Am J Physiol Renal Physiol 292: F1645–F1651, 2007.
First published January 30, 2007; doi:10.1152/ajprenal.00060.2006.
Furosemide increases water content in renal tissue
Michael Pedersen,1,2 Zsolt Vajda,3 Hans Stødkilde-Jørgensen,1 Søren Nielsen,3 and Jørgen Frøkiær2,3
1
MR Research Centre, Aarhus University Hospital, and 2Institute of Clinical Medicine
and 3The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark
Submitted 22 February 2006; accepted in final form 13 January 2007
Pedersen M, Vajda Z, Stødkilde-Jørgensen H, Nielsen S,
Frøkiær J. Furosemide increases water content in renal tissue. Am J
Physiol Renal Physiol 292: F1645–F1651, 2007. First published
January 30, 2007; doi:10.1152/ajprenal.00060.2006.—The present
study was designed to evaluate the short-term effects of intravenous
administration of furosemide on key functions in the kidney cortex
and the outer and inner medulla of rats by using magnetic resonance
imaging (MRI). Renal tissue water content, renal tissue oxygenation
(in relation to the magnetic resonance spin-spin relaxation rate), the
apparent diffusion coefficient (ADC) of water, and volume of renal
blood flow were measured. Furosemide administration resulted in an
increased water content in all regions of the kidney. In parallel with
this, we found a significant reduction in ADC in the cortex (2.7 ⫾
0.1 ⫻ 10⫺3 to 2.3 ⫾ 0.1 ⫻ 10⫺3 mm2/s; P ⬍ 0.01) and in the outer
medulla (2.3 ⫾ 0.1 ⫻ 10⫺3 to 2.0 ⫾ 0.1 ⫻ 10⫺3 mm2/s; P ⬍ 0.01),
indicating that the intra- to extracellular volume fraction of water
increased in response to furosemide administration. Furosemide also
decreased the blood oxygenation in the cortex (49.1 ⫾ 2.9 to 40.9 ⫾
2.0 s⫺1; P ⬍ 0.01), outer medulla (41.9 ⫾ 2.8 to 33.2 ⫾ 1.6 s⫺1; P ⬍
0.01) and in the inner medulla (37.1 ⫾ 2.9 to 26.7 ⫾ 1.8 s⫺1; P ⬍
0.01), indicating an increased amount of oxygenated Hb in the renal
tissue. Moreover, renal blood flow decreased in response to furo-
semide (6.9 ⫾ 0.2 to 4.4 ⫾ 0.2 ml/min; P ⬍ 0.001). In conclusion,
furosemide administration was associated with increased renal water
content, an increase in the intra- to extracellular volume fraction of
water, an increased oxygen tension, and a decrease in the renal blood
flow. Thus MRI provides an integrated evaluation of changes in renal
function, leading to decreased renal water and solute reabsorption in
response to furosemide, and, in addition, MRI provides an alternative
tool to monitor noninvasively changes at the cellular level.
magnetic resonance imaging; furosemide; loop diuretic; kidney cor-
tex; kidney medulla; renal function; oxygenation
DIURETICS PROMOTE THE RENAL excretion of both water and
solutes from the body. Furosemide is a powerful diuretic acting
on the thick ascending limb of the loop of Henle, where ⬃25%
of all sodium is reabsorbed. Furosemide exerts its action by
inhibition of the Na⫹-K⫹-2Cl⫺ apical membrane cotransporter
NKCC2 or BSC-1, which is the most dominating apical NaCl
transport protein located to this segment (36). The direct effect
of the furosemide-induced volume depletion is an increase in
the isoosmotic urine production (by) impairing the concentrat-
ing capacity of the kidney. Thus furosemide may secondarily
be associated with marked changes in the overall renal water
and sodium content and may be associated with changes in
water diffusion between different compartments in the kidney.
Magnetic resonance imaging (MRI) allows direct quantifi-
cation of the renal water content, which is based on measure-
ment of longitudinal relaxation time (T1) of the tissue and a
Address for reprint requests and other correspondence: J. Frøkiær, The
Water and Salt Research Center and Institute of Clinical Medicine, Univ. of
Aarhus, Aarhus Univ. Hospital, Skejby, 8200 Aarhus N, Denmark (e-mail:
jf@ki.au.dk).
