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Kidney International, Vol. 51 (1997, pp. 393—401

Sites of erythropoietin production

PATRICK H. MAXWELL, DAVID J.P. FERGUSON, LYNN G. NICHOLLS, JOHN P. IREDALE,
CHRISTOPHER W. PUGH, MARTIN H. JOHNSON, and PETER J. RATCLIFFE

The Institute of Molecular Medicine and The Electronmicroscopy Unit, John Radcliffe Hospital, Oxford; University Medicine, Southampton General
Hospital, Southampton; and Department of Anatomy, Cambridge University, Cambridge, England, United Kingdom

Erythropoietin is a circulating hormone that governs the rate of However, it is less clear under what circumstances this system is
red blood cell production and hence the oxygen-carrying capacity activated in vivo. In the whole animal erythropoietin is modulated
of the blood. In response to anemia or hypoxemia circulating by minor changes, such as donating a single unit of blood [23].
levels of erythropoietin can increase a thousand-fold; the regula- While it is quite likely that erythropoietin production is also
tory mechanisms underlying this offer an important model system regulated by hypoxia-responsive mechanisms other than HIF-1,
for oxygen-sensing at both the physiological and molecular levels. this strongly suggests that the HIF-1 system is controling gene
It is now almost 40 years since Jacobson and colleagues showed expression in the kidney in response to minor alterations in the
that removal of the kidneys prevented the erythropoietic response oxygen supply. Similarly, the observation that erythropoietin gene
to hemorrhage or cobalt, whereas ablation of a number of other expression in several other organs is modulated by anemia and/or
organs did not [11. A wealth of evidence has confirmed that the hypoxia suggest that the HIF-1 system is activated in these tissues
kidneys are the principal site of erythropoietin production in the in response to such stimuli.
adult, and the cells responsible have now been identified as the Although the mechanism and function of oxygen-sensing in
fibroblast-like population [2, 3]. In addition to the kidneys, it is other tissues is of great interest, this manuscript will focus on
established that the adult liver is an important source of erythro- erythropoietin gene expression in the kidney and liver. In these
poietin [4], and the cell populations involved have been identified organs it has been an important goal to determine which cells are
as the hepatocytes [5, 6] and the Ito cells [71. involved in erythropoietin production, how they sense changes in
Apart from kidney and liver, erythropoietin mRNA has been oxygen supply and why this mechanism should fail in renal
demonstrated in a range of other normal organs using sensitive disease.
and specific techniques (RNase protection or reverse transcrip-
tase-PCR). These studies are summarized in Table 1 [2, 8—11]. Identification of renal erythropoietin producing cells
Erythropoietin expression has also been documented in early In situ hybridization for erythropoietin mRNA demonstrated
postimplantation mouse embryos [12], human placenta [13] and a that the gene was expressed in cortical peritubular cells [24, 25].
variety of tumors [14—19]. Returning to the normal adult animal,
Precise identification of the cells as the cortical and outer
it is not established that erythropoietin mRNA is translated into
medullary fibroblasts has recently been achieved by ourselves
erythropoietin in organs other than the liver and kidney, although
(using a marker gene strategy) and by others (combining in situ
cultured astrocytes do produce immunoreactive erythropoietin
[201. Interestingly, the total amount of mRNA in the testis and hybridization for erythropoietin mRNA with immunolabeling) [2,
3]. Our own approach was to place a marker gene (SV4O T
brain of unstimulated rodents is in the region of 30% and 10%, antigen) in the context of extensive DNA (16.5 kb) from the
respectively, of the level in the kidney. The blood-brain barrier erythropoietin locus. This Epo-TAg fusion construct was injected
makes it likely that any role of brain erythropoietin gene expres-
into mouse zygotes and eight transgenic lines were generated. In
sion is local rather than systemic. Thus the functional significance
one of these lines, EpoTAgF, homologous recombination be-
(if any) of erythropoietin gene expression in these tissues remains tween the erythropoietin locus and the marker gene had occurred,
to he ascertained. What is clear is that in tissues outside the so that the marker gene was placed in the context of the native
kidney and liver the level of erythropoietin niRNA is generally erythropoietin locus [2]. In Epo-TAg1' mice the marker gene is
increased (usually 2- to 3-fold) by anemia and/or hypobaric correctly placed relative to all erythropoietin control sequences
hypoxia (Table 1). This was one of the first clues that hypoxic and, as might be anticipated, the pattern of expression in terms of
regulation of gene expression was not simply a feature of special- organ distribution and inducibility is very similar to that of the
ized erythropoietin producing cells in the liver and kidney, hut was
more widespread. We now know that the HIF-1 system of hypoxic native gene [2].
In response to anemia or hypoxia expression of the Epo-TAg
regulation operates widely in tissue culture cells [21, 22] and has
gene is induced, and SV4O T antigen accumulates in the nucleus
been implicated in the control of a broad range of genes, at least
of cells expressing the gene (Fig. 1). Immunolabeling for SV4O T
in cultured cell lines (see other other articles in this volume). antigen showed a recruitment pattern of interstitial cells that was
qualitatively and quantitatively similar to that described previ-
ously using in Situ hybridization for erythropoietin mRNA [24.-.
© 1997 by the International Society of Nephrology 26]. By using double immunolabeling (Fig. 2A), and also by
393
394 Maxwell et al: Sites of e,ythropoietin production

