- Email: [email protected]
Measurement of renal vein blood flow in rats by high-field magnetic resonance
Kidney International Vol. 52 (1997), pp. 1359—1363
VASCULAR BIOLOGY - HEMODYNAMICS - HYPERTENSION
Measurement of renal vein blood flow in rats by high-field magnetic resonance STEFFEN RINGGAARD, THORA CHRISTIANSEN, MARTIN BAJ, ERIK M. PEDERSEN, HANS STØDKLLDE-JORGENSEN, and ALLAN FLYVBJERG Magnetic Resonance Research Centre, Aarhus University Hospital, Institute of Experimental Clinical Research, Aarhus University, and Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Aarhus, Denmark
Measurement of renal vein blood flow in rats by high-field magnetic resonance. The aim of the present study was to examine whether magnetic resonance imaging (MRI) based method for non-invasive in vivo measurement of vein blood flow in rats could be used to estimate renal blood flow (RBF). Measurements were performed using a high-field (7 Tesla) MRI scanner with a short echo time phase contrast velocity measurement pulse sequence. The method was evaluated in vitro by flow measurements in an acrylic pipe and in vivo by recording left renal vein blood flow in normal
and unilaterally nephrectomized rats. In a subset of animals RBF was measured by a direct method using '4C-tetraethylammoniumbromide. In vitro a high accuracy was found between applied and MRI measured flow rates in the range from 0.5 to 33 ml/min (r = 0.997; P < 0.001). In vivo the MRI measured left renal vein blood flow was 70% higher in unilaterally nephrectomized animals compared to control animals (3.4 0.4 mI/min/ 100 g body wt vs. 2.0 0.1 ml/min/100 g body wt, P < 0.001). Direct measurements of RBF revealed comparable values (3.4 0.3 ml/min/l00 g body wt vs. 2.3 0.4 ml/min/100 g body wt, P = 0.05). In addition, the left kidney volume was recorded by MRI with an increase amounting to 40% (1.18 0.05 ml vs. 0.84 0.02 ml; P < 0.001) in the nephrectomized
aspects of renal (patho)physiology. Recently, evidence was pro-
vided that MRI can be used for the estimation of glomerular number in situ [7]. MRI is routinely being used in humans and large animals for quantification of blood flow [8] and has also been shown to be applicable to flow measurement in rat popliteal vessels [91. This opens the possibility that MRI may be used for blood flow measurements in kidney related vessels in rodents.
Accordingly, the aim of the present work was to establish a non-invasive MRI-based method for the measurement of renal perfusion in rats. METHODS
MIII measurement system
The velocity measurement system was implemented on a highfield (7 Tesla) experimental MRI scanner (SISCO 300; Varian, Palo Alto, CA, USA) with a bore diameter of 10 cm. The scanner between left renal vein blood flow and MRI measured renal volume (r = was equipped with a 10 gauss/cm field gradient system with a rise 0.91; P < 0.001). In summary, MRI is a non-invasive tool by which measurement of renal vein blood flow can be performed, and it is time of 0.1 ms. A 35 mm diameter dedicated surface RF-coil was concluded that MRI-based renal vein flow measurements can be used to used for optimizing the signal-to-noise ratio. The pulse program estimate RBF in small rodents. was based on a modified phase contrast sequence called FAcE (FID-Acquired-Echo) [10]. This sequence allows short echo times by acquiring left and right echo half-parts separately. The two Changes in renal blood flow and volume are inherent features half-parts are concatenated before a standard two-dimensional of various physiological and pathophysiological conditions, includFourier Transform reconstruction is applied. The through-plane ing pregnancy, diabetes mellitus, ureteral obstruction and con- component of the velocity vector is encoded in the phase part of tralateral nephrectomy [1—6]. Most experimental studies pub- the image by performing two acquisitions with opposite sign of the lished today dealing with the functional and structural changes in velocity sensitivity and subtracting the reconstructed phase imthese conditions are based on invasive methods and examinations ages. The measurement time was 17 mm, repetition time (TR) of tissue in vitro. and echo time (TE) were 250 and 3.5 ms, respectively, slice The applicability of magnetic resonance imaging (MRI) as a thickness 1.5 mm, acquisition matrix 256 X 256, in-plane resolureliable non-invasive monitor of changes in kidney, cortex, me- tion 0.23 x 0.23 mm2 and flip-angle 70°. The velocity sensitivity is dulla and pelvis volume in experimental diabetes was recently defined by the phase-shift/velocity relation: a velocity of 4 cm/ described [1]. The continued development of MRI methods has second introduced a phase-shift of 180° in each of the two opened an even broader usefulness of the method in various encodings. Four signal averages were acquired. The measurement gradient was velocity compensated and the gradient strengths were kept below 6 gauss/cm in order to minimize phase coherence group compared to controls. Finally, a positive correlation was seen
spread across the slice caused by non-ideal response of the
Key words: blood flow, magnetic resonance imaging, renal vein, uninephrectomy, kidney volume. Received for publication December 16, 1996 and in revised form May 30, 1997 Accepted for publication July 7, 1997
gradient amplifier. After locating the renal vein by a set of images with different orientations, five sagittal measurement slices were placed orthogonal to the vessel (Fig. 1). The measurement time was the same for acquisition of up to five slices, as the slices were
© 1997 by the International Society of Nephrology
acquired in an interleaved fashion. The measurements were 1359
1360
Ringgaard et al: Renal blood flow measurement in rats by MRI
0.5 1.0 1.5
2.0 2.5
4.0 4.5 5.0 5.5 6.0
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1 .0 0.5 0.0 Fl, cm
Fig. 1. MRI image of the left kidney and left renal vein in a unincphrectomized rat. The position of 5 measurement planes orthogonal to the vein are indicated with vertical lines. The MRI signal intensity from the renal vein is increased due to flow effects.
