Cortical and medullary betaine-GPC modulated by osmolality
independently of oxygen in the intact kidney
Gary J.
Cowin,
Stuart
Crozier,
Zoltan H.
Endre,
I. Anne
Leditschke, and
Ian M.
Brereton
Renal Research Unit, Department of Medicine and Centre for Magnetic
Resonance, University of Queensland, Brisbane, Queensland 4029, Australia
 |
ABSTRACT |
Renal osmolyte concentrations are reduced during reflow
following ischemia. Osmolyte decreases may follow oxygen
depletion or loss of extracellular osmolality in the medulla.
Image-guided volume-localized magnetic resonance (MR) microspectroscopy
was used to monitor regional osmolytes during hyposmotic shock and hypoxia in the intact rat kidney. Alternate spectra were acquired from
24-µl voxels in cortex and medulla of the isolated perfused kidney.
There was a progressive decrease in the combined
betaine-glycerophosphorylcholine (GPC) peak intensity of 21% in cortex
and 35% in medulla of normoxic kidneys between 60 and 160 min after
commencing perfusion. Hypoxia had no significant effect on the
betaine-GPC peak intensity in cortex or medulla, despite a dramatic
reduction in tubular sodium, potassium, and water reabsorption. The
results suggest that cortical and medullary intracellular osmolyte
concentrations depend on osmotically regulated channels that are
insensitive to oxygen and dissociated from the oxygen-dependent
parameters of renal function, the fractional excretion of sodium, the
fractional excretion of potassium, and urine-to-plasma inulin
concentration ratio.
renal osmolytes; volume-localized magnetic resonance spectroscopy; isolated perfused rat kidney; hyposmotic shock
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INTRODUCTION |
RENAL MEDULLARY CELLS are subject to high and variable
osmolality as a consequence of the high medullary interstitial
osmolality generated by the countercurrent multiplier mechanism (35).
The medullary cells balance the extracellular osmolality by
concentrating organic osmolytes (9, 43), which protect the cells from
high ionic concentrations in the interstitial space. These osmolytes include the methylamines, glycerophosphorylcholine (GPC) and betaine, the polyols, inositol and sorbitol, and amino acids such as taurine (19). In high concentrations, these osmolytes have no effect on protein
function in contrast to high concentrations of ions such as
Na+,
K+, and
Cl
. In addition, osmolytes
stabilize proteins against the high intracellular concentrations of
urea present in medullary cells (8).
Cellular regulation of osmolyte concentrations in response to osmotic
variation has been extensively studied in regional samples of excised
kidney (2, 4, 5, 16, 23, 32, 37), freshly isolated medullary cells (6,
39), and in renal cell lines (30) such as Madin-Darby canine kidney
(MDCK) (31) and PAP-HT25 (20) and GRB-PAP1 (3) derived from rabbit
papilla. The mechanisms by which cellular osmolyte
concentration is regulated include the active uptake of betaine and
inositol, the synthesis of sorbitol from glucose by aldose reductase,
and the degradation of GPC by GPC:choline phosphodiesterase (8). The
response to increased extracellular osmolality is slow, requiring hours
to days for upregulation of mRNA for transporters and enzymes (8). The
initial response to increased interstitial osmolality is cell shrinkage, resulting in a nonspecific increase in intracellular osmolality. It has been suggested that amino acids contribute to
balancing the intracellular osmolality during this initial response
(8).
In contrast to the slow response to hyperosmolality, intracellular
osmolytes are lost rapidly following a reduction in extracellular osmolality (29) or acute induction of diuresis (42). For example, an
instantaneous decrease in the osmolality of the incubation medium of
isolated inner medullary collecting duct cells from 600 to 300 mosmol/kg, so called "hyposmotic shock," results in a rapid
influx of water and cell swelling (to 152 ± 15% of control) followed by a return to the original cell volume within 15 min (22).
The initial cell swelling is accompanied by a decrease in intracellular
Na+,
K+, and
Cl
. Although no recovery of
the cellular content of Na+ and
Cl
occurs over 1 h,
cellular K+ content recovers by 10 min and then gradually increases, restoring the sum of the cellular
Na+,
K+, and
Cl
concentrations after 60 min of incubation (22). This is consistent with a minor role for these
ions in balancing the reduction in extracellular osmolality. The rapid
efflux of osmolytes is followed by slow downregulation of the four
mechanisms of osmolyte accumulation described above (31, 39). The
initial rapid release of osmolytes is thought to result from osmotic
regulation of channels (25) rather than from nonspecific cell damage
(39).
