F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.174653.1 Research Article Articles The Potential Mitigating Effect of Curcumin Root Extract Against Synthetic Anabolic Steroid Boldenone-Induced Kidney Injury in Male Rats

[version 1; peer review: 2 approved with reservations]

 Neamah Asmaa M. Validation https://orcid.org/0000-0002-8232-7917 1 Alkadhimy Sura M. Data Curation Methodology https://orcid.org/0000-0002-5235-886X 2 Abbas Ruwaidah A.R. Investigation Resources https://orcid.org/0000-0002-8125-6053 3 Al-Hamadani Mohammed Y.I. Formal Analysis Writing – Review & Editing https://orcid.org/0009-0003-4957-2069 a 4 Yousef Mokhtar Ibrahim Project Administration Supervision 5 Department of Environment, College of Science, University of Al-Qadisiyah, Al Diwaniyah, Iraq Department of Biology, College of Science, University of Karbala, Karbala, Iraq Department of Pathological Analysis, College of Applied Sciences, University of Fallujah, Al-Fallujah, Iraq Department of Biology, College of Education, University of Fallujah, Al-Fallujah, Iraq Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt mohammed.y.issa@uofallujah.edu.iq

No competing interests were disclosed.

 16 1 2026 2026 15 64 5 1 2026 Copyright: © 2026 Neamah AM et al. 2026 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

The anabolic-androgenic steroid boldenone is widely abused and is strongly linked to nephrotoxicity, primarily mediated through oxidative stress and inflammation. Curcumin, a natural polyphenol, is renowned for its potent antioxidant and anti-inflammatory properties, but its protective role against boldenone-induced renal injury is not fully elucidated. This study aimed to investigate the potential nephroprotective effects of curcumin against boldenone-induced kidney damage in a rat model, focusing on biochemical parameters, oxidative stress markers, inflammatory response, and histological changes.

 Methods

Thirty-two male Wistar rats were divided into four groups (n=8): control, curcumin-only (300 mg/kg/day), boldenone-only (5 mg/kg/week), and boldenone + curcumin. Over the experimental period, body weight, kidney weight, food/water intake, and serum renal function markers (urea, creatinine, electrolytes) were monitored. Renal tissue was analyzed for oxidative stress markers (MDA, GSH, CAT, SOD), the inflammatory cytokine TNF-α, and histological alterations.

 Results

Boldenone administration significantly increased body and kidney weights, serum urea, creatinine, potassium, and chloride, while decreasing sodium and calcium. It induced marked oxidative stress (elevated MDA, depleted GSH, CAT, SOD) and inflammation (elevated TNF-α), resulting in significant tubular necrosis and hypertrophy. Co-treatment with curcumin substantially mitigated these abnormalities, normalizing serum markers, restoring antioxidant defenses and electrolyte balance, reducing TNF-α expression, and ameliorating histological damage.

 Conclusion

Curcumin exerts significant nephroprotection against boldenone-induced toxicity through its antioxidant, anti-inflammatory, and cytoprotective mechanisms. These findings justify its exploration as a potential adjunct therapy to prevent steroid-associated kidney injury. Future studies should focus on elucidating the precise molecular signaling pathways involved and validating these effects in clinical scenarios of AAS abuse.

 Anabolic-androgenic steroids Boldenone Curcumin root extract Kidney injury Oxidative stress TNFα expressions Nephroprotection Male Wistar rats Renal toxicity Antioxidant therapy Steroid-induced nephrotoxicity. The author(s) declared that no grants were involved in supporting this work. Introduction

Anabolic-androgenic steroids (AASs) are artificial analogs of testosterone that increase its anabolic effects and reduce androgenic properties ( Fadah et al., 2023; Hussain et al., 2024). Their ability to stimulate muscle protein synthesis and increase lean body mass has enabled them to gain popularity among athletes who are interested in performance enhancement. In addition to sports, AASs have also found applications in veterinary medicine to stimulate muscle development in animals (racehorses and working dogs) ( Borodi et al., 2020; Islam et al., 2022; Ayubi et al., 2023).

