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Role of the renin-angiotensin system in the response of the rat kidney to hypothermia
J. therm. Biol. Vol. 5. pp. 17 to .~0
Pergamon Press Lid 1980. Printed in Great Britain
ROLE OF THE RENIN-ANGIOTENSIN SYSTEM IN THE RESPONSE OF THE RAT KIDNEY TO HYPOTHERMIA A. R. NOBLE, M. A. WtNCH and K. A. MUNDAY Department of Physiologyand Pharmacology,The University, Southampton, England (Receired 20 July 1979; in revised form 3 September 1979)
Abstract--I. Changes in renal excretion of sodium, potassium and water after cooling to 27°C were monitored in three groups of rats. 2. Immunization with acetylated rat renin (Group B) reduced plasma renin activity by 51% compared to animals immunized with unmodified renin (Group A) and to saline injected control animals (Group Cg 3. In the two control groups (A and C) a hypothermic diuresis and natriuresis was seen but this was abolished in the renin-suppressed rats. 4. Potassium excretion was substantially reduced on cooling all three groups of animals and was not affected by reduction of plasma renin activity. 5. An equivalent reduction in mean arterial blood pressure was found in all three groups.
Endogenous circulating renin activity was reduced in rats by immunizing them over a period of 5 wks with acetylated rat renin. This procedure has been previously shown to generate antisera which react in vitro with both homologous and acetylated renin and would, it was assumed, reduce circulating renin activity (Deodhar et al., 1964). Renal function was investigated before and after cooling to 27°C in these renin-suppressed rats and in two groups of control animals.
INTRODUCTION
OF "mE many changes in physiological function which follow reduction in body temperature, those which involve the circulation and kidney function have received considerable attention. Hypothermia reduces systemic arterial blood pressure and this is accompanied by a decrease in renal blood flow and glomerular filtration rate. There is however, no simple relation between these variables. For example, renal blood flow is disproportionately reduced on cooling as a consequence of intrarenal vasoconstriction and increased blood viscosity (Chapman et al., 1975). The distribution of blood flow within the kidney is also altered as outer cortical blood flow is less affected by cooling than the other regions (Willson et at., 1978). Hypothermia directly affects tubular transport mechanisms and as a consequence of all these effects a diuresis is often seen (Kanter, 1962; Johns & Munday 1967). Other workers (Blatteis & Horvath, 1958; Hong & Boylan, 1959) have failed to demonstrate a consistent diuresis and the confusion probably results from variations in experimental technique. Decreased tubular reabsorption of sodium, potassium, chloride, bicarbonate and phosphate (Walker et al., 1966) and reduced tubular transport of glucose (Kanter, 1959) have also been reported. The role of control mechanisms in these responses is poorly understood. At normal body temperature tubular handling of sodium and potassium, the intrarenal distribution of blood flow and systemic arterial blood pressure are all influenced by the renin-angiotensin system. In the dog hypothermia increases the rate of secretion of renin by the kidney leading to elevated circulating plasma renin levels (Munday & Noble, 1970). Inactivation of renin by the liver is also reduced on cooling (Harris et al., 1977). We have, therefore, examined the possibility that the renin system may be linked with the altered handling of water, sodium and potassium in the kidneys of hypothermic rats. T.a. 5 / I - - e
MATERIALS AND M E T H O D S
Three groups of six female Wistar albino rats (2(X)-290g) were used. Group A was injected with homologous, nonimmunogenic, rat renin. This was prepared as described below and 1330 renin units, in I ml saline,was emulsified with an equal volume of Freunds Complete Adjuvant (Difco)and given i.p.on alternatedays for 5 wks, In Group B an equivalent amount of acetylated renin was used in an otherwise identicalprotocol. Group C animals were given saline injectionsinstead of the renin-adjuvant mixture and therefore formed an additional control group. At the end of the 5 wk immunization schedule the animals were anaesthetized with sodium pentobarbitone (Nembutal, Abbott. 60mg/kg b.w., i.p.)an oesophogeal thermistor probe inserted and a tracheostomy performed. A carotid artery cannula allowed continuous monitoring of the arterialblood pressure and was used for the collection of blood samples. An infusion of Tyrode's solution was given via a femoral vein cannula. For these cannulations, and also for the urethral cannula used for urine collection, polyethylene tubing (Portex pp 25) was used. O n completion of the surgical procedures an 0.6 ml blood sample was retained for plasma renin activityassay and replaced immediately with an equal volume of physiological saline containing 3 % bovine serum albumin Fr.V (Pentax, Miles-Seravac).A continuous infusion of Tyrode's solution, at a rate of 6.5 ml/kg, h, was started and the animals allowed to equilibrate for 2 h. The bladder was
emptied by gentle supra-pubic palpation and urine produced in three subsequent 30 min periods was collected in weighed specimen tubes. During this 90 min normothcrmic 17
18
A. R. NOBLE. M. A. WtnCH and K. A. ML'NDAY
period, arterial blood pressure and oesophageal temperature were continuously monitored. The rats were then cooled from 37-38:C to 26-27°C by application of ice to the abdomen. The ice was removed when the !emperature had fallen to 29~C thus allowing for an undershoot and avoiding cooling below 25°C. Shivering was avoided by adjustment of anaesthesia before applying the ice. On completion of the 1 h cooling period the bladder was again emptied and three further 30 rain urine collections made. Urine sodium and potassium concentrations were estimated by flame photometry. Blood samples taken from the carotid artery were placed in ice-cooled tubes containing I mg tetra-sodium EDTA, centrifuged at 1000 0 for 10 rain and the packed cell volume recorded. The plasma was retained and stored at -20~C until assayed for plasma renin activity by radioimmunoassay ofangiotensin 1 generated from endogenous substrate during incubation at pH 7.4 using dimercaprol and 8-OH quinoline as converting enzyme and angiotensinase inhibitors {Haber et al., 1969). One renin activity unit is defined as the amount of renin which will generate I ng angiotensin I/ml of plasma.h when incubated at 37°C.