Only one study has examined the regional response of osmolytes to
ischemia, a combined electron microprobe-HPLC study by Beck et
al. (4) in rat kidney following renal artery clamping. A change in
medullary osmolyte concentrations was not detected during ischemia. Osmolyte concentrations were decreased following 1 h of reperfusion and did not recover after 24 h of reperfusion (4). In
contrast, osmolytes in the cortex changed minimally and had returned to
control values at 24 h. The extracellular ionic concentration decreased
during ischemia. This would have caused or accompanied cell
swelling. Such a hyposmotic response should cause an efflux of
osmolytes as discussed above. However, because the hyposmotic response
was caused by restricting oxygen delivery, the direct effect of oxygen
supply upon regulation of osmolyte concentration could not be
differentiated from the effect of reduced extracellular osmolality. The
present study was designed to address this question in cortical and
medullary regions of the intact kidney.
Proton magnetic resonance spectroscopy
(1H-MRS) can detect osmolytes in
the intact kidney. This technique was critical in identifying osmolytes
in early studies in cortical and medullary protein-free extracts (23).
More recently, localized 1H-MRS
has allowed the nondestructive monitoring of osmolytes in the human
kidney in vivo (13). A localization volume of 15.6 cm3 enabled an average intensity
of combined osmolyte signals to be determined for the whole kidney. The
intensity of the osmolyte region of the spectrum increased with mild
dehydration and decreased following rehydration (13). The same pattern
of change occurred with the urine osmolality and suggested a
correlation between renal osmolality and osmolyte concentration.
However, the volumes monitored are too large to allow regional
differences in osmolyte concentrations to be observed. Similarly,
31P-MRS of the whole kidney in
vivo has been used to investigate the response of GPC to various
methods of acute diuresis in the rabbit (41, 42), without regional
discrimination of osmolyte concentrations necessary to differentiate
osmolyte responses unique to the cortex and medulla.
Using MR microspectroscopy in the isolated perfused rat kidney (IPRK),
we recently achieved regional localization of osmolytes within the
kidney at much greater spectral resolution than is possible in vivo
(10). This was possible because the IPRK is unaffected by artifacts due
to movement and sample inhomogeneity as occur in vivo. The IPRK is a
widely used model, which allows plasma flow to be regulated
independently of oxygen delivery. By combining
1H-MR microimaging and
volume-localized MR microspectroscopy, we acquired
1H spectra from both the cortex
and the medulla of the IPRK (10). The medullary spectrum was dominated
by a large peak at 3.2 ppm containing superimposed peaks from betaine
and GPC and peaks at 3.4 and 3.5 ppm that contain a contribution from
inositol. Osmolytes were less prominent in the more complex cortical
spectrum, presumably reflecting the greater metabolic diversity of the cortex.
The present study aimed to compare the effects of hyposmotic shock and
oxygen deprivation as stimuli for osmolyte release. Image-guided
1H-MR microspectroscopy was used
to monitor time-dependent changes in osmolyte concentrations in the
cortex and medulla in the intact perfused kidney in the presence and
absence of hypoxia. The hypoxic IPRK model allowed separation of
osmotic and oxygen-dependent changes that occur simultaneously with
ischemia. The differentiated regional response of osmolytes to
osmotic and oxygen availability were then correlated with parameters of
global renal function.
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MATERIALS AND METHODS |
Kidney perfusion. Right kidneys
(average weight 0.7 g) from male Sprague-Dawley rats (weighing
150-170 g) were perfused at 37°C with Krebs-Henseleit buffer
containing bovine serum albumin (6.7 g/100 ml), glucose, 20 amino acids (17), 1 µCi of
[14C]inulin and gassed
with 95% O2-5%
CO2 as previously described (11,
33). Renal artery perfusion pressure was measured directly within the
perfusion cannula via a polyethylene line to a pressure transducer
(Abbott Australasia). Kidney perfusion pressure was held constant
between 90 and 110 mmHg by a process controller (model 2073; West
Instruments, Brighton, UK) that regulated the speed of the peristaltic
pump (model 503U; Smith and Nephew, Watson-Marlow, Cornwall, UK).
Perfusate flow was monitored by wide-beam ultrasound using a Transonic
model T206 flow meter with an inline cannulating flow
probe (model SN22; Transonic Systems, Ithaca, NY). Outputs from the
pressure transducer and the flow meter were connected to a Biopac data
acquisition system and monitored and recorded using AcqKnowledge
software (Biopac Systems, Goleta, CA).
