PfliJgers Archiv
Pflfigers Arch. 370, 81-85 (1977)
EuropeanJourrml
of Physk~bgy 9 by Springer-Verlag 1977
Autoregulation of Renal Blood Flow in the Rat JII~[ H E L L E R a n d V L A D I S L A V H O R A C E K with technical assistance of JANA HOLLYOV,/~ Institute for Clinical and Experimental Medicine, CS-14622 Prague-KrG Czechoslovakia
Summary. R e n a l b l o o d flow was m e a s u r e d in rats a n a e s t h e t i z e d with p e n t o b a r b i t a l s o d i u m b y e m p l o y ing the P A H c l e a r a n c e m e t h o d a n d the 133Xe w a s h o u t technique. B o t h m e t h o d s were in g o o d a g r e e m e n t . I n 17 rats a 25 ~ d e c r e m e n t o f b l o o d p r e s s u r e was attained through haemorrhage; RBF measured by PAH c l e a r a n c e d e c l i n e d b y 3 ~ , a c c o r d i n g to 133Xe w a s h o u t b y 2.7 ~ . I n f u r t h e r 21 rats a n increase o f b l o o d pressure b y an a v e r a g e o f 26 ~ was achieved b y o c c l u s i o n o f the c a r o t i d a r t e r i e s ; R B F m e a s u r e d by P A H c l e a r a n c e d e c l i n e d b y 3 ~ , a c c o r d i n g to 133Xe w a s h o u t b y 1.1 ~ . R B F was t h e r e f o r e a l m o s t perfectly a u t o r e g u l a t e d d u r i n g b o t h e x p e r i m e n t a l m a n o e u v r e s ; glom e r u l a r filtration rate (inulin clearance) was a u t o r e g u l a t e d in a s i m i l a r m a n n e r . W a t e r a n d s o d i u m e x c r e t i o n c h a n g e d in p r o p o r t i o n to c h a n g e s o f b l o o d pressure. Key words." R e n a l
b l o o d flow -
Autoregulation of
r e n a l b l o o d flow.
INTRODUCTION T h e p h e n o m e n o n o f the a u t o r e g u l a t i o n o f r e n a l b l o o d flow ( R B F ) , i.e., its r e m a r k a b l e c o n s t a n c y d u r i n g c h a n g e s o f b l o o d p r e s s u r e in the r a n g e o f 80 to 180 m m Hg, is well p r o v e n in d o g s a n d h u m a n beings ( H i n s h a w , 1964; Selkurt, 1963; T h u r a u , 1964). D a t a o b t a i n e d f r o m rats, however, are less consistent. W e i s s et al. (1959) d e s c r i b e d the presence o f a u t o r e g u l a t i o n in i s o l a t e d r a t k i d n e y s p e r f u s e d with artificial c o l l o i d a l solutions. O h l e r et al. (1959) d i d n o t o b serve a n y a u t o r e g u l a t i o n in k i d n e y s p e r f u s e d in situ with b l o o d f r o m the a o r t a o f a n o t h e r rat. Studies h a v e been p u b l i s h e d recently e m p l o y i n g m o d e r n m e t h o d s f o r m e a s u r i n g R B F in rats. A r e n d s h o r s t et al. (1975) u s e d a n e l e c t r o m a g n e t i c f l o w m e t e r
s i m u l t a n e o u s l y w i t h P A H c l e a r a n c e (they f o u n d a n excellent a g r e e m e n t o f b o t h m e t h o d s ) a n d d e s c r i b e d a very effective a u t o r e g u l a t i o n . K ~ l l s k o g et al. (1975) measured RBF employing radioactive microspheres. A l t h o u g h a u t o r e g u l a t i o n was p r e s e n t in s o m e cases, the p e r c e n t a g e o f rats w h i c h failed to p r o d u c e a u t o r e g u l a t i o n for s o m e u n k n o w n r e a s o n was high. I n o r d e r to clarify this d i s c r e p a n c y we m e a s u r e d R B F in i n d i v i d u a l rats e m p l o y i n g s i m u l t a n e o u s l y the t33Xe w a s h o u t t e c h n i q u e a n d the P A H c l e a r a n c e m e t h o d after a p p l y i n g p r o c e d u r e s w h i c h b r o u g h t a b o u t c h a n g e s in a r t e r i a l pressure. W e f o u n d a g o o d a g r e e m e n t b e t w e e n b o t h m e t h o d s a n d a perfect a u t o r e g u l a t i o n o f R B F in all cases. METHODS Rats from a Wistar strain, of both sexes, weighing from 180-220 g, were anaesthetized with Pentobarbital sodium, 50 mg/kg BW i.p. A cannula was introduced into the jugular vein, and 2.4 mg PAH and 4 mg of inulin in 0.2 ml isotonic saline were injected. Then an infusion of 0.12 mg PAH and 0.2 mg of inulin in 10 gl saline solution per rain was started. With this dosage the PAH plasma concentration ranged from 1.4 to 2.1 mg ~. Subsequently, a cannula was inserted into the trachea, and into a femoral artery and vein for blood pressure registration and collection of blood samples respectively; further cannulae were introduced into both ureters via a small suprapubic incision. The left kidney was prepared by a flank incision, placed into a plastic dish and covered with liquid paraffin at a temperature of 37~C. The entire rat except for the vicinity of the kidney was screened off with lead plates. A small hypodermic needle, fitted to the end of a polyethylene tubing, was inserted in the initial experiments into the left renal artery; lateron the needle was placed into the aorta proximal to the renal arteries. As the curves obtained were identical, the route of administration is not differentiated in the further text. A scintillating crystal, 2.5 x 3 cm in diameter (Tesla P~'emy~leni) with a cylindrical collimator 3.3 cm long, was fixed closely above the kidney. The crystal was connected with a DM 160 counter by means of an integrator DR 213 (ACEC, Belgium); the washout curves were registered on a recorder with a time constant of J s;,the rate of paper shifting amounted to 60 cm/min. Approximately t00 gCi 133Xe (Berlin-Buch) in 0.1 ml saline were injected rapidly into the aorta. The registered tracings were read and calculated
82
Pflfigers Arch. 370 (1977)
according to the method of Thorburn et al. Out of the 4 components of the washout curve thus obtained only the first was evaluated in these experiments. Two control clearance periods of 20 rain each were carried out 5 0 - 6 0 rain after the prime of PAH and inulin. In the middle of each period, 0.15 ml of blood was withdrawn from the arterial cannula; this blood loss was replaced immediately by the same amount of isotonic saline i.v. After termination of the second clearance period, the procedure leading to the change of blood pressure was performed. As soon as the blood pressure values became stabilized, three further clearance periods of 20 rain each ensued. 133Xe was injected regularly at the 5th rain of each period. A decrease of blood pressure was achieved by haemorrhage in a total of 33 rats; initially 2 ml blood were withdrawn (1% BW); because the blood pressure frequently declined below the expected limit of autoregulation (80 mmHg), in further experiments only 1.5 ml were withdrawn. Only those experiments (i.e. 17 rats) were included into results where the pressure was reduced by more than 10 mm Hg but still remaining within the limits of autoregulation. An increase of blood pressure was obtained in 21 rats through the ligature of the carotid arteries. Only animals with an increase of blood pressure higher than 10 mmHg were included into our results.
