Evaluation of Early and Delayed Meloxicam Treatment Against Regulated Cell Death Pathways and ERK1/2 Phosphorylation in a Rat Model of Renal Ischemia–Reperfusion Injury

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates whether meloxicam, a COX-2-selective NSAID, protects the rat kidney against ischemia–reperfusion (I/R) injury, and whether the timing of administration (at the onset vs. 1 h after the onset of reperfusion) affects the outcome. Using a 40 min bilateral ischemia / 120 min reperfusion model in four groups (Sham, IR, Melox+IR, Melox+IR1; n = 6 each), the authors report reduced histological tubular injury, lower serum creatinine and TOS, suppression of IL-6/IL-1β with preserved IL-10, and reduced immunolabeling of KIM-1, Caspase-3, RIPK3, MLKL and pERK1/2, with the early-administration group generally showing the stronger effect. The topic fits the journal scope and the breadth of endpoints is a strength. However, several issues affect the central claims and should be addressed.

Major points

1. Ferroptosis is not adequately demonstrated. Ferroptosis is assessed solely through ERK1/2 and pERK1/2. pERK1/2 is a general MAPK signaling node, not a specific or canonical marker of ferroptosis, which is defined by iron-dependent lipid peroxidation. Without direct evidence (e.g., GPX4, SLC7A11/xCT, ACSL4, FTH1, tissue iron, GSH/GSSG, or lipid-peroxidation products such as MDA or 4-HNE), the conclusion that meloxicam “modulates ferroptosis” is unsupported. Please add appropriate ferroptosis readouts or substantially temper the ferroptosis claims in the title, abstract, results and conclusions.
2. The definition and timing of the treatment groups are internally contradictory. Section 2.1 labels the groups “1st-Hour Reperfusion” (Melox+IR) and “2nd-Hour Reperfusion” (Melox+IR1), whereas the abstract and Figure 1 state Melox+IR received the drug at the onset of reperfusion and Melox+IR1 at 1 h after onset. More importantly, page 4 states that “immediately before clamp removal at the end of the ischemic period, rats in the treatment groups received meloxicam … following meloxicam administration, the clamps were removed to initiate reperfusion,” implying that BOTH treatment groups were dosed before reperfusion began — which contradicts the delayed-dosing definition of Melox+IR1. The dosing time in each group must be stated unambiguously and made consistent across all sections. The label “IR1” is also non-intuitive; renaming to reflect timing (e.g., Melox-0h vs. Melox-1h) would help.
3. The mechanistic evidence rests entirely on semi-quantitative (0–4) IHC/IF grading. No orthogonal quantitative method (Western blot densitometry, qPCR, or ELISA) is provided for any cell-death or signaling marker. For claims about three distinct regulated cell-death pathways, single-modality ordinal scoring is limited. At minimum this limitation should be stated; ideally, key markers (RIPK3/MLKL, cleaved Caspase-3, pERK1/2) should be confirmed by an independent quantitative assay. The title (“Modulation of … Pathways”) overstates what correlative immunolabeling can establish.
4. Statistical reporting is incomplete and partly inappropriate. (a) The comparisons underlying the significance symbols (*, **, #, ##) are never defined in the legends (“significant differences between groups” — relative to which group?); each comparison must be specified. (b) The Methods state biochemical data were analyzed with “Student’s t-test and one-way ANOVA”; for four groups, repeated pairwise t-tests inflate the type I error rate. ANOVA with a clearly named post-hoc multiple-comparison test should be used, and the exact test producing each p-value stated. (c) No normality/variance testing is reported to justify parametric vs. non-parametric choices. (d) With n = 6/group, please report whether the sample size was justified, give exact p-values, and ideally overlay individual data points on the bar graphs.
5. The pharmacological rationale needs strengthening, particularly the COX-2 paradox. COX-2-derived prostaglandins contribute to renal hemodynamic autoregulation, and NSAIDs/COX-2 inhibitors are classically associated with reduced renal perfusion and nephrotoxicity, especially in the stressed kidney. The manuscript should explicitly address why COX-2 inhibition is protective rather than harmful here and discuss the proposed COX-independent/pleiotropic mechanisms with greater specificity.
6. The conclusions overstate the translational implications. The study uses a single dose (1 mg/kg), a single short reperfusion window (120 min), and no dose–response or longer-term functional/survival follow-up. Describing meloxicam as a “promising, accessible, and potent pharmacological strategy” for high-risk surgery and transplantation is not supported by these data and should be tempered. A dedicated limitations paragraph is needed (short reperfusion, single dose/time points, semi-quantitative readouts, absence of urinary biomarkers, COX-2 nephrotoxicity context).
7. The BUN/creatinine discrepancy is not convincingly explained. BUN did not improve with treatment while creatinine did, within the same 120 min window. The explanation that 120 min was “insufficient” is internally inconsistent, since creatinine changed in that same window. A more coherent interpretation should be offered (e.g., BUN is confounded by extrarenal factors such as protein catabolism, volume status, and anesthesia).
8. TAS/TOS interpretation should match the per-group data, and the assay description should be corrected. From Figure 6, TOS in Melox+IR1 appears comparable to IR and the TAS differences are modest with wide error bars; the claim that meloxicam “shifts the balance toward TAS” holds mainly for Melox+IR, not Melox+IR1. Also, TAS/TOS measured with Rel Assay kits are colorimetric/spectrophotometric (Erel method) assays, not ELISA as stated in Section 2.2; please correct.

