Expression of Lysyl Oxidase-Related Protein and Effect of Lysyl Oxidase Inhibition in Cyclosporine-Induced Nephropathy Mouse Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, authors have investigated the contribution of Lysyl oxidase-like 2 (LOXL2) inhibitor to cyclosporine A (CsA)-induced nephropathy, in male CD-1 mice as a well-established model of progressive tubulointerstitial fibrosis, after administration of CsA.

The results obtained with appropriate methods seem to indicate that LOXL2 inhibitor treatment can attenuate CsA nephropathy progression and therefore conclude that LOXL2 inhibitor may be a potential therapeutic target in CsA nephropathy.

I only have a few points that could be improved.

For example, it would be better to indicate in the figure captions the number of samples analyzed and subjected to statistical analysis, i.e., the number of mice used for each experiment.

Which LOXL2i, lysyl oxidase-like 2 inhibitor, is this?

It's always referred to simply as LOXL2i, lysyl oxidase-like 2 inhibitor, without a specific name or acronym, not even in the Drugs and Chemicals section, which itself isn't present at all. Is it patented? Is there already a reference bibliography for this inhibitor? Please specify in the text.

 

Author Response

Comment 1: For example, it would be better to indicate in the figure captions the number of samples analyzed and subjected to statistical analysis, i.e., the number of mice used for each experiment.

Answer: We thank the reviewer for pointing out this important issue. Accordingly, we have revised all relevant figure legends to indicate the number of mice used in each experiment. Specifically, the number of animals per group has been added to the figure captions for all figures.

 

Comment 2: Which LOXL2i, lysyl oxidase-like 2 inhibitor, is this? It's always referred to simply as LOXL2i, lysyl oxidase-like 2 inhibitor, without a specific name or acronym, not even in the Drugs and Chemicals section, which itself isn't present at all. Is it patented? Is there already a reference bibliography for this inhibitor? Please specify in the text.

Answer: We thank the reviewer for this important issue. We agree that the identity and background information of the LOXL2 inhibitor used in this study were insufficiently described in the original manuscript. The inhibitor used throughout this study is Compound #765 (6-amino-5-(4-methoxybenzoyl)indolizine-7-carbonitrile), a novel indolizine derivative previously synthesized and characterized by our group [1]. Compound #765 was selected based on its potent and selective inhibitory activity against LOXL2 enzymatic activity, as previously demonstrated in our study [1]. Regarding patent status, Compound #765 is protected under Korean Patent No. 10-2631074 (Application No. 10-2021-0046557; filed April 9, 2021; registered January 25, 2024), entitled "Novel indolizine derivatives and compositions for the prevention or treatment of fibrosis containing the same," assigned to the Industry-Academic Cooperation Foundation of Yonsei University, Seoul, Republic of Korea. To address the reviewer’s concern, we revised the manuscript to clearly specify the inhibitor and added a dedicated “Drugs and Chemicals” subsection in the Materials and Methods section describing the full chemical name, synthesis reference, stock preparation, and storage conditions of Compound #765. We also added the corresponding article in the reference section.

 