analyzed with a dedicated program, that removes offset in the animals had free access to standard rat chow and tap water phase images by subtraction of a plane fitted through stationary throughout the experiment. tissue and removes phase noise from regions with low signal To investigate if the renal vein flow changed over time, five intensity [11]. The flow was determined in the left-most slice control rats were subjected to consecutive flow measurements for entirely free of the kidney. The area of the renal vein and the 21/2 hours. The first measurement was initiated 30 minutes after velocity in the center of the vessel was also calculated from the anesthesia followed by measurements every 20 minutes. Left renal vein blood flow and left kidney volume were meavelocity images. The intra-observer accuracy of the analysis procedure was sured in a group of 8 normal control rats and 8 right-side verified by double analysis of all rat flow measurements by the nephrectomized animals. The control animals were sham-opersame person and a coefficient of variance was determined. ated by flank incision and manipulation of the kidney. All MRI measurements in vitro The flow measurement system was evaluated in vitro by measurements on a 3 mm diameter acrylic pipe flushed with viscosity
operations were performed under anesthesia with sodium barbital (50 mg/kg body weight i.p.). Both groups were MRI scanned 44 days after operation under anesthesia with sodium barbital. Flow
measurements were performed approximately 30 minutes after
increased water. The viscosity was adjusted to 4 centipoise anesthesia and kidney volume measurements were performed (blood-like) with carboxylic methyl cellulose. The flow was driven by a non-pulsatile centrifugal pump and the flow rate, controlled by a valve, covered the range from 0.5 mI/mm to 33 mI/mm. The
applied flow rate was measured by accumulation of the water during the MRI acquisition and weighing it afterwards. Five measurements were performed for each flow rate for the determination of measurement variation and the acquired images were submitted to an analysis procedure as described above. To evaluate the dependence on the angle between the vessel and the slice plane a series of in vitro measurements with angles ranging from 0 to 60° in steps of 10° was performed. MRI measurements in vivo Adult female Wistar rats (Møllegaards Avlslab., Eiby, Denmark) with a mean body weight of 201 2 g were studied. Rats were housed three per cage in a room with 12:12 hours artificial 2°C and humidity 55 2%. The light cycle, temperature 21
immediately after the flow measurement under the same anesthesia [lj. Gadolinium (100 d; Gd-DTPA, Schering, Germany) was
injected for contrast enhancement and Ti weighted spin echo images were obtained in 10 minutes with TR/TE = 500/10 msec. The in-plane resolution was 0.3 X 0.3 mm2 and slice thickness I mm. Sixteen slices covered the kidney with slice gaps varying from 0.5 to 1.0 mm. The volume was calculated by summation of kidney
areas in each slice after manually contouring the kidney [1]. Due to poor image quality in one of the control rats, the left kidney volume was not measured in that animal.
Direct measurements of RBF To compare the MRI measured values of renal vein blood flow with direct measurements of RBF a subset of animals (5 shamoperated and 5 right-side nephrectomized rats) was applied to direct measurements. The clearance studies were performed in animals chronically cannulated with arterial, venous and bladder
1361
Ringgaard et al: Renal blood flow measurement in rats by MRI
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Applied flow rate, mi/rn/n
—2
Fig. 3. Typical distribution of velocities across the rat renal vein in vivo. The shown distribution in cells corresponds to the acquisition resolution (cell side = 0.23 mm). The vessel diameters range from 1.5 to 2.5 mm. The parabolic profile indicates fully developed laminar flow.
Fig. 2. MRI measurements of flow in 3 mm acrylic pipe. Each applied
flow was measured 5 times and the mean measured flow rate plotted
6
against the applied flow rate is shown (dashed line: the identity line).
catheters as previously described [12, 13]. Animals were kept under the same conditions as described in the first experiment and were comparable to these animals in respect to sex, body weight
and procedures during sham-operation and uninephrectomy. Fourty-four days after the operation the animals were anesthetized with sodium barbital as described above and flow measurements performed after anesthesia for 20 to 30 minutes. RBF was estimated by measuring the renal clearance of '4C-tetraethylammoniumbromide (TEA), a marker of the effective renal plasma flow (ERPF) [12, 131 as follows:
.