The perfused kidney was placed in a modified (10) 20-mm nuclear
magnetic resonance (NMR) tube (Wilmad, Buena, NJ). The cannula, perfusion line, and tubing from the ureter were attached to a Perspex
holder that supported one side of the kidney to facilitate insertion
into the NMR tube and positioning of the kidney in close proximity to
the surface coil (modified after a holder described in the unpublished
observations of K.-P. Fichtner, Z. H. Endre, W. Kriz, U. Werner, S. Pomer, E. Ritz, and W. E. Hull). This also positioned the kidney in the center of the sensitive volume of the
15-cm bore magnet. The kidney was submerged in the venous effluent,
which was recirculated via two return lines. All lines that passed in
and out the top of the NMR tube and probe were water-jacketed from a
temperature-regulated water bath. The water bath temperature (41°C)
was precalibrated to maintain a temperature of 37°C for the
perfusate delivered to the kidney within the magnet. For these
experiments, the ureter was cannulated with a piece of 0.28 mm (ID) × 0.61 mm (OD) polyethylene tubing ~40 mm in length, which was
attached to ~1 m of 0.58 mm (ID) × 0.96 mm (OD) polyethylene tubing that passed out the bottom of the NMR tube through a glass tube
fused into the base of the NMR tube, which rose above the height of the
venous effluent (10). The larger bore tubing enabled the urine to flow
by gravity so there was no back pressure on the kidney. Perfusate
samples were collected to coincide with the start of urine collections
(10- to 15-min periods). Perfusion pressure and flow were monitored
continuously. Kidney function was estimated from
[14C]inulin clearance
[measurement of glomerular filtration rate (GFR)] and
electrolyte excretion (Na+ and
K+).
[14C]inulin activity
was measured in an LKB 1217 Rackbeta scintillation counter.
Na+ and
K+ were measured by flame
photometry (FLM3; Radiometer, Copenhagen, Denmark). Renal function was
assessed from urine flow, ratio of [14C]inulin in urine
to plasma
[(U/P)inulin], GFR,
fractional excretion of sodium
(FENa), and the fractional
excretion of potassium (FEK).
Two groups of kidneys (each n = 5)
were studied. Control kidneys were perfused for ~160-170 min
without perturbation. Hypoxia was induced by switching the gassing
mixture from 95% O2-5%
CO2 to 95%
N2-5%
CO2. The time of induction of
hypoxia varied slightly between kidneys because of modest differences
in experimental setup time (actual mean hypoxia start time was 81.4 ± 12 min). To facilitate comparison of renal function between
kidneys after the induction of hypoxia, the time of start of hypoxia in
each experiment was adjusted to coincide with 80 min. The functional data for each experiment were plotted against time, and a line of best
fit was applied. Functional data taken from the line of best fit at
10-min intervals between 40 min and 160 min were used to analyze
differences between groups. The data from five control and five
hypoxia experiments were analyzed and represented as means ± SD.
The initial values of each functional parameter for control and hypoxia
experiments were not significantly different (Student's
t-test, Table
1). All functional data were then
normalized to the average of the first three data points to highlight
the changes induced by hypoxia.
Assessment of osmolality in the IPRK.
To demonstrate that both hypoxic and control kidneys were similarly
exposed to hyposmotic shock, the technique of Knepper (26) was used to
measure regional osmolality in a number of rat kidneys that had been
perfused for varying lengths of time (20 min and 120 min) and after
hypoxia (perfused for 140 min after induction of hypoxia at 80 min). We compared these with unperfused kidneys. The kidneys were rapidly cooled
in liquid nitrogen, and a central core from cortex to papilla was
dissected while frozen and then sectioned serially in 0.5-mm segments.
The osmolality of these similar-sized segments was then measured on a
vapor-pressure osmometer (model 5500; Wescor, Logan, UT). The urine and
perfusate samples collected during the final 10 min of perfusion were
also measured. These results are shown in Table
2. Serial urinary and plasma osmolality
measurements in the 120 min control and 140 min hypoxic kidney from
Table 2 are compared in Table 3 with serial
(U/P)inulin ratios. Urine and
perfusate osmolalities from the last urine and perfusate sample of each
experiment are also shown in Table 2. For the in vivo experiment, urine
was sampled from the bladder and serum was analyzed instead of
perfusate.
