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An ontogenetic and interspecific study of the renin-angiotensin system in australian anuran amphibia
Camp. Biochem Physiol. Vol. 73A. No. 2. pp. 187 to 191. 1982 Printed in Great Britain.
0300-9629;82/[email protected]~0 0 1982 Pergamon Press Ltd
AN ONTOGENETIC AND INTERSPECIFIC STUDY OF THE RENIN-ANGIOTENSIN SYSTEM IN AUSTRALIAN ANURAN AMPHIBIA P. TAYLOR, G. C. SCROOP, M. J. TYLER and M. DAVIES Departments of Physiology and Zoology, University of Adelaide, Box 498 G.P.O., Adelaide, S.A. 5001, Australia (Received
19 January
1982)
Abstract-l. The relationships between renal renin content and the parameters of body weight, lifestyle, and stage of development have been examined in 26 species of Australian frogs. 2. A significant inverse relationship between body weight and renal renin content was found in both adult and juvenile frogs and tadpoles. 3. An apparent relationship with ontogenesis was found in one of two species studied where renal renin content fell with progressive development from tadpole to adult frog. 4. No relationship could be demonstrated between renal renin content and lifestyle.
INTRODUCTION
The presence of renin or of renin-like activity has been confirmed in representatives of all vertebrate classes, and its phylogenetic appearance coincides with the evolution of the kidney. In fact Sokabe (1974) has suggested that one of the trends in vertebrate evolution has been the localisation of renin-containing cells at or near the glomerulus. Renin is a proteolytic enzyme which initiates the formation of a series of small polypeptides termed angiotensin I. II and III. The latter two are regarded to have important roles in water and electrolyte homeostasis both by direct action on renal function and, indirectly, by stimulating aldosterone release (Freeman & Davis, 1979; Blair-West rf a/., 1980). In most species of the Amphibia there is a fundamental dichotomy in exposure to environmental factors influencing the achievement of electrolyte homeostasis. This is associated with the major ecological shift in the course of metamorphosis from a transitory, but entirely aquatic, stage (the tadpole) to the terrestrial or other non-aquatic lifestyle. It could be anticipated that, in general, electrolyte homeostasis would be far more difficult to achieve in the aquatic than in the terrestrial environment. unless high ambient temperatures or other factors accelerated water loss in the latter. In anuran Amphibia (frogs and toads) renin activity has been confirmed in the kidney and plasma of many species including Ram catesheiana (Sokabe et nl., 1972), R. pipiens (Capelli et cd.. 1970) and Bufo areII~W~ (Nolly & Fasciolo, 197 I ). However. each investigation has tended to use a single amphibian species as a subject, and we are unaware of any major comparative study. Similarly we have been unable to locate any studies on the renin-angiotensin system of tadpoles, nor any investigation of the nature of reninangiotensin levels through the rapid morphological changes from the tadpole through metamorphic climax to juvenile frog.
Here we present evidence of a major shift in reninangiotensin activity during metamorphosis, and also examine 24 species of adult Australian frogs seeking any association between the levels of renal renin and the systematic position or lifestyles of the respective species. MATERIAL AND
METHODS
Selection of species for study was based on their availability or association with other studies in the herpetology laboratory, but we attempted to include representatives of a wide range of genera and lifestyles. Fewer small species were included because of the great difficulty of tissue collection. The species studied, together with their systematic and ecological positions, are shown in Table 1. The impossibility of collecting sufficient blood for assay from individuals of the smaller species led to the decision to collect kidneys and measure renal renin content. After collection animals were maintained in vivaria in a controlled 30°C laboratory with a fixed photoperiod. Immediately prior to sampling the animals were either decerebrated and spinalised or killed by partial immersion in l-3% solutions of chloral hydrate. The whole animal was weighed; the kidneys were removed through a mid-ventral incision, freed of adhering connective tissue, lightly blotted, weighed and placed in a buffered solution for storage at -20°C. Renal extracts for estimation of renal renin content were prepared in the following manner. The kidneys from each individual (or in large animals 0.1 g kidney tissue) were placed in 2.0 ml phospho-saline buffer (pH 7.5) and homogenised using an ultra Tyrostat. The homogenate was spun for 30min at 80OOrpm at 4°C. The supernatant was transferred to a dialysis bag and dialysed at 4°C for 24 hr against a pH 3.3 glycine buffer and then for a further 24 hr against a pH 7.5 phosphate buffer. The dialysate was then transferred to capped storage tubes and frozen until used for assay. Renin in the extracts was not measured directly. Rather, the capacity of the extract to generate angiotensin I when incubated with sheep substrate was measured, and renal renin activity was expressed in terms of angiotensin I generated per gram of kidney. For the incubation step two tubes were set up for each renal extract, containing 400 ~1 187
IXX
P. TAYLOUVTal. Table
Family.