Boldenone is one of these substances widely used as a growth promoter in animal rearing to increase weight gain and lower breeding expenses, which has questioned the ethical and safety issues about food quality and human exposure ( El-Moghazy et al., 2012; Tousson et al., 2012, 2016; Getabalew et al., 2020). Despite the boldenone being a Schedule III controlled substance rated to be used in veterinary practice, it has been used by athletes and bodybuilders illegally to build muscle. This abuse results in high systemic boldenone concentrations, which are frequently irreversible or irreparable organ damage especially nephrotoxicity.

As the abuse of AASs is increasingly becoming a widespread issue, the need to find natural compounds and other drugs with the ability to overcome their negative effects is on the rise ( Abd El-Zaher et al., 2025; Abosharaf et al., 2025). Phytochemicals possessing strong antioxidant and anti-inflammatory activities have been of particular interest as to their therapeutic purpose ( Essawy et al., 2022; Shalaby et al., 2023; Hasan et al., 2024). Curcumin is one of such compounds that are polyphenolic pigments of Curcuma longa (turmeric) which have extensive biological effects, such as anti-inflammatory, antioxidant, antimicrobial, and anticancer impacts ( Wang et al., 2012; Alotaibi et al., 2021; Islam et al., 2022; Radwan et al., 2024).

The cytoprotective and antioxidant properties of curcumin involve the scavenging of reactive oxygen species (ROS), the control of the inflammatory response, and the stimulation of home antioxidant defense mechanisms ( Samarghandian et al., 2017; Damiano et al., 2020). These attributes indicate that curcumin is possible to relieve boldenone-induced kidney oxidative stress and damage.

Thus, the hypothesis of the present research is that curcumin root extract will help to prevent oxidative stress and kidney damage as caused by boldenone in male rats. It is aimed at testing the possible nephroprotective effect of curcumin and giving information about the possible ways of its use in the treatment of anabolic steroid-induced nephrotoxicity.

 Materials and methods Animal model and housing

Thirty-two healthy male Wistar albino rats (180–200 g, 12–16 weeks old) were obtained from the Drug Control Center in Baghdad, Iraq. Animals were acclimatized for two weeks under controlled laboratory conditions (22 ± 2 °C, 50–70% relative humidity, and a 12-hour light/dark cycle). During acclimation, rats were provided with a standard laboratory diet and water ad libitum.

All procedures were performed in accordance with the International Council for Laboratory Animal Science (ICLAS) Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Fallujah University, College of Applied Sciences (Ethical approval no. UOF.CAS.06-240311).

Experimental design

Rats were randomly assigned to four equal groups (n = 8):

Group 1 (Control): Received no treatment.

Group 2 (Curcumin): Administered curcumin orally (300 mg/kg body weight/day for 2 weeks) following Radwan et al. (2023).

Group 3 (Boldenone): Received intramuscular injections of boldenone (5 mg/kg body weight/week for 4 weeks) according to Alm-Eldeen and Tousson (2012).

Group 4 (Boldenone + Curcumin): Exposed to boldenone for 4 weeks, followed by 2 weeks of oral curcumin supplementation (300 mg/kg/day).

Throughout the experiment, body weight, food intake, and water consumption were recorded weekly. The following formula was used to get the relative kidney weight (RKW): RKW = Kidney weight Body weight × 100

Blood and tissue collection

After overnight fasting (10–12 h), rats were anesthetized with pentobarbital and euthanized. To ensure total sedation of the rats before euthanasia, all rats were anesthetized by injecting them with pentobarbital at a concentration of 50 mg/kg body weight into the peritoneal cavity. Loss of pedal reflex and loss of corneal reflex were used to confirm the depth of anesthesia. After anesthesia, animals were exsanguinated by cardiac puncture. Every practice was carried out with compliance with the American Veterinary Medical Association (AVMA) Guidelines to the Euthanasia of Animals (2020) and the Institutional Animal Care and Use Committee (IACUC) of Fallujah University, College of Applied Sciences (Ethical approval no. UOF.CAS.06-240311).

