Clinical and Experimental Pharmacology and Physiology (1976) 3,13-93.
REVIEW ARTICLE
Regulation of renin release and intrarenal formation of angiotensin. Studies in the isolated perfused rat kidney K. G. Hofbaner, H. Zschiedrich and F. Gross Department of Pharmacology, University of Heidelberg, Heidelberg, Germany (Received 23 May 1975)
SUMMARY
1. Isolated rat kidneys were perfused at a constant pressure of 90 mmHg in a single-pass system with either a cell-free medium or a suspension of washed bovine red blood cells, free of the components of the renin-angiotensin system. In red blood cell perfused kidneys renal haemodynamics and sodium reabsorption corresponded closer to values observed in the intact rat than in cell-free perfused kidneys. 2. In red blood cell-perfused kidneys in the absence of plasma renin substrate autoregulation of renal blood flow was almost complete at pressures above 90 mmHg, provided that perfusion pressure was changed rapidly. 3. Renin release varied inversely with perfusion pressure within a pressure range from 50 to 150 mmHg; the greatest changes of renin release occurred, when perfusion pressure was reduced from 90 to 70 mmHg; maximal stimulation of renin release was observed at 50 mmHg. After reduction of perfusion pressure, renin release immediately started to rise and reached a new level within 5 min. Local reduction of perfusion pressure in small arteries and arterioles by the injection of microspheres induced a short-lasting decrease in renal plasma flow and a transient stimulation of renin release. 4. High concentrations of furosemide stimulated renin release by a direct intrarenal mechanism. 5. Isoproterenol stimulated renin release in low concentrations without a concomitant vasodilation, whereas high concentrations induced an increase in both renal plasma flow and renin release. The effects of isoproterenol were completely blocked by propranolol. 6. Sodium nitroprusside induced similar increases in renal plasma flow, as did high concentrations of isoproterenol, but only a small and slow increase in renin release was observed. 7. Angiotensin I1 (AII) suppressed renin release in concentrations corresponding to plasma levels measured in the intact rat independently of its vasoconstrictor Correspondence:Professor F. Gross, PharmakologischesInstitut der Universitlt Heidelberg,6900 Heidelberg 1, Im NeuenheimerFeld 366, W. Germany.
73
74
K. G . Hofbauer, H. Zschiedrich and F. Gross effects, whereas vasopressin in antidiuretic concentrations did not affect renin release. 8. AII, AI, synthetic tetradecapeptide renin substrate (TDP), crude and purified rat plasma renin substrate induced a dose-dependent reduction in renal plasma flow. SQ 20 881, a competitive inhibitor of converting enzyme, and low doses of l-Sar-8-Ala-A11(saralasin), a competitive antagonist of AII, did not change renal plasma flow, whereas high concentrations of saralasin had a vasoconstrictor effect on their own. 9. Saralasin inhibited the vasoconstrictor effects of A11 and TDP to a similar degree. SQ 20 881 inhibited the vasoconstrictor effects of A1 and purified renin substrate, but did not influence the actions of TDP and the crude renin substrate preparation. 10. From these data it is concluded, that A1 is converted into A11 within the kidney at a rate of 1-2%. The vasoconstriction induced by the crude renin substrate probably does not involve the A11 receptors. TDP may act by itself on the A11 receptors or via the direct intrarenal formation of AIT. The vasoconstriction induced by purified renin substrate is probably due to the intrarenal formation of A1 and its subsequent conversion to AII. Key words : antidiuretic hormone, angiotensin I1 antagonist, converting enzyme inhibitor, furosemide, isoproterenol, microspheres, renal autoregulation, renal haemodynamics, plasma renin substrate, tetradecapeptide substrate, vasodilation.