genus and species
I. Angiotensin
I levels in adult frogs
Body wt ?&SD (g)
Kidney wt X + SD
42.01 & 10.95 IO.31 rt 3.43 35.12 32.99 + 5.22 9:55 3.07 k 1.77 16.04 + 0.41 3.80 2.22 * 0.95 I.06 & 0.23 26.98 ) 5.05 0.78 k 0.11 16.86 8.88 5.21 2688
0.222 + 0.078 0.045 + 0.018 0.135 0.135 * 0.053 0.056 0.018 f 0.015 0.064 + 0.014 0.015 0.009 _ir 0.003 0.007 + 0.002 0.104 + 0.012 0.003 f 0.000 0.077 0.043 0.020 0.337
0.679 + 0.296 0.209 ? 0.016 0.259 0.477 & O.! 1I 0.182 0.077 f 0.024 0.267 k 0.031 0.227 0.180 I_ 0.046 0.113 + 0.066 0.457 + 0.276 0.127 + 0.015 0.282 0.408 0.218 0.745
4.381 + 2.799 3.25 5.965 + 3.684 4.330 * 1.443 15.66 18.626 f 8.088 16.825 + 3.430 4.590 f 3.210 43.80 f 7.255 3.69 9.49 IO.90 2.21
IO.54 i: 5.46
0.050 & 0.028
0.213 + 0.195
3.750 2 1.768
8.55 4.44 * I.11 14.85 ?I 1.06 20.38 19.46 _+ 4.22 0.94 z!z0.16 11.51 I 7.79
0.023 0.024 * 0.01 0.050 ‘I 0.011 0.046 0.114 + 0.048 0.004 rt 0.002 0.023 + 0.005
0.244 0.201 f 0.09 0.214 f 0.022 0.163 0.350 & 0.064 0.125 f 0.063 0.354 + 0.065
10.84 7.842 ) 3.429 4.430 f 1.443 3.54 3.267 + 0.770 43.993 + 38.534 16.608 rf: 5.315
ng Aljhr Y&SD
(g)
assay buffer. 100 1118-hydroxy qninolone, 400 ,uI sheep substrate and 100~1 renal extract. They were incubated for I hr. one at 37’ C, the other at 4°C. The 37’C reaction was stopped by cooling to 4’C. The angiotensin I content of each incubate was determined immediately after incubation trsing the r~~dioimmunoassay method of Johnston et t/l. ( 1969). Results obtained from the adult frogs led us to investigate the renin levels in earlier stages of the life cycle, in juveniles and tadpoles. A limited number of specimens of several species was avaihrbie for study, using animals reared from spawn in the laboratory. Tadpoles were staged accordmg to Gosner (1960). The earliest stage used was stage 27. but most were between stages 40 and 42, that is, they were undergoing metamorphosis. The same procedure
Table 2. Angiotensin
ng AI/g kidney!hr YkSD
n (adult frogs)
Habit
I
Fossorial Fossorial Fossorial Arboreal Arboreal Scansorial Terrestrial Arboreal Arboreal Arboreal Terrestrial Scansorial Scansorial Arboreal Arboreal Arborcal
2
Terrestrial
3.267 + 1.239 5.405 & 2.645
1.92
2 2 2 1 1
Fossorial Terrestrial Fossorial Fossorial Fossorial Terrestrial Aquatic
for tissue collection and renin estimation niles and tadpoles as for adults.
was used for juve-
RESULTS The results of the investigation of adult frogs demonstrated the existence of high variability in AI production expressed as nanograms per hour. particularly when the production is corrected for individual kidney weights (Table I). Although there is di~~~ulty in classifying frogs in terms of their basic lifestyle (a species fossorial for much of the year may be aquatic during the breeding season). we are unable to detect any distinct association between renal renin content
I levels in tadpoles
and juvenile
frogs
Body wt ?+SD (g)
Kidney wt S & SD
ng AI!hr
(E)
.r f SD
ng AI:g kidney!hr S i: SD
n
0.876 _+ 0.4701 0.459 + 0. I24
0.005 + 0.0023 0.003 + 0.0004
0.15 li: 0.0783 0.156 f 0.0301
34.50 i: 15.9425 64.447 If: 19.2465
14 7
1.198 + 0.3972
0.004 f 0.0015
0.095 + 0.0609
23.386 k 14.8835
9
0.007 0.006 0.004 + 0.0016 0.003 f 0.001
0.058 0.202 0. I55 t 0.0909 0.082 + 0.0142
8.92 36.73 45.282 i 32.4176 29.14 i_ 9.1508
I 1 6 3
0.40
1.29 0.755 f 0.3427 0.651 _e 0.03
Renin-angiotensin 100
1 . so
x
\i
Body
Weight
(g)
Fig. 1. The relationship between renal renin content (ng Al/g kidney/hr) and body weight (g) in 68 adult (0) and 11 juvenile
(x
) frogs.
