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Department of Physiology, Monash University, Melbourne, Victoria, Australia
ABSTRACT
The aim of the current study was to determine whether renal medullary oxygenation is independent of the level of cortical blood flow by testing responses to stimuli that selectively reduce blood flow in either the cortex or medulla. In anesthetized rabbits, renal arterial infusion of [Phe2,Ile3,Orn8]-vasopressin selectively reduced medullary perfusion and PO2 (54 ± 24 and 50 ± 10%, respectively) but did not significantly affect cortical perfusion or tissue oxygenation. In contrast, stimulation of the renal nerves resulted in renal cortical ischemia with reductions in total renal blood flow (76 ± 3% at 4 Hz), cortical perfusion (57 ± 17%), and cortical PO2 (44 ± 12%). Medullary tissue PO2 was reduced by 70 ± 5% at 4 Hz, despite medullary perfusion being unaffected and distal tubular sodium reabsorption being reduced (by 83.3 ± 1.2% from baseline). In anesthetized rats, in which renal perfusion pressure was maintained with an aortic constrictor, intravenous infusion of ANG II (0.55 μg?kg1?min1) dose dependently reduced cortical perfusion (up to 65 ± 3%; P < 0.001) and cortical PO2 (up to 57 ± 4%; P < 0.05). However, medullary perfusion was only significantly reduced at the highest dose (5 μg?kg1?min1; by 29 ± 6%). Medullary perfusion was not reduced by 1 μg?kg1?min1 ANG II, but medullary PO2 was significantly reduced (12 ± 4%). Thus, although cortical and medullary blood flow may be independently regulated, medullary oxygenation may be compromised during moderate to severe cortical ischemia even when medullary blood flow is maintained.
acute renal failure; acute tubular necrosis; hypoxia; ischemia; laser-Doppler flowmetry; fluorescence oximetry; oxygen; rabbits; rats; renal circulation
THE UNIQUE VASCULAR architecture of the kidney allows differential regulation of cortical and medullary blood flow (7, 10, 14). Indeed, many vasoconstrictor factors that profoundly reduce total renal blood flow (TRBF) and cortical blood flow appear to have considerably less impact on medullary blood flow (6). Thus the renal medulla may be at least partially protected from hypoxic insults during periods of reduced overall (total) renal blood flow, if medullary blood flow, and so presumably oxygen delivery to this region, is maintained. However, this hypothesis is predicated on renal cortical and medullary oxygenation being relatively independent. Changes in glomerular filtration, tubular sodium reabsorption, and/or the possibility of oxygen loss from preglomerular arterial blood (27) may render medullary oxygenation dependent on the level of cortical blood flow.
The aim of the current study was to examine the relationship between tissue oxygenation and perfusion in the cortex and medulla. We tested the hypothesis that medullary oxygenation is independent of cortical blood flow. To achieve this, we employed a variety of vasoactive stimuli to selectively reduce blood flow in either the renal cortex or renal medulla, while simultaneously recording tissue PO2 (fluorescence oximetry and/or Clarke electrodes) and perfusion (laser-Doppler flowmetry) in both regions. Stimuli comprised stimulation of the renal nerves and renal arterial infusion of ANG II, which selectively reduce cortical perfusion, and renal arterial infusion of the V1 receptor agonist [Phe2,Ile3,Orn8]-vasopressin, which selectively reduces medullary perfusion. A range of doses/frequencies was tested so we could determine the relevance of our findings to both physiological and pathophysiological conditions. The stimuli we tested are likely to be relevant to physiological stressors that increase the risk of tubular necrosis and acute renal failure (26). For example, dehydration is associated with increased circulating levels of arginine vasopressin (8, 9) and acute hemorrhage is associated with increased circulating levels of ANG II (13) and increased renal sympathetic nerve activity (20).