Blood samples were collected via the inferior vena cava into non-heparinized tubes, allowed to clot, and centrifuged at 860 × g for 20 min. Serum was separated and stored at –80 °C for biochemical assays.

Kidneys were excised, washed in chilled 0.9% saline, and cleared of fat and connective tissue. A 10% (w/v) tissue homogenate was prepared using a Potter–Elvehjem homogenizer in ice-cold 0.01 M sodium phosphate buffer (pH 7.4) containing 1.15% KCl. Homogenates were centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatant was stored at –20 °C for further biochemical analysis.

Kidney function and electrolyte assessment

Serum creatinine and urea were estimated according to Patton and Crouch (1977). Electrolytes (sodium, potassium, calcium, and chloride) were measured using commercial diagnostic kits (Sensa-core, India) as described by Abd Eldaim et al. (2019).

Antioxidant and oxidative stress assays

Renal oxidative stress and antioxidant parameters were determined as follows:

Malondialdehyde (MDA): Estimated as a marker of lipid peroxidation using a Biodiagnostic kit (Egypt) following Mesbah et al. (2004).

Reduced Glutathione (GSH): Measured by the method of Teare et al. (1993) using 5,5′-dithiobis-(2-nitrobenzoic acid) and expressed as μmol GSH/g tissue.

Catalase (CAT): Activity determined spectrophotometrically by monitoring the decomposition of H 2O 2 at 240 nm, following Saggu et al. (2014).

Superoxide Dismutase (SOD): Activity assessed using the inhibition of adrenaline auto-oxidation according to Misra and Fridovich (1972).

 Histopathological examination

Kidney specimens from different groups were isolated, fixed in 10% neutral buffered formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin after Tousson (2016).

 Immuno-histochemical detection

Following Tousson et al. (2025) immuno-labeling of tumor necrosis factor alpha (TNFα) were 5 μm kidney sections onto positively charged slides. Distribution of TNFα stained cytoplasm were examined in deparaffinized sections using an Avidin–Biotin–Peroxidase immunohistochemical method (Elite–ABC, Vector Laboratories, CA, USA) with TNFα monoclonal antibody (dilution 1:100; DAKO Japan Co, Tokyo, Japan).

 Statistical analysis

Data were expressed as mean ± standard error (SE). Statistical comparisons among groups were performed using one-way analysis of variance (ANOVA), followed by appropriate post-hoc testing. Differences were considered statistically significant at p < 0.05.

In figures and tables, symbols (*) and (#) denote significant differences compared to the control and boldenone-treated groups, respectively.

 Results

Table 1 presents the effects of boldenone (ASB) administration and curcumin treatment on body weight, relative kidney weight, and feed and water consumption. Rats treated with boldenone showed a significant increase in body and relative kidney weights, as well as in feed and water intake, compared to the control and curcumin-only groups. However, post-treatment with curcumin (ASB + Cur) markedly ameliorated these alterations, demonstrating a normalizing effect and indicating that curcumin mitigates the physiological disturbances induced by boldenone.

Effects of Curcumin and Boldenone, Alone and in Combination, on Physiological Parameters in Rats: Water Intake, Feed Intake, Relative Body Weight (RBW), and Relative Kidney Weight (RKW).
Parameter Experimental groups
Control Cur ASB ASB + Cur
Water intake (ml/rat/day) 27.4 # ± 1.68 29.2 # ± 1.55 42.4* ± 2.38 33.6 # * ± 1.84
Food intake (g/rat/day) 12.6 # ± 0.71 12.4 # ± 0.84 19.4* ± 1.09 12.9 # ± 0.87
RBW (g/100 g) 27.0 # ± 1.41 25.3 # ± 1.45 44.0* ± 2.81 38.3 # * ± 1.75
RKW (g/100 g) 2.46 # ± 0.16 2.39 # ± 0.16 4.14* ± 0.19 2.70 # ± 0.20

The findings are shown using the mean ± SE. * and # denote significant differences from the ASB group (p < 0.05) and a significant change from the control group (p < 0.05), respectively.