and the predominant environmental parameters. Similarly there is no constant trend in families. nor in those genera in which more than one species have been examined. The results obtained from each individual adult (and the juveniles listed in Table 2) have been pooled and plotted in Fig. 1 to test the existence of any relationship between frog body weight and renal renin. The highest levels of renal renin was found in frogs weighing less than 0.5 g and declined progressively with increasing body weight. Data correlations were attempted with eight curve fitting models (Griffith, 1978) and statistically significant correlations were obtained with each (Table 3). The highest r value was obtained with a power curve function. Two individuals that are clearly exceptional within the last category are both members of the aquatic species Rheohatruchus silus. Figure 2 plots the renal renin content of individual tadpoles against their body weight. As in the study of adult and juvenile frogs plotted in Fig. 1, smaller individuals tended to exhibit a higher level of renal renin, and the largest individuals clearly had the lowest levels. Data correlations with the eight curve fitting models again yielded significant relationships with highest r value being obtained from the logarithmic function curve (Table 3). However, when results of all tadpoles of all species were plotted in an ontogenetic sequence (Fig. 3) no association was apparent. When ontogenetic data are examined for a single species, there is on occasion an apparent relationship between the developmental stage attained by the individual and the renal renin content (for example in
in Australian
frogs
P.
TAYLOR
IOO-
IOO-
so-
90.
SO
et al.
.
1
.
.
..
70
60 !
.
SO-
70-
.. 1
.
..
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; 50i 2 e j z ,CT40 k 1
. l
.
.
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.
.
. .
.
.
.
. .
.
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. ..
:*
.
20.
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.
. l
f
20
.
:
IO-
.
IO i
.
0I 0
20
IO
Body
Weight
(g)
tug. 2. The relationship between renal renin content (ng A1 g kldne) hr) and body weight (g) in 31 tadpoles.
OJ
.
r
25
30 Developmental
35 Stage
40
45
Number
Fig. 3. The relationship between renal renin content (ng Al/g kidney.hr) and developmental stage in 23 tadpoles.
/.;rr~~,irr~,.orj~~l~,,t~c,r~,\is in Fig. 4). Hovvcvcr. such a trend Hence in such a respect our juveniles may vary in the 1101 so readil! detectable in the case of C~~/O~LIIILI extent to which they retain remnant. larval. renal ~,uI~/I/I(,,\(Fig. 5). in which the level peaks following characters of a structural nature. and this might contribute to the observed variability in renin levels at c,~lnplction of metamorphic climax. these latter stages. Certainly it demonstrates a closer association between renin and the larval pronephros DISCL SSION than with the adult mesonephros. but we infer nothing further. The most interesting result from this study is demThe fact that the species of frogs which attain small onstt-ation of the existence in frogs of a developmental progression from higher levels of renal renin in early body weight (~2.5 g) have high renal renin levels stages of the lift cycle to substantially lower levels in comparable to juvenile frogs of large species, suggests the adult. The data for L. ~c,o?j~r/~rn~t,n.sis and C. lon~,I,Y\ dilfcr only shghtly in the attainment of peak 1:~ILIc\: thou provide an interesting parallel with JUVENILES TADPOLES ADULTS (7) (31 (2) i ~wlt~ ~huined from a number of mammalian species 00, . \I hrch h;~vc high lcvcls of plasma renin in the fetus. a pxh~n~ of plasma renin activity at birth, followed by aI* . ;I gudual reduction to adult levels (Fleischman et al., 1975). Similarly the mammalian fetus is bathed in a .. liquid environment until shortly before birth. Developmental stages are defined principally upon the naturc of changes in metamorphosis as these affect cxtcrnal features. In the latter stages internal cllxn~c‘~ include radical alteration to the kidney from II\ lc~r\;~l to adult condition. but Fox (1963) reports I 11.11 V)IIIC renal changes are not complete at terminaII~~II 01 external metamorphosis. For example de25 30 Developmental Stage Number ZL~IIC’I at~trn of the pronephros in European Runu few ,vjrffrff~ commcnccs at a stage equivalent to 41 of Fig. 4. The relationship between renal renin content (~nl\ncr (1960). and continues until after metamor(ng Al:g kidney./hr) in tadpoles. juveniles and adults of phosis (in apecimcns that here we term “juveniles”). Litoritr ~~otjlilur~l~n.sis. is
Renin-angiotensin TADPOLES (14)
JUVENILES 5)
(
in Australian
ADULTS (41
I 9I
frogs
A~kno~c~l~,dyut,~~,nt.\~~ We arc grateful for the linancial by the Australian Kidney Fountl:ction and National Heart Foundation of Australia. WC v,~\h IO thank Julie Marker and Chris Miller for technical ,I\~,Iante.