Minor points

1. “Acute renal damage (ARD)” is non-standard; please use “Acute Kidney Injury (AKI)” per KDIGO and standardize throughout. The abbreviation also appears before it is defined.
2. The corresponding-author email is a placeholder (“e-posta@cumhuriyet.edu.tr”) and must be replaced with a valid address.
3. The IL-10 pattern (highest in Sham, near-absent in IR) deserves comment: please clarify physiologically why uninjured tissue shows the highest IL-10 grade and in what sense meloxicam “preserves” it.
4. KIM-1 is assessed only by tissue IHC; consider acknowledging the absence of urinary KIM-1, its more established compartment.
5. Several mechanistic claims are supported by conference abstracts (e.g., refs 35 and 41). Please replace with peer-reviewed primary sources where possible.
6. Please proofread for stray non-English characters and typos (e.g., “literatüre”; the dotted Turkish “İ” in the ELISA/NSAID entries of the abbreviation list). A careful language edit is recommended.
7. Please verify that every representative IF/IHC image matches the reported grade and the corresponding bar graph (e.g., IL-6 in Melox+IR1, described as severe).
8. Please tabulate all primary antibodies (target, host, vendor, catalog number, dilution) for reproducibility; currently only the secondary detection system is given.
9. Please state randomization and blinding for the biochemical analyses (blinding is described only for histology/IF) and report any animal mortality or exclusions.
10. The Figure 1 schematic appears to carry a “NotebookLM” watermark. Please confirm the originality/licensing of this figure, remove the watermark, and ensure the depicted directionality reflects data actually generated in this study.

Overall assessment

The question is relevant and the breadth of endpoints is a clear strength, and the core observations (reduced tubular injury and lower creatinine with meloxicam, and a generally stronger effect of early administration) are internally consistent across the histological, biochemical and immunolabeling endpoints. The points above are intended to be constructive: the two that most affect the central message are the ferroptosis interpretation (point 1) and the contradictory description of the treatment-group dosing times (point 2), and addressing these—together with the statistical reporting and a more measured framing of the conclusions—would substantially strengthen the manuscript.

Author Response

RESPONSE TO THE RESPECTED REVIEWER 1

Dear Reviewer,

We would like to express our sincere gratitude for your highly constructive comments, valuable insights, and precise suggestions regarding our manuscript. We deeply appreciate and value every single point raised during the peer-review process. We firmly believe that your expert feedback has significantly improved the scientific quality, clarity, and overall depth of our study.

Accordingly, we have carefully addressed and implemented all the requested revisions in a point-by-point manner. To facilitate your evaluation, all revisions, corrections, and quantitative additions (including the requested percentage and fold-change values) in the revised manuscript have been highlighted in yellow.

This research is of immense importance to us, representing a project into which we have poured a substantial amount of personal effort, dedication, and intensive laboratory work. We are fully aware of the inherent limitations of our study. It is important to note that this investigation was conducted entirely within our own individual capacities and institutional means, without the support of any external funding or financial grants. Despite these resource constraints, we have strived to maintain the highest possible methodological and ethical standards to ensure a meaningful contribution to the literature.

Below, please find our point-by-point responses to your specific comments and queries. Once again, we thank you for your time, effort, and invaluable guidance in helping us elevate the standard of our work.

Sincerely,

Dr. Mahmut Şahin

On behalf of all authors

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a clinically relevant topic and presents interesting findings on the potential renoprotective effects of meloxicam in renal I/R injury. However, important methodological clarifications, a more balanced interpretation of the results, and a more critical discussion of the study limitations are required.