5
E
0 0 0 C
>3a)
C
ERPF UThA V/PThA RBF = ERPF/(1
hct) 1/0.9
a) a)
where UT,-A and TjA are activities of TEA in urine and plasma; V the urine flow rate; hct the hematocrit and 0.9 the extraction fraction of TEA in the rat kidney [12, 13]. After measurement of
ERPF the kidneys were retrogradely perfusion fixed with 4% formaldehyde through the aorta. The perfusion lasted for four minutes with a constant pressure of 120 mm Hg and by the end of the perfusion weights of the left kidneys were measured.
Statistics All results are given as mean
SEM. Correlation between in vitro MRI measured flow-rate and applied flow was analyzed using
20
40
100 120 140 60 80 Time after anaesthesia, minutes
160
Fig. 4. Left renal vein blood flow as function of time after anesthesia with sodium barbital (50 mg/kg body weight i.p.) in five animals. The left renal vein blood flow was measured every 20 minutes for 2½ hours.
ments were less than 2 standard deviations from the applied flow.
linear regression. Measurements on the nephrectomized and The in vitro test of dependence on angled slice planes showed the control groups were compared with unpaired Mann-Whitney rank sum tests and correlation between kidney volume and flow was tested with Pearson's product momentum. RESULTS
In vitro measurements A close correlation (r = 0.997, P < 0.001) was found between applied and MRI measured flow (Fig. 2) in vitro and all measure-
following deviations at different angles: 10°:0.5%, 20°:3.0%, 30°: 11.2%, 40°:18.3%, 50°:19.3%, 60°:20.3%.
In vivo measurements The velocity distribution across the renal vein of the rats was almost parabolic as expected under stable flow conditions (Fig. 3). The vessels had a diameter of 2 to 3 mm and thus the number of pixels across the vessels were 8 to 12. The coefficient of variance
1362
Ringgaard ci al. Renal blood flow measurement in rats by MR/
B
Kidney volume, ml
12
(1)
A
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Velocity, cm/sec
0.06
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r'a
sham-operated (E) and uninephrectomized () rats. Data are given as mean + SEM. *p < 0.05,
0
0.00
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Fig. 5. Left renal vein blood flow, kidney volume, vessel area and maximum velocity in
0
0.02
a
'4
CU
< 0.001. NS: not significant.
uninephrectomized group when compared to the control group
-
0.8 mI/mm vs. 4.2 0.2 ml/min, P < 0.001) or by 70% when expressed relative to body weight (3.4 0.4 mI/mm/IOU g body wt vs. 2.0 0.1 mI/mm/lOU g body wt, P < 0.001). Furthermore, the (7.5
.
.
1.4
cross sectional area of the renal vein had increased by 35% 0.004 cm2 vs. 0.031 0.004 cm2, P < 0.05) in the (0.042
1.3
uninephrectomized group compared to the control group, while no significant change was seen in the velocity in the centre of the 0.5 cm/second vs. 4.0 0.3 cm/second, NS). The vein (4.6 kidney volume increased by 40% in uninephrcctomized animals compared to the control group, (Fig. 5). A tight correlation (r = 0.91, P < 0.001) was found between left renal vein blood flow and kidney volume, (Fig. 6). Direct measurements of RBF in a subset of uninephrectomized and control animals revealed single kidney RBF values of 8.0 (1.7 mI/mm vs. 5.6 1.0 mI/mm (P = 0.09), respectively, or when expressed relative to body weight to 3.4 0.3 ml/min/100 g body wt versus 2.3 0.4 ml/min/l00 g body wt, (P = 0.05). In addition, perfusion fixed left kidney weight increased by 50% in uninephrectomized animals compared to the control group (P < 0.01).
..
1.1
>, ci)
1.0
0.9 0.8 0.7
2
4
6
8
10
12
14
Left renal blood flow, mi/mm
DISCUSSION
Fig. 6. Correlation between left renal vein blood flow and kidney volumes The aim of this study was to establish a MRI based method for in sham-operated (ts) and uninephrectomized (•) animals (r = 0.91, P < renal vein blood flow measurements in rats. The primary advan0.001). tage of the present technique in comparison with previously
described methods for blood flow measurements [3, 4, 6, 14, 15j is that it is non-invasive and an uncomplicated tool for investigators
found by double analysis of the blood flow measurements was with availability to MRI facilities. The method requires some 11% (range 0.08% to 22%). The left renal vein blood flow rate did not change significantly during the first hour after anesthesia (Fig. 4). After 60 minutes the flow decreased in all animals. The total decline over the 2½ hours of measurement varied between 11% and 58%.
The left renal vein blood flow rate increased by 79% in the
operator experience to locate the renal vessels on the MRI images, to position the slices for measurement correctly on the planning images and to trace the vein accurately during analysis. However, having gained that experience renal vein blood flow measurements can he recorded within thirty minutes and the data analysis performed within fifteen minutes.