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Innovative Methodology
subsequent conversion to percentage of water (13, 41). Previ-
ously, this technique has been applied in Long Evans rats,
where tissue water content was examined after cerebral ische-
mia, showing a close correlation between in vivo MR-mea-
sured water content in the brain and ex vivo with wet/dry
measurements (41). Moreover, the relationship between intra-
and extracellular diffusion can be measured by the so-called
apparent diffusion coefficient (ADC), a technique that is in-
herently sensitive to diffusion by the random Brownian motion
of protons. Importantly, ADC depends on the intra- and extra-
cellular volume fraction and, to some extent, the cell size and
the membrane permeability (39), whereas a decrease in ADC
can reflect in cellular swelling and vice versa (24, 25).
Recently, measurement of the oxygen levels in the renal
tissue has become possible using a technique referred to as
blood oxygen level-dependent (BOLD) MRI (30). The tech-
nique has been applied to various conditions in the brain and
has also been demonstrated to be promising in quantifying the
oxygenation of the human kidney (11, 18, 31). Recently, this
technique showed that furosemide is associated with an in-
crease in medullary oxygenation in young women, whereas an
unchanged medullary oxygenation was found in older women
(11). This discrepancy in the effect of furosemide may reflect
age-related changes in renal prostaglandin synthesis or renal
hemodynamics. Consequently, to separate these effects, it is
important to measure potential changes in renal blood flow
(RBF) after furosemide administration.
Noninvasive measurement of blood flow velocity is per-
formed routinely with MRI using a phase subtraction technique
in which one-dimensional velocity encoding is generated by a
gradient echo phase-contrast sequence (11). The high RBF in
conjunction with rapid molecular diffusion of water between
intra- and extracellular compartments in the kidney makes
changes in the renal intra- and extracellular compartments
sensitive to hemodynamics. Additionally, because furo-
semide acts primarily in the thick ascending limb, medullary
oxygen content is affected when the Na⫹-K⫹-2Cl⫺ cotrans-
porter is inhibited, increasing the medullary oxygen concen-
tration (3, 30).
Furosemide administration is generally not thought to be
associated with marked changes in the vascular system, e.g., no
change in the mean arterial blood pressure and a minimal fall
in blood volume within 1 h after administration (2). However,
several studies have shown that the RBF is slightly reduced
after administration of furosemide (3, 7, 14, 15), supporting the
view that furosemide acts as a renal vasocontrictor.
The aim of the present study was to examine whether MRI
could detect changes in key renal function parameters in
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F1645
Innovative Methodology
F1646 FUROSEMIDE INCREASES WATER CONTENT IN RENAL TISSUE
response to furosemide. This was achieved by measuring
dynamic changes in the tissue water content, ADC, renal
oxygen consumption, and RBF before and after administration
of furosemide in intact rats. Having established a noninvasive
method to investigate such important mechanisms would stim-
ulate MRI to become a more prominent tool in the understand-
ing of basal physiological principles in the kidney.
MATERIALS AND METHODS
Experimental procedure. Male Wistar rats weighing 350 – 400 g
(120 –130 days of age) were anaesthetized with Mebumal (100 mg/kg
body wt; Nycomed, Copenhagen, Denmark). The rats were main-
tained on a dry pellet diet with free access to water before the
experiment. For drug administration, a polyurethane catheter (0.015
in. Tygo; Norton Performance Plastics, Taunton, MA) was implanted
in the femoral vein. MRI was performed with a Philips NT 1.5 T
system (Philips Medical Systems, Best, The Netherlands) using a
small surface coil of 4 cm in diameter. The animal was placed supine
with the kidneys placed above the imaging coil. The fully anaesthe-
tized rat was then subjected to an imaging protocol as shown in
Fig. 1. After the scout images obtaining and the optimal positions
were found, a group of baseline scans including BOLD, ADC, RBF,
and T1 measurements was initially acquired, followed by intravenous
administration of 0.1 mg/kg body wt furosemide (injected in a volume
of 1 ml/kg; Furix; Benzon Pharma, Copenhagen, Denmark; n ⫽ 6) or
a similar volume of saline (n ⫽ 5). MRI was subsequently performed,
ending with a T1 measurement 57 min after the injection. Rectal
temperature was monitored throughout the experimental protocol. All
animals were cared for according to the local ethical standards of
laboratory animals, and approval was obtained before experimental
procedures.
