PROSTAGLANDINS

ANGIOTENSIN II AND NOREPINEPHRINE AFTER INDOMETHACIN IN ISOLATED PERFUSED CANINE KIDNEYS. TACHYPHYLAXIS VS. MODULATOR

EFFECT OF PROSTAGLANDINS

Byron Ellis and Harold D. Itskovitz, M.D.

Department of Medicine Medical College of Wisconsin

Milwaukee, Wisconsin 53226

ABSTRACT

High levels of radioimmunoassayable PGE were measured in the perfusate of isolated kidneys. Indomethacgn inhibited PGE release in this system. Small reductions in the pressor effects o 1 norepi- nephrine (NE) were associated with increasing perfusate levels of PGE2; a large increase in the pressor effect of NE followed additions of indomethacin and reductions in perfusate PGE2 levels. A marked reduction in pressor responsiveness to angiotensin II (AII) was meas- ured in the isolated kidney which could not be prevented or reversed by indomethacin. It is believed that tachyphylaxis was responsible for the marked reduction in pressor responsiveness to AI1 and that this is independent of alterations in prostaglandin metabolism. How- ever prostaglandins appeared to modulate the pressor effects of AI1 as they did NE in the isolated perfused kidney.

INTRODUCTION

Vasodilator prostaglandins, in particular PGE2, have been shown to attenuate the vasoconstrictive effects of the pressor hormones AI1 and NE (1,2,3,4). In contrast inhibition of prostaglandin synthesis with indomethacin has been reported to enhance their vasoconstrictor activities (5,6,7,8). During the course of studies of renal hemody- namics in isolated blood perfused canine kidneys we have noted a con- sistent time dependent dimunition of pressor responses to injected NE and especially to AII. Since isolated perfused kidneys release large amounts of prostaglandins we considered the possibility that the ob- served diminished responses to NE and AI1 might be a consequence of prostaglandin modulation of their vasoconstrictor effects. Therefore we attempted to maintain or increase the vasoconstrictor actions of NE and AI1 by inhibition of prostaglandin synthesis. The addition of indomethacin enhanced the pressor effects of these two constrictors. Despite indomethacin, however, the vasoconstrictor action of AI1 re- mained far less than control whereas the vasoconstrictor action of NE superceded control. In the present report we have summarized our data and attempted to explain these results in terms of modulation and tachyphylaxis. Whereas our data lead us to believe that prostaglandins modulate the pressor effects of NE and AI1 they do not support a role for prostaglandins in tachyphylaxis to AII.

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PROSTAGLANDINS

METHODS

13 male mongrel dogs weighing 20-25 kg were anesthetized with pentobarbital (65 mg/kg) . The left kidney of each dog was removed surgically and transferred to a lucite organ chamger where it was perfused as described in previous reports (9,lO) with one liter of autologous heparinized blood (400 Udkg) within 2 minutes of nephrec- tomy. The perfusate was kept at 37 C. by passage through a coil im- mersed in a constant temperature water bath. The kidneys were per- fused in a pulsatile fashion (70 beats/min) using a Waters pump oxy- genator system. Stroke volume was adjusted to maintain a constant systolic pressure of 130 mmHg, Blood pressure was monitored contin- uously using a Tycos manometer and a Narco electronic pressure trans- ducer. Blood flow to the kidney was measured by timed collections of the renal venous effluent and by an electronic flow probe incorporated into the arterial line and connected to a Carolina Instruments flow meter. Blood flows and perfusion pressures were recorded on a Grass Instruments chart recorder. ered to a membrane oxygenator