As expected, regional osmolality (Table 2) declined rapidly in this
model, particularly in medulla. Even 20 min of perfusion created a
model of medullary hyposmotic shock, so that the degree of hyposmotic
shock was comparable between control and hypoxic groups at the start of
hypoxia (80 min). Hypoxia made no difference to the already low
medullary osmolality. In the control perfused kidney, the
(U/P)inulin was constant and high
while the urine osmolality fell rapidly and remained hypotonic,
indicating an intact diluting segment. After hypoxia, the
(U/P)inulin decreased while the
urine osmolality increased, approaching the perfusate levels.
Therefore, perfusion produced a comparable degree of hyposmotic shock
in control and hypoxic kidneys, and hypoxia produced no change in
tissue osmolality (Table 2). As anticipated in this model, urine
osmolality was largely dissociated from medullary osmolality.
MR techniques. Imaging and localized
spectroscopy were performed on a Bruker AMX300 console interfaced to a
7-T, 15-cm vertical bore magnet. The magnet system was
equipped with shielded gradients capable of 20 G/cm, with a bird cage
resonator as the transmission coil and a 15 mm × 20 mm elliptical, actively decoupled surface coil as the receiver coil.
All gradients and radiofrequency probes were custom
designed at the Centre for Magnetic Resonance, University of
Queensland. Scout images for voxel localization were acquired using the
RARE pulse sequence (23a) with the following parameters: a field of view of 3 cm, a 2562
matrix, 500-µm slices, and 8 echo acquisitions and a total
acquisition time for 4 slices of 2 min.
Regional 1H microspectroscopy was
performed using the VOSY pulse sequence (24). This
allowed a voxel (~24 µl in volume) to be located exclusively within
the cortex and medulla of each kidney as shown in Fig.
1 and described previously (10). Spectra
from renal cortex and medulla were then acquired alternately at
intervals of 12 min during each experiment, except in hypoxic
experiments when the same region was acquired before and immediately
following hypoxia. The first spectrum of each experiment was alternated between cortex and medulla. Typical VOSY parameters were: voxel dimensions 2 × 4 × 3 mm (24 µl), recycle time (TR) = 2 s, and echo time (TE) = 12 ms. TM and TE
crusher gradients of 8 G/cm were used for flow and
unwanted echo suppression. Water suppression was carried out using a
three-directional SUBMERGE pulse sequence with 20-ms, 5-cycle sinc
(sinx/x) shaped pulses and 10-ms, 4 G/cm gradients in each direction (14). Typically, 320 transients were
collected with a recycle time of 2 s. A line-broadening of 8 Hz was
applied to each spectrum prior to transformation. The integral of the
betaine-GPC peak in the first spectrum of the cortex and medulla of
each experiment was assigned as 1.0; subsequent peak integrals were
determined relative to the first peak. Hence, comparison of the
betaine-GPC peak intensities between experiments is not possible; only
changes within the experiments can be compared, assuming that the
biophysical state (e.g., protein binding) of betaine or GPC did not
change during the experiment. Because of the differences in setup time
described above, the first spectrum in each series of control cortical
and medullary spectra was adjusted to a start time of 60 min to define
time-dependent changes within groups. The effect upon the scatter by
shifting the timing of the MR data was trivial. Spectral peak
assignment has been previously described (10). Assignment of the large
peak centered at 3.2 ppm to betaine-GPC was confirmed (10).

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Fig. 1.
Transverse RARE scout image of the isolated perfused rat kidney (IPRK)
illustrating representative positioning of a cortical and medullary
voxel.
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Swelling of the perfused kidney was approximated from serial MR
images in two additional hypoxia group experiments by
measuring the total area and medullary area in the same slice
throughout each experiment using the region of interest tool (ROI) in
the Bruker Paravision package.
Statistical analysis. Differences
between functional data within an experiment and between control and
hypoxic experiments at each time point were analyzed by Student's
t-test. Time-dependent changes in NMR
spectral peaks were analyzed using the variance ratio method (15).
 |
RESULTS |
Initial parameters of renal function were assessed at 40 min; there was
no difference between hypoxic and control experimental groups (Table
2). Mean initial GFR for the whole group was 0.46 ml · min
1 · g
kidney
1, using an average
kidney weight of 0.7 g for rats of 150 g body weight in
our laboratory. Parameters of renal function were well-maintained in
control kidneys during perfusion for 160 min as shown by the near
constant values of (U/P)inulin,
FENa, and
FEK (Figs. 3 and 4). Urine flow
and GFR decreased during the control experiments, whereas renal
vascular resistance did not change (Figs. 2
and 3). Hypoxia caused a rapid decrease in
(U/P)inulin (Fig.
