September 10, 2010
Dear BioMed Central Editorial Team,
Thank you for submitting our manuscript for review by your experts. Our responses to
their concerns are outlined below in blue font. In addition, changes to the manuscript
itself are easily delineated in Track changes, and in some areas, in changed font color.
We hope that you find our responses have improved the quality of our paper and make
the paper suitable for publication in BMC Complementary and Alternative Medicine.
Sincerely,
Sharon Adler, MD, on behalf of all of the authors
Response to Reviewers:
Reviewer 1
Version: 1 Date: 7 August 2010
Reviewer: Alaaeldin Hamza
Reviewer's report:
Comments to the Author
This study deals with the very timely and important subject of diabetic
nephropathy and the use of curcumin to prevent or minimize the renal
dysfunction. I found this document to be overall very well informed, objective and
well written. I think that it is largely acceptable but I have a number of comments
for author consideration.
My comments are as the following:
1-Although the subject is important, the authors offer very little if anything that is new.
Previews work has shown that curcumin can ameliorate some of the
nephropathy associated with diabetes. Tikoo et al (2008) confirmed that
treatment of diabetic rats with curcumin significantly decreased blood urea
nitrogen and creatinine and increased albumin; variables associated with the
development of diabetic nephropathy. They suggested that protection against
development of diabetic nephropathy by curcumin treatment involved changes in
post-translational modifications of histone H3, expression of heat shock
protein-27 (HSP-27) and mitogen-activated protein kinase p38 in diabetic kidney.
K Tikoo, R L Meena, D G Kabra, A B Gaikwad, Change in post-translational
modifications of histone H3, heat-shock protein-27 and MAP kinase p38
expression by curcumin in streptozotocin-induced type I diabetic nephropathy.
British Journal of Pharmacology (2008) 153, 1225–1231.
We believe that the Reviewer has missed the point if the conclusion is that “very little if
anything is new” in this paper. All other published papers show a purported benefit for
curcumin in diabetic nephropathy. We point out shortcomings in the experimental
approaches of these previously published works, raising some doubt regarding their
conclusions, and then show, that at least in this mouse model, curcumin did not
attenuate the cardinal manifestation of early diabetic nephropathy, albuminuria. In
addition, the use of timed urine collections for HPLC measurements of urine
curcuminoid/creatinine ratios to quantify curcumin bioavailability represents a completely
novel aspect of this work.
Our paper urges a healthy skepticism concerning the benefit reported by others. As
nephrologists, it is our position that the published parameters used by other authors to
study curcumin in diabetic nephropathy were frequently inadequate. One novel aspect of
our paper is that we used criteria for the diagnosis of diabetic nephropathy in mice that
would be used in patients. By these criteria, these mice did not benefit. We would hope
that publication bias would not prevent publication of this negative, but significant result
because, in fact, it IS different than the published literature on this topic to date.
2- The second concern is the dose of curcumin in animal study. The authors fed mice at
dosage 5000 ppm amd 7500 ppm which have been used in mouse model of Alzheimer
disease. I think these doses are too low to induce effect in diabetic mice. Chiu et.al.
(2009) found that curcumin at dosage level 150mg/kg was effective to prevent diabetesassociated abnormalities in kidney of rats. In another study, 75 mg•kg–1•day–1curcumin
ameliorated renal failure in mice. Suresh Babu1 and Srinivasan (1998) fed rats with
curumin (0.5% in the diet) to ameliorate renal lesion in diabetic rats.
The Reviewer believes that we did not see an effect because the dose that we chose was too low. We
are not in agreement with the Reviewer concerning this point for the following reasons:
1). We chose doses that were high enough to show benefit for curcumin in another disease model,
e.g. Alzheimer disease.
2) We obtained curcumin from the same source as the Alzheimer study.
3) We used the same compounder to make the mouse food.
4) We demonstrated by collecting urine and making HPLC measurements that curcumin and
curcuminoid were present in urine, thereby demonstrating pharmacodynamically that the target organ
(eg the kidney) was exposed to drug. The latter is actually unique in this field, since no other study has
demonstrated this.