Preparation of rat renin Rat kidneys (160 g} were cleared of fat and connective tissue and renin extracted using the first four stages of the method of Peart et al (1966}. The slight precipitate which formed during the final dialysis was removed by centrifugation and the supcrnatant lyophilized. The yield of protein was 288mg with a renin activity of 1480 renin units/rag. This was assessed using sheep renin substrate in the procedure described as a renin "concentration" assay by Skinner {1967). Acetylation of rat renin A sample of the renin preparation described above (90 rag) was dissolved in 45 ml of 0.9% saline at 0°C and 60 ml of 50% saturated (0°C} sodium acetate added. Acetic anhydride (1.65 ml) was added dropwise over a 5rain period and the mixture stirred at 0°C for I h. It was then dialysed against running tap water for 24h, centrifuged and the supcrnatant lyophilized. Protein yield was 92 mg with a renin activity of 300 units/rag protein. This represents a reduction of 80% in renin activity after acctylation. Statistical analysis of results was by an unpaired Student's t-test. Results for urine flow and urinary sodium and potassium concentration and excretion rate are expressed as the mean + SEM of three observations at each temperature range in each of six rats.
RESULTS
Group A - - R a t s immunized with unmodified rat renin Mean plasma renin activity was 6.8 + 3.3 units/ml and the packed cell volume was 43.3 5- 1.2~/~ Urine flow at 37°C was 58.7 5- 8.7 #l/min.kg and this rose to 1 1 3 . 2 5 - l l . 6 p l / m i n . k g alter cooling to 27°C (P < 0.01), an increase of 93~/~ The urine atxlium concentration was 100.1 + 13.3mM at 37"C and 126.9 5- 8.6 mM after cooling, Although this was not statistically significant the difference of 27*//, contributed to a sodium excretion rate 87% above normothermic values at 13.5 5- 1.35/tmol$/min.kg (P < 0.05). Urine potassium concentration fell from 220.0 5- 23.2 m M at 37°C to 40.1 + 6.3 m M at 27°C (P < 0.001), a decrease of g27/~ Despite the diuresis, potassium excretion rate was significantly reduced after cooling with a 67% decrease from
13.0 +__ 1.2/amols/min.kg to 3.3 +_ 0.5/~mols/min.kg
(P < 0.001). Mean arterial blood pressure fell from 108.9 + 2.1 mm Hg to 92.0 + 3.3 mm Hg after cooling (P < 0.001).
Group B---Rats immunized with acetylated renin preparation In this group plasma renin activity was 3.3 + 1.0 units/mE This represents a reduction of 51% compared to the animals immunized with non-acetylated rat renin {Group A). Mean packed cell volume was 44.1 + 1.4%. Urine flow at 37°C was 33.5 + 3.9/A/ min.kg` a lower value than the G r o u p A animals (P < 0.01) and there was no diuresis on cooling to 27°C when urine flow remained unchanged at 33.0 + 4.0 #l/rain. kg. The sodium concentration of the urine at 37°C was 96.8 + 17.1 mM and was not significantly different to the group A rats. Again, urine sodium concentration, at 139. I + 12.9 mM following cooling, was 44% above normothermic levels {not significant). As no hypothermic diuresis occurred in this group of animals, sodium excretion rate was not significantly altered by cooling being 3.9 + 1.2/~mols,;min. kg at 37°C and 5.4 + 0.9/~mols/min.kg at 27°C. The pattern of potassium excretion in the group B animals was very similar to that in the group A rats. The urine potassium concentration ol 258.9 + 13.4mM at 37°C fell to 55.7 + 5.3raM at 27°C (P < 0.001), a decrease of 78%. Renal potassium excretion rate decreased from 8.4 + i.0/Jmois/min, kg to 1.7 + 0.2pmols/min.kg (P < 0.001). Although a similar response to hypothermia was observed in the groups A and B rats it is interesting that there was a significant difference (P < 0.01) in the normothermic potassium excretion rate between the two groups. G r o u p B with a lower circulating plasma renin activity, excreted 35% less potassium than the group A animals. Mean systemic arterial blood pressure was 115.9 + 3.0 mm Hg at 37°C and 82.6 + 5.0 nun Hg at 27°C (P < 0.001), a similar pattern to that found in the group A rats.
Group C--Rats oiven saline injections but no renin or adjuvant The plasma renin activity in this group was 6.4 + 0.7 units/ml and compared closely to the rats immunized with non-aeetylated rat renin {group A). A very similar pattern in urine flow was also obsm~ed with the normothermic flow rate of 63.4 + 7.g~l/ min.kg changing to a hypothermic diuresis of 136.5 + 24.2/A/min.kg on cooling to 270C (P < 0.05). The group C animals differed from both other groups in that the normothermic urine sodium concentration of 184.4 5- l l . g m M was substantially higher (P < 0.001 both eases). On cooling this fell to 126.7 5- 9.2 raM. The trend on cooling is therefore the reverse of that found in the other two groups but the concentration of sodium in the urine at 27"C in all three groups is very similar. Total sodium excretion, in common with the group A animals, did n o w a natriuresis on cooling, rising from 11.8 5- 1.6/~mol/ min.kg at 37°C to 17.3 5- 1.8/anols/min,kg at 27°C (P < 0.02).
