modulating the tubuloglomerular feed-back mechanism in the canine kidney; Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, U.S.A.

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1 J. Physiol. (1986), 380, pp With 3 text-figures Printed in Great Britain RENAL VASOCONSTRICTOR RESPONSE TO HYPERTONIC SALINE IN THE DOG: EFFECTS OF PROSTAGLANDINS, INDOMETHACIN AND THEOPHYLLINE BY JOHN G. GERBER AND ALAN S. NIES From the Division of Clinical Pharmacology, C-237, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, U.S.A. (Received 22 November 1985) SUMMARY 1. The role of prostaglandins in the mechanism of the tubuloglomerular feed-back was examined in the anaesthetized dog using the infusion of hypertonic saline to increase renal plasma sodium concentration by 30 mmol/l as the stimulus to activate the tubuloglomerular feed-back. 2. Two sequential infusions of hypertonic saline into the renal artery for 10 min separated by 90 min resulted in equivalent reductions in renal blood flow ( to ml/min first time; to 200 ± 15 ml/min second time). 3. Administration of indomethacin, 8 mg/kg, between the two infusions did not alter the renovascular response to hypertonic saline ( to ml/min vs to ml/min). 4. Infusion of either prostaglandin E2 (PGE2) or prostaglandin '2 (PGI2) during the second hypertonic saline infusion period also resulted in no change in the renovascular response ( to ml/min vs to ml/min; and to ml/min vs to ml/min, respectively). 5. Intrarenal theophylline, to a concentration of 40,sg/ml, however, totally abolished the renovascular response to hypertonic saline (279 ± 20 to ml/min control vs to ml/min during theophylline). 6. The systemic hypertensive response during the infusion of hypertonic saline was unaltered by indomethacin or prostaglandins but was totally abolished by theophylline. 7. Our data suggest that prostaglandins do not have a role in either mediating or modulating the tubuloglomerular feed-back mechanism in the canine kidney; however, intrarenal adenosine has a critical role in the mediation of renal vasoconstriction as well as the systemic hypertension during the activation of the tubuloglomerular feed-back by intrarenal infusion of hypertonic saline. INTRODUCTION Renal prostaglandins have been demonstrated to play an important role in the modulation of renal vascular resistance, in the stimulation of renin release and in the modulation of the effects of vasopressin (Gerber, Olson & Nies, 1981; Gerber, 2-2

2 36 J. G. GERBER AND A. S. NIES Anderson, Schrier & Nies, 1982). The ability of the distal tubular fluid composition to modulate glomerular filtration by altering afferent vascular tone, termed tubuloglomerular feed-back, is thought to be an important function in preventing excessive salt and water loss by the nephron if the proximal tubule fails (Thurau & Boylen, 1976; Briggs & Wright, 1979). The role of prostaglandins in the initiation and maintenance of tubuloglomerular feed-back has not been studied extensively. Schnermann and co-workers using micropuncture in rats have reported that indomethacin pre-treatment blunted the tubuloglomerular feed-back response and that this response would be restored by the infusion of prostaglandin E2 (PGE2) or prostaglandin '2 (PGJ2) into the abdominal aorta (Schnermann, Schubert, Hermle, Herbst, Stowe, Yarimizu & Weber, 1979; Schnermann & Weber, 1982). However, studies from other laboratories have not been able to confirm these findings in rats (Hahne, Selen & Persson, 1984; Boberg, Hane & Persson, 1984). Because of these conflicting reports, we decided to examine the role of prostaglandins in the tubuloglomerular feed-back response in dogs using the renal vascular response to an intrarenal infusion of hypertonic saline as a measure of tubuloglomerular feed-back. We have previously demonstrated that intrarenal infusion of hypertonic NaCl in salt-depleted dogs resulted in renal vasoconstriction and a decrease in glomerular filtration rate (Gerber, Branch, Nies, Hollifield & Gerkens, 1979). This vasoconstriction could be abolished by salt loading of the dogs or by treating dehydrated dogs with intravenous furosemide. In addition, a filtering kidney was necessary for this effect of hypertonic sodium chloride to occur. All these data suggested that the mechanism of renal vasoconstriction during hypertonic saline infusion was through the activation of tubuloglomerular feed-back. Subsequently, Schnermann, Briggs & Wright (1981) using micropuncture in rat kidneys to interrupt distal sodium chloride delivery, demonstrated that the reduction of glomerular filtration rate during hypertonic saline infusion was indeed secondary to the activation oftubuloglomerular feed-back. Thus we feel that the vascular response to the infusion of hypertonic saline into the renal artery is a good in vivo model of the tubuloglomerular feed-back mechanism. METHODS Thirty-four mongrel dogs of either sex weighing kg were used to assess the renal haemodynamic effect of an intrarenal hypertonic sodium chloride infusion. All the dogs were prepared similarly and were modestly dehydrated by exclusion of food and water for 36 h. We have previously demonstrated that this level of dehydration is necessary to see a consistent vasoconstrictor response to an intrarenal hypertonic saline infusion (Gerber et al. 1979). Under pentobarbitone anaesthesia the dogs were intubated and respirated using a positive pressure respirator at a rate of 16 breaths/min. We maintained a stable level of anaesthesia with supplemental pentobarbitone as necessary so that the dogs were unresponsive to pain. Femoral arterial and venous cannulae were placed for blood pressure monitoring and drug administration, respectively. Through a flank incision, the right kidney was exposed and the ureter and the renal vein were cannulated. The renal artery was isolated and two 25-gauge infusion needles were placed in the renal artery, one for the infusion of hypertonic saline and the other for the infusion of prostaglandins or theophylline. An electromagnetic flow probe was placed around the renal artery, and blood flow was constantly monitored using a flowmeter. Group I (n = 7). The effect ofprostaglandin E2 (PGE2) on the renal vascular response to hypertonic 8aline infusion. After a 15 min control period during which urinary volume and sodium concentra-

