kidney cortex slices: effect of sodium

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ACTIVE AND INACTIVE RENIN RELEASE FROM RABBITKIDNEY CORTEX SLICES: EFFECT OF SODIUM CONCENTRATION

AND OF FUROSEMIDE

BY K. A. MUNDAY, A. R. NOBLE AND H. K. RICHARDSFrom the Department of Physiology and Pharmacology, University of Southampton,

Southampton S09 3TU

(Received 4 January 1982)

SUMMARY

1. Active and inactive renin release by rabbit kidney cortex slices was investigated.Inactive renin was estimated as the increase in renin activity after acidification(pH 2 8) of slice supernatant solutions.

2. Active renin release was increased when incubation medium [Na+] was reduced.This relationship was linear (r2 = 0 96) over the range [Na+] = 23-133 mM.

3. For the same range of [Na+] inactive renin secretion decreased when medium[Na+] was reduced (r2 = 0 92). Therefore, the proportion of total renin which was inthe inactive form decreased linearly as [Na+] was reduced (r2 = 0-97).

4. Chloride ions did not appear to be important in altering the secretion of eitheractive or inactive renin.

5. Adding furosemide to the incubation medium in concentrations up to 40 ,ug/ml.did not change secretion ofeither form of renin. The action of furosemide on secretionof active and inactive renin in vivo is therefore secondary to altered renal function.

6. Regulation of the relative amount of active and inactive renin in plasma couldbe entirely an intrarenal event. It is not essential to invoke a plasma activatingenzyme for inactive renin in order to explain changes in plasma levels of the two formsof renin.

7. This paper supports the hypothesis that release of inactive renin by the kidneyis controlled by a sodium-sensitive mechanism.

INTRODUCTION

The existence of both 'active' and 'inactive' forms of renin has been widelyestablished (Overturf, Druilhet & Kirkendall, 1979; Leckie, 1981). An agreed, precisebiochemical definition of these two forms remains elusive despite extensive studies.The physiological role of inactive renin is similarly poorly understood. However, thepresence of substantial amounts of inactive renin in both plasma and kidney extracts,together with observations that plasma levels of the two forms respond in differentways to differing stimuli, does suggest the basis for a new site for physiological controlof the renin system.The macula densa hypothesis for the control of renin secretion proposes a link

J. Physiol. (1982), 328, pp. 421-430 421

K. A. MUNDA Y, A. R. NOBLE AND H. K. RICHARDS

between sodium entering the distal tubule of the nephron and the secretion of reninby the granular cells. Several aspects of this hypothesis are still contentious, but thebalance of evidence seems to favour an inverse correlation between macula densasodium load and renin secretion (Vander, 1967; Keeton & Campbell, 1980). Thepresence of inactive forms of renin was not considered in the formulation of thishypothesis as much of the experimental work was done before the existence ofinactive forms of renin was recognized.

In studies using the rabbit as an experimental model, furosemide diuresis withoutvolume depletion (Richards, Lush, Noble & Munday, 1981 a) and the phase of dietarysodium depletion in which the animals were in negative sodium balance (Grace,Munday & Noble, 1979a; Grace, Munday, Noble & Richards, 1979b), were bothassociated with raised plasma active renin concentrations. Plasma levels of inactiverenin, however, became unmeasurable. This is in direct contrast to situations suchas haemorrhage (Richards, Grace, Noble & Munday, 1979), ,i-adrenoceptor stimulationwith isoprenaline (Richards, Noble & Munday, 1981 b), and the hypotension associatedwith urethane anaesthesia (Grace et al. 1979b) which increased plasma levels of bothactive and inactive renin. This suggests to us that plasma levels of inactive renin couldalso be regulated by a sodium-sensitive mechanism.The control of the relative amounts of active and inactive renin in plasma could

be effected intrarenally. This may be based on either two independent renin secretioncontrol processes or regulatable interconversion of the two forms of renin prior tosecretion. A further alternative is that changes in the relative amounts of circulatingactive and inactive renin could reflect the activity of a proteolytic enzyme in plasma.We have therefore investigated secretion of the two forms of renin in vitro using

a rabbit kidney cortex slice preparation. The response to changes in incubationmedium [Na+] and also to furosemide was studied. A preliminary report of this workhas already been published (Munday, Noble & Richards, 1980).