5) We demonstrated other biological effects of the administered curcumin on the kidney (eg a
decrement in renal cortical HSP25 and an increase in urine 12-HETE excretion).
6) In the paper by Babu and Srinivasan (Mol Cell Biochem 1811-2): 87-96, 1998) to which the
Reviewer refers above, the dose of 0.5% that was administered is equivalent to 5000 ppm, which is
the lower of the 2 doses that we administered.
Therefore, there is abundant evidence that the curcumin that was given actually reached the target
organ and was administered was present in sufficient quantity to achieve other biological responses at
the target organ. It nevertheless did not reduce albuminuria.
However, we agree with the Reviewer that under other conditions and possibly at an even higher
dose, this therapy may have been more effective. In fact, the manuscript already acknowledges this in
the last paragraph, which states: “While strain, species, and dosing issues may be responsible for this
negative result, the biological responses of HSP25 and 12/15-LO to curcumin may underlie this
failure. Thus, despite encouraging in vitro effects, these data do not confirm prior published in vivo
work and suggest that curcumin is not universally useful in ameliorating DN”. Note that we did not
state that curcumin does not work under any condition. We do state, and we underscore, that the
dose we gave was adequate to achieve other biological responses in the kidney, and that under these
conditions, curcumin did not attenuate the key manifestation of diabetic nephropathy, albuminuria.
References
Chiu J, Khan ZA, Farhangkhoee H, Chakrabarti S. Nutrition. 2009
Sep;25(9):964-72. Curcumin prevents diabetes-associated abnormalities in the
kidneys by inhibiting p300 and nuclear factor-kappaB.
P. Suresh Babu1 and K. Srinivasan (1998).Amelioration of renal lesions
associated with diabetes by dietary curcumin in streptozotocin diabetic rats.
Molecular and Cellular Biochemistry, 181, (1-2)
3-The paper is quit long, this is uncomfortably large for many people. I would like
you omit any non-essential materials from conclusion.
We went through the text and tried to shorten where we could.
4- Figure 5 needs to represented, the data of blood glucose, changes in body
weight and food intake must be added in table.
The data on food intake/4 days is already shown in Figure 5, and a tabular form would
probably be redundant. We removed the blood glucose values from the text and now
display them as a Table added to Figure 3. We do not have the body weights.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Reviewer 2. Version: 1 Date: 17 August 2010
Reviewer: Sae kwang Ku
With all due respect, the authors refute the allegation of the Reviewer that the
manuscript cannot be understood due to grammatical errors. In fact, it is this review, and
not the paper, that is written with grammatical errors that obfuscate. Indeed, I had
difficulty in understanding some of the comments of this Reviewer due to this problem,
and therefore could address most, but not all, of the comments.
Reviewer's report:
This manuscript presents results of a study designed to examine the ability
Curcumin to the podocyte p38MAPK-HSP pathway in vitro. However, author(s)
did not revealed any favorable effects on streptozotocin-induced diabetic
nephropathy in vivo.
This reviewer concluded as “Major Compulsory Revisions (which the author must
respond to before a decision on publication can be reached)”
Despite the potential interest of this manuscript to the field, it is not possible to
provide an adequate review of the manuscript in its current form due to the
number of grammatical errors and some results are difficult to understanding.
The author(s) are encouraged to describe or answers as following questions
Major comment: Why used streptozotocin-induced diabetic nephropathy in vivo
only. There are numerous other nephropathy animal models; Before concluded
Curcumin did not attenuate streptozotocin-induced diabetic nephropathy,
author(s) should be test on other animal models, especially STZ-induced Rats or
db/db mice. In addition, it seems to be need more detail explains on the Material
and methods.