Ringgaard Ct a!: Renal blood flow measurement in rats by MRJ
When using MRI based methods the achievement of sufficient signal-to-noise ratio is often a limiting factor for spatial resolution and reliability. In the present study the signal-to-noise ratio was optimized by use of a specially designed RF surface coil with a diameter of 35 mm, which allowed signal extraction from central regions of the rats.
1363
can be used as an estimate of RBF in small rodents. Finally, the method may be used for assessment of vessel flow in other veins of small rodents. ACKNOWLEDGMENTS The study was supported by grants from The Danish Heart Foundation,
The accuracy of the system was tested in vitro by MRI mea- the Danish Diabetes Association, the Danish Medical Research Council, surements of known flow in a thin acrylic pipe. The in vivo the Ruth Kønig Petersen Foundation, the Novo Foundation, the Nordic conditions were mimicked by using a pipe with diameter similar to the rat renal vein, water with blood-like viscosity and flow rates covering the physiologically interesting range. In this in vitro setup a high accuracy was achieved between applied and measured flow.
Insulin Foundation, the Aage Louis Petersen Foundation, Institute of Experimental Clinical Research, Aarhus University and the Karen Elise Jensen Foundation. Prof. J.C. Djurhuus and Prof. H. are thanked for critically reading the manuscript, and research technician Preben Daugbrd is
The flow measurements were not influenced significantly by gratefully acknowledged for designing the RF coil. measurement planes not being exactly orthogonal to the pipe (that is, for angling up to 200) as the decrease in the through-plane
component of the velocity due to angulation is compensated for
by an increase in intersection area. This is crucial for in vivo measurements, where the vessels may be irregularly twisted.
Blood flow measurements were performed in the renal vein instead of the artery for two reasons. First, the diameter of the renal vein is 2 to 3 mm and therefore easier to locate than the artery, which is only around 0.5 mm. Secondly, the vein blood flow
is almost non-pulsatile, which means that cardiac gating is not necessary. The decrease in renal vein blood flow over time, observed in the
present study, may be due to either a gradual decrease in body temperature during the stay in the magnet or an effect of sodium barbital anesthesia. It is noteworthy, however, that the renal vein blood flow was stable within one hour after induction of anaesthesia and that the MRI measurement is completed within a time span of thirty minutes. As stated above the main purpose of the present study was to establish an MRI based method for measuring renal vein blood
flow that could be used as an estimate of RBF in various (patho)physiological conditions in rodents. This aim was achieved as the absolute values of single renal vein flow rates in normal rats
Reprint requests to Steffen Ringgaard, M. Sc., Centre of Magnetic Resonance, Skejhy Sygehus, Brendstrupgaardsvej, DK-8200 Aarhus N. Denmark. E-mail: [email protected]
REFERENCES I. CI-IRISTIANSEN T, RASCH R, STØDKILDE-JØRGENSEN
H, FLYVBJERG A:
Relationship between MRI and morphometric measurements of kidney size, cortex, medulla and pelvis volumes in diabetic and
non-diabetic rats. Kidney mt 51:50—56, 1997 2. HAYSLEI-r JP: Effect of age on compensatory renal growth. Kidney mt 23:599—602, 1983 3. JENSEN PK, SANDAHL CHRISTIANSEN J, STEVEN K, PARVING H-H: Renal function in streptozotocin-diabetic rats. Diahetologia 21:409 — 414, 1981 4. SEvER-HANSEN K, HANSEN J, GUNDERSEN HJG: Renal hypertrophy in
experimental diabetes. Diabetologia 18:501—505, 1980 5. FINE L: The biology of renal hypertrophy. (Editorial review) Kidney mt 29:619—634, 1986 6. TUCKER BJ, MENDONCA MM, BLANTZ RC: Contrasting effects of
acute insulin infusion on renal function in awake nondiabetic and diabetic rats. JAm Soc Nephrol 3:1686—1693, 1993 7. BASGEN JM, STEFFES MW, STILLMAN AE, MAUER SM: Estimating
glomerular number in situ using magnetic resonance imaging and biopsy. Kidney lot 45:1668—1672, 1994 8. SZOLAR DH, SAKUMA H, HIGGINS CB: Cardiovascular applications of
magnetic resonance flow and velocity measurements. J Magn Reson Imaging 6:78—89, 1996 9. MEYER RA, COOPER TG, POTCHEN EJ: Measurement of blood flow in
were comparable both to the RBF measurements performed in the present study and to previously published measurements of rat popliteal vessels by phase contrast MR angiography. (abstract) Society of Magnetic Resonance Annual Meeting, San Francisco, 1994, p RBF. In the present study renal vein blood flow in normal rats 992 averaged 2.0 ml/minhlOO g body wt, which is comparable to the 10. SCHEIDEGGER MB, MALER SE, BOSIGER P: FID-Acquired-Echos single kidney RBF values obtained in the present and previous (FAcE): A short echo time imaging method for flow artefact suppresstudies by measurements both in anesthetized [3, 15] and consion. Magn Reson Imaging 9:517—524, 1991 scious [6] animals with values ranging from 2 to 2½ mI/mm/IOU g