3 Pus AND TUBULOGLOMERULAR FEED-BACK tion, renal blood flow, and systemic blood pressure were monitored, hypertonic sodium chloride, calculated to elevate renal plasma sodium concentration by 30 mmol/l, was infused into the renal artery for 10 min. During the 10 min infusion, renal blood flow was monitored continuously, and the hypertonic infusion rate was adjusted so that the 30 mmol/l increase in sodium concentration remained constant as previously described (Gerber et al. 1979). In addition, urinary volume and sodium concentration were monitored during the infusion. After the infusion was stopped, the dogs were allowed to restabilize for the next 90 min. The first part of the study was then repeated except that an intrarenal infusion of PGE2 (10 ng/kg. min) was initiated 15 min prior to the hypertonic saline infusion and maintained throughout the hypertonic saline infusion. We have previously demonstrated that this dose of PGE2 is a potent stimulator of renin secretion without producing any systemic effect (Gerber, Branch, Nies, Gerkens, Shand, Hollifield & Oates, 1978). Group II (n = 5). The effect of PGI2 on the renal vascular response to hypertonic saline. This study was identical to the study in group I except that PGI2 (10 ng/kg. min) was used in place of PGE2 during the second infusion of hypertonic saline. We have previously demonstrated that this dose of PGI2 is a potent stimulator of renin release without systemic side effects (Gerber et al. 1978). Group III (n = 10). The effect ofprostaglandin inhibition on the renal vascular response to hypertonic saline. In these dogs, two intrarenal infusions of hypertonic saline were performed in a manner similar to that described in groups I and II, except that indomethacin, 8 mg/kg I.v., was administered between the two infusion periods. This dose of indomethacin results in a high level of prostaglandin inhibition in the canine kidney (Gerber, Nies & Olson, 1981). The importance of an intact prostaglandin system to observe the tubuloglomerular feed-back was tested in these experiments. Group IV (n = 6). The effect of the first hypertonic saline infusion on the response to the second infusion. In these dogs, two infusions of hypertonic saline were performed in a similar manner as described in the previous sections except that only the vehicles for the drugs were administered between the infusion periods. These dogs served as time controls for the two infusions of hypertonic saline into the renal artery. Group V (n = 6). The effect of theophylline on the renal vascular response to hypertonic saline. In these dogs, two infusions of hypertonic saline were performed in a similar manner as described in the previous section except that a continuous intrarenal infusion of theophylline, 7-10 mg/min, was administered 15 min prior to and during the second hypertonic saline infusion period. We calculated this dose to achieve a renal plasma concentration of theophylline of about 40 #sg/ml based on the renal plasma flow. In preliminary experiments we have demonstrated that this concentration of theophylline is required to block the vasoconstrictor response to intrarenal adenosine infusion. Statistics. The renal haemodynamic data were analysed by an analysis of variance using Dunnett's method for multiple comparisons with a control. The urinary sodium excretion data and the blood pressure data were analysed using Student's t test for paired data comparing the control period to the infusion period. A P value of less than 0 05 was considered significant. RESULTS An intrarenal infusion of hypertonic saline resulted in a transient increase in renal blood flow followed by a sustained decrease in renal blood flow, as we have described previously (Gerber et al. 1979). The sustained decrease in renal blood flow is proposed to be secondary to activation of the tubuloglomerular feed-back mechanism. Two infusions of hypertonic saline, separated by 90 min, gave almost identical responses whether PGE2 or PGI2 was infused during the second hypertonic saline infusion or whether indomethacin or vehicle was administered prior to the second infusion (Figs. 1 and 2). This finding suggests that prostaglandins are not required for an active tubuloglomerular feed-back response. However, an intrarenal infusion of theophylline, calculated to maintain a concentration of 40 jug/ml in renal arterial plasma, resulted in a complete abolition of the decrease in renal blood flow to hypertonic saline (Fig. 3). These data support the recent hypothesis that adenosine may be the 37

