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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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Table 1.   Initial parameters of renal function

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.

                              
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Table 2.   Regional renal osmolality


                              
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Table 3.   Comparison of urinary and plasma osmolality

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.

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (open circle ) and 20 min (black-triangle) 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.

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).

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 (open circle ) were compared with control () data excluding the first (prehypoxia) data point for each experiment. Cortical data were not linearly correlated.

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.

                              
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Table 4.   Representative total and regional renal areas before and after induction of hypoxia


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Physiol 277(3):F338-F346
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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