support provided
.
I
.
REFKRKYCES
.. .. 20
i
,’
l
. 2;
3b
i5
Developmental Stage
: 4b
Number
Fig. 5. The relationship between renal (ngAl:g kidney;hr) in tadpoles. juveniles C.vclortr,lrr loi1~/ipc.s.
i
d
renin content and adults of
that such small species are exhibiting paedomorphosis: namely that they retain in the adult stage a juvenile characteristic. In the three species involved (Litoria fu//ux, L. meiriana and Ranideh signijka) there are other clearly paedomorphic traits in cranial osteology, such as persistence of a large frontoparietal fontanelle in the adult. Hence these species may be viewed as precocious adults, and their high renal renin levels possibly attributable to an arrest of the development of the kidney. Unfortunately the choice of method of measuring renal renin was dictated by animal size and by the limited number of frogs available. It does not permit extrapolation to circulating levels of renin and renin turnover rates, which may be of greater physiological significance. It is also possible that, according to their habitat and habits, species may differ in their sensitivity to AI1 which is regarded as the major effector substance of the renin-angiotensin system. Flinn (1977) found a differential sensitivity to the effect of AI1 on drinking behaviour in birds, according to their natural habitat.
BLAIR-WESTJ. R., COGHLALJ. P.. DI NIO~ D. A.. L.rI L). I’. W.. HARDY K. J.. Scoc~cx~s B. A. & WKIGHI R. D. (1980) A dose-response comparison of the actions of angiotonsin II and angiotensin III in sheep. J. Ent/~vrir~r~/. X7. 409-4 17. CAPELLI J. P.. WESSON L. G. & AI~ONII G. F. (14701 ,\ phylogenetic study of the renin angiotensin \>\tcm .I,~I. J. PhjWl. 218, 1171 I1 78. FLEISCHMAN A. R.. 0~1:s G. K.. bsrr IN M. b.. C‘,\1 I K. _I. & CHEZ R. A. (1975) Plasma renin activit! during o\lnc pregnancy. Am J. Ph!~aiol. 228, 901 Y04. FLINN R. B. (1977) Climate. thirst and renin. ,-l,,1cvil,tr,l
Clinic,trl cmd Cli,?ltrfo/o(/i~trI.il.w~c~itrttonT,,[rrl.\i,c.ti~~il\. Vol. 89, pp. 130 140. 19th Annual Meeting Colorado
Springs U.S.A. FOX H. (1963) The amphibian pronephros. Q. Rec. Hioi. 38(l), 1.25. FRMMANR. H. & DAVIS J. 0. (19791 Phq~iological act~vn\ of angiotensin II on the kidney. Fcrlr~ PRK. I,cl/~r .IuI. Sots r\-p. Bid. 2276-2279. GOSNER K. L. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Iic,r/lc’to/o@ 16, 183.-190. GRIFFITH E. N. (1978) Program number 208013B. Data lit to eight curves: PPX59 software catalog: Texas Instruments Incorporated. JOHNSTONC. I., MENDELSOHN F. & CASLFY D. ( 19691 Plasma renin determination employing a radioimmunoassay of angiotensin I. Proc,. Au.st. Sock. ,Mvt/. RL~.T.2. 27 I. NOLLY H. L. & FASC‘IOLOJ. C. (1971) The renin anglotcnsm system In Bufo trrrncmrrn and Bufo ,~~,~~,~‘,~~‘,~~~~,~. (‘oI)I/I. Biochcw. Physiol. 39, 823-83 I, SOKAIW H. (1974) Phylogeny of the renal cni.ct\ of ,~ngiotensin. Kit//ley 1!1r. 6. 263-27 I. SOKABI H.. NISHIMI~KA. H.. KA\~AHI K.. Tr \biohl S. & ARAI T. (1972) Plasma renin activity in varying hydrated states in the bullfrog. 4n1. J. Ph~xiol. 222, 132 116.
