Autoregulation of renal blood flow (RBF), glomerular capillary pressure, and glomerular filtration rate (GFR) are essential for the maintenance of normal renal function. During arterial pressure reduction, this is accomplished by a decrease in renal vascular resistance, predominantly through dilation of preglomerular resistance vessels. In our study, we investigated two conditions that might interfere with the ability to vasodilate: first, abnormalities in the intrarenal vessels resulting from hypertension, and second, the renal vasodilatory action of antihypertensive drugs.
Previous studies showed that glomerulosclerosis, vascular lesions, interstitial fibrosis, and tubular atrophy are typical findings in the juxtamedullary cortex of the 40-week-old spontaneously hypertensive rats (SHRs), whereas the superficial cortex reveals only minor changes (1-3). Nevertheless, we found that autoregulation of RBF as well as total GFR was well preserved in 10- and 40-week-old SHRs, and if anything, that the autoregulation of GFR was more efficient in the inner than in the outer cortex. It should be noted, though, that the range of autoregulation was reset to a higher pressure range, corresponding to the prevailing arterial pressure in SHRs (4).
In this study, we wanted to investigate autoregulatory vasodilatation in short-term and long-standing hypertension after dilating of the resistance vessels by antihypertensive drugs. Iversen et al. (4) found that resetting of RBF autoregulation to the normal pressure range could be obtained after 7 days of treatment with captopril, whereas only incomplete normalization of autoregulation was observed in 40-week-old SHRs.
Angiotensin II has a vasoconstrictive effect on both afferent and efferent arterioles (5), and inhibition or blockade of the angiotensin II effect will reduce pre- and postglomerular resistance. Calcium channel blockers dilate mainly preglomerular vessels and thereby reduce the capability for RBF autoregulation (6). Despite the impaired RBF autoregulation and reduced blood pressure, calcium channel blockers are reported to increase GFR, probably because of their main effect on the preglomerular vessels. Our working hypothesis was therefore that GFR autoregulation should be better preserved in hypertensive animals during treatment with a calcium channel blocker than with a converting-enzyme inhibitor or an angiotensin II-receptor antagonist. Furthermore, such a difference in response to total GFR might be different in deep and superficial glomeruli, resulting from inhomogeneity of the angiotensin II effect among the various layers of the cortex. This study was therefore undertaken to examine the effect of an angiotensin I-converting enzyme inhibitor (enalapril), an angiotensin II type 1 receptor antagonist (losartan), and a calcium channel blocker (nifedipine) on GFR autoregulation in SHRs at age 10 and 40 weeks by using 10-week-old Wistar-Kyoto rats (WKYs) as controls.
Studies of GFR in different cortical layers have been possible by the recently developed aprotinin (Ap) method, which permits repeated GFR measurements without urine samples and without interfering with the tubuloglomerular feedback mechanism (7). The theoretic and experimental basis for the method has been described by Tenstad et al. (8).
MATERIALS AND METHODS
Experiments were done in 10-week-old male WKY rats, and in 10- and 40-week-old male SHRs. They were performed in accordance with, and under the approval of, the Norwegian State Board for Biological Experiments with Living Animals. The rats had free access to water and were fed ordinary rat chow (B&K Universal A/S, Oslo, Norway) containing 0.30% sodium, 0.70% potassium, 0.88% calcium, and 18% crude protein.
Treatment of hypertension
All animals were treated once daily by gavage for 5 days. The drugs were dissolved in 0.5 ml sterile water immediately before gavage. Daily doses of 15 mg/kg body weight of losartan and 10 mg/kg body weight of enalapril were given in normotensive WKYs (n = 13) and in 10-week-old (n = 14) and 40-week-old SHRs (n = 13). Because of the pronounced hypotensive effect of nifedipine in normotensive rats, they were treated with doses of 5 mg/kg body weight (n = 6), whereas the hypertensive rats were given 15 mg/kg body weight (n = 13). Untreated animals were given 0.5 ml sterile water (n = 24).