Renal water content. Accurate and noninvasive measurements of
the kidney water content were performed by determining T1. Even
though the kidney demonstrates a large blood volume, a linear
increase in T1 vs. percent water content from cortex to inner medulla
has been found in rats (23) and rabbits (20). The reciprocal of the total
water content (W), defined as grams of water per grams of tissue, is
linearly dependent on the reciprocal of T1, expressed by 1/W ⫽
A ⫹ B/T1, where A and B are constants depending on field strength
and physical conditions (13, 41). The constants A and B were deter-
mined using tissue-mimicking gelatine standards of different water
content (68 –90%), and the results have shown to correlate to the
percentage of water within 1% (13). Having established the measure-
ment methodology with the in vitro work, in vivo correlation was
performed between the relaxation time and the amount of tissue water
present. T1 measurements were performed by a MIX-mode sequence
provided by the Philips software. The following time parameters were
used: 50 –350 ms echo time (TE), a 370-ms inversion recovery delay,
a 760-ms repetition time (TR), and a 2,500-ms delay between the
excitation pulse in the inversion recovery part and the previous
excitation in the spin-echo part. Quantitative T1 maps were calculated
from pixel-by-pixel analysis using a nonlinear least-squares fit to the
magnitude image data and were then converted to water content maps
Fig. 1. Study design. Investigation of the effect of iv furosemide administra-
tion on renal functions in the anaesthetized rat by magnetic resonance imaging
(MRI). The time scale shows the different MRI procedures performed before
and after injection of furosemide.
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using determined constants A and B. Other relevant parameters were
13 ⫻ 13-cm field-of-view, 128 ⫻ 128 matrix, 2-mm slice thickness,
and three transients.
Relative water diffusion (ADC). To obtain specific information on
relative intra- to extracellular water diffusion, diffusion weighted
images were acquired using a spin-echo-based echo planar imaging
(EPI) sequence that combines the diffusion-sensitizing gradients be-
fore and after the 180° pulse with a fast EPI readout. The diffusion-
sensitizing gradients were applied in three orthogonal gradients (x, y,
and z). Seven different gradient strengths were applied corre-
sponding to diffusion-weighting factors (b values) between 0 and
1,000 s2/mm. With the use of pixel-by-pixel nonlinear least squares
fit, the signal intensities were fitted to the exponential expression,
S(b) ⫽ S(0)exp(⫺b 䡠 ADCdirection), where S(b) is the signal inten-
sity at diffusion-weighting factor b, S(0) is the signal intensity at b ⫽
0, and ADCdirection is the apparent diffusion constant at individual
directions. The factor b was given by: b ⫽ ␥2␦2(⌬ ⫺ ␦/3)G2, where
␥ is the gyromagnetic constant, ␦ is the duration of the individual
gradient pulses, ⌬ is the time separation between the leading edges of
the two gradient pulses, and G is the strength of the diffusion field
gradient. The ADC map was approximated by the mean diffusivity,
given by (ADCx ⫹ ADCy ⫹ ADCz)/3 (38). The size, thickness,
number of the slices, and resolution were the same as in the water
content measurement. Other parameters were TR ⫽ 1,200 ms and
TE ⫽ 31 ms.
Renal oxygen changes (BOLD MRI). BOLD MRI was acquired to
obtain specific information on the renal oxygen concentration. BOLD
MRI is sensitive to changes in regional oxygenation and is based on
the fact that oxyhemoglobin is diamagnetic, whereas deoxyhemoglo-
bin is paramagnetic. Reduction in deoxyhemoglobin concentration
therefore causes an increase in the relative spin-spin relaxation rate
(R2*), the so-called BOLD contrast. A double-echo gradient-echo
sequence was used to determine R2*, which is directly proportional to
the tissue content of deoxyhemoglobin (9, 30) and is thereby inversely
proportional to the tissue concentration of oxygen. BOLD scans were
performed in three coronal slices to image both kidneys. Using TE of
4 and 40 ms in combination with a 40° flip angle and a TR ⫽ 100 ms
resulted in heavily 1/R2* weighted images. As for ADC scans, the
position, size, resolution, and thickness are equal to the image param-
eters chosen for the measurements of the tissue water content. The
slope of fitted Log(intensity) vs. TE was performed to derive a R2*
map on a pixel-by-pixel frame.