METHODS
General. Experiments were performed in 13 male New Zealand White rabbits (2.84 ± 0.05 kg) and 17 male Sprague-Dawley rats (393 ± 27 g). Two experimental protocols were performed in separate groups of rabbits. In protocol 1, the left renal nerves were stimulated at frequencies of 0.5, 1, 2, and 4 Hz in random order (n = 7). In protocol 2, incremental doses of the V1-agonist [Phe2,Ile3,Orn8]-vasopressin (1, 5, and 25 ng?kg1?min1) were infused into the left renal artery (n = 6). Protocols 3 and 4 were performed in rats. In protocol 3, incremental doses of ANG II (0.5, 1, and 5 μg?kg1?min1) were infused into the jugular vein (n = 11). In protocol 4, we made a direct comparison between fluorescence oximetry and Clarke electrode measurements of kidney tissue PO2 at a range of depths below the cortical surface (n = 6). All procedures were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved in advance by the Animal Ethics Committee of the Department of Physiology, Monash University.
Surgical preparations for protocols 1 and 2 (rabbits). Rabbits were anesthetized with pentobarbital sodium (90150 mg plus 3050 mg/h; Nembutal; Merial, NSW, Australia), intubated, and artificially ventilated. A solution of compound sodium lactate (Baxter Health Care, Toongabbie, NSW, Australia) was infused at a rate of 0.18 ml?kg1?min1 to replace fluid loss, and esophageal temperature was maintained at 37.5°C. The left kidney was placed in a micropuncture cup for stability, the left ureter was catheterized, and a transit-time ultrasound flow probe was placed around the left renal artery for measurement of TRBF (type 2SB, Transonic Systems, Ithaca, NY). Renal nerves were isolated and placed on a set of hooked electrodes (protocol 1) (10, 14), or the kidney was denervated and a catheter was placed in the renal artery via the ileolumbar artery (protocol 2) (3, 25). To measure medullary laser-Doppler flux (MLDF) and medullary tissue oxygenation (MPO2), a needle laser-Doppler flow probe (MNP110XP, tip diameter 500 μm, Oxford Optronix, Oxford, UK) and PO2 probe (BF/OT, tip diameter 350 μm, Oxford Optronix) were simultaneously advanced into the kidney using a micromanipulator so that their tips lay 9 mm apart, 1012 mm below the midregion of the lateral surface of the kidney [i.e., within the inner medulla (10)]. For measurement of cortical tissue oxygenation (CPO2) and cortical laser-Doppler flux (CLDF), a second PO2 probe was inserted 2 mm into the left kidney and a laser-Doppler probe (MSP310XP, Oxford Optronix) was placed on the kidney surface. After instrumentation, a bolus dose of [3H]inulin (4 μCi, New England Nuclear Research Products, Sydney, NSW, Australia) was given intravenously and the maintenance infusion was replaced with a solution containing four parts compound sodium lactate to one part of a 10% polygeline/electrolyte solution (Haemacell, Hoechst, Melbourne, Victoria, Australia). This solution also contained [3H]inulin (300 nCi/ml) and 40 μg/ml LiCl (Merck, Darmstadt, Germany) so that arterial plasma Li+ concentration averaged 60 ± 10 μM across the two protocols. Respiratory rate was then adjusted so that arterial PO2 was 90110 mmHg (ABL 310, Radiometer, Copenhagen, Demark). Experimental manipulations commenced after a 90-min equilibration period.
Protocol 1: renal nerve stimulation in rabbits. The renal nerves were stimulated using purpose-written software in the LabVIEW graphical programming language (National Instruments, Austin, TX) coupled to a LabPC+ data-acquisition board (National Instruments). Each train of stimuli was preceded by a 10-min control period during which urine was collected and a 0.5-ml arterial blood sample was taken at the midpoint to allow calculation of [3H]inulin, Na+, and Li+ clearance. The renal nerves were then stimulated at either 0.5, 1, 2, or 4 Hz at 8 V with a pulse width of 2 ms for 12 min, and blood and urine samples were collected during the final 10 min. Ten minutes were allowed between stimulation periods. The order of presentation of the four frequencies was randomized.
Protocol 2: [Phe2,Ile3,Orn8]-vasopressin infusions in rabbits. [Phe2,Ile3,Orn8]-vasopressin was administered directly into the left renal artery in sequential infusions of 0, 1, 5, and 25 ng?kg1?min1 for 40 min each. Urine was collected during the final 20 min of each period. A 0.5-ml arterial blood sample was taken at the midpoint of each period for clearance measurements.