METHODS

Preparation of kidney cortex slicesNew Zealand White rabbits weighing 3-0-4-0 kg were obtained from the breeding colony

maintained in the University Animal House. These animals received a standard laboratory chow(RAG - Christopher Hill Division of RHM) from weaning and tap water ad libitum. The animalswere killed by neck fracture or with a lethal dose of pentobarbitone sodium. Both kidneys wereexcised, placed on ice, and the poles and medulla removed. Eleven transverse slices of renal cortexwere hand-cut from each kidney with a razor blade. Slices were approximately 200 ,um thick andweighed 50-100 mg. After accurate weighing, and retention of one slice as a non-incubated control,each slice was incubated in a stoppered flask with 5 ml. of oxygenated (02+Go2, 95:5, v/v)Krebs-Ringer bicarbonate buffer (pH 7-4) containing 10 mM-glucose. This basic medium wasmodified as appropriate to the experiments described. Flasks were incubated for 90 min at 37 'C.The medium was oxygenated by bubbling the gas mixture through the incubation medium forapproximately 5 sec every 10 min. After incubation active and inactive renins were assayed in theslice supernatant as described below. The non-incubated control slice was immediately frozen at-20 'C and subsequently ground in a Potter homogenizer in 2 ml. of buffer. The renins extractedinto the buffer were estimated.The time course and the effect of the oxygenation protocol on release of active renin by kidney

cortex slices was investigated. In the first experiment slices were incubated for up to 2 hr in theunmodified Krebs-Ringer bicarbonate medium. One set offlasks was oxygenated as described above

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SODIUM AND RENIN RELEASE

and for the other set this was omitted. Every 20 min a 50 ,ul. aliquot of the incubation mediumwas removed for renin assay. Results are shown in Table 1. Renin release was consistently 20-25 %higher in the group which was regularly re-oxygenated. This was not statistically significant whencomparisons were made at each time interval. Renin release in the oxygenated medium was linearover the 2 hr time course studied. In the set for which the medium was only oxygenated prior tothe incubation, renin release departed from linearity only in the final sample collected. Althoughthis was beyond the normal time course of our experiments (90 min), a protocol which includedregular oxygenation was adopted.

TABLE 1. Time course of release of active renin by kidney cortex slices

Time (min) 20 40 60 80 100 120

Active renin (p-mole angiotensin I/hr. mg wet weight tissue)Oxygenated during 1P2+0-4 2-8+0-7 4-1+0-8 57+10 7-0+141 8-1+1P4incubationNo oxygenation 1P0+0-3 2-3+0-8 3-3+1-0 4-5+14 5-5+±17 6-1+1-8during incubationActive renin release in each 20 min incubation period (p-mole angiotensin I/hr. mg wet weight

tissue)Oxygenated during 1-2+004 1P6+0 5 1P2+0 3 1-6+003 1-2+003 1P2+004incubationNo oxygenation 10±+03 1-3+05 1-0+03 1-2+03 10±+03 0-5+0-2during incubationActive renin was measured in aliquots of incubation medium removed every 20 min. One set

of flasks was oxygenated every 10 min throughout the incubation and the other set only at thestart. In the lower half of this table renin release per 20 min incubation period is shown. This iscalculated from the data in the upper half of the table (n = 6).