0300-9629;82/[email protected]~0 0 1982 Pergamon Press Ltd
AN ONTOGENETIC AND INTERSPECIFIC STUDY OF THE RENIN-ANGIOTENSIN SYSTEM IN AUSTRALIAN ANURAN AMPHIBIA P. TAYLOR, G. C. SCROOP, M. J. TYLER and M. DAVIES Departments of Physiology and Zoology, University of Adelaide, Box 498 G.P.O., Adelaide, S.A. 5001, Australia (Received
19 January
1982)
Abstract-l. The relationships between renal renin content and the parameters of body weight, lifestyle, and stage of development have been examined in 26 species of Australian frogs. 2. A significant inverse relationship between body weight and renal renin content was found in both adult and juvenile frogs and tadpoles. 3. An apparent relationship with ontogenesis was found in one of two species studied where renal renin content fell with progressive development from tadpole to adult frog. 4. No relationship could be demonstrated between renal renin content and lifestyle.
INTRODUCTION
The presence of renin or of renin-like activity has been confirmed in representatives of all vertebrate classes, and its phylogenetic appearance coincides with the evolution of the kidney. In fact Sokabe (1974) has suggested that one of the trends in vertebrate evolution has been the localisation of renin-containing cells at or near the glomerulus. Renin is a proteolytic enzyme which initiates the formation of a series of small polypeptides termed angiotensin I. II and III. The latter two are regarded to have important roles in water and electrolyte homeostasis both by direct action on renal function and, indirectly, by stimulating aldosterone release (Freeman & Davis, 1979; Blair-West rf a/., 1980). In most species of the Amphibia there is a fundamental dichotomy in exposure to environmental factors influencing the achievement of electrolyte homeostasis. This is associated with the major ecological shift in the course of metamorphosis from a transitory, but entirely aquatic, stage (the tadpole) to the terrestrial or other non-aquatic lifestyle. It could be anticipated that, in general, electrolyte homeostasis would be far more difficult to achieve in the aquatic than in the terrestrial environment. unless high ambient temperatures or other factors accelerated water loss in the latter. In anuran Amphibia (frogs and toads) renin activity has been confirmed in the kidney and plasma of many species including Ram catesheiana (Sokabe et nl., 1972), R. pipiens (Capelli et cd.. 1970) and Bufo areII~W~ (Nolly & Fasciolo, 197 I ). However. each investigation has tended to use a single amphibian species as a subject, and we are unaware of any major comparative study. Similarly we have been unable to locate any studies on the renin-angiotensin system of tadpoles, nor any investigation of the nature of reninangiotensin levels through the rapid morphological changes from the tadpole through metamorphic climax to juvenile frog.
Here we present evidence of a major shift in reninangiotensin activity during metamorphosis, and also examine 24 species of adult Australian frogs seeking any association between the levels of renal renin and the systematic position or lifestyles of the respective species. MATERIAL AND
METHODS
Selection of species for study was based on their availability or association with other studies in the herpetology laboratory, but we attempted to include representatives of a wide range of genera and lifestyles. Fewer small species were included because of the great difficulty of tissue collection. The species studied, together with their systematic and ecological positions, are shown in Table 1. The impossibility of collecting sufficient blood for assay from individuals of the smaller species led to the decision to collect kidneys and measure renal renin content. After collection animals were maintained in vivaria in a controlled 30°C laboratory with a fixed photoperiod. Immediately prior to sampling the animals were either decerebrated and spinalised or killed by partial immersion in l-3% solutions of chloral hydrate. The whole animal was weighed; the kidneys were removed through a mid-ventral incision, freed of adhering connective tissue, lightly blotted, weighed and placed in a buffered solution for storage at -20°C. Renal extracts for estimation of renal renin content were prepared in the following manner. The kidneys from each individual (or in large animals 0.1 g kidney tissue) were placed in 2.0 ml phospho-saline buffer (pH 7.5) and homogenised using an ultra Tyrostat. The homogenate was spun for 30min at 80OOrpm at 4°C. The supernatant was transferred to a dialysis bag and dialysed at 4°C for 24 hr against a pH 3.3 glycine buffer and then for a further 24 hr against a pH 7.5 phosphate buffer. The dialysate was then transferred to capped storage tubes and frozen until used for assay. Renin in the extracts was not measured directly. Rather, the capacity of the extract to generate angiotensin I when incubated with sheep substrate was measured, and renal renin activity was expressed in terms of angiotensin I generated per gram of kidney. For the incubation step two tubes were set up for each renal extract, containing 400 ~1 187
IXX
P. TAYLOUVTal. Table
Family.
