Effect of Free Radical Scavenger (Tempol) on IntrarenalOxygenation in Hypertensive Rats as Evaluated by BOLD MRI

Lu-Ping Li, PhD1,*, Belinda S. Y. Li, PhD2, Pippa Storey, PhD1, Laura Fogelson1, Wei Li,MD1, and Pottumarthi Prasad, PhD11Department of Radiology, Evanston Northwestern Healthcare, Evanston, Illinois, andNorthwestern University Medical School, Chicago, Illinois2GE Healthcare, Evanston, Illinois

AbstractPurpose—To demonstrate a differential response following administration of a free radicalscavenger, tempol, in kidneys of hypertensive compared to normotensive rats.

Material and Methods—Data were obtained in spontaneously hypertensive rats (SHR, N = 5).Wistar-Kyoto rats (WKY, N = 6) were used as normotensive controls.

Results—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), whileWKY 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 becomparable to pre-tempol values in WKY rats, suggesting an improved medullary oxygenation inSHRs.

Conclusion—Based on both baseline R2* values and the differential effect of the free radicalscavenger on renal medullary oxygenation, BOLD MRI can distinguish hypertensive from normalkidney in rats.

KeywordsBOLD MRI; kidney; medulla; free radical scavenger; nitric oxide; spontaneously hypertensive rats

MANY STUDIES HAVE indicated that renal medullary blood flow (MBF) plays animportant 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 isblunted compared with normotensive Wistar Kyoto (WKY) rats (3).

Renal medullary blood flow is typically measured using invasive laser Doppler probes inanimal models (4), and to date there is no non-invasive alternative of direct measurement toextend these observations to humans. Significant advances have been made in understandingthe causes of essential hypertension through the use of invasive measurements of regionalblood flow and oxygenation in animal models. It has proven difficult to extend the researchto 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

© 2005 Wiley-Liss, Inc.*Address reprint requests to: L.-P.L., Radiology Department/Center for Advanced Imaging, Walgreen Building, Suite G507, EvanstonHospital, 2650 Ridge Avenue, Evanston, IL 60201. lli@enh.org.

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Published in final edited form as:J Magn Reson Imaging. 2005 March ; 21(3): 245–248. doi:10.1002/jmri.20260.

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under normal conditions and during physiologic and pharmacologic stresses may providevaluable information in terms of the pathophysiology of hypertension.

Blood oxygenation level dependent magnetic resonance imaging (BOLD MRI) techniquehas been demonstrated to be effective in monitoring the changes in renal oxygenationfollowing the administration of various pharmacologic agents that either increase (5) ordecrease (6) intrarenal blood flow in normal rats and following the administration of nitricoxide (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 NOin hypertensive kidneys. Oxidative stress in hypertension has been attributed partly to thedecreased 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). TheO2

− reacts with NO to form peroxynitrite, which effectively depletes NO in vascularendothelial 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 betweensuperoxide and NO and thus release the inactivated NO. Short- and long-term administrationof tempol has been shown to increase MBF in SHR by 35–50% and reduce mean arterialpressure (MAP) by ~20 mmHg compared with untreated SHR as evaluated by invasivetechnique (11–14). The increase in MBF in SHR is consistent with the suggestion thattempol reduces oxidative stress and enhances the activity of the NO system (11,13). Thetempol effect appears to be selective for SHR, in as much as it does not reduce MAP inWKY rats (11,12).

The goal of our current work is to determine whether BOLD MRI can document differencesin kidneys of hypertensive subjects and normotensive controls following administration oftempol.

MATERIALS AND METHODSAll experiments were performed in anesthetized rats (Inactin, 100 mg/kg) and wereapproved by the Institutional Animal Care and Use Committee. The studies were performedon 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 HarlanLaboratories (Madison, WI) and the SHRs were at least nine weeks old, the age by whichthey are known to become hypertensive (15). The jugular vein was catheterized foradministration of the free radical scavenger tempol.

MRI studies were performed on a 1.5-T Twin Speed scanner equipped with Excitetechnology (GE Healthcare, Milwaukee, WI) using a research sequence, multiple gradientecho (mGRE) (TR/TE/flip angle/bandwidth [BW] = 75 msec/8–50.4 msec/20°/42 kHz) toacquire 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 over12 repetitions (i.e., number of excitations [NEX] = 12). A standard quadrature extremity coilwas used for signal reception and the animal was positioned in a right lateral position inorder to minimize susceptibility artifacts from bowel gas. The signal intensity vs. time datawere 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 (GEHealthcare, Milwaukee, WI). R2* was used as a semi-quantitative measure of relative tissueoxygenation (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

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were obtained every 3 minutes for 45 minutes. Regions of interest (ROIs) containing at leastfour pixels were placed on the medulla and cortex to obtain values for the mean and SD ofR2*. The statistical significance of the differences between pre- and post-tempol R2* valueswas assessed using the two-tailed paired Student’s t-test.