Renin and renal function in hypothermia The rate of renal sodium excretion in the group C rats was higher than in the other two groups at both temperatures but with potassium excretion the converse was found. Urine potassium concentration at 37°C was 119.3 + ! 1.7 mM and fell to 22.4 _+ 2.7 mM on cooling to 27°C (P < 0.001). Although this drop of 81% exactly mirrors the results found in groups A and B, the actual concentrations of potassium at both temperatures were significantly lower than in the other two groups of animals. As urine flow in this group was correspondingly higher, the overall potassium excretion rate, 7.1 -4- 1.1 ~mols/min.kg at 37°C and 2.4 _ 0.3/zmols/min.kg at 27°C, was comparable to the other two groups. The group C rats therefore differed from the other two groups in having a higher urine sodium concentration and lower potassium concentration at normal temperatures. As plasma renin activity in group A was similar to that of Group C it is suggested that this is part of the response of the rats to repeated injections of Freunds Complete Adjuvant. Systemic arterial blood pressure in the group C rats was 111.7 + 3.4 mm Hg at 37°C and 75.9 + 2.6 mm Hg at 27°C (P < 0.001) and was equivalent to the other two groups of animals. DISCUSSION
Immunisation of rats against acetylated rat renin as a way of suppressing circulating renin levels has been reported elsewhere (Deodhar et al., 1964). In the original study it was found that acetylation reduced the specific activity of rat renin by 65~/~ comparable to the 80% inhibition found in the present study, but no data was presented to show that circulating renin levels were indeed reduced by this procedure. Our data shows a reduction of 48% in plasma renin levels in the group B animals when compared with the group given only saline injections (group C). There was a good correlation between renin levels in group C rats and the group A animals immunized with nonacetylated renin and so it is concluded that, as in the original study, no significant antirenin titre developed in these animals. The two non renin-suppressed groups of rats (groups A and C) both showed a substantial hypothermic diuresis. In group A urine flow increased by 93% and in group C by 115% after cooling to 27°C. This data is consistent with the 107% increase reported for rats cooled to 25°C and given an intravenous saline infusion (Johns & Munday, 1967). In some studies on dogs, a hypothermic diuresis has also been found (Segar et al., 1956; Kanter, 1962), but in others (Hong & Boylan, 1959) no frank diuresis was demonstrated. A substantial reduction in GFR was however found by the latter authors and calculation of the percentage of filtered water which was excreted reveals decreased water reabsorption during hypothermia. In the work reported here, the hypothermic diuresis and natriuresis responses were abolished in the group of animals in which circulating renin levels were suppressed (group B). Exactly what the role of the renin system is in the genesis of the hypothermic diuresis is not clear. The postulated role of angiotensin II causing increased secretion of ADH (Mouw et aL, 1971)
19
would appear to act in opposition to the observed changes. Similarly it is difficult to envisage a mechanism involving aldosterone or some other mineraiocorticoid especially as neither adrenalectomy nor hypophysectomy abolish the diuretic response to hypothermia in the rat (Johns & Munday--unpublished observations). The hypothermic diuresis might have occurred as a result of a change in the intrarenal distribution of blood flow. The nephrons of the outer cortex are thought to reabsorb less of the filtered sodium and water than those of the inner cortex (de Rouffignac & Bonvalet, 1974). In a study in hypothermic dogs (Withey et al., 1976), it was found that blood flow to the outer renal cortex was reduced by 34% on cooling to 27°C whereas flow to the iner cortex was reduced by 72%. Similar results were obtained in the rat (Willson et al., 1978). It has been suggested that this accounts for the altered sodium and water handling by the kidney in hypothermia. The role of the renin system in the control of the blood flow distribution under these circumstances is not known. In normothermic animals, during infusion of angiotensin II, the changes in regional renal blood flow may be correlated with changes in renal function (Cambridge et al., 1976). Low doses of angiotensin cause a reduction in urine flow and high doses give a diuresis response. Although this has obvious analogies to the situation in the hypothermic rats described in the current paper, not enough is known of the sensitivity of the kidney to angiotensin after cooling to make a firm hypothesis. In addition, the dose of angiotensin needed to give a diuretic response in normotbermic rats is probably outside the physiological range. It is also not possible at the moment to distinguish betwen the effects of angiotensin altering renal function by changing intrarenal distribution of blood flow and direct actions of the hormone on tubular transport processes. Tubular transport of sodium is known to be temperature dependent (Torelli et al., 1973) and also to be under the control of angiotensin (Munday et al., 1972). In the two non renin-suppressed groups of rats (groups A and C) a natriuretic response was observed after cooling. Such a response has been consistently reported by others (Walker et al., 1966; Johns & Munday 1967). In the renin-suppressed group B animals there was no significant increase in sodium excretion rate after cooling. The mechanisms for this are intimately linked with those already discussed for the diuresis response. A precise answer to the question why suppression of endogenous renin should make the hypothermic kidney conserve water and sodium cannot at the moment be given. The group B animals did not show a different response to the other groups in terms of their potassium handling after cooling. In all cases there was a substantial reduction in the potassium excretion rate. Again, this has been consistently reported elsewhere. The dissociation of the effects on sodium and potassium excretion after cooling the renin suppressed rats could be taken to suggest that the mechanism does not have mineralocorticoid involvement. Some care must be exercised in interpretation, however, as it cannot be assumed that all three groups of animals were in the same state of sodium and potassium
20
A, R. NOBLL M. A. Wl.'qCu and K. A. MU.~DAV
balance after the different immunization schedules. An indication of this is given by the observation that during the period before cooling started there was a significant difference in urine flow and potassium excretion between the animals immunized with acetylated (group B) and non-acetylated renin (group A). Only two of the three groups of animals in this study were given adjuvant injections and this provides an interesting comparison for the effects of Freund's Complete Adjuvant on renal function. Although plasma renin activity was similar in groups A and C, the group C animals, which had not received adjuvant, had a higher urine sodium concentration and a lower potassium concentration at normal temperature than either of the other two groups. Circulating plasma renin activity in the rat can therefore be suppressed by immunization with rat renin. This has provided a useful means of investigating the physiological role played by the renin system in hypothermia. Suppression of renin inhibits the diuresis and natriuresis responses to hypothermia but does not appear to interfere with the inhibition of potassium excretion commonly observed in these circumstances.
