BritishJournal .fHaematology, 1975,29, 129.
The Subcellular Localization of the Erythrostimulant Factors Present in the Kidney and Spleen of the Rabbit ELVIOGIOVANNINI AND GABRIELLA ROSI
Istituto di Biologia Generale, Perugia University (Received 25 March 1974; accepted f o r publication 5 July 1974) SUMMARY. The subcellular localization of erythrostimulaiit factors contained in the kidney and spleen of the rabbit have been studied. This was done by subjecting homogenates to fractionation by centrifugation and assessing the erythrostimulant capacity of the hypotonic extracts prepared from the various fractions thus obtained. The results of these studies show that the erythrostiinulant factors are present in the light mitochondrial and microsomal fractions of the normal kidney. In conditions of anaemia they are to be found not only in the above-mentioned fractions but also in tlie mitochondria1 fraction. By contrast, in the spleen such factors are present only in the mitochondrial fraction. It is likely that the different localization of the erythrostimulant factors in the kidney and in the spleen of the rabbit may be closely connected with the different fimctions performed by these organs in the humoral regulation of erythropoiesis.
A study has been made of the distribution of tlie erythrostimulant factors in the various subcellular fractions of tlie kidney of normal and bled rabbits. These studies were extended also to the spleen, in which the eryathrostimulalit factors of renal origin are at least partly deposited (Liotti &- Giovannini, 1g7oa). Kuratowska (1965) reported an erythropoietic factor in the nuclear and mitochondria1 fractions of the kidney of anaemic or CoCl, treated rabbits, whereas in the normal animal this factor is present only in the nuclear fraction. Contrera et al (1966) have pointed out the presence of a renal erythropoictic factor (REF) in the light mitochondria1 fraction of rat kidney in conditions of alioxic hypoxia. Wong et al (1968) and Cantor et a1 (1969), using a similar technique, have noted the existence of the REF not only in the light niitochoiidrial fraction, but also in the microsomal fraction, in the kidney of the rat. MATERIAL AND METHOD Studies have been carried out on the variations in the reticulocyte levels in the peripheral blood, produced in normal rabbits by administering hypotonic extracts of the subcellular fractions obtained from the kidneys of normal or bled animals and from the spleen of normal animals. Correspondence: Dr Elvio Giovannini, Istituto di Biologia Gencrale, Facolti di Mcdicina e Chirurgia dell' Universiti, Via del Giochetto, 06100 Perugia, Italy.
129
Elvio Giovannini and Gabriella Rosi
130
The extracts were given by two intraperitoneal injections of 20 ml each on successive 20 ml of blood per kg of body weight were taken off by cardiac puncture. Before the treatment the rabbits were subjected, on alternate days, to 10 successive counts of the reticulocyte concentration and of the number of erythrocytes. Only those animals having erythrocyte levels of between 5 800 ooo and 6 200 ooo erythrocyteslpl. and with reticulocyte levels of between 1.5% and 3%, such as to give a standard error of not more than 0.1,relative to the 10 values measured, were used. The reticulocyte count was repeated on the second, third, fourth and sixth days after the beginning of the treatment. The reticulocyte level was determined on the basis of 2000 erythrocytes. The organs removed from the animals sacrificed were homogenized in a Potter homogenizer, using a 0.25 M saccharose solution in the proportion of 9 ml/g of tissue. The homogenates were then subjected to fractionated centrifugation, using a Christ Omega I1 refrigerated centrifuge operating at a temperature of 5OC. The fracticnation scheme shown below was followed. It is similar to those reported by Contrera et al (1966) and by Cantor et al (1969).