Renal arterial pressure (RAP), RBF, and GFR were measured 4 h after the drug administration on the fifth day of antihypertensive therapy. The rats were anesthetized with intraperitoneal pentobarbital (40-50 mg/kg body weight), and after tracheotomy (PE-260), they were placed on a servo-controlled heating pad to keep the body temperature constant at 37°C. A PE-50 catheter was placed in the right jugular vein for infusion of 5% bovine albumin in 0.9% sodium chloride solution at a rate of 1.0 ml/100 g body weight per hour throughout the experiment. The right carotid artery was cannulated for blood sampling.
Arterial pressure was measured through a PE-50 catheter in the left femoral artery with a Hewlett-Packard pressure transducer connected to a Gould TA 4000 recorder. This pressure was assumed to be equal to RAP. RBF was measured in the left renal artery by a 2-mm diameter flowprobe connected to a transit-time flowmeter (Transonic) and a Gould recorder. The probe was calibrated on isolated perfused renal arteries as described previously (9). A screw clamp was placed on the aorta between the renal arteries for adjustment of the left RAP. After surgery, the rats were allowed to recover for 30 min. Thereafter, RAP was reduced in steps of 5-10 mm Hg, and RBF was recorded for 1-2 min after each reduction. This relation between RBF and RAP was recorded from control pressure to ≈60 mm Hg in normotensive and to 80 mm Hg in hypertensive rats. The lower pressure limit of RBF autoregulation was defined as the lowest RAP that could be reached without reducing RBF (designated RAP3 in tables). GFR was measured 2-6 mm Hg above this limit (RAP2 in tables) to be sure that this measurement was done within the range of unaltered RBF. During nifedipine treatment, RBF autoregulation was greatly impaired. In this group, GFR was therefore measured at control pressure and at a pressure 2-6 mm Hg above the lower pressure limit of RBF autoregulation during enalapril treatment.
Aprotinin (Sigma A 4529, MW 6,500) was dissolved in 0.05 M Na-phosphate buffer at pH 7.5 and labeled with 125I or 131I by IODO-GEN technic (Pierce Chemical Company, Rockford, IL, U.S.A.) at the Institute for Energy Technology, Kjeller, Norway. The initial specific activity of labeled Ap was ≈60 MBq/mg Ap, corresponding to ≈3 × 106 counts/min/μg.
Measurements of GFR
Total and zonal GFR were obtained by measuring the tubular uptake of radiolabeled Ap as described by Tenstad et al. (7). After taking blood samples for hematocrit and background radioactivity measurements, 10 μl 125I-labeled aprotinin (125I-Ap) was injected in the jugular vein in the course of 5-6 s. Arterial blood samples (0.1 ml/sample) were then collected at 15 s and 1, 3, 5, 10, and 15 min, respectively, after starting the injection of 125I-Ap. After collection of each blood sample, 0.1-0.2 ml of 5% bovine serum albumin was injected to compensate for the blood loss. Fifteen minutes after infusion of 125I-Ap, the RAP was reduced and maintained at 2-6 mm Hg above the lowest pressure limit of RBF autoregulation in the remaining part of the experiment. Five minutes after pressure reduction, a bolus of 131I-Ap was injected iv., and arterial blood samples were collected at 15 s and 1, 3, 5, and 8 min in the same way as after 125I-Ap injection. Thereafter both renal pedicles were ligated. The kidneys were removed, frozen in isopentane prechilled to -20°C, and put into preweighed plastic tubes, reweighed, and immediately counted in a gamma counter (COBRA II AUTO-GAMMA) for 1 min, before being frozen again.
The blood samples were centrifuged for 10 min at 5,000 r/min, and 10 μl plasma was taken from each sample for analysis of the total protein concentration with a refractometer (American Optical). Another 10 μl of plasma was diluted in 1 ml 0.9% NaCl, and radioactivity was counted for 5 min.