Major concerns

• In the introduction section, the authors should revise the description of ferroptosis as ERK1/2 activation is not a defining feature, support the knowledge gap with more recent references (3-5 references) and expand the discussion of the crosstalk among apoptosis, necroptosis, and ferroptosis. Why established ferroptotic markers were not used (e.g. GPX4, SLC7A11, ACSL4, Fe2⁺ iron content).
• Provide additional methodological details, including blinding procedures, antibody information and the statistical analyses. For example,

The body temperature 40 ⁰C appears unusually high for rats as normothermia is typically maintained at 37-38⁰C. Justify the selected temperature.

The authors should provide the catalog number of all assay kits used.

The level of significance between groups in Figures is not clear (*, **, # denote significant differences between groups; ns: Not significant), especially the authors did not describe whether post hoc multiple tests were performed.

Specify the post hoc test used after ANOVA or Kruskal-Wallis test. Report whether data were represented as mean ± SD or mean ± SE.

Clarify if the histopathological and immunostaining evaluations were performed by blinded histopathologist.  

• Moderate the mechanistic conclusions throughout the Discussion, as the data demonstrate associations rather than direct inhibition of specific cell death pathway. Revise the conclusion and avoid causal language such as meloxicam inhibits necroptosis, ferroptosis, and apoptosis.
• The authors should avoid overstating in conclusion. The data demonstrates associations but do not establish that meloxicam directly inhibits necroptosis, ferroptosis, or apoptosis. For example, the claim that meloxicam act as a “powerful modulator” or pleiotropic agent should be softened unless supported by mechanistic experiments.
• As previously mentioned, the discussion of ferroptosis is not sufficiently supported. ERK1/2 and pERK1/2 are not specific or established markers for ferroptosis, therefore conclusions regarding ferroptosis should be interpreted cautiously.
• Expand the comparisons with previous studies investigation on meloxicam or other COX2 inhibitors in Renal I/R injury, highlighting agreements and discrepancies.
• Indicate a dedicated paragraph discussing the study limitations, including reliance on semi-quantitative immunostaining, absence of canonical ferroptosis markers or mechanistic experiments. Additionally, consider discussing limitations related to dose selection, timing of administration, and lack of long-term functional outcomes.
• Describe the potential mechanisms by which meloxicam may regulate oxidative stress and inflammation more critically rather than attributing all observed effects directly to COX-2 inhibition.
• The explanation for the lack of significant BUN reduction is speculative. Consider alternative explanations or support this interpretation with relevant literature.
• Several statements repeat the Results section. The discussion should focus on interpreting the findings and their biological significance rather than restating the data.

Minor concerns

• The English language should be revised throughout the manuscript to correct minor grammatical errors and ensure consistent terminology.
• Update the introduction with more recent references and strengthen the study rationale in the introduction.
• Reduce repetition in the discussion and strengthen comparisons with previous studies.
• Several references should be revised and replaced with more relevant references. Use references that are directly related to renal I/R. Include more recent original studies to strengthen the scientific background.

Comments on the Quality of English Language

The English language should be revised throughout the manuscript to correct minor grammatical errors and ensure consistent terminology.

Author Response

RESPONSE TO THE RESPECTED REVIEWER 2

Dear Reviewer,

We would like to express our sincere gratitude for your highly constructive comments, valuable insights, and precise suggestions regarding our manuscript. We deeply appreciate and value every single point raised during the peer-review process. We firmly believe that your expert feedback has significantly improved the scientific quality, clarity, and overall depth of our study.

Accordingly, we have carefully addressed and implemented all the requested revisions in a point-by-point manner. To facilitate your evaluation, all revisions, corrections, and quantitative additions (including the requested percentage and fold-change values) in the revised manuscript have been highlighted in turquase.

This research is of immense importance to us, representing a project into which we have poured a substantial amount of personal effort, dedication, and intensive laboratory work. We are fully aware of the inherent limitations of our study. It is important to note that this investigation was conducted entirely within our own individual capacities and institutional means, without the support of any external funding or financial grants. Despite these resource constraints, we have strived to maintain the highest possible methodological and ethical standards to ensure a meaningful contribution to the literature.

Below, please find our point-by-point responses to your specific comments and queries. Once again, we thank you for your time, effort, and invaluable guidance in helping us elevate the standard of our work.

Please see the attachment

Sincerely,

Dr. Mahmut Şahin

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study by Sahin M. et al. investigates the renoprotective effects of the nonsteroidal anti-inflammatory drug meloxicam in a rat model of bilateral renal ischemia-reperfusion (I/R) injury. The authors place particular emphasis on investigating the mechanisms of renal tubular epithelial injury associated with inflammation, apoptosis, necroptosis, and ferroptosis. Although the renoprotective effects of meloxicam are well known, the authors attempted to further investigate the mechanisms of renal injury and evaluate the protective effects of meloxicam in this model. This research area is highly relevant because of the high incidence of ischemic kidney injury. While reading the manuscript, I identified several important issues that I would like to bring to the authors' attention.