genus and species
I. Angiotensin
I levels in adult frogs
Body wt ?&SD (g)
Kidney wt X + SD
42.01 & 10.95 IO.31 rt 3.43 35.12 32.99 + 5.22 9:55 3.07 k 1.77 16.04 + 0.41 3.80 2.22 * 0.95 I.06 & 0.23 26.98 ) 5.05 0.78 k 0.11 16.86 8.88 5.21 2688
0.222 + 0.078 0.045 + 0.018 0.135 0.135 * 0.053 0.056 0.018 f 0.015 0.064 + 0.014 0.015 0.009 _ir 0.003 0.007 + 0.002 0.104 + 0.012 0.003 f 0.000 0.077 0.043 0.020 0.337
0.679 + 0.296 0.209 ? 0.016 0.259 0.477 & O.! 1I 0.182 0.077 f 0.024 0.267 k 0.031 0.227 0.180 I_ 0.046 0.113 + 0.066 0.457 + 0.276 0.127 + 0.015 0.282 0.408 0.218 0.745
4.381 + 2.799 3.25 5.965 + 3.684 4.330 * 1.443 15.66 18.626 f 8.088 16.825 + 3.430 4.590 f 3.210 43.80 f 7.255 3.69 9.49 IO.90 2.21
IO.54 i: 5.46
0.050 & 0.028
0.213 + 0.195
3.750 2 1.768
8.55 4.44 * I.11 14.85 ?I 1.06 20.38 19.46 _+ 4.22 0.94 z!z0.16 11.51 I 7.79
0.023 0.024 * 0.01 0.050 ‘I 0.011 0.046 0.114 + 0.048 0.004 rt 0.002 0.023 + 0.005
0.244 0.201 f 0.09 0.214 f 0.022 0.163 0.350 & 0.064 0.125 f 0.063 0.354 + 0.065
10.84 7.842 ) 3.429 4.430 f 1.443 3.54 3.267 + 0.770 43.993 + 38.534 16.608 rf: 5.315
ng Aljhr Y&SD
(g)
assay buffer. 100 1118-hydroxy qninolone, 400 ,uI sheep substrate and 100~1 renal extract. They were incubated for I hr. one at 37’ C, the other at 4°C. The 37’C reaction was stopped by cooling to 4’C. The angiotensin I content of each incubate was determined immediately after incubation trsing the r~~dioimmunoassay method of Johnston et t/l. ( 1969). Results obtained from the adult frogs led us to investigate the renin levels in earlier stages of the life cycle, in juveniles and tadpoles. A limited number of specimens of several species was avaihrbie for study, using animals reared from spawn in the laboratory. Tadpoles were staged accordmg to Gosner (1960). The earliest stage used was stage 27. but most were between stages 40 and 42, that is, they were undergoing metamorphosis. The same procedure
Table 2. Angiotensin
ng AI/g kidney!hr YkSD
n (adult frogs)
Habit
I
Fossorial Fossorial Fossorial Arboreal Arboreal Scansorial Terrestrial Arboreal Arboreal Arboreal Terrestrial Scansorial Scansorial Arboreal Arboreal Arborcal
2
Terrestrial
3.267 + 1.239 5.405 & 2.645
1.92
2 2 2 1 1
Fossorial Terrestrial Fossorial Fossorial Fossorial Terrestrial Aquatic
for tissue collection and renin estimation niles and tadpoles as for adults.
was used for juve-
RESULTS The results of the investigation of adult frogs demonstrated the existence of high variability in AI production expressed as nanograms per hour. particularly when the production is corrected for individual kidney weights (Table I). Although there is di~~~ulty in classifying frogs in terms of their basic lifestyle (a species fossorial for much of the year may be aquatic during the breeding season). we are unable to detect any distinct association between renal renin content
I levels in tadpoles
and juvenile
frogs
Body wt ?+SD (g)
Kidney wt S & SD
ng AI!hr
(E)
.r f SD
ng AI:g kidney!hr S i: SD
n
0.876 _+ 0.4701 0.459 + 0. I24
0.005 + 0.0023 0.003 + 0.0004
0.15 li: 0.0783 0.156 f 0.0301
34.50 i: 15.9425 64.447 If: 19.2465
14 7
1.198 + 0.3972
0.004 f 0.0015
0.095 + 0.0609
23.386 k 14.8835
9
0.007 0.006 0.004 + 0.0016 0.003 f 0.001
0.058 0.202 0. I55 t 0.0909 0.082 + 0.0142
8.92 36.73 45.282 i 32.4176 29.14 i_ 9.1508
I 1 6 3
0.40
1.29 0.755 f 0.3427 0.651 _e 0.03
Renin-angiotensin 100
1 . so
x
\i
Body
Weight
(g)
Fig. 1. The relationship between renal renin content (ng Al/g kidney/hr) and body weight (g) in 68 adult (0) and 11 juvenile
(x
) frogs.