The kidney was placed in a constantly cooled petri dish containing isopentane and was first divided transversely in three 3- to 4-mm thick slices. Then samples of outer, middle, and inner cortex were cut from each of these slices under the dissection microscope at ×16 magnification, as previously described (1). Each piece of cortical tissue, weighing ≈5 mg, was placed in a preweighed plastic tube, reweighed, and the radioactivity was counted for 5 min.
The total and local renal clearances of Ap (CAp) were calculated as the amount of 125I-Ap or 131I-Ap accumulated in 1 g kidney or tissue sample (Q) divided by the time-integrated plasma concentration (∫PAp): eqn. (1)
To obtain GFR, the clearance was corrected for an average plasma protein-bound fraction of Ap (Pb) of 0.07, and for a Donnan distribution (r) across the glomerular membrane of 0.63-0.68, depending on the plasma concentration of protein (8).
Thus total and local GFR were calculated as eqn. (2)
Zonal GFR was obtained as the average GFR in five tissue samples in each zone.
The renal filtration fraction (FF) was calculated as eqn. (3)
where RPF is renal plasma flow and Hct is hematocrit.
Renal vascular resistance (RVR) was calculated as eqn. (4)
Numeric estimate of RBF and GFR autoregulation. To quantify of the degree of RBF and GFR autoregulation, we calculated the “fractional compensation” (10), defined as eqn. (5)
where ΔRBFauto and ΔRBF0 are flow changes resulting from a change in renal arterial pressure (ΔRAP), with (auto) and without (0) autoregulation. In the absence of autoregulation, RBF was assumed to decrease in proportion to RAP. A fractional compensation of 0.90 means that a reduction of RAP decreased RBF by only 10% of that expected without autoregulation.
In calculating fractional compensation of GFR, we assumed that, without autoregulation, GFR would decrease linearly with reduced pressure, extrapolating to zero at an arterial pressure of 60 mm Hg (10,11). eqn. (6)
where GFR1 is GFR at control arterial pressure, and ΔGFR is the difference between GFR at control and reduced pressure.
The results are presented as mean ± SEM. Differences between groups were assessed by one-way analysis of variance with the use of Bonferroni's corrections. Where significant differences were found, the groups were compared by Student's t test. Differences between cortical layers were analyzed by paired comparisons. A value of p < 0.05 was considered to be statistically significant.
Effects of losartan, enalapril, and nifedipine on hemodynamic parameters
Blood pressure. In 10-week-old WKYs, losartan and enalapril treatment did not influence the RAP. Because of the pronounced effect of 10 and 15 mg/kg/day of nifedipine on the arterial pressure in these rats, the dose was reduced to 5 mg/kg/day to keep the blood pressure >90 mm Hg (Table 1). A reduction of arterial pressure was observed in 10- and 40-week-old SHRs during treatment with all drugs but significantly more after nifedipine and enalapril treatment compared with losartan therapy (Tables 2 and 3).
RBF and GFR. In 10-week-old WKYs, RBF was significantly increased after losartan, enalapril, and nifedipine therapy, whereas GFR remained constant (Table 1). Enalapril and nifedipine and increased RBF and GFR in 10-week-old SHRs, whereas losartan had no significant effect on RBF and GFR (Table 2). RBF was increased in 40-week-old SHRs after all drug regimens, whereas an increase in GFR was induced by enalapril and nifedipine but not by losartan treatment (Table 3).
Renal vascular resistance. Renal vascular resistances (RVR) were significantly higher in hypertensive rats (141-143% vs. 100%) than in normotensive WKY rats. Losartan, enalapril, and nifedipine markedly reduced RVRs in 10- and 40-week-old SHRs. A more pronounced effect on RVR was observed in 10- and 40-week-old SHRs during enalapril and nifedipine treatment than after losartan therapy (Tables 2 and 3).
