*Brifish]oumaf ofHuernafology, 1976, 32, 10s.
Hepatic Erythropoietin: Enhanced Production in Anephric Rats with Hyperplasia of Kupffer Cells CESARE PESCHLE, GIANNIMARONE, ARTUROGENOVESE, CRISTINA MAGLIAND MARIO CONDORELLI
Institute of Medical Pathology, II Faculty of Medicine and Surgery, University of Nuples, Naples, Italy (Received
10
March 197s; acceptedfor publication 28 M a y 1975)
Erythropoietin (Ep) levels were assayed in serum of adult male rats subjected sequentially to (I) administration of colloidal carbon, Zymosan or their vehicles, (2) .sham operation or bilateral iiephrectomy with and without subtotal hepatectomy, and (3) hypoxia (0.4s-0.40atmospheres of air for 6 h starting I h after the operation). In aiiephric rats these agents induced a significant potentiation of hypoxic Ep activity. Since they did not apparently modify the kinetics of exogenous Ep, it is postulated that this phenomenon is mediated by enhanced extrarenal Ep production. Both colloidal carbon and Zymosan induced hyperplasia of the reticuloendothelial system (RES).Moreover, subtotal hepatectomy almost abolished the Ep response to hypoxia evoked by Zymosan. The correlation between hyperpIasia of hepatic RES and enhanced Ep production in aiiephric rats primed with these agents suggests that Kupffer cells constitute a major source for extrarenal Ep. Additionally, it is of interest that colloidal carbon and Zymosan did not significantly modify the renal production of Ep. It is well established that the kidney is the main site of erythropoietin (Ep) production and/or activation. It has been postulated that the biogenesis of Ep involves interaction of the renal erythropoietic factor (REF),also termed erythrogenin (Eg), with a serum component (Gordon et al, 1967). The REF may function either as an enzyme cleaving Ep from a serum substrate (Zanjani et al, 1971) or a pro-Ep rendered active upon incubation with a normal serum component (Peschle et at, 1974). In spite of extensive investigation, the renal tissue(s) involved in production of Ep and/or Eg has not yet been elucidated. While fluorescence in conjugated anti-Ep serum selectively interacted with the glomerular tufts (Fisher et al, 1965), Zanjani et al (1967) extracted significant levels of Eg from both glomeruli and tubules of hypoxic kidneys. The mechanism of Ep production in the anephric rat has recently attracted considerable interest (Fried, 1972; Peschle et ul, 1972, 1973 ; Schooley & Mahlmann, 1972; Kaplan et a!, 1973). In nephrectomized rats subjected to hypoxia, Ep levels in serum are approximately 8090% lower than in normal controls (Schooley & Mahlmann, 1972; Peschle et ul, 1972). Furthermore, the liver apparently plays a prominent role in extrarenal Ep production (Fried, 1972), possibly via hepatic Eg (Peschle et al, 1973 ;Kaplaii et ul, 1973). These studies, however, Correspondence: Dr Cesare Peschle, Istituto Patologia Medica, Nuovo Policlinico, Via S. Panshi, 8013 I Napoli, Italy. 105
Cesare Peschle et al
I 06
do not demonstrate thc extrarcnal tissue involved in production of Ep. The present investigation has been undertaken in an attcinpt to evaluate tlie role of the reticulociidothclial system (RES) as a possible source for Ep and/or Eg. Thus, Ep production was assessed in sham-operated, nephrectoinizcd and/or hepatectomized rats primed with agents which modify both the phagocytic function of tlic RES a i d enhance its proliferation, as evaluated on the basis of RES hyperplasia. In thcse studies, both a ‘stimulant’ and a ‘dcpressaiit’ of the RES were employed, i.e. colloidal carbon and Zymosan respcctivcly.
MATERIAL AND METHODS 20-25 g CF I female mice and 200-250 g male Wistar rats were used. The animals were maintained on a diet of standard pellets and tap water ad libitum. A minimum of either five rats or six mice per group was employed.
(I) Rat Experiments: (A)Administration ($Colloidal Agents, (B) Sham-operation or Nephrectoniy, Associated or not with Subtotal Hepatectonzy, (C)Hypoxia In the first series of experiments, tlie rats received intravenously 1.0 ml/25o g body weight ~ IPelikaii Werke, Hannover), its vehicle (4.3% fish of either colloidal carbon ( C I I / I ~A, glue and 1% carbolic acid in H,O) or sterile physiological saline daily for four consecutive
days. 24 h after the last injection the animals were subjected to sham-operation or bilateral nephrectomy under light ether anaesthesia, and thereafter to a period of hypoxia (0.40 atmospheres of air for 6 h starting I 11 after the operation). In further experiments, the rats received either colloidal carbon or its vehicle, as indicated above, on days 1-4. O n day 5 , 10 iu of Ep/rat (Step I sheep plasma Ep, Connaught Medical Research Laboratories, Toronto, diluted in 0.5 ml of sterile physiological saline) were injected intravenously 2 h after nephrectomy. The animals were sacrificed 2 or 4 h after Ep. In a second series of studies, the rats were injected iiitraveiiously with Zymosan (Sigma Co:, St Louis) at tlie dosage of 5 mg/200 g body weight, once on days I and 2, twice on day 3. The control group received an equivalent volume of physiological salinc. On day 4 the animals were operated on and exposed to hypoxia as dcscribcd above. In similar cxperimeiits the animals primed with Zyinosan or its vehicle wcre subjected to nephrectomy with or without 80-90% hepatectoniy, and then to thc standard hypoxic stimulus. In all studies, hepariiiizcd blood was collected by cardiac puiicturc under light ether anaesthesia immediately after the end of hypoxia. The scrum obtained from each group was pooled, dialysed against physiological saline for 24 li at 4’C and stored at -zo”C until assayed for Ep activity. (2) Ep
Assay
The Ep activity of test niatcrials was assessed in ex-hypoxic polycythaeinic mice, according to a slight modification o f a previously reported procedure (Peschle et al, 1972).Test materials were injected subcutaneously on days 3 and 4 post-hypoxia, radioiron (0.5 PCi [ 5 gFe]citrate) intravenously on day 5. Rcsults are cxpressed in terms of either 48 h % RBC-59Fe incorporation or equivalent units of either the first or second International Refercnce Preparation,
Erythropoietiii in Aticphric Rafs
107
National Institute for Mcdical Research, Loiidoii (IRP I or 11) of Ep. In this regard, tlie log dose/resyonse rcgrcssioii liiic for Ep is significant between 0.05 and up to at lcast 1.5 iu. RESULTS Aiiepliric rats priiiicd with colloidal carbon showcd a iiiarkcd potciitiatioii of serum Ep activity evoked by die hypoxic exposurc. O n the otlicr hand, this agcnt did not enhance scrum Ep titres in sliani-operated animals, subjcctcd siniultancously to liypoxia (Table I). TABLE I. Ep levels in serum of rats subjected scqucntially to (I) administration of colloidal carbon or its vehicle, (2) bilateral
Group No.