Major Comments

1. In experimental studies of kidney diseases in rats and mice, various doses of meloxicam ranging from several milligrams to several tens of milligrams per kilogram have been used. Therefore, it is very important that the authors justify the selection of the 1 mg/kg dose used in the present study. The authors cite reference [27] (line 135), but they do not justify why this particular dose was selected. In addition, Section 2.1 does not indicate whether the Sham and untreated IR groups received a placebo treatment, for example, intravenous physiological saline. This point should be clarified for the Sham and IR groups that did not receive meloxicam treatment.
2. The authors used commercially available ELISA kits to assess total antioxidant status (TAS) and total oxidant status (TOS) (lines 147–150). However, it is very important for readers to understand the details of the specific substrates used in these assays and the biological material in which the analysis was performed (serum and/or kidney tissue homogenates). These details are essential for understanding which antioxidants and oxidation products were evaluated using these tests. Do these assays take into account the activity of antioxidant enzymes? Which antigens are specifically detected in the TAS-TOS ELISA assays?
3. Section 2.4 (Histopathology) contains very general criteria for the assessment of pathological changes in kidney tissue sections. I believe that more precise criteria should be provided for the scoring of each grade of histopathological alteration. The criteria described in line 157 as "absent (0), mild (1), moderate (2), and severe (3)" are rather general and require more detailed explanation. Evaluation of only six microscopic fields appears to be a relatively small number for quantitative or semi-quantitative analysis. It is also important to indicate whether the histopathological evaluation was performed independently by more than one pathologist.
4. The Results section (Chapter 3) is largely descriptive and does not provide quantitative comparisons of the observed effects expressed as percentages or fold changes relative to the control group. The results should be described in greater detail with a more comprehensive quantitative analysis of the observed effects.
5. Some of the results presented in the manuscript are not described or discussed in the Results section. In particular, the authors do not describe the findings regarding total antioxidant status (TAS) and total oxidant status (TOS) in the serum of rats subjected to renal I/R and treated with meloxicam, as presented in Figure 6 (page 8). It is also important to indicate whether similar changes in TAS/TOS were observed in kidney tissue homogenates.

Minor Comments

1. In Figure 1 (page 3), meloxicam appears to be administered intramuscularly (into the thigh) or into the dorsal region of the rat. However, the authors state that the drug was administered intravenously via the lateral tail vein. The route of administration shown in the figure should be corrected to avoid confusion regarding the method of meloxicam administration.
2. The authors do not report whether postoperative mortality occurred during the study. Please indicate whether any animals died after surgery or whether survival was 100% in all experimental groups.
3. The figure legends should be supplemented with information specifying the biological material used for the biochemical analyses, as well as the corresponding experimental conditions. Providing only the name of the assay is insufficiently informative for the reader and should be clarified.

Author Response

RESPONSE TO THE RESPECTED REVIEWER 3

Dear Reviewer,

We would like to express our sincere gratitude for your highly constructive comments, valuable insights, and precise suggestions regarding our manuscript. We deeply appreciate and value every single point raised during the peer-review process. We firmly believe that your expert feedback has significantly improved the scientific quality, clarity, and overall depth of our study.

Accordingly, we have carefully addressed and implemented all the requested revisions in a point-by-point manner. To facilitate your evaluation, all revisions, corrections, and quantitative additions (including the requested percentage and fold-change values) in the revised manuscript have been highlighted in green.

This research is of immense importance to us, representing a project into which we have poured a substantial amount of personal effort, dedication, and intensive laboratory work. We are fully aware of the inherent limitations of our study. It is important to note that this investigation was conducted entirely within our own individual capacities and institutional means, without the support of any external funding or financial grants. Despite these resource constraints, we have strived to maintain the highest possible methodological and ethical standards to ensure a meaningful contribution to the literature.

Below, please find our point-by-point responses to your specific comments and queries. Once again, we thank you for your time, effort, and invaluable guidance in helping us elevate the standard of our work.

Please see the attachment

Sincerely,

Dr. Mahmut Şahin

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for their careful, point-by-point response. The revision addresses the major concerns from my first review.

