Renin in thymus, gut, hindlimb, and adrenal of (mRen-2)27 and normal rats: secretion and content studies

Pei Rong, Jennifer L. Wilkinson-Berka, and Sandford L. Skinner

Department of Physiology, The University of Melbourne, Parkville, Victoria 3052, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thymic ablation and assay of organ renin revealed that one-third of the increasing plasma level of active renin after removal of kidneys and adrenals from Ren-2 rats originates from the thymus. Splanchnic arteriovenous difference and renin content indicate that gut can account for the remainder. Secretion of active renin from these sites correlated significantly with increasing plasma potassium. Prorenin was not secreted from these sites or from hindlimb in amounts sufficient to raise the plasma level, and yet plasma prorenin remained higher than active renin throughout the 12-h protocol. The source of prorenin that accounts for the high plasma prorenin phenotype of the intact conscious Ren-2 rat was not specifically identified. When sensitive assays were used, a low level of active renin secretion from thymus and gut was also apparent 12 h after removal of kidneys and adrenals in normal Sprague-Dawley rats, and plasma prorenin was at this time higher than active renin. A likely source of this extrarenal, extra-adrenal renin is the macrophage.

tissue renin; prorenin; hypertension; macrophage renin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

WE RECENTLY CONFIRMED the adrenal origin of increasing plasma prorenin after binephrectomy (BNx) in the (mRen-2)27 rat (9, 12) and showed that the parallel increase in plasma active renin was from a separate unidentified source (9). The secretion of both active renin and prorenin from these extrarenal tissues of the Ren-2 rat was stimulated by potassium, and similar but much weaker responses were detected in normal Sprague-Dawley rats (SDR). In earlier arteriovenous (a-v) difference studies we did detect active renin secretion from Ren-2 adrenal (8) and concluded incorrectly that the adrenals were probably the main source of the increasing level of plasma active renin as well as prorenin after BNx. But in more recent work it has become clear that the BNx-stimulated secretion of active renin is from another source in Ren-2 rats, from which secretion can rise to >= 5 times that from normal SDR kidneys and which is potassium sensitive (9). This level of secretion makes it likely that the tissue(s) of origin could be identified by conventional ablation and secretion methods. Furthermore, because the tissues in question are capable of processing and secreting active Ren-2 renin, they may also be able to express and secrete the natural rat renin at a lower level, and this might be demonstrable with sensitive assays in normal SDR. Such a finding could then have relevance to the pathophysiology of the renin-angiotensin system (RAS) in normal animals and humans. Expression of the renin gene, whether as the Ren-2 mouse transgene or as the normal structural renin gene of the rat, occurs in various organs (4, 14) from which fully processed active renin might be secreted. Accordingly, experimental protocols appropriate to the thymus, eye, ovary, gut, liver, and limb in addition to the adrenal were established in Ren-2 rats, and comparisons were made with SDR.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Animals

The rat colonies and general procedures have been described previously (8, 9). Homozygous (HMZ) female Ren-2 rats, 12-15 wk old, from which maintenance angiotensin-converting enzyme inhibitor (ACEI) treatment (lisinopril, 10 mg/ml in drinking water) had been withdrawn 3 wk earlier, were used in these studies. The routine of lisinopril withdrawal returned plasma active renin to normal for the Ren-2 rat (from the higher levels induced by lisinopril), and blood pressure rose but not to the levels of untreated Ren-2 rats (9). In conscious rats, systolic blood pressure was estimated by the tail-cuff method (model PE-300, Narco Bio-Systems). SDR were also adult females 12-15 wk old but were not given ACEI unless otherwise stated. Ren-2 breeding pairs and weanlings received a pellet diet containing 0.76% potassium and 0.25% sodium (GR2+ pellets, Clark King, Melbourne, Australia). SDR and spontaneously hypertensive rats (SHR) were fed a pellet diet containing only 0.0002% potassium and 0.4-0.6% sodium (Norco, Lismore, Australia). Rats were housed at 19-21°C with a 12:12-h light-dark cycle. Experiments were approved by the Animal Experimentation Ethics Committee of The University of Melbourne.