nephrectomy or shaiii opcration, and (3) hypoxia
Trcattiient of dotlor rats
R a t scrwit/ assay moiise
(m4 I 2
3
I 2
3
+
Saline+nephrectomy hypoxia Vehicle +nephrectomy+ hypoxia Colloidal carbon nephrectoiny hypoxia
+
+ +
+
0.S 0.5
0.5 Standards (itijectcd in assay inicc) Saline 0.05 iu Ep 0.20 iu Ep
+
Saliiie sham opcration hypoxia Vehicle sham operation +hypoxia Colloidal carbon+ sham operation+ hypoxia
0.2 0.2
0.2
Standards (injected in assay iiiicc) Saline 0.10iu Ep 0.80 iu Ep
M e a n % [59Fe]RBC Equivalent incorporation in polyi t 4 / m l of cythaemic mice+ SEM rat serum 13.73 k 1.28 14.46+ 1.90 26.4812.03*
0.26 0.30
0.80
1.07+ 0.24 6.77k 1.53 16.50k1.89 I2.44k 1.191 9.22.k1.05$ I3.42k 1.8s?,$ 1.08k 0.42 7.52f 1.40 25.45 f 2 . 3 3
A iniiiiiiiuni of fivc rats atid six iriicc pcr group. and 2. f Not significant when coriiparcd with groups I and 2. :! Sincc the diffcrciice bctwccii thcse groups is not significant, corrcsponding Ep lcvels wcrc not cvaluatcd.
* P< 0.01when compared with groups I
Similarly, administration of Zyiiiosaii in iicplirectoiiiizcd rats causcd a sharp iiicrcasc of liypoxic Ep activity ovcr coiitrol values (Tablc 11). A potciitiatioii of the Ep rcspoiise to liypoxia was not obscrved, liowcvcr, in sham-opcratcd aiiiiiials priiiicd with Zyiiiosaii (Table 11).
Tablc 111 shows tliat tlic kinetics of cxogciious Ep in aiicpliric rats was iiot sigiiificaiitly modified by adiiiiiiistratioii of colloidal carbon. Although the results are not preseiitcd here, a similar iicgativc result has becii observcd after Zyiiiosaii treatment. These results indicate that tlie potentiation of liypoxic Ep activity in aiiephric rats primed with these agents is mediated by an cnhaiiciiig effect 011 Ep productioii rather than an iiiflueiice on Ep kiiictics. Hepatectoiiiy in aiiepliric rats treated with Zymosan resulted in almost coinpletc abolition of tlie Ep response to liypoxia (Table IV). Liver weights were sigiiificaiitly augmented in colloidal carbon- or Zyinosaii-trcated rats ovcr coiitrol levels (Table V). In this regard, it is
Cesare Peschle et a1
I08
TABLE 11. Ep levels in serum of rats subjected sequentially to (I) administration of Zymosan or its vehicle, (2) bilateral nephrectomy or sham operation, and (3) hypoxia
Grogp No. I 2
I 2
Mean % [59Fe]RBC Equivalent incorporation in polyiulml of cythaemic mice+ SEM rat semm
Rat serum/ assay mouse (ml)
Treatment
Vehicle +sham operation+ hypoxia 0.10 Zymosan+ sham operation+ hypoxia 0.10 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
7-12k 047t S.3Of o.7It 1.85 20.22 5.76 If:0.56 14.58k 1.14
Vehicle+nephrectomy+ hypoxia 0.5 Zymosan+nephrectomy+ hypoxia 0.5 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
~
9.08 f0.82 15.62f ~ . o g *
0.10 0.30
2.25k0.53
8.89 k 2.22 I9.09f 1.25 ~
~
~ _ _ _
A minimum of five rats and six mice per group. * P< 0.01when compared with group I. t Since the difference between these groups is not significant, corresponding Ep levels were not evaluated.
well established that administration of these agents leads to an increase of liver and spleen weight, caused by hyperplasia, and secondarilyhypertrophy, of the RES in these organs (Benacerraf & Sebesteyen, 1957; Kelly et a!, 1962; Wooles et al, 1962; Stuart, 1970). Histologic evidence of RES hyperplasia in liver of anephric rats following colloidal carbon administration is presented in Fig I . Similarly, marked hyperplasia of hepatic RES was observed in anephric rats primed with Zymosan. TABLE 111. Erythropoietic activity in serum of rats subjected sequentially to ( I ) administration of colloidal carbon or its vehicle, ( 2 ) bilateral nephrectomy, (3) injection of Ep (10 iu of Step I sheep plasma Ep), (4) sacrifice at 2 or 4 h after Ep
Group Treatment of donor rats
No.