3) and increases in
FENa and
FEK (Fig.
4).

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Fig. 2.
Hypoxia and renal function: urine flow and renal vascular resistance
(RVR). Each value represents the mean ± SD of 5 experiments.
Differences between corresponding control (solid line) and hypoxic
(broken line) data points were significant as follows:
* P < 0.05, ** P < 0.01, and
*** P < 0.001.
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Fig. 3.
Hypoxia and renal function: urine-to-plasma
[14C]inulin
concentration ratio
[(U/P)inulin] and
glomerular filtration rate (GFR; inulin clearance). Each value
represents the mean ± SD of 5 experiments for control (solid line)
and hypoxic (dotted line) experiments. Recalculated GFR values with
data transposed 10 min ( ) and 20 min ( ) are also shown.
Differences between corresponding control and hypoxic data points were
significant as follows: * P < 0.05, ** P < 0.01, and
*** P < 0.001.
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Fig. 4.
Hypoxia and renal function: fractional excretion of sodium
(FENa) and potassium
(FEK). Each value represents the
mean ± SD of 5 experiments. Differences between corresponding
control (solid line) and hypoxic (dotted line) data points were
significant as follows: * P < 0.05, ** P < 0.01, and
*** P < 0.001.
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Induction of hypoxia initially increased both GFR and urine flow (Figs.
2 and 3). GFR in kidneys following hypoxia was greater than in control
experiments between 80 and 100 min and less than in control kidneys
when the perfusion time exceeded 140 min. Hypoxia increased urine flow
until 150 min. The increased GFR during the initial stage of hypoxia is
an artifact arising from a combination of the intrarenal and extrarenal
urinary "dead space" and the initial increase in urine flow.
Immediately prior to the initiation of hypoxia, the urine dead space
was filled with concentrated, low-sodium urine. The
(U/P)inulin was therefore maximal
for the particular GFR and urine flow. Increased urine flow, caused by hypoxic inhibition of sodium and water reabsorption, first expels dead
space urine with a high inulin concentration. Samples of urine taken
during this phase had greater volume but maintained high
(U/P)inulin, leading to an
increased calculated GFR. Once the urine with high
(U/P)inulin was cleared, the
calculated GFR decreased as a result of the decreased
(U/P)inulin, now coupled with a
decreasing urine flow probably from tubular swelling following hypoxia
(Fig. 2). At an initial flow rate of 0.04 ml/min, the long ureteric
line of volume 0.26 ml would have required at least 6.5 min for the
urine to be completely replaced. Coupled with the effective dead space
within the kidney, this suggests an approximate urine clearance time of
10 min for the total dead space. Transposing the
(U/P)inulin collection period
(Ui) by 10 min (to
Ui + 10) and recalculating the GFR
from the initial flow (Vi)
substantially eliminated the hypoxic increase in GFR (Fig. 3).
Transposition by 20 min reduced GFR below normal, indicating that the
correct clearance time was intermediate between 10 and 20 min.
Betaine-GPC decreased progressively in both the cortex and medulla
during normoxic perfusions (Fig. 5 and
6). Data fitting by linear regression
indicated that betaine-GPC decreased at a greater rate in medulla than
in cortex. The effect of hypoxia on the time-dependent reduction of
betaine-GPC in the IPRK was analyzed by comparing posthypoxic data with
corresponding control data by the variance ratio method (15). The
medullary betaine-GPC peak will be considered first. Combining the
control and hypoxic data demonstrated a linear decrease in the
betaine-GPC peak with time (Fig. 7).
Separating the control and hypoxic data did not reduce the variance
compared with the combined data when using a linear model with variance
analysis. However, analysis of the mean betaine-GPC peaks by the
variance ratio method and Student's t-test demonstrated that separating
the control and hypoxic data was significantly different from the
combined data (P < 0.05). Thus,
although the mean values were lower, hypoxia had no significant effect
on the rate of reduction in medullary betaine-GPC in the IPRK.

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Fig. 5.
Time dependence of proton magnetic resonance
(1H-MR) spectra in medulla
(A) and cortex
(B). A typical series of 10-min
1H spectra acquired from two
24-µl voxels located in medulla and cortex. Acquisition of medullary
spectra 1,
3, 5,
and 7 commenced at 60, 76, 102, and
128 min, respectively. Acquisition of cortical spectra
2, 4,
6, and
8 commenced at 48, 89, 115, and 141 min, respectively. This shows the decrease in intensity of the
betaine-glycerophosphorylcholine (betaine-GPC) peak (3.2 ppm) with time
in both medulla and cortex. Peak assignments are as follows: betaine
(B), 3.9 ppm; inositol (I), 3.5 and 3.6 ppm; perfusate (P), 3.8, 3.5, and 3.0 ppm; and glutamate-glutamine (G), 2.1 and 2.4 ppm.