RBF. Volume blood flow was measured using a phase-contrast gra-
dient echo sequence equipped with a flow-sensitizing bipolar gradient
in the slice direction. The strengths of the flow-encoding gradients
were set according to RBF values from the literature (35). Ten slices
of 3 mm thickness were prescribed perpendicular to the renal veins.
Each slice had an 11 ⫻ 11-cm field of view and a resolution of 256 ⫻
256 pixels to ensure appropriate pixels to find and derive the blood
flow in the renal veins. Other parameters were TE ⫽ 4 ms, TR ⫽ 25
ms, 40° flip angle, and four transients. The phase images were
subtracted, and quantitative RBF, in units of milliliters per milliliter,
was determined by multiplication with the renal vein area.
Data analysis. For each time frame, T1 scan with TE ⫽ 50 ms,
ADC scan with b ⫽ 0, and BOLD scan using TE ⫽ 4 ms were used
as anatomic templates to facilitate placements of regions of interest
(ROI). Slices with clearly visualized cortex, inner medulla, and outer
medulla were chosen for our analysis. ROIs were manually inserted to
encompass those three regions and were then duplicated to the water
map, ADC map, and BOLD map, which were made using Osiris
Medical Software (Geneva University Hospital, Geneva, Switzerland)
and Photoshop (Adobe Systems, San Jose, CA) graphical software.
RBF values were summarized for all slices in which the renal vein
was clearly seen. The mean values and SEs were calculated for all
measurements. The following statistical analysis was performed:
ANOVA of data from one observation time to another for the same
parameter was performed by a one-way ANOVA model. Where
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 Innovative Methodology
FUROSEMIDE INCREASES WATER CONTENT IN RENAL TISSUE F1647
ANOVA indicated that statistical significance existed, Dunnett’s mul-
tiple comparison was used to determine statistical significance among
baseline and treated values. Comparisons between furosemide and
saline groups were evaluated by Student’s t-test for unpaired data.
Differences were considered statistically significant at the 0.05 level.
The saline group was considered as the control.

RESULTS

The gradient echo images used as an anatomical template

revealed excellent spatial information of the rat kidney. Only
minimal artifacts from respiratory movements were seen, and
cortex as well as inner and outer medulla were easily recog-
nized in both kidneys (Fig. 2). Pixel-by-pixel color maps of the
effect of furosemide and saline on tissue water content, ADC,
R2*, and RBF are presented in Fig. 3. Changes are indicated by
color shifts, with red shift indicating a relative decrease and
blue shift indicating a relative increase. Data are presented and
as relative changes (index) compared with data obtained before
intravenous administration of furosemide or saline (Figs. 4 –7).
Furosemide increases the renal water content. The renal
water content increased significantly in all regions of the
kidney following administration of furosemide. In the kidney
cortex, the water content increased from 78.5% ⫾ 0.7% at
baseline to 81.9% ⫾ 1.0% (P ⫽ 0.009) after furosemide. The
outer medullary water content increased from 81.8% ⫾ 0.9% at
baseline to 85.8% ⫾ 0.8% (P ⫽ 0.002) after furosemide, and
the inner medullary water content showed a similar increase,
from 87.2% ⫾ 0.9% at baseline to 89.4% ⫾ 0.6% (P ⫽ 0.007)
after furosemide. The renal water content of cortex, outer
medulla, and inner medulla did not change significantly in
response to saline administration. Thus furosemide induced a
marked increase in renal water content. Comparison of the
Fig. 3. Pixel-by-pixel maps of different physiological parameters [tissue water
content (A), apparent diffusion coefficient (ADC; B), and blood oxygen
level-dependent (BOLD ;C)], of the rat kidneys before and after administration
of furosemide or saline. The maps are layered on an anatomical image acquired
during the functional scan. The color schemes indicate a relative increase or
decrease in the tissue water content, ADC, or oxygenation, e.g., pixels that are
shifted red indicate a reduced level of oxygenation and pixels that are shifted
blue indicate an increased level of oxygenation.