days. In the experiments in which the donors were bled,
Centrijiigal acceleration (g) (outer radius)
Fraction Nuclear Mitochondria1 Light mitochondrial* Microsomal Soluble
2500
Time (min) IS
7.500
IS
I 5000
20
35000
60
* The light mitrochondrial fraction of rat kidney, obtained by Cantor et a1 (1969) by centrifuging at 21 ooo g for 20 min, contains a few mitochondria, some lysosomes and most of the De Duve peroxysomes or ‘microbodies’, with which the REF would be particularly associated. Each of the various subcellular fractions of corpuscular structure was resuspended in bidistilled water in the proportion of 3 ml/g of tissue; the whole was then frozen at - 20°C and brought back to 5°C after 24 h, in order to obtain complete disintegration of the subcellular particles so as to favour the solubilization in the hypotonic medium of the erythrostimulant factors present in them. This material was then centrifuged at 20 ooo rpm (42 930 g) for 15 min. The supernatant thus obtained was diluted to 40 ml by the addition of bidistilled water and made isotonic with NaC1. The soluble fraction obtained from the ultra-centrifugation of a spleen homogenate was brought to a volume of 40 ml by the addition of a 0.25 M saccharose solution before being used in the treatment. In view of the fact that the volume of the soluble fraction obtained from the ultracentrifugation of the homogenate of a pair of kidneys was incompatible with the treatment of a single animal, its volume was reduced to 40 nil by concentration in a vacuum, after dialysis against bidistilled water in order to remove the large quantity of saccharose dissolved in it.
Subcellular Localization ofErythrostimulant Factors
131
The various materials to be injected, after the addition of antibiotics, were kept at - 20°C up to the moment of use. In all, three series of experiments were performed. In the first series, a study was made of the distribution of the erythrostimulant factors in the various subcellular fractions of the kidney of normal rabbits. In the second series the experiments were repeated by using the kidneys of rabbits bled 18 h previously, the time at which they are found to be most rich in erythrostimulant factors (Liotti & Giovannini, 197ob). In the third series the experiments were performed by using the spleens of normal rabbits. In each series of experiments, six donors were used. From each donor, five subcellular fractions were obtained (nuclear, mitochondrial, light mitochondrial, microsonial and soluble fractions), taken from the kidneys or from the spleen, according to the case. The extract of each fraction was given to a recipient animal. Consequently, in each series of experiments, 30 animals were used, five recipients for each of the six donors.
RESULTS
The experiments of the first series (see Table I), carried out by using the various subcellular fractions of normal kidneys, show that in this organ the crythrostimulant factors are coiicentrated in the light mitochondrial and microsomal fractions. Treatment with extracts of the light mitochondrial (Group I-C) and microsonial (Group I-D) fractions produces a highly significant increase in the number of reticulocytes circulating in the peripheral blood of the recipient animals. O n the other hand, the administration of extracts of the nuclear (Group I-A), mitochondrial (Group I-B) and soluble (Group I-E) fractions does not result in any significant variation in the reticulocyte count. The experiments of the second series (see Table II), performed by treating with extracts of the various subcellular fractions of the kidneys removed from bled animals, showed that the anaemic kidney contains erythrostimulant factors not only in the liglit mitochondrial and microsomal fractions, but also in the mitochondrial fraction. Treatment with extracts of the mitochondrial (Group 11-B), liglit mitochondrial (Group 11-C) and microsomal (Group 11-D) fractions produces a highly significant increase in the number of circulating reticulocytes, whereas the administration of extracts prepared from the nuclear (Group 11-A) and soluble (Group 11-E) fractions does not produce any variation in the reticulocyte count. The third series of experiments (see Table III), performed by using the various subcellular fractions of the spleens removed from normal donors, show that the erythrostiniulant factors in this organ are concentrated in the mitochondrial fraction. Treatment carried out with the extracts prepared from this fraction (Group 111-B) produces a highly significant increase in the number of the circulating reticulocytes. Administration of extracts prepared from the other subcellular fractions (Groups 111-A, 111-C, 111-D and 111-E) does not result in any significant variation in the reticulocyte count. In animals treated with extracts of the various active fractions increases are also observed in the number of erythrocytes.
Initial After 2 days After 3 days After 4 days After 6 days
Time
2.3 +_0.1065 2.35+0.1996 2.z3+_0.1308 2.38&0.1641 2.27+0.1358
M+ SE t t t t = 0.221 = 0.4152 = 0.4089 = 0.174
Comparison with initial value
Group I-A
t 2.2 k0.1506 t 2.4 k0.0816 t 2.3 +0.1506 t
2.33 k0.1283 2 . 5 50.1826
M k SE
I
t = 6.743* t = 8.303* t = 5.692* t = 1.762
MkSE
-+ 0.1176 3.4550.1335 3.7 +0.1291 3.IS&O.IOs7 2.55 0.1232 2.25
Comparison with initial value
I
Group I-C
* Highly significant difference.