INTRODUCTION The mechanisms participating in the regulation of renin release have been extensively studied (Vander, 1967; Davis, 1971). From studies in the whole animal it became obvious, that the interpretation of results obtained in vivo is difficult, since various factors which stimulate or suppress renin release in the kidney, as well as extrarenal factors, may interfere with each other. Accordingly, a simplified experimental model, such as the isolated kidney, should be a suitable tool to study the regulation of renin release under defined and controlled conditions. We have developed a method for the perfusion of isolated rat kidneys with a medium, which is free of any component of the renin-angiotensin system. Since the perfusate is not recirculated, renin, which is released during the perfusion, does not re-enter the kidney. Moreover the lack of renin substrate in the perfusion fluid prevents the formation of angiotensin within the renal vascular bed; thus, changes of renin release are without influence on renal function. By means of this experimental model we have studied the effects of various stimuli and measures which are known to affect the renin-angiotensin system on renin release and on renal function. Furthermore the intrarenal formation of A11 from renin substrate and A1 was investigated to obtain information on the role which the reninangiotensin system may play in the regulation of renal haemodynamics. METHODS Preparation Male Sprague-Dawley rats weighing 180-240 g were anaesthetized with sodium pento-
Renin-angio tensin system in the isolated kidney
75
barbitone (Nembutal) 50 mg/kg i.p. The right kidney was prepared and connected to the perfusion system avoiding a period of ischaemia, according to Schurek et al. (1972). Perfusion medium Either a modified Krebs-Henseleit solution containing 35 g/1 of a gelatine preparation (Haemaccel, Behringwerke) was used or a 38 vol. % suspension of washed bovine red blood cells. The composition of the perfusion medium and the preparation of the red blood cells have been described elsewhere (Hofbauer et al., 1973b, 1974). Perfusion system The perfusion fluid is transported from a reservoir to a plastic disc oxygenator and equilibrated with a mixture of 95 vol. % O2 and 5 vol. % C 0 2 at atmospheric pressure. From the oxygenator the medium is delivered to the kidney by means of a peristaltic pump (Harvard App. No. 1203) via a bubble trap and a windkessel. Mean perfusion pressure is continuously measured by a pressure transducer (Statham P23Db) and kept constant at 90 mmHg by a pump speed modulator (Harvard App. No. 550). The venous perfusate passes a drop counter and renal plasma flow is recorded as number of drops/lO s. The venous effluent is not recirculated. Urine is collected for 5 or 10 min periods; urine flow/min is calculated from the weight of the sample and the period of the collection. The reservoir, the oxygenator, the kidney receptacle and all tubes are kept at a constant temperature of 37°C. Analytical methods In samples of the perfusate and of urine inulin was determined by the method of Schreiner (1950), PAH was measured by the reaction of Bratton & Marshall (1939) and sodium concentration by flame photometry (Zeiss, PMQ 11). In samples of venous perfusate renin concentration was determined by two different methods. In the control experiments, and in those with furosemide, the perfusate was incubated for 12 h after addition of rat plasma renin substrate according to the method of Boucher, MCnard & Genest (1967). In the other experiments samples of 50 p1 were incubated for 30 and 60 min (pH 7.2,37"C) with 200 pl of a solution containing 60 mg/ml of a rat plasma substrate preparation, specific concentration about 75 ng AI/mg (Boucher et al., 1967). As inhibitors of angiotensinases and converting enzyme 3 mM 8-hydroxyquinoline, 5 mM Na,EDTA and 1.6 mM dimercaprol had been added. The reaction was stopped by immediate cooling in ice water. At the end of both incubation procedures the amount of angiotensin I was measured by radioimmunoassay (Haber et al., 1969). The rate of angiotensin I formation per hour was higher with the second than with the first method. Plasma renin concentration is expressed as the amount of angiotensin I formed at 37°C during incubation periods of 12 h or 1 h, respectively. Kidney renin content is expressed as pg angiotensin 1formed during an incubation period of 10 min according to the method of Orth et al. (1971). Statistical analysis All values given in the text, Figures and Table are means fs.e.m. ; Student's t-test, t-test for paired data and linear regression analysis were used (Sachs, 1972). All doses are expressed as concentrations of the substances per ml of the perfusion medium. RESULTS Function of the isolated kidney Cell-free perfusion. Kidneys were perfused with the cell-free medium for 100-120 min.