Rat serum/ assay mouse (ml)
Mean % [59Fe]RBC incorporation in polycytkaemic mice_+SEM ~
I 2
3
4
+ +
Vehicle nephrectomy + Ep +2 h 0.5 Colloidal carbon +nephrectomy +Ep + 2 h 0.5 Vehicle nephrectomy + Ep +4 h 1.0 Colloidal carbon+ nephrectomy Ep + 4 h 1.0 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
+
-
I4.32f 1.58 17.81i-2.44 18.60k2.59 21.05 f 1.44 1.88k0.48 7.92 f 0.69 17.02k 1.37
A minimum of five rats and six mice per group. Since the difference between radioiron incorporation values in vehicle- or colloidal carbon-treated groups is not significant, Ep levels are not calculated here.
Erythropoietin in Anephric Rats
FIG I. Liver histology in rats subjected to hypoxia (0.45 atmospheres of airj6 h, starting after thc opcration) after priming with colloidal carbon (above) or its vehicle (below). x 216. (Fucing p 108)
Erythropoietin in Anephric Rats
109
TABLE IV. Ep levels in serum of rats subjected to (I) administration of Zymosan or its vehicle, ( 2 ) bilateral nephrectomyf So-go% hepatectomy, and (3) hypoxia
Group NO.
Rat serum/ assay mouse
Treatment of donor rats
(ml) I
z
3 4
+
Vehicle nephrectomy +hypoxia 0.5 Vehicle+nephrectomy and subtotal hepatectomy hypoxia 0.5 Zymosan+nephrectomy +hypoxia 0.5 Zymosan+nephrectomy and subtotal hepatectomy hypoxia 0.5 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
+
+
Mean % [59Fe]RBC Equivalent incorporation in poly- iu/ml of cythaemic mice SEM rat serum 5.63 f 0.21
0.09
1.29f 0.18 10.37f I.s3*
N.D.
3.89 f 0.64
0.07
0.20
1.53fo.31 6.5821.45 17.46+ 1.33
A minimum of five rats and six mice per group. N.D.: non-detectable activity. * P< 0.01when compared with groups I, 2 and 3 .
DISCUSSION The present studies indicate that administration of either colloidal carbon or Zymosan induced simultaneously hyperplasia of liver and spleen RES and elevation of the extrarenal Ep response to hypoxia. Although the results are not presented here, preliminary evidence indicated that other agents, which modify RES function but do not induce RES hypcrplasia (i.e., Gadolinium, etc.), did not potentiate the extrarenal Ep activity following hypoxia. Thus, a direct correlation may be established in anephric rats between hyperplasia of hepatoTABLE V. Liver weight values (g/Ioo g ofbody weight) in rats ( I ) primed with colloidal carbon, Zymosan or their respective vehicles, (2) subjected to nephrectomy, and (3) to hypoxia
Group No.
Liver weight (mean g/Ioo g body weight fSEW
Treatment of donor rats
Experiment I
2
3 Experiment I
2
I
+ +
Saline+ nephrectomy hypoxia Vehicle+ nephrectomy hypoxia Colloidal carbon +nephrectomy +hypoxia 2
+ +
Vehicle+nephrectomy hypoxia Zymosan+ nephrectomy hypoxia
3.61f0.11 3.50fo.12 4.5420.19* 4.34fO.19 5.52 2 0.13*
I ~
~~~~~~~
~
A significant increase of real liver weight values (not expressed as ratio between liver weight/Ioo g body weight) was observed in colloidal carbon- or Zymosan-treated animals over those in vehicle-treated rats. * P< 0.01when compared with the control group(s).
II0
Cesave Peschle et a!
splenic RES and enhanced Ep production, suggesting that the RES plays a significant role in this function. As previously indicated, it is thought that the liver is the major source of extrarenal Ep (Fried, 1972). In view of the present findings it may be further postulated that in nephrectomized animals the Kupffer cells are involved in tlie biogeiiesis of Ep. In line with this concept, subtotal hepatectomy in aiiephric rats primed with Zymosaii almost abolished the extrareiial Ep response to hypoxia. The experiments with fluorescein conjugated anti-Ep serum by Fisher et al(1964) indicated that the glomerular tufts are apparciitly a source for renal Ep. The present studies accordingly suggest that liver ‘littorial’ cells (i.e. Kupffer cells; Kelly et al, 1962) play a significant role in tlie hepatic production of Ep. In ancphric animals primed with these agciits the pliagocytic activity of the RES was not correlated with extrareiial Ep levcls. Under tlie present experimental conditions, we have coiifirmcd that colloidal carbon and Zymosan depressed or stimulated respectively the pliagocytic activity of tlie RES (unpublished observations). However, both agciits cnhanccd tlie extrareiial production of Ep. This lack of corrclatioii is not at variaiicc with tlie concept postulated here, i.e. generation of Ep by thc livcr RES. Distinct metabolic pathways may underlie phagocytic activity and Ep productioii in Kupffer cells, resulting in the lack of correlation between these functions. It was coiisidercd possible that hyperplasia of the RES caused iiicrcascd production of Ep through hypoxia of hepatocytes caused by compression of siiiusoids by tlie Kupffer cells. However, histologic observations showed that hyperplasia of the RES did not lead to siiiusoidal compression. Potentiation of tlie Ep response to hypoxia by colloidal carbon or Zymosan, although demonstrated in aiiephric rats, was not observed in sham-operated animals. This phenomenon is possibly related to lack of a significant iiiflueiice of these agents on glomerular cclls. It should also be noted that Eg activity in livcr and spleen, although elevated in aiiephric rats exposed to hypoxia, is not detected in sham-operated controls (Peschle et al, 1973 ; Kaplan et a!, 1973), thus indicating that the extrareiial source of Ep ‘switches in’ when rciial production is shut OK The possibility also exists that in shamoperated animals the large amount of Ep derived from the kidney obscured the enhancing action of these agents on extrareiial Ep activity. In aiiephric rodents similar levels of Ep activity were observed following administration of either lead acetate (Schooley & Mahlmaiiii, 1g74), colloidal carbon or Zymosaii. In this regard, since the former agent modifies RES function (Trejo et al, 1972), its stimulatory effect on the extrareiial sources for Ep may be also mediated by the RES. Finally, these experimental studies might prove of .clinical significance as reduccd Ep production is a major factor in renal anaemia (Erslev, 1972). Thus, administration of lion-toxic agents inducing RES hyperplasia may prove beneficial in thc management of this condition. Conclusive demonstration of the role played by Kupffer cells in extrarenal Ep production requires further investigations, such as the culture of Kupffer cells and immuiiofluorescent observations with anti-Ep serum, currently in progress in our laboratory. However, these studies provide the first indication that in iiephrectomized rats colloidal agents enhancing RES proliferation sharply potentiate hepatic Ep production.