RBF and total GFR autoregulation
In WKYs, the antihypertensive drugs did not change the lower pressure limit of RBF autoregulation. In untreated 10-week-old SHRs, the range of RBF autoregulation was shifted to the right, the lower pressure limit being increased to 108 ± 4 mm Hg (Fig. 1). Losartan and enalapril normalized the range of RBF autoregulation, but only enalapril reduced the lower pressure limit to a level not significantly different from that observed in normotensive rats. In untreated 40-week-old SHRs, the range of RBF autoregulation was shifted to higher pressure levels, and the lower pressure limit was 113 ± 2 mm Hg, which was reduced significantly by losartan and enalapril (losartan, 98 ± 3 mm Hg; enalapril, 104 ± 3 mm Hg). None of these drugs normalized RBF autoregulatory range completely. However, within the observed range, the fractional compensation of RBF was ≥0.94 in all losartan and enalapril groups, not different from that obtained in untreated animals, whereas nifedipine impaired RBF autoregulation in all groups (Fig. 1, Table 4).
Fractional compensation of total GFR was lower than the compensation of RBF in untreated WKY rats and during enalapril or losartan treatment in all groups, whereas nifedipine caused a higher autoregulatory compensation for GFR than for RBF in WKYs and in 10-week-old SHRs (Table 4). Fractional compensation of total GFR was reduced in all treated rats, but statistical significance was reached only in 40-week-old SHRs with all types of treatment and in 10-week-old SHRs during losartan therapy (Table 4).
Zonal GFR autoregulation
The main impression from the zonal pressure-GFR curves shown in Fig. 2 is the similarity to that obtained for the whole kidney and the fairly uniform pattern in the three zones. Thus zonal GFR autoregulation in all zones, as judged by fractional compensation, was significantly reduced during losartan treatment in 10-week-old SHRs and in all treated 40-week-old SHRs, such as obtained from total GFR. However, on average the zonal GFR compensation was somewhat lower than that calculated for the whole kidney (Table 4). Moreover, fractional compensation tended to increase from outer to the inner cortex, suggesting better autoregulation of GFR in deep than in juxtamedullary cortex (Fig. 3). The values showed the same trend in all groups, suggesting that this pattern was not accidental. Thus ratio of fractional compensation between inner and outer cortex, as well as the ratio between inner and middle cortex, was all different from 1.00 (fractional compensation: IC/OC = 1.25 ± 0.03; MC/OC = 1.15 ± 0.02) By using paired analysis, the fractional compensation in inner cortex was significantly higher than that in the outer and middle cortex in all groups (p < 0.05), with the exception of untreated WKYs.
The main finding in this study was the impaired autoregulation of GFR within the range of complete RBF autoregulation in 40-week-old SHRs during antihypertensive treatment. In contrast, only a slight dissociation between RBF and GFR autoregulation was found in normotensive animals and in untreated SHRs. Thus our data suggest that the antihypertensive drugs are responsible for the selective effect on GFR in 40-week-old hypertensive rats.
We have previously shown that the renal abnormalities in 40-week-old SHRs are not homogeneously distributed in the kidney (1). Glomerulosclerosis, vascular hypertrophy, and tubular atrophy is more pronounced in the juxtamedullary than in the superficial cortex (1,2). Interestingly, and in apparent conflict with these findings, antihypertensive treatment impaired autoregulation of GFR less in deep than in superficial cortex. These data suggest that the hypertrophy of the interlobular artery may affect autoregulation more in superficial than in juxtamedullary cortex in hypertensive rats.
In the normotensive rat, renal blood flow is kept constant during pressure reduction mainly by preglomerular vasodilatation, which will keep GFR constant if this mechanism is acting alone. However, as shown in a previous study (1), GFR is reduced by 10-12% despite unaltered RBF during reduction of arterial pressure in normotensive rats, as well as in young and old SHRs. A similar difference between RBF and GFR autoregulation also has been found in the dog (12). The decrease in GFR during pressure reduction suggests a reduction in postglomerular resistance and glomerular capillary pressure, or a decline in the filtration coefficient (Kf), but our data cannot distinguish between these possibilities. A constrictor effect on the postglomerular vessels might result from increased angiotensin II formation during pressure reduction (13-15). The impaired GFR autoregulation during losartan and enalapril treatment, but not in the untreated animals, suggests that the renin-angiotensin system might be involved in hypertensive rats, and that inhibition of the renin-angiotensin system may reduce the ability to keep GFR relatively constant during arterial pressure reduction, at least in 40-week-old SHRs. These observations are in line with those of Kastner et al. (16), who demonstrated that intrarenal formation of angiotensin II may play an important role in the control of GFR.