HARRIS P. J., NOBLEA. R. & MUNDAYK. A. (1977) Inactivation of renin by the isolated perfused rat liver: effect of reduced temperature. Biochem. Med. 17, 158-163. HoNG S. K. & BOYLAN J. W. (1959) Renal concentrating operation in hypothermic dogs. Am. J. Physiol. 196, 1150-1154.
JOH,~S E. J. & MUND^V K. A. (1967) Hormonal influences on urinary ionic changes in hypothermia. Acta Endocr. Suppl. !19, 102. KANTER G. S. (1959) Renal clearance of glucose in hypothermic dogs. Am. J. Physiol. 196, 866--872. KANTERG. S. (1962) Renal clearance of sodium and potassium in hypothermia. Can. J. Biochem. Physiol. 40, 113-122. Mouw D., BONJOUR J-P., MALVIN R. L. & VANDER A. (1971) Central action of angiotensin in stimulating ADH release. Am. J. Physiol. 220, 239-242. Mtnqo^Y K. A. & NoRt.E A. R. (1970) Renin secretion in the hypothermic dog. J. Physiol. 206, 38P-39P. MUNDAY K. A., PARSONSB. J. & POATJ. A. (1972) Studies on the mechanism of action of angiotensin on ion transport by kidney cortex slices. J. Physiol. 224, 195-206. PEART W. S., LLOYDA. M., TUATCUERG. N., LEVERA. F., PAVNE N. & STONEN. (1966) Purification of pig renin. Biochem. J. 99, 708-716. ROUFI:IGNACC. DE • BONVALET J. P. (1974) Heterogeneity of nephron population. In Kidney and Urinary Tract Physiology (Edited by THUR^U K.) 1st edn Ch. 12. pp. 391--409. MTP International Review of Science, Physiology series 1. Butterworths, London. REFERENCES SEG^R W. E., RILEY P. A. & BAalLAT. G. (1956) Urinary composition during hypothermia. Am. J. Physiol. 185, BLATTEISC. M. & HORVATH S. M. (1958) Renal cardio528--532. vascular and respiratory responses and their interrelaSglNN~ S. L. (1967) Improved assay methods for renin tions during hypothermia. Am. J. Physiol. 192, 357-363. "concentration" and "activity" in human plasma. CircuCAMnltX;E D., CH^PM^N B. J. & MUNVAV K. A. (1976) lation Res. 20, 391--402. Regional renal blood flows in the rat during infusion of natriuretic and anti-natriuretic doses of angiotensin II. J. TORELLI G., MILL^ E., KLEINMANL. I. & FAELLIA. (1973) Effect of hypothermia on renal sodium reabsorption. Endocr. 65, 9P-10P. Pfliigers Arch. ges. Physiol. 342, 219-230. CHAPMAN B. J., Wm-IEY W. R. & MUNDAY K. A. (1975) Autoregulation of renal blood flow in dogs at normal WALKER A. W., SMITH G. R, FRAZER S. C. (1966) Renal responses to hypothermia. Clinica chira. Acta 14, body temperature and at 27°C. Clin. Sci. Mol. Med. 462-474. 501-508. DEODHAR S. D., HaAS E. & GOLDBLATTH. (1964) Produc, WILLSON R. A., CH^PMAN B. J. & MUNDA¥ K. A. (1978) The effect of hypothermia on the distribution of blood tion of antirenin to homologous renin and its effect on flow within the rat kidney. J. therm. Biol. 3, 43-47. experimental renal hypertension. 3. exp. Med. !19, WI'tBEY W. R., CHAP:~tANB. J. & MUNDAY K. A. (1976) 425-432. Distribution of blood flow in the hypothermic (27°C) HA~ER E., K o ~ T~ PAGEL. B., KLIMANB. St. PURNOII~ dog kidney. Clin. Sci. Mol. Med. 51,583-588. A.( 1969)Application ofradioimmunoauay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. clin. £ndocr. Metab. Key Word lndex--Hypothermia; diuresis; natriureis; 29, 1349-1355. renin ; renin-antibodies.