BP rnmHr 160 9
9
9
140
9
00 9 O9 000 9 00
9
9
00
120
9 9 00
100
9D
9 9
9 9 9
9
o~149149
~
9 9 99 9 9 9 - 0 ~ 0 - gO 000' 000 u 9 9 00000
. 9
9 0
9 9 9 ~ 9
0000 000 9 0 0 0 09 9 OO ~ 0 0 9 9 9
9 o
80
00"0000 OO 0 O 9 O 0:0000
9
9
9 9
9
RESULTS Figure 1 demonstrates a summary of PAH clearance measurements in 155 rats from our previous experiments and the relation of this value (calculated from the haematocrit on the total RBF) to the arterial pressure. These are initial control periods of our various experiments always performed under identical conditions. The considerable scatter in the basal levels of blood pressure is rather remarkable although all rats under observation were of the same weight (180-220 g) and had been anaesthetized in the same manner (50 mg/kg of pentobarbital sodium i.p.); the cause of this fact is not quite apparent. A decrease of blood pressure (Tables 1 and 3) by 25% from the mean value of 124 to 96mmHg, achieved by withdrawal of 1.5 or 2 ml of blood, was accompanied by a decrease of urine flow rate from 5.7 to 4.2 gl/min/g KW (26.3 %) and of sodium excretion from 0.44 to 0.3 I.tEq/min/g KW (32 %). The inulin clearance decreased only by 4 % (from 1.05 to 1.00 ml/min/g KW) and RBF calculated from PAH clearance and haematocrit declined by 3 % (from 5.3 to 5.13 ml/min/g KW). Similarly, RBF calculated from the 1st component of the washout curve of 133Xe declined only by 2.7% (from 5.34 to 5.20 ml/min/g KW).
60 i
i
i
RBF rnI/min.gKW "~ Fig. 1. Relationship between blood pressure (BP mm Hg) and renal blood flow values (RBF) calculated from PAH clearance and haematocrit. Previous experiments
Table 1.
Analytical Methods. Inulin was determined by a modified diphenylamine method (Little, 1949); PAH according to Smith et al. (1954); sodium on a flame photometer (Zeiss Jena, GDR). Haematocrit was measured in the heparinized blood at the end of the experiment.
An increase of blood pressure (Tables 2 and 3) by 26.6% from 108.7 to 136.1 mmHg induced by a ligature of carotid arteries led to an increase in urine flow rate by 31.4% (from 3.95 to 5.03 gl/min/g KW) and to an increase in sodium excretion by 38.8 % (from 0.35 to 0.47 laEq/min/g KW). Inulin clearance increased only by less than 2 % (from 1.05 to 1.07 ml/
Renal function in rats after a decrease of blood pressure BP mmHg
n
17
V
UNaV
Cin
RBF from CPAH RBF from 133Xe
17
17
17
17
17
Controls (mean _ S.E,M.)
123,71 1.62
5.7 0.26
0,44 0.02
1.05 0.08
5.30 0.08
5.34 0.41
After haemorrhage (mean ___ S.E.M.)
92.53 2.04
4.16 0.17
0.30 0.02
1,00 0.02
5.13 0.05
5.20 0.80
Mean ~ change
25.22
26.27
33.97
4.41
3.09
2.68
N.S.
N.S.
N.S.
P (t-test)
0,001
0.001
0.001
A hhreviations: BP mm Hg = blood pressure; V = urine flow rate in ~tl/min/g KW; UN~V = sodium excretion in p,Eq/min/g KW; C~n = inulin clearance, in ml/min/g KW, RBF from CpAH = renal blood flow calculated from CpA~ + haematocrit, ml/min/g KW; RBF from 133Xe = renal blood flow calculated from the 1st component of the washout curve, ml/min/g KW; n = number of animals; S.E.M. = standard error of means; P = significance according to the Student's t-test; N.S. = non-significant difference
83
J. Heller and V. Hor~t6ek: Autoregulation of Renal Blood Flow in the Rat Table 2.
Renal function in rats after an increase of blood pressure BP mmHg
n
UNaV
C~n
21
21
RBF from CpAH RBF from 12~Xe
9
9
Controls (mean _+ S.E.M.)
108.7 2.19
3.9 0.2
0.37 0.02
1.05 0.03
5.45 0.19
5.49 0.63
Carotid lig. (mean + S.E.M.)
136.1 1.9
5.0 0.2
0.47 0.03
1.07 0.02
5.54 0.18
5.54 0.62
31.40
38.83
Mean ~ change P (t-test)
21
V
26.59 0.001
0.001
0.001
21
1.91
1.83
1.06
N.S.
N.S.
N.S.
Abbreviations." See Table 1
min/g KW); RBF calculated from PAH clearance and haematocrit increased from 5.45 to 5.54, RBF calculated from 133Xewashout from 5.49 to 5.53 ml/min/g KW, i.e. by 1 ~o.