The authors agree that the study of additional models of diabetic nephropathy would
have been ideal. However at this point, since the funds for the project have been
completely expended, that is not an option. The use of streptozotocin for induction of
diabetic nephropathy in the DBA2j mouse has been advocated by members of the
National Institutes of Health-funded “mouse consortium” (Qi Z, Fujita H, Jin J, Davis LS,
Wang Y, Fogo AB, Breyer MD: Characterization of Susceptibility of Inbred Mouse
Strains to Diabetic Nephropathy. Diabetes 54:2628-2637, 2005). Indeed the divergent
result in this animal model compared to the result in rats may reflect underlying
differential responses to therapy based on strain/species. This has been stated clearly in
the manuscript, and is worthwhile documenting. It is important to note that we did not
“conclude that curcumin did not attenuate diabetic nephropathy” as the Reviewer stated
above. In our conclusion, we stated that “these data do not confirm prior published in
vivo work and suggest that curcumin is not universally useful in ameliorating DN”.
Statistical Analysis
This reviewer strongly suggest that used more sensitive statistical method such
as ANOVA test, especially on In Vivo
As stated in the Statistical analysis section, all the data were analyzed by ANOVA,
followed by Student t test to discern within group differences.
Details
1. Seems to be need to change the ‘in vitro, in vivo’ with Italic. As requested by the
Reviewer, we have italicized “in vitro” and “in vivo”.
2. P3. Line 14. You said “previously showed in vitro that short-term incubation of
podocytes in medium with a high glucose concentration resulted in
phosphorylation of p38MAPK and downstream HSP25.” But we did not know
when/where you said(quote the origin) and what time is short-term?
We apologize regarding confusion concerning this reference. Our work is described not
only in the sentence to which the Reviewer refers, but continuously in the next 8 lines.
The reference therefore is at the end of the sentence on the eighth line, and is reference
18. I have removed references 5, 7, and 8 in this citation to eliminate any confusion.
In the current manuscript referring to reference 18 alluded to above, the incubation time
was inadvertently left out of the manuscript and we thank the Reviewer for pointing out
this omission. The sentence in question has been corrected as follows: “We previously
shown in vitro that short-term incubation of podocytes in medium with a high glucose
concentration (up to 4 hours) resulted in phosphorylation of p38MAPK and downstream
HSP25, associated with maintenance of the actin cytoskeleton. Incubation of podocytes
in high glucose medium for as briefly as 4 hours with a p38MAPK inhibitor attenuated
downstream HSP25 phosphorylation, inducing F- to G-actin cleavage, and cytoskeletal
disruption”.
3. P4. Podocyte culture
Podocyte culture: briefly write the method of ‘conditionally immortalized mouse
podocytes’
It seems to be need to write the ‘thermosensitive SV 40 transgene’ method: what
kind(gene sequence) of promoter, terminator and others did you use. And how
do you check the transgene well-done.
Reference 23 is use ‘100 U/ml of the #-IFN’ but you use ‘50 U/ml’, explain why
the dose is difference.
Reviewer 1 complained that the manuscript was too long, so we believe that the addition
of the information requested by Reviewer 2 here will make the manuscript even longer,
and is unnecessary. Reference 23 provides all of the information the Reviewer wishes.
This cell line requires no additional validation. It is an extremely well established and
accepted cell line for the study of podocyte biology. If one goes to Google Scholar and
searches the name “Mundel” (referring to Peter Mundel, who developed the cell line) and
“SV40 podocytes”, there are 342 hits. If one omits Mundel’s name in the search, there
are 945 hits. The vast majority of those 945 hits are by investigators using the Mundel
podocytes. This underscores how much these cells are utilized, and how well-known the
cells are by investigators in the field of glomerular biology. The interested Alternative
Medicine investigator can find the details regarding this cell line quite easily in the
published literature.
While the Reviewer’s comment concerning the interferon concentration in this reference
is correct (Ref 23), the difference between the 50 and 100 U/ml concentration of
interferon is not critical. For example, in the publication entitled “Podocyte expression of
the CDK-inhibitor p57 during development and disease” published in Kidney
International (2001) 60:2235–2246, which includes Mundel as an author, an interferon
concentration of 50 U/ml was used, as was used in our submitted work. In order to
address the Reviewer’s concerns, we added the words “with minor modifications” to
cover this small difference. The sentence now reads: “Conditionally immortalized mouse
podocytes (Pods), carrying a thermosensitive SV40 transgene, were obtained from Dr.