Mixtures of air, 02 and CO2 were deliv- at rates sufficient to maintain blood

pH, pO2 and pCO2 at physiologic levels. Electrolyte solutions (sod- ium, potassium, calcium, chloride, phosphate, sulfate), glutamine, urea, inulin, creatinine, acetate, pyruvate and glucose were added to the perfusate at rates to provide sufficient metabolic substrates and maintain physiologic concentrations of serum electrolytes. This was accomplished by adjusting the infusion rates of different electro- lyte solutions on the basis of repeated blood gas and biochemical analyses of the perfusate performed every 30 minutes during the course of isolated perfusion. Insulin and antidiuretic hormone were infused at rates of 2 UU/min and 1 uU/min respectively. Renin was measured by the radioimmunoassay technique of Haber et al (11) and PGE2 was meas- ured by radioimmunoassay as described by Dray and modified by Dunn et al (12). Pressor responses of the kidney were assessed every 30 min- utes using intraarterial injections of AI1 amide (Hypertensin, Ciba) at two dose levels ( 25 and 50 ng) and NE (Sigma) at dose levels of 100 and 200 ng. These doses were selected since they produced readily measurable pressor responses of similar degrees during initial minutes of perfusion. Indomethacin (1 mg) was added to the venous side of the system in 9 perfused kidneys after 150 minutes of perfusion. By this time the vascular responses to AI1 were minimal. In 4 additional dogs, indomethacin (2 mg/kg) was injected in vivo one-half hour before anes- thesia. Statistical significance was determined using students t-test or the Wilcoxon two sample rank test. Data are expressed as mean +_ standard error.

RESULTS

The renal hemodynamic data of the 9 untreated kidneys (no indometh- acin in vivo) are summarized in Table IA. Renal blood flow was adjusted manually to maintain perfusion pressure constant and as a result of this technique reached levels approximating 3-4 ml/gm tissue. The mean inulin clearance was 17-18 ml/min which is approximately 60% of that expected for similar sized kidneys in vivo.

982 DECEMBER 198tiVOL.20NO.6

Table 1A: Group 1

(No Pretreatment)

Renal function of 9 isolated kidneys given indomethacin at 150 minutes of perfusion

Post Indomethacin ( 1 mg)

Initial (< 50 min)

150 min

30 min

100 min

BP (mmHg)

105?0.8

101+1.1

10321.3

107k2.1

RBF (ml/min)

154+11

229t19

187+22

130+-20

C In (ml/min)

18.8tO.9

17.1k2.0

10.7+1.5

6.3f1.5

F.E. H20

0.037'0.015

0.060+0.019

0.058+0.015

0.061+0.014

F.E. Na

0.033'0.013

0.023'0.012

0.018+0.007

0.027'0.009

U/P osm

1.74kO.17

1.10+0.21

1.04?rO.18

1.2620.16

Table 1B: Group 2

(Pretreated with Indomethacin 2 mg/kg)

Renal function of 4 isolated kidneys from dogs pretreated with indomethacin

one-half hour before anesthesia

Initial

(< 50 min)

150 min

BP (mmHg)

103k3.0

lOlf1.9

RBF (ml/min)

164?30

188+30

C In (mUmin

13.722.2

15.2k2.6

F.E. H20

0.022+0.007

0.026+0.013

F.E. Na

0.025'0.009

0.013r0.003

U/P osm

2.23tO.31

1.87?0.44

PROSTAGLANDINS

984 DECEMBER 1980VOL.20N0.6

PROSTAGLANDINS

The kidneys were able to reabsorb 96% of the filtered water and sod- ium and produce a concentrated urine. Following the addition of indo- methacin to the perfusate after 150 minutes, blood flow and inulin clearance diminished but other renal functions remained unchanged. In the second series of 4 experiments (Table 1B) dogs were given indo- methacin one-half hour before anesthesia. Kidneys from these animals demonstrated little change in blood flow over 150 minutes of perfus- ion. These kidneys reabsorbed a greater percentage of filtered salt and water than the previous group and urine osmolality was maintained in a more concentrated state.