DISCUSSION From Figure 1, which summarizes our control measurements in previous experiments, a considerable scatter in the basal levels of blood pressure is quite evident. Furthermore it is apparent that a direct relationship exists between the blood pressure level and RBF and finally that the scatter of the values is enormous. At an arterial pressure of 100 mmHg, for example, the values found for RBF fluctuated between 3.08 and 7.1 ml/min/g KW. The interpretation of this phenomenon is not clear. Although at prima facie this observation does not testify for the presence of RBF autoregulation in rats, it is possible t h a t - i n spite of considerably different interindividual v a l u e s - a perfect autoregulation might exist intraindividually. The present experiments confirm this assumption. The presence of autoregulation of renal blood flow in rats as determined in these experiments agrees with the results of Arendshorst et al. (1975). The existence of autoregulation has now been proved by means of the following three methods: PAH clearance, electromagnetic flowmeter, and Xe washout. Although the first component of the washout curve which we used and which was not confirmed by autoradiography yields the comparatively widest scatter of values (Table 3), the mean value still agrees well with PAH clearance. Results by Kgllskog et al. (1975), however, who employed radioactive microspheres, are not quite uniform. According to these authors, 6 rats out of 25 did not show any autoregulation. As demonstrated in Table 1 of their study, however, only in 14 rats a reduction of blood pressure within the autoregulatory range (if 70 mmHg is considered as the lowest limit)
was achieved. In the remaining 11 rats, the blood pressure values decreased below this limit after the clamping of the aorta. For the solution of the problem whether the autoregulation was present or absent, these 11 rats can therefore not be taken into account. In this case, 6 rats out of 14, represent almost half of the total number, in which the presence of autoregulation was not found. Studies in which the hydrostatic pressure was measured in intrarenal blood vessels at various levels of blood pressure also contribute to our knowledge of the existence of renal blood flow autoregulation. It is remarkable that even here the results are not uniform although the methods used are frequently very similar. In 1962 Thurau and Wober measured the hydrostatic pressure in the peritubular capillaries and in the tubules of rats before and after occlusion of both carotids; the changes in blood pressure ranged between 80 and 164 mmHg while the pressure in the intrarenal structures remained constant. Gertz et al. (1966) measured the hydrostatic pressure in the glomerular capillaries indirectly by means of the intratubular stopflow pressure method. Although this technique yields absolute values which are probably too high (Kfillskog et al., 1975), the fact that this pressure remained constant within the range of blood pressure values between 90 and 160 mmHg confirms the autoregulatory response. B.onvalet et al. (1972) measured glomerular filtration rate in single glomeruli (SNGFR) with labelled ferrocyanide and found no significant changes of values when the decrease of the perfusion pressure, induced by a clamp, amounted to 68 turn Hg; below this value the SNGFR decreased. Similarly, Bonvalet et al. (1973) did not find any changes of SNGFR within the range of autoregulation in the course of acute hypertension. Knox et al. (1974) measured SNGFR at a blood pressure of 130 and 100 mmHg; the values were always identical. K/illskog et al. (1975) measured the pressure in glomerular capillaries according to the method of Gertz as well
84
Pfl/igers Arch. 370 (1977)
Table 3. Comparison of individual values for RBF as measured by PAH clearance and 133Xe washout Increase of blood pressure
Decrease of blood pressure
n
Mean S.E.M.
RBF from CpAn
RBF from laaxe
RBF from CpAn
RBF from 133Xe
control
exper,
control
exper,
control
exper.
control
exper.
5.12 4.83 6.17 4.79 5.01 5.76 5.02 5.12 5.96 6.07 5.22 4.93 5.12 5.82 4.73 5.23 5.12 . . . .
5.01 4.63 5.87 4.29 5.27 5.34 4.97 5.01 5.68 5.79 5.27 4.78 4.99 5.61 4.66 5.03 5.03
5.13 3.92 6.14 3.17 5.31 7.03 4.88 4.98 5.97 5.71 5.83 3.87 6.27 5.99 4.91 5.11 5.41 . . . .
5.00 3.71 6.07 3.91 5.03 5.87 4.88 4.71 5.69 5.98 5.80 4.01 6.07 5.66 4.39 5.27 5.33
5.07 4.67 6.12 5.76 6.34 6.52 4.73 5.23 4.73 --
5.12 4.71 6.18 5.87 6.40 6.40 5.12 5.18 4.89 --
3.43 3.88 6.83 3.42 6.12 7.13 4.60 4.23 6.13 6.27 4.82 3.93 6.12 6.23 5.71 5.22 4.72 5.28 6.43 6.97 4.73
5.31 4.93 6.67 3.81 6.18 7.42 4.92 4.41 6.23 6.38 4.72 3.81 6.27 6.27 5.70 5.44 4.87 5.31 6.64 6.07 4.87
9 5.45 0.19
9 5.54 0.18
21 5.49 0.63
21 5.54 0.62
17 5.30 0.08
. . . .
. . . . 17 5,13 0.05
17 5.34 0.41
. . . .
. . . . 17 5.20 0.80
as the pressures in the welling points, in the perit u b u l a r capillaries, a n d in the p r o x i m a l tubuli. A l l pressures w e r e c o n s t a n t w i t h i n the r a n g e o f a u t o r e g u lation. C o n t r a r y to these e x p e r i m e n t s s u p p o r t i n g R B F a u t o r e g u l a t i o n , studies h a v e been p u b l i s h e d y i e l d i n g results p r i m a r i l y i n c o m p a t i b l e w i t h the existence o f a u t o r e g u l a t i o n . S c h o e p p e a n d F r e y (1966) f o u n d a n e l e v a t i o n o f the i n t r a t u b u l a r p r e s s u r e f r o m 11 to 23 m m H g l i n e a r with t h e increase o f b l o o d p r e s s u r e f r o m 60 to 140 m m H g . K o c h et al. (1968) a n d B a n k et al. (1970) e l e v a t e d the b l o o d p r e s s u r e in rats b y e m p l o y i n g a c l a m p r o u n d the c a r o t i d s a n d b y c u t t i n g the v a g a l nerves o r b y a n infusion o f e p i n e p h r i n e . T h e p r e s s u r e i n c r e a s e d every t i m e in the efferent arteriole, in the p e r i t u b u l a r capillaries a n d in the tubuli. W h e n , h o w e v e r , the r e n a l p e r f u s i o n p r e s s u r e was k e p t constant b y e m p l o y i n g a c l a m p o n the a o r t a these changes d i d n o t o c c u r after the increase in systemic b l o o d pressure. T h e o b s e r v a t i o n s o f A l l i s o n et al. (1972) m i g h t be p l a c e d in the s a m e c a t e g o r y . T h e y f o u n d a significant positive c o r r e l a t i o n b e t w e e n the p r o x i m a l h y d r o s t a t i c p r e s s u r e a n d the b l o o d p r e s s u r e after a n infusion o f m a n n i t o l . A similar c o r r e l a t i o n was f o u n d b e t w e e n the pressures in efferent arterioles o r peri-
t u b u l a r c a p i l l a r i e s a n d b l o o d pressure. A c c o r d i n g to these a u t h o r s , h o w e v e r , " s i n c e the statistical significance o f these differences m a y be r e l a t e d to the n u m b e r & o b s e r v a t i o n s . . . t h e i r p h y s i o l o g i c a l significance remains uncertain". A similar difference in results exists also in the dogs. F o r instance, M c N a y a n d A b e (1970) m e a s u r e d R B F with a n e l e c t r o m a g n e t i c f l o w m e t e r as well as b y m e a n s o f r a d i o a c t i v e m i c r o s p h e r e s , a n d c h a n g e d the r e n a l p e r f u s i o n p r e s s u r e within the r a n g e f r o m 81 to u p to 136 m m H g . W h i l e the t o t a l flow was a l t e r e d o n l y slightly the e x t e r n a l q u a r t e r o f the c o r t i c a l tissue d i d n o t s h o w a n y a u t o r e g u l a t i o n a n d the two i n t e r n a l q u a r t e r s d i s p l a y e d an inverse r e l a t i o n b e t w e e n pressure a n d flow. L o y n i n g (1971), h o w e v e r , using the m e t h o d o f local h y d r o g e n gas clearance, f o u n d a small decrease in b l o o d flow in the o u t e r as well as in the inner h a l f o f the c o r t e x at a decrease o f b l o o d p r e s s u r e f r o m 131 to 76 m m H g , i.e., n o evidence for a b l o o d flow r e d i s t r i b u t i o n . I n c o n c l u s i o n , the a u t o r e g u l a t i o n o f t o t a l r e n a l b l o o d flow in a n a e s t h e t i z e d rats seems to have been p r o v e n b y a n u m b e r o f i n d e p e n d e n t m e t h o d s even if the c h a n g e s o f the b l o o d p r e s s u r e were achieved b y various means.