TABLE 2. Time course of release of active renin by kidney cortex slices

Time (min) 0-20 20-40 40-60 60-80 80-100 100-120Active renin (p-mole angiotensin I/hr. mg wet weight tissue)

Oxygenated during 1-2+0 4 1-2+0 4 1P3+0 4 1-2+0 4 1P2+0 4 1P2+004incubationNo oxygenation 1-3+05 1P2+05 1-2+05 1P1+04 1P2+04 11+04during incubationSlices were transferred to fresh oxygenated incubation medium every 20 min. One set of flasks

was re-oxygenated after 10 min. The remaining set was not re-oxygenated. Active renin releasedin each 20 min period is shown. (n = 6).

In a second series of control experiments, slices were incubated in media which were changedevery 20 min. Alterations in renin release as a result of build-up of the hormone in the incubationmedium were therefore minimized. Results are shown in Table 2. Active renin release by the kidneycortex slices was again found to be linear over the time course studied. The amount of renin releasedin each 20 min period was very similar to the previous experiment in which secreted renin wasallowed to accumulate in the incubation medium (Table 1). With the introduction of freshincubation media every 20 min, further intermediate oxygenation of the medium did not now alterthe rate of renin secretion.The kidney cortex slice preparation described was therefore considered to be a viable model for

studying experimental manipulations of renin release.

Measurement of active and inactive renin8Inactive renin can only be measured after activation. This was achieved by acidification.

Supernatant samples were dialysed against pH 2-8 glycine-HCl buffer (170 mM) for 24 hr and thenredialysed topH 7*5 against a phosphate buffer (175 mM) for a further 24 hr. Subsequent renin assay

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yielded a value for total renin (active+ inactive). Active renin alone was estimated in aliquots ofsupernatant which had been dialysed against pH 7-5 buffer for 48 hr. Inactive renin was thereforedetermined as the difference between these two sets of measurements.

Active renin was measured by radioimmunoassay of generated angiotensin I (Stockigt, Collins& Biglieri, 1971) after incubation of dialysed supernatant samples with excess sheep renin substrate(Skinner, 1967). Details of the evaluation of these assay procedures for active and inactive reninhave been published previously (Richards et al. 1981 a). Results are presented as renin released permg wet weight tissue during the 90 min incubation.

Statistical analysis of resultsThe renin activities in each slice supernatant with and without acidification were compared using

a non-parametric paired test, the Wilcoxon signed-rank test. For analysis of the data obtained fromcalculation of the percentage increase in renin activity after acidification, a paired Student's t testwas used. Linear regression analysis was used to relate [renin] to buffer [Na+].

RESULTS

Effect of buffer [Na+] on release of active and inactive reninsFor the complete Krebs-Ringer bicarbonate buffer, containing 10 mM-glucose,

[Na+] = 133 mm. A series of four buffers with lower [Na+] containing, respectively,106 mm, 78 mm, 51 mm and 23 mM-Na+ was prepared. The NaCl was replaced withequimolar amounts of choline chloride. All other constituents of the basic bufferremained unchanged. Thus, any changes in [Cl-] in the series of buffers were small,an important aspect of the design of the experiments in view of the proposed rolefor Cl- ions in the control of renin secretion (Kotchen, Galla & Luke, 1978). In Table3 results are shown for active and inactive renin released during incubation of kidneycortex slices in each of the buffers. The percentage increase in renin activity followingacidification is also shown.

TABLE 3. Active and inactive renin release by rabbit kidney cortex slices: effect of [Na+]Sodium concentration (mM) 133 106 78 51 23

Total renin (p-mole angiotensin I/hr. mg wet weight tissue)11-7+3-2* 13-9+2-2* 17-9+4-6 22-0+5-4 29-7+7-4

Active renin (p-mole angiotensin I/hr. mg wet weight tissue)t9-9+2-4 12-8+2-2 16-8+4-0 22-4+5-6 31-1+8-2

Inactive renin (p-mole angiotensin I/hr. mg wet weight tissue)1-8+0-6 1 1+0-3 1 1+0-6 -0-4+0-2 -1-4+0-8

Increase in renin activity after acidification (%)§17-8+2-4 9-0+2-9 6-9+2-1 -1-7 +1-2 -4 5+1-3