= 0.1516
= 0.7618 = 0.6572 = 0.4603
Comparison with initial value
Group I-B
I
2.45 2 0.1478
M_+SE
Cornpariron w i f h initial value
Group I-D
2.35fO.1727 2.45k0.1544 2.4 +O.Ig49 2.4 +o.2145 2.45f0.211
M+ SE
f = 0.4317 t = 0.192 t = 0.1816 t z 0.3668
Comparison with initial value
Croup I-E
count is expressed as the reticulocytes/Ioo ewthrocytes. standard error (Mk , (Means+ . .~ SE). Comparison between the means of the initial and successive values by means of Student's t (degrees of freedom = IO).)
TABLE I. Variations with time in the reticulocyte counts ofrabbits treated with hypotonic extracts of the following fractions: nuclear (Group I-A), mitochondria1 (Group LB),light mitochondria1 (Group I-C), microsoma1 (Group I-D) or soluble (Group I-E) obtained from the kidneys of normal donors. The reticulocyte
n
c
0
Q
5
h,
W
Initial After 2 days After 3 days After 4 days After 6 days
Time
I
2.6 ko.0966 2.7 k0.1673 2.75k0.1384 2.8 +0.1291 2.9 20.1633
M+ SE t = 0.5176 t = 0.8885 t = 1.24 t = 1.581
Comparison with initial value
Group II-A
I
I
MfSE
I
M+ SE
Mf SE 2.45 f0.133 S t = 6.106* 2.5550.1176 t = 6.852* 2.65+0.1088 t = 4.427* 2.7S&O.I3IO t = 0.6238 2.6550.1232
Comparison with initial value
Group II-D
2.5 k0.1438 t = 10.47* 3.8 50.1570 t = I O . 2 2 * 4.0Sf0.1747 t = 6.866* 3.4 fo.1438 t = 1.203 2.65+0.1928
Comparison with initial value
Group II-C
2.65k0.1118 4.1550.0847 t = 11.61* 4.1 to.0816 4.4 kO.1125 t = II.82* 4.3Sf0.1232 3.7 50.1461 t = 6.314* 3.75fo.1147 2.7 k0.1183 t = 0.9333 2.8550.1232 2.55+0.1088
M+ SE
Comparison with initial value
Group II-B
t = 0.5619 f = 1.161 t = 1.604 t = 1.101
with initial value
Comparison
TABLE 11. Variations with time in the reticulocyte counts of rabbits treated with hypotonic extracts of the following fractions: nuclear (Group 11-A), mitochondrial (Group 11-B),light mitochondria1 (Group 11-C), microsomal (Group 11-D) or soluble (Group 11-E)obtained from the kidneys of donors bled 18 h before. The reticulocyte count is expressed as the reticulocytes/roo erythrocytes. (Meansf standard error ( M t SE). Comparison between the means of the initial and successive values by means of Student’s t (degrees of freedom = ID).)
2 CP
w
W
H
5
3
%. 2
trj T
g2.
F
5
Initial After 2 days After 3 days After 4 days After 6 days
Time
I
2.45fo.1945 2.35f0.1857 2.4 +0.205 2.45k0.1522
2.3 f0.177
Mf SE t t t t
I M f SE
t t t t
2.4
2.3 k0.1693 2.3 k0.139
= 0.1808 = 0.5571 = 0.5937
+0.22~1
2.35 k0.1875 2.45 _+ 0.2012
M + SE 2.5
*0.1211
I
I
t t t t
= 0.2526 = 0.7851 = 1.054
= 1.168
Comparison with initial
Group III-E
t = 0.3635 2.7 +O.IZII t = 0.1707 2.5~+0.1565 t = 0.1979 2.65f0.1478 t = 0.2142 2.7 ko.1461
Comparison with initial
Group III-D
= 0.8262
* Highly significant difference.
