Title: Effects of thyroid hormone analogue and leukotrienes pathway blocker on renal ischemia/reperfusion injury in a mouse model.

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1 Author's response to reviews Title: Effects of thyroid hormone analogue and leukotrienes pathway blocker on renal ischemia/reperfusion injury in a mouse model. Authors: Najah R hadi (drnajahhadi@yahoo.com) Fadhil G al-amran (fadhil.al-amran@ucdenver.edu) ayad a hussein (ruaa_fsl@yahoo.com) Version: 5 Date: 18 September 2011 Author's response to reviews: Author's response to reviews Effects of thyroid hormone analogue and a leukotrienes pathway-blocker on renal ischemia/reperfusion injury in mice Authors: Fadhil G. Yousif (Fadhil.Al-amran@ucdenver.edu) Najah R. Hadi Ayad A. Hussein (ruaa_fsl@yahoo.com) Version: 1 Date: The Biomed Central Editorial Team Object: MS: Effects of thyroid hormone analogue and a leukotrienes pathway-blocker on renal ischemia/reperfusion injury in mice. Thank you for consideration of our manuscript for publication in your journal. We have reviewed the above manuscript according to your reviewer s comments. Author's response to reviews: see over Reviewer's report Reviewer: Geurt Stokman. Major compulsory revisions 1. An important point of concern is that this study lacks the proper controls. Sham-operated animals injected with MK-886 or DITPA should have been included. Both compounds are dissolved in potentially harmful vehicle solutions

2 (one with a ph of 9 and one with 2% ethanol) which, when injected IP, may result in intestinal injury or otherwise affect the outcome of IR injury. Actually, the sham-operated animals were injected with the two vehicles solutions but this information was mistakenly not found in the manuscript. 2. The authors conclude that MK-886 protects against IR injury by reducing inflammatory and oxidative processes. The tissue sections of animals subjected to renal IR injury do not seem to contain many inflammatory cells at all (compare figure 4A to B). Does this mean that not enough damage was induced to achieve an inflammatory response? Serum levels of IL-6 and TNF-# are reduced by MK-886, but to establish that this is also associated with decreased inflammatory cell influx, neutrophils or other inflammatory cells should be quantified either by by immunostaining or, alternatively, by scoring the inflammatory cell influx using the HE stainings. As the authors acknowledge in the discussion (page 12), neutrophils/pmns enter the damaged kidney within the first 24 hours of reperfusion. If MK-866 is thought to act primarily on activation and migration of these cells, then why didn t the authors examine renal function and histology at this time point? In another I/R model, Sharyo et al (2009)42 showed that 30 min renal ischemia caused significant elevation in serum urea, creatinine and IL-6 after 48 hr of reperfusion. So we depended on this study in our work for examining renal function. 3. How do the authors know that the concentrations and the administration of compounds are actually sufficient to induce a biological effect in vivo? Was this tested in a pilot-study? MK-886 administration does seem to have an effect on renal function during IR injury, but DITPA clearly not. A recent study by Li et al. (Moll Cell Endocrinol, 2011) shows that T3 administration does prevent renal failure and tissue injury during IR injury. Maybe the dosage and time points of administration were not correct in this study. The discrepancy between both studies and why DITPA treatment did not protect against IR injury in this study should be discussed. In the present study, DITPA administration was only 30 min prior to ischemia induction to see if there was any anti-inflammatory action for DITPA like that proved for MK-886 (before this study). This may be short time course to exert preconditioning effect on oxidative stress similar to that induced in hepatic and renal I/R injury in which T3 administration was 36 hr and 48 hr respectively prior to ischemia induction67,68 and this may give suggestion for plane future in studying DITPA in I/R model.

3 4. In the discussion section, the authors share their view on how MK-886 effects inflammation and ROS production. Again, this section contains many errors and should be carefully rewritten. I will just provide a few examples that explain what I mean. The authors describe that neutrophils enter the kidney in the first hours after ischemia (page 11). IL-6 and TNF-# are T cell or macrophage-associated products and these cells enter the kidney at days 2 5 after ischemia (page 12). IL-6 and TNF-# promote migration of neutrophils (PMNs) to the injured kidney (page 14). How does that match? More likely, neutrophils are attracted by chemokines such as CXCL1 or CXCL2 which are highly expressed up to 24 hours after ischemia.. The mechanism of I/R is not fully understood. For this example, IL-6 might be produced by endothelial cells due to injuries stimuli. IL-6 regulates the expression of adhesion molecules and other cytokines in endothelial cells including IL-1# and TNF-# which in turn potently enhance the inflammatory response62which lead to chemotaxis of PMNs, which in turn produce inflammatory mediaters. On page 14, the authors refer to a paper on a collagen-induced arthritis model in which TNF-# and IL-6 levels were also reduced. Interesting, but I don t think that a comparison of this model and the renal IR injury model is relevant at this point. In that study, they mentioned that MK-886 act on IL-6 and TNF-# by the same mechanism supposed in this study although in different models Minor essential revisions 1. Affiliations of the authors are not present. Please provide these 2. done. Introduction 2. Page 2: The definition ARF is superseded by acute kidney injury (AKI) 3. Page 2: An oxidative burst by a neutrophil does not (necessarily) provoke ROS production in target cells. Rather, IR-induced ROS production in tubular epithelial cells appears to result from metabolic abnormalities. Methods 4. Page 5: What was the age of the animals? 12 weeks 5. Page 5: What volume was used for IP administration? Not more than 0.1 ml