Table 2 shows a remarkable increase in serum creatinine and urea levels of the rat subjected to boldenone relative to the control and curcumin-treated rats indicating a failure of the kidneys to perform their functions. Conversely, simultaneous administration of curcumin with boldenone (ASB + Cur) decreased both parameters substantially as compared to the ASB group implying a nephroprotective effect of curcumin on boldenone-induced kidney malfunction.

Alterations in the serum urea and creatinine levels of rats given boldenone (ASB), curcumin (Cur.), or both.
Parameter Experimental groups
Control Cur ASB ASB+Cur
Creatinine (mg/dl) 0.61 # ± 0.05 0.59 # ± 0.03 0.98* ± 0.05 0.65 # ± 0.04
Urea (mg/dl) 23.0 # ± 0.90 24.5 # ± 0.89 37.2* ± 1.15 27.0 # ± 1.26

The findings are shown using the mean ± SE. * and # denote significant differences from the ASB group (p < 0.05) and a significant change from the control group (p < 0.05), respectively.

Table 3 shows the impact of boldenone and curcumin on serum levels of electrolytes. The administration of boldenone resulted in a significant reduction in sodium and calcium and a significant increase in potassium and chloride and a significant decrease in chloride levels, compared to the control and curcumin groups. On the other hand, ASB + Cur co-treatment leveled these electrolytes significantly, increasing the levels of sodium and calcium, decreasing those of potassium and chloride levels compared to the ASB. These results show that curcumin is a good solution to the electrolyte imbalances which boldenone causes.

Changes in the levels of electrolytes of rats treated with curcumin (Cur.), boldenone (ASB), and their combination.
Parameter Experimental groups
Control Cur ASB ASB + Cur
K + (mmol/l) 3.85 # ± 0.16 3.65 # ± 0.21 4.35* ± 0.18 4.04* # ± 0.22
Na + (mmol/l) 138.5 # ± 5.42 139.9 # ± 4.41 131.0* ± 6.12 138. 5 # ± 5.25
Ca ++ (mmol/l) 1.22 # ± 0.10 1.21 # ± 0.09 1.16* ± 0.08 1.21 # ± 0.09
Cl - (mmol/l) 102.0 # ± 8.36 101.5 # ± 9.01 112.5* ± 8.55 102.0 # ± 9.45

The findings are displayed using the mean ± SE. A significant difference from the ASB group (p < 0.05) is shown by #, and a significant change from the control group (p < 0.05) is indicated by *.

According to Table 4, the boldenone treatment had a significant effect on the renal malondialdehyde (MDA) levels, significantly reduced the concentration of glutathione (GSH) and activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) in comparison with the control and curcumin groups. Nevertheless, the co-treatment with curcumin (ASB + Cur) had a significant effect on decreasing the lipid peroxidation rates by lowering the level of MDA and, at the same time, returning the GSH, CAT, and SOD levels to almost normal levels in comparison with the ASB group. These findings validate the assumptions that curcumin has strong antioxidant and cytoprotective effects in boldenone-induced oxidative renal injury.

Changes in enzymatic and non-enzymatic antioxidant parameters of rats treated with curcumin (Cur.), boldenone (ASB), and their combination.
Parameter Experimental groups
Control Cur ASB ASB+Cur
MDA (nmole/g tissue) 30.5 # ± 1.25 26.8 # ± 1.50 60.5* ± 2.75 33.4 # ± 1.31
GSH (mmol/mg protein) 3.25 # ± 0.11 3.38 # ± 0.09 1.18* ± 0.11 3.12 # ± 0.12
CAT (U/mg protein) 36.3 # ± 1.45 42.5 # ± 2.91 18.5* ± 1.04 33.5 # ± 1.33
SOD (U/mg protein) 40.0 # ± 2.40 44.8 # ± 2.35 20.5* ± 1.45 38.5 # ± 2.16

The findings are displayed using the mean ± SE. A significant difference from the ASB group (p < 0.05) is shown by #, and a significant change from the control group (p < 0.05) is indicated by *.