76
K . G . Hofbauer, H . Zschiedrich and F. Gross
During the first hour renal plasma flow, glomerular filtration rate (GFR) and urinary sodium excretion (UNaV)increased ; these values remained constant for the second hour of the perfusion. Sixty minutes after the beginning of the perfusion, renal plasma flow was higher, whereas GFR was lower than values measured in the intact rat (Fig. 1). Fractional sodium reabsorption (RNa%)was 81% and extraction of PAH (EpAH) was 34%. At the end of
I
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RPF
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GFR
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FF
80
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c ._
E u 40
W
4 0
FIG.I . Renal plasma flow (RPF), glomerular filtrationrate (GFR), filtrationfraction (FF), net sodium reabsorption (RNs),fractional sodium reabsorption (RN~%), and extraction of PAH (ENs), in kidneys perfused with the cell-free medium (white columns, n = 14) or the red blood cell (RBC) suspension (hatched columns, n = 7) (means+ s.e.m.).
the experiment the weight of the perfused kidney was higher than that of the contralateral kidney removed and weighed immediately before the isolation of the perfused kidney (1252f27 v. 895+26 mg). Red blood cellperfusion. In these experiments kidneys were initially perfused with the cellfree medium for 20-30 min and subsequently for 70-80 min with the red blood cell suspension. Sixty minutes after connecting the kidney to the perfusion system, renal plasma flow was one-third of that obtained in cell-free perfused kidneys, whereas GFR was almost twice as high as in the former group. Consequently, filtration fraction was increased. UNaV was similar to that measured in cell-free experiments; but net sodium reabsorption was twice as high and RNa% was 90%. E p A H amounted to 80% and corresponded to values obtained in vivo (Fig. 1). During the course of the perfusion, renal plasma flow, GFR and UNaVdecreased slightly, whereas RNa% and E p A H remained unchanged. At the end of the experiment the weight of the perfused kidneys was increased similarly to that of cell-free perfused kidneys (1271 +23 v. 930f 30 for the contralateral kidney). Renin release Control experiments. In cell-free perfused kidneys mean renin release was constant during
Renin-angiotensin system in the isolated kidney
77
the whole period of observation. In red blood cell experiments renin release decreased during the first hour, but subsequently remained constant in all kidneys. After perfusion for 1 h, venous renin concentration was similar in both series of experiments, but as a consequence of the higher flow rate in cell-free perfused kidneys renin release was higher in that group (2412f 340 ng AI/min v. 942f 128 ng AI/min). In studies with cell-free perfusion the renin content of the perfused kidneys was equal to or lower than the amount measured in the contralateral, non-perfused kidney, but the means (n = 6) did not differ significantly from each other (146 & 19 v. 182& 34 pg AI). In red blood cell experiments, the renin content of the perfused and the non-perfused kidneys was similar (169f 12 v. 170f 11 pg AI). No changes in the juxtaglomerular apparatus were seen in slices from the perfused kidneys stained by the modified trichrome method (Endes, Gomba & DBvCnyi, 1969). Effects of changes in perfusion pressure. In kidneys perfused with the red blood cell suspension perfusion pressure was repeatedly changed between 30 and 210 mmHg. Elevation or reduction of perfusion pressure over the whole pressure range was performed within 3-5 min. At pressures above 90 mmHg autoregulation of renal plasma flow was almost complete in five out of six kidneys (Fig. 2). When perfusion pressure was lower than 90 mmHg, renal plasma flow changed in parallel with perfusion pressure.
0‘
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30
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I 190
rnrnHg
3-5min
c
FIG.2. Autoregulation of renal blood flow (RBF) during rapid changes of perfusion pressure (PP) in kidneys perfused with the RBC suspension (n = 5, means5s.e.m.).