Table 1. Postnatal erythropoietin gene expression outside the kidney and liver
Basal expression Induction Induction
Organ Species (relative to kidney) by anemia" by hypoxia Reference
Lung R NO Increased Unchanged [8, 101
Spleen R,Mo 20% 2-fold Increased [8, 101
Brain H,M,R,Mo 10% 20-fold 3-fold [2,9, 11]
Testes R,Mo 30% 3-fold NT [2, 9]
Preputial gland Mo NQ 3-fold NT [2]
Ovaiy Mo NQ NT NT [2]
Quantitative estimates are for the total erythropoietin mRNA in the organ relative to that in the kidneys in unstimulated animals. The response to
anemia or hypoxia is much lower than that in kidney or liver, so the contribution of these organs to total erythropoietin mRNA is insignificant in
stimulated animals. Abbreviations are: H, human; M, monkey; R, rat; Mo, mouse; NO, not quantified; NT, not tested. In the studies cited the following
tissues were found not to Contain detectable erythropoietin mRNA: bone marrow, stomach, intestine, skeletal muscle, heart and salivary gland.
a
Includes induction of functional anemia with CO exposure

two quite different populations (fibroblasts and dendritic cells),

Fig. 1. Section of a kidney from an acutely anemic Epo-TAg" mouse
immunolabeled for SV4O T antigen. The transverse section shows the full
thickness of the cortex and the outer stripe of the Outer medulla, and a
part of the inner stripe of the outer medulla. Nuclei of cells expressing the
Epo-TAg gene are visible as black dots lying between the tubules, and arc
present through the entire depth of the cortex and in the outer stripe of
the outer medulla. Abbreviation G is glomeruli (magnification X50).
immunoelectron microscopy (Fig. 3) we were able to characterize hypoxia is rather limited, so the mechanism behind this striking
the erythropoietin-producing cells as the fibroblasts of the cortical recruitment pattern remains uncertain. What is clear is that
and outer medullary interstitium. This cell population forms the gradients of oxygen tension in the cortex are rather steep (up to 42
major part of the morphologically-defined Type 1 cells of the
interstitium [27]. However, the term Type 1 interstitial cells also
includes the "dendritic" antigen-presenting cells, which are dis-
tinct from the fibroblasts and have different ultrastructural and
antigenic features [28]. Therefore, the term Type I cells includes
and is probably best avoided. Further, it seems reasonable to refer
to the resident cells of the interstitium as fibroblasts, rather than
"fibroblastoid" or "fibroblast-like" [29].
Within the interstitium the surface enzyme eeto-5'-nucleotidase
is restricted to the fibroblasts [30]; however, a substantial propor-
tion of fibroblasts (for instance those of the outer medulla) do not
bear this marker. This demonstrates that there is functional
heterogeneity in the fibroblast population. In response to severe
stimulation most, but not all of the fibroblasts of the deep cortex
express the Epo-TAg gene (Fig. 1) [2, 3, 26], demonstrating that
erythropoietin production is not a property of a small subpopu-
lation. That erythropoietin production and regulation of the
oxygen carrying capacity of the blood should be the response of
the renal fibroblasts might be regarded as surprising. However, it
is plausible that their resident location between the capillaries and
the oxygen-consuming tubules, with extensive processes for mon-
itoring local conditions, offers a prime site for measuring available
oxygen. It would, of course, be of great interest to relate local
oxygen tension to gene expression by individual cells; unfortu-
nately this has not been achieved. It is interesting that erythro-
poietin production occurs in the fibroblasts of the cortex and the
outer medulla (including the inner stripe in response to severe
stimulation); it is not produced in the inner medulla, although this
is the most hypoxic region in the kidney due to the countercurrent
exchange of oxygen in the medullary vasa recta [31]. In under-
standing this apparent paradox it is first important to note that the
fibroblasts of the inner medulla are morphologically distinct from
those of the cortex and outer medulla [29]. Second, local hypoxia
certainly occurs in the cortex, at least in part because of significant
preglomerular shunt diffusion [32].
An intriguing observation is that fibroblasts appear to be
recruited to express the erythropoietin gene in an all-or-none
fashion, with recruitment spreading outwards from the eortieo-
medullary boundary [261. Unfortunately, knowledge about how
oxygen levels in the kidney change in response to anemia or
mm Hg per 10 jim [32]), and so it is possible that the apparent
all-or-none recruitment is due to sharp changes in local oxygen-
ation. Overall, it is interesting to speculate that in some way the
very complexity of renal oxygenation makes the renal interstitiuni
a suitable site for sensing changes in the availability of oxygen.
 Maxwell et al: Sites of etythropoietin production 395