Erythropoietitz in Aticphric Rats
I11
ACKNOWLEDGMENTS
This work was supported in part by a Grant from the CNR, Italy. We wish to thank Mr A. Di Costanzo, Mr E. Vaccaro and Mr P. Ciaglia for their excellent technical help. REFERENCES BENACERRAF, B. & SEBESTEYEN, M.M. (1957) Effect of bacterial endotoxins on the rcticuloendothelial system. Federation Proceedings, 16, 860. ERSLEV, A.J. (1972) Anemia of chronic renal failure. Hematology (ed. by W. J. Williams, E. Beutlcr, A. J. Erslev and 11. W. Rundles), p 237. McGrawHill, Ncw York. G. & PORTEOUS, D.D. (1965) FISHER,J.W., TAYLOR, Localization of erythropoietin in glomeruli of shecp kidney by fluorescent antibody technique. Natiire, 205, 611. FRIED,W. (1972) The liver as a sourcc of extrarcnal erythropoietin production. Blood, 40, 671. GORDON, A.S., COOPER, G.W. & ZANJANI,E.D. (1967) The kidney and crythropoicsis. Serninarr in Hernafology, 4, 337. KAPLAN,S.M., ROTHMANN,S.A., GORDON,A.S., RAPPAPORT, LA., CAMISCOLI, J.F. & PESCHLE,C. (1973) Extrarenal sites of erythrogenin production. Proceedinp of the Sociefyfor Experimental Biology and Medicine, 143, 310. KELLY,L.S., BROWN,B.A. & DOBSON,E.L. (1962) Cell division and phagocytic activity in liver reticuloendothclial cells. Proceedings oftke Socictyfor Experinrental Biology and Medicine, 110, 5 5 5 . PESCHLE, C., SASSO, G.F., RAPPAPORT, LA. & CONDORELLI, M. (1972) Erythropoictin production in ncphrcctomized rats: possible role of the rcnal erythropoietic factor. Jonrnal 4 Laboratory and Clinical Ahdicine, 79,950. PESCHLE,C., D'AVANZO, A., RAPPAPORT,LA., RUSSOLILLO, S., MARONE,G. & CONDORELLI, M. (1973) Kolc of erythrogenin from liver and spleen in erythropoictin production in the anephric rat.
Nafrirc, 243. s39. C., MARONE, G., GENOVESE, A,, QUATTRIN, PESCHLE, S. & CONDORELLI, M. (1974) Studies on the erythrogenin (Eg)-crythropoietin (Ep) system. Infernafional Syriryosiriiir on Erythnynicsis, Tokyo. SCHOOLEY, J.C. & MAHLMANN, L.J. (1972) Erythropoictin production in the anephric rat. I. Rclationship between nephrectomy, time of hypoxic exposure, and erythropoictin production. Blood, 39, 3 I. SCHOOLEY, J.C. & MAHLMANN, L.J. (1974) Hepatic crythropoictin production in the lead-poisoned rat. Blood, 43, 425. Sysfrw, STUART,A.E. (1970) The R~ticrrIo-~r~dot/rcIial p 89. Livingstone, Edinburgh. TREJO, R.A., DI LUZIO,N.R., LOOSE,L.D. & HOFFMAN, E. (1972) Rcticuloendothelial and hepatic functional alterations following lead acetate administration. Expriincntal and ILfolccif/nr Pathology, 17, 14s. WOOLES,W.R., ELKO,E.E. & DI LUZIO,N.R.(1962) Iiiflucncc of pre- and post-irradiation zyniosan administration on rcticuloendothclial function. Radiation Research, 16, 548. G.W., GORDON, A.S., WONC, ZANJANI,E D . , COOPER, K.K. & SCRIBNER, V.A. (1967) The rcnal crythropoictic factor (REF). IV. Distribution in mammalian kidneys. Proceedings of fhe Society for Espeuinrcnfal Biology and Medicine, 126, 540. ZANJANI,E.D., MCLAURIN,W.D., GORDON,A.S., RAPPAPORT, I.A., GIBBS,J.M. & GmARr, A.S. (1971) Biogenesis of crythropoictin: role of the substrate for erythrogenin. Jonrnal of Laboratory and Clinical Medicine, 77, 751.