Total renin (active and inactive) was measured after dialysis to pH 2-8 and back to pH 7-5 toactivate inactive renin. Samples for active renin estimation were dialysed at pH 7-5. Results aremeans + S.E.M. (n = 10). Significance of differences: * P < 0 05 for paired comparison of acidified(total renin) and non-acidified (active renin) supernatant samples.

t Active renin: correlation coefficient (r2) vS. [Na+] = 0-96.T Inactive renin: correlation coefficient (r2) vs. [Na+] = 0-92.§ Increase in renin activity (%) after acidification: correlation coefficient (r2) vs. [Na+] = 0-97.

A negative correlation (r2 = 0 96) was found between active renin release and buffer[Na+]. Active renin released when buffer [Na+] = 23 mm was 2130 higher than forslices incubated at the highest sodium concentration ([Na+] = 133 mm) (P < 0 01).Acidification of supernatant samples yielded significant increases in renin activity

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only in the buffers containing the two highest sodium concentrations ([Na+] 133and 106 mM). At the two lowest sodium concentrations ([Na+] = 51 and 23 mM)acidification had a negative effect on renin activity. This we attribute to a small lossofrenin activity during acid dialysis and is discussed elsewhere (Richards et al. 1981 a).Inactive renin release was calculated from the data for active and total renin. Apositive correlation (r2 = 0-92) was found between inactive renin and [Na+].

TABLE 4. Active and inactive renin release by rabbit kidney cortex slices: effect of varyingreplacement compound for [Na+] in low [Na+] incubation media

Buffer [Na+] (mM) 133 23 23 23 23Replacement compound Choline Mannitol Tris(110 mM) chloride

Total renin (p-mole angiotensin I/hr. mg wet weight tissue)108+ 2.3* 21-1 + 36 22-4+5-0 19-9+4-1 355±+9-8

Active renin (p-mole angiotensin I/hr. mg wet weight tissue)9-0+2-0 21-8+3-8 22-8+5-1 20-5+41 38-2+11P8

Inactive renin (p-mole angiotensin I/hr. mg wet weight tissue)18+0-4 -07±+0A4 -0 4+0A4 -0-5+0-2 -2-7+P17

Increase in renin activity after acidification (%)20-5+4-3 -3-2+1-3 -2-0+1-4 -2-6±0-6 -70+±1-6

Total renin (active and inactive) was measured after dialysis to pH 2-8 and back to pH 7-5 toactivate inactive renin. Samples for active renin estimation were dialysed at pH 7*5. Results aremeans +s.E.M. (n = 10). Significance of differences: * P < 0-05 for paired comparison of acidified(total renin) and non-acidified (active renin) supernatant samples.

Thus, a decrease in buffer [Na+] was associated with increased release ofactive reninand a decrease in inactive renin.

In non-incubated control slices, extractable kidney renin content was 280-5 + 18-2 p-mole angiotensin I/hr. mg wet weight tissue (n = 40). Acidification led to an increasein renin activity of31-2+ 2-2 %. Renin release over a 90 min period by a group (n = 40)of control slices incubated in a buffer with [Na+] = 133 mm was 10-0+009 p-moleangiotensin I/hr. mg wet weight tissue. This represents 3-6% of the extractable reninactivity.The possibility existed of a direct agonist role on renin release for the choline

chloride used to replace sodium chloride in these experiments. This was ruled out inthe next series of experiments.

Relative effects ofcholine chloride, mannitol and Tris as a replacementfor sodium chloridein incubation buffers

In this series, the control buffer again had [Na+] = 133 mm. A series of buffers wasprepared with [Na+] = 23 mm using equimolar amounts of either choline chloride orTris or mannitol to replace the rest of the NaCl. In a further group no replacementwas made. This resulted in a substantial drop in osmotic strength ofthe buffer. Kidneycortex slices were incubated, as before, in each of these buffers, and results for reninsecretion are shown in Table 4.