Protocol 3: ANG II infusion in rats. Rats were anesthetized with pentobarbital sodium (1530 mg plus 15 mg/h), tracheostomized, and artificially ventilated. Body temperature was maintained at 37.5°C using a servo-controlled infrared heating lamp (Cole Parmer, Digisense, Chicago, IL). Throughout, a solution of bovine serum albumin (2% wt/vol in 0.9% wt/vol NaCl; Sigma, St. Louis, MO) was infused at 2 ml?kg1?h1. Rats were prepared as previously described (4). In brief, the right carotid artery, femoral artery, bladder, left ureter, and jugular vein were catheterized, and an aortic constrictor was placed around the abdominal aorta above the left renal artery. The left kidney was placed in a stabilized micropuncture cup and prepared as for protocols 1 and 2, except that probes were inserted to a depth of 4.5 mm (inner stripe of the outer medulla) and 1 mm (cortex) and a Clarke type electrode (Ox-10, Unisense, tip diameter <10 μm, Denmark) was inserted into the renal cortex rather than a fluorescence probe. In some rats (n = 5), both a fluorescence PO2 probe and a Clarke type electrode were inserted into the renal medulla so that measurement of tissue PO2 using the two techniques could be compared. Following instrumentation, a bolus dose of [3H]inulin (1 μCi, New England Nuclear Research Products) was given intravenously and [3H]inulin was added to the maintenance infusion. Respiratory rate was then adjusted so that arterial PO2 was 90110 mmHg (ABL310, Radiometer, Denmark), and 20 min were allowed before experimental manipulations commenced.
ANG II was administered into the jugular vein in sequential doses of 0, 0.5, 1, and 5 μg?kg1?min1. Each dose was infused for 12 min. Renal perfusion pressure (monitored via the femoral artery catheter) was held constant by adjusting an aortic constrictor placed above the level of the left renal artery. Urine was collected and a 0.5-ml blood sample was taken at the midpoint during the final 10 min of each control (0 μg?kg1?min1) period for determination of [3H]inulin and Na+ clearance.
Protocol 4: comparison of renal tissue PO2 measurement between techniques. Rats in protocol 4 were prepared as for protocol 3, except that an aortic constrictor was not placed around the abdominal aorta. A Clarke-type PO2 electrode and a fluorescent oximetry PO2 probe were simultaneously advanced through the renal tissue in 1-mm increments. Tissue PO2 at each level (from 16 mm below the renal surface) was recorded for 2 min.
Measurement of hemodynamic variables and determination of renal function. Arterial pressure (mmHg) was measured via an ear artery catheter (rabbits) or carotid/femoral artery catheters (rats). Laser-Doppler flux and PO2 probes were connected to a laser-Doppler flowmeter/tissue oximetry system (Oxylite/Oxyflo, Oxford Optronix), which allowed continuous measurement of laser-Doppler flux (units) and tissue PO2 (mmHg). Clarke-type electrodes were connected to a picoammeter (PA-2000, Unisense). The electrode was precalibrated at 37°C with a solution of 154 mM NaCl bubbled with 21% O2 and a solution containing sodium ascorbate (0.1 M) and NaOH (0.1 M). Analog signals were digitized and recorded by a data-acquisition program (Universal Acquisition, University of Auckland, Auckland, New Zealand). Baseline levels of CLDF and MLDF, obtained after animals were killed, were subtracted from experimental data before subsequent analysis. The position of each of the intrarenal probes was also checked postmortem.
[3H]inulin clearance was used to estimate glomerular filtration rate (15). Sodium and lithium concentrations in plasma and urine were determined by atomic absorption spectrophotometry (Avanta, GBC Scientific Equipment, Dandenong, Victoria, Australia), and proximal and distal tubular sodium reabsorption was estimated as previously described (29). At the conclusion of the experiment, animals were killed with an intravenous overdose of pentobarbital sodium.