+
M k SE
Comparison with initial value
Group III-C
2.3 f0.188 t = 7.246* 2.5 f0.1525 t = 9.346* 2.35 0.2029 t = 6.41* 2.45 f 0.1928 t = 2.016 2.45f0.1688
Comparison with initial value
GYOUP III-B
2.4 kO.1342 3.8 fo.139 = 0.1949 4.3 +0.1528 = 0.3693 3.85f0.1821 = 0.6425 2.85k0.1784 = 0.5703
Comparison with initial value
Groidp III-A
drial (Group 111-B),light mitochondria1 (Group 111-C), microsomal (Group 111-D) or soluble (Group 111-E) obtained from the spleens of normal donors. The reticulocyte count is expressed as reticulocytes/Ioo erythrocytes. (Meansf standard error ( M t SE). Comparison between the means of the initial and successive values by means of Student's t (degrees of freedom = IO).)
TABLE 111. Variations with time in the reticulocyte counts ofrabbits treated with hypotonic extracts of the following fractions: nuclear (Group 111-A), mitochon-
H
CP
c 5 *8.
3-
2.
F;'
23
E. 0
9
w P
Subcellular Localization of Erythrostimulant Factors
I3
s
DISCUSSION The localization of the erythrostimulant factors in the light mitochondrial and microsonial fractions of the kidney of normal rabbits appears to be in agreement with that observed by Cantor et al(1969) in the kidney of normal rats. The presence of the erythrostimulant factors in the microsomal fraction of the kidney can be explained by the facts that (I) the kidney is the organ that produces these substances and ( 2 ) the microsomal fraction is rich in material coming from the ergastoplasmatic structures in which the factors are produced. The erythrostimulant factors probably pass into the light mitochondrial fraction before being released into the circulation. If the production of these factors increases considerably, they probably also accumulate in the mitochondria1 fraction. This could explain the appearance of the crythrostiinulant factors in this fraction in the anaemic kidney. The fact that the erythrostimulant substances contained in the spleen are present only in the mitochondrial fraction strengthens the theory that this fraction is the site in which, at cellular level, these factors tend to accumulate. In fact, the spleen merely acts as a deposit of erythrostimulant factors of renal origin (Liotti & Giovannini, 197oa). It is unlikely that the presence of the erythrostimulant factors at the level of the mitochondrial fraction can be due simply to adsorption on the surface of the mitochondria themselves. This is suggested by the fact that the mitochondrial factions reveal their activity only if disintegrated through freezing and being kept in hypotonic medium in order to favour the solubilization of the factors contained in them. Moreover, the mitochondrial fractions preservc their activity even when they are subjected to repeated washings before the hypotonic treatment. It is probable, therefore, that the presence of the erythrostimulant factors in the mitochondrial fraction of the anaemic kidney and the normal spleen is related to very precise mechanisms that regulate the release of these factors on demand. REFERENCES CANTOR,L.N., ZANJANI,E.D., WONG, K.K. & GORDON,A.S. (1969) The renal erythropoietic factor (REF). IX. Its subcellular distribution.
Proceedings of the Society4r Experimental Biology and Medicine, 130, 950. J.F., GORDON,A S . & WEINTRAUB, A.H. CONTRERA, (1966) Extraction of an erythropoietin-producing factor from a particulate fraction of rat kidney. Blood, 28, 330. KURATOWSKA, Z. (1965) O n the subcellular localization of the erythropoietic renal factor. Bulletin de I’AcadCmie Polonaise des Sciences, 13, 385.
E. (197oa) Studi c ricerche LIOTTI,F.S. & GIOVANNINI, in tema di rapporti tra rene e milza nel controllo dell’eritropoiesi. Haematologica, 55, 681. E. (1g7ob) Attivith LIOTTI, F.S. & GIOVANNINI, eritrostimolante di estratti renali di coniglio normale o salassato. Rivista di Biologia, 63, 3 0 1 . WONG, K.K., ZANJANI,E.D., COOPER,G.W. & GORDON,A.S. (1968) The renal erythropoietic factor. V. Studies on its purification. Proceedings of the Society for Experimental Biology and Medicine, 128,67.