The ferroptosis claim has been appropriately toned down — the title and abstract now frame the findings around ERK1/2/pERK1/2 signaling rather than asserting ferroptosis modulation without specific markers (GPX4, SLC7A11/ACSL4, etc.), which is a more defensible interpretation of the data. The previously contradictory description of meloxicam dosing timing between Melox+IR and Melox+IR1 has also been clarified and is now internally consistent.

On the statistics, the authors have replaced repeated pairwise t-tests with ANOVA/Tukey and Kruskal-Wallis/Dunn's as appropriate, and added quantitative fold-change values that strengthen the semi-quantitative results. The TAS/TOS methodology has been correctly described as colorimetric (Erel method) rather than ELISA, and a Limitations section now transparently acknowledges the semi-quantitative approach, single-dose design, and lack of urinary KIM-1 data. Minor issues (abbreviation standardization, placeholder email, Figure 1 disclosure) are also resolved.

The discussion of the COX-2 paradox and the BUN–creatinine discrepancy is still a bit thin and could be tightened further, but this doesn't rise to the level of blocking publication.

Overall, the manuscript has been sufficiently improved to warrant publication in Biomedicines.

Author Response

Dear Reviewer,

We are profoundly grateful for your careful re-evaluation of our manuscript and for your encouraging final assessment. Your constructive feedback during the initial review round was truly pivotal in reshaping and significantly strengthening the scientific foundation, terminology, and methodological clarity of our study.

We sincerely appreciate your concluding remarks regarding the discussion section. While we have maintained the current text to preserve the streamlined structure approved in this final stage, we fully acknowledge your valuable perspective on the complexities of the COX-2 paradox and the BUN–creatinine dynamics. These insights will undoubtedly serve as excellent guidance for our future research directions.

Thank you once again for your immense contribution, critical insights, and for endorsing our manuscript for publication in Biomedicines. We wish you continued success in your professional endeavors.

Sincerely,

Dr. Mahmut Şahin

On behalf of all authors

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been sufficiently improved and suitable for publication

Author Response

We would like to express our deepest gratitude for your valuable time, meticulous evaluation, and highly constructive feedback throughout the peer-review process. Your expert guidance during the initial round of reviews has been instrumental in significantly elevating the scientific quality, accuracy, and overall presentation of our manuscript.

We are truly honored and encouraged by your final endorsement and positive recommendation for publication in Biomedicines. Thank you once again for your immense contribution to our work, and we wish you continued success in your academic and professional endeavors.

Sincerely,

Dr. Mahmut Şahin

On behalf of all authors

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have provided comprehensive and well-reasoned responses to all of the comments raised in my previous review. In my opinion, the manuscript has been substantially improved following revision.

I have only one remaining minor comment regarding the quality of the histological images presented in Figure 3 (page 8). The hematoxylin and eosin (H&E)-stained sections exhibit an atypical staining pattern. In particular, the hematoxylin staining appears insufficient, as the cell nuclei are poorly visualized, while the sections are predominantly stained in various shades of pink by eosin. If possible, I would recommend replacing these images with more representative H&E micrographs or improving the color rendering of the existing images, provided that this issue is related to image processing rather than to the original staining protocol.

Apart from this minor point, I have no further comments. I would like to thank the authors for their constructive responses and for the considerable improvements made to the manuscript. I wish them every success with their work.

Author Response

We sincerely thank the Reviewer for their encouraging final assessment and for their exceptionally keen eye regarding the histological presentation in Figure 3.

As the Reviewer accurately identified, the atypical, predominantly pink appearance was indeed an unintended consequence of digital image post-processing and formatting adjustments, rather than any deficiency in the underlying H&E staining protocol.

In strict accordance with your excellent recommendation, we have maintained the exact same original and representative fields of view but have carefully optimized the digital saturation and contrast parameters of the micrographs in Figure 3. Through this fine-tuning, the color rendering has been successfully corrected: the hematoxylin-stained cell nuclei are now clearly and distinctively visualized in their proper blue/purple tones, establishing a balanced contrast with the eosin counterstaining.

Please note that this updated figure has been fully integrated into page 8 of the revised manuscript, and the high-resolution standalone image file has also been successfully uploaded directly to the journal submission system.

However, if the Reviewer feels that this digital adjustment is still insufficient to meet the journal's highest standards, we remain fully prepared to completely re-photograph these specific sections from our original slides and replace the figures entirely.

Thank you once again for your invaluable guidance that has greatly elevated the visual and professional quality of our work, and for your kind wishes for our success.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

I have carefully reviewed the revised manuscript. The authors have presented Figure 3 on page 8 in a new version with satisfactory quality suitable for publication. Therefore, I recommend manuscript biomedicines-4404728 for publication.