Statistical analysis. Mean values for all control periods are presented as absolute values ± SE. Unpaired t-tests were used to determine whether baseline levels differed between the rabbits studied in protocols 1 and 2, and between the rats studied in protocols 3 and 4. Responses to renal nerve stimulation and infusions of vasoconstrictor peptides were calculated from the average values during each clearance period and are presented as a percentage of control values ± SE. We tested for dose- or frequency-dependent effects of each stimulus using one-way ANOVA. Specific contrasts were made by partitioning one-way ANOVA. Two-way ANOVA was used to test for differences in responses, of regional kidney (cortical vs. medullary) laser-Doppler flux and PO2 and sodium reabsorption (proximal vs. distal tubular), to the various vasoactive stimuli. The risk of type 1 error was controlled by conservatively adjusting P values using the Ryan-Holm step-down procedure (Sidak inequality) (19). Model II regression analysis was used to determine the relationship between tissue PO2 measurements made with Oxylite probes and Clarke-electrodes (18).
RESULTS
Baseline cardiovascular and renal variables. Baseline systemic and renal hemodynamics were similar in rabbits studied in protocols 1 and 2 (Table 1) and in rats studied in protocols 3 and 4 (Table 2). Fractional sodium excretion was considerably less in rats than in rabbits (Tables 1 and 2).
Protocol 1: renal nerve stimulation in rabbits. Stimulation of the renal nerves resulted in immediate and marked reductions in both TRBF and CLDF [see typical recording (Fig. 1)]. CLDF was reduced by 57 ± 17% at 4 Hz (Fig. 2A). In contrast, MLDF was not significantly altered by renal nerve stimulation (Fig. 2A). As expected, reductions in renal CLDF were accompanied by reductions in CPO2, which was reduced by 44 ± 12% at 4 Hz (Fig. 2B). Marked reductions in MPO2 were observed during renal nerve stimulation (Fig. 1). These averaged 23 ± 10 and 70 ± 5% from baseline at 2 and 4 Hz, respectively (Fig. 2B).
Renal nerve stimulation was associated with frequency-dependent reductions in glomerular filtration rate (by 6 ± 9, 2 ± 30, 59 ± 10, and 86 ± 3%, respectively, at 0.5, 1, 2, and 4 Hz; P < 0.001); urine flow (by 24 ± 9, 22 ± 21, 76 ± 5, and 89 ± 2%, respectively, at 0.5, 1, 2, and 4 Hz; P < 0.001); and urinary sodium excretion (by 24 ± 5, 25 ± 26, 56 ± 18, and 84 ± 2%, respectively, at 0.5, 1, 2, and 4 Hz; P < 0.001). There was also a frequency-dependent reduction in total sodium reabsorption (by 5 ± 9, 22 ± 12, 59 ± 9, and 84 ± 3%, respectively, at 0.5, 1, 2, and 4 Hz; P < 0.001), with proportionately similar reductions in proximal and distal tubular reabsorption, as indicated by Li+ clearance. Thus proximal tubular sodium reabsorption was reduced by 10 ± 16, 11 ± 21, 64 ± 7, and 82 ± 4%, respectively, at 0.5, 1, 2, and 4 Hz (P < 0.001), while distal tubular sodium reabsorption was reduced by 1 ± 4, 30 ± 9, 66 ± 6, and 83 ± 1%, respectively. Neither mean arterial pressure nor heart rate was significantly altered by renal nerve stimulation.
Protocol 2: responses to infusion of [Phe2,Ile3,Orn8]-vasopressin in rabbits. Infusion of [Phe2,Ile3,Orn8]-vasopressin into the renal artery caused dose-dependent and sustained reductions in MLDF and MPO2. Although [Phe2,Ile3,Orn8]-vasopressin markedly reduced MLDF and MPO2, no significant changes in TRBF, CLDF, or CPO2 were observed (Fig. 3, A and B). [Phe2,Ile3,Orn8]-vasopressin did not significantly affect glomerular filtration rate, urine flow, urinary sodium excretion, or tubular sodium reabsorption at the doses administered. Mean arterial pressure and heart rate were also not significantly altered by [Phe2,Ile3,Orn8]-vasopressin.