In all four groups of slices incubated in a low sodium buffer ([Na+] = 23 mM) activerenin release was again much higher than in the control group ([Na+] = 133 mM).Results for incubations in which sodium chloride was replaced with choline chloride,

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Tris or mannitol were almost identical. In the group in which no replacement was madefor the missing sodium chloride, a substantial further increase in release of renin wasobserved. This is probably associated with osmotic rupture of the tissue as the mediabecame turbid during incubation.No increase in renin activity was found following acidification of any of the slice

supernatants containing the low sodium concentration again illustrating the failureof the slices to secrete inactive renin under these conditions.

TABLE 5. Active and inactive renin release by rabbit kidney cortex slices: effect of addingfurosemide to incubation medium

[Furosemide] (,ug/ml.) 0 5 10 20 40

Total renin (p-mole angiotensin I/hr. mg wet weight tissue)15.6+2.8* 14-5+3-4* 13.5+3.2* 15-4+3.8* 14-5+3-2*

Active renin (p-mole angiotensin I/hr. mg wet weight tissue)13-5+2-3 1282+2X9 11P9+2-9 13-5+3-5 12'8+3-0

Inactive renin (p-mole angiotensin I/hr. mg wet weight tissue)2-1+0-6 2-3+0-6 1P6+0-4 1-9+0-4 1P7+0-3

Increase in renin activity after acidification (%)15-3+2-2 19-0+3-6 12-9+2-1 14-3+2-3 13-3+±18

Total renin (active and inactive) was measured after dialysis to pH 2-8 and back to pH 7-5 toactivate inactive renin. Samples for active renin estimation were dialysed at pH 7-5. Results aremeans+ s.E.M. (n = 10). Significance of differences: * P < 0-05 for paired comparison of acidified(total renin) and non-acidified (active renin) supernatant samples.

We conclude that the use of choline chloride to replace sodium chloride in the buffersolutions for the previous experiments did not per se have any significant effect onthe results obtained.

Effect offurosemide on release of active and inactive reninsWe have proposed the existence of a sodium-sensitive mechanism controlling the

relative amounts of active and inactive renin in plasma. Part of the evidence tosupport this hypothesis is a study offurosemide diuresis in vivo in the rabbit (Richardset al. 1981 a). Changes in active renin secretion in response to furosemide are generallyconsidered to be secondary to altered tubular sodium and chloride re-absorption butcould conceivably be mediated by a direct effect of the diuretic on renin releasemechanisms. This possibility was therefore investigated.Kidney slices were prepared as before and incubated in the full Krebs-Ringer

bicarbonate buffer ([Na+] = 133 mM). Furosemide was added to the incubationmedium in concentrations up to 40 ,ug/ml. (122 gM). Results are shown in Table 5.Furosemide at all concentrations studied had no significant effect on release of eitheractive or inactive renin compared to controls. All slice supernatants produced asignificant increase in renin activity following acidification. The percentage increasein activity was not related to the concentration of furosemide in the buffer.Furosemide-induced changes in secretion of both forms of renin in vivo thereforeappear to be secondary to altered renal function.

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DISCUSSION

The hypothesis that the kidney has a sodium-sensitive receptor located at themacula densa which regulates the secretion ofrenin remains popular, but contentious.The basic conflict is over whether such a receptor responds to high or low sodiumload or concentration (Thurau, Schnermann, Nagel, Horster & Wahl, 1967; Vander,1967; Keeton & Campbell, 1980). In vitro studies using kidney slices have providedevidence in support of both high and low sodium as the trigger. Several studies usingrat kidneys (Oelkers, Molzahn, Samwer, Kreiser & Kutschke, 1970; Hammersen,Karsunky, Fischinger, Rosenthal & Taugner, 1971; Lyons & Churchill, 1975a) haveshown that renin release is directly related to medium [Na+]. However, an inverserelationship has been reported from studies using dog kidneys (Michelakis, 1971), ratrenal cortical cell suspensions (Lyons & Churchill, 1975b) and superfused glomeruli(Holdsworth, McLean, Morris, Dax & Johnston, 1976). The data reported here forrabbit kidneys support the latter concept in so far as one considers the secretion ofthe active form of renin. As medium [Na+] was reduced to 23 mm there was a 213 %increase in active renin release compared to controls ([Na+] = 133 mM), with aninverse linear relationship between the two variables (r2 = 096) (Table 3). However,a direct linear relationship (r2 = 0 92) was found between inactive renin release and[Na+]. Thus, in response to changes in [Na+] the alterations in release of active andinactive renin occur in opposite directions. This is in contrast to the in vitro effectsof isoprenaline which increased the secretion of active renin whilst inactive reninremained unchanged (Richards et al. 1981 b).

