hypothesis that regulation of TAL sodium reabsorption, including NKCC2 abundance, plays a significant role in different pathophysiological conditions with impaired renal sodium handling (1, 21, 31, 32, 35, 41). Detailed studies of the role of RSNs for the regulation of TAL function in these pathophysiological conditions, which include congestive heart failure, hypertension, and sepsis, are warranted.
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ACKNOWLEDGEMENTS |
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The technical assistance of Iben Nielsen, Barbara Seider, and Haya Holmegaard is acknowledged.
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FOOTNOTES |
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This work received financial support from The Danish Medical Research Council, the Danish Heart Foundation, The Novo Nordic Foundation, The Eva and Robert Voss Hansen Foundation, The Ruth Kønig-Petersen Foundation, and The Helen and Ejnar Bjørnow Foundation.
1 Renal clearance experiments were performed during the inactive period of the rat (i.e., during the daytime), when sodium- and water-retaining mechanisms are maximally activated. To get stable urine production under these conditions, all rats were slightly water loaded by infusion of a hypotonic glucose solution (2.5 ml/h). Therefore, as previously described, baseline levels of urine flow rate (V) were similar (i.e., clamped) in all four groups as previously demonstrated (23, 25). Furthermore, in accordance with previous studies performed during the daytime and with a low sodium infusion rate (infusion rate: 32.5 µmol Na/h) (23-25), renal sodium handling was similar in all four groups.
Address for reprint requests and other correspondence: T. E. N. Jonassen, Dept. of Pharmacology, Univ. of Copenhagen, 3 Blegdamsvej, Bldg. 18.5, DK-2200 Copenhagen N, Denmark (E-mail: fitj{at}farmakol.ku.dk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 19, 2002;10.1152/ajprenal.00258.2002
Received 18 July 2002; accepted in final form 6 November 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alvarez-Guerra, M,
and
Garay RP.
Renal Na-K-Cl cotransporter NKCC2 in Dahl salt-sensitive rats.
J Hypertens
20:
721-7,
2002[ISI][Medline].
2.
Andersen, SE,
and
Bie P.
Continuous servo-controlled replacement of urinary sodium loss in conscious dogs.
Am J Physiol Regul Integr Comp Physiol
259:
R313-R326,
1990
3.
Bailly, C,
Imbert-Teboul M,
Roinel N,
and
Amiel C.
Isoproterenol increases Ca, Mg, and NaCl reabsorption in mouse thick ascending limb.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F1224-F1231,
1990
4.
Barajas, L,
and
Powers K.
Innervation of the renal proximal convoluted tubule of the rat.
Am J Anat
186:
378-388,
1989[ISI][Medline].
5.
Barajas, L,
Powers K,
and
Wang P.
Innervation of the renal cortical tubules: a quantitative study.
Am J Physiol Renal Fluid Electrolyte Physiol
247:
F50-F60,
1984
6.
Barajas, L,
Powers K,
and
Wang P.
Innervation of the late distal nephron: an autographic and ultrastructural study.
J Ultrastruct Res
92:
146-157,
1985[ISI][Medline].
7.
Bajaras, L,
and
Powers KV.
Innervation of the thick ascending limb of Henle.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F340-F348,
1988
8.
Bankir, L,
Fischer C,
Fischer S,
Jukkula K,
Specht HC,
and
Kriz W.
Adaptation of the rat kidney to altered water intake and urine concentration.
Pflügers Arch
412:
42-53,
1988[ISI][Medline].
9.
Biemesderfer, D,
Pizzonia J,
Abu-Alfa A,
Exner M,
Reilly R,
Igarashi P,
and
Aronson PS.
NHE3: a Na+/H+ exchanger isoform of renal brush border.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F736-F742,
1993
10.
Bouby, N,
Bankir L,
Trinh-Trang-Tan M-M,
Minuth WW,
and
Kriz W.
Selective ADH-induced hypertrophy of the medullary thick ascending limb in Brattleboro rats.
Kidney Int
28:
456-466,
1985[ISI][Medline].
11.
DiBona, GF.
Neurogenic regulation of renal tubular sodium reabsorption.