When perfusion pressure was raised stepwise by 20 mmHg increments from 90 to 190 mmHg at 5 min intervals, renal plasma flow and GFR increased, the least changes occurring between 110 and 150 mmHg. UN,Vrose with perfusion pressure in a nearly linear function, whereas RNa%fell. When perfusion pressure was lowered similarly from 90 to 30 mmHg a linear reduction of renal plasma flow, GFR and U,,V occurred (Fig. 3), whereas RNa% was slightly enhanced. Rising perfusion pressure up to 150 mmHg decreased renin release, but no further diminution was observed when perfusion pressure was raised from 150 to 190 mmHg. Reduction of perfusion pressure from 90 to 70 mmHg markedly stimulated renin release; the maximal stimulation occurred, when perfusion pressure was further decreased to 50 mmHg (Fig. 3). Lowering of perfusion pressure from 90 to 70 mmHg elicited an increase in renin release within 1 min and the maximum response was obtained within 5 min. When perfusion pressure was re-established at 90 mmHg, renin release fell
78
K. G. Hofbauer, H. Zschiedrich and F. Gross
i
2 -I
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r
0.2
00
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50r
c
i 2000
400
E
o
30 50 70 90
90 110 130 150 170 190
Perfusion Pressure (rnrnHg)
FIG.3. Renal plasma flow (RPF), glomerular filtration rate (GFR), urinary sodium excretion (U,.V) and renin release (RR) in RBC perfused kidneys during changes of perfusion pressure (PP) by steps of 20 mm Hg each at intervals of 5 min (n = 5 ; n = 4 for RR, means s.e.m.).
4000r
a I000 I
30
1
I
1
I
50
70
90
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I10
Time (min)
FIG.4. Renal plasma flow (RPF) and renin release (RR) during two periods of reduction in perfusion pressure (PP) in a RBC perfused kidney. Renin concentration in the perfusate was measured at intervals of 1 min.
Renin-angiotensin system in the isolated kidney
79
towards control levels ;similar observations were made after reduction of perfusion pressure from 90 to 50 mmHg (Fig. 4). When, in cell-free perfused kidneys, perfusion pressure was decreased from 90 to 45 mmHg for 20 or 30 min, renal vascular resistance remained unchanged and consequently, renal plasma flow was reduced by approximately 50%. Renin release increased two-fold for the whole period of pressure reduction. After perfusion pressure had been restored, renin release also returned towards control levels. Non-radioactive microspheres (M) (diameter 35 pm, 3 M Corp.), as a 0.05% suspension (0.5 mg/kidney), injected into the renal artery induced an immediate decrease in renal plasma flow. Within the following 15 min renal plasma flow rose towards control values. During the first 2 min after the injection renin release decreased slightly but increased thereafter reaching a maximum at 7 min; subsequently renin release declined gradually towards control levels. Effects of furosemide. When furosemide (0.5 mg/ml) was infused over a 30 min period in red blood cell perfused kidneys, renal plasma flow increased during the administration of Furosemide
~
30 40
~
l
50 60 7 0 80
t
l
90
100 110
l
l
Time ( r n i n )
FIG.5. Renal plasma flow (RPF), glomerular filtration rate (GFR), urinary sodium excretion (U,.V), and renin release (RR) before, during, and after the infusion of furosemide in RBC perfused kidneys (n = 5, means 5 s.e.m.).