J. Heller and V. Hor~ek: Autoregulation of Renal Blood Flow in the Rat REFERENCES Allison, M. E. M., Lipham, E. M., Gottschalk, C. W. : Hydrostatic pressure in the rat kidney. Amer. J. Physiol. 223, 975 - 9 8 3 (1972) Arendshorst, W. J., Finn, W. F., Gottschalk, C. W.: Autoregulation of blood flow in the rat kidney. Amer. J. Physiol. 228, 127133 (1975) Bank, N., Aynedjian, H. S., Bansal, V. K., Goldman, D. M. : Effect of acute hypertension on sodium transport by the distal nephron. Amer. J. Physiol. 219, 275- 280 (1970) Bonvaiet, J. P., Bencsfith, P., de Rouffignac, C. : Glomerular filtration rate of superficial and deep nephrons during aortic constriction. Amer. J. Physiol. 222, 599-606 (1972) Bonvalet, J. P., Bencsfith, P., de Rouffignac, C.: Glomerular filtration rate of superficial and juxta-medullary nephrons in the rat during different types of arterial hypertension. Pfliigers Arch. 340, 133 - 144 (1973) Gertz, K. H., Mangos, J. A., Braun, G., Pagel, H. D.: Pressure in the glomerular capillaries of the rat kidney and its relation to arterial blood pressure. Pflfigers Arch. ges. Physiol. 288, 369374 (1966) Hinshaw, L. B." Mechanism of renal autoregulation: Role of tissue pressure and description of a multifactor hypothesis. Circulat. Res. 15, Suppl. I, 120-131 (1964) K/illskog, ()., Lindbom, L.O., Ulfendahl, H.R., Wolgast, M. : The pressure-flow relationship of different nephron populations in the rat. Acta physiol, scand. 94, 2 8 9 - 300 (1975) Knox, F. G., Ott, C., Cuche, J. L., Gasser, J., Haas, J. : Autoregulation of single nephron filtration rate in the presence and the absence of flow to the macula densa. Circulat. Res. 34, 836-842 (1974) Koch, K. M., Aynedjian, H. S., Bank, N. : Effect of acute hypertension on sodium reabsorption by the proximal tubule. J. clin. Invest. 47, 1696-1709 (1968) Little, J. M. : A modified diphenylamine procedure for the determination of inulin. J. biol. Chem. 180, 747-752 (1949)
85
Loyning, E. W. : Effect of reduced perfusion pressure on intrarenal distribution of blood flow in dogs. Acta physiol, scand. 83, 191 202 (1971) McNay, J. L., Abe, Y. : Pressure-dependent heterogenity of renal cortical blood flow in dogs. Circulat. Res. 27, 571-587 (/970) Ohler, W., Harth, O., Kreienberg, W. : Die Abhfingigkeit der Nierendurchblutung vom arteriellen Druck bei der Ratte. Pfltigers Arch. ges. Physiol. 269, 274-281 (1959) Schoeppe, W. E., Frey, J.: Pressure in the proximal tubule of the rat kidney and its relation to arterial blood pressure. Proc. &the IIIrd Inter. Nephrol. Congress, 268 (1966) Selkurt, E. E. : The renal circulation. In: Handbook of Physiology, Circulation, Sect. 2, vol. II, 1457--I5/6. Washington, D.C.: Am. Physiol. Soc., Ed. 1963 Smith, H. W., Finkelstein, N., Aliminosa, L., Crawford, B., Graber, M. : The renal clearances of substituted hippuric acid derivatives and other aromatic acids in dog and man. J. clin. Invest. 24, 388-404 (/945) Thorburn, G.D., Kopald, H.H., Herd, J.A., Hollenberg, M., O'Morchoe, C. C. C., Barger, A, C. : Intrarenal distribution of nutrient blood flow determined with Krypton s5 in the unanaesthetized dog. Circulat. Res. 13, 290-307 (1963) Thnrau, K., Weber, E.: Zur Lokalisation der autoregulativen Widerstandsfinderungen in der Niere. M~kropunktionsmessnngen der Drucke in Tubuli und peritubul~ren Capillaren der Rattenniere bei Anderungen des arteriellen Druckes. Pfltigers Arch. ges. Physiol. 274, 553-566 (1962) Thurau, K.: Renal hemodynamics. Amer. J. Med. 36, 698-719 (1964) Weiss, C., Passow, H., Rothstein, A. : Autoregulation of flow in isolated rat kidney in the absence of red ceils. Amer. J. Physiol. 169, 1115-1118 (1959)
Received October 18, 1976
Pflfigers Arch. 370, 81-85 (1977)
EuropeanJourrml
of Physk~bgy 9 by Springer-Verlag 1977
Autoregulation of Renal Blood Flow in the Rat JII~[ H E L L E R a n d V L A D I S L A V H O R A C E K with technical assistance of JANA HOLLYOV,/~ Institute for Clinical and Experimental Medicine, CS-14622 Prague-KrG Czechoslovakia
Summary. R e n a l b l o o d flow was m e a s u r e d in rats a n a e s t h e t i z e d with p e n t o b a r b i t a l s o d i u m b y e m p l o y ing the P A H c l e a r a n c e m e t h o d a n d the 133Xe w a s h o u t technique. B o t h m e t h o d s were in g o o d a g r e e m e n t . I n 17 rats a 25 ~ d e c r e m e n t o f b l o o d p r e s s u r e was attained through haemorrhage; RBF measured by PAH c l e a r a n c e d e c l i n e d b y 3 ~ , a c c o r d i n g to 133Xe w a s h o u t b y 2.7 ~ . I n f u r t h e r 21 rats a n increase o f b l o o d pressure b y an a v e r a g e o f 26 ~ was achieved b y o c c l u s i o n o f the c a r o t i d a r t e r i e s ; R B F m e a s u r e d by P A H c l e a r a n c e d e c l i n e d b y 3 ~ , a c c o r d i n g to 133Xe w a s h o u t b y 1.1 ~ . R B F was t h e r e f o r e a l m o s t perfectly a u t o r e g u l a t e d d u r i n g b o t h e x p e r i m e n t a l m a n o e u v r e s ; glom e r u l a r filtration rate (inulin clearance) was a u t o r e g u l a t e d in a s i m i l a r m a n n e r . W a t e r a n d s o d i u m e x c r e t i o n c h a n g e d in p r o p o r t i o n to c h a n g e s o f b l o o d pressure. Key words." R e n a l
b l o o d flow -
Autoregulation of