Protocol 3: responses to infusion of ANG II in rats. Intravenous infusion of ANG II dose dependently reduced TRBF and CLDF (Fig. 4A). MLDF was maintained at control levels at 0.5 and 1 μg?kg1?min1. ANG II infusion reduced MLDF in most (but not all) rats at 5 μg?kg1?min1.
ANG II-induced reductions in CLDF were associated with significant reductions in CPO2 and also MPO2 at both 1 and 5 μg?kg1?min1 (Fig. 4, A and B). ANG II infusion significantly increased mean arterial pressure (up to 48 ± 7% at 5 μg?kg1?min1, P < 0.001), but renal perfusion pressure was maintained at control levels by adjustment of a suprarenal aortic occluder (P < 0.01).
Protocols 3 and 4: comparison of tissue PO2 measurement methods. Values of MPO2 measured using fluorescence oximetry were highly correlated with those obtained using a Clarke-type electrode in protocol 3 (Fig. 4A). The slope of this relationship did not differ significantly from unity, and the ordinal intercept did not differ significantly from zero (Fig. 4A). Nevertheless, when tissue PO2 was measured at various depths below the cortical surface in protocol 4, values obtained using fluorescence oximetry were systematically 2.78.0 mmHg less than those obtained with a Clarke-type electrode (Fig. 4B). Renal tissue PO2 in rats was found to be greatest close to the cortical surface (1 mm; 40 ± 5 mmHg), whereas it averaged <30 mmHg at depths between 2 and 6 mm below the cortical surface (Fig. 4B). Reliable measurements of tissue PO2 at 1 mm below the renal surface could not be obtained using the fluorescence oximetry probes.
DISCUSSION
Stimulation of the renal nerves at frequencies 4 Hz in rabbits decreased TRBF and CLDF but had little or no influence on MLDF, consistent with our previous observations (10, 14). Similarly, intravenous infusion of ANG II in rats reduced TRBF and CLDF at doses without effect on MLDF (3, 4). In contrast, renal arterial infusion of the V1 agonist [Phe2,Ile3,Orn8]-vasopressin in rabbits caused selective reductions in MLDF without significant changes in TRBF or CLDF (7, 25). Thus we were able to produce graded ischemia within the cortex or medulla without significantly altering perfusion of the alternate region. This enabled us to test whether local oxygenation within each of these kidney regions is dependent on blood flow within the other.
Our major finding was that MPO2 is dependent on the level of cortical blood flow (Fig. 6A). Figure 6 shows three-dimensional plots of the data obtained from protocols 1, 2, and 3. Figure 6A shows that MPO2 was reduced during reductions in perfusion in either the renal cortex or renal medulla. As expected, selective reductions in MLDF during infusion of [Phe2,Ile3,Orn8]-vasopressin were accompanied by reductions in MPO2. Interestingly, however, reductions in CLDF induced by stimulation of the renal nerves in rabbits, or infusion of ANG II in rats, were also associated with marked reductions in MPO2. These reductions could be observed even when MLDF was completely maintained. In contrast, Fig. 6B shows that CPO2 is independent of the level of medullary perfusion. Infusion of the V1 agonist [Phe2,Ile3,Orn8]-vasopressin, which resulted in marked reductions in MLDF, had no significant effect on CPO2.
Our results indicate that even relatively modest reductions in cortical perfusion and oxygenation can lead to a reduction in MPO2. Inner MPO2 was reduced by 23 ± 10% by 2-Hz renal nerve stimulation in rabbits, which reduced CLDF and CPO2 by 34 ± 8 and 15 ± 11%, respectively. Similarly, tissue PO2 within the medullary inner stripe was reduced by 12 ± 4% by 1 μg?kg1?min1 ANG II in rats, which reduced CLDF and CPO2 by 46 ± 3 and 22 ± 8%, respectively. However, a threshold of cortical ischemia and/or hypoxia seems to be required before CPO2 or MPO2 is reduced. Neither CPO2 nor MPO2 was reduced by infusion of ANG II at 0.5 μg?kg1?min1, which reduced CLDF in rats by 38 ± 3%. Furthermore, renal PO2 was not reduced by 1-Hz renal nerve stimulation, which reduced CLDF in rabbits by 20 ± 6%.