Table 2 summarizes mean prostaglandin E2 concentrations in the blood perfusate before and after indomethacin. During initial min- utes of perfusion, pg/ml .

the mean PGE2 concentration was less than 500 By 150 minutes without indomethacin, the mean PGE2 levels in-

creased significantly to more than 2600 pg/ml. The perfusate from dogs pretreated with indomethacin showed no significant change during 250 minutes of perfusion. Following the addition of indomethacin in the former series (no pretreatment), reductions in PGE2 concentrations were observed to levels not significantly different from initial val- ues and similar to those levels obtained at corresponding time inter- vals in the pretreated group. Data for perfusate renin activity are shown also in Table 2. Renin activity decreased after indomethacin in the first group but the change was not statistically significant in this series when compared with studies where no indomethacin was given. Here, too, there was a gradual increase in renin activity dur- ing the first 150 minutes of perfusion followed by a decline during the last 100 minutes. A typical physiograph recording of pressor respon- ses to AI1 and NE is shown in Fig. 1. During initial minutes of per- fusion the responses to both drugs were of a similar degree; by 150 minutes the angiotensin response was almost undetectable whereas there was only a slight dimunition of the NE effect. Following indomethacin there was only a slight recovery in the AI1 response but a greater increase in the effect of NE.

The mean data of 9 kidneys are summarized in Table 3. The re- sponse to the two doses of AI1 decreased over 150 minutes of perfusion to 9% and 13% respectively of original values. Both reductions were significant. The corresponding dimunitions in response to NE were to 58% and 80% of initial values and was significant only for the 100 ng dose. Indomethacin caused a slight increase in AI1 responses for both doses. The increases were significant when compared to the preindo- methacin pressor responses, but the pressor effects after indomethacin were still significantly less than those initially measured. On the other hand the pressor effect of NE after indomethacin was significant- ly higher than both pre-indomethacin and initial values. Table 3 illustrates also the results from 4 kidneys pretreated with indometh- acin. The purpose was to determine whether AI1 would lose its pressor effect even in cases where PGE2 never increased in the perfusate. In this case the response to NE did not change over 150 minutes of per- fusion whereas once again the response to AI1 decreased significantly to 13% and 29% of original values.

DECEMBER 1980VOL.20N0.6 985

50 m

in

150 m

in

I80

min

--+

---A

--

30

min

P

OS

T

IN30

ME

THA

CIN

250

min

100

min

P

OS

T

IND

OM

ETH

AC

JN

, lm

ln

,

200 n

g N

E

A A

Fig. 1 Effects of indomethacin on pressure response of an isolated

perfused kidney to intra-arterial injections of NE and AII.

Indomethacin was given after 150 minutes of perfusion.

I 40

mm

H

g

Table 3 -

Initial

150 Min

Post Indo

(30 min)

Post Indo

(100 min)

Initial

150 min

9 Isolated Kidneys - No Pretreatment with Indomethacin: Pressure Changes mmHg

AI1

NE

25 ng

50 ng

100 ng

200 ng

18.3k5.3

30.3k8.0

15.423.6

29.1t5.1

1.7*0.5**

3.9+0.9x*

8.921.9"

23.2k3.8

4.7+-0.8$t

7.0+1.1;

18.6f2.0++

39.1+3.9+;

4.0?0.8;?

7.0Mf+

23.1+2.7;?

48.8-cS.2;;