4 38 J. G. GERBER AND A. S. NIES mediator of the tubuloglomerular feed-back mechanism (Osswald, Nabakowski & Hermes, 1980). Systemic arterial pressure increased significantly, concomitant with renal vasoconstriction during the hypertonic saline infusion. Theophylline blocked the increase 400 c E E II I I I E Z- Es m: II Fig. 1. The abscissa represents the time in minutes. 0 is the control and 1-10 are the minutes of hypertonic saline infusion into the renal artery. The ordinate is renal blood flow (R.B.F.) in ml/min. The left side is the first infusion period and the right side is the second infusion period with hypertonic saline. The upper panel is the control dog where only a vehicle was administered between the two infusion periods. The lower panel represents dogs given 8 mg indomethacin/kg between the. two infusion periods. The asterisk signifies that those numbers are significantly different from the control number (0). in blood pressure as well as the renal vascular response, suggesting that saline loading alone could not be responsible for the change in blood pressure (Table 1). The hypertonic saline infusion resulted in at least a tenfold increase in urinary sodium excretion. Although PGE2, PGJ2 and theophylline infusions also resulted in a natriuresis, hypertonic saline caused a marked increase in sodium excretion even during the infusion of PGE2, PGI2 or theophylline.

5 PGs AND TUBULOGLOMERULAR FEED-BACK 39 DISCUSSION Tubuloglomerular feed-back is a physiologic mechanism by which the distal tubular composition of the nephron controls the single-nephron glomerular filtration rate by altering the afferent arteriolar tone. Numerous micropuncture studies have E E cn C m 400 E 300 i- E Fig. 2. The abscissa represents the time in minutes. On the left panel, 0 is the control and 1-10 are the minutes of hypertonic saline infusion into the renal artery. On the right panel, -1 represents the point with no drugs, 0 represents the control period with either PGE2 (top panel) or PGJ2 (bottom panel) infused, and 1-10 are the minutes of hypertonic saline infusion with continued prostaglandin infusion. The ordinate is the renal blood flow (R.B.F.) in ml/min. The asterisk signifies that those numbers are significantly different from the control number (0). demonstrated the existence of this mechanism in the single nephron. We have recently described that an intrarenal infusion of hypertonic saline into the renal artery resulted in a decrease in renal blood flow and glomerular filtration rate suggestive of activation of the tubuloglomerular feed-back mechanism in the entire kidney (Gerber et al. 1979). The effect of hypertonic saline on renal blood flow was inhibited by expanding the vascular volume with normal saline, administering