Organ Ablation Protocols

The experimental plan and rationale were the same as for our previous study, in which the profiles of plasma active renin and prorenin were followed in the pentobarbitone-anesthetized rat for 12 h (9). The standard protocol of BNx with biadrenalectomy (BNx+BADRx) was compared with previously thymectomized (THYMx) rats or with rats from which the ovaries were removed (BOVAx) and eyes enucleated (BOCULx). BNx+BADRx procedures have been previously described and include the administration of replacement maintenance glucocorticoid as dexamethasone (DEX) (9). Adult THYMx was performed 1 wk before the acute BNx+BADRx experiment, with animals under pentobarbitone anesthesia, following standard aseptic procedures. Ribs 3 and 4 were severed on the left side of the sternum, both lobes of the thymus were removed after ligation of the base, and the wound was rapidly closed. This procedure can cause pneumothorax that may lead to respiratory insufficiency and death within 6 h. The survival rate in our study was 67% (10 of 15). BOVAx was performed after BNx+BADRx by ligation and excision of the attachments of the ovaries to their oviducts. BOCULx was performed 10 h after BNx+ BADRx+BOVx by posterior ligation and enucleation of the orbit, with animals under continuing pentobarbitone anesthesia. During surgical procedures, carotid artery mean pressure was monitored continuously (Maclab/8, Analog Digital Instruments, Sydney, Australia).

Tissue Collection for Renin Content Assay from Ren-2 Rats Before and 12 h After BNx

Two groups of HMZ female Ren-2 rats 3 wk off ACEI were anesthetized with pentobarbitone. One group [conscious systolic blood pressure (BP) 147 ± 8 mmHg, n = 5] was perfused immediately via the inferior vena cava with PBS, and the other group (BP 142 ± 3 mmHg, n = 5) was perfused 12 h after BNx. Both adrenals, both lobes of thymus, and a section of duodenum (~200 mg) were excised, halved, and snap-frozen in appropriate buffers (8, 9) in preparation for active and prorenin assays.

Arteriovenous Difference Protocols

With animals under pentobarbitone anesthesia, hindlimb blood was collected from a femoral vein via a polyethylene catheter and portal and hepatic venous samples were collected by direct puncture with 25-gauge needles attached to flexible tubing. Rats were administered heparin (80 U/100 g body wt) as anticoagulant. In various protocols as presented in RESULTS, portal, hepatic, and femoral venous blood samples were collected simultaneously with arterial blood from anesthetized Ren-2 rats and SDR. In all protocols the blood removed was replaced immediately with the same volume of isotonic saline without the red blood cells. Each blood sample was aliquoted into two tubes, one for assay of active renin and one for total renin (see Tissue and Plasma Renin Assays). The volume of each sample was 0.2-0.4 ml. The maximum amount of blood removed for all purposes over the 12-h period was 2.5 ml.

Tissue and Plasma Renin Assays

The quantitative assays of active renin and total renin in rat plasma and tissues have been described in detail previously (1, 8, 9, 13). The assays adhere to enzyme kinetic principles in which active renin (or total renin after trypsin activation of prorenin) is allowed to react with an exogenous natural rat renin substrate under physiological conditions at pH 7.4 in the presence of protease inhibitors. At collection, samples for active renin were placed immediately in the protease inhibitor mixture, but for total renin no inhibitors were used before trypsin treatment. Generated angiotensin (ANG) I was estimated by RIA, and renin values are expressed as Goldblatt Units (GU), referable to the international hog renin standard distributed by The National Standards Laboratory, Holly Hill, London, UK. In interpreting the present results it has not been necessary to distinguish between Ren-2 renin and natural rat renin in the Ren-2 rats (2). It is assumed that in these specific protocols the high levels of active renin and prorenin in the transgenics are due to the transgene. Angiotensinogen (aogen) was estimated as ANG I released by an excess of mouse salivary gland renin, as previously described (13).