80
K. G . Hofbauer, H . Zschiedrich and F. Gross
furosemide (Pc0.02, n = 5, paired t-test) and fell after the infusion had been stopped. GFR remained unchanged throughout the perfusion period. Within the first 10 min of the infusion UNaVrose, reached a maximum after 20 min, and decreased towards control levels after the infusion of the drug had been stopped. During furosemide administration R N a % fell from 90% to about 60%. In the individual experiments renin release increased within 20 min by two- to six-fold and declined in parallel with UNaVtowards control values after the furosemide infusion had been discontinued (Fig. 5). Renin content of the perfused kidneys was similar to that of the contralateral, non-perfused kidneys (131 +28 v . 132+ 19 pg AI). A lower dose of furosemide (0.05 mglml), which increased U,,V two-fold, had no significant effect on renin release. Effects of isoproterenol. In cell-free perfused kidneys isoproterenol was infused at various doses for 5 min periods. Intervals of 10 or 15 min were inserted between the single infusions. The lowest dose of isoproterenol given (0.5 ng/ml) increased renin release by 79% (P
REVIEW ARTICLE
Regulation of renin release and intrarenal formation of angiotensin. Studies in the isolated perfused rat kidney K. G. Hofbaner, H. Zschiedrich and F. Gross Department of Pharmacology, University of Heidelberg, Heidelberg, Germany (Received 23 May 1975)
SUMMARY
1. Isolated rat kidneys were perfused at a constant pressure of 90 mmHg in a single-pass system with either a cell-free medium or a suspension of washed bovine red blood cells, free of the components of the renin-angiotensin system. In red blood cell perfused kidneys renal haemodynamics and sodium reabsorption corresponded closer to values observed in the intact rat than in cell-free perfused kidneys. 2. In red blood cell-perfused kidneys in the absence of plasma renin substrate autoregulation of renal blood flow was almost complete at pressures above 90 mmHg, provided that perfusion pressure was changed rapidly. 3. Renin release varied inversely with perfusion pressure within a pressure range from 50 to 150 mmHg; the greatest changes of renin release occurred, when perfusion pressure was reduced from 90 to 70 mmHg; maximal stimulation of renin release was observed at 50 mmHg. After reduction of perfusion pressure, renin release immediately started to rise and reached a new level within 5 min. Local reduction of perfusion pressure in small arteries and arterioles by the injection of microspheres induced a short-lasting decrease in renal plasma flow and a transient stimulation of renin release. 4. High concentrations of furosemide stimulated renin release by a direct intrarenal mechanism. 5. Isoproterenol stimulated renin release in low concentrations without a concomitant vasodilation, whereas high concentrations induced an increase in both renal plasma flow and renin release. The effects of isoproterenol were completely blocked by propranolol. 6. Sodium nitroprusside induced similar increases in renal plasma flow, as did high concentrations of isoproterenol, but only a small and slow increase in renin release was observed. 7. Angiotensin I1 (AII) suppressed renin release in concentrations corresponding to plasma levels measured in the intact rat independently of its vasoconstrictor Correspondence:Professor F. Gross, PharmakologischesInstitut der Universitlt Heidelberg,6900 Heidelberg 1, Im NeuenheimerFeld 366, W. Germany.
73
74
K. G . Hofbauer, H. Zschiedrich and F. Gross effects, whereas vasopressin in antidiuretic concentrations did not affect renin release. 8. AII, AI, synthetic tetradecapeptide renin substrate (TDP), crude and purified rat plasma renin substrate induced a dose-dependent reduction in renal plasma flow. SQ 20 881, a competitive inhibitor of converting enzyme, and low doses of l-Sar-8-Ala-A11(saralasin), a competitive antagonist of AII, did not change renal plasma flow, whereas high concentrations of saralasin had a vasoconstrictor effect on their own. 9. Saralasin inhibited the vasoconstrictor effects of A11 and TDP to a similar degree. SQ 20 881 inhibited the vasoconstrictor effects of A1 and purified renin substrate, but did not influence the actions of TDP and the crude renin substrate preparation. 10. From these data it is concluded, that A1 is converted into A11 within the kidney at a rate of 1-2%. The vasoconstriction induced by the crude renin substrate probably does not involve the A11 receptors. TDP may act by itself on the A11 receptors or via the direct intrarenal formation of AIT. The vasoconstriction induced by purified renin substrate is probably due to the intrarenal formation of A1 and its subsequent conversion to AII. Key words : antidiuretic hormone, angiotensin I1 antagonist, converting enzyme inhibitor, furosemide, isoproterenol, microspheres, renal autoregulation, renal haemodynamics, plasma renin substrate, tetradecapeptide substrate, vasodilation.