r e n a l b l o o d flow.
INTRODUCTION T h e p h e n o m e n o n o f the a u t o r e g u l a t i o n o f r e n a l b l o o d flow ( R B F ) , i.e., its r e m a r k a b l e c o n s t a n c y d u r i n g c h a n g e s o f b l o o d p r e s s u r e in the r a n g e o f 80 to 180 m m Hg, is well p r o v e n in d o g s a n d h u m a n beings ( H i n s h a w , 1964; Selkurt, 1963; T h u r a u , 1964). D a t a o b t a i n e d f r o m rats, however, are less consistent. W e i s s et al. (1959) d e s c r i b e d the presence o f a u t o r e g u l a t i o n in i s o l a t e d r a t k i d n e y s p e r f u s e d with artificial c o l l o i d a l solutions. O h l e r et al. (1959) d i d n o t o b serve a n y a u t o r e g u l a t i o n in k i d n e y s p e r f u s e d in situ with b l o o d f r o m the a o r t a o f a n o t h e r rat. Studies h a v e been p u b l i s h e d recently e m p l o y i n g m o d e r n m e t h o d s f o r m e a s u r i n g R B F in rats. A r e n d s h o r s t et al. (1975) u s e d a n e l e c t r o m a g n e t i c f l o w m e t e r
s i m u l t a n e o u s l y w i t h P A H c l e a r a n c e (they f o u n d a n excellent a g r e e m e n t o f b o t h m e t h o d s ) a n d d e s c r i b e d a very effective a u t o r e g u l a t i o n . K ~ l l s k o g et al. (1975) measured RBF employing radioactive microspheres. A l t h o u g h a u t o r e g u l a t i o n was p r e s e n t in s o m e cases, the p e r c e n t a g e o f rats w h i c h failed to p r o d u c e a u t o r e g u l a t i o n for s o m e u n k n o w n r e a s o n was high. I n o r d e r to clarify this d i s c r e p a n c y we m e a s u r e d R B F in i n d i v i d u a l rats e m p l o y i n g s i m u l t a n e o u s l y the t33Xe w a s h o u t t e c h n i q u e a n d the P A H c l e a r a n c e m e t h o d after a p p l y i n g p r o c e d u r e s w h i c h b r o u g h t a b o u t c h a n g e s in a r t e r i a l pressure. W e f o u n d a g o o d a g r e e m e n t b e t w e e n b o t h m e t h o d s a n d a perfect a u t o r e g u l a t i o n o f R B F in all cases. METHODS Rats from a Wistar strain, of both sexes, weighing from 180-220 g, were anaesthetized with Pentobarbital sodium, 50 mg/kg BW i.p. A cannula was introduced into the jugular vein, and 2.4 mg PAH and 4 mg of inulin in 0.2 ml isotonic saline were injected. Then an infusion of 0.12 mg PAH and 0.2 mg of inulin in 10 gl saline solution per rain was started. With this dosage the PAH plasma concentration ranged from 1.4 to 2.1 mg ~. Subsequently, a cannula was inserted into the trachea, and into a femoral artery and vein for blood pressure registration and collection of blood samples respectively; further cannulae were introduced into both ureters via a small suprapubic incision. The left kidney was prepared by a flank incision, placed into a plastic dish and covered with liquid paraffin at a temperature of 37~C. The entire rat except for the vicinity of the kidney was screened off with lead plates. A small hypodermic needle, fitted to the end of a polyethylene tubing, was inserted in the initial experiments into the left renal artery; lateron the needle was placed into the aorta proximal to the renal arteries. As the curves obtained were identical, the route of administration is not differentiated in the further text. A scintillating crystal, 2.5 x 3 cm in diameter (Tesla P~'emy~leni) with a cylindrical collimator 3.3 cm long, was fixed closely above the kidney. The crystal was connected with a DM 160 counter by means of an integrator DR 213 (ACEC, Belgium); the washout curves were registered on a recorder with a time constant of J s;,the rate of paper shifting amounted to 60 cm/min. Approximately t00 gCi 133Xe (Berlin-Buch) in 0.1 ml saline were injected rapidly into the aorta. The registered tracings were read and calculated
82
Pflfigers Arch. 370 (1977)
according to the method of Thorburn et al. Out of the 4 components of the washout curve thus obtained only the first was evaluated in these experiments. Two control clearance periods of 20 rain each were carried out 5 0 - 6 0 rain after the prime of PAH and inulin. In the middle of each period, 0.15 ml of blood was withdrawn from the arterial cannula; this blood loss was replaced immediately by the same amount of isotonic saline i.v. After termination of the second clearance period, the procedure leading to the change of blood pressure was performed. As soon as the blood pressure values became stabilized, three further clearance periods of 20 rain each ensued. 133Xe was injected regularly at the 5th rain of each period. A decrease of blood pressure was achieved by haemorrhage in a total of 33 rats; initially 2 ml blood were withdrawn (1% BW); because the blood pressure frequently declined below the expected limit of autoregulation (80 mmHg), in further experiments only 1.5 ml were withdrawn. Only those experiments (i.e. 17 rats) were included into results where the pressure was reduced by more than 10 mm Hg but still remaining within the limits of autoregulation. An increase of blood pressure was obtained in 21 rats through the ligature of the carotid arteries. Only animals with an increase of blood pressure higher than 10 mmHg were included into our results.