Statistical Analyses

Experiments were conducted with >= 5 rats to a group. Quantitative values are expressed as means ± SE, and significance was tested using one- or two-way ANOVA (Minitab 10.51), where appropriate, followed by the Newman-Keuls test. The Kruskal-Wallis test was used for nonparametric data. Student's t-test was used for paired data as indicated.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies in Ren-2 Rats

Effect of adult THYMx on plasma renin post-BNx+BADRx in female Ren-2 rats. One week after THYMx, systolic BP of conscious Ren-2 rats had fallen from 170 ± 6.9 to 143 ± 7.1 mmHg (n = 5, P < 0.01), but body weight (229 ± 9 to 234 ± 9 g) and conscious resting plasma renin levels were not affected. Active renin after THYMx was 0.14 ± 0.03 mGU/ml, and prorenin was 6.41 ± 1.9 mGU/ml vs. control active renin 0.16 ± 0.02 mGU/ml and prorenin 5.02 ± 0.93 mGU/ml. However, as seen in Fig. 1, the expected post-BNx+BADRx rise in plasma active renin was significantly attenuated throughout the 12-h period by prior THYMx. The control active renin profile in Fig. 1 is the reference data from our previous publication (9). This is appropriate, because these were matched, experimental groups with protocols conducted at approximately the same time. The mean area under the active renin curve was reduced to 63% of control, whereas prorenin remained unchanged at the level expected because BADRx removes its major source (9, 12). It should be noted that the concentration of prorenin remained at normal unstressed levels and was at all times higher than that of active renin, indicating continuing secretion from tissues other than the adrenals. This normal resting plasma level of prorenin in Ren-2 rats is greatly in excess of prorenin in normal rats (~20-fold; Ref. 9). The increasing level of aogen was little affected by THYMx (Fig. 1).


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Fig. 1.   Effect of thymectomy (THYMx) 1 wk before binephrectomy and biadrenalectomy (BNx+BADRx) on renin profiles (active renin, A; prorenin, B; angiotensinogen, C) in anesthetized female homozygous (HMZ) Ren-2 rats. DEX, dexamethasone (4 µg/kg sc); GU, Goldblatt Units. open circle , BNx+BADRx time control without THYMx. , THYMx+BNx+BADRx. Values are means ± SE, each n = 5. * P < 0.05, ** P < 0.001 vs. time-matched control by two-way ANOVA followed by Newman-Keuls test.

Relationship of plasma potassium to plasma renin post-THYMx+BNx+BADRx in female Ren-2 rats. Figure 2 shows a positive linear correlation between active renin and plasma potassium over the 12-h experimental period, with a significantly lower regression slope than for non-THYMx rats. For prorenin, no similar relationship or correlation existed, and the data were not different from BNx+BADRx without THYMx.


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Fig. 2.   Relationships of plasma renin to plasma potassium after THYMx+BNx+BADRx in female HMZ Ren-2 rats. For active renin, correlation is significant (r = 0.781, P < 0.001). Slope of regression with prior THYMx (, y = 1.248+0.378x) is lower than that for non-THYMx rats (open circle , y = -2.47+0.84x), shown as interrupted line (P < 0.05, confidence interval analysis).

BOVAx and BOCULx on plasma renin post-BNx+BADRx in female Ren-2 rats. Figure 3 shows the consequence of performing BNx+BADRx and then BOVAx on the renin profiles during the subsequent 12-h experimental period. As shown in Fig. 3, BOCULx was added to the protocol 10 h after the initial surgery in an additional group. These ablation procedures were performed to remove two further known sites of renin synthesis, but in each case the result was not a decrease but a significant increase in the active renin profile. Prorenin remained unchanged from the relatively constant normal level associated with BADRx.


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Fig. 3.   Effect of ovariectomy (BOVAx) with and without subsequent removal of eyes (BOCULx) in female HMZ Ren-2 rats on post-BNx+BADRx renin profiles (active renin, A; prorenin, B) compared with BNX+BADRx alone. open circle , BNx+BADRx (reference data from Fig. 1); , BNx+BADRx + BOVAx; black-triangle, BNx+BADRx + BOVAx + BOCULx. DEX, 4 µg/kg sc. ** P < 0.01; *** P < 0.001 vs. time-matched BNx+BADRx by two-way ANOVA followed by Newman-Keuls test.