INTRODUCTION The mechanisms participating in the regulation of renin release have been extensively studied (Vander, 1967; Davis, 1971). From studies in the whole animal it became obvious, that the interpretation of results obtained in vivo is difficult, since various factors which stimulate or suppress renin release in the kidney, as well as extrarenal factors, may interfere with each other. Accordingly, a simplified experimental model, such as the isolated kidney, should be a suitable tool to study the regulation of renin release under defined and controlled conditions. We have developed a method for the perfusion of isolated rat kidneys with a medium, which is free of any component of the renin-angiotensin system. Since the perfusate is not recirculated, renin, which is released during the perfusion, does not re-enter the kidney. Moreover the lack of renin substrate in the perfusion fluid prevents the formation of angiotensin within the renal vascular bed; thus, changes of renin release are without influence on renal function. By means of this experimental model we have studied the effects of various stimuli and measures which are known to affect the renin-angiotensin system on renin release and on renal function. Furthermore the intrarenal formation of A11 from renin substrate and A1 was investigated to obtain information on the role which the reninangiotensin system may play in the regulation of renal haemodynamics. METHODS Preparation Male Sprague-Dawley rats weighing 180-240 g were anaesthetized with sodium pento-
Renin-angio tensin system in the isolated kidney
75
barbitone (Nembutal) 50 mg/kg i.p. The right kidney was prepared and connected to the perfusion system avoiding a period of ischaemia, according to Schurek et al. (1972). Perfusion medium Either a modified Krebs-Henseleit solution containing 35 g/1 of a gelatine preparation (Haemaccel, Behringwerke) was used or a 38 vol. % suspension of washed bovine red blood cells. The composition of the perfusion medium and the preparation of the red blood cells have been described elsewhere (Hofbauer et al., 1973b, 1974). Perfusion system The perfusion fluid is transported from a reservoir to a plastic disc oxygenator and equilibrated with a mixture of 95 vol. % O2 and 5 vol. % C 0 2 at atmospheric pressure. From the oxygenator the medium is delivered to the kidney by means of a peristaltic pump (Harvard App. No. 1203) via a bubble trap and a windkessel. Mean perfusion pressure is continuously measured by a pressure transducer (Statham P23Db) and kept constant at 90 mmHg by a pump speed modulator (Harvard App. No. 550). The venous perfusate passes a drop counter and renal plasma flow is recorded as number of drops/lO s. The venous effluent is not recirculated. Urine is collected for 5 or 10 min periods; urine flow/min is calculated from the weight of the sample and the period of the collection. The reservoir, the oxygenator, the kidney receptacle and all tubes are kept at a constant temperature of 37°C. Analytical methods In samples of the perfusate and of urine inulin was determined by the method of Schreiner (1950), PAH was measured by the reaction of Bratton & Marshall (1939) and sodium concentration by flame photometry (Zeiss, PMQ 11). In samples of venous perfusate renin concentration was determined by two different methods. In the control experiments, and in those with furosemide, the perfusate was incubated for 12 h after addition of rat plasma renin substrate according to the method of Boucher, MCnard & Genest (1967). In the other experiments samples of 50 p1 were incubated for 30 and 60 min (pH 7.2,37"C) with 200 pl of a solution containing 60 mg/ml of a rat plasma substrate preparation, specific concentration about 75 ng AI/mg (Boucher et al., 1967). As inhibitors of angiotensinases and converting enzyme 3 mM 8-hydroxyquinoline, 5 mM Na,EDTA and 1.6 mM dimercaprol had been added. The reaction was stopped by immediate cooling in ice water. At the end of both incubation procedures the amount of angiotensin I was measured by radioimmunoassay (Haber et al., 1969). The rate of angiotensin I formation per hour was higher with the second than with the first method. Plasma renin concentration is expressed as the amount of angiotensin I formed at 37°C during incubation periods of 12 h or 1 h, respectively. Kidney renin content is expressed as pg angiotensin 1formed during an incubation period of 10 min according to the method of Orth et al. (1971). Statistical analysis All values given in the text, Figures and Table are means fs.e.m. ; Student's t-test, t-test for paired data and linear regression analysis were used (Sachs, 1972). All doses are expressed as concentrations of the substances per ml of the perfusion medium. RESULTS Function of the isolated kidney Cell-free perfusion. Kidneys were perfused with the cell-free medium for 100-120 min.