The Subcellular Localization of the Erythrostimulant Factors Present in the Kidney and Spleen of the Rabbit ELVIOGIOVANNINI AND GABRIELLA ROSI
Istituto di Biologia Generale, Perugia University (Received 25 March 1974; accepted f o r publication 5 July 1974) SUMMARY. The subcellular localization of erythrostimulaiit factors contained in the kidney and spleen of the rabbit have been studied. This was done by subjecting homogenates to fractionation by centrifugation and assessing the erythrostimulant capacity of the hypotonic extracts prepared from the various fractions thus obtained. The results of these studies show that the erythrostiinulant factors are present in the light mitochondrial and microsomal fractions of the normal kidney. In conditions of anaemia they are to be found not only in the above-mentioned fractions but also in tlie mitochondria1 fraction. By contrast, in the spleen such factors are present only in the mitochondrial fraction. It is likely that the different localization of the erythrostimulant factors in the kidney and in the spleen of the rabbit may be closely connected with the different fimctions performed by these organs in the humoral regulation of erythropoiesis.
A study has been made of the distribution of tlie erythrostimulant factors in the various subcellular fractions of tlie kidney of normal and bled rabbits. These studies were extended also to the spleen, in which the eryathrostimulalit factors of renal origin are at least partly deposited (Liotti &- Giovannini, 1g7oa). Kuratowska (1965) reported an erythropoietic factor in the nuclear and mitochondria1 fractions of the kidney of anaemic or CoCl, treated rabbits, whereas in the normal animal this factor is present only in the nuclear fraction. Contrera et al (1966) have pointed out the presence of a renal erythropoictic factor (REF) in the light mitochondria1 fraction of rat kidney in conditions of alioxic hypoxia. Wong et al (1968) and Cantor et a1 (1969), using a similar technique, have noted the existence of the REF not only in the light niitochoiidrial fraction, but also in the microsomal fraction, in the kidney of the rat. MATERIAL AND METHOD Studies have been carried out on the variations in the reticulocyte levels in the peripheral blood, produced in normal rabbits by administering hypotonic extracts of the subcellular fractions obtained from the kidneys of normal or bled animals and from the spleen of normal animals. Correspondence: Dr Elvio Giovannini, Istituto di Biologia Gencrale, Facolti di Mcdicina e Chirurgia dell' Universiti, Via del Giochetto, 06100 Perugia, Italy.
129
Elvio Giovannini and Gabriella Rosi
130
The extracts were given by two intraperitoneal injections of 20 ml each on successive 20 ml of blood per kg of body weight were taken off by cardiac puncture. Before the treatment the rabbits were subjected, on alternate days, to 10 successive counts of the reticulocyte concentration and of the number of erythrocytes. Only those animals having erythrocyte levels of between 5 800 ooo and 6 200 ooo erythrocyteslpl. and with reticulocyte levels of between 1.5% and 3%, such as to give a standard error of not more than 0.1,relative to the 10 values measured, were used. The reticulocyte count was repeated on the second, third, fourth and sixth days after the beginning of the treatment. The reticulocyte level was determined on the basis of 2000 erythrocytes. The organs removed from the animals sacrificed were homogenized in a Potter homogenizer, using a 0.25 M saccharose solution in the proportion of 9 ml/g of tissue. The homogenates were then subjected to fractionated centrifugation, using a Christ Omega I1 refrigerated centrifuge operating at a temperature of 5OC. The fracticnation scheme shown below was followed. It is similar to those reported by Contrera et al (1966) and by Cantor et al (1969).