BP rnmHr 160 9
9
9
140
9
00 9 O9 000 9 00
9
9
00
120
9 9 00
100
9D
9 9
9 9 9
9
o~149149
~
9 9 99 9 9 9 - 0 ~ 0 - gO 000' 000 u 9 9 00000
. 9
9 0
9 9 9 ~ 9
0000 000 9 0 0 0 09 9 OO ~ 0 0 9 9 9
9 o
80
00"0000 OO 0 O 9 O 0:0000
9
9
9 9
9
RESULTS Figure 1 demonstrates a summary of PAH clearance measurements in 155 rats from our previous experiments and the relation of this value (calculated from the haematocrit on the total RBF) to the arterial pressure. These are initial control periods of our various experiments always performed under identical conditions. The considerable scatter in the basal levels of blood pressure is rather remarkable although all rats under observation were of the same weight (180-220 g) and had been anaesthetized in the same manner (50 mg/kg of pentobarbital sodium i.p.); the cause of this fact is not quite apparent. A decrease of blood pressure (Tables 1 and 3) by 25% from the mean value of 124 to 96mmHg, achieved by withdrawal of 1.5 or 2 ml of blood, was accompanied by a decrease of urine flow rate from 5.7 to 4.2 gl/min/g KW (26.3 %) and of sodium excretion from 0.44 to 0.3 I.tEq/min/g KW (32 %). The inulin clearance decreased only by 4 % (from 1.05 to 1.00 ml/min/g KW) and RBF calculated from PAH clearance and haematocrit declined by 3 % (from 5.3 to 5.13 ml/min/g KW). Similarly, RBF calculated from the 1st component of the washout curve of 133Xe declined only by 2.7% (from 5.34 to 5.20 ml/min/g KW).
60 i
i
i
RBF rnI/min.gKW "~ Fig. 1. Relationship between blood pressure (BP mm Hg) and renal blood flow values (RBF) calculated from PAH clearance and haematocrit. Previous experiments
Table 1.
Analytical Methods. Inulin was determined by a modified diphenylamine method (Little, 1949); PAH according to Smith et al. (1954); sodium on a flame photometer (Zeiss Jena, GDR). Haematocrit was measured in the heparinized blood at the end of the experiment.
An increase of blood pressure (Tables 2 and 3) by 26.6% from 108.7 to 136.1 mmHg induced by a ligature of carotid arteries led to an increase in urine flow rate by 31.4% (from 3.95 to 5.03 gl/min/g KW) and to an increase in sodium excretion by 38.8 % (from 0.35 to 0.47 laEq/min/g KW). Inulin clearance increased only by less than 2 % (from 1.05 to 1.07 ml/
Renal function in rats after a decrease of blood pressure BP mmHg
n
17
V
UNaV
Cin
RBF from CPAH RBF from 133Xe
17
17
17
17
17
Controls (mean _ S.E,M.)
123,71 1.62
5.7 0.26
0,44 0.02
1.05 0.08
5.30 0.08
5.34 0.41
After haemorrhage (mean ___ S.E.M.)
92.53 2.04
4.16 0.17
0.30 0.02
1,00 0.02
5.13 0.05
5.20 0.80
Mean ~ change
25.22
26.27
33.97
4.41
3.09
2.68
N.S.
N.S.
N.S.
P (t-test)
0,001
0.001
0.001
A hhreviations: BP mm Hg = blood pressure; V = urine flow rate in ~tl/min/g KW; UN~V = sodium excretion in p,Eq/min/g KW; C~n = inulin clearance, in ml/min/g KW, RBF from CpAH = renal blood flow calculated from CpA~ + haematocrit, ml/min/g KW; RBF from 133Xe = renal blood flow calculated from the 1st component of the washout curve, ml/min/g KW; n = number of animals; S.E.M. = standard error of means; P = significance according to the Student's t-test; N.S. = non-significant difference
83
J. Heller and V. Hor~t6ek: Autoregulation of Renal Blood Flow in the Rat Table 2.
Renal function in rats after an increase of blood pressure BP mmHg
n
UNaV
C~n
21
21
RBF from CpAH RBF from 12~Xe
9
9
Controls (mean _+ S.E.M.)
108.7 2.19
3.9 0.2
0.37 0.02
1.05 0.03
5.45 0.19
5.49 0.63
Carotid lig. (mean + S.E.M.)
136.1 1.9
5.0 0.2
0.47 0.03
1.07 0.02
5.54 0.18
5.54 0.62
31.40
38.83
Mean ~ change P (t-test)
21
V
26.59 0.001
0.001
0.001
21
1.91
1.83
1.06
N.S.
N.S.
N.S.
Abbreviations." See Table 1
min/g KW); RBF calculated from PAH clearance and haematocrit increased from 5.45 to 5.54, RBF calculated from 133Xewashout from 5.49 to 5.53 ml/min/g KW, i.e. by 1 ~o.