Hepatic Erythropoietin: Enhanced Production in Anephric Rats with Hyperplasia of Kupffer Cells CESARE PESCHLE, GIANNIMARONE, ARTUROGENOVESE, CRISTINA MAGLIAND MARIO CONDORELLI
Institute of Medical Pathology, II Faculty of Medicine and Surgery, University of Nuples, Naples, Italy (Received
10
March 197s; acceptedfor publication 28 M a y 1975)
Erythropoietin (Ep) levels were assayed in serum of adult male rats subjected sequentially to (I) administration of colloidal carbon, Zymosan or their vehicles, (2) .sham operation or bilateral iiephrectomy with and without subtotal hepatectomy, and (3) hypoxia (0.4s-0.40atmospheres of air for 6 h starting I h after the operation). In aiiephric rats these agents induced a significant potentiation of hypoxic Ep activity. Since they did not apparently modify the kinetics of exogenous Ep, it is postulated that this phenomenon is mediated by enhanced extrarenal Ep production. Both colloidal carbon and Zymosan induced hyperplasia of the reticuloendothelial system (RES).Moreover, subtotal hepatectomy almost abolished the Ep response to hypoxia evoked by Zymosan. The correlation between hyperpIasia of hepatic RES and enhanced Ep production in aiiephric rats primed with these agents suggests that Kupffer cells constitute a major source for extrarenal Ep. Additionally, it is of interest that colloidal carbon and Zymosan did not significantly modify the renal production of Ep. It is well established that the kidney is the main site of erythropoietin (Ep) production and/or activation. It has been postulated that the biogenesis of Ep involves interaction of the renal erythropoietic factor (REF),also termed erythrogenin (Eg), with a serum component (Gordon et al, 1967). The REF may function either as an enzyme cleaving Ep from a serum substrate (Zanjani et al, 1971) or a pro-Ep rendered active upon incubation with a normal serum component (Peschle et at, 1974). In spite of extensive investigation, the renal tissue(s) involved in production of Ep and/or Eg has not yet been elucidated. While fluorescence in conjugated anti-Ep serum selectively interacted with the glomerular tufts (Fisher et al, 1965), Zanjani et al (1967) extracted significant levels of Eg from both glomeruli and tubules of hypoxic kidneys. The mechanism of Ep production in the anephric rat has recently attracted considerable interest (Fried, 1972; Peschle et ul, 1972, 1973 ; Schooley & Mahlmann, 1972; Kaplan et a!, 1973). In nephrectomized rats subjected to hypoxia, Ep levels in serum are approximately 8090% lower than in normal controls (Schooley & Mahlmann, 1972; Peschle et ul, 1972). Furthermore, the liver apparently plays a prominent role in extrarenal Ep production (Fried, 1972), possibly via hepatic Eg (Peschle et al, 1973 ;Kaplaii et ul, 1973). These studies, however, Correspondence: Dr Cesare Peschle, Istituto Patologia Medica, Nuovo Policlinico, Via S. Panshi, 8013 I Napoli, Italy. 105
Cesare Peschle et al
I 06
do not demonstrate thc extrarcnal tissue involved in production of Ep. The present investigation has been undertaken in an attcinpt to evaluate tlie role of the reticulociidothclial system (RES) as a possible source for Ep and/or Eg. Thus, Ep production was assessed in sham-operated, nephrectoinizcd and/or hepatectomized rats primed with agents which modify both the phagocytic function of tlic RES a i d enhance its proliferation, as evaluated on the basis of RES hyperplasia. In thcse studies, both a ‘stimulant’ and a ‘dcpressaiit’ of the RES were employed, i.e. colloidal carbon and Zymosan respcctivcly.
MATERIAL AND METHODS 20-25 g CF I female mice and 200-250 g male Wistar rats were used. The animals were maintained on a diet of standard pellets and tap water ad libitum. A minimum of either five rats or six mice per group was employed.
(I) Rat Experiments: (A)Administration ($Colloidal Agents, (B) Sham-operation or Nephrectoniy, Associated or not with Subtotal Hepatectonzy, (C)Hypoxia In the first series of experiments, tlie rats received intravenously 1.0 ml/25o g body weight ~ IPelikaii Werke, Hannover), its vehicle (4.3% fish of either colloidal carbon ( C I I / I ~A, glue and 1% carbolic acid in H,O) or sterile physiological saline daily for four consecutive
days. 24 h after the last injection the animals were subjected to sham-operation or bilateral nephrectomy under light ether anaesthesia, and thereafter to a period of hypoxia (0.40 atmospheres of air for 6 h starting I 11 after the operation). In further experiments, the rats received either colloidal carbon or its vehicle, as indicated above, on days 1-4. O n day 5 , 10 iu of Ep/rat (Step I sheep plasma Ep, Connaught Medical Research Laboratories, Toronto, diluted in 0.5 ml of sterile physiological saline) were injected intravenously 2 h after nephrectomy. The animals were sacrificed 2 or 4 h after Ep. In a second series of studies, the rats were injected iiitraveiiously with Zymosan (Sigma Co:, St Louis) at tlie dosage of 5 mg/200 g body weight, once on days I and 2, twice on day 3. The control group received an equivalent volume of physiological salinc. On day 4 the animals were operated on and exposed to hypoxia as dcscribcd above. In similar cxperimeiits the animals primed with Zyinosan or its vehicle wcre subjected to nephrectomy with or without 80-90% hepatectoniy, and then to thc standard hypoxic stimulus. In all studies, hepariiiizcd blood was collected by cardiac puiicturc under light ether anaesthesia immediately after the end of hypoxia. The scrum obtained from each group was pooled, dialysed against physiological saline for 24 li at 4’C and stored at -zo”C until assayed for Ep activity. (2) Ep
Assay
The Ep activity of test niatcrials was assessed in ex-hypoxic polycythaeinic mice, according to a slight modification o f a previously reported procedure (Peschle et al, 1972).Test materials were injected subcutaneously on days 3 and 4 post-hypoxia, radioiron (0.5 PCi [ 5 gFe]citrate) intravenously on day 5. Rcsults are cxpressed in terms of either 48 h % RBC-59Fe incorporation or equivalent units of either the first or second International Refercnce Preparation,
Erythropoietiii in Aticphric Rafs
107
National Institute for Mcdical Research, Loiidoii (IRP I or 11) of Ep. In this regard, tlie log dose/resyonse rcgrcssioii liiic for Ep is significant between 0.05 and up to at lcast 1.5 iu. RESULTS Aiiepliric rats priiiicd with colloidal carbon showcd a iiiarkcd potciitiatioii of serum Ep activity evoked by die hypoxic exposurc. O n the otlicr hand, this agcnt did not enhance scrum Ep titres in sliani-operated animals, subjcctcd siniultancously to liypoxia (Table I). TABLE I. Ep levels in serum of rats subjected scqucntially to (I) administration of colloidal carbon or its vehicle, (2) bilateral
Group No.