Tissue renin content and organ weight before and after BNx in female Ren-2 rats. Figure 4 compares the tissue contents of renin and prorenin in adrenal, thymus, and duodenum before and 12 h after BNx. Both forms of renin were present in adrenal and were increased markedly post-BNx. Thymus and duodenum contained little if any prorenin, and these amounts were unchanged by BNx. However, the considerable amounts of active renin in thymus decreased and in gut increased with BNx. The total organ wet weights of two adrenals (65 ± 2 mg, n = 5) and thymus (368 ± 18 mg, n = 5) did not change during the 12-h period, but in a further group (n = 6) taken to 24-h post-BNx, thymic tissue had completely disappeared and only capsule remained. When the calculation was made from organ weights, the content of active renin in whole thymus before BNx (18.9 ± 4 mGU) was greater than for two adrenals (8.6 ± 1.2 mGU), but total renin contents (active + prorenin) were equivalent because of the prorenin in adrenal. After BNx (12 h), thymic active renin content fell to 12.3 ± 1 mGU, but in the adrenals both forms of renin increased (active renin from 8.6 ± 1.2 to 36.6 ± 2.3 mGU and prorenin from 12.1 ± 1.2 to 164 ± 7.1 mGU).


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Fig. 4.   Effect of 12-h BNx on active renin (A) and prorenin (B) plasma level and tissue renin content (per wet wt) in female HMZ Ren-2 rats (n = 5). * P < 0.05; *** P < 0.001 vs. non-BNx control (C) by two-way ANOVA followed by Newman-Keuls test.

Renin a-v differences across gut, liver, kidney, and leg in female Ren-2 rats. Secretion of active renin was detected from the splanchnic region (portal and hepatic veins) in sham-operated intact anesthetized female Ren-2 rats (Fig. 5). For kidney and leg, the sample numbers were too small for statistical testing in this protocol, but for leg, secretion was tested separately (see remainder of paragraph). The a-v differences for prorenin across gut, liver, kidney, and leg were not significant (Fig. 5). Although this experiment involved only a sham BNx procedure, it nevertheless resulted in a small rise in arterial plasma active renin and a considerable rise in prorenin. Thus arterial plasma prorenin rose to 40.4 ± 10.3 mGU/ml at 12 h postoperation compared with the conscious resting level of 11.1 ± 1.1 mGU/ml. In a further experiment, active renin secretion from the leg was examined specifically in 6 female Ren-2 rats 12 h after BNx+BADRx, when circulating levels were higher than for the sham experiment. In this experiment the femoral venous active renin level was 17% higher than arterial (venous 3.0 ± 0.6, arterial 2.54 ± 0.5 mGU/ml, P < 0.05, paired t-test). Renin a-v difference across the adrenal under these conditions is significant for both active renin and prorenin in Ren-2 rats; this observation has been published previously (8).


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Fig. 5.   Arterial (open bars) and venous (solid bars) concentrations of renin-angiotensin system (RAS) components (active renin, A; prorenin, B; angiotensinogen, C) across various regions in intact sham-operated (SNx) female HMZ Ren-2 rats 12 h after induction of anesthesia. Nos. in parentheses indicate nos. of rats from which paired arteriovenous (a-v) samples were collected. * P < 0.05, ** P < 0.01 by paired t-test.

Studies in SDR

Effect of adult THYMx on plasma renin post-BNx+BADRx in female SDR. Renin profiles after BNx+BADRx were studied 1 wk after THYMx in the same protocol as for Ren-2 rats, which was illustrated in Fig. 1. Figure 6 shows the expected decreasing renin profile in SDR. THYMx itself had no effect on the normal resting level of active renin before surgery, but at 12 h post-BNx+BADRx, active renin was significantly lower in the THYMx group (note log plot of Fig. 6). In this experiment, prorenin levels were only estimated between 6 and 10 h postsurgery and were unaffected by THYMx. At 8 and 10 h, plasma active renin concentration had fallen below prorenin, as noted in a previous study (9).


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Fig. 6.   Effect of adult THYMx 1 wk before BNx+BADRx in anesthetized female Sprague-Dawley rats (SDR). Active renin is plotted on a log scale for active renin (A), prorenin (B), and angiotensinogen (C). DEX was given at 4 µg/kg sc. , THYMx; open circle , time-matched non-THYMx control, as previously presented (9). Values significantly different between groups (each n = 5): ** P < 0. 01 with original analog data at each time interval by nonparametric Kruskal-Wallis test.

BOVAx and BOCULx on plasma renin post-BNx+BADRx in female SDR. This protocol was conducted in the same manner as for Ren-2 rats, as illustrated in Fig. 3. Neither BNx+BADRx+BOVAx nor BNx+BADRx+BOVAx with BOCULx 10 h later (each n = 6) had any additional effect on the decreasing profiles of active renin or prorenin compared with BNx+BADRx alone in SDR (data not shown).