The renal medulla, particularly the renal outer medulla, is inherently sensitive to hypoxic stimuli (2). This is chiefly due to the fact that medullary oxygen consumption is relatively high, particularly in the medullary thick ascending limb (2), yet oxygen delivery to the renal medulla is limited by the counter- current arrangement of the medullary vasculature (33). Consequently, any stimulus that disturbs the balance between medullary oxygen delivery and oxygen consumption, such as a reduction in local blood flow or an increase in tubular oxygen demand, may result in medullary hypoxia.
The possibility that increased medullary oxygen consumption contributes to the development of medullary hypoxia, during renal nerve stimulation in rabbits and ANG II infusion in rats, is consistent with the fact that these stimuli can enhance tubular sodium reabsorption (3, 11). On the other hand, our finding of reduced sodium reabsorption in both the proximal and distal tubule during renal nerve stimulation argues against this proposal, at least with respect to our observations in rabbits. Nevertheless, direct measurements of juxtamedullary filtration and sodium reabsorption within the renal medulla are required to resolve this issue.
MLDF was not significantly reduced by electrical stimulation of the renal nerves in rabbits or by intravenous infusion of ANG II at 1 μg?kg1?min1 in rats. Thus it seems unlikely that the reductions in MPO2 observed during these stimuli resulted from reduced medullary perfusion. However, within the kidney, and particularly within the renal medulla with its looplike vascular architecture (21), tissue oxygen delivery is not solely dependent on the level of perfusion but also depends on the oxygen content of inflowing blood, which in turn is dependent on the magnitude of upstream oxygen losses (33). The fact that the medullary vascular supply arises within the renal cortex provides an opportunity for cortical hypoxia to reduce the oxygen content of blood perfusing the medulla. It is now well accepted that diffusional shunting of oxygen occurs not only within the medullary capillaries but also between conduit arteries and veins in the kidney (1, 16, 21, 27, 31). Cortical hypoxia will increase the gradients for diffusion of oxygen from arterial blood destined for the juxtamedullary glomeruli to either hypoxic cortical tissue or venous blood draining the hypoxic cortex. Cortical ischemia, and so increased erythrocyte transit time, should further facilitate this process (30). That is, much of the oxygen within the renal arterial system may be lost before the level of the juxtamedullary afferent arterioles, so that medullary oxygenation may be reduced even though medullary perfusion is maintained. Direct measurements of PO2 within the vessels supplying the renal medulla are required to resolve this issue.
Medullary hypoxia is thought to be important in the pathogenesis of tubular necrosis and acute renal failure (12). Therefore, the pathophysiological significance of our findings depends on whether endogenous stimuli that reduce renal perfusion can cause medullary hypoxia. This seems likely during some physiological stressors, such as hemorrhage and dehydration, that increase the risk of acute renal failure (26). For example, in mild water deprivation, reductions in medullary blood flow result from relatively small increases in circulating levels of arginine vasopressin (8, 9). Furthermore, activation of renal sympathetic nerve activity and the renin-angiotensin system during acute blood loss likely causes considerable renal vasoconstriction (13, 20). Studies of the roles of these neural and hormonal factors in the control of medullary oxygenation during hemorrhage and water deprivation should clarify this issue.
In the current study, medullary PO2 and laser-Doppler flux were assessed in both the inner medulla (rabbits) and inner stripe of the outer medulla (rats). Consequently, our conclusions must be confined to these regions of the kidney. Deeper regions of the renal medulla were chosen in preference to the outer stripe of the outer medulla, chiefly to avoid the potentially confounding effects of changes in the renal cortical environment. Thus we can be confident that measurements obtained within the renal medulla reflected only changes in medullary perfusion and PO2. Nevertheless, it must be borne in mind that changes in O2 consumption in the outer medulla are likely to influence inner medullary oxygenation, as the vascular segments that supply the inner medulla traverse the outer medulla as capillaries (21). Conversely, the countercurrent arrangement of ascending and descending vasa recta provides a basis for changes in oxygen consumption in the inner medulla to alter oxygenation within the outer medulla (33).