DISCUSSION From Figure 1, which summarizes our control measurements in previous experiments, a considerable scatter in the basal levels of blood pressure is quite evident. Furthermore it is apparent that a direct relationship exists between the blood pressure level and RBF and finally that the scatter of the values is enormous. At an arterial pressure of 100 mmHg, for example, the values found for RBF fluctuated between 3.08 and 7.1 ml/min/g KW. The interpretation of this phenomenon is not clear. Although at prima facie this observation does not testify for the presence of RBF autoregulation in rats, it is possible t h a t - i n spite of considerably different interindividual v a l u e s - a perfect autoregulation might exist intraindividually. The present experiments confirm this assumption. The presence of autoregulation of renal blood flow in rats as determined in these experiments agrees with the results of Arendshorst et al. (1975). The existence of autoregulation has now been proved by means of the following three methods: PAH clearance, electromagnetic flowmeter, and Xe washout. Although the first component of the washout curve which we used and which was not confirmed by autoradiography yields the comparatively widest scatter of values (Table 3), the mean value still agrees well with PAH clearance. Results by Kgllskog et al. (1975), however, who employed radioactive microspheres, are not quite uniform. According to these authors, 6 rats out of 25 did not show any autoregulation. As demonstrated in Table 1 of their study, however, only in 14 rats a reduction of blood pressure within the autoregulatory range (if 70 mmHg is considered as the lowest limit)
was achieved. In the remaining 11 rats, the blood pressure values decreased below this limit after the clamping of the aorta. For the solution of the problem whether the autoregulation was present or absent, these 11 rats can therefore not be taken into account. In this case, 6 rats out of 14, represent almost half of the total number, in which the presence of autoregulation was not found. Studies in which the hydrostatic pressure was measured in intrarenal blood vessels at various levels of blood pressure also contribute to our knowledge of the existence of renal blood flow autoregulation. It is remarkable that even here the results are not uniform although the methods used are frequently very similar. In 1962 Thurau and Wober measured the hydrostatic pressure in the peritubular capillaries and in the tubules of rats before and after occlusion of both carotids; the changes in blood pressure ranged between 80 and 164 mmHg while the pressure in the intrarenal structures remained constant. Gertz et al. (1966) measured the hydrostatic pressure in the glomerular capillaries indirectly by means of the intratubular stopflow pressure method. Although this technique yields absolute values which are probably too high (Kfillskog et al., 1975), the fact that this pressure remained constant within the range of blood pressure values between 90 and 160 mmHg confirms the autoregulatory response. B.onvalet et al. (1972) measured glomerular filtration rate in single glomeruli (SNGFR) with labelled ferrocyanide and found no significant changes of values when the decrease of the perfusion pressure, induced by a clamp, amounted to 68 turn Hg; below this value the SNGFR decreased. Similarly, Bonvalet et al. (1973) did not find any changes of SNGFR within the range of autoregulation in the course of acute hypertension. Knox et al. (1974) measured SNGFR at a blood pressure of 130 and 100 mmHg; the values were always identical. K/illskog et al. (1975) measured the pressure in glomerular capillaries according to the method of Gertz as well
84
Pfl/igers Arch. 370 (1977)
Table 3. Comparison of individual values for RBF as measured by PAH clearance and 133Xe washout Increase of blood pressure
Decrease of blood pressure
n
Mean S.E.M.
RBF from CpAn
RBF from laaxe
RBF from CpAn
RBF from 133Xe
control
exper,
control
exper,
control
exper.
control
exper.
5.12 4.83 6.17 4.79 5.01 5.76 5.02 5.12 5.96 6.07 5.22 4.93 5.12 5.82 4.73 5.23 5.12 . . . .
5.01 4.63 5.87 4.29 5.27 5.34 4.97 5.01 5.68 5.79 5.27 4.78 4.99 5.61 4.66 5.03 5.03
5.13 3.92 6.14 3.17 5.31 7.03 4.88 4.98 5.97 5.71 5.83 3.87 6.27 5.99 4.91 5.11 5.41 . . . .
5.00 3.71 6.07 3.91 5.03 5.87 4.88 4.71 5.69 5.98 5.80 4.01 6.07 5.66 4.39 5.27 5.33
5.07 4.67 6.12 5.76 6.34 6.52 4.73 5.23 4.73 --
5.12 4.71 6.18 5.87 6.40 6.40 5.12 5.18 4.89 --
3.43 3.88 6.83 3.42 6.12 7.13 4.60 4.23 6.13 6.27 4.82 3.93 6.12 6.23 5.71 5.22 4.72 5.28 6.43 6.97 4.73
5.31 4.93 6.67 3.81 6.18 7.42 4.92 4.41 6.23 6.38 4.72 3.81 6.27 6.27 5.70 5.44 4.87 5.31 6.64 6.07 4.87
9 5.45 0.19
9 5.54 0.18
21 5.49 0.63
21 5.54 0.62
17 5.30 0.08
. . . .
. . . . 17 5,13 0.05
17 5.34 0.41
. . . .
. . . . 17 5.20 0.80
as the pressures in the welling points, in the perit u b u l a r capillaries, a n d in the p r o x i m a l tubuli. A l l pressures w e r e c o n s t a n t w i t h i n the r a n g e o f a u t o r e g u lation. C o n t r a r y to these e x p e r i m e n t s s u p p o r t i n g R B F a u t o r e g u l a t i o n , studies h a v e been p u b l i s h e d y i e l d i n g results p r i m a r i l y i n c o m p a t i b l e w i t h the existence o f a u t o r e g u l a t i o n . S c h o e p p e a n d F r e y (1966) f o u n d a n e l e v a t i o n o f the i n t r a t u b u l a r p r e s s u r e f r o m 11 to 23 m m H g l i n e a r with t h e increase o f b l o o d p r e s s u r e f r o m 60 to 140 m m H g . K o c h et al. (1968) a n d B a n k et al. (1970) e l e v a t e d the b l o o d p r e s s u r e in rats b y e m p l o y i n g a c l a m p r o u n d the c a r o t i d s a n d b y c u t t i n g the v a g a l nerves o r b y a n infusion o f e p i n e p h r i n e . T h e p r e s s u r e i n c r e a s e d every t i m e in the efferent arteriole, in the p e r i t u b u l a r capillaries a n d in the tubuli. W h e n , h o w e v e r , the r e n a l p e r f u s i o n p r e s s u r e was k e p t constant b y e m p l o y i n g a c l a m p o n the a o r t a these changes d i d n o t o c c u r after the increase in systemic b l o o d pressure. T h e o b s e r v a t i o n s o f A l l i s o n et al. (1972) m i g h t be p l a c e d in the s a m e c a t e g o r y . T h e y f o u n d a significant positive c o r r e l a t i o n b e t w e e n the p r o x i m a l h y d r o s t a t i c p r e s s u r e a n d the b l o o d p r e s s u r e after a n infusion o f m a n n i t o l . A similar c o r r e l a t i o n was f o u n d b e t w e e n the pressures in efferent arterioles o r peri-
t u b u l a r c a p i l l a r i e s a n d b l o o d pressure. A c c o r d i n g to these a u t h o r s , h o w e v e r , " s i n c e the statistical significance o f these differences m a y be r e l a t e d to the n u m b e r & o b s e r v a t i o n s . . . t h e i r p h y s i o l o g i c a l significance remains uncertain". A similar difference in results exists also in the dogs. F o r instance, M c N a y a n d A b e (1970) m e a s u r e d R B F with a n e l e c t r o m a g n e t i c f l o w m e t e r as well as b y m e a n s o f r a d i o a c t i v e m i c r o s p h e r e s , a n d c h a n g e d the r e n a l p e r f u s i o n p r e s s u r e within the r a n g e f r o m 81 to u p to 136 m m H g . W h i l e the t o t a l flow was a l t e r e d o n l y slightly the e x t e r n a l q u a r t e r o f the c o r t i c a l tissue d i d n o t s h o w a n y a u t o r e g u l a t i o n a n d the two i n t e r n a l q u a r t e r s d i s p l a y e d an inverse r e l a t i o n b e t w e e n pressure a n d flow. L o y n i n g (1971), h o w e v e r , using the m e t h o d o f local h y d r o g e n gas clearance, f o u n d a small decrease in b l o o d flow in the o u t e r as well as in the inner h a l f o f the c o r t e x at a decrease o f b l o o d p r e s s u r e f r o m 131 to 76 m m H g , i.e., n o evidence for a b l o o d flow r e d i s t r i b u t i o n . I n c o n c l u s i o n , the a u t o r e g u l a t i o n o f t o t a l r e n a l b l o o d flow in a n a e s t h e t i z e d rats seems to have been p r o v e n b y a n u m b e r o f i n d e p e n d e n t m e t h o d s even if the c h a n g e s o f the b l o o d p r e s s u r e were achieved b y various means.