4 Isolated Kidneys Pretreated with Indomethacin (2 mg/kg)

15.025.6

2.0+-1.7*

* Significantly different

** Significantly different

t Significantly different

tt Significantly different

22.5r7.2

8.5k1.3

19.5f2.6

6.5+2.8*

8.320.9

19.8k4.0

from initial response

from initial response

from 150 minute response

from 150 minute response

PC.05

PC.01

PC.05

PC.01

PROSTAGLANDINS

DISCUSSION

Previously, we used a bioassay technique for measurement of prostaglandins to show that isolated blood perfused canine kidneys released large quantities of PGE2 and PGF2o and that the high level of prostaglandin release in this system could be inhibited by addi- tions of indomethacin or meclofenamate (13). The present study con- firms our previous findings using a radioimmunoassay technique for measuring PGE2 (Table 2) and PGF2o (data not shown). Our data also indicate that kidneys from dogs pretreated with indomethacin prior to anesthesia and nephrectomy maintain low levels of PGE2 release for more than 4 hours of isolated perfusion. In this situation, the renal blood flow remained at a more constant level and urine osmolality was greater than in perfused kidneys not pretreated with indomethacin. Our results are consistent with those of Terragno et al (14) who found that levels of PGE in renal venous blood rose significantly after surgical stress. 1 n this situation renal blood flow was supported by the high level release of PGE since the addition of indomethacin re- duced renal blood flow marked y 1 and increased mean aortic blood pres- sure. These results also are consistent with previous reports that intrarenal prostaglandins can affect renal hemodynamics, antidiuretic hormone activity and the urine concentrating mechanism (2,15). Iso- lated perfused kidneys release large quantities of renin as well as prostaglandins (Table 2). However, our experience indicates that a high level of renin release can be maintained only for 3-4 hours in this system. Thereafter perfusate levels of renin activity begin to decline. PGI (2,16) and in

& . . and PGE2 have been shown to stimulate renin release ibition of prostaglandin synthesis by indomethacin can

result in diminished release of renin (17-19). Although the perfusate levels of renin activity declined in these studies after treatment with indomethacin, we could not establish a statistical relationship for this effect in view of the tendency for renin release to decrease in our system even without such treatment. However a more recent and extended experience with such measurements indicates that indomethacin given at different time intervals of isolated perfusion can reduce renin release in this system as in other preparations though the lev- els continue to remain elevated.

It has been well established that the pressor effects of vasocon- strictor substances are diminished in the presence of high PGE2 acti- vity and increased in its absence (1). Present results following in- jections of NE and AI1 before and after indomethacin are consistent with previous experiences. However we observed only a small effect on AI1 responsiveness in the isolated kidney when compared with NE. The marked response to indomethacin in the case of NE may be a consequence of the effects of prostaglandins and indomethacin to modulate the up- take, storage, and release of NE by the nerve ending (20) Thereby indo- methacin or inhibition of prostaglandin synthesis may increase the free NE available for activation of receptor sites. On the other hand an- alyses of our results indicate that to a large measure the loss of pressor activity to angiotensin in the isolated perfused kidney is in- dependent of the prostaglandin system since it occurs in kidneys from animals pretreated with indomethacin in the absence of enhanced prosta- glandin activity. Also the degree of loss of responsiveness to AI1

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PROSTAGLANDINS

which was far greater than the case with NE is out of proportion to that which could be expected on the basis of prostaglandin modulation alone. In view of the continued responsiveness to NE, the high renin activity in the perfusate probably resulting in high levels of circu- lating angiotensin and the absence of usual degradative mechanisms for angiotensin in the isolated system, a tachyphylactic mechanism would appear to be a logical explanation for loss of responsiveness to AII. A continued exposure to high levels of angiotensin I or II could sat- urate, block, or alter angiotensin receptors and render them unrespon- sive to additional exogenous angiotensin (21). If this is the case then it would appear from our data that the prostaglandins are not responsible for the phenomenon of angiotensin tachyphylaxis since manipulation of this system had little effect on angiotensin respon- siveness.

Our results and interpretations differ slightly from those of Aiken who studied AI1 tachyphylaxis in isolated coeliac and mesenteric ar- teries (22). Aiken was able to induce tachyphylaxis in this prepara- tion following repetitive doses of AII. He observed two phases of tachyphylaxis: a short acting phase which persisted in the presence of indomethacin and appeared to be on a basis of receptor saturation; a long acting phase which could be prevented by indomethacin and was attributed to AI1 induced release of prostaglandins. We believe that we observed this second phase also in our system as evidenced by the small but significant changes in pressor responsiveness to AI1 meas- ured before and after indomethacin (Table 3). However we have termed this a modulating effect of prostaglandins on AI1 rather than a tachy- phylactic phenomenon.

ACKNOWLEDGEMENTS

This work was supported by grant HL-12677 from the National Institutes of Health. We wish to thank Ms. Judy Affeldt and Ms. Joan Meller for technical assistance and Ms. Anne Szatkiewicz for prep- aration of this manuscript.

REFERENCES

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Editor: Alan S. Nies Received: 6-19-80 Revision to reviewers: 9-4-80 Accepted: g-18-80

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