days. In the experiments in which the donors were bled,
Centrijiigal acceleration (g) (outer radius)
Fraction Nuclear Mitochondria1 Light mitochondrial* Microsomal Soluble
2500
Time (min) IS
7.500
IS
I 5000
20
35000
60
* The light mitrochondrial fraction of rat kidney, obtained by Cantor et a1 (1969) by centrifuging at 21 ooo g for 20 min, contains a few mitochondria, some lysosomes and most of the De Duve peroxysomes or ‘microbodies’, with which the REF would be particularly associated. Each of the various subcellular fractions of corpuscular structure was resuspended in bidistilled water in the proportion of 3 ml/g of tissue; the whole was then frozen at - 20°C and brought back to 5°C after 24 h, in order to obtain complete disintegration of the subcellular particles so as to favour the solubilization in the hypotonic medium of the erythrostimulant factors present in them. This material was then centrifuged at 20 ooo rpm (42 930 g) for 15 min. The supernatant thus obtained was diluted to 40 ml by the addition of bidistilled water and made isotonic with NaC1. The soluble fraction obtained from the ultra-centrifugation of a spleen homogenate was brought to a volume of 40 ml by the addition of a 0.25 M saccharose solution before being used in the treatment. In view of the fact that the volume of the soluble fraction obtained from the ultracentrifugation of the homogenate of a pair of kidneys was incompatible with the treatment of a single animal, its volume was reduced to 40 nil by concentration in a vacuum, after dialysis against bidistilled water in order to remove the large quantity of saccharose dissolved in it.
Subcellular Localization ofErythrostimulant Factors
131
The various materials to be injected, after the addition of antibiotics, were kept at - 20°C up to the moment of use. In all, three series of experiments were performed. In the first series, a study was made of the distribution of the erythrostimulant factors in the various subcellular fractions of the kidney of normal rabbits. In the second series the experiments were repeated by using the kidneys of rabbits bled 18 h previously, the time at which they are found to be most rich in erythrostimulant factors (Liotti & Giovannini, 197ob). In the third series the experiments were performed by using the spleens of normal rabbits. In each series of experiments, six donors were used. From each donor, five subcellular fractions were obtained (nuclear, mitochondrial, light mitochondrial, microsonial and soluble fractions), taken from the kidneys or from the spleen, according to the case. The extract of each fraction was given to a recipient animal. Consequently, in each series of experiments, 30 animals were used, five recipients for each of the six donors.
RESULTS
The experiments of the first series (see Table I), carried out by using the various subcellular fractions of normal kidneys, show that in this organ the crythrostimulant factors are coiicentrated in the light mitochondrial and microsomal fractions. Treatment with extracts of the light mitochondrial (Group I-C) and microsonial (Group I-D) fractions produces a highly significant increase in the number of reticulocytes circulating in the peripheral blood of the recipient animals. O n the other hand, the administration of extracts of the nuclear (Group I-A), mitochondrial (Group I-B) and soluble (Group I-E) fractions does not result in any significant variation in the reticulocyte count. The experiments of the second series (see Table II), performed by treating with extracts of the various subcellular fractions of the kidneys removed from bled animals, showed that the anaemic kidney contains erythrostimulant factors not only in the liglit mitochondrial and microsomal fractions, but also in the mitochondrial fraction. Treatment with extracts of the mitochondrial (Group 11-B), liglit mitochondrial (Group 11-C) and microsomal (Group 11-D) fractions produces a highly significant increase in the number of circulating reticulocytes, whereas the administration of extracts prepared from the nuclear (Group 11-A) and soluble (Group 11-E) fractions does not produce any variation in the reticulocyte count. The third series of experiments (see Table III), performed by using the various subcellular fractions of the spleens removed from normal donors, show that the erythrostiniulant factors in this organ are concentrated in the mitochondrial fraction. Treatment carried out with the extracts prepared from this fraction (Group 111-B) produces a highly significant increase in the number of the circulating reticulocytes. Administration of extracts prepared from the other subcellular fractions (Groups 111-A, 111-C, 111-D and 111-E) does not result in any significant variation in the reticulocyte count. In animals treated with extracts of the various active fractions increases are also observed in the number of erythrocytes.
Initial After 2 days After 3 days After 4 days After 6 days
Time
2.3 +_0.1065 2.35+0.1996 2.z3+_0.1308 2.38&0.1641 2.27+0.1358
M+ SE t t t t = 0.221 = 0.4152 = 0.4089 = 0.174
Comparison with initial value
Group I-A
t 2.2 k0.1506 t 2.4 k0.0816 t 2.3 +0.1506 t
2.33 k0.1283 2 . 5 50.1826
M k SE
I
t = 6.743* t = 8.303* t = 5.692* t = 1.762
MkSE
-+ 0.1176 3.4550.1335 3.7 +0.1291 3.IS&O.IOs7 2.55 0.1232 2.25
Comparison with initial value
I
Group I-C
* Highly significant difference.