J. Heller and V. Hor~ek: Autoregulation of Renal Blood Flow in the Rat REFERENCES Allison, M. E. M., Lipham, E. M., Gottschalk, C. W. : Hydrostatic pressure in the rat kidney. Amer. J. Physiol. 223, 975 - 9 8 3 (1972) Arendshorst, W. J., Finn, W. F., Gottschalk, C. W.: Autoregulation of blood flow in the rat kidney. Amer. J. Physiol. 228, 127133 (1975) Bank, N., Aynedjian, H. S., Bansal, V. K., Goldman, D. M. : Effect of acute hypertension on sodium transport by the distal nephron. Amer. J. Physiol. 219, 275- 280 (1970) Bonvaiet, J. P., Bencsfith, P., de Rouffignac, C. : Glomerular filtration rate of superficial and deep nephrons during aortic constriction. Amer. J. Physiol. 222, 599-606 (1972) Bonvalet, J. P., Bencsfith, P., de Rouffignac, C.: Glomerular filtration rate of superficial and juxta-medullary nephrons in the rat during different types of arterial hypertension. Pfliigers Arch. 340, 133 - 144 (1973) Gertz, K. H., Mangos, J. A., Braun, G., Pagel, H. D.: Pressure in the glomerular capillaries of the rat kidney and its relation to arterial blood pressure. Pflfigers Arch. ges. Physiol. 288, 369374 (1966) Hinshaw, L. B." Mechanism of renal autoregulation: Role of tissue pressure and description of a multifactor hypothesis. Circulat. Res. 15, Suppl. I, 120-131 (1964) K/illskog, ()., Lindbom, L.O., Ulfendahl, H.R., Wolgast, M. : The pressure-flow relationship of different nephron populations in the rat. Acta physiol, scand. 94, 2 8 9 - 300 (1975) Knox, F. G., Ott, C., Cuche, J. L., Gasser, J., Haas, J. : Autoregulation of single nephron filtration rate in the presence and the absence of flow to the macula densa. Circulat. Res. 34, 836-842 (1974) Koch, K. M., Aynedjian, H. S., Bank, N. : Effect of acute hypertension on sodium reabsorption by the proximal tubule. J. clin. Invest. 47, 1696-1709 (1968) Little, J. M. : A modified diphenylamine procedure for the determination of inulin. J. biol. Chem. 180, 747-752 (1949)
85
Loyning, E. W. : Effect of reduced perfusion pressure on intrarenal distribution of blood flow in dogs. Acta physiol, scand. 83, 191 202 (1971) McNay, J. L., Abe, Y. : Pressure-dependent heterogenity of renal cortical blood flow in dogs. Circulat. Res. 27, 571-587 (/970) Ohler, W., Harth, O., Kreienberg, W. : Die Abhfingigkeit der Nierendurchblutung vom arteriellen Druck bei der Ratte. Pfltigers Arch. ges. Physiol. 269, 274-281 (1959) Schoeppe, W. E., Frey, J.: Pressure in the proximal tubule of the rat kidney and its relation to arterial blood pressure. Proc. &the IIIrd Inter. Nephrol. Congress, 268 (1966) Selkurt, E. E. : The renal circulation. In: Handbook of Physiology, Circulation, Sect. 2, vol. II, 1457--I5/6. Washington, D.C.: Am. Physiol. Soc., Ed. 1963 Smith, H. W., Finkelstein, N., Aliminosa, L., Crawford, B., Graber, M. : The renal clearances of substituted hippuric acid derivatives and other aromatic acids in dog and man. J. clin. Invest. 24, 388-404 (/945) Thorburn, G.D., Kopald, H.H., Herd, J.A., Hollenberg, M., O'Morchoe, C. C. C., Barger, A, C. : Intrarenal distribution of nutrient blood flow determined with Krypton s5 in the unanaesthetized dog. Circulat. Res. 13, 290-307 (1963) Thnrau, K., Weber, E.: Zur Lokalisation der autoregulativen Widerstandsfinderungen in der Niere. M~kropunktionsmessnngen der Drucke in Tubuli und peritubul~ren Capillaren der Rattenniere bei Anderungen des arteriellen Druckes. Pfltigers Arch. ges. Physiol. 274, 553-566 (1962) Thurau, K.: Renal hemodynamics. Amer. J. Med. 36, 698-719 (1964) Weiss, C., Passow, H., Rothstein, A. : Autoregulation of flow in isolated rat kidney in the absence of red ceils. Amer. J. Physiol. 169, 1115-1118 (1959)
Received October 18, 1976