Fig. 2. A. Section from perfusion-fixed renal cortex of an acutely anemic Epo-TAg° mouse, immunostained for 5V40 T antigen (brown, nuclear) and
ecto-5'-nucleotidase (red). Some interstitial cells are seen to label for both antigens (arrows). Abbreviations are: t, tubule; Ca, capillary (magnification
X500). B. Low power view of a renal tumor from an Epo-TAg° homozygote stained with hematoxylin and eosin. Tumor cells are seen infiltrating
between occasional tubules and larger cystic structures (Cy) (magnification X75). C. Renal tumor from an Epo-TAg" homozygote, immunolabeled for
ecto-5'-nucleotidase (brown). Ecto-5'-nucleotidase is evident in the widened iflterstitium. The epithelium lining a cyst (Cy) does not label for
ecto-5 '-nucleotidase. Abbreviation t is tubules (magnification >< 150). D. Renal tumor from an Epo-TAg' homozygote, double labeled for SV4O T
antigen (brown) and ecto-5'-nucleotidase (red). Many of the cells in the widened interstitium are seen to contain SV4O T antigen in their nuclei. There
is extensive surface labeling for ecto-5'-nucleotidase, and some of the tumor cells label for both antigens (arrows). Abbreviation t is tubule
(magnification X 900).

Attempts to obtain renal erythropoietin-producing cells in expression is increased in response to anemia. The cells expressing
culture Epo-TAg in the preputial gland lie in the interstitium of the gland
Despite numerous attempts, renal cell lines producing erythro- between the epithelial structures, but we have not characterized
poietin in a regulated fashion have remained elusive. Renal them further.
fibroblastoid cell lines are available, but they do not produce The normal renal morphology in Epo-TAg mice and the
erythropoietin. Expression of SV4O T antigen has been used to sporadic tumors at other sites suggested that expansion and
derive cell lines expressing other hormones [33], and a second aim immortalization of the renal cells would require a higher level of
of the Epo-TAg transgenic experiments was to selectively expand expression of the gene in the kidney and increased tissue speci-
and immortalize erythropoietin producing cells from the kidney. ficity. Epo-TAg5T mice were used to obtain the high levels of tissue
In unstimulated Epo-TAg mice (including heterozygous Epo- specificity observed for expression of the endogenous erythropoi-
TAg'1 mice) the interstitium was normal, as anticipated, since in etin gene. In order to obtain sustained high-level expression in the
the unstimulated situation relatively few cells express the etyth- kidney, Epo-TAg' mice were rendered anemic. This was achieved
ropoietin and Epo-TAg genes. That the SV4O T antigen was by crossing with a mouse strain which has an autosomal dominant
biologically active was clearly demonstrated by an increase in the hemolytic anemia (Nan [341) or by feeding with an iron-deficient
size of the preputial gland in almost all male Epo-TAg mice, and diet from the time of weaning. Additionally, we studied homozy-
by the development of extrarenal tumors expressing T antigen gous Epo-TAg" mice. These mice have no normal erythropoietin
(Table 2). The unexpected hyperplasia of the preputial gland led gene and have a severe but incomplete deficiency of erythropoi-
us to examine whether this gland expresses the erythropoietin etin, the low level of erythropoietin being derived from the
gene in normal mice. We found that the preputial gland contains unaltered erythropoietin sequence in the Epo-TAg fusion gene
a similar amount of mRNA to that found in the testes and that [7]. In the anemic EpoTAgF! mice there was a two- to threefold
396 Maxwell et al: Sites of etythropoietin production