In most earlier reports only one form of renin was considered. Renin measurementsreported may have represented just active renin, or possibly a mixture of active andinactive renins, depending on the way the samples were processed prior to assay. Afurther complication with respect to interpretation of earlier data relates to thespecies used. In several species including human, dog, sheep and pig, inactive plasmarenins have been widely reported (Overturf et al. 1979; Leckie, 1981). Most in vitrowork on renin release has used the rat kidney as a model. There is disagreement overwhether the rat kidney does (Morris & Johnston, 1976; Vandongen, Poessee, Strang& Birkenhager, 1977), or does not (Lauritzen, Damsgaard, Rubin & Lauritzen, 1976;Nakane, Nakane, Corvol & Menard, 1980) secrete inactive renin at all.The data reported here are compatible with our earlier suggestion that the relative

amount of active and inactive renin secreted by the kidney, and present in plasma,is regulated by a sodium-sensitive mechanism (Richards et al. 1981 a).Plasma kallikrein (Sealey, Atlas, Laragh, Silverberg & Kaplan, 1979) and plasmin

(Osmond, Lo, Loh, Zingg & Hedlin, 1978) have been widely investigated as possibleactivating enzymes for inactive renin. However, two pieces of evidence from datareported here suggest to us that control of plasma levels of active and inactive reninsis primarily an intrarenal event. The first observation is that release of active andinactive renin from kidney slices changes in opposite directions in response to changesin medium [Na+]. A degree of independence between the secretary mechanisms forthe two renins is therefore indicated. Secondly, the renin released by control kidneyslices increased by 17-8 % (Table 3), 20-5 % (Table 4) and 15-3 % (Table 5), followingacidification of supernatant samples. This correlates well with values we have

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reported for normal rabbit plasma renin measurements (Grace et al. 1979b; Richardset al. 1979, 1981 a, b). Renin activity extracted from non-incubated kidney slicesincreased by 31P2 % following acidification. This is higher than we have observed forany rabbit plasma sample. The mixture ofrenins secreted does not then merely reflectwhat is stored in the kidney. This does not of course preclude the simultaneousexistence of an activating system for inactive renin in plasma but it is not essentialto postulate that such a mechanism exists in order to explain changes in the relativeamounts of the different renins in plasma.How might independent secretion control for active and inactive renins be

achieved? One possibility could be a sodium-sensitive step regulating activation ofinactive renin prior to secretion. An alternative would be the existence oftwo separatepools of renin within the kidney with independent secretion control. We cannotcurrently rule out either of these possibilities, but some studies using rat kidneysprovide support for the general concepts. Gillies & Morgan (1978) isolated individualglomeruli and their associated tubular tissue by microdissection. The active renincontent of these glomeruli was increased by salt depletion and by raised distal tubularsodium load. It was suggested that the increased renin concentration may haveresulted from activation of inactive renin, as the total renin content did not alter.A study of renin storage and release in the rat by de Senarclens, Pricam, Banichahi& Vallotton (1977) showed that acute reversal of sodium depletion was associatedwith increased kidney content of acid-activatable (inactive) renin. Simultaneously,secretary granules with crystalline cores appeared in the epitheloid cells. The authorsspeculated that these granules might contain inactive renin as a storage form.