Am J Physiol Renal Fluid Electrolyte Physiol
233:
F73-F81,
1977
12.
DiBona, GF,
and
Sawin LL.
Role of renal nerves in sodium retention of cirrhosis and congestive heart failure.
Am J Physiol Regul Integr Comp Physiol
260:
R298-R305,
1991
13.
DiBona, GF,
Sawin LL,
and
Jones SY.
Characteristics of renal sympathetic nerve activity in sodium retaining disorders.
Am J Physiol Regul Integr Comp Physiol
271:
R295-R302,
1996
14.
Ecelbarger, CA,
Terris J,
Hoyer JR,
Nielsen S,
Wade JB,
and
Knepper MA.
Localization and regulation of the rat renal Na+-K+-2Cl cotransporter, BSC-1.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F619-F628,
1996
15.
Ecelbarger, CA,
Yu S,
Lee AJ,
Weinstein LS,
and
Knepper MA.
Decreased renal Na-K-2Cl cotransporter abundance in mice with heterozygous disruption of the Gs gene.
Am J Physiol Renal Physiol
277:
F235-F244,
1999
16.
Elalouf, JM,
Buhler JM,
Tessoit C,
Bellanger AC,
Dublineau I,
and
de Rouffignac C.
Predominant expression of 1-adrenergic receptor in the thick ascending limb of rat kidney. Absolute mRNA quantitation by reverse transcription and polymerase chain reaction.
J Clin Invest
91:
264-272,
1993[ISI][Medline].
17.
Gundersen, HJ,
Bendtsen TF,
Korbo L,
Marcussen N,
Møller A,
Nielsen K,
Nyengaard JR,
Pakkenberg B,
Sørensen FB,
Vesterby A,
and
West MJ.
Some new, simple and efficient stereological methods and their use in pathological research and diagnosis.
APMIS
96:
379-394,
1988[ISI][Medline].
18.
Herman, PJ,
Sawin LL,
and
DiBona GF.
Role of renal nerves in renal sodium retention of nephrotic syndrome.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F823-F829,
1989
19.
Janjua, N,
Jonassen TEN,
Langhoff S,
Thomsen K,
and
Christensen S.
The role of sodium depletion for the acute antidiuretic effect of bendroflumethiazide in rats with nephrogenic diabetes incipidus.
J Pharm Exp Ther
299:
1-7,
2001
20.
Jonassen, TEN,
Christensen S,
Sørensen AM,
Marcussen N,
Flyvbjerg A,
Andreasen F,
and
Petersen JS.
Effects of chronic octreotide treatment on renal changes during liver cirrhosis in rats.
Hepatology
29:
1387-1395,
1999[ISI][Medline].
21.
Jonassen, TEN,
Græbe M,
Nielsen S,
Promeneur D,
Christensen S,
and
Olsen NV.
Lipopolysaccharide-induced acute renal failure in rats: effects of specific phosphodiesterase type 3 and 4 inhibition.
J Pharm Exp Ther
303:
1-11,
2002
22.
Jonassen, TEN,
Marcussen N,
Haugan K,
Skyum H,
Christensen S,
Andreasen F,
and
Petersen JS.
Functional and structural changes in the thick ascending limb of Henle's loop in rats with liver cirrhosis.
Am J Physiol Regul Integr Comp Physiol
273:
R568-R577,
1997
23.
Jonassen, TEN,
Nielsen S,
Christensen S,
and
Petersen JS.
Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis.
Am J Physiol Renal Physiol
275:
F216-F225,
1998
24.
Jonassen, TEN,
Petersen JS,
Sørensen AM,
Andreasen F,
and
Christensen S.
Aldosterone receptor-blockade inhibits increased furosemide-sensitive sodium transport in the thick ascending limb of Henle's loop in rats with liver cirrhosis.
J Pharmacol Exp Ther
287:
931-936,
1998
25.
Jonassen, TEN,
Promeneur D,
Christensen S,
Petersen JS,
and
Nielsen S.
Decreased vasopressin-mediated renal water reabsorption in rats with chronic aldosterone-receptor blockade.
Am J Physiol Renal Physiol
278:
F246-F256,
2000
26.