A ata *1

.1

' i..;'
ft

•aa.

Fig. 3. Immunoelectron microscopy of the renal interstitium in an anemic Epo-TAg" mouse. A. Electron micrograph of an interstitial libroblast which is
undergoing mitosis (M). Adjacent endothelium (En) and tubular cells (T) are indicated. Bar is 0.5 /.Lm. B. Detail of the enclosed area in A,
immunolaheled for SV4O T antigen. Gold particles (arrows) are seen over the electron lucent region of the nucleus. Bar is 0.2 .rm. C. Serial section to
that shown in B, immunolabeled for ecto-5'-nucleotidase showing gold particles (arrows) along the plasmalemma. Bar is 0.2 ttm.

Table 2. Incidence of tumors in Epo-TAg mice

Tumors
Animals N (all Sites) Kidney Liver Lymph Eye Jaw Other
Nontransgenic 250 3.2% 0.4% 0.4% 2.0% — —
Other Epo-TAg lines 158 17.7% — 1.9% 0.6% — 12.6%
Epo-TAg1' Heterozygotes 175 12.6% 0.6% 6.3% 4.0% 2.3%
F.poTAgH X Nan 54 11.1% — 3.7% 3.7% 3.7%
Epo-TAg" iron deficient 10 10.0% — — — 10.0% —
Epo-TAg° homozygotes 125 11.2% 8.0% 1.6%
—
0.8% 0.8% 0.8%
All mice in the colony surviving more than 155 days are included. In each group the number of mice at risk (N) is given, and the proportion of these
developing tumors. Renal tumors were very rare except in homozygous Epo-TAg° mice, which have sustained high level expression of the Epo-TAg
gene; ten of these mice developed tumors in the kidney.
Testis
Intestine

increase in the number of interstitial cells compared to anemic noelectron microscopy of occasional mitotic cells in the intersti-
control mice. The ability of renal fibroblasts both to express SV4O tium, which showed the presence of both SV4O T antigen and
T antigen and to proliferate was further demonstrated by immu- ecto-5'-nucleotidase (Fig. 3).
 Maxwell et a!: Sites of etythropoietin production 397