and the predominant environmental parameters. Similarly there is no constant trend in families. nor in those genera in which more than one species have been examined. The results obtained from each individual adult (and the juveniles listed in Table 2) have been pooled and plotted in Fig. 1 to test the existence of any relationship between frog body weight and renal renin. The highest levels of renal renin was found in frogs weighing less than 0.5 g and declined progressively with increasing body weight. Data correlations were attempted with eight curve fitting models (Griffith, 1978) and statistically significant correlations were obtained with each (Table 3). The highest r value was obtained with a power curve function. Two individuals that are clearly exceptional within the last category are both members of the aquatic species Rheohatruchus silus. Figure 2 plots the renal renin content of individual tadpoles against their body weight. As in the study of adult and juvenile frogs plotted in Fig. 1, smaller individuals tended to exhibit a higher level of renal renin, and the largest individuals clearly had the lowest levels. Data correlations with the eight curve fitting models again yielded significant relationships with highest r value being obtained from the logarithmic function curve (Table 3). However, when results of all tadpoles of all species were plotted in an ontogenetic sequence (Fig. 3) no association was apparent. When ontogenetic data are examined for a single species, there is on occasion an apparent relationship between the developmental stage attained by the individual and the renal renin content (for example in
in Australian
frogs
P.
TAYLOR
IOO-
IOO-
so-
90.
SO
et al.
.
1
.
.
..
70
60 !
.
SO-
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.. 1
.
..
.
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. l
.
.
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. .
.
.
.
. .
.
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. ..
:*
.
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.
. l
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.
:
IO-
.
IO i
.
0I 0
20
IO
Body
Weight
(g)
tug. 2. The relationship between renal renin content (ng A1 g kldne) hr) and body weight (g) in 31 tadpoles.
OJ
.
r
25
30 Developmental
35 Stage
40
45
Number
Fig. 3. The relationship between renal renin content (ng Al/g kidney.hr) and developmental stage in 23 tadpoles.
/.;rr~~,irr~,.orj~~l~,,t~c,r~,\is in Fig. 4). Hovvcvcr. such a trend Hence in such a respect our juveniles may vary in the 1101 so readil! detectable in the case of C~~/O~LIIILI extent to which they retain remnant. larval. renal ~,uI~/I/I(,,\(Fig. 5). in which the level peaks following characters of a structural nature. and this might contribute to the observed variability in renin levels at c,~lnplction of metamorphic climax. these latter stages. Certainly it demonstrates a closer association between renin and the larval pronephros DISCL SSION than with the adult mesonephros. but we infer nothing further. The most interesting result from this study is demThe fact that the species of frogs which attain small onstt-ation of the existence in frogs of a developmental progression from higher levels of renal renin in early body weight (~2.5 g) have high renal renin levels stages of the lift cycle to substantially lower levels in comparable to juvenile frogs of large species, suggests the adult. The data for L. ~c,o?j~r/~rn~t,n.sis and C. lon~,I,Y\ dilfcr only shghtly in the attainment of peak 1:~ILIc\: thou provide an interesting parallel with JUVENILES TADPOLES ADULTS (7) (31 (2) i ~wlt~ ~huined from a number of mammalian species 00, . \I hrch h;~vc high lcvcls of plasma renin in the fetus. a pxh~n~ of plasma renin activity at birth, followed by aI* . ;I gudual reduction to adult levels (Fleischman et al., 1975). Similarly the mammalian fetus is bathed in a .. liquid environment until shortly before birth. Developmental stages are defined principally upon the naturc of changes in metamorphosis as these affect cxtcrnal features. In the latter stages internal cllxn~c‘~ include radical alteration to the kidney from II\ lc~r\;~l to adult condition. but Fox (1963) reports I 11.11 V)IIIC renal changes are not complete at terminaII~~II 01 external metamorphosis. For example de25 30 Developmental Stage Number ZL~IIC’I at~trn of the pronephros in European Runu few ,vjrffrff~ commcnccs at a stage equivalent to 41 of Fig. 4. The relationship between renal renin content (~nl\ncr (1960). and continues until after metamor(ng Al:g kidney./hr) in tadpoles. juveniles and adults of phosis (in apecimcns that here we term “juveniles”). Litoritr ~~otjlilur~l~n.sis. is
Renin-angiotensin TADPOLES (14)
JUVENILES 5)
(
in Australian
ADULTS (41
I 9I
frogs
A~kno~c~l~,dyut,~~,nt.\~~ We arc grateful for the linancial by the Australian Kidney Fountl:ction and National Heart Foundation of Australia. WC v,~\h IO thank Julie Marker and Chris Miller for technical ,I\~,Iante.
support provided
.