body wt. In addition, the increase in renal vein blood flow obtained in unilaterally nephrectomized animals by this method
corroborated well with the present and previously published values, both in respect to absolute and relative changes [2, 4, 16, 17]. Furthermore, an expected tight correlation between renal vein blood flow and kidney volume was observed. Finally, the increase in renal vein blood flow in unilaterally nephrectomized animals was due mainly to an increase in vessel diameter with insignificant changes in blood velocity. The importance of this observation is still unknown. In conclusion, the present data indicate that MRI is a reliable non-invasive tool for measuring renal vein blood flow in rats. The
accuracy of the method was tested in vitro and in vivo in a
11. WALKER PG, CRANNEY GB, SCHEIDEGGER MB, WASELESKI G, Po-
HOST GM, YOGANATHAN AP: Semiautomated method for noise
reduction and background phase error correction in MR phase velocity data. J Magn Reson Imaging 3:52 1—530, 1993 12. PETERSEN JS, CHRISTENSEN S: Superiority of tetraethylammonium to
p-amino-hippurate as a marker for plasma flow during furosemide diurcsis. Renal Physiol 10:102—109, 1987 13. RASMUSSEN SN, SHALMI M, HANSEN M, CHRISTENSEN S: Tetraethyl-
ammonium and p-aminohippurate as clearance markers for renal plasma flow in the rat during saline and glucose infusion. Renal Physiol Biochem 13:314—323, 1990 14. HAYLOR J, SINGH I, EL NAHAS MA: Nitric oxide synthesis inhibitor
prevents vasodilation by insulin-like growth factor I. Kidney mt 39:333—335, 1991
15. HIRSCHBERG RR, KOPPLE JD: Evidence that insulin-like growth factor
I increases renal plasma flow and glomerular filtration rate in fasted rats. J Clin Invest 83:326—330, 1989
well-known model of renal hyperperfusion. The renal vein blood 16. F0TINO S: The solitary kidney: A model of chronic hyperfiltration in humans. Am J Kidney Dis 13:88—98, 1989 flow rates obtained both in normal and uninephrectomized rats 17. KAUFMAN JM, SIEGEL. NJ, HAYSLE1T JP: Functional and hemodywere similar to RBF levels obtained by direct, invasive methods, namic adaptation to progressive renal ablation. Circ Re.s 36:286—293, and it is concluded that MRI based renal vein flow measurements 1975
VASCULAR BIOLOGY - HEMODYNAMICS - HYPERTENSION
Measurement of renal vein blood flow in rats by high-field magnetic resonance STEFFEN RINGGAARD, THORA CHRISTIANSEN, MARTIN BAJ, ERIK M. PEDERSEN, HANS STØDKLLDE-JORGENSEN, and ALLAN FLYVBJERG Magnetic Resonance Research Centre, Aarhus University Hospital, Institute of Experimental Clinical Research, Aarhus University, and Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Aarhus, Denmark
Measurement of renal vein blood flow in rats by high-field magnetic resonance. The aim of the present study was to examine whether magnetic resonance imaging (MRI) based method for non-invasive in vivo measurement of vein blood flow in rats could be used to estimate renal blood flow (RBF). Measurements were performed using a high-field (7 Tesla) MRI scanner with a short echo time phase contrast velocity measurement pulse sequence. The method was evaluated in vitro by flow measurements in an acrylic pipe and in vivo by recording left renal vein blood flow in normal
and unilaterally nephrectomized rats. In a subset of animals RBF was measured by a direct method using '4C-tetraethylammoniumbromide. In vitro a high accuracy was found between applied and MRI measured flow rates in the range from 0.5 to 33 ml/min (r = 0.997; P < 0.001). In vivo the MRI measured left renal vein blood flow was 70% higher in unilaterally nephrectomized animals compared to control animals (3.4 0.4 mI/min/ 100 g body wt vs. 2.0 0.1 ml/min/100 g body wt, P < 0.001). Direct measurements of RBF revealed comparable values (3.4 0.3 ml/min/l00 g body wt vs. 2.3 0.4 ml/min/100 g body wt, P = 0.05). In addition, the left kidney volume was recorded by MRI with an increase amounting to 40% (1.18 0.05 ml vs. 0.84 0.02 ml; P < 0.001) in the nephrectomized
aspects of renal (patho)physiology. Recently, evidence was pro-
vided that MRI can be used for the estimation of glomerular number in situ [7]. MRI is routinely being used in humans and large animals for quantification of blood flow [8] and has also been shown to be applicable to flow measurement in rat popliteal vessels [91. This opens the possibility that MRI may be used for blood flow measurements in kidney related vessels in rodents.