nephrectomy or shaiii opcration, and (3) hypoxia
Trcattiient of dotlor rats
R a t scrwit/ assay moiise
(m4 I 2
3
I 2
3
+
Saline+nephrectomy hypoxia Vehicle +nephrectomy+ hypoxia Colloidal carbon nephrectoiny hypoxia
+
+ +
+
0.S 0.5
0.5 Standards (itijectcd in assay inicc) Saline 0.05 iu Ep 0.20 iu Ep
+
Saliiie sham opcration hypoxia Vehicle sham operation +hypoxia Colloidal carbon+ sham operation+ hypoxia
0.2 0.2
0.2
Standards (injected in assay iiiicc) Saline 0.10iu Ep 0.80 iu Ep
M e a n % [59Fe]RBC Equivalent incorporation in polyi t 4 / m l of cythaemic mice+ SEM rat serum 13.73 k 1.28 14.46+ 1.90 26.4812.03*
0.26 0.30
0.80
1.07+ 0.24 6.77k 1.53 16.50k1.89 I2.44k 1.191 9.22.k1.05$ I3.42k 1.8s?,$ 1.08k 0.42 7.52f 1.40 25.45 f 2 . 3 3
A iniiiiiiiuni of fivc rats atid six iriicc pcr group. and 2. f Not significant when coriiparcd with groups I and 2. :! Sincc the diffcrciice bctwccii thcse groups is not significant, corrcsponding Ep lcvels wcrc not cvaluatcd.
* P< 0.01when compared with groups I
Similarly, administration of Zyiiiosaii in iicplirectoiiiizcd rats causcd a sharp iiicrcasc of liypoxic Ep activity ovcr coiitrol values (Tablc 11). A potciitiatioii of the Ep rcspoiise to liypoxia was not obscrved, liowcvcr, in sham-opcratcd aiiiiiials priiiicd with Zyiiiosaii (Table 11).
Tablc 111 shows tliat tlic kinetics of cxogciious Ep in aiicpliric rats was iiot sigiiificaiitly modified by adiiiiiiistratioii of colloidal carbon. Although the results are not preseiitcd here, a similar iicgativc result has becii observcd after Zyiiiosaii treatment. These results indicate that tlie potentiation of liypoxic Ep activity in aiiephric rats primed with these agents is mediated by an cnhaiiciiig effect 011 Ep productioii rather than an iiiflueiice on Ep kiiictics. Hepatectoiiiy in aiiepliric rats treated with Zymosan resulted in almost coinpletc abolition of tlie Ep response to liypoxia (Table IV). Liver weights were sigiiificaiitly augmented in colloidal carbon- or Zyinosaii-trcated rats ovcr coiitrol levels (Table V). In this regard, it is
Cesare Peschle et a1
I08
TABLE 11. Ep levels in serum of rats subjected sequentially to (I) administration of Zymosan or its vehicle, (2) bilateral nephrectomy or sham operation, and (3) hypoxia
Grogp No. I 2
I 2
Mean % [59Fe]RBC Equivalent incorporation in polyiulml of cythaemic mice+ SEM rat semm
Rat serum/ assay mouse (ml)
Treatment
Vehicle +sham operation+ hypoxia 0.10 Zymosan+ sham operation+ hypoxia 0.10 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
7-12k 047t S.3Of o.7It 1.85 20.22 5.76 If:0.56 14.58k 1.14
Vehicle+nephrectomy+ hypoxia 0.5 Zymosan+nephrectomy+ hypoxia 0.5 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
~
9.08 f0.82 15.62f ~ . o g *
0.10 0.30
2.25k0.53
8.89 k 2.22 I9.09f 1.25 ~
~
~ _ _ _
A minimum of five rats and six mice per group. * P< 0.01when compared with group I. t Since the difference between these groups is not significant, corresponding Ep levels were not evaluated.
well established that administration of these agents leads to an increase of liver and spleen weight, caused by hyperplasia, and secondarilyhypertrophy, of the RES in these organs (Benacerraf & Sebesteyen, 1957; Kelly et a!, 1962; Wooles et al, 1962; Stuart, 1970). Histologic evidence of RES hyperplasia in liver of anephric rats following colloidal carbon administration is presented in Fig I . Similarly, marked hyperplasia of hepatic RES was observed in anephric rats primed with Zymosan. TABLE 111. Erythropoietic activity in serum of rats subjected sequentially to ( I ) administration of colloidal carbon or its vehicle, ( 2 ) bilateral nephrectomy, (3) injection of Ep (10 iu of Step I sheep plasma Ep), (4) sacrifice at 2 or 4 h after Ep
Group Treatment of donor rats
No.