renal water content in the furosemide-treated rats with the

saline-treated rats showed that the change was primarily lo-
cated to the renal outer medulla (P ⫽ 0.016) and secondarily to
cortex (P ⫽ 0.09) and inner medulla (P ⫽ 0.22; Fig. 4).
Furosemide decreases the ADC. Indexed ADC values are
presented in Fig. 5. Furosemide induced an abrupt decrease in
the ADC inside the kidney. ANOVA for repeated measure-
ments comparing ADC values from the furosemide group with
the saline group showed no significant differences at 8 or 26
min after administration (8 min: P ⬎ 0.316; 27 min: P ⬎
0.086). However, at 46 min after furosemide administration,
the relative change in the cortical ADC was significantly larger
than the relative change after administration of saline (⫺13 ⫾
3% vs. 1 ⫾ 5%; P ⫽ 0.01), indicating that furosemide induced
a shift in the intra- to extracellular volume fraction ratio.
Similar reduction in the outer medullary ADC was found in the
furosemide group compared with the saline group (⫺11 ⫾ 6%
Fig. 2. A coronal magnetic resonance (MR) image of the rat kidneys achieved vs. 1 ⫾ 6%; P ⫽ 0.036). The relative change in ADC of the
from a standard gradient-echo sequence. Cortex, outer medulla, and inner
medulla are distinguished by gray scale. The images were performed using a inner medulla remained unchanged during the protocol, and no
conventional clinical MR system equipped with microcoils designed for significant differences between groups were found at any time
measurements in small animals. (P ⬎ 0.419).
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Innovative Methodology
F1648 FUROSEMIDE INCREASES WATER CONTENT IN RENAL TISSUE

RBF were measured in response to a furosemide bolus using

MRI. The main finding was that furosemide induced a signif-
icant increase in the renal water content, predominantly located
to the cortex and the outer medulla. In parallel, a significant
reduction in the diffusion of the tissue water molecules was
demonstrated in the cortex and in the outer medulla, indicating
an increase in the intra- to extracellular water compartment
ratio. BOLD MRI revealed that blood oxygenation in the outer
and inner medulla was increased following administration of
furosemide, indicating increased levels of blood oxygenated
Hb, i.e., reduced oxygen uptake by the renal tissue. Further-
more, furosemide administration was associated with an im-
mediate reduction in RBF.
Renal water content increases in response to furosemide.
Furosemide induced a marked increase in the overall kidney
water content. From the MRI approach used in the present
Fig. 4. Effect of furosemide or saline on the renal tissue water content in
cortex, outer medulla, and inner medulla. Data reflect the percentage of change
between baseline and treated kidneys; *P ⬍ 0.05. The outer medullary water
content increased significantly after administration of furosemide.

Furosemide decreases renal energy expenditure. Figure 6

shows absolute and relative BOLD MRI (R2*) data. For all
regions, the response to furosemide showed a significant dif-
ference between baseline and treated R2* values (P ⬍ 0.01).