76
K . G . Hofbauer, H . Zschiedrich and F. Gross
During the first hour renal plasma flow, glomerular filtration rate (GFR) and urinary sodium excretion (UNaV)increased ; these values remained constant for the second hour of the perfusion. Sixty minutes after the beginning of the perfusion, renal plasma flow was higher, whereas GFR was lower than values measured in the intact rat (Fig. 1). Fractional sodium reabsorption (RNa%)was 81% and extraction of PAH (EpAH) was 34%. At the end of
I
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RPF
I
-
GFR
I
FF
80
I
c ._
E u 40
W
4 0
FIG.I . Renal plasma flow (RPF), glomerular filtrationrate (GFR), filtrationfraction (FF), net sodium reabsorption (RNs),fractional sodium reabsorption (RN~%), and extraction of PAH (ENs), in kidneys perfused with the cell-free medium (white columns, n = 14) or the red blood cell (RBC) suspension (hatched columns, n = 7) (means+ s.e.m.).
the experiment the weight of the perfused kidney was higher than that of the contralateral kidney removed and weighed immediately before the isolation of the perfused kidney (1252f27 v. 895+26 mg). Red blood cellperfusion. In these experiments kidneys were initially perfused with the cellfree medium for 20-30 min and subsequently for 70-80 min with the red blood cell suspension. Sixty minutes after connecting the kidney to the perfusion system, renal plasma flow was one-third of that obtained in cell-free perfused kidneys, whereas GFR was almost twice as high as in the former group. Consequently, filtration fraction was increased. UNaV was similar to that measured in cell-free experiments; but net sodium reabsorption was twice as high and RNa% was 90%. E p A H amounted to 80% and corresponded to values obtained in vivo (Fig. 1). During the course of the perfusion, renal plasma flow, GFR and UNaVdecreased slightly, whereas RNa% and E p A H remained unchanged. At the end of the experiment the weight of the perfused kidneys was increased similarly to that of cell-free perfused kidneys (1271 +23 v. 930f 30 for the contralateral kidney). Renin release Control experiments. In cell-free perfused kidneys mean renin release was constant during
Renin-angiotensin system in the isolated kidney
77
the whole period of observation. In red blood cell experiments renin release decreased during the first hour, but subsequently remained constant in all kidneys. After perfusion for 1 h, venous renin concentration was similar in both series of experiments, but as a consequence of the higher flow rate in cell-free perfused kidneys renin release was higher in that group (2412f 340 ng AI/min v. 942f 128 ng AI/min). In studies with cell-free perfusion the renin content of the perfused kidneys was equal to or lower than the amount measured in the contralateral, non-perfused kidney, but the means (n = 6) did not differ significantly from each other (146 & 19 v. 182& 34 pg AI). In red blood cell experiments, the renin content of the perfused and the non-perfused kidneys was similar (169f 12 v. 170f 11 pg AI). No changes in the juxtaglomerular apparatus were seen in slices from the perfused kidneys stained by the modified trichrome method (Endes, Gomba & DBvCnyi, 1969). Effects of changes in perfusion pressure. In kidneys perfused with the red blood cell suspension perfusion pressure was repeatedly changed between 30 and 210 mmHg. Elevation or reduction of perfusion pressure over the whole pressure range was performed within 3-5 min. At pressures above 90 mmHg autoregulation of renal plasma flow was almost complete in five out of six kidneys (Fig. 2). When perfusion pressure was lower than 90 mmHg, renal plasma flow changed in parallel with perfusion pressure.
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30
50
70
90
110
130
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170
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I 190
rnrnHg
3-5min
c
FIG.2. Autoregulation of renal blood flow (RBF) during rapid changes of perfusion pressure (PP) in kidneys perfused with the RBC suspension (n = 5, means5s.e.m.).