Accordingly, the aim of the present work was to establish a non-invasive MRI-based method for the measurement of renal perfusion in rats. METHODS
MIII measurement system
The velocity measurement system was implemented on a highfield (7 Tesla) experimental MRI scanner (SISCO 300; Varian, Palo Alto, CA, USA) with a bore diameter of 10 cm. The scanner between left renal vein blood flow and MRI measured renal volume (r = was equipped with a 10 gauss/cm field gradient system with a rise 0.91; P < 0.001). In summary, MRI is a non-invasive tool by which measurement of renal vein blood flow can be performed, and it is time of 0.1 ms. A 35 mm diameter dedicated surface RF-coil was concluded that MRI-based renal vein flow measurements can be used to used for optimizing the signal-to-noise ratio. The pulse program estimate RBF in small rodents. was based on a modified phase contrast sequence called FAcE (FID-Acquired-Echo) [10]. This sequence allows short echo times by acquiring left and right echo half-parts separately. The two Changes in renal blood flow and volume are inherent features half-parts are concatenated before a standard two-dimensional of various physiological and pathophysiological conditions, includFourier Transform reconstruction is applied. The through-plane ing pregnancy, diabetes mellitus, ureteral obstruction and con- component of the velocity vector is encoded in the phase part of tralateral nephrectomy [1—6]. Most experimental studies pub- the image by performing two acquisitions with opposite sign of the lished today dealing with the functional and structural changes in velocity sensitivity and subtracting the reconstructed phase imthese conditions are based on invasive methods and examinations ages. The measurement time was 17 mm, repetition time (TR) of tissue in vitro. and echo time (TE) were 250 and 3.5 ms, respectively, slice The applicability of magnetic resonance imaging (MRI) as a thickness 1.5 mm, acquisition matrix 256 X 256, in-plane resolureliable non-invasive monitor of changes in kidney, cortex, me- tion 0.23 x 0.23 mm2 and flip-angle 70°. The velocity sensitivity is dulla and pelvis volume in experimental diabetes was recently defined by the phase-shift/velocity relation: a velocity of 4 cm/ described [1]. The continued development of MRI methods has second introduced a phase-shift of 180° in each of the two opened an even broader usefulness of the method in various encodings. Four signal averages were acquired. The measurement gradient was velocity compensated and the gradient strengths were kept below 6 gauss/cm in order to minimize phase coherence group compared to controls. Finally, a positive correlation was seen
spread across the slice caused by non-ideal response of the
Key words: blood flow, magnetic resonance imaging, renal vein, uninephrectomy, kidney volume. Received for publication December 16, 1996 and in revised form May 30, 1997 Accepted for publication July 7, 1997
gradient amplifier. After locating the renal vein by a set of images with different orientations, five sagittal measurement slices were placed orthogonal to the vessel (Fig. 1). The measurement time was the same for acquisition of up to five slices, as the slices were
© 1997 by the International Society of Nephrology
acquired in an interleaved fashion. The measurements were 1359
1360
Ringgaard et al: Renal blood flow measurement in rats by MRI
0.5 1.0 1.5
2.0 2.5
4.0 4.5 5.0 5.5 6.0
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1 .0 0.5 0.0 Fl, cm
Fig. 1. MRI image of the left kidney and left renal vein in a unincphrectomized rat. The position of 5 measurement planes orthogonal to the vein are indicated with vertical lines. The MRI signal intensity from the renal vein is increased due to flow effects.
analyzed with a dedicated program, that removes offset in the animals had free access to standard rat chow and tap water phase images by subtraction of a plane fitted through stationary throughout the experiment. tissue and removes phase noise from regions with low signal To investigate if the renal vein flow changed over time, five intensity [11]. The flow was determined in the left-most slice control rats were subjected to consecutive flow measurements for entirely free of the kidney. The area of the renal vein and the 21/2 hours. The first measurement was initiated 30 minutes after velocity in the center of the vessel was also calculated from the anesthesia followed by measurements every 20 minutes. Left renal vein blood flow and left kidney volume were meavelocity images. The intra-observer accuracy of the analysis procedure was sured in a group of 8 normal control rats and 8 right-side verified by double analysis of all rat flow measurements by the nephrectomized animals. The control animals were sham-opersame person and a coefficient of variance was determined. ated by flank incision and manipulation of the kidney. All MRI measurements in vitro The flow measurement system was evaluated in vitro by measurements on a 3 mm diameter acrylic pipe flushed with viscosity
operations were performed under anesthesia with sodium barbital (50 mg/kg body weight i.p.). Both groups were MRI scanned 44 days after operation under anesthesia with sodium barbital. Flow
measurements were performed approximately 30 minutes after
increased water. The viscosity was adjusted to 4 centipoise anesthesia and kidney volume measurements were performed (blood-like) with carboxylic methyl cellulose. The flow was driven by a non-pulsatile centrifugal pump and the flow rate, controlled by a valve, covered the range from 0.5 mI/mm to 33 mI/mm. The
applied flow rate was measured by accumulation of the water during the MRI acquisition and weighing it afterwards. Five measurements were performed for each flow rate for the determination of measurement variation and the acquired images were submitted to an analysis procedure as described above. To evaluate the dependence on the angle between the vessel and the slice plane a series of in vitro measurements with angles ranging from 0 to 60° in steps of 10° was performed. MRI measurements in vivo Adult female Wistar rats (Møllegaards Avlslab., Eiby, Denmark) with a mean body weight of 201 2 g were studied. Rats were housed three per cage in a room with 12:12 hours artificial 2°C and humidity 55 2%. The light cycle, temperature 21
immediately after the flow measurement under the same anesthesia [lj. Gadolinium (100 d; Gd-DTPA, Schering, Germany) was
injected for contrast enhancement and Ti weighted spin echo images were obtained in 10 minutes with TR/TE = 500/10 msec. The in-plane resolution was 0.3 X 0.3 mm2 and slice thickness I mm. Sixteen slices covered the kidney with slice gaps varying from 0.5 to 1.0 mm. The volume was calculated by summation of kidney
areas in each slice after manually contouring the kidney [1]. Due to poor image quality in one of the control rats, the left kidney volume was not measured in that animal.