Jonassen, TEN,
Sørensen AM,
Petersen JS,
Andreasen F,
and
Cristensen S.
Increased natriuretic efficiency of furosemide in rats with carbon tetrachloride induced liver cirrhosis.
Hepatology
31:
1224-1230,
2000[ISI][Medline].
27.
Kim, GH,
Ecelbarger CA,
Mitchell C,
Packer RK,
Wade JB,
and
Knepper MA.
Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop.
Am J Physiol Renal Physiol
276:
F96-F103,
1999
28.
Kountouras, J,
Billing BH,
and
Scheuer PJ.
Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat.
Br J Exp Pathol
65:
305-311,
1984[ISI][Medline].
29.
Leyssac, PP,
Frederiksen O,
Holstein-Rathlou NH,
Alfrey AC,
and
Christensen P.
Active lithium transport by the rat renal proximal tubule: a micropuncture study.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F86-F93,
1994
30.
Lykkegaard, S,
and
Poulsen K.
Ultramicroassay for plasma renin concentration in the rat using the antibody trapping technique.
Anal Biochem
75:
250-259,
1976[ISI][Medline].
31.
Manning, J,
Beutler K,
Knepper MA,
and
Vehaskari MV.
Upregulation of renal BSC1 and TSC in prenatally programmed hypertension.
Am J Physiol Renal Physiol
283:
F202-F206,
2002
32.
Marumo, R,
Kaizuma S,
Nogae S,
Kanazawa M,
Kimura T,
Saito T,
Ito S,
and
Matsubara M.
Differential upregulation of rat Na-K-Cl cotransporter, rBSC1, mRNA in the thick ascending limb of Henle in different pathological conditions.
Kidney Int
54:
877-888,
1998[ISI][Medline].
33.
Mohuczy-Dominiak, D,
and
Garg LC.
[]-2 Adrenoceptors in medullary thick ascending limbs of the rabbit kidney.
J Pharmacol Exp Ther
266:
279-287,
1993[Abstract].
34.
Nielsen, S,
Maunsbach AB,
Ecelbarger CA,
and
Knepper MA.
Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and macula densa of rat kidney.
Am J Physiol Renal Physiol
275:
F885-F893,
1998
35.
Nogae, S,
Michimata M,
Kanazawa M,
Honda S,
Ohta M,
Imai Y,
Ito S,
and
Matsubara M.
Cardiac infarcts increase sodium transporter transcripts (rBSC1) in the thick ascending limb of Henle.
Kidney Int
57:
2055-2063,
2000[ISI][Medline].
36.
Orlowski, J,
Kandasamy RA,
and
Shull GE.
Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.
J Biol Chem
267:
9331-9339,
1992
37.
Petersen, JS,
and
DiBona GF.
Effects of renal denervation on sodium balance and renal function during chronic furosemide administration in rats.
J Pharmacol Exp Ther
262:
1103-1109,
1992[Abstract].
38.
Petersen, JS,
Shalmi M,
Lam HR,
and
Christensen S.
Renal response of furosemide in conscious rats: effects of acute instrumentation and peripheral sympathectomy.
J Pharmacol Exp Ther
258:
1-7,
1991[Abstract].
39.
Slick, GL,
Aguilera AJ,
Zambraski EJ,
DiBona GF,
and
Kaloyanides GJ.
Renal neuroadrenergic transmission.
Am J Physiol
229:
60-65,
1975
40.
Spannow, J,
Thomsen K,
Petersen JS,
Haugan K,
and
Christensen S.
Influence of renal nerves and sodium balance on the acute antidiuretic effect of bendroflumethiazide in rats with diabetes insipidus.
J Pharmacol Exp Ther
282:
1155-62,
1997
41.
Staahltoft, D,
Nielsen S,
Janjua NR,
Christensen S,
Marcussen N,
Skøtt O,
and
Jonassen TEN
Chronic losartan treatment normalizes renal water handling in rats with congestive heart failure.
Am J Physiol Renal Physiol
282:
F307-F315,
2002
42.
Thomsen, K,
and
Shirley DG.
The validity of lithium clearance as an index of sodium and water delivery from the proximal tubules.
Nephron
77:
125-138,
1997[ISI][Medline].