Pergamon Press Lid 1980. Printed in Great Britain
ROLE OF THE RENIN-ANGIOTENSIN SYSTEM IN THE RESPONSE OF THE RAT KIDNEY TO HYPOTHERMIA A. R. NOBLE, M. A. WtNCH and K. A. MUNDAY Department of Physiologyand Pharmacology,The University, Southampton, England (Receired 20 July 1979; in revised form 3 September 1979)
Abstract--I. Changes in renal excretion of sodium, potassium and water after cooling to 27°C were monitored in three groups of rats. 2. Immunization with acetylated rat renin (Group B) reduced plasma renin activity by 51% compared to animals immunized with unmodified renin (Group A) and to saline injected control animals (Group Cg 3. In the two control groups (A and C) a hypothermic diuresis and natriuresis was seen but this was abolished in the renin-suppressed rats. 4. Potassium excretion was substantially reduced on cooling all three groups of animals and was not affected by reduction of plasma renin activity. 5. An equivalent reduction in mean arterial blood pressure was found in all three groups.
Endogenous circulating renin activity was reduced in rats by immunizing them over a period of 5 wks with acetylated rat renin. This procedure has been previously shown to generate antisera which react in vitro with both homologous and acetylated renin and would, it was assumed, reduce circulating renin activity (Deodhar et al., 1964). Renal function was investigated before and after cooling to 27°C in these renin-suppressed rats and in two groups of control animals.
INTRODUCTION
OF "mE many changes in physiological function which follow reduction in body temperature, those which involve the circulation and kidney function have received considerable attention. Hypothermia reduces systemic arterial blood pressure and this is accompanied by a decrease in renal blood flow and glomerular filtration rate. There is however, no simple relation between these variables. For example, renal blood flow is disproportionately reduced on cooling as a consequence of intrarenal vasoconstriction and increased blood viscosity (Chapman et al., 1975). The distribution of blood flow within the kidney is also altered as outer cortical blood flow is less affected by cooling than the other regions (Willson et at., 1978). Hypothermia directly affects tubular transport mechanisms and as a consequence of all these effects a diuresis is often seen (Kanter, 1962; Johns & Munday 1967). Other workers (Blatteis & Horvath, 1958; Hong & Boylan, 1959) have failed to demonstrate a consistent diuresis and the confusion probably results from variations in experimental technique. Decreased tubular reabsorption of sodium, potassium, chloride, bicarbonate and phosphate (Walker et al., 1966) and reduced tubular transport of glucose (Kanter, 1959) have also been reported. The role of control mechanisms in these responses is poorly understood. At normal body temperature tubular handling of sodium and potassium, the intrarenal distribution of blood flow and systemic arterial blood pressure are all influenced by the renin-angiotensin system. In the dog hypothermia increases the rate of secretion of renin by the kidney leading to elevated circulating plasma renin levels (Munday & Noble, 1970). Inactivation of renin by the liver is also reduced on cooling (Harris et al., 1977). We have, therefore, examined the possibility that the renin system may be linked with the altered handling of water, sodium and potassium in the kidneys of hypothermic rats. T.a. 5 / I - - e
MATERIALS AND M E T H O D S
Three groups of six female Wistar albino rats (2(X)-290g) were used. Group A was injected with homologous, nonimmunogenic, rat renin. This was prepared as described below and 1330 renin units, in I ml saline,was emulsified with an equal volume of Freunds Complete Adjuvant (Difco)and given i.p.on alternatedays for 5 wks, In Group B an equivalent amount of acetylated renin was used in an otherwise identicalprotocol. Group C animals were given saline injectionsinstead of the renin-adjuvant mixture and therefore formed an additional control group. At the end of the 5 wk immunization schedule the animals were anaesthetized with sodium pentobarbitone (Nembutal, Abbott. 60mg/kg b.w., i.p.)an oesophogeal thermistor probe inserted and a tracheostomy performed. A carotid artery cannula allowed continuous monitoring of the arterialblood pressure and was used for the collection of blood samples. An infusion of Tyrode's solution was given via a femoral vein cannula. For these cannulations, and also for the urethral cannula used for urine collection, polyethylene tubing (Portex pp 25) was used. O n completion of the surgical procedures an 0.6 ml blood sample was retained for plasma renin activityassay and replaced immediately with an equal volume of physiological saline containing 3 % bovine serum albumin Fr.V (Pentax, Miles-Seravac).A continuous infusion of Tyrode's solution, at a rate of 6.5 ml/kg, h, was started and the animals allowed to equilibrate for 2 h. The bladder was
emptied by gentle supra-pubic palpation and urine produced in three subsequent 30 min periods was collected in weighed specimen tubes. During this 90 min normothcrmic 17
18
A. R. NOBLE. M. A. WtnCH and K. A. ML'NDAY
period, arterial blood pressure and oesophageal temperature were continuously monitored. The rats were then cooled from 37-38:C to 26-27°C by application of ice to the abdomen. The ice was removed when the !emperature had fallen to 29~C thus allowing for an undershoot and avoiding cooling below 25°C. Shivering was avoided by adjustment of anaesthesia before applying the ice. On completion of the 1 h cooling period the bladder was again emptied and three further 30 rain urine collections made. Urine sodium and potassium concentrations were estimated by flame photometry. Blood samples taken from the carotid artery were placed in ice-cooled tubes containing I mg tetra-sodium EDTA, centrifuged at 1000 0 for 10 rain and the packed cell volume recorded. The plasma was retained and stored at -20~C until assayed for plasma renin activity by radioimmunoassay ofangiotensin 1 generated from endogenous substrate during incubation at pH 7.4 using dimercaprol and 8-OH quinoline as converting enzyme and angiotensinase inhibitors {Haber et al., 1969). One renin activity unit is defined as the amount of renin which will generate I ng angiotensin I/ml of plasma.h when incubated at 37°C.