Peter Mundel and cultured as described with minor modifications (23). Conditionally
immortalized mouse podocytes (Pods), carrying a thermosensitive SV40 transgene,
were obtained from Dr. Peter Mundel and cultured as described with minor modifications
(23). Conditionally immortalized mouse podocytes (Pods), carrying a thermosensitive
SV40 transgene, were obtained from Dr. Peter Mundel and cultured as described with
minor modifications by Mundel et al (23)”.
4. P6. DNase 1 inhibition assay for the measurement of F/G actin ratio
You could better changing number(24,25,18) to researchers’ name(blikstad I,
papakonstanti EA, Dai T)
We changed the sentence as follows: Pod F- and G-actin were measured using the
methods of others (24, 25) and as previously utilized and reported by us (18).
Need to write the full name of GA
Thank you. We have changed GA to G-actin.
5. Experimental animals
Need to write the detailed animal conditions (ages, donator or where do you
purchased)
Thank you. We added to the manuscript that the mice were purchased from Jackson
Laboratories, Bar Harbor, Maine, USA, and that they weighed 20-22 gm and were 2
months old at the time of streptozotocin injection.
For the collecting the podocyte you must to kill the animals, so have to write the
anesthesia method and IRB name & approval number.
The podocytes are a cell line, as discussed above and as stated in the paper. They were
provided by Dr Peter Mundel (Ref 23), and no mice were killed to obtain podocytes. The
mice, however, were killed to do the kidney tissue Western blots. The manuscript
already states: “All animal studies were performed under a protocol approved by the Los
Angeles Biomedical Research Institute Animal Use Committee”. Our IRB issues
approval in the form of a letter to the investigator. There is no approval number. At the
request of the Reviewer, we have now added that the mice were killed by
exsanguination under general anesthesia.
Discretionary revisions: When you added the materials in food, it may be difficult
to control the same dosage (mg/kg body weight). We suggest you to administer
the materials using gastric gavage (Zondae) for accurate dosage
We agree with this comment, and considered this approach when we designed the
project. Unfortunately, we did not have the expertise to perform gavage.
6. P7. In experimental I, you measured urine items on day 9 and 15. Why you
measured on that day? And clear define the day of 9 and 15, define the day 1.
Also in experiment 2, define the weeks 2, 4 and 7
Mice were injected with streptozotocin daily for 5 days. One week later, fasting blood
glucose was measured to ascertain for diabetes. In Experiment 1, Days 9 and 15 refer to
the number of days of confirmed diabetes. These days were chosen because they were
the earliest time points at which we detected albuminuria.
In Experiment 2, weeks 2, 4, and 7 refer to the number of weeks with confirmed
diabetes.
This clarification has been added to the manuscript.
7. P7. Numbers of animals in groups are different. Why do you have different
number in your each group? And you measure the on 9 and 15 day but you did
not write how many animal you kill on each days (9, 15)
Some mice did not become diabetic after streptozotocin injections. Some mice died.
8. P8. The sentence ‘to determine ~ subsequent experiments’ previously, and it
is seems to be as a repetition stationery
I cannot understand this.
9. P8. The pods changes in cur 60-70 min stimulation but we don’t know
what/how the podocytes changes. You need to write the detail changes.
We eliminated the clause “and induced quantifiable biological changes in Pods”
10. P10.
Line 3, check the DMcur0 value (2.8±07) and figure 3 does not indicate the 2.8
level
Thank you. This was an error and is now corrected to read 2.23±07 in the text.
Line 20. You wrote the measurement were made from 5-11weeks but you just on
one time values which is don’t know what weeks values
These were repeat measures during this time period. Mice were in steady state at this
time, and their values did not differ from one week to the next, so the values were
aggregated.
line 9. You did high concentration cur food experiment cause by thinking low
dosage. But we think many cause ex) short-period administration, nullified effect,
irregular cur-administration etc (P.18).
We originally intended the study to be carried out for at least 12 weeks. However, in
Experiment 1, by nine days it was clear that the diabetic mice already had significant
albuminuria, and that curcumin did not attenuate the albuminuria. Therefore, the study
was short because there was no benefit, not the other way around. I do not know what
you mean by nullified effect.