RESULTSFigure 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 brightercompared with that of WKY rat implying that SHR is poorly oxygenated. The relativelydarker medulla in the post-R2* images of the SHRs indicate that the oxygenation wasimproved, probably due to increased blood flow. No appreciable increase in medullarybrightness is observed post-tempol in the WKY rats, implying the oxygenation change isminimal.

Figure 2 is the comparison of the R2* time course from medulla of representative SHR andWKY 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 administrationof tempol, while the R2* in the WKY rats remained relatively stable over the entire dataacquisition 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 acquiredmore 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 forpost-tempol, the changes in WKY rats were minimal and did not reach statisticalsignificance in any of the kidney tissues. The reduction in R2* after tempol administrationwas observed in all individual SHRs in the medulla and cortex. The changes in cortex maybe due to partial volume effects. SHRs have higher baseline R2* than WKY rats, suggestinglower basal medullary oxygenation status. The magnitude of the changes in SHRs is about12 second−1 (~30%) in the medulla.

DISCUSSIONThe difference in baseline values between the two strains of rats should be noted. The higherbaseline R2* in the SHRs is consistent with the report by other groups due to inherentreduced bio-availability of NO in the kidneys of hypertensive rats (17). The baseline R2* inthe medulla and cortex in SHRs is higher than our previous study (7), probably due to thefact 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 theSHR 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 veryclose to that reported in our previous study (7).

The data presented here demonstrate the utility of BOLD MRI in distinguishinghypertensive from normal kidneys, based on the effect of the free radical scavenger tempolon 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) butsignificantly decreased R2* in SHR (40.56 ± 0.66 second−1 to 28.58 ± 0.6 second−1 inmedulla). The degree of R2* changes is in qualitative agreement with the observedmedullary blood flow and MAP changes induced by tempol administration assessed byinvasive measurement (11). Previous investigations based on invasive techniques haveshown that the blood flow in SHRs increases as much as 35–50% with tempol treatment

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(13). The MAP decreased by 28% in SHRs and the renal vascular resistance decreased by29% (11). It is interesting to note that our study showed that the R2* dropped about 30% inthe medulla after tempol administration (Fig. 3). Also, the post-tempol R2* in SHRs is closeto that in WKY rats, suggesting that tempol normalizes the renal blood flow in SHRs. It isalso possible that free radical scavenging may have a direct effect on medullary pO2 assuggested by a recent report (18). Either way, the present results suggest that BOLD MRImay be a useful tool in evaluating the potential effectiveness of antihypertensivepharmacologic therapies, especially treatments based on administration of oxygen freeradical scavengers (12,13).

Experiments analogous to the one reported here should be easily translated to human studieswith a careful choice of vasoactive substances and oxygen free radical scavengers. This factcombined 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 ofhypertension directly in humans.

AcknowledgmentsContract grant sponsor: National Institutes of Health; Contract grant number: RO1DK-53221.

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Figure 1.Representative pre- and post-tempol R2* maps from a SHR and a WKY rat. Note therelatively darker medulla in the post-tempol R2* map as compared to pre-tempol R2* map,signifying an increase in medullary oxygenation in SHR rats. The window and level settingsfor all the maps are the same. The trend of decreased R2* values as an indicator of increasedblood flow in the medulla post-tempol is consistent with previous published data (9,11).There is no appreciable decrease in medullary brightness in post-tempol R2* in the WKYrat, implying no significant change in medullary oxygenation in WKY rats.

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Figure 2.Plot shows R2* in outer medulla vs. time from medulla of representative SHRs and WKYrats. Note that the SHR shows a significant response to tempol (administered at time zero).However, the response of R2* to tempol in the WKY rat is minimal. The baseline R2* inSHR is higher than that of the WKY rat, which implies that the hypertensive kidney ispoorly oxygenated. The error bar indicates the SD in each ROI measurement.

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Figure 3.Illustration of individual changes post-Tempol in SHRs and WKY rats. Average (mean ±SE) of all points acquired at least 20 minutes after tempol administration was used as post-tempol R2*. Mean R2* values pre- and post-tempol in the renal medulla and cortex wereaveraged over all rats of each strain. SHRs showed significant response to tempol in medullaand cortex as evaluated by BOLD MRI measurements. WKY rats did not show significantresponse to tempol.

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