6 40 J. G. GERBER AND A. S. NIES furosemide or rendering the kidneys non-filtering thereby preventing fluid flow at the macula densa. These are also conditions that will inhibit the tubuloglomerular feed-back in the micropuncture studies in rats. In addition, using micropuncture studies, Schnermann et al. (1981) have recently reported that indeed the decrease in glomerular filtration rate during infusion of hypertonic saline was mediated by the E 300 m250 * * l III II IFT III I I, FFF, Fig. 3. The abscissa represents the time in minutes. On the left panel, 0 is th, control and 1-10 are the minutes of hypertonic saline infusion into the renal artery. On the eight panel, -1 represents the point with no drugs, 0 represents the control period with theophylline being infused, and 1-10 are the minutes of hypertonic saline with a bac.ground of theophylline infusion. The ordinate is the renal blood flow (R.B.F.) in ml/min. The asterisk signifies that those numbers are significantly different from the control number (0). tubuloglomerular mechanism. These studies were performed by interrupting the distal tubular delivery of sodium chloride in some nephrons while keeping it intact in others. In nephrons where there was no distal delivery of sodium chloride, hypertonic saline infusion did not change the single-nephron glomerular filtration rate. In the intact nephron, hypertonic saline infusion reduced the single-nephron glomerular filtration rate. Since tubuloglomerular feed-back is secondary to afferent arteriolar constriction, measuring total renal blood flow is a good monitor of total kidney tubuloglomerular feed-back during renal infusion of hypertonic saline. Using the hypertonic saline infusion model, we tested the hypothesis that prostaglandins mediate or modulate the tubuloglomerular feed-back in dogs. This hypothesis was proposed by Schnermann and co-workers who reported that indomethacin inhibited the tubuloglomerular feed-back response in the rat kidney (Schnermann et al. 1979). In addition, Schnermann and Weber later reported that PGI2 and PGE2 infusions restored the tubuloglomerular feed-back mechanism in indomethacin treated rats, but that prostaglandin (PGF2.Z) was inactive (Schnermann & Weber, 1982). These data suggested that prostaglandins (E2 or '2) have a permissive role in

7 PGs AND TUBULOGLOMERULAR FEED-BACK the tubuloglomerular feed-back mechanism but are probably not the mediator of the response. In contrast, Hahne et al. (1984) reported that indomethacin did not affect tubuloglomerular feed-back in the rat and Boberg et al. (1984) and Persson, Hahne & Selen (1983) reported that PGI2 reduced the sensitivity of the tubuloglomerular feed-back in the rat kidney. However, Persson et al. (1983) found that PGE2 and 41 TABLE 1. Blood pressure (mmhg) C 10 C 10 Control Indomethacin * * * * C 10 C C' 10 PGE * n.s. * PG * n.s. * Theophylline * * n.s. Sodium excretion (csmol/min) C 10 C 10 Control * * Indomethacin * * C 10 C C' 10 PGE * ±19 403±114 * * PG * * * Theophylline * * * C is the measurement at the control point. 10 is the measurement at the 10 min infusion point of the hypertonic saline. C' is the measurement during the infusion of either PGE2, PGI2 or theophylline but before the onset of the hypertonic saline infusion. * indicates that the difference between C and 10 is statistically significant. PGF2. enhanced the sensitivity of tubuloglomerular feed-back. We found the tubuloglomerular feed-back response in the dog to be resistant to treatment with high doses of indomethacin administered acutely in a dose that we have previously shown to inhibit renal prostaglandin synthesis almost totally. In addition, infusion of PGI2 and PGE2 into the renal artery at a dose that resulted in physiologic effects as measured by renal vasodilation and natriuresis did not affect the vasoconstrictor response to hypertonic saline infusion. In contrast, intrarenal theophylline, which