= 0.1516
= 0.7618 = 0.6572 = 0.4603
Comparison with initial value
Group I-B
I
2.45 2 0.1478
M_+SE
Cornpariron w i f h initial value
Group I-D
2.35fO.1727 2.45k0.1544 2.4 +O.Ig49 2.4 +o.2145 2.45f0.211
M+ SE
f = 0.4317 t = 0.192 t = 0.1816 t z 0.3668
Comparison with initial value
Croup I-E
count is expressed as the reticulocytes/Ioo ewthrocytes. standard error (Mk , (Means+ . .~ SE). Comparison between the means of the initial and successive values by means of Student's t (degrees of freedom = IO).)
TABLE I. Variations with time in the reticulocyte counts ofrabbits treated with hypotonic extracts of the following fractions: nuclear (Group I-A), mitochondria1 (Group LB),light mitochondria1 (Group I-C), microsoma1 (Group I-D) or soluble (Group I-E) obtained from the kidneys of normal donors. The reticulocyte
n
c
0
Q
5
h,
W
Initial After 2 days After 3 days After 4 days After 6 days
Time
I
2.6 ko.0966 2.7 k0.1673 2.75k0.1384 2.8 +0.1291 2.9 20.1633
M+ SE t = 0.5176 t = 0.8885 t = 1.24 t = 1.581
Comparison with initial value
Group II-A
I
I
MfSE
I
M+ SE
Mf SE 2.45 f0.133 S t = 6.106* 2.5550.1176 t = 6.852* 2.65+0.1088 t = 4.427* 2.7S&O.I3IO t = 0.6238 2.6550.1232
Comparison with initial value
Group II-D
2.5 k0.1438 t = 10.47* 3.8 50.1570 t = I O . 2 2 * 4.0Sf0.1747 t = 6.866* 3.4 fo.1438 t = 1.203 2.65+0.1928
Comparison with initial value
Group II-C
2.65k0.1118 4.1550.0847 t = 11.61* 4.1 to.0816 4.4 kO.1125 t = II.82* 4.3Sf0.1232 3.7 50.1461 t = 6.314* 3.75fo.1147 2.7 k0.1183 t = 0.9333 2.8550.1232 2.55+0.1088
M+ SE
Comparison with initial value
Group II-B
t = 0.5619 f = 1.161 t = 1.604 t = 1.101
with initial value
Comparison
TABLE 11. Variations with time in the reticulocyte counts of rabbits treated with hypotonic extracts of the following fractions: nuclear (Group 11-A), mitochondrial (Group 11-B),light mitochondria1 (Group 11-C), microsomal (Group 11-D) or soluble (Group 11-E)obtained from the kidneys of donors bled 18 h before. The reticulocyte count is expressed as the reticulocytes/roo erythrocytes. (Meansf standard error ( M t SE). Comparison between the means of the initial and successive values by means of Student’s t (degrees of freedom = ID).)
2 CP
w
W
H
5
3
%. 2
trj T
g2.
F
5
Initial After 2 days After 3 days After 4 days After 6 days
Time
I
2.45fo.1945 2.35f0.1857 2.4 +0.205 2.45k0.1522
2.3 f0.177
Mf SE t t t t
I M f SE
t t t t
2.4
2.3 k0.1693 2.3 k0.139
= 0.1808 = 0.5571 = 0.5937
+0.22~1
2.35 k0.1875 2.45 _+ 0.2012
M + SE 2.5
*0.1211
I
I
t t t t
= 0.2526 = 0.7851 = 1.054
= 1.168
Comparison with initial
Group III-E
t = 0.3635 2.7 +O.IZII t = 0.1707 2.5~+0.1565 t = 0.1979 2.65f0.1478 t = 0.2142 2.7 ko.1461
Comparison with initial
Group III-D
= 0.8262
* Highly significant difference.