Reference

1. Shim DH, Kim MJ, Chung HW, Kim MN, Sohn MH, Lee S, et al. Targeting Lysyl Oxidase-like 2: A Therapeutic Strategy for Idiopathic Pulmonary Fibrosis with a Novel Indolizine Derivative. Pharmaceutics. 2026;18(5):554. PubMed PMID: doi:10.3390/pharmaceutics18050554.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1.The manuscript investigates the role of LOXL2 inhibition in cyclosporine A (CsA)-induced nephropathy using a murine model. The study is relevant and addresses an important gap in targeting extracellular matrix remodeling in kidney fibrosis. The experimental design is generally sound, and the results are promising. However, several aspects require clarification, deeper analysis, and improvement before the manuscript can be considered for publication.
2. While the study demonstrates the beneficial effects of LOXL2 inhibition, the mechanistic insight remains limited. The authors primarily report associations (e.g., reduced TGF-β signaling and MAPK pathway activity) without establishing causality. Additional experiments (e.g., pathway inhibition/activation studies or in vitro validation) would strengthen mechanistic conclusions. 
3. The study includes appropriate control groups; however, the rationale for selecting the LOXL2 inhibitor dose (10 mg/kg/day) is not clearly justified.
Information regarding randomization and blinding procedures during treatment allocation and analysis is insufficient, though partial mention of blinded histological analysis is present. Sample size (n=18 total) appears relatively small; power analysis is not provided.
4. The statistical methods (ANOVA with Tukey post hoc test and t-test) are appropriate, but there is no mention of normality testing or variance homogeneity.
Exact p-values are not consistently reported; reliance on significance markers (*, #) limits interpretability.
5. Figures lack sufficient quantitative detail. For example Western blot results should include full-length blots or uncropped images. Densitometric analysis methods are not described in detail. Units and scales in some graphs are unclear or insufficiently labeled (e.g., Figure 2 panels) .
6. The authors conclude that LOXL2 inhibition may be a therapeutic strategy; however, translational relevance is overstated. The discussion does not adequately address conflicting literature where LOXL2 inhibition showed limited or variable effects in different models .Potential off-target effects or safety concerns of LOXL2 inhibition are not discussed.
7. The study reports reduction in F4/80-positive cells and TUNEL-positive cells; however. quantification methods need clearer description. Additional markers (e.g., caspase activation, cytokine panels) would strengthen conclusions.
8. Several grammatical errors and typographical issues are present throughout the manuscript (e.g., spacing, punctuation, inconsistent use of abbreviations). Terms such as “aĴenuated” appear with formatting issues. Some abbreviations are inconsistently defined (e.g., LOXL2i vs LOXL2 inhibitor). Ensure all abbreviations are defined at first use.
9. Figure legends are generally descriptive but could be improved by including sample size (n), clarifying statistical comparisons, avoiding repetition. More detail is needed for antibody validation and sources, RT-qPCR efficiency and normalization strategy and criteria for histological scoring
10. In page 3–4 under results section, the increase in LOXL2 expression is clearly shown, but fold-change values should be explicitly stated rather than inferred from figures.
11. In page 5–6 under histology, quantification of fibrosis using MT and PSR staining is appropriate; however, inter-observer variability is not addressed.
12. In page 9–10 (signaling pathways), the reduction in p-p38, p-Smad3, and p-ERK1/2 is interesting, but upstream regulators are not explored.

Author Response

Responses to Reviewer 2

The manuscript investigates the role of LOXL2 inhibition in cyclosporine A (CsA)-induced nephropathy using a murine model. The study is relevant and addresses an important gap in targeting extracellular matrix remodeling in kidney fibrosis. The experimental design is generally sound, and the results are promising. However, several aspects require clarification, deeper analysis, and improvement before the manuscript can be considered for publication.

 

Comment 1: While the study demonstrates the beneficial effects of LOXL2 inhibition, the mechanistic insight remains limited. The authors primarily report associations (e.g., reduced TGF-β signaling and MAPK pathway activity) without establishing causality. Additional experiments (e.g., pathway inhibition/activation studies or in vitro validation) would strengthen mechanistic conclusions.  

Answer: We thank the reviewer for this valuable comment. We agree that mechanistic validation was needed to strengthen the causal relationship between LOXL2 inhibition and the observed anti-fibrotic effects. To address this concern, we performed additional in vitro experiments using HK-2 cells. HK-2 cells were treated with CsA (5 µM) in the presence or absence of Compound #765 (LOXL2 inhibitor) at 1 µM, mirroring the four experimental groups used in our in vivo study (Control / Compound #765 / CsA / CsA+ Compound #765). The mRNA expression of key fibrosis-related markers — TGF-β1, α-SMA, and Collagen — was assessed by quantitative real-time PCR and Compound #765 (LOXL2 inhibitor) significantly attenuated this upregulation.

             In line with our in vivo findings, CsA treatment significantly upregulated the mRNA expression of TGF-β1, α-SMA, and Collagen type 1A in HK-2 cells. Importantly, co-treatment with Compound #765 significantly attenuated the CsA-induced upregulation of these fibrosis-related genes. These results provide mechanistic support for the antifibrotic effects observed in our murine model and suggest that Compound #765 exerts its protective effects, at least in part, through direct inhibition of fibrosis-related gene expression in tubular epithelial cells.

These additional in vitro experimental details have been incorporated into the Methods section, the corresponding results are described in the Results section, and the supporting data are presented in the Supplementary Figures.

 

Comment 2:  The study includes appropriate control groups; however, the rationale for selecting the LOXL2 inhibitor dose (10 mg/kg/day) is not clearly justified. Information regarding randomization and blinding procedures during treatment allocation and analysis is insufficient, though partial mention of blinded histological analysis is present. Sample size (n=18 total) appears relatively small; power analysis is not provided.

Answer: We thank the reviewer for