I agree with the Reviewer’s comments that gavage would have provided a more stable
dose and would have been the preferred method of drug delivery. Unfortunately, we did
not have the technical expertise to do this. Instead, we have offered evidence that the
drug was present at the target organ, and that it succeeded in inducing a biological effect
there. Our HPLC measurements clearly show that curcuminoid was delivered to the
kidney, and our Western analyses of kidney lysate show a biologic effect (low HSP25
and high urine 12-HETE/creatinine) of that curcuminoid in the kidney. Despite that, there
was no attenuation of albuminuria. In my view, I believe that this may be a strain effect.
As stated in the manuscript: “The burden of explaining why curcumin failed to
ameliorate albuminuria in these mice remains, and one can only speculate. A unique
response in this mouse strain cannot be ruled out, as it is well-appreciated that genetic
backgrounds influence both disease susceptibility and response to treatments.”
11. P11.
Line 23 how did you calculate ‘food intake was only ~50% higher’. In figure 5. I
think the food intake volume is too much by a mouse.
Sorry for the confusion. The Legend reads that the food intake was measured on Day 4,
but omitted that the measurement reflected the food intake of the first four days of the
protocol. This has now been clarified in the legend.
Line 24,25, check the unit of creatinine ‘mcg’
“mcg” was intended to be micrograms. Sometimes when one uses a symbol, it is
changed during pdf transformation or when viewed with a different version of WORD. A
 has been inserted in the text to replace mc as requested.
12. P12.
Line 2. I think you collect the sample from the day 9 to 15 (during a week). If you
do that, your samples have no consistency. Correctly explain when did you
collect the renal sample.
These were timed samples collected overnight on day 9 and day 15.
Line 9. U 12-HETE/cr -> urinay 12-HETE/cr. Done.
13. P15.
Line 3,9 You need to quote reference. I am not sure to what this refers.
Line 23. You argued Babu et al did not analyze the histological changes. But you
also did not offer the histological analysis (glomeruli histomorphometry etc)
Our concern with Babu’s work is not that they failed to analyze histological change, but
rather that they analyzed it poorly. As stated in the manuscript: “a description of how the
histologic analyses were performed is lacking, and the photomicrographs provided are of
very low magnification and not easily interpretable by the reader”.
We did not perform histological evaluation because we already knew that the treatment
did not work in our experimental mice based on the measured urine albumin/creatinine
values.
14. P17. Line 15. (6,93,94)(95) -> (6,93-95)
Thank you. We made this change.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Reviewer 3. Version: 1 Date: 18 August 2010
Reviewer: Kulbhushan Tikoo
Reviewer's report:
In the present study the authors had explored the potential of curcumin as a
treatment regimen against diabetes mellitus (DM) both in vitro and in vivo. It is
interesting study in a way as it raises doubts about other studies which shows
protective role of curcumin in diabetic nephropathy. But before the acceptance of
this manuscript, there are certain critical points to be addressed.
Major revisions
1) My major concern is with the quality of the western blots. The blots shown in
the manuscript are not from the single blot for different treatment groups. This
makes it inconvenient to compare the results for different treatment groups and
thus pose a question on the reliability of the data. Level of p38 and hsp25 in
normal control animals should be included in figure 6.
The Reviewer is correct regarding the Western blots. Unfortunately, there were too many
samples, so the samples could not be run together on a single gel.
With regard to the second, equally valid point, our limited goal in this experiment was to
determine if curcumin had an effect on p38MAPK or HSP25 in diabetic mice. Therefore,
we compared the results in diabetic mice who received dietary curcumin with diabetic
mice who did not receive curcumin. Our goal was not to compare diabetic mice to nondiabetic mice, even though that would be of some interest as well. Unfortunately,
although we would like to provide the Reviewer with this information, we no longer have
cell lysate from these mice, and cannot do new blots, We also have no additional
funding to support an entirely new set of mice for this purpose.