 Impact of Cur on histopathological examination

Normal architecture with normal renal tubules and glomeruli were detected in kidney sections in both control and Cur groups ( Figure 1A & 1B). Conversely, kidney sections in treated rats with ASB revealed severe hypertrophy in tubular cells and marked necrotic tubular cells ( Figure 1C). Treated rats with Cur after ASB treatments (ASB+Cur) showed a good degree of kidney improvements with only mild hypertrophy in glomeruli and tubular cells ( Figure 1D).

Photo-micrographs of H&E -stained kidney sections.

A&B: Normal structure of glomeruli (G) and renal tubules (RT) in control and Cur groups. C: Severe hypertrophy in tubular cells and marked necrotic tubular cells in treated rats with ASB. D: Only mild hypertrophy in glomeruli and tubular cells in ASB+Cur.

 Impact of Cur on TNFα expressions

Figure 2 revealed the changes in TNFα expressions in kidney sections in different groups. Negative or faint positive reactions were detected in control and Cur groups while a moderate positive reaction was detected in ASB group ( Figure 2A- 2C). On the other hand; treated rats with Cur after ASB treatments (ASB+Cur) showed mild positive reaction for TNFα ( Figure 2D).

Photo-micrographs of TNFα expressions in kidney sections (arrows).

A&B: Faint expression of TNFα in the control and Cur groups. C: Moderate reaction in ASB group. D: Mild reaction treated rats with Cur after ASB treatments (ASB+Cur).

 Discussion

The increase in body weight gain observed following boldenone administration in this study aligns with the findings of El-Moghazy et al. (2012), who reported similar effects in male rabbits. This growth was coupled with increasing feed and water consumption and relative kidney weight, which is in agreement with the present findings. Equally, Hoseini et al. (2009) resulted that the body and kidney weights of mice in the study had risen due to the administration of nandrolone decanoate. Conversely, Chang et al. (2021) also established a body and kidney weight increase reduction of curcumin root extract over the control, which confirms our observation of mitigating effect of curcumin. Moreover, Ali et al. (2018) revealed that the treatment with curcumin reduced the relative kidney weight, water consumption, urine production, and weight gain, which additionally confirmed our data.

The changes in serum calcium, sodium, creatinine, urea, potassium and chloride in the current study all point to the fact that boldenone undecylenate growth promoter abuses cause structural and functional impairment of the kidney, which may cause secondary and progressive kidney failure. These results are in agreement with El-Moghazy et al. (2012) and Tousson et al. (2016) studies which found that the use of boldenone led to a marked increase in serum urea and creatinine concentration in rabbits, and that boldenone was indeed nephrotoxic.

In a similar study, Alm-Eldeen and Tousson (2012) showed that the injection of boldenone had significant effect on increasing the serum total protein, urea, and creatinine level and reducing the albumin/globulin (A/G) ratio in male rabbits. These observations are supported by our results and are consistent with the study conducted by Tousson et al. (2016), who demonstrated that hepatic and renal toxicity were severe following the administration of boldenone.

Mechanistically, high concentrations of anabolic steroid have been reported to suppress the Na+/K+ -ATPase activity thus causing calcium and sodium ion to build up in the cell, hence interference with the electrolyte homeostasis. The electrolyte imbalances reported in this study are in line with those reported by Tousson et al. (2018) and Elmasry et al. (2018). There are even suggestions that AASs may be able to damage renal glomeruli directly through the induction of glomerular hyperfiltration and hypertrophy ( Sessa et al., 2020).

The protective effect of curcumin that was realized in this study is in line with what has been reported before. Venkatesan et al. (2000) also showed that curcumin prevented adriamycin-induced nephrotoxicity in rats and Ueki et al. (2013) established that curcumin inhibited renal inflammation and cisplatin-induced nephrotoxicity in mice. Equally, Damiano et al. (2020) stated that curcumin rescued rat kidneys exposed to ochratoxin-induced injury, which also indicates the broad renoprotective nature of curcumin.