Renin a-v differences across gut, liver, and leg in female SDR before and after ACEI. Figure 7 illustrates the arterial and splanchnic venous renin levels in untreated female SDR 12 h after BNx+BADRx. There is a significant secretion of active renin and removal of aogen by the gut. These differences were not apparent after the blood had passed through the liver. In a separate protocol, secretion of renin from the femoral bed was studied in SDR at 8, 10, and 12 h post-BNx, but only at 8 h was a small difference detected (venous 31.6 ± 3.7 vs. arterial 28.5 ± 2.4 µGU/ml, P < 0.05, paired t-test, n = 6). Femoral a-v difference was also studied after raising plasma renin by pretreatment with enalapril for 7 days (30 mg · kg-1 · day-1 in drinking water). Arterial plasma levels were 40-fold higher than normal (1 mGU/ml vs. 25 µGU/ml) at 12-h post-BNx+BADRx, but a significant difference in active renin was not detected across the leg. Prorenin was not assayed in this experiment.


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Fig. 7.   Arterial and venous concentrations of RAS components (active renin, A; prorenin, B; angiotensinogen, C) across gut and liver in anesthetized female SDR 12 h after BNx+BADRx. Values are arterial (open bars), portal venous (solid bars), and hepatic (gray bars) concentrations (means ± SE of 6/group). Significant differences, portal vs. carotid: * P < 0.05, ** P < 0.01; portal vs. hepatic: # P < 0.05 by paired t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These experiments were planned to elucidate whether, in the rat, particular organs or tissues other than kidney secrete locally synthesized renin into the circulation. Renin is widely expressed in the body, and it would be surprising if locally synthesized renin remained exclusively within its tissue of origin under all circumstances. It is clear that detectable amounts of fully processed active renin do enter the circulation in anephric humans and mice (3, 11), but the tissues of origin have not been identified. We used the transgenic Ren-2 rat as a first step in approaching this question because of its enhanced tissue renin expression. We then made comparisons with nontransgenics after the likely tissues were identified and the methodology refined.

Contrary to normal rats, the plasma levels of active renin and prorenin in Ren-2 rats increase after BNx, and the increasing prorenin but not active renin is abolished by BADRx (9, 11). Recognizing the relative abundance of renin mRNA in various Ren-2 tissues (14), together with our separate finding of active renin in thymic epithelial cells and macrophages (unpublished observations), we predicted that the thymus would be a likely source of active renin secretion, and this was confirmed here by an ablation study. However, THYMx in Ren-2 rats did not remove all of the increasing active renin in plasma after BNx, and a further source(s) exists that also displays a significant positive correlation with increasing plasma potassium. Although the correlation does not prove potassium as the stimulus in this circumstance, our earlier finding of a direct dependence of increasing active renin (and prorenin) on plasma potassium after BNx in Ren-2 rats (9) makes it likely that secretion of active renin from all tissue sources is potassium sensitive.

In seeking these further sources of active renin secretion in the BNx Ren-2 rat, we ablated the ovaries and eyes, both of which express renin (1, 14), but we did not find any reduction in the post-BNx levels as occurred after THYMx. Indeed, the renin levels increased significantly, which would be consistent with the more extensive acute surgical intervention in this experiment. Plasma potassium was not estimated in this latter protocol, but because in previous work even sham procedures raised potassium in Ren-2 rats (9), it remains likely that potassium is the common factor in this enhanced active renin response. It is important to note that, within this standard protocol, whereas plasma active renin rose with ablation of the ovaries and eyes and fell with prior THYMx, the prorenin level was unaffected. This increases our confidence in the replicate validity of the protocol.

Thymus contains considerable amounts of active renin in Ren-2 rats, and the total content is actually higher than for adrenal. This is also true for the normal SDR, as presented in a separate publication (unpublished observations). For the gut, we cannot make similar comparisons because our study of the entire gut is not yet complete. However, by extrapolation from the active renin content of a small duodenal sample, the entire gut might contain even more active renin than either adrenal or thymus. The effect of BNx on renin content at these sites was of interest, because it reveals information about feedback control of synthesis. Adrenal and duodenal levels of active renin both increased at 12 h post-BNx, but thymic levels were significantly depleted. It is known that the thymus undergoes acute involution with stress due to activation of the pituitary-adrenal axis (10), and in the present experiments by 24 h post-BNx, thymic tissue other than capsule had completely disappeared. At 12 h post-BNx, the decrease in thymic renin probably reflects the beginning of this process with the loss of renin-containing cellular elements, particularly macrophages.