In a previous study, it was demonstrated that resetting of RBF autoregulation in SHRs occurs with increasing blood pressure, and that 7 days of captopril treatment normalizes RBF autoregulation in young SHRs but not in 40-week-old SHRs (4). Similarily, in our study, both losartan and enalapril reset RBF autoregulation to lower pressure levels in 10- and 40-week-old SHRs, but complete normalization of the lower pressure limit of RBF autoregulation was obtained only in enalapril-treated 10-week-old SHRs. These observations indicate that the resistance was reduced by the antihypertensive treatment before RAP was reduced for testing autoregulation, leaving less ability for additional dilatation. In our study, the impaired GFR autoregulation was found in 10- and 40-week-old SHRs during losartan treatment and in enalapril-treated 40-week-old SHRs (i.e., in the groups in which the lower limit of RBF autoregulation was partially normalized). Thus the reduced GFR autoregulation in these groups might be the result of both restricted preglomerular dilatation and reduced increase of efferent resistance caused by less effect of angiotensin II on this segment. As GFR was not different between 10- and 40-week-old SHRs, the effect of GFR per se seems not to play a role. However, FF was significantly lower in 40-week-old SHRs, an observation that might suggest that efferent angiotensin II constriction was impaired in these rats. An additional effect on Kf might also contribute to the impaired GFR autoregulation.
The mechanism by which nifedipine interfered with GFR autoregulation in 40-week-old SHRs is probably different from that induced by enalapril and losartan. Nifedipine selectively dilates preglomerular vessels, causing increased glomerular capillary pressure even after blood-pressure reduction (17). This mechanism may explain the relatively well preserved GFR autoregulation in spite of the greatly impaired RBF autoregulation induced by nifedipine (18).
As stated, renal anatomic pathology is not homogeneously distributed in the renal cortex. The juxtamedullary cortex reveals more glomerulosclerosis, vascular hypertrophy, and tubular atrophy than do the superficial layers. Nevertheless, paired comparison showed significantly better autoregulation in juxtamedullary than in superficial cortex. This might be the result of hypertrophy and reduced ability to dilate the interlobular arteries, which are responsible for a large fraction of preglomerular resistance of superficial nephrons and for the autoregulation of glomerular pressure in these nephrons (19). Alternatively, an increased pressure decrease along the interlobular arteries in hypertensive animals might induce a permanent dilation of downstream resistance vessels and thereby impair the potential for further reduction of resistance during pressure reduction.
When we consider small differences in fractional compensation, it is clearly pertinent to look into possible methodologic errors, especially the contribution of extratubular Ap. Whereas all filtered Ap is accumulated in the proximal tubular cells in renal cortex, the renal interstitium and plasma will have an Ap concentration close to that of arterial plasma at the time of kidney excision, which will be higher for the second than for the first tracer. Because the medulla has a larger extratubular space, the inclusion of nonfiltered medullary Ap could add considerably to whole kidney Ap content and give an excessively high GFR per gram in the second clearance period. This may explain the apparently more efficient autoregulation of whole kidney GFR than that in the cortical zones, as evident in Fig. 3 and Table 4. The effect is exaggerated when the amount of filtered and tubular accumulation of Ap is reduced during reduction of arterial pressure in the second clearance period.