Rat serum/ assay mouse (ml)
Mean % [59Fe]RBC incorporation in polycytkaemic mice_+SEM ~
I 2
3
4
+ +
Vehicle nephrectomy + Ep +2 h 0.5 Colloidal carbon +nephrectomy +Ep + 2 h 0.5 Vehicle nephrectomy + Ep +4 h 1.0 Colloidal carbon+ nephrectomy Ep + 4 h 1.0 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
+
-
I4.32f 1.58 17.81i-2.44 18.60k2.59 21.05 f 1.44 1.88k0.48 7.92 f 0.69 17.02k 1.37
A minimum of five rats and six mice per group. Since the difference between radioiron incorporation values in vehicle- or colloidal carbon-treated groups is not significant, Ep levels are not calculated here.
Erythropoietin in Anephric Rats
FIG I. Liver histology in rats subjected to hypoxia (0.45 atmospheres of airj6 h, starting after thc opcration) after priming with colloidal carbon (above) or its vehicle (below). x 216. (Fucing p 108)
Erythropoietin in Anephric Rats
109
TABLE IV. Ep levels in serum of rats subjected to (I) administration of Zymosan or its vehicle, ( 2 ) bilateral nephrectomyf So-go% hepatectomy, and (3) hypoxia
Group NO.
Rat serum/ assay mouse
Treatment of donor rats
(ml) I
z
3 4
+
Vehicle nephrectomy +hypoxia 0.5 Vehicle+nephrectomy and subtotal hepatectomy hypoxia 0.5 Zymosan+nephrectomy +hypoxia 0.5 Zymosan+nephrectomy and subtotal hepatectomy hypoxia 0.5 Standards (injected in assay mice) Saline 0.05 iu Ep 0.20 iu Ep
+
+
Mean % [59Fe]RBC Equivalent incorporation in poly- iu/ml of cythaemic mice SEM rat serum 5.63 f 0.21
0.09
1.29f 0.18 10.37f I.s3*
N.D.
3.89 f 0.64
0.07
0.20
1.53fo.31 6.5821.45 17.46+ 1.33
A minimum of five rats and six mice per group. N.D.: non-detectable activity. * P< 0.01when compared with groups I, 2 and 3 .
DISCUSSION The present studies indicate that administration of either colloidal carbon or Zymosan induced simultaneously hyperplasia of liver and spleen RES and elevation of the extrarenal Ep response to hypoxia. Although the results are not presented here, preliminary evidence indicated that other agents, which modify RES function but do not induce RES hypcrplasia (i.e., Gadolinium, etc.), did not potentiate the extrarenal Ep activity following hypoxia. Thus, a direct correlation may be established in anephric rats between hyperplasia of hepatoTABLE V. Liver weight values (g/Ioo g ofbody weight) in rats ( I ) primed with colloidal carbon, Zymosan or their respective vehicles, (2) subjected to nephrectomy, and (3) to hypoxia
Group No.
Liver weight (mean g/Ioo g body weight fSEW
Treatment of donor rats
Experiment I
2
3 Experiment I
2
I
+ +
Saline+ nephrectomy hypoxia Vehicle+ nephrectomy hypoxia Colloidal carbon +nephrectomy +hypoxia 2
+ +
Vehicle+nephrectomy hypoxia Zymosan+ nephrectomy hypoxia
3.61f0.11 3.50fo.12 4.5420.19* 4.34fO.19 5.52 2 0.13*
I ~
~~~~~~~
~
A significant increase of real liver weight values (not expressed as ratio between liver weight/Ioo g body weight) was observed in colloidal carbon- or Zymosan-treated animals over those in vehicle-treated rats. * P< 0.01when compared with the control group(s).
II0
Cesave Peschle et a!
splenic RES and enhanced Ep production, suggesting that the RES plays a significant role in this function. As previously indicated, it is thought that the liver is the major source of extrarenal Ep (Fried, 1972). In view of the present findings it may be further postulated that in nephrectomized animals the Kupffer cells are involved in tlie biogeiiesis of Ep. In line with this concept, subtotal hepatectomy in aiiephric rats primed with Zymosaii almost abolished the extrareiial Ep response to hypoxia. The experiments with fluorescein conjugated anti-Ep serum by Fisher et al(1964) indicated that the glomerular tufts are apparciitly a source for renal Ep. The present studies accordingly suggest that liver ‘littorial’ cells (i.e. Kupffer cells; Kelly et al, 1962) play a significant role in tlie hepatic production of Ep. In ancphric animals primed with these agciits the pliagocytic activity of the RES was not correlated with extrareiial Ep levcls. Under tlie present experimental conditions, we have coiifirmcd that colloidal carbon and Zymosan depressed or stimulated respectively the pliagocytic activity of tlie RES (unpublished observations). However, both agciits cnhanccd tlie extrareiial production of Ep. This lack of corrclatioii is not at variaiicc with tlie concept postulated here, i.e. generation of Ep by thc livcr RES. Distinct metabolic pathways may underlie phagocytic activity and Ep productioii in Kupffer cells, resulting in the lack of correlation between these functions. It was coiisidercd possible that hyperplasia of the RES caused iiicrcascd production of Ep through hypoxia of hepatocytes caused by compression of siiiusoids by tlie Kupffer cells. However, histologic observations showed that hyperplasia of the RES did not lead to siiiusoidal compression. Potentiation of tlie Ep response to hypoxia by colloidal carbon or Zymosan, although demonstrated in aiiephric rats, was not observed in sham-operated animals. This phenomenon is possibly related to lack of a significant iiiflueiice of these agents on glomerular cclls. It should also be noted that Eg activity in livcr and spleen, although elevated in aiiephric rats exposed to hypoxia, is not detected in sham-operated controls (Peschle et al, 1973 ; Kaplan et a!, 1973), thus indicating that the extrareiial source of Ep ‘switches in’ when rciial production is shut OK The possibility also exists that in shamoperated animals the large amount of Ep derived from the kidney obscured the enhancing action of these agents on extrareiial Ep activity. In aiiephric rodents similar levels of Ep activity were observed following administration of either lead acetate (Schooley & Mahlmaiiii, 1g74), colloidal carbon or Zymosaii. In this regard, since the former agent modifies RES function (Trejo et al, 1972), its stimulatory effect on the extrareiial sources for Ep may be also mediated by the RES. Finally, these experimental studies might prove of .clinical significance as reduccd Ep production is a major factor in renal anaemia (Erslev, 1972). Thus, administration of lion-toxic agents inducing RES hyperplasia may prove beneficial in thc management of this condition. Conclusive demonstration of the role played by Kupffer cells in extrarenal Ep production requires further investigations, such as the culture of Kupffer cells and immuiiofluorescent observations with anti-Ep serum, currently in progress in our laboratory. However, these studies provide the first indication that in iiephrectomized rats colloidal agents enhancing RES proliferation sharply potentiate hepatic Ep production.