I
.
REFKRKYCES
.. .. 20
i
,’
l
. 2;
3b
i5
Developmental Stage
: 4b
Number
Fig. 5. The relationship between renal (ngAl:g kidney;hr) in tadpoles. juveniles C.vclortr,lrr loi1~/ipc.s.
i
d
renin content and adults of
that such small species are exhibiting paedomorphosis: namely that they retain in the adult stage a juvenile characteristic. In the three species involved (Litoria fu//ux, L. meiriana and Ranideh signijka) there are other clearly paedomorphic traits in cranial osteology, such as persistence of a large frontoparietal fontanelle in the adult. Hence these species may be viewed as precocious adults, and their high renal renin levels possibly attributable to an arrest of the development of the kidney. Unfortunately the choice of method of measuring renal renin was dictated by animal size and by the limited number of frogs available. It does not permit extrapolation to circulating levels of renin and renin turnover rates, which may be of greater physiological significance. It is also possible that, according to their habitat and habits, species may differ in their sensitivity to AI1 which is regarded as the major effector substance of the renin-angiotensin system. Flinn (1977) found a differential sensitivity to the effect of AI1 on drinking behaviour in birds, according to their natural habitat.
BLAIR-WESTJ. R., COGHLALJ. P.. DI NIO~ D. A.. L.rI L). I’. W.. HARDY K. J.. Scoc~cx~s B. A. & WKIGHI R. D. (1980) A dose-response comparison of the actions of angiotonsin II and angiotensin III in sheep. J. Ent/~vrir~r~/. X7. 409-4 17. CAPELLI J. P.. WESSON L. G. & AI~ONII G. F. (14701 ,\ phylogenetic study of the renin angiotensin \>\tcm .I,~I. J. PhjWl. 218, 1171 I1 78. FLEISCHMAN A. R.. 0~1:s G. K.. bsrr IN M. b.. C‘,\1 I K. _I. & CHEZ R. A. (1975) Plasma renin activit! during o\lnc pregnancy. Am J. Ph!~aiol. 228, 901 Y04. FLINN R. B. (1977) Climate. thirst and renin. ,-l,,1cvil,tr,l
Clinic,trl cmd Cli,?ltrfo/o(/i~trI.il.w~c~itrttonT,,[rrl.\i,c.ti~~il\. Vol. 89, pp. 130 140. 19th Annual Meeting Colorado
Springs U.S.A. FOX H. (1963) The amphibian pronephros. Q. Rec. Hioi. 38(l), 1.25. FRMMANR. H. & DAVIS J. 0. (19791 Phq~iological act~vn\ of angiotensin II on the kidney. Fcrlr~ PRK. I,cl/~r .IuI. Sots r\-p. Bid. 2276-2279. GOSNER K. L. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Iic,r/lc’to/o@ 16, 183.-190. GRIFFITH E. N. (1978) Program number 208013B. Data lit to eight curves: PPX59 software catalog: Texas Instruments Incorporated. JOHNSTONC. I., MENDELSOHN F. & CASLFY D. ( 19691 Plasma renin determination employing a radioimmunoassay of angiotensin I. Proc,. Au.st. Sock. ,Mvt/. RL~.T.2. 27 I. NOLLY H. L. & FASC‘IOLOJ. C. (1971) The renin anglotcnsm system In Bufo trrrncmrrn and Bufo ,~~,~~,~‘,~~‘,~~~~,~. (‘oI)I/I. Biochcw. Physiol. 39, 823-83 I, SOKAIW H. (1974) Phylogeny of the renal cni.ct\ of ,~ngiotensin. Kit//ley 1!1r. 6. 263-27 I. SOKABI H.. NISHIMI~KA. H.. KA\~AHI K.. Tr \biohl S. & ARAI T. (1972) Plasma renin activity in varying hydrated states in the bullfrog. 4n1. J. Ph~xiol. 222, 132 116.