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Fig. 6.
Betaine-GPC peak intensity in cortex and medulla of control kidneys.
Individual data points from 5 experiments are plotted for medullary
voxels (squares) and cortical voxels (solid circles). Equations
obtained from linear regression of the data were as follows: from
medulla, y = 1.180 0.00342x
(R = 0.837, n = 20, P < 0.001, broken line); and from
cortex, y = 1.077 0.00202x
(R = 0.517, n = 19, 0.01 < P < 0.05, solid line).
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Fig. 7.
Effect of hypoxia on medullary betaine-GPC levels. Peak intensities
following hypoxia ( ) were compared with control ( ) data excluding
the first (prehypoxia) data point for each experiment. Hypoxia had no
effect on the rate of decline in medullary betaine-GPC (see
RESULTS). Linear regression to the
combined data yielded y = 0.996 0.00226x
(R = 0.460, n = 31, P < 0.01, solid line).
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In the cortex, the betaine-GPC peak intensities showed greater scatter
than in the medulla. This relatively greater scatter arises partly from
the lower peak intensity (and signal-to-noise ratio) of betaine-GPC in
cortical spectra. Possibly because of this scatter, the combined
cortical control and hypoxic betaine-GPC peaks were not linearly
correlated (Fig. 8), although the control data alone were correlated (Fig. 6). Separating the control and hypoxic
betaine-GPC peaks did not reduce the variance compared with the
combined betaine-GPC peak. Therefore, hypoxia did not induce a
detectable change in the betaine-GPC concentration in the cortex of the
IPRK.

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Fig. 8.
Effect of hypoxia on cortical betaine-GPC levels. Peak intensities
following hypoxia ( ) were compared with control ( ) data excluding
the first (prehypoxia) data point for each experiment. Cortical data
were not linearly correlated.
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Whole kidney and medullary area estimates are shown in Table
4 as serial measurements of the same area
in consecutive image slices. These data demonstrate small and
inconsistent changes in kidney area during the perfusion period studied
and suggest minimal changes in regional volume over this time. This
would have limited effect on the intensity of the osmolyte peaks during the perfusion. Significant renal swelling during the initiation of
perfusion and before placement of the kidney into the magnet could have
occurred. Wolff et al. (40-42) observed medullary
swelling following saline diuresis in vivo, which resulted in a
medullary tissue volume increase of ~34%. This would be equivalent
to the start of perfusion. However, there was clearly little change in medullary volume during the observation period.
 |
DISCUSSION |
Betaine-GPC was readily identified in 24-µl voxels localized to
cortex or medulla (Fig. 5), although the sensitivity and resolution of
the MRS method in the intact kidney is much lower than MRS or HPLC
analysis of in vitro extracts. A contribution from inositol was also
seen in medulla. Osmolytes were less prominent in cortex. During
normoxic perfusion, betaine-GPC declined progressively in both cortex
and medulla (Fig. 6).
This is best explained as a response to hyposmotic shock caused by a
progressive washout of the corticomedullary osmotic gradient secondary
to the high perfusate flow present in the IPRK, compared with the
kidney in vivo, when the IPRK is perfused without erythrocytes (28).
Alternatively, this could be explained by increased permeability or
injury to cortical and medullary cells in this model. Although selective hypoxic injury to medullary thick ascending limb cells is
recognized in this preparation (see below), proximal tubules are well
preserved during normoxic perfusion. Since both cortical and medullary
betaine-GPC decline progressively, injury-related changes in
betaine-GPC appear an unlikely explanation. It is possible that the
large hyposmotic effect observed may have masked a much smaller
oxygen-dependent decrease in renal osmolyte concentration. However, the
results presented are consistent with the conclusion that hyposmotic
shock was the major cause of osmolyte reduction observed in the IPRK.
The high perfusate flow in this preparation occurs mainly as a
consequence of the lower viscosity of the perfusate in the absence of
erythrocytes, which helps to maintain an adequate oxygen delivery (28).