R2* values of control kidneys did not change significantly after
administration of saline (7.3 ⫾ 0.4; P ⬎ 0.2). However, when
comparing the responses to furosemide with the responses to
saline at 3 min after the administration, we found no significant
differences between relative BOLD values in the cortex (⫺9 ⫾
7% vs. 0 ⫾ 5%; P ⫽ 0.065) nor in the outer medulla (⫺15 ⫾
6% vs. ⫺5 ⫾ 4%; P ⫽ 0.116). Inner medullary BOLD values
differed significantly between the furosemide group and con-
trols (⫺18 ⫾ 4% vs. ⫺1 ⫾ 7%; P ⫽ 0.023). Repeating the
scan at 21 min showed a significant difference between relative
BOLD values of furosemide and saline groups in the outer
medulla (⫺26 ⫾ 7% vs. ⫺7 ⫾ 10%; P ⫽ 0.029) and in the
inner medulla (⫺20 ⫾ 8% vs. 0 ⫾ 4%; P ⫽ 0.019). However,
relative cortical values remained unchanged at 21 min (⫺13 ⫾
8% vs. 0 ⫾ 5%; P ⫽ 0.1). At 39 min after administration, a
marked reduction in the relative BOLD values of the furo-
semide group was observed compared with the saline group,
and statistics showed a significant difference for cortex
(⫺20 ⫾ 5% vs. ⫺2 ⫾ 3%; P ⬍ 0.001), outer medulla (⫺25 ⫾
5% vs. ⫺1% ⫾ 3%; P ⬍ 0.001), and inner medulla (⫺32 ⫾
6% vs. ⫺11 ⫾ 8%; P ⫽ 0.007).
Furosemide decreases RBF. Relative changes in RBF in
response to furosemide are shown in Fig. 7. RBF decreased
significantly after administration of furosemide (⫺30 ⫾ 14%;
P ⬍ 0.001), whereas RBF was unchanged in the saline-treated
rats (14 ⫾ 9%; P ⫽ 0.385). Relative RBF did not differ
between furosemide and saline treatment at 16 min (⫺31 ⫾ 6%
vs. ⫺13 ⫾ 13%; P ⫽ 0.086) or at 34 min (⫺32 ⫾ 5% vs.
⫺20 ⫾ 9%; P ⫽ 0.095). However, at 52 min, the relative RBF
was significantly reduced in the furosemide group (⫺36 ⫾ 4%
vs. ⫺16 ⫾ 5%; P ⫽ 0.021). Fig. 5. Effect of furosemide or saline on ADC in cortex (A), outer medulla (B),
and inner medulla (C). Data are indexed values relative to baseline values;
DISCUSSION *P ⬍ 0.05. ANOVA for repeated measurements showed a significant differ-
ence between baseline and treated values for cortex (at 44 min) and outer
During the present study, changes in renal tissue water medulla (at 44 min) but not in the inner medulla. Decreased ADC can be
content, molecular water diffusion, intrarenal oxygenation, and interpreted as an increase in the intra- to extracellular volume fraction.

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FUROSEMIDE INCREASES WATER CONTENT IN RENAL TISSUE
Fig. 6. Effect of furosemide or saline on renal tissue oxygenation [associated
with spin-spin relaxation rate (R2*)] in cortex (A), medulla (B), and inner
medulla (C). Lower R2* implies relative higher blood PO2, hence higher tissue
PO2. Data are indexed values relative to baseline values; *P ⬍ 0.05. At 39 min,
inner medullary R2* differed significantly from the baseline level (at 3, 21, and
39 min). Similarly, outer medullary R2* demonstrated a significant decrease
(at 21 and 39 min). At 39 min after administration, a significant reduction of
R2* was observed in the cortex and in the outer and inner medulla.
study, tissue water content can be derived by measuring the
spin-lattice relaxation rate, a constant that corresponds to the
percentage of water in the tissue. This demonstrated that the renal
water content increased in all regions of the kidney after furo-
semide administration. Thus it may be anticipated that handling of
water in the different nephron segments changes dramatically
immediately after administration of furosemide. The urinary con-
centrating process depends on the coordinated function of the loop
of Henle and the collecting duct. In the thick ascending limb of
the loop of Henle, reabsorption of sodium and chloride
powers the countercurrent multiplier process responsible for
generation of the corticomedullary osmotic gradient, whereas
the collecting ducts, under the control of vasopressin, allow
variable degrees of osmotic equilibration, resulting in variable
water excretion and a reciprocal relationship between urinary
flow and urinary osmolality. Furosemide specifically binds to
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Innovative Methodology
F1649
and inhibits the Na⫹-K⫹-2Cl⫺ apical membrane cotransporter
NKCC2 responsible for the active chloride reabsorption in the
thick ascending limb of the loop of Henle in the mammalian
renal tubule (10). Thus the direct effect of this inhibitor is a
reduction in the active reabsorption of chloride in this segment,
resulting in a decreased driving force. It is well established that
this rapidly leads to an increased diuresis. The increased renal
water content after administration of furosemide may therefore
primarily represent an increase in the amount of water present
in the lumen of the distal tubule and collecting ducts. Charac-
teristically, in a previous study, immunohistochemical local-
ization of rat kidney NKCC2 was restricted to the medullary
and cortical thick ascending limb segments (10). Thus the
marked increase in renal water content demonstrated in the
cortex and outer medulla may reflect that the density of
NKCC2 is high in this area. Correspondingly, administration of
furosemide leads to an increased amount of water remaining in
the renal tubular lumen of the thick ascending limb and
collecting duct. Our current MRI system did not allow us to
identify which exact cellular segments the increased water
content was located to. To perform such examinations, we have
to use either a tracer or drug that is characterized by being
located specifically to specific segments or by a very high field
magnet providing a much higher spatial resolution.