Even though the extratubular space is much smaller in the cortex than in the medulla, a similar mechanism might contribute to the apparently more efficient autoregulation of GFR in the inner cortex because samples from this zone may contain some outer medullar tissue. On the other hand, this kind of methodologic bias could not explain the higher compensation in the middle than in the outer cortex. We therefore consider it most likely that the higher compensation in inner cortical layers does reflect more efficient autoregulation of GFR in juxtamedullary than in superficial cortex. This is also in line with our previous observation (1).
In conclusion, the results show that GFR autoregulation is impaired after losartan, enalapril, and nifedipine treatment in 40-week-old SHRs. The autoregulatory ability of the GFR is better preserved in juxtamedullary cortex in hypertensive animals during antihypertensive treatment, probably because of reduced ability to dilate the interlobular arteries.
Acknowledgment: This work was supported by a grant from MSD, Norway. We also thank MSD for the gift of AT1-receptor antagonist, losartan (DuP 753).
1. Wang X, Aukland K, Ofstad J, Iversen BM. Autoregulation of zonal glomerular filtration rate and renal blood flow
in spontaneously hypertensive rats. Am J Physiol
2. Olson JL, Wilson SK, Heptinstall RH. Relation of glomerular injury to preglomerular resistance in experimental hypertension. Kidney Int
3. Iversen BM, Horvei G, Ofstad J. Hypertrophy and increased glomerular capillary pressure (PG) in deep cortical glomeruli in SHR [Abstract]. J Hypertens
1994;12 (suppl 3):S36.
4. Iversen BM, Sekse I, Ofstad J. Resetting of renal blood flow
autoregulation in spontaneously hypertensive rats. Am J Physiol
5. Schnermann J, Briggs JP, Weber PC. Tubuloglomerular feedback, prostaglandins, and angiotensin in the autoregulation of glomerular filtration rate. Kidney Int
6. Loutzenhiser R, Epstein M. The renal hemodynamic effects of calcium antagonists. In: Epstein M, Loutzenhiser R, eds. Calcium antagonists and the kidney
. Philadephia: Hanley & Belfus, 1990;33-73.
7. Tenstad O, Williamson HE, Aukland K. Repeatable measurement of local and zonal GFR in the rat kidney with aprotinin. Acta Physiol Scand
8. Tenstad O, Williamson HE, Clausen G, Øien A, Aukland K. Glomerular filtration and tubular absorption of the basic polypeptide aprotinin. Acta Physiol Scand
9. Iversen BM, Kvam FI, Mørkrid L, et al. Effect of mesangiolysis on autoregulation of renal blood flow
and glomerular filtration rate in rats. Am J Physiol
10. Aukland K, Øien AH. Renal autoregulation: models combining tubuloglomerular feedback and myogenic response. Am J Physiol
11. Moore LC. Interaction of tubuloglomerular feedback and proximal nephron reabsorption in autoregulation. Kidney Int
12. Kirchheim HR, Ehmke H, Hackenthal E, Löwe W, Persson P. Autoregulation of renal blood flow
, glomerular filtration rate and renin release in conscious dogs. Pflugers Arch
13. Hall JE, Guyton AC, Cowley AW. Dissociation of renal blood flow
and filtration rate autoregulation by renin depletion. Am J Physiol
14. Eide I, Løyning E, Kiil F. Evidence for hemodynamic autoregulation of renin release. Circ Res
15. Conrad KP, Brinck-Johnsen T, Gellai M, Valtin H. Renal autoregulation in chronically catheterized conscious rats. Am J Physiol
16. Kastner RP, Hall JE, Guyton AC. Control of glomerular filtration rate: role of intrarenally formed angiotensin II. Am J Physiol
17. Kvam FI, Iversen BM, Ofstad J. Glomerular capillary pressure is dependent on systemic blood pressure during treatment with calcium channel blockers [Abstract]. J Am Soc Nephrol
18. Carmines PK, Navar LG. Disparate effects of Ca channel blockade on afferent and efferent arteriolar responses to ANG II. Am J Physiol
19. Heyeraas KJ, Aukland K. Interlobular arterial resistance: influence of renal arterial pressure and angiotensin II. Kidney Int