Of particular interest is the fact that duodenum contains only active renin, indicating that, as for thymus, locally synthesized Ren-2 renin can be fully processed. It has been shown that the entire gastrointestinal tract of the Ren-2 rat expresses Ren-2 renin (14) and that Ren-1 message can be detected at low levels in normal SDR jejunum/ileum (14). Taken together with the present considerable a-v difference for active renin across the Ren-2 rat gut and liver, and with recognition of the high blood flow in the hepatic vein, it would seem a safe prediction that gut is the remaining main source of potassium-sensitive active renin secretion after BNx. The cellular origin of renin in gut has yet to be identified, but it is interesting to speculate that this might again be the macrophage. Renin protein and message have been identified in rodent macrophages (5-7), and we have separately confirmed this in SDR thymus (unpublished observations).

Concerning prorenin, although the tissue sources of the remaining plasma level after BNx+BADRx were not identified, several tissues can be tentatively excluded. Thymus and duodenum contain and secrete only active renin, whereas across the leg an a-v difference for prorenin was not detected at a time when plasma levels were elevated. Thus in the Ren-2 rat, tissue sources outside of kidney, adrenal, thymus, duodenum, limbs, and probably liver (no a-v difference) secrete prorenin at a rate that maintains the normal resting level. As in our previous study, contrary to active renin, prorenin secretion from these unknown sources is not sensitive to plasma potassium.

It is interesting to reflect on whether, in the Ren-2 rat, uptake from plasma might contribute to the renin content in some tissues and/or its subsequent secretion. However, the tissues of interest in our study (adrenal, thymus, and gut) can each express both the transgene and the natural gene and can process the renin to its active form. This does not deny the possibility of tissue uptake, but it makes it unnecessary as an explanation of the findings in these tissues. On the other hand, the unidentified post-BNx+BADRx source(s) of prorenin could have resulted from prior uptake, although the constancy of the plasma level would argue against this and is more consistent with continuing constitutive release.

After these observations and conclusions in the Ren-2 rat, we performed the more difficult experiments with the normal SDR and obtained evidence from both thymic ablation and gut a-v difference studies of phenomena similar to Ren-2 rats but at much lower levels of renin secretion. It is likely that, had we studied these events at times later than 12 h post-BNx, evidence for thymic and possibly gut secretion of active renin would not have been apparent. This follows from the fact that circulatory renin levels continue to fall beyond 12 h post-BNx, and the thymus undergoes involution. It is therefore possible that the remaining tissue sources of active renin are also depleted. Nevertheless, it can be concluded from our 12-h data that extrarenal and extra-adrenal tissues of the normal rat can secrete active renin in measurable amounts into the circulation. Whereas this is unlikely ever to have a conventional endocrine effect, should one source prove to be the tissue macrophage, it would be consistent with a monocyte-macrophage cell-mediated RAS endocrine system having important pathophysiological properties in situ.


    ACKNOWLEDGEMENTS

We thank Bronwyn Rees for expert technical assistance and for maintenance of the transgenic rat colony, which was established in 1993 following the gift of breeding pairs from Dr. Detlev and Ursula Ganten, Max-Delbruck Centrum (Berlin-Buch, Germany). Zeneca Ltd (UK) kindly donated the oral lisinopril used for the maintenance of the Ren-2 colony. We are indebted to Professor J. A. F. Miller, Walter and Eliza Hall Institute, The University of Melbourne, for instruction with the procedure for thymectomy in adult rats.


    FOOTNOTES

This work was conducted under a grant from the National Health and Medical Research Council of Australia and was sanctioned by the Animal Experimentation Ethics Committee of The University of Melbourne.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for correspondence and reprint requests: J. L. Wilkinson-Berka, Dept. of Physiology, The Univ. of Melbourne, Parkville 3052, Australia (E-mail: j.berka{at}physiology.unimelb.edu.au).

Received 7 January 1999; accepted in final form 26 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(4):E639-E646
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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