The results of this study on the oxidative stress are also consistent with the results reported by earlier researchers that anabolic steroids cause redox imbalance and lipid peroxidation. Dornelles et al. (2017) showed that steroid exposure was associated with heightened hepatic and renal oxidative stress, and Frankenfeld et al. (2014) also described multi-organ damage (liver, heart, kidneys) caused by nandrolone decanoate treatment. The renal toxicity here also favors the nephrotoxic effects of Tsitsimpikou et al. (2016) and cardiac oxidative stress of Kara et al. (2018) after stanozolol use.

The high antioxidant activity exhibited by Curcumin in this case as indicated by the increase of GSH, CAT, and SOD activity and decrease of MDA levels prove that this compound can prevent lipid peroxidation and oxidative stress. It is in line with Tokaç et al. (2013) who emphasized the curcumin action of protecting renal tissue against oxidative and DNA damage. Equally, Samarghandian et al. (2017) and Ali et al. (2018) found similar renoprotective effects in chronic kidney disease and oxidative stress models. Together, these findings support the idea that the curcumin has a potent nephroprotective impact in terms of antioxidant, anti-inflammatory, and cytoprotective properties.

The current paper proves the protective action of Cur against the ASB-induced renal damage through the histopathological changes and the TNF changes in the expression. The ASB exposure led to significant distortion of normal renal architecture with severe tubular hypertrophy and massive necrotic alterations. The results are corroborated by the existing literature that shows that ASB as well as other analogous chemical stressors cause oxidative stress and inflammation, which eventually result in tubular damage and cellular loss of integrity. This is also in accordance with Samarghandian Tousson al. (2025) who concludes that; nandrolone decanote as steroids caused renal toxicity and inflammation in male rats.

 Conclusion

This research shows that the use of boldenone causes serious oxidative stress, biochemical imbalance and kidney dysfunction in male rats. On the other hand, curcumin root extract is capable of overcoming these side effects by improving the antioxidant defenses, decreasing the lipid peroxidation and healing the renal functions. These results imply that curcumin has a high nephroprotective ability as against renal toxicity induced by anabolic steroid. Future studies should also focus on the elucidation of the molecular mechanisms that are involved in the protective effect of curcumin, especially its regulation of oxidative stress, inflammatory, and apoptotic pathways of renal tissue. Such investigations could advance the development of curcumin-based adjunct therapies for preventing anabolic steroid–associated nephrotoxicity in humans.

 Consent to participate

Not applicable.

 Declaration of generative AI Use in the writing process

In the course of drafting this manuscript, the authors engaged the generative AI tool Perplexity during the middle editorial stage to help with the phrasing, and we revisited the text to improve the clarity, fluency, and readability to the text. After AI-assisted revisions, authors reviewed, changed, and sanctioned all the texts to make sure they were correct and complete. Responsibility for the text lies with the author, and the use of AI has not affected the scientific quality and conclusions of the work.

 Data availability statement

The datasets supporting the conclusions of this study, including the completed ARRIVE 2.0 checklist and raw experimental data, are publicly available in the Zenodo repository.

Repository: Zenodo

Title: ARRIVE Checklist and Supporting Data for The Potential Mitigating Effect of Curcumin Root Extract Against Synthetic Anabolic Steroid Boldenone-Induced Kidney Injury in Male Rats

DOI: https://doi.org/10.5281/zenodo.18133897 Neamah et al. (2026)

License: Creative Commons Zero (CC0 1.0 Public Domain Dedication)

The dataset includes:

ARRIVE 2.0 Author Checklist (PDF)

Raw experimental data files (Excel and PDF)

All data are freely accessible without restriction.

 Acknowledgements

This is not applicable for that specific section. The author declares that no additional support, assistance, or acknowledgments are applicable for this study.

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Competing interests: No competing interests were disclosed.