Renal intra- to extracelullar volume fraction increases in
response to furosemide. Furosemide administration was asso-
ciated with a significant reduction in the ADC index both in the
outer medulla and cortex of the kidney. The origin of the ADC
reduction is complex as water diffusion in the renal tissue is
affected by a heterogeneous intraRBF distribution, glomerular
filtration, tubular secretion and reabsorption, and urine flow
(42). Anderson et al. (1) have used a simplified tissue model of
osmotically driven cell volume changes on ADC in the rat
optic nerve. They found that the changes in membrane polar-
ization and cellular energy were not sufficient to induce sig-
nificant changes in ADC, whereas changes in the cellular
volume fraction induced by cell membrane depolarization are
sufficient.
The furosemide-induced reduction in ADC may therefore
correspond to an increase in the intra- to extracellular water
compartment ratio, indicating that the relative intracellular
Fig. 7. Relative renal blood flow (RBF) measured by MRI using an estimate
of the renal vein flow before and after administration of furosemide or saline.
Data are relative to baseline values; *P ⬍ 0.05. RBF demonstrated a significant
reduction 52 min after administration of furosemide, whereas RBF was not
markedly reduced following administration of saline.
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Innovative Methodology
F1650 FUROSEMIDE INCREASES WATER CONTENT IN RENAL TISSUE
volume of water increases compared with the extracellular
volume. Thus the ADC reduction could be a result of an acute
intact apical water transport in the principal cells of the cortical
and outer medullary collecting ducts increasing the cellular
volume in these segments of the kidney. The furosemide-
induced impairment of the osmotic gradient would compro-
mise water reabsorption to the interstitial compartment and
vasa recta. This finding supports the view that furosemide does
not influence short-term regulation of aquaporin 2 in the
principal cells. Thus it is anticipated that the trafficking of
aquaporin 2 and apical water reabsorption is intact in these
segments, whereas the reduced driving force may account for
the inhibition of basolateral water transport.
Whether molecules need to cross a membrane or are hin-
dered by different extracellular environments to diffuse in a
given direction makes a major difference for ADC measure-
ments (13, 22, 39). In the kidney, the vascular structures
supplying the cortex, outer medulla, and inner medulla are
different. In the cortex, many glomeruli have long efferent
arterioles, which extend to the medulla where they divide,
forming vascular bundles, and, in the inner medulla, the cores
of these bundles break up into a capillary plexus. Another
regional difference concerns the interstitial cells, which be-
come more numerous and larger as one descends from cortex
to papilla (40). Changes in ADC should therefore either be a
result of hemodynamic changes or be a result of changes in the
cellular environment. Because ADC is a scalar obtained from
three directions, changes in ADC do not fully reveal noniso-
tropic reorganization of the intracellular compartment. Differ-
ent experimental approaches have resulted in slightly different
ADC values in the rat model, which could be caused by
different diffusion-encoding techniques (5, 6, 21, 42). In gen-
eral, because the structure of the kidney is anisotropic, a full
description of the self-diffusion of water inside the kidney
should be investigated by more advanced methods, such as
application of diffusion tensor imaging (34).