2) From the present results, it seems that curcumin at higher dose is actually
increasing the plasma glucose level and urinary 12-HETE/cr excretion in vivo.
The authors should explain the possible reason for the same in discussion.
In Experiment 1, the fasting blood glucose measurements reported were taken one week
after the last of five streptococci injections. The diets were initiated the next day.
Therefore, in Experiment 1, the glucose measures did not reflect any influence of the
diet. In Experiment 2, the diets were assigned one week prior to the start of the five daily
streptozotocin injections, and the fasting blood glucose measurements were taken one
week after the last of the five injections. Therefore, in Experiment 2, the glucose
measurements did reflect the assigned diet. There was a statistically significant
increment in fasting blood glucose in the mice getting curcumin in Experiment 2. This
was noted for both the diabetic and the non-diabetic groups. Because of this difference,
noted 1 week after the last Stz dose, we monitored the blood glucose at later time
points, including in some mice who were kept for up to 11 weeks for the purpose alone.
These values showed no difference. These data are now presented in tabular form as
requested by the Reviewer. In addition, a statement has been added to acknowledge
that early differences in glycemic control may have also contributed to the failure of
curcumin to attenuate diabetic nephropathy in these mice. (“In addition, at lest in
Experiment 2, fasting blood glucose was higher at week 1 in mice receiving curcumin, a
finding that was not replicated in measures taken at later weeks. These early differences
were statistically significant, but their biological significance is uncertain. Nevertheless,
we cannot exclude that this apparently transient and relatively small increment in blood
glucose early in disease development contributed to the lack of apparent efficacy of
curcumin to attenuate albuminuria.” )
We have no explanation for this difference in glycemic control, and are aware (and cite
in the paper) evidence by others of an opposite effect of curcumin on glycemic control
after Stz injections.
As the Reviewer points out, these data do show an increase in urine 12-HETE in the
curcumin treated mice. Indeed, it is the belief of the authors that this increment may in
part explain the deleterious impact of curcumin on albuminuria as detected under these
experimental conditions. The Discussion section of the manuscript delves into this in
some detail. (As stated, “Curcumin has been reported to inhibit lipoxygenases by one
group of investigators (87), but was found to be a substrate of lipoxygenases by another
group (88). Urine 12-HETE is a reliable measure of activation of the 12/15-LO pathway
in vivo (96), and in these curcumin-treated mice, it was increased. In prior studies
performed to inhibit 12/15-LO in Stz-DN rats, our published work showed that chemical
inhibition of 12/15-LO is only transiently effective, and that tachyphylaxis occurs rapidly
(97). In the rats receiving the chemical inhibitor, a linear relationship between urine 12-
HETE excretion and albuminuria was observed (97). The failure of curcumin to suppress
activation of 12/15-LO may have contributed to the albuminuria observed in the
curcumin-treated diabetic DBA2J mice”). Unfortunately, we have no further insight with
regard to this issue.
3) In statistical analysis, for various results, if the authors have set the value of
NG treatment group as 1 every time and then comparing the effects in other
treatment groups relative to NG, then they should not have a SEM for NG. The
authors should explain these calculations in detail.
The mean value was set as one for the control group(s), but even in the controls, there is
variance around that mean. The error bars reflect the variance around the mean in the
control group.
Minor revisions
1) There are some contradictory statements in the results, like
“there was no measurable curcuminoid in mice fed Cur0 or Cur5,000 diets.
Urinary curcuminoid was abundantly detected in mice fed the Cur5,000 diet”
The authors should rectify the same.
Thank you for picking this up! We had also given a diet lower in curcumin than the
Cur5000 diet (Cur160 ppm), in which curcuminoid could not be detected in urine.
Because of its non-detectability and its lack of efficacy, after initially including it in the
manuscript, we decided to remove it just prior to submission. This was a vestigial
reference to it that we forgot to expunge, coupled to a typographical error! We have
corrected the sentence.
2) There are too many values in the results. The inclusion of tables for comparing
values like blood glucose etc., makes the reader to easily understand the results
We removed the blood glucose values from the text and added it as a Table in Fig 3a.