 30 3 2026 Copyright: © 2026 Jiang D 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript investigates the protective effects of curcumin root extract against kidney injury induced by the anabolic-androgenic steroid boldenone in male Wistar rats. The study addresses a highly relevant toxicological issue, utilizing a comprehensive approach that includes biochemical, oxidative stress, inflammatory, and histopathological markers. However, there are significant discrepancies in the reporting of the experimental design, as well as typographical issues in the data tables that must be addressed to ensure scientific accuracy.

 

 There is a major contradiction between the described methodology and the interpretation of the results. In the "Experimental design" section, Group 4 is described as being exposed to boldenone for 4 weeks, followed by 2 weeks of oral curcumin supplementation. This makes it a post-treatment or therapeutic model. However, the Abstract and Results sections refer to this as a "Co-treatment" and a "simultaneous administration". The authors must clarify whether the treatments were given simultaneously or sequentially, and strictly align the terminology throughout the manuscript.

 The statistical notations within Tables 1 through 4 are highly irregular and confusing.

 In the Discussion, the authors state that high concentrations of anabolic steroids suppress Na/K-ATPase activity, leading to electrolyte imbalances. Since the authors did not actually evaluate Na/K-ATPase expression or activity in their methodology, this claim should be moderated and clearly stated as a hypothesis drawn from existing literature, rather than a direct conclusion of this study.

 The "Animal model and housing" and "Experimental design" sections lack details regarding the curcumin root extract. The authors should add a brief clarification regarding the standardization, source, and purity of the curcumin extract used, as this is vital for reproducibility.

 The photomicrographs in Figure 1 have a strong yellow tint, which may indicate issues with color balance or white balance during image capture.

 The internal textual marks ("G" and "RT") placed directly over the tissue structures in Figure 1 obscure the cellular details. It is recommended to use arrows or asterisks instead, explaining them in the figure legend.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Drug development for organ injury, in vivo drug delivery, nanomedicine, biomedical engineering, radiopharmaceuticals, molecular imaging

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.192576.r458721 Reviewer response for version 1 Trujillo Daniela Joyce 1 Referee Instituto Potosino de Investigación Científica y Tecnológica (DMA-IPICYT), San Luis, Mexico

Competing interests: No competing interests were disclosed.

 23 3 2026 Copyright: © 2026 Trujillo DJ 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript evaluates the potential protective effect of curcumin root extract against boldenone-induced kidney injury in male rats through biochemical, oxidative stress, inflammatory, and histological parameters. The topic is relevant, and the results are, in general, consistent with the proposed objective. However, some aspects of the introduction, data presentation, image quality, and discussion should be improved before indexing. Therefore, major revisions are required.

Major points

In the Introduction, the authors should explain in more detail the mechanisms by which boldenone or anabolic steroids may induce nephrotoxicity. In addition, it will strengthen the rationale if they mention whether systemic biomarkers of this toxicity have been previously reported.

The statistical presentation in tables is confusing. The use of the same symbols for all columns makes it difficult to identify the exact comparisons. The statistical notation should be clarified and better explained in each table legend.

The quality of the microphotographs in Figure 1 should be improved. The background appears too yellow, and the authors should clarify whether the contrast or color balance was excessively modified. It is also suggested to remove the internal marks “G” and “RT” from the images.

In the Results section, it would have been valuable if the authors had evaluated Na⁺/K⁺-ATPase expression, especially because this mechanism is later mentioned in the Discussion. If this was not evaluated, the discussion should be moderated accordingly.

In the Discussion, the authors should better justify why studying compounds that may counteract the harmful effects of steroid abuse is scientifically relevant. This would strengthen the translational importance of the study.

The manuscript should more clearly distinguish whether curcumin was used as a co-treatment or as a post-treatment strategy, since this affects the interpretation of the protective effect.

 Minor points

The manuscript would benefit from improvement in English writing and sentence structure in several sections.

The authors should revise the consistency of terms and descriptions across the Results and Discussion sections.

It is suggested to improve the explanation of the histological findings in the figure legends.

The conclusions should be slightly moderated and limited to the experimental model.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

kidney phisiology and metabolic diseases

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.