It has been suggested that Cl- ions rather than Na+ ions are important in maculadensa mediated renin release (Kotchen et al. 1978). Such a proposal is not supportedby our experiments with rabbit kidney cortex slices. Replacement of sodium chloridein the medium with choline chloride or Tris or mannitol (Table 4) produced verysimilar results for both active and inactive renin release.The effect of furosemide on kidney slices was also studied. No significant changes

in either form of renin were found in the concentration range 0-0-12 mm. This wasa range which we considered to be relevant for comparison with dose ranges used invivo. Desaulles & Schwartz (1979) failed to record altered renin secretion at a doseof 0 15 mm but did report stimulation at does levels 10 and 50 times greater. Otherauthors have also reported that furosemide had no effect on renin release from kidneyslices (Corsini, Crossland & Bailie, 1974; Lyons & Churchill, 1975 a). Corsini et al.(1974) suggested that the reason for this was that the slices were already secretingmaximally. Our data are not in accord with this suggestion. Reducing medium [Na+]resulted in a much higher renin secretion rate than was achi-eved in any of the seriesusing furosemide. We conclude that the effects of furosemide in vivo on active andinactive renin release (Richards et al. 1981a) are secondary to changes in tubularfunction rather than a direct action of furosemide on any part of the juxtaglomerularapparatus.

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REFERENCES

CORSINI, W. A., CROSSLAN, K. L. & BAILLIE, M. D. (1974). Renin secretion by rat kidney slices invitro. Proc. Soc. exp. Biol. Med. 145, 403-406.

DESAULLES, E. & SCHWARTZ, J. (1979). A comparative study of the action of frusemide andmethyclothiazide on renin release by rat kidney slices and the interaction with indomethacin.Br. J. Pharmac. 65, 193-196.

GILLIES, A. & MORGAN, T. (1978). Renin content of individual juxtaglomerular apparatuses andthe effect of diet, changes in nephron flow rate and in vitro acidification on the renin content.Pfluger8 Arch. 375, 105-110.

GRACE, S. A., MUNDAY, K. A. & NOBLE, A. R. (1979a). Sodium potassium and water metabolismin the rabbit: the effect of sodium depletion and repletion. J. Physiol. 292, 407-420.

GRACE, S. A., MUNDAY, K. A., NOBLE, A. R. & RICHARDS, H. K. (1979b). Plasma active andinactive renin in the rabbit: effect of dietary sodium depletion and repletion. J. Physiol. 292,421-428.

HAMMERSEN, G., KARSUNKY, K.-P., FISCHINGER, J., ROSENTHAL, J. & TAUGNER, R. (1971).Influence of sodium concentration on the release of renin from kidney cortex slices and isolatedglomeruli. Pfluigers Arch. 328, 344-355.

HOLDSWORTH, S., MCLEAN, A., MORRIS, B. J., DAX, E. & JOHNSTON, C. I. (1976). Renin releasefrom isolated rat glomeruli. Clin. Sci. 51, 97-99s.

KEETON, T. K. & CAMPBELL, W. B. (1980). The pharmacologic alteration of renin release.Pharmac. Rev. 31, 81-227.

KOTCHEN, T. A., GALLA, J. H. & LUKE, R. G. (1978). Contribution of chloride to the inhibition ofplasma renin by sodium chloride in the rat. Kidney Int. 13, 201-207.

LAURITZEN, M., DAMSGAARD, J. J., RUBIN, I. & LAURITZEN, E. (1976). A comparison of theproperties of renin isolated from pig and rat kidney. Biochem. J. 155, 317-323.

LECKIE, B. J. (1981). Inactive renin: an attempt at a perspective. Clin. Sci. 60, 119-130.LYONS, H. J. & CHURCHILL, P. C. (1975a). The influence of ouabain on in vitro renin secretion and

intracellular sodium. Nephron 14, 442-450.LYONS, H. J. & CHURCHILL, P. C. (1975b). Renin secretion from rat renal cortical cell suspensions.Am. J. Phyeiol. 228, 1835-1839.