Direct measurements of RBF To compare the MRI measured values of renal vein blood flow with direct measurements of RBF a subset of animals (5 shamoperated and 5 right-side nephrectomized rats) was applied to direct measurements. The clearance studies were performed in animals chronically cannulated with arterial, venous and bladder
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Ringgaard et al: Renal blood flow measurement in rats by MRI
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20
30
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Applied flow rate, mi/rn/n
—2
Fig. 3. Typical distribution of velocities across the rat renal vein in vivo. The shown distribution in cells corresponds to the acquisition resolution (cell side = 0.23 mm). The vessel diameters range from 1.5 to 2.5 mm. The parabolic profile indicates fully developed laminar flow.
Fig. 2. MRI measurements of flow in 3 mm acrylic pipe. Each applied
flow was measured 5 times and the mean measured flow rate plotted
6
against the applied flow rate is shown (dashed line: the identity line).
catheters as previously described [12, 13]. Animals were kept under the same conditions as described in the first experiment and were comparable to these animals in respect to sex, body weight
and procedures during sham-operation and uninephrectomy. Fourty-four days after the operation the animals were anesthetized with sodium barbital as described above and flow measurements performed after anesthesia for 20 to 30 minutes. RBF was estimated by measuring the renal clearance of '4C-tetraethylammoniumbromide (TEA), a marker of the effective renal plasma flow (ERPF) [12, 131 as follows:
.
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E
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>3a)
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ERPF UThA V/PThA RBF = ERPF/(1
hct) 1/0.9
a) a)
where UT,-A and TjA are activities of TEA in urine and plasma; V the urine flow rate; hct the hematocrit and 0.9 the extraction fraction of TEA in the rat kidney [12, 13]. After measurement of
ERPF the kidneys were retrogradely perfusion fixed with 4% formaldehyde through the aorta. The perfusion lasted for four minutes with a constant pressure of 120 mm Hg and by the end of the perfusion weights of the left kidneys were measured.
Statistics All results are given as mean
SEM. Correlation between in vitro MRI measured flow-rate and applied flow was analyzed using
20
40
100 120 140 60 80 Time after anaesthesia, minutes
160
Fig. 4. Left renal vein blood flow as function of time after anesthesia with sodium barbital (50 mg/kg body weight i.p.) in five animals. The left renal vein blood flow was measured every 20 minutes for 2½ hours.
ments were less than 2 standard deviations from the applied flow.
linear regression. Measurements on the nephrectomized and The in vitro test of dependence on angled slice planes showed the control groups were compared with unpaired Mann-Whitney rank sum tests and correlation between kidney volume and flow was tested with Pearson's product momentum. RESULTS
In vitro measurements A close correlation (r = 0.997, P < 0.001) was found between applied and MRI measured flow (Fig. 2) in vitro and all measure-
following deviations at different angles: 10°:0.5%, 20°:3.0%, 30°: 11.2%, 40°:18.3%, 50°:19.3%, 60°:20.3%.
In vivo measurements The velocity distribution across the renal vein of the rats was almost parabolic as expected under stable flow conditions (Fig. 3). The vessels had a diameter of 2 to 3 mm and thus the number of pixels across the vessels were 8 to 12. The coefficient of variance
1362
Ringgaard ci al. Renal blood flow measurement in rats by MR/
B
Kidney volume, ml
12
(1)
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0.06
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sham-operated (E) and uninephrectomized () rats. Data are given as mean + SEM. *p < 0.05,
0
0.00
1.5
Fig. 5. Left renal vein blood flow, kidney volume, vessel area and maximum velocity in
0
0.02
a
'4
CU
< 0.001. NS: not significant.
uninephrectomized group when compared to the control group
-
0.8 mI/mm vs. 4.2 0.2 ml/min, P < 0.001) or by 70% when expressed relative to body weight (3.4 0.4 mI/mm/IOU g body wt vs. 2.0 0.1 mI/mm/lOU g body wt, P < 0.001). Furthermore, the (7.5
.
.