Despite this proliferation, renal tumors were relatively infre- 30

quent, occurring in only 8% of the EpoTAgH homozygotes, and C)
in none of the other two anemic groups (Table 2). In the tumors
00.
that did occur, malignant cells were seen to have replaced the
w
majority of the renal tissue. Within the tumors there were some 0)
surviving tubules and cysts that were were lined by cuboidal c 20
epithelium (Fig. 2B). Immunostaining revealed that the tumor
cells expressed SV4O T antigen and ecto-5'-nucleotidase (Figs. 2 ><C\j
C, D). The epithelial lining of the cysts did not express either of
these markers, and presumably represented residual tubular
epithelial tissue. Although cultured cells from these tumors did
o. 10
0
express SV4O T antigen, expression of the Epo-TAg gene was not
increased by hypoxia. Analysis of Epo-TAg mRNA showed that in .0
E
some of the tumors transcripts were aberrantly initiated although
in others they were correctly initiated. z
0
We also obtained primary cell cultures from kidneys without
Outer Inner cortex and
tumors, either by perfusion-digestion or by outgrowth from small cortex outer medulla
fragments cut from the renal cortex. Supernatants from such
cultures derived from heterozygous Epo-TAgH mice were Fig. 4. The effect of ureteric ligation on the capacity for etythropoietin gene
expression. One ureter of a heterozygous Epo-TAg' mouse was ligated,
screened for erythropoietin expression after hypoxic exposure, but and eight days later kidney sections were prepared and labeled for SV4O
were negative. Limited further experiments have examined pri- T antigen. Prior to sacrifice the mouse was exposed to hypobaric hypoxia
mary cell cultures from Epo-TAg mice on matrix, and co-cultures to induce Epo-TAg expression. The histogram shows the number of cells
of functioning epithelial cells with a fibroblastoid cell line from an labeled for SV4O T antigen in the control (LII) and obstructed kidney (U).
Epo-TAg mouse have been screened for erythropoietin produc- For each kidney strips of width 500 zm including the full depth of the
cortex and the outer medulla were assessed. The strip was subdivided into
tion (in collaboration with Prof. C. Bauer and Dr. H. Marti). two halves on the basis of depth ("outer cortex" and "inner cortex/outer
Finally, we have also screened conditionally immortalized fibro- medulla"). The number of labeled nuclei (mean SCM, 5 observations)
blastoid cell lines derived from the kidneys of adult and embryonic per unit area (0.25 mm2) is given.
mice bearing a transgene encoding a temperature sensitive variant
of SV4O T antigen under the control of an MHC Class I promoter
[35]. This allowed SV4O T antigen (and thereby the proliferative
stimulus) to be removed from the cells, allowing "fibroblasts" to poietin gene expression at a cellular level. Localization of the
be studied in the quiescent state. Again, on hypoxic exposure potential for erythropoietin gene expression using Epo-TAg trans-
erythropoietin mRNA was not detected. genic mice has the substantial advantage that visualization of the
From these experiments we can conclude that the erythropoi- stable marker, SV4O T antigen, can be combined with labeling for
etin-producing cells in the renal interstitium do have the capabil- other antigens.
ity to divide (that is, they are not post-mitotic). However, it Heterozygous Epo-TAg'1 mice were subjected to one of three
appears that this cell population is relatively resistant to the different forms of renal injury to the right kidney (ureteric
transforming effect of the viral oncogene SV4O T antigen. This obstruction, ischemia-reperfusion or a needlestick pass through
suggests that an additional signal, which is infrequent or absent in the kidney). Prior to sacrifice Epo-TAg expression was induced
renal fibroblasts, would be required in addition for malignant either by rendering the animals anemic (using phenylhydrazine)
transformation. In this context, it is of interest that the develop- or hypoxic (in a hypobaric chamber). Kidney sections were labeled
ment of beta cell pancreatic islet tumors in mice expressing an for SV4O T antigen to identify cells expressing the Epo-TAg gene,
insulin-SV4O T antigen fusion gene correlates with IGF-II expres- and for markers associated with different interstitial cell popula-
sion in the islets, and in IGF-I1 —I— mice fewer malignant tumors tions. For the global forms of injury (ureteric obstruction and
develop [36]. Perhaps more importantly, the consistent failure of ischemia-reperfusion) the injured and control kidneys were la-
ourselves and others to obtain renal erythropoietin producing beled in parallel.
cells in culture strongly suggests that changes in the fibroblasts Following renal injury there was a marked reduction in the
themselves, or the absence of some feature of the renal interstitial number of cells expressing the Epo-TAg gene compared to the
environment precludes erythropoietin gene expression. Teleogi- control kidney (Fig. 4). Expression remained restricted to fibro-
cally this might be appropriate, since it would be potentially blasts. With more severe stimulation the reduction in the number
harmful if erythropoietin production was activated by local hyp- of cells expressing the Epo-TAg gene in the injured kidney was
oxia due to causes other than reduced oxygen delivery, such as less marked. As expected from previous studies (such as [39, 40]),
local inflammation. morphological changes were observed in the interstitium, and iii
particular, in each form of injury the fibroblasts showed myofi-
Effects of renal injury on renal erythropoietin production broblastoid changes. One possibility would be that erythropoietin
A striking feature of almost all forms of renal disease, whether production in the abnormal interstitium is restricted to "normal"
acute or chronic, is the relative deficiency of erythropoietin fibroblasts which have not developed myofibroblast features. This
resulting in anemia [37, 381. The mechanisms underlying this have turns out not to be the case, since cells with myofibroblast
not been explored extensively. We were therefore interested in characteristics were shown to express Epo-TAg.
studying the effects of injury on the renal potential for erythro- Thus in response to renal injury the fibroblasts appear to alter
398 Maxwell et al: Sites of erythropoietin production