MICHELAKIS, A. M. (1971). The effect of sodium and calcium on renin release in vitro. Proc. Soc. exp.Biol. Med. 137, 833-836.

MORRIS, B. J. & JOHNSTON, C. I. (1976). Isolation of renin granules from rat kidney cortex andevidence for an inactive form of renin (prorenin) in granules and plasma. Endocrinology 98,1466-1474.

MUNDAY, K. A., NOBLE, A. R. & RICHARDS, H. K. (1980). In vitro release of active and inactiverenin by rabbit kidney cortex slices. J. Phy8iol. 301, 28P.

NAKANE, H., NAKANE, Y., CORVOL, P. & MENARD, J. (1980). Sodium balance and renin regulationin rats: role of intrinsic renal mechanisms. Kidney Int. 17, 607-614.

OELKERS, W., MOLZAHN, M., SAMWER, K. F., KREIsER, H. & KUTSCHKE, H. (1970). Renin releasefrom kidney slices. Pfluigers Arch. 321, 67-82.

OSMOND, D. H., Lo, E. K., LOH, A. Y., ZINGG, E. A. & HEDLIN, A. H. (1978). Kallikrein andplasmin as activators of inactive renin. Lancet ii, 1375-1376.

OVERTURF, M. L., DRUILHET, R. E. & KIRKENDALL, W. M. (1979). Renin: multiple forms andprohormones. Life Sci., Oxford 24, 1913-1924.

RICHARDS, H. K., GRACE, S. A., NOBLE, A. R. & MUNDAY, K. A. (1979). Inactive renin in rabbitplasma: effect of haemorrhage. Clin. Sci. 56, 105-108.

RICHARDS, H. K., LUSH, D. J., NOBLE, A. R. & MUNDAY, K. A. (1981 a). Inactive renin in rabbitplasma: effect of frusemide. Clin. Sci. 60, 393-398.

RICHARDS, H. K., NOBLE, A. R. & MUNDAY, K. A. (1981 b). Isoprenaline-induced secretion of activeand inactive renin in anaesthetised rabbits and by kidney cortex slices. Clin. Sci. 61, 679-684.

SEALEY, J. E., ATLAS, S. A., LARAGH, J. H., SILVERBERG, M. & KAPLAN, A. P. (1979). Initiation

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430 K. A. MUNDA Y, A. R. NOBLE AND H. K. RICHARDS

of plasma prorenin activation by Hageman factor-dependent conversion of plasma prekallikreinto kallikrein. Proc. natn. Acad. Sci. U.S.A. 76, 5914-5918.

DE SENARCLENS, C. F., PRICAM, C. E., BANICHAHI, F. D. & VALLOTTON, M. B. (1977). Reninsynthesis, storage and release in the rat: a morphological and biochemical study. Kidney Int.11, 161-169.

SKINNER, S. L. (1967). Improved assay methods for renin 'concentration' and 'activity' in humanplasma. Circulation Res. 20, 391-402.

STOCKIGT, J. R., COLLINS, R. D. & BIGLIERI, E. G. (1971). Determination of plasma reninconcentration by angiotensin I. immunoassay. Circulation Res. 28-29 supply . II), 175-189.

THURAU, K., SCHNERMANN, J., NAGEL, W., HORSTER, M. & WAHL, M. (1967). Composition oftubularfluid in the macula densa segment as a factor regulating the function of the juxtaglomerularapparatus. Circulation Res. 20-21 (suppl II), 79-89.

VANDER, A. J. (1967). Control of renin release. Phy8iol. Rev. 47, 359-382.VANDONGEN, R., POESSEE, M., STRANG, K. D. & BIRKENHAGER, W. H. (1977). Evidence that

'inactive' renin is produced outside the kidney of the rat. Clin. Sci. 53, 189-191.

kidney cortex slices: effect of sodium

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