Preparation of rat renin Rat kidneys (160 g} were cleared of fat and connective tissue and renin extracted using the first four stages of the method of Peart et al (1966}. The slight precipitate which formed during the final dialysis was removed by centrifugation and the supcrnatant lyophilized. The yield of protein was 288mg with a renin activity of 1480 renin units/rag. This was assessed using sheep renin substrate in the procedure described as a renin "concentration" assay by Skinner {1967). Acetylation of rat renin A sample of the renin preparation described above (90 rag) was dissolved in 45 ml of 0.9% saline at 0°C and 60 ml of 50% saturated (0°C} sodium acetate added. Acetic anhydride (1.65 ml) was added dropwise over a 5rain period and the mixture stirred at 0°C for I h. It was then dialysed against running tap water for 24h, centrifuged and the supcrnatant lyophilized. Protein yield was 92 mg with a renin activity of 300 units/rag protein. This represents a reduction of 80% in renin activity after acctylation. Statistical analysis of results was by an unpaired Student's t-test. Results for urine flow and urinary sodium and potassium concentration and excretion rate are expressed as the mean + SEM of three observations at each temperature range in each of six rats.
RESULTS
Group A - - R a t s immunized with unmodified rat renin Mean plasma renin activity was 6.8 + 3.3 units/ml and the packed cell volume was 43.3 5- 1.2~/~ Urine flow at 37°C was 58.7 5- 8.7 #l/min.kg and this rose to 1 1 3 . 2 5 - l l . 6 p l / m i n . k g alter cooling to 27°C (P < 0.01), an increase of 93~/~ The urine atxlium concentration was 100.1 + 13.3mM at 37"C and 126.9 5- 8.6 mM after cooling, Although this was not statistically significant the difference of 27*//, contributed to a sodium excretion rate 87% above normothermic values at 13.5 5- 1.35/tmol$/min.kg (P < 0.05). Urine potassium concentration fell from 220.0 5- 23.2 m M at 37°C to 40.1 + 6.3 m M at 27°C (P < 0.001), a decrease of g27/~ Despite the diuresis, potassium excretion rate was significantly reduced after cooling with a 67% decrease from
13.0 +__ 1.2/amols/min.kg to 3.3 +_ 0.5/~mols/min.kg
(P < 0.001). Mean arterial blood pressure fell from 108.9 + 2.1 mm Hg to 92.0 + 3.3 mm Hg after cooling (P < 0.001).
Group B---Rats immunized with acetylated renin preparation In this group plasma renin activity was 3.3 + 1.0 units/mE This represents a reduction of 51% compared to the animals immunized with non-acetylated rat renin {Group A). Mean packed cell volume was 44.1 + 1.4%. Urine flow at 37°C was 33.5 + 3.9/A/ min.kg` a lower value than the G r o u p A animals (P < 0.01) and there was no diuresis on cooling to 27°C when urine flow remained unchanged at 33.0 + 4.0 #l/rain. kg. The sodium concentration of the urine at 37°C was 96.8 + 17.1 mM and was not significantly different to the group A rats. Again, urine sodium concentration, at 139. I + 12.9 mM following cooling, was 44% above normothermic levels {not significant). As no hypothermic diuresis occurred in this group of animals, sodium excretion rate was not significantly altered by cooling being 3.9 + 1.2/~mols,;min. kg at 37°C and 5.4 + 0.9/~mols/min.kg at 27°C. The pattern of potassium excretion in the group B animals was very similar to that in the group A rats. The urine potassium concentration ol 258.9 + 13.4mM at 37°C fell to 55.7 + 5.3raM at 27°C (P < 0.001), a decrease of 78%. Renal potassium excretion rate decreased from 8.4 + i.0/Jmois/min, kg to 1.7 + 0.2pmols/min.kg (P < 0.001). Although a similar response to hypothermia was observed in the groups A and B rats it is interesting that there was a significant difference (P < 0.01) in the normothermic potassium excretion rate between the two groups. G r o u p B with a lower circulating plasma renin activity, excreted 35% less potassium than the group A animals. Mean systemic arterial blood pressure was 115.9 + 3.0 mm Hg at 37°C and 82.6 + 5.0 nun Hg at 27°C (P < 0.001), a similar pattern to that found in the group A rats.
Group C--Rats oiven saline injections but no renin or adjuvant The plasma renin activity in this group was 6.4 + 0.7 units/ml and compared closely to the rats immunized with non-aeetylated rat renin {group A). A very similar pattern in urine flow was also obsm~ed with the normothermic flow rate of 63.4 + 7.g~l/ min.kg changing to a hypothermic diuresis of 136.5 + 24.2/A/min.kg on cooling to 270C (P < 0.05). The group C animals differed from both other groups in that the normothermic urine sodium concentration of 184.4 5- l l . g m M was substantially higher (P < 0.001 both eases). On cooling this fell to 126.7 5- 9.2 raM. The trend on cooling is therefore the reverse of that found in the other two groups but the concentration of sodium in the urine at 27"C in all three groups is very similar. Total sodium excretion, in common with the group A animals, did n o w a natriuresis on cooling, rising from 11.8 5- 1.6/~mol/ min.kg at 37°C to 17.3 5- 1.8/anols/min,kg at 27°C (P < 0.02).