Fig. 5. Immunoelectronmicroscopy of the liver of an anemic Epo-TAg" mouse. A. Electron micrograph through an Ito cell lying in the space of Disse (SD)
subtended by hepatocytes (H) and a sinusoid (S). Note the extraction of the lipid droplets (L). Bar is I jim. B. Detail of the enclosed area in A,
immunostained for SV4O T antigen and showing labeling with gold particles (arrows) over the euchromatin of the cell nucleus. Abbreviations are: N,
nucleus; L, lipid droplet; NM, nuclear membrane. Bar is 0.2 jim.

their threshold for erythropoietin expression, and become par- population involved has not been elucidated. In adult life, al-
tially refractory to stimulation by anemia or hypobaric hypoxia. though the liver is a significant secondary source, the kidney is the
The rather similar changes in the fibroblasts and their reduced principal site of production. This switch from predominant liver to
capacity for Epo-TAg expression following different forms of kidney gene expression is unlikely to be related to the alterations
injury are consistent with clinical observations that the great in oxygenation that occur at around the time of birth, since it
majority of patients with renal failure (regardless of the underly- occurs at quite different times in different species [43—46] and is
ing pathology) have a relative deficiency of erythropoietin [37, 38]. not altered by transplantation of the fetal liver into an adult
The striking exception to this is autosomal dominant polycystic environment [47].
kidney disease (ADPKD) [41]. Since erythropoietin production is In the adult liver two different populations of cells express the
also relatively preserved in ESRF patients who develop acquired erythropoietin gene, the hepatocytes and a non-parenchymal
cystic disease of the kidneys [421, it is unlikely that it is a direct population. We have characterized the latter population in ane-
consequence of the genetic defect in ADPKD patients. This mic Epo-TAg'1 mice as the Ito cells (also known as lipocytes,
observation should provide a valuable clue to understanding how stellate cells or perisinusoidal cells), using double immunolaheling
erythropoietin production is permitted in the normal kidney, and
inactivated in renal disease. Overall, it seems likely that the and immunoelectron microscopy (Fig. 5) [1 These cells have a
number of similarities to the renal fibroblasts, besides producing
refractory behavior of erythropoietin producing cells in renal erythropoietin. First, they both lie between the capillaries and
disease and the difficulty in obtaining satisfactory cell culture lines
parenchynial cells and both possess extensive networks of pro-
relate to similar alterations in erythropoietin gene regulation in
cesses [29, 48]. Second, they are both believed to be the predom-
both circumstances. Thus it appears that the particular circum-
inant matrix-synthesizing cells in their respective organs. Third, in
stances allowing erythropoietin production can he met, at least
partially, in the injured or diseased kidney but rarely (if ever) in response to injury both proliferate and develop myofibroblastoid
isolated cultured cells. features including increased expression of the intermediate fila-
ment desmin [39, 48, 49]. Fourth, they both label with the rat
Erythropoietin production by the liver monoclonal antibody ER-TR7 and bear the surface enzyme
It is clearly established that the liver is the major site of ccto-5'-nucleotidase, expression of the latter being augmented in
erythropoietin gene expression in fetal life, although the cell response to anemia [7, 50]. Finally erythropoietin gene expression
 Maxwell et al: Sites of eiythropoietin production 399
A B
N H N H N Co CN H

— EPO

Plastic Matrix Matrix

Fig. 6. Eythropoietin gene expression in cultured rat Ito cells. A. Ito cells were isolated from a rat by perfusion-digestion, followed by density gradient
centrifugation and elutriation. They were then cultured on plastic (left hand lanes) or EHS matrix (right hand lanes). After seven days cells were
incubated in parallel for 16 hours in 21% (N) or 1% oxygen (H). Total RNA was prepared and 20 xg analyzed by RNase protection assay. Erythropoietin
mRNA (EPO) was detected in RNA from hypoxic cells, and was more abundant in cells cultured on matrix. B. Ito cells were isolated and cultured on
matrix. After seven days cells were exposed in parallel to 21% oxygen (N), 100 xM cobaltous chloride (Co), I mi potassium cyanide (CN) or 1% oxygen
(H). Twelve micrograms of RNA was analyzed from each culture. Erythropoietin mRNA was only detected in the cells exposed to hypoxia.