The current study was performed in both rats and rabbits, and changes in tissue PO2 in response to stimuli were confirmed using two separate techniques. In contrast to previous findings in conscious rabbits (28), basal MPO2 was greater than basal CPO2 in the anesthetized rabbits we studied. This likely reflects differences between the studies, in the type of probes used to measure CPO2 and the positioning of the PO2 probes within the cortex. In the study of Strauss et al. (28), modified Clarke-type electrodes were used to determine CPO2, and these probes were positioned close to the cortical surface, the region of the kidney with the greatest tissue PO2. In protocols 1 and 2 in the current study, however, CPO2 was measured using fluorescence oximetry probes. To avoid exposure to air, and obtain reliable PO2 measurements within cortical tissue, we found that these probes had to be inserted at least 2 mm into the cortex, such that the probe tips were located within the midcortical region of the kidney. Numerous studies have reported that in addition to a corticomedullary PO2 gradient, a PO2 gradient exists within the renal cortex. In fact, in two recent studies, Palm et al. (22) and Welch et al. (31, 32) reported PO2 in the midcortical region of rats to be similar to the values of PO2 observed in rabbits in the current study (22, 32). In these previous reports, midcortical PO2 was reported to be less than 30 mmHg, much lower than tissue PO2 in more superficial cortical regions and less than that in the outer medulla. We confirmed these observations in the current study. Importantly, we also showed that responses of MPO2 to infusion of ANG II observed using Oxylite probes and Clarke electrodes were indistinguishable within the renal medulla. Our finding of a small systematic difference in baseline tissue PO2 values obtained using these two methods may be a consequence of the probe types, in that the smaller Clarke electrodes may bias PO2 measurements toward intravascular rather than parenchymal regions (30) where PO2 is known to be higher (17). Nevertheless, we can be confident that this small systematic difference in PO2 measurements provided by these two techniques did not confound our major conclusions from the current study.
A possible limitation of our current study relates to the use of laser-Doppler flowmetry to estimate cortical and medullary perfusion. Laser-Doppler flux is directly proportional to the velocity of erythrocytes (10), but in highly perfused tissues such as the kidney it is not necessarily responsive to changes in blood flow mediated by changes in the number of perfused capillaries (5). However, this limitation is unlikely to have confounded our present observations using laser-Doppler flowmetry, as they are consistent with those of previous studies using local hydrogen clearance, which on theoretical grounds should reflect changes in bulk flow (23, 24). For example, Parekh and colleagues (23, 24) observed reductions in cortical blood flow in responses to adrenergic stimulation (norepinephrine infusion) and infusion of ANG II, in the absence of substantive changes in medullary blood flow, in anesthetized rats. Moreover, we were able to show dose-dependent reductions in medullary perfusion in response to [Phe2,Ile3,Orn8]-vasopressin using laser-Doppler flowmetry.
In conclusion, our results indicate that, under the conditions of the current study, cortical tissue oxygenation is independent of medullary blood flow and oxygenation, being chiefly determined by the local level of blood flow in the cortex. Medullary tissue PO2 in both the inner medulla (rabbits) and inner stripe of the outer medulla (rats), in contrast, appears to be dependent on the levels of both of cortical and medullary perfusion. Thus the relative insensitivity of medullary blood flow to many vasoconstrictor factors (6) may not necessarily protect the medulla from hypoxia during renal ischemia. At present, the mechanisms that underlie the dependence of medullary oxygenation on cortical perfusion remain unclear.
GRANTS
This work was funded by National Health and Medical Research Council of Australia Grants 143785 and 143603; Ramaciotti Foundation Grants A6370, RE159/98, and RA032/01; and Monash University (Small Grant 62/2003). P. O'Connor was a recipient of an Australian Postgraduate Scholarship.
Present address of P. M. O'Connor: Dept. of Physiology, Medical College of Wisconsin, Milwaukee, WI 53266.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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