Progressive cell swelling following a reduction in interstitial
osmolality may be the cause of the progressive decrease in urine flow
(Fig. 2) during normoxic perfusion. Swelling and associated increases
in weight of the perfused kidney (but not a reduction in urine flow)
are alleviated by increasing perfusate oncotic pressure (36). However,
the MR imaging (MRI) microscopy estimates of regional areas (Table 4)
suggest that significant changes in regional volume did not occur.
Continued high perfusate flow ensures washout of the medullary
interstitial osmotic gradient. Osmolytes exiting the cells are washed
out of the kidney by the increased perfusate flow, decreasing the
intensity of the betaine-GPC peak. Consequently, the IPRK rapidly loses
the ability to concentrate urine as a consequence of the loss of the
corticomedullary osmotic gradient. This could be addressed in future
experiments by addition of red blood cells and antidiuretic hormone
to the perfusate (27).
The possibility that MRI microspectroscopy may have been insufficiently
sensitive to detect small hypoxia-induced changes in the rate of
betaine-GPC efflux was addressed by determining the difference in
efflux rate that could have been detected by MR under the conditions
used in this study. This was done by varying the slope of the
regression through the hypoxia data as a multiple of the control
regression while simultaneously retaining the absolute error arising
from each data point. This transformation was performed by adding the
observed difference between each data point and the original regression
through the hypoxia data to the recalculated line. For each multiple of
the control slope, the new hypoxia data and control data were analyzed
using the variance ratio (F) test
(15). The calculated F value was then
plotted against the multiple of the slope of the hypoxic data (Fig.
9). An F
value greater than 3.35 indicates that fitting the data to the separate control and hypoxia group linear regression lines provides a
significantly better fit (P < 0.05)
than fitting the data to a single regression line of the combined data.
Differences between the control and hypoxia groups were significant
when the slope of the hypoxia group was varied by more than
0.3
to 0.3 times the control slope.

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Fig. 9.
Detection limits for rate of change in medullary betaine-GPC by volume
localized MR microspectroscopy. Data transformation was used to assess
the detection limits observable by MR under the conditions utilized in
the study. Significance differences in osmolyte efflux were observed
between hypoxic and control groups when the slope of the hypoxic group
was varied by more than 0.3 times the control slope
(F value greater than 3.35).
|
|
Therefore, despite the inherent scatter in the volume-localized
magnetic resonance data acquired from the medulla, differences in
efflux rates (equivalent to slope) of betaine-GPC greater
than 0.3 times the control rate were detectable using the pooled data. This indicates that the magnetic resonance method was sufficiently sensitive to detect biologically important changes in efflux of renal
osmolytes from the medulla of the intact kidney.
The characteristics, mechanisms, and control of the rapid release of
osmolytes following a hyposmotic insult are not completely understood.
The response of medullary cells to acute hyposmotic changes has been
investigated primarily in renal cell lines, since these allow rapid
changes in extracellular osmolality by replacing the incubation medium
with fresh medium of a different osmolality (31). These studies have
demonstrated that rapid efflux of the osmolytes is greatest in the
first 15 min following the switch to a medium of lower osmolality. In
the GRB-PAP1 cell line, ~50% of sorbitol was lost in the first 15 min (3). In comparison, 15% and 30% betaine and inositol,
respectively, were released from the MDCK cell line in the first 15 min
(31). Specific channel activation has been implicated in osmolyte
release (25). Increased intracellular
Ca2+ initiated by activation of G
proteins has been implicated in the release of sorbitol (25). However,
intracellular Ca2+ does not appear
to be involved in the activation of osmolyte release of other osmolytes
(25). Betaine permeability is reported to be G protein dependent and
Ca2+ independent, whereas efflux
of inositol, GPC, and taurine are independent of both
Ca2+ and G protein (25). Whether
osmolyte release occurs via specific transporters for each osmolyte or
via a common transporter receptive to various signals is not known.
Osmolytes in medullary cells were not changed during ischemia
itself, although reperfusion decreased osmolyte content in the in vivo
study of Beck et al. (4). However, the effect of reduced oxygen
delivery and the resultant decrease in osmolality on osmolyte concentrations could not be differentiated in this model. In
comparison, medullary interstitial osmotic changes that occur because
of perfusion itself (see below) preceded a reduction in oxygen
availability in IPRK. The rate of decrease in renal betaine-GPC in
oxygenated kidneys was not modified by hypoxia (Figs. 7 and 8). This is
consistent with cellular regulation of osmolytes being predominantly a
consequence of osmotic balance and not directly affected by oxygen
availability in this preparation. Loss of membrane integrity by a
majority of cells could be expected to increase the release of betaine and GPC from the cells. However, the similarity between the normoxic and hypoxic experiments suggests membrane integrity during the hypoxic
period was comparable to normoxia in these experiments. Thus the
results are consistent with the release of osmolytes through specific
channels in response to hyposmolality (21) with little contribution
from nonspecific loss of membrane integrity.