Renin and renal function in hypothermia The rate of renal sodium excretion in the group C rats was higher than in the other two groups at both temperatures but with potassium excretion the converse was found. Urine potassium concentration at 37°C was 119.3 + ! 1.7 mM and fell to 22.4 _+ 2.7 mM on cooling to 27°C (P < 0.001). Although this drop of 81% exactly mirrors the results found in groups A and B, the actual concentrations of potassium at both temperatures were significantly lower than in the other two groups of animals. As urine flow in this group was correspondingly higher, the overall potassium excretion rate, 7.1 -4- 1.1 ~mols/min.kg at 37°C and 2.4 _ 0.3/zmols/min.kg at 27°C, was comparable to the other two groups. The group C rats therefore differed from the other two groups in having a higher urine sodium concentration and lower potassium concentration at normal temperatures. As plasma renin activity in group A was similar to that of Group C it is suggested that this is part of the response of the rats to repeated injections of Freunds Complete Adjuvant. Systemic arterial blood pressure in the group C rats was 111.7 + 3.4 mm Hg at 37°C and 75.9 + 2.6 mm Hg at 27°C (P < 0.001) and was equivalent to the other two groups of animals. DISCUSSION
Immunisation of rats against acetylated rat renin as a way of suppressing circulating renin levels has been reported elsewhere (Deodhar et al., 1964). In the original study it was found that acetylation reduced the specific activity of rat renin by 65~/~ comparable to the 80% inhibition found in the present study, but no data was presented to show that circulating renin levels were indeed reduced by this procedure. Our data shows a reduction of 48% in plasma renin levels in the group B animals when compared with the group given only saline injections (group C). There was a good correlation between renin levels in group C rats and the group A animals immunized with nonacetylated renin and so it is concluded that, as in the original study, no significant antirenin titre developed in these animals. The two non renin-suppressed groups of rats (groups A and C) both showed a substantial hypothermic diuresis. In group A urine flow increased by 93% and in group C by 115% after cooling to 27°C. This data is consistent with the 107% increase reported for rats cooled to 25°C and given an intravenous saline infusion (Johns & Munday, 1967). In some studies on dogs, a hypothermic diuresis has also been found (Segar et al., 1956; Kanter, 1962), but in others (Hong & Boylan, 1959) no frank diuresis was demonstrated. A substantial reduction in GFR was however found by the latter authors and calculation of the percentage of filtered water which was excreted reveals decreased water reabsorption during hypothermia. In the work reported here, the hypothermic diuresis and natriuresis responses were abolished in the group of animals in which circulating renin levels were suppressed (group B). Exactly what the role of the renin system is in the genesis of the hypothermic diuresis is not clear. The postulated role of angiotensin II causing increased secretion of ADH (Mouw et aL, 1971)
19
would appear to act in opposition to the observed changes. Similarly it is difficult to envisage a mechanism involving aldosterone or some other mineraiocorticoid especially as neither adrenalectomy nor hypophysectomy abolish the diuretic response to hypothermia in the rat (Johns & Munday--unpublished observations). The hypothermic diuresis might have occurred as a result of a change in the intrarenal distribution of blood flow. The nephrons of the outer cortex are thought to reabsorb less of the filtered sodium and water than those of the inner cortex (de Rouffignac & Bonvalet, 1974). In a study in hypothermic dogs (Withey et al., 1976), it was found that blood flow to the outer renal cortex was reduced by 34% on cooling to 27°C whereas flow to the iner cortex was reduced by 72%. Similar results were obtained in the rat (Willson et al., 1978). It has been suggested that this accounts for the altered sodium and water handling by the kidney in hypothermia. The role of the renin system in the control of the blood flow distribution under these circumstances is not known. In normothermic animals, during infusion of angiotensin II, the changes in regional renal blood flow may be correlated with changes in renal function (Cambridge et al., 1976). Low doses of angiotensin cause a reduction in urine flow and high doses give a diuresis response. Although this has obvious analogies to the situation in the hypothermic rats described in the current paper, not enough is known of the sensitivity of the kidney to angiotensin after cooling to make a firm hypothesis. In addition, the dose of angiotensin needed to give a diuretic response in normotbermic rats is probably outside the physiological range. It is also not possible at the moment to distinguish betwen the effects of angiotensin altering renal function by changing intrarenal distribution of blood flow and direct actions of the hormone on tubular transport processes. Tubular transport of sodium is known to be temperature dependent (Torelli et al., 1973) and also to be under the control of angiotensin (Munday et al., 1972). In the two non renin-suppressed groups of rats (groups A and C) a natriuretic response was observed after cooling. Such a response has been consistently reported by others (Walker et al., 1966; Johns & Munday 1967). In the renin-suppressed group B animals there was no significant increase in sodium excretion rate after cooling. The mechanisms for this are intimately linked with those already discussed for the diuresis response. A precise answer to the question why suppression of endogenous renin should make the hypothermic kidney conserve water and sodium cannot at the moment be given. The group B animals did not show a different response to the other groups in terms of their potassium handling after cooling. In all cases there was a substantial reduction in the potassium excretion rate. Again, this has been consistently reported elsewhere. The dissociation of the effects on sodium and potassium excretion after cooling the renin suppressed rats could be taken to suggest that the mechanism does not have mineralocorticoid involvement. Some care must be exercised in interpretation, however, as it cannot be assumed that all three groups of animals were in the same state of sodium and potassium
20
A, R. NOBLL M. A. Wl.'qCu and K. A. MU.~DAV
balance after the different immunization schedules. An indication of this is given by the observation that during the period before cooling started there was a significant difference in urine flow and potassium excretion between the animals immunized with acetylated (group B) and non-acetylated renin (group A). Only two of the three groups of animals in this study were given adjuvant injections and this provides an interesting comparison for the effects of Freund's Complete Adjuvant on renal function. Although plasma renin activity was similar in groups A and C, the group C animals, which had not received adjuvant, had a higher urine sodium concentration and a lower potassium concentration at normal temperature than either of the other two groups. Circulating plasma renin activity in the rat can therefore be suppressed by immunization with rat renin. This has provided a useful means of investigating the physiological role played by the renin system in hypothermia. Suppression of renin inhibits the diuresis and natriuresis responses to hypothermia but does not appear to interfere with the inhibition of potassium excretion commonly observed in these circumstances.