+
M k SE
Comparison with initial value
Group III-C
2.3 f0.188 t = 7.246* 2.5 f0.1525 t = 9.346* 2.35 0.2029 t = 6.41* 2.45 f 0.1928 t = 2.016 2.45f0.1688
Comparison with initial value
GYOUP III-B
2.4 kO.1342 3.8 fo.139 = 0.1949 4.3 +0.1528 = 0.3693 3.85f0.1821 = 0.6425 2.85k0.1784 = 0.5703
Comparison with initial value
Groidp III-A
drial (Group 111-B),light mitochondria1 (Group 111-C), microsomal (Group 111-D) or soluble (Group 111-E) obtained from the spleens of normal donors. The reticulocyte count is expressed as reticulocytes/Ioo erythrocytes. (Meansf standard error ( M t SE). Comparison between the means of the initial and successive values by means of Student's t (degrees of freedom = IO).)
TABLE 111. Variations with time in the reticulocyte counts ofrabbits treated with hypotonic extracts of the following fractions: nuclear (Group 111-A), mitochon-
H
CP
c 5 *8.
3-
2.
F;'
23
E. 0
9
w P
Subcellular Localization of Erythrostimulant Factors
I3
s
DISCUSSION The localization of the erythrostimulant factors in the light mitochondrial and microsonial fractions of the kidney of normal rabbits appears to be in agreement with that observed by Cantor et al(1969) in the kidney of normal rats. The presence of the erythrostimulant factors in the microsomal fraction of the kidney can be explained by the facts that (I) the kidney is the organ that produces these substances and ( 2 ) the microsomal fraction is rich in material coming from the ergastoplasmatic structures in which the factors are produced. The erythrostimulant factors probably pass into the light mitochondrial fraction before being released into the circulation. If the production of these factors increases considerably, they probably also accumulate in the mitochondria1 fraction. This could explain the appearance of the crythrostiinulant factors in this fraction in the anaemic kidney. The fact that the erythrostimulant substances contained in the spleen are present only in the mitochondrial fraction strengthens the theory that this fraction is the site in which, at cellular level, these factors tend to accumulate. In fact, the spleen merely acts as a deposit of erythrostimulant factors of renal origin (Liotti & Giovannini, 197oa). It is unlikely that the presence of the erythrostimulant factors at the level of the mitochondrial fraction can be due simply to adsorption on the surface of the mitochondria themselves. This is suggested by the fact that the mitochondrial factions reveal their activity only if disintegrated through freezing and being kept in hypotonic medium in order to favour the solubilization of the factors contained in them. Moreover, the mitochondrial fractions preservc their activity even when they are subjected to repeated washings before the hypotonic treatment. It is probable, therefore, that the presence of the erythrostimulant factors in the mitochondrial fraction of the anaemic kidney and the normal spleen is related to very precise mechanisms that regulate the release of these factors on demand. REFERENCES CANTOR,L.N., ZANJANI,E.D., WONG, K.K. & GORDON,A.S. (1969) The renal erythropoietic factor (REF). IX. Its subcellular distribution.
Proceedings of the Society4r Experimental Biology and Medicine, 130, 950. J.F., GORDON,A S . & WEINTRAUB, A.H. CONTRERA, (1966) Extraction of an erythropoietin-producing factor from a particulate fraction of rat kidney. Blood, 28, 330. KURATOWSKA, Z. (1965) O n the subcellular localization of the erythropoietic renal factor. Bulletin de I’AcadCmie Polonaise des Sciences, 13, 385.
E. (197oa) Studi c ricerche LIOTTI,F.S. & GIOVANNINI, in tema di rapporti tra rene e milza nel controllo dell’eritropoiesi. Haematologica, 55, 681. E. (1g7ob) Attivith LIOTTI, F.S. & GIOVANNINI, eritrostimolante di estratti renali di coniglio normale o salassato. Rivista di Biologia, 63, 3 0 1 . WONG, K.K., ZANJANI,E.D., COOPER,G.W. & GORDON,A.S. (1968) The renal erythropoietic factor. V. Studies on its purification. Proceedings of the Society for Experimental Biology and Medicine, 128,67.