1.4
cross sectional area of the renal vein had increased by 35% 0.004 cm2 vs. 0.031 0.004 cm2, P < 0.05) in the (0.042
1.3
uninephrectomized group compared to the control group, while no significant change was seen in the velocity in the centre of the 0.5 cm/second vs. 4.0 0.3 cm/second, NS). The vein (4.6 kidney volume increased by 40% in uninephrcctomized animals compared to the control group, (Fig. 5). A tight correlation (r = 0.91, P < 0.001) was found between left renal vein blood flow and kidney volume, (Fig. 6). Direct measurements of RBF in a subset of uninephrectomized and control animals revealed single kidney RBF values of 8.0 (1.7 mI/mm vs. 5.6 1.0 mI/mm (P = 0.09), respectively, or when expressed relative to body weight to 3.4 0.3 ml/min/100 g body wt versus 2.3 0.4 ml/min/l00 g body wt, (P = 0.05). In addition, perfusion fixed left kidney weight increased by 50% in uninephrectomized animals compared to the control group (P < 0.01).
..
1.1
>, ci)
1.0
0.9 0.8 0.7
2
4
6
8
10
12
14
Left renal blood flow, mi/mm
DISCUSSION
Fig. 6. Correlation between left renal vein blood flow and kidney volumes The aim of this study was to establish a MRI based method for in sham-operated (ts) and uninephrectomized (•) animals (r = 0.91, P < renal vein blood flow measurements in rats. The primary advan0.001). tage of the present technique in comparison with previously
described methods for blood flow measurements [3, 4, 6, 14, 15j is that it is non-invasive and an uncomplicated tool for investigators
found by double analysis of the blood flow measurements was with availability to MRI facilities. The method requires some 11% (range 0.08% to 22%). The left renal vein blood flow rate did not change significantly during the first hour after anesthesia (Fig. 4). After 60 minutes the flow decreased in all animals. The total decline over the 2½ hours of measurement varied between 11% and 58%.
The left renal vein blood flow rate increased by 79% in the
operator experience to locate the renal vessels on the MRI images, to position the slices for measurement correctly on the planning images and to trace the vein accurately during analysis. However, having gained that experience renal vein blood flow measurements can he recorded within thirty minutes and the data analysis performed within fifteen minutes.
Ringgaard Ct a!: Renal blood flow measurement in rats by MRJ
When using MRI based methods the achievement of sufficient signal-to-noise ratio is often a limiting factor for spatial resolution and reliability. In the present study the signal-to-noise ratio was optimized by use of a specially designed RF surface coil with a diameter of 35 mm, which allowed signal extraction from central regions of the rats.
1363
can be used as an estimate of RBF in small rodents. Finally, the method may be used for assessment of vessel flow in other veins of small rodents. ACKNOWLEDGMENTS The study was supported by grants from The Danish Heart Foundation,
The accuracy of the system was tested in vitro by MRI mea- the Danish Diabetes Association, the Danish Medical Research Council, surements of known flow in a thin acrylic pipe. The in vivo the Ruth Kønig Petersen Foundation, the Novo Foundation, the Nordic conditions were mimicked by using a pipe with diameter similar to the rat renal vein, water with blood-like viscosity and flow rates covering the physiologically interesting range. In this in vitro setup a high accuracy was achieved between applied and measured flow.
Insulin Foundation, the Aage Louis Petersen Foundation, Institute of Experimental Clinical Research, Aarhus University and the Karen Elise Jensen Foundation. Prof. J.C. Djurhuus and Prof. H. are thanked for critically reading the manuscript, and research technician Preben Daugbrd is
The flow measurements were not influenced significantly by gratefully acknowledged for designing the RF coil. measurement planes not being exactly orthogonal to the pipe (that is, for angling up to 200) as the decrease in the through-plane
component of the velocity due to angulation is compensated for
by an increase in intersection area. This is crucial for in vivo measurements, where the vessels may be irregularly twisted.
Blood flow measurements were performed in the renal vein instead of the artery for two reasons. First, the diameter of the renal vein is 2 to 3 mm and therefore easier to locate than the artery, which is only around 0.5 mm. Secondly, the vein blood flow
is almost non-pulsatile, which means that cardiac gating is not necessary. The decrease in renal vein blood flow over time, observed in the
present study, may be due to either a gradual decrease in body temperature during the stay in the magnet or an effect of sodium barbital anesthesia. It is noteworthy, however, that the renal vein blood flow was stable within one hour after induction of anaesthesia and that the MRI measurement is completed within a time span of thirty minutes. As stated above the main purpose of the present study was to establish an MRI based method for measuring renal vein blood
flow that could be used as an estimate of RBF in various (patho)physiological conditions in rodents. This aim was achieved as the absolute values of single renal vein flow rates in normal rats
Reprint requests to Steffen Ringgaard, M. Sc., Centre of Magnetic Resonance, Skejhy Sygehus, Brendstrupgaardsvej, DK-8200 Aarhus N. Denmark. E-mail: [email protected]
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