8 42 J. G. GERBER AND A. S. NIES also resulted in renal vasodilation and natriuresis, totally inhibited the renal vasoconstriction produced by the infusion of hypertonic saline. Our theophylline data agree with previous studies that have proposed adenosine as the mediator of the tubuloglomerular feed-back (Osswald et al. 1980; Gerkens, Heidemann, Jackson & Branch, 1983). It is interesting that hypertonic saline infusion into the renal artery resulted in systemic hypertension that was also blocked by theophylline. The mediator of this hypertension is unknown but it is unlikely to be adenosine as adenosine vasodilates most vascular beds except the kidneys. From our present data, we can conclude that prostaglandins are unlikely to play a role in the tubuloglomerular feed-back mechanism in the dog. However, it is possible that this may not be the case in the rat. There is precedence for a species difference in the renovascular response to prostaglandins, PGE2 can be a vasodilator in both the dog and the rat, but under some conditions PGE2 can be a vasoconstrictor in the rat (Chang, Splawinski, Oates & Nies, 1975; Malik & McGiff, 1978). However, theophylline appears to inhibit the tubuloglomerular feed-back in both species suggesting that renal adenosine production may play a critical role in the renal vascular response during tubuloglomerular feed-back. This work was supported by Public Health Service Grant NIH HL Dr Gerber is a Burroughs Wellcome Scholar in Clinical Pharmacology. REFERENCES BOBERG, U., HAHNE, B. & PERSSON, A. E. G. (1984). The effect of intraarterial infusion of prostacyclin on the tubuloglomerular feedback control in the rat. Acta physiologica scandinavica 121, BRIGGS, J. P. & WRIGHT, F. S. (1979). Feedback control of glomerular filtration rate; site of the effector mechanism. American Journal of Physiology 236, F CHANG, L. C. T., SPLAWINSKI, J. A., OATES, J. A. & NIES, A. S. (1975). Enhanced renal prostaglandin production in the dog. II. Effects on intrarenal hemodynamics. Circulation Research 36, GERBER, J. G., ANDERSON, R. J., SCHRIER, R. W. & NIES, A. S. (1982). Prostaglandins and the regulation of renal circulation and function. In Advances in Prostaglandin, Thromboxane and Leukotriene Research, vol. 10, ed. OATES, J. A., pp New York: Raven Press. GERBER, J. G., BRANCH, R. A., NIES, A. S., GERKENS, J. F., SHAND, D. G., HOLLIFIELD, J. & OATES, J. A. (1978). Prostaglandin and renin release: assessment of renin secretion following infusion of PGI2, E2 and D2 into the renal artery of anesthetized dogs. Prostaglandins 16, GERBER, J. G., BRANCH, R. A., NIEs, A. S., HOLLIFIELD, J. W. & GERKENS, J. F. (1979). Influence of intrarenal infusion of hypertonic saline on renal blood flow and renin release in the dog. American Journal of Physiology 237, F GERBER, J. G., NIES, A. S. & OLSON, R. D. (1981). Control of canine renin release: macula densa requires prostaglandin synthesis. Journal of Physiology 319, GERBER, J. G., OLSON, R. D. & NIES, A. S. (1981). Interrelationship between prostaglandins and renin release. Kidney International 19, GERKENS, J. F., HEIDEMANN, H. T., JACKSON, E. K. & BRANCH, R. A. (1983). Aminophylline inhibits renal vasoconstriction produced by intrarenal hypertonic saline. Journal of Pharmacology and Experimental Therapeutics 225, HAHNE, B., SELEN, G. & PERSSON, E. G. (1984). Indomethacin inhibits renal functional adaptation to nephron loss. Renal Physiology 7, MALIK, K. U. & MCGIFF, J. C. (1975). Modulation by prostaglandins of adrenergic transmission on the isolated perfused rabbit and rat kidney. Circulation Research 36,

9 Pus AND TUBULOGLOMERULAR FEED-BACK 43 OSWALD, H., NABAKOWSKI, G. & HERMES, H. (1980). Adenosine as a possible mediator of metabolic control of glomerular filtration rate. International Journal of Biochemi8try 12, PERSSON, A. E. G., HAHNE, B. & SELEN, G. (1983). The effect of tubular perfusion with PGE2, PGF2a and PGI2 on the tubuloglomerular feedback control in the rat. Canadian Journal of Physiology and Pharmacology 61, SCHNERMANN, J., BRIGGS, J. & WRIGHT, F. S. (1981). Feedback-mediated reduction of glomerular filtration rate during infusion of hypertonic saline. Kidney International 20, SCHNERMANN, J., SCHUBERT, G., HERMLE, M., HERBST, R., STOWE, N. T., YARIMIZU, S. & WEBER, P. C. (1979). The effect of inhibition of prostaglandin synthesis on tubuloglomerular feedback in the rat kidney. Pfliuger8 Archiv 379, SCHNERMANN, J. & WEBER, P. C. (1982). Reversal of indomethacin-induced inhibition of tubuloglomerular feedback by prostaglandin infusion. Pro8taglandins 24, THURAU, K. & BOYLAN, J. W. (1976). Acute renal success. The unexpected logic of oliguria in acute renal failure. American Journal of Medicine 61,