4 6. Page 6: Please provide more information on the ELISA procedure. Specificity of the antibodies, dilutions, composition of blocking buffer or diluents, chromogen. For IL-6, Samples and calibrators were incubated in the microtiter plate coated with the first monoclonal antibody anti-il-6, in presence of the second anti-il-6 monoclonal antibody linked to acetylcholinesterase (ACE). After incubation, the wells were washed and the bound enzymatic activity is detected by addition of a chromogenic substrate. The intensity of the coloration is proportional to the IL-6 concentration in the sample or calibrator. -Calibrator: one vial contains lyophilized bovine serum albumin -IL-6 ACE conjugate: one vial contains lyophilized bovine serum albumin -Diluent 1: one 25 ml vial contains bovine serum albumin. -Diluent 2: one vial contains lyophilized material of human origin -Wash solution (20x): one 50 ml vial -Substrate: one vial -Stop solution: Stop solution is a tacrine solution. For TNF-#, Samples and calibrators are incubated in the microtiter plate coated with the first monoclonal antibody anti-tnf-#, in presence of the second anti-tnf-# monoclonal antibody linked to alkaline phosphatase. After incubation, the wells were washed and the bound enzymatic activity is detected by addition of a chromogenic substrate. -Calibrator: one vial contains lyophilized bovine serum albumin -TNF-# conjugate: one vial contains lyophilized bovine serum albumin -Diluent 1: one 25 ml vial contains bovine serum albumin. -Diluent 2: one vial contains lyophilized material of human origin. -Wash solution (20x): one 50 ml vial -Substrate buffer: one 30 ml vial of diethanolamine-hcl solution. -Substrate: two tablets -Stop solution: one 6 ml vial of NaOH 1N solution. 7. Page 6: Creatinine measurements using colorimetric assays often use the Jaffe method. It is well established that rodent serum contains chromogens that interfere with the read-out, and exaggerate creatinine levels due to high background levels. Which method was used here? According to the kit, it is Jaffe method but this effect was precluded by the use of sham group and so the increase in creatinine level occurred in all groups of study. 8. Page 6: Please provide more details on the method used to measure MDA,

5 such as reagents, incubation steps, wavelength used for measurements etc. Preparation of TBA reagent: gm of TBA was added to 75 ml of DW gm of trichloroacetic acid (TCA) were added to DW ml of 11.9 N Hydrochloric acid (HCL) were added to DW. - The solution completed up to 100ml of DW. Procedure: -1 ml of kidney homogenate was added to 2ml of TBA reagent and mixing. -The mixture was heated by water bath at (100 o C) for (15 min). -Cooled and then centrifuged at 3000 rpm for (10 min). - Light absorbance of clear supernatant was determined at 535 nm against blank using spectrophotomer. Calculation The concentration of MDA = : Extinction coefficient = M-1 Cm-1 D: Dilution factor The results were expressed as nmol MDA/g tissue. Results 9. Figure 2: The right figure does not show creatinine concentrations. Units in the figure legend are incorrect (pg/ml instead of mg/dl or um). 10. Figure 4: Figure 4A shows part of the cortex (glomeruli) whereas the other figures show sections of the corticomedullary area. Please show a similar section of the kidney as the other figures. Level of interest: An article whose findings are important to those with closely related research interests Quality of written English: Not suitable for publication unless extensively edited done 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 All were done Reviewer: Silvio Tucci, Jr. Reviewer's report:.

6 Minor essential revisions: 1 - Introduction - very extensive; it should be reduced. Some paragraphs were reduced 2 - Results - Figure 2 (error bar chart) it looks that MDA chart is displayed twice; the same chart is displayed in Figure a and Figure 3, and the creatinine chart is not displayed. 3 - Page 9 - Figure 8: there is no Figure 8; is it Figure 5? 4 - reference list - very extensive (93 references). This is OK for a review article. I believe that the list can and must be reduced. Some references were deleted Level of interest: An article whose findings are important to those with closely related research interests Quality of written English: Needs some language corrections before being Published done 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 interest's Reviewer: Hossein Ali Arab Reviewer's report: Minor Essential Revisions A: Abstract 1- The abstract is not organized by the style of the Journal. It must be structured into separate sections of Background, Methods, Results and Conclusions 2- The abstract is more than 350 words in length 3- The introduction (background) is too long and it must be reduced to 3-4 Sentences B: Introduction 1- Generally, the authors are used a complicated literature. It s better to use simple and clear statements for witting, ie: a. 1th Paragraph: second sentence is too long and ambiguous, it should be separated into 2 sentences

7 b. 2th paragraph: the first sentence is also too long and must be changed more than one sentences c. etc.,,,,,,,, 2-2th paragraph, line 11: change neutrophil to Neutrophi Changed 3th paragragh, line 7: it is stated that Prevention or reduction of nitric oxide generation reduces.. It is necessary to clear the source of NO generation (inos or cnos) inos 3- The reason for choosing thyroid hormone analogue and a leukotrienes pathway blocker together is not explained in any part of the manuscript. We try to see If DITPA have any anti-inflammatory effect and so we use it together with MK-886 which has a proven anti-inflammatory action. C:Results 1- Change the title of "Effects on kidney parenchyma" to "Effects on histology" 2- Omit the title of grouping finding and summerize this section (effects on histology) into 1-2 paragraph 3- Error bar charts is not drawn appropriate, the chart of different groups should be separated from each other 4- Put Photomicrographs from each groups in In fig.4 D:Discussion 1- This part is too long and presented in many separated sections that is not appropriate. It should be combined into a single section or maximum 2-3 related section 2- There is not any conclusion at the end of discussion E: References: There is too many references, the numbers should be reduced All were done - Discretionary Revisions The recommendation for title is: Effects of thyroid hormone analogue and a leukotrienes pathway blocker on renal ischemia/reperfusion injury in mice Level of interest: An article of importance in its field

8 Quality of written English: Needs some language corrections before being published 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' below

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