Review for 10.1186/1471-230X-9-26 (2009)

Reviewer's report
Title:No evidence for protective erythropoietin alpha signalling in rat hepatocytes
Version: 1Date:16 October 2008
Reviewer: Constance Noguchi
Reviewer's report:
In light of reports of protection from ischemia-reperfusion injury in rodents by
erythropoietin (EPO), the authors examine the potential for EPO to directly
protect hepatocytes from hypoxia-reoxygenation and cold-induced injury.
Expression of EPO receptor (EPOR) mRNA is detected by PCR in cultures of
hepatocytes isolated from rat livers. Incubation with EPO under select conditions
reduced cell injury by hypoxia as determined by lactate dehydrogenase (LDH)
release, but this was also observed with EPO treated with heat-inactivation
(attributed to glycine in EPO treatment). No EPO induction of Bcl-2 and EPO
specific reduction in LDH release by cold is observed, and activation of JAK2 and
STAT5 in EPO perfused rat livers was not detected. Therefore, the authors
conclude that there is no evidence for a direct EPO protective effect on rat
hepatocytes.
Critique:
Major Compulsory Revisions:
The EPO protective activity for hepatic ischemia-reperfusion injury reported in
several animal models suggest EPOR expression and function beyond
hematopoietic tissues. Of particular interest is the mechanism of EPO protection
via direct or indirect activity in non-hematopoietic tissue that express low level of
EPOR compared with erythroid progenitor cells. The evidence for EPOR mRNA
expression in hepatocytes presented here are incomplete. In addition, Pinto et al.
(Blood 2008) reported that EPO mediated regulation of hepcidein expression via
EPOR signaling and C/EBPalpha expression in primary mouse hepatocytese.
Although they observed no statistical differences in viability with EPO treatment,
their work provides evidence for functional EPOR production in primary
hepatocytes and should be included in the discussion. The data presented here
extends the culture conditions to hypoxia-reoxygenation and cold-induced injury.
A positive control (glycine) is included for hypoxic injury and was useful in
explaining the protection observed with heat-inactivated EPO, although it is
unclear why protection is greater with EPO heat-inactivation. However, no
positive control is provided for cold-induced injury. Controls for pJAK2 and
pSTAT5 are also lacking. The authors should discuss the possible indirect
mechanisms that may account for EPO protective activity in liver.
Specifics:
1. Figure 5: Data on EPOR expression in hepatocytes is incomplete. The PCR in
(A) only shows a portion of the EPOR transcript. Is the mature erythroid form of
the EPOR identical to the transcript produced in hepatocytes? Has this been
shown previously? How does the level of expression indicated in (B) compare
with levels produced in an erythroid control such as erythroid progenitor cells,
bone marrow or UT-7 cells?
2. Figure 1: Why does heat-inactivation increase the protective effect of EPO
observed in Figure 1?
3. A positive control for protection to cold-induced injury should be included.
4. Figure 7: What are the bands close to pJAK2 and pSTAT5 in the perfused rat
liver extracts? Which are the correct pJAK2 and pSTAT5 bands? The
comparison with total JAK2 and STAT5 should be provided, unless these
Western blots are intended to show complete absence of pJAK2 and pSTAT5. If
this is the case, then some treatment to show that activation of JAK2 and STAT5
can be detected by some other treatment should be included.
5. If EPO does not stimulate hepatocytes directly, what are the other possible
mechanisms for the reported protection from hypoxia-reoxygenation in the liver?
Minor Revisions:
1. Figure 5: What was the time in culture prior to analysis of the hepatocytes
used in this study?
Discretionary Revisions:
1. Figure 1: The protein content of recombinant human EPO sold commercially
consists primarily of albumin as carrier. Therefore, if the protection shown in
figure 1 is non-specific and related to glycine, is it also present with addition of
albumin alone in an analogous buffer similarly treated?
2. Figure 5: Pinto et al., (Blood 2008) examined primary mouse hepatocytes
immediately following isolation. Does EPOR increase, decrease or remain
unchanged when analyzed immediately following isolation? What is the level of
EPOR in the perfused liver?
3. Figure 3: The protection by glycine should also be included here for the
analysis of nuclear morphology.
4. Figure 6: Josefsen et al. (Stem Cells 2000) showed expression of Bcl-xL in
CD34(+) hematopoietic progenitor cell cultures at 4 days of EPO treatment which
then increased with culture time. Does longer EPO treatment of hepatocytes
beyond 12 hours affect the results presented here?
5. Figure 7. The results for cultured hepatocytes should be shown.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.