in both these populations appears to be dependent on similar adequate eiythropoiesis in response to more severe stimuli, and
cis-acting sequence(s) in transgenic mice, in a manner that is some evidence for this comes from experimental studies in the rat
different from the hepatocyte population [51]. [9]. This differential response in the two organs might relate to
Given these similarities, cultured Ito cells might provide a different patterns of oxygenation or it could reflect differences in
valuable model for the renal erythropoietin producing cells. A the cellular and molecular mechanisms involved in recruitment.
major advantage of Ito cells is that they can be isolated in Other mechanisms could also be important. Thus it is plausible
substantial numbers directly from rat liver. The separation proce- that hepatic erythropoietin mRNA is less efficiently translated or
dure relies on their buoyant density which is low due to the that erythropoietin produced by the liver is either not released
presence of lipid droplets. This is in marked contrast to the into the circulation or has a shorter circulating half-life than renal
situation with preparations of "renal fibroblasts," which require erythropoietin.
many cell divisions making their relationship to the fibroblasts of
the normal renal interstitium uncertain. One week after explan- Conclusions
tation, we found that primary cultures of Ito cells express the Many of the recent advances in understanding regulation of the
erythropoietin gene in a hypoxically-inducible fashion. Interest- erythropoietin gene have involved cultured hepatoma cells. While
ingly, the level of hypoxically-induced expression was substantially much valuable insight has been gained, this model system cannot
higher if the cells had been cultured on matrix, rather than on represent the complex environment of the functioning kidney.
plastic (Fig. 6). Culturing Ito cells on plastic is asssociated with the Major oustanding questions (which overlap) are the circum-
development of myofibroblast features [48], so this forms an stances resulting in HIF-1 activation in vivo, local oxygen tension
interesting parallel with the behavior of fibroblasts in injured in the environment of the erythropoietin producing cells, and the
kidneys. Since cobalt induces eiythropoietin expression both in precise circumstances which allow erythropoietin production to
vivo [52] and by cultured hepatoma cells [531, we also tested occur.
whether it induces erythropoietin gene expression in cultured Ito
cells. In this experiment no induction was observed. Interestingly, Acknowledgments
a lack of induction by cobalt in cells which produce erythropoietin This work was supported by the Welicome Trust. We are grateful for
in response to hypoxia has also been reported for primary generous gifts of antibodies (J. Gannon, B. Kaissling, M. Thisted and A.
hepatocytes [54] and cultured astrocytes [11]. Vecchi) and conditionally immortalized murine renal fibroblasts (A.
Returning to the whole animal, it is intriguing that in response Woolf). We also thank M. Rychetnik (electron microscopy) and J. Gentry
to liver injury both humans and rodents produce more eiythro- (isolation of Ito cells) for technical assistance.
poietin [55, 56], in contrast to the situation in the kidney. Which Reprint requests to Patrick H. Maxwell, Room 420, Institute of Molecular
cells in the liver are producing erythropoietin in this setting Medicine, John Radcliffe Hospital, Oxford 0X3 9DU, England, United
remains to be established, but this differential response to injury is Kingdom.
intriguing and may be helpful in understanding the regulation of E-mail: pmaxwell@immsvr.jr2.ox.ac.uk
erythropoietin production. Another feature of the liver is that
oxygen gradients are relatively well understood compared to those References
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to the central vein [57]. In response to stimulation both hepato- 1. JACOBSON LO, GOLDWASSER E, FRIED W, PLZAK L: Role of the kidney
in erythropoiesis. Nature 179:633—634, 1957
cytes and Ito cells are recruited in a pattern spreading outwards 2. MAXWELL PH, OSMOND MK, PUGH CW, HERYET A, NICHOLLS LG,
from the central vein. This pattern is consistent with local P°2 TAN CC, DOE BG, FERGUSON DJP, JOHNSON MH, RATCLIFFE PJ:
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