Erythropoietitz in Aticphric Rats
I11
ACKNOWLEDGMENTS
This work was supported in part by a Grant from the CNR, Italy. We wish to thank Mr A. Di Costanzo, Mr E. Vaccaro and Mr P. Ciaglia for their excellent technical help. REFERENCES BENACERRAF, B. & SEBESTEYEN, M.M. (1957) Effect of bacterial endotoxins on the rcticuloendothelial system. Federation Proceedings, 16, 860. ERSLEV, A.J. (1972) Anemia of chronic renal failure. Hematology (ed. by W. J. Williams, E. Beutlcr, A. J. Erslev and 11. W. Rundles), p 237. McGrawHill, Ncw York. G. & PORTEOUS, D.D. (1965) FISHER,J.W., TAYLOR, Localization of erythropoietin in glomeruli of shecp kidney by fluorescent antibody technique. Natiire, 205, 611. FRIED,W. (1972) The liver as a sourcc of extrarcnal erythropoietin production. Blood, 40, 671. GORDON, A.S., COOPER, G.W. & ZANJANI,E.D. (1967) The kidney and crythropoicsis. Serninarr in Hernafology, 4, 337. KAPLAN,S.M., ROTHMANN,S.A., GORDON,A.S., RAPPAPORT, LA., CAMISCOLI, J.F. & PESCHLE,C. (1973) Extrarenal sites of erythrogenin production. Proceedinp of the Sociefyfor Experimental Biology and Medicine, 143, 310. KELLY,L.S., BROWN,B.A. & DOBSON,E.L. (1962) Cell division and phagocytic activity in liver reticuloendothclial cells. Proceedings oftke Socictyfor Experinrental Biology and Medicine, 110, 5 5 5 . PESCHLE, C., SASSO, G.F., RAPPAPORT, LA. & CONDORELLI, M. (1972) Erythropoictin production in ncphrcctomized rats: possible role of the rcnal erythropoietic factor. Jonrnal 4 Laboratory and Clinical Ahdicine, 79,950. PESCHLE,C., D'AVANZO, A., RAPPAPORT,LA., RUSSOLILLO, S., MARONE,G. & CONDORELLI, M. (1973) Kolc of erythrogenin from liver and spleen in erythropoictin production in the anephric rat.
Nafrirc, 243. s39. C., MARONE, G., GENOVESE, A,, QUATTRIN, PESCHLE, S. & CONDORELLI, M. (1974) Studies on the erythrogenin (Eg)-crythropoietin (Ep) system. Infernafional Syriryosiriiir on Erythnynicsis, Tokyo. SCHOOLEY, J.C. & MAHLMANN, L.J. (1972) Erythropoictin production in the anephric rat. I. Rclationship between nephrectomy, time of hypoxic exposure, and erythropoictin production. Blood, 39, 3 I. SCHOOLEY, J.C. & MAHLMANN, L.J. (1974) Hepatic crythropoictin production in the lead-poisoned rat. Blood, 43, 425. Sysfrw, STUART,A.E. (1970) The R~ticrrIo-~r~dot/rcIial p 89. Livingstone, Edinburgh. TREJO, R.A., DI LUZIO,N.R., LOOSE,L.D. & HOFFMAN, E. (1972) Rcticuloendothelial and hepatic functional alterations following lead acetate administration. Expriincntal and ILfolccif/nr Pathology, 17, 14s. WOOLES,W.R., ELKO,E.E. & DI LUZIO,N.R.(1962) Iiiflucncc of pre- and post-irradiation zyniosan administration on rcticuloendothclial function. Radiation Research, 16, 548. G.W., GORDON, A.S., WONC, ZANJANI,E D . , COOPER, K.K. & SCRIBNER, V.A. (1967) The rcnal crythropoictic factor (REF). IV. Distribution in mammalian kidneys. Proceedings of fhe Society for Espeuinrcnfal Biology and Medicine, 126, 540. ZANJANI,E.D., MCLAURIN,W.D., GORDON,A.S., RAPPAPORT, I.A., GIBBS,J.M. & GmARr, A.S. (1971) Biogenesis of crythropoictin: role of the substrate for erythrogenin. Jonrnal of Laboratory and Clinical Medicine, 77, 751.