HARRIS P. J., NOBLEA. R. & MUNDAYK. A. (1977) Inactivation of renin by the isolated perfused rat liver: effect of reduced temperature. Biochem. Med. 17, 158-163. HoNG S. K. & BOYLAN J. W. (1959) Renal concentrating operation in hypothermic dogs. Am. J. Physiol. 196, 1150-1154.
JOH,~S E. J. & MUND^V K. A. (1967) Hormonal influences on urinary ionic changes in hypothermia. Acta Endocr. Suppl. !19, 102. KANTER G. S. (1959) Renal clearance of glucose in hypothermic dogs. Am. J. Physiol. 196, 866--872. KANTERG. S. (1962) Renal clearance of sodium and potassium in hypothermia. Can. J. Biochem. Physiol. 40, 113-122. Mouw D., BONJOUR J-P., MALVIN R. L. & VANDER A. (1971) Central action of angiotensin in stimulating ADH release. Am. J. Physiol. 220, 239-242. Mtnqo^Y K. A. & NoRt.E A. R. (1970) Renin secretion in the hypothermic dog. J. Physiol. 206, 38P-39P. MUNDAY K. A., PARSONSB. J. & POATJ. A. (1972) Studies on the mechanism of action of angiotensin on ion transport by kidney cortex slices. J. Physiol. 224, 195-206. PEART W. S., LLOYDA. M., TUATCUERG. N., LEVERA. F., PAVNE N. & STONEN. (1966) Purification of pig renin. Biochem. J. 99, 708-716. ROUFI:IGNACC. DE • BONVALET J. P. (1974) Heterogeneity of nephron population. In Kidney and Urinary Tract Physiology (Edited by THUR^U K.) 1st edn Ch. 12. pp. 391--409. MTP International Review of Science, Physiology series 1. Butterworths, London. REFERENCES SEG^R W. E., RILEY P. A. & BAalLAT. G. (1956) Urinary composition during hypothermia. Am. J. Physiol. 185, BLATTEISC. M. & HORVATH S. M. (1958) Renal cardio528--532. vascular and respiratory responses and their interrelaSglNN~ S. L. (1967) Improved assay methods for renin tions during hypothermia. Am. J. Physiol. 192, 357-363. "concentration" and "activity" in human plasma. CircuCAMnltX;E D., CH^PM^N B. J. & MUNVAV K. A. (1976) lation Res. 20, 391--402. Regional renal blood flows in the rat during infusion of natriuretic and anti-natriuretic doses of angiotensin II. J. TORELLI G., MILL^ E., KLEINMANL. I. & FAELLIA. (1973) Effect of hypothermia on renal sodium reabsorption. Endocr. 65, 9P-10P. Pfliigers Arch. ges. Physiol. 342, 219-230. CHAPMAN B. J., Wm-IEY W. R. & MUNDAY K. A. (1975) Autoregulation of renal blood flow in dogs at normal WALKER A. W., SMITH G. R, FRAZER S. C. (1966) Renal responses to hypothermia. Clinica chira. Acta 14, body temperature and at 27°C. Clin. Sci. Mol. Med. 462-474. 501-508. DEODHAR S. D., HaAS E. & GOLDBLATTH. (1964) Produc, WILLSON R. A., CH^PMAN B. J. & MUNDA¥ K. A. (1978) The effect of hypothermia on the distribution of blood tion of antirenin to homologous renin and its effect on flow within the rat kidney. J. therm. Biol. 3, 43-47. experimental renal hypertension. 3. exp. Med. !19, WI'tBEY W. R., CHAP:~tANB. J. & MUNDAY K. A. (1976) 425-432. Distribution of blood flow in the hypothermic (27°C) HA~ER E., K o ~ T~ PAGEL. B., KLIMANB. St. PURNOII~ dog kidney. Clin. Sci. Mol. Med. 51,583-588. A.( 1969)Application ofradioimmunoauay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. clin. £ndocr. Metab. Key Word lndex--Hypothermia; diuresis; natriureis; 29, 1349-1355. renin ; renin-antibodies.