When perfusion pressure was raised stepwise by 20 mmHg increments from 90 to 190 mmHg at 5 min intervals, renal plasma flow and GFR increased, the least changes occurring between 110 and 150 mmHg. UN,Vrose with perfusion pressure in a nearly linear function, whereas RNa%fell. When perfusion pressure was lowered similarly from 90 to 30 mmHg a linear reduction of renal plasma flow, GFR and U,,V occurred (Fig. 3), whereas RNa% was slightly enhanced. Rising perfusion pressure up to 150 mmHg decreased renin release, but no further diminution was observed when perfusion pressure was raised from 150 to 190 mmHg. Reduction of perfusion pressure from 90 to 70 mmHg markedly stimulated renin release; the maximal stimulation occurred, when perfusion pressure was further decreased to 50 mmHg (Fig. 3). Lowering of perfusion pressure from 90 to 70 mmHg elicited an increase in renin release within 1 min and the maximum response was obtained within 5 min. When perfusion pressure was re-established at 90 mmHg, renin release fell
78
K. G. Hofbauer, H. Zschiedrich and F. Gross
i
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r
0.2
00
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50r
c
i 2000
400
E
o
30 50 70 90
90 110 130 150 170 190
Perfusion Pressure (rnrnHg)
FIG.3. Renal plasma flow (RPF), glomerular filtration rate (GFR), urinary sodium excretion (U,.V) and renin release (RR) in RBC perfused kidneys during changes of perfusion pressure (PP) by steps of 20 mm Hg each at intervals of 5 min (n = 5 ; n = 4 for RR, means s.e.m.).
4000r
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I10
Time (min)
FIG.4. Renal plasma flow (RPF) and renin release (RR) during two periods of reduction in perfusion pressure (PP) in a RBC perfused kidney. Renin concentration in the perfusate was measured at intervals of 1 min.
Renin-angiotensin system in the isolated kidney
79
towards control levels ;similar observations were made after reduction of perfusion pressure from 90 to 50 mmHg (Fig. 4). When, in cell-free perfused kidneys, perfusion pressure was decreased from 90 to 45 mmHg for 20 or 30 min, renal vascular resistance remained unchanged and consequently, renal plasma flow was reduced by approximately 50%. Renin release increased two-fold for the whole period of pressure reduction. After perfusion pressure had been restored, renin release also returned towards control levels. Non-radioactive microspheres (M) (diameter 35 pm, 3 M Corp.), as a 0.05% suspension (0.5 mg/kidney), injected into the renal artery induced an immediate decrease in renal plasma flow. Within the following 15 min renal plasma flow rose towards control values. During the first 2 min after the injection renin release decreased slightly but increased thereafter reaching a maximum at 7 min; subsequently renin release declined gradually towards control levels. Effects of furosemide. When furosemide (0.5 mg/ml) was infused over a 30 min period in red blood cell perfused kidneys, renal plasma flow increased during the administration of Furosemide
~
30 40
~
l
50 60 7 0 80
t
l
90
100 110
l
l
Time ( r n i n )
FIG.5. Renal plasma flow (RPF), glomerular filtration rate (GFR), urinary sodium excretion (U,.V), and renin release (RR) before, during, and after the infusion of furosemide in RBC perfused kidneys (n = 5, means 5 s.e.m.).
80
K. G . Hofbauer, H . Zschiedrich and F. Gross
furosemide (Pc0.02, n = 5, paired t-test) and fell after the infusion had been stopped. GFR remained unchanged throughout the perfusion period. Within the first 10 min of the infusion UNaVrose, reached a maximum after 20 min, and decreased towards control levels after the infusion of the drug had been stopped. During furosemide administration R N a % fell from 90% to about 60%. In the individual experiments renin release increased within 20 min by two- to six-fold and declined in parallel with UNaVtowards control values after the furosemide infusion had been discontinued (Fig. 5). Renin content of the perfused kidneys was similar to that of the contralateral, non-perfused kidneys (131 +28 v . 132+ 19 pg AI). A lower dose of furosemide (0.05 mglml), which increased U,,V two-fold, had no significant effect on renin release. Effects of isoproterenol. In cell-free perfused kidneys isoproterenol was infused at various doses for 5 min periods. Intervals of 10 or 15 min were inserted between the single infusions. The lowest dose of isoproterenol given (0.5 ng/ml) increased renin release by 79% (P