The absence of a role for oxygen in the immediate regulation of
osmolyte concentrations is supported by the dissociation of oxygen-dependent parameters of physiological function from osmolyte concentrations in the intact kidney. In control kidneys,
(U/P)inulin, FENa, and
FEK were well maintained
throughout perfusion in control kidneys (Figs. 3 and 4) despite washout
of the osmolytes. Initiation of hypoxia resulted in a rapid decrease in
(U/P)inulin (Fig. 3) and increase
in FENa and
FEK (Fig. 4). The sensitivity of
medullary thick ascending limb cells to injury in the
erythrocyte-free IPRK (1, 7, 34) and preservation of proximal tubule
viability (12, 28, 34) suggest the proximal tubule is the primary site
of sodium and water transport measured in this model. The maintenance
of FENa,
FEK, and
(U/P)inulin, despite the decrease in osmolyte concentrations during normoxic perfusion, and the dramatic
decrease in Na+,
K+, and
H2O reabsorption with initiation
of hypoxia are consistent with a dominant role of the cortical tubules
in IPRK transport. GFR declined progressively in control kidneys as a
result of decreasing urine flow while
(U/P)inulin was maintained. After
hypoxia GFR declined further as the
(U/P)inulin decreased. The initial
artifactual increase that accompanied the increased urine volume
illustrates that calculated GFR results must be interpreted with
caution when a significant urinary "dead space" is present within
or outside the kidney.
Other MRS spectroscopy studies have been used to examine osmolytes in
kidney. 31P-MRS studies were used
to monitor GPC during diuresis in rabbit kidneys in vivo (40, 41). In
these studies, the GPC peak was an average signal from the whole kidney
rather than a localized region within the kidney, and other osmolytes
were not detected. A decrease in rabbit renal GPC content was not
observed following diuresis induced by infusion of saline alone, saline
plus furosemide, or saline plus furosemide and antidiuretic hormone
(41). However, GPC decreased after glucose infusion. It is tempting to
conclude that the changes in the combined betaine-GPC peak reported in our study are primarily changes in betaine concentrations. However, intravoxel resolution of betaine and GPC peaks is presently
insufficient to determine a unique integral for each peak.
Improvements in MR quantification through advances in data acquisition
and spectral analysis will reduce the inherent uncertainty in isolated
tissue and in vivo studies (3, 33, 38). Automated spectral analysis
removes operator variation of the position of the integral limits and
enables separation of overlapping peaks not possible with
operator-defined integrals. These methods will improve the
quantification of broad peaks with low peak intensity superimposed on a
broad baseline distortion such as demonstrated by the cortical
betaine-GPC data (Fig. 8) compared with the medullary data (Fig. 7).
In conclusion, image-guided, volume-localized MR microspectroscopy in
the IPRK has enabled changes in regional renal osmolyte concentrations
to be contrasted with functional data in the intact kidney. This study
indicates that washout of medullary osmolality in the erythrocyte-free
perfused rat kidney occurs primarily as a consequence of hyposmotic
shock and is independent of oxygen availability. As expected,
parameters of tubular function measured in this study were highly
oxygen dependent and primarily reflected proximal tubular transport.
GFR measurements in the IPRK need to be interpreted with caution
especially following a perturbation. However, osmolyte concentrations
in both medulla and cortex were insensitive to oxygen and dissociated
from parameters of oxygen-dependent renal function.
 |
ACKNOWLEDGEMENTS |
We are grateful to R. G. Duggleby for expert assistance in data
analysis. This research was supported by the National Health and
Medical Research Council (Australia).
 |
FOOTNOTES |
Results of these experiments were presented in part at the Fourth
International Conference on Magnetic Resonance Microscopy, Albuquerque,
NM, September 1997 and the XIVth International Congress of Nephrology,
Sydney, Australia, May 1997.
Address for reprint requests and other correspondence: Z. H. Endre,
Dept. of Medicine, Univ. of Queensland, Clinical Sciences Bldg.,
Royal Brisbane Hospital, Brisbane, 4029, Australia (E-mail:
z.endre{at}medicine.uq.edu.au).
Received 22 December 1997; accepted in final form 10 May 1999.
 |
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