Renal energy expenditure is reduced in response to furo-
semide. The BOLD technique readily detects the reduction in
medullary oxygenation that follows administration of furo-
semide. We have recently demonstrated that the value of R2*
correlates inversely with the tissue content of oxygen (27),
meaning that a decreased R2* implies an increase in the
oxyhemoglobin concentration, which subsequently results in a
proportional increase in blood and tissue PO2. The BOLD MRI
technique does not allow distinguishing changes in oxygen-
ation produced by alternations in the supply of oxygen from
those produced by changes in oxygen consumption (30). The
present study showed that furosemide induces an overall de-
crease in R2*, especially in the medullary regions. These result
values are consistent with previous studies in humans (30) and
in rats (32, 33). Similar observations were found using invasive
oxygen-sensitive microelectrodes (3), from which furosemide
was concluded to enhance medullary oxygen availability.
The improved level of medullary oxygen may be explained
by the decreased consumption of oxygen necessary to main-
tain the medullary transport of ions, and therefore the
equilibrium from oxy- to deoxyhemoglobin is shifted be-
cause furosemide acts by reducing regional tubular oxygen
consumption for reabsorptive transport of sodium (30). We
demonstrated that R2* was mostly decreased in the inner
medulla, although one would think that furosemide mainly
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would act by decreasing the active transport in cells lining
the medullary thick ascending limb. This is consistent with
previous findings demonstrating that changes induced by
furosemide are fully dominated by changes in R2* and that
the BOLD effect is not dependent on changes in the tissue
water content (30).
RBF decreases in response to furosemide. A close correla-
tion between the percentage change of relative RBF and the
percentage change of ADC has recently been demonstrated
(28, 29), suggesting that a decrease in ADC followed by
administration of furosemide was mediated by hemodynamic
changes. Our data showed that furosemide induced a signifi-
cant decrease in RBF, supporting the view that furosemide
causes renal vasoconstriction (3). These effects may therefore
add to the change in ADC, since renal vasoconstriction may be
associated with a decrease in the intravascular volume. Fur-
thermore, volume depletion has effects on renal hemodynam-
ics, including a sustained decrease in glomerular filtration rate
and the blood perfusion of the kidney (4, 14, 16, 18, 43), which
has been shown to depend linearly on RBF (35). Furosemide
has in addition been demonstrated to block the tubuloglomeru-
lar feedback response, followed by significant decreases in
renal interstitial concentrations of ATP (26). Furthermore,
furosemide elicited a marked impairment of RBF and glomer-
ular filtration rate autoregulatory efficiency as well as the
autoregulation-associated alterations in RVR, as previously
reported by other investigators (8, 17). RBF in the sham-
operated rats decreased slightly with time, suggesting that
anesthesia may reduce the systemic blood flow. However, the
decrease was not statistically significant compared with base-
line. Importantly, the present study demonstrated that RBF was
reduced at all time points following intravenous administration
of furosemide.
In conclusion, in vivo magnetic resonance monitoring of
kidney tissue water content following administration of furo-
semide revealed increased cortical and medullary water con-
tent. Diffusion-weighted MRI was able to detect a significant
decrease in cortical and medullary ADC after administration of
furosemide, indicating an increase in the intra- to extracellular
volume fraction ratio. Consistent with previous studies, BOLD
imaging revealed increased medullary tissue oxygenation.
Thus MRI is a noninvasive method providing direct visualiza-
tion of the kidney and simultaneous estimation of essential
renal physiological parameters in vivo.
ACKNOWLEDGMENTS
We authors thank Dr. Steffen Ringgaard, MR Research Centre, Aarhus
University Hospital, for help and assistance in the computing of the image
analysing program.
Present address for M. Pedersen: MR Research Centre and Institute of
Clinical Medicine, Aarhus University Hospital, Skejby, 8200 Aarhus N,
Denmark (e-mail: michael@mr.au.dk).
GRANTS
The Water and Salt Research Centre at the University of Aarhus is
established and supported by the Danish National Research Foundation (Dan-
marks Grundforskningsfond). Support for this study was provided by The
Danish Medical Research Council and the WIRED program (Nordic Council
and the Nordic Centre of Excellence Program in Molecular Medicine).
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