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To demonstrate a differential response following administration of a free radical scavenger, tempol, in kidneys of hypertensive compared to normotensive rats.
Data were obtained in spontaneously hypertensive rats (SHR, N = 5). Wistar-Kyoto rats (WKY, N = 6) were used as normotensive controls.
Consistent with prior reports, SHRs show a significant response to tempol (R2*decreased from 40.56 ± 0.66 second−1 to 28.58 ± 0.6 second−1 in medulla, P < 0.05), while WKY rats exhibit a minimal change (R2* measuring 22.36 ± 4.38 second−1 pre-tempol and 21.57 ± 4.78 second−1 post-tempol, in medulla). The post-tempol R2* in SHR was found to be comparable to pre-tempol values in WKY rats, suggesting an improved medullary oxygenation in SHRs.
Based on both baseline R2* values and the differential effect of the free radical scavenger on renal medullary oxygenation, BOLD MRI can distinguish hypertensive from normal kidney in rats.
MANY STUDIES HAVE indicated that renal medullary blood flow (MBF) plays an important role in the control of arterial pressure (1,2). In spontaneously hypertensive rat (SHR), a commonly used animal model of essential hypertension, regulation of MBF is blunted compared with normotensive Wistar Kyoto (WKY) rats (3).
Renal medullary blood flow is typically measured using invasive laser Doppler probes in animal models (4), and to date there is no non-invasive alternative of direct measurement to extend these observations to humans. Significant advances have been made in understanding the causes of essential hypertension through the use of invasive measurements of regional blood flow and oxygenation in animal models. It has proven difficult to extend the research to humans because of the lack of suitable monitoring techniques. The availability of a non-invasive technique to monitor the renal medullary oxygenation and/or blood flow in humans under normal conditions and during physiologic and pharmacologic stresses may provide valuable information in terms of the pathophysiology of hypertension.
Blood oxygenation level dependent magnetic resonance imaging (BOLD MRI) technique has been demonstrated to be effective in monitoring the changes in renal oxygenation following the administration of various pharmacologic agents that either increase (5) or decrease (6) intrarenal blood flow in normal rats and following the administration of nitric oxide (NO) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in SHRs and WKY rats (7). The results in SHR were particularly consistent with the reduced bioavailability of NO in hypertensive kidneys. Oxidative stress in hypertension has been attributed partly to the decreased bioavailability of potent vasodilator NO due to the generation of free radicals (8,9). The major vascular oxygen-derived free radical is superoxide anion (O2−) (8). The O2− reacts with NO to form peroxynitrite, which effectively depletes NO in vascular endothelial cells (10). The renal medulla is particularly sensitive to oxidative stress (9).
Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidinoyl), a stable, membrane permeable, metal-independent superoxide dismutase mimetic, can inhibit the aforementioned reaction between superoxide and NO and thus release the inactivated NO. Short- and long-term administration of tempol has been shown to increase MBF in SHR by 35–50% and reduce mean arterial pressure (MAP) by ~20 mmHg compared with untreated SHR as evaluated by invasive technique (11–14). The increase in MBF in SHR is consistent with the suggestion that tempol reduces oxidative stress and enhances the activity of the NO system (11,13). The tempol effect appears to be selective for SHR, in as much as it does not reduce MAP in WKY rats (11,12).
The goal of our current work is to determine whether BOLD MRI can document differences in kidneys of hypertensive subjects and normotensive controls following administration of tempol.
All experiments were performed in anesthetized rats (Inactin, 100 mg/kg) and were approved by the Institutional Animal Care and Use Committee. The studies were performed on SHRs (N = 5; weight: 344.6 ± 14.6 g, age: 22.2 ± 0.8 weeks) and WKY rats (N = 6, weight: 316.3 ± 16.5 g, age: 21.5 ± 3.3 weeks). All animals were purchased from Harlan Laboratories (Madison, WI) and the SHRs were at least nine weeks old, the age by which they are known to become hypertensive (15). The jugular vein was catheterized for administration of the free radical scavenger tempol.
MRI studies were performed on a 1.5-T Twin Speed scanner equipped with Excite technology (GE Healthcare, Milwaukee, WI) using a research sequence, multiple gradient echo (mGRE) (TR/TE/flip angle/bandwidth [BW] = 75 msec/8–50.4 msec/20°/42 kHz) to acquire 16 T2*-weighted images. The field of view was 13 cm, with a matrix size of 256 × 256 and slice thickness of 1.6 mm. Due to the small voxel size, the signal was averaged over 12 repetitions (i.e., number of excitations [NEX] = 12). A standard quadrature extremity coil was used for signal reception and the animal was positioned in a right lateral position in order to minimize susceptibility artifacts from bowel gas. The signal intensity vs. time data were fitted to a single decaying exponential function to determine the value of R2* (= 1/T2*) using a custom implemented analysis module in FUNCTOOL on an AW workstation (GE Healthcare, Milwaukee, WI). R2* was used as a semi-quantitative measure of relative tissue oxygenation (16). A decrease in R2* indicates an increase in tissue pO2.
After obtaining a set of baseline T2*-weighted images, the tempol (Sigma-Aldrich, St. Louis, MO) solution (180 µmol/kg) was administered intravenously. The BOLD signals were obtained every 3 minutes for 45 minutes. Regions of interest (ROIs) containing at least four pixels were placed on the medulla and cortex to obtain values for the mean and SD of R2*. The statistical significance of the differences between pre- and post-tempol R2* values was assessed using the two-tailed paired Student’s t-test.
Figure 1 shows R2* maps from one representative SHR and one representative WKY rat, pre- and post- administration of tempol. The medulla in baseline R2* map of SHR is brighter compared with that of WKY rat implying that SHR is poorly oxygenated. The relatively darker medulla in the post-R2* images of the SHRs indicate that the oxygenation was improved, probably due to increased blood flow. No appreciable increase in medullary brightness is observed post-tempol in the WKY rats, implying the oxygenation change is minimal.
Figure 2 is the comparison of the R2* time course from medulla of representative SHR and WKY rats. Note the elevated baseline R2* in the SHR as compared with the WKY rats. Further, the R2* in the SHRs showed a rapid and significant drop following administration of tempol, while the R2* in the WKY rats remained relatively stable over the entire data acquisition period. The post-tempol R2* in SHR approached those in WKY rats.
The individual and mean R2* values in medulla and cortex for all of the rats, pre- and post-tempol, are shown in Figure 3. For statistical analysis, the average of all points acquired more than 20 minutes after tempol administration were used as post-tempol R2*. While R2* in the medulla and in the cortex in the SHRs showed a statistically significant decrease for post-tempol, the changes in WKY rats were minimal and did not reach statistical significance in any of the kidney tissues. The reduction in R2* after tempol administration was observed in all individual SHRs in the medulla and cortex. The changes in cortex may be due to partial volume effects. SHRs have higher baseline R2* than WKY rats, suggesting lower basal medullary oxygenation status. The magnitude of the changes in SHRs is about 12 second−1 (~30%) in the medulla.
The difference in baseline values between the two strains of rats should be noted. The higher baseline R2* in the SHRs is consistent with the report by other groups due to inherent reduced bio-availability of NO in the kidneys of hypertensive rats (17). The baseline R2* in the medulla and cortex in SHRs is higher than our previous study (7), probably due to the fact that the average age of the SHRs was older in this study compared to our previous study (22 vs. 12 weeks). It has been reported that the blood pressure gradually builds up as the SHR get older and the medullary blood flow decreases gradually compared with WKY rats (2). The baseline R2* in the medulla and cortex in WKY rats, on the other hand, is very close to that reported in our previous study (7).
The data presented here demonstrate the utility of BOLD MRI in distinguishing hypertensive from normal kidneys, based on the effect of the free radical scavenger tempol on medullary oxygenation. Tempol had no effect on the R2* in WKY rats (from 22.36 ± 4.38 second−1 pre-tempol and 21.57 ± 4.78 second−1 post-tempol in medulla) but significantly decreased R2* in SHR (40.56 ± 0.66 second−1 to 28.58 ± 0.6 second−1 in medulla). The degree of R2* changes is in qualitative agreement with the observed medullary blood flow and MAP changes induced by tempol administration assessed by invasive measurement (11). Previous investigations based on invasive techniques have shown that the blood flow in SHRs increases as much as 35–50% with tempol treatment (13). The MAP decreased by 28% in SHRs and the renal vascular resistance decreased by 29% (11). It is interesting to note that our study showed that the R2* dropped about 30% in the medulla after tempol administration (Fig. 3). Also, the post-tempol R2* in SHRs is close to that in WKY rats, suggesting that tempol normalizes the renal blood flow in SHRs. It is also possible that free radical scavenging may have a direct effect on medullary pO2 as suggested by a recent report (18). Either way, the present results suggest that BOLD MRI may be a useful tool in evaluating the potential effectiveness of antihypertensive pharmacologic therapies, especially treatments based on administration of oxygen free radical scavengers (12,13).
Experiments analogous to the one reported here should be easily translated to human studies with a careful choice of vasoactive substances and oxygen free radical scavengers. This fact combined with the results presented here and in our previous study on NO inhibition (7) suggest the possible role for BOLD MRI to elucidate pathophysiologic mechanisms of hypertension directly in humans.
Contract grant sponsor: National Institutes of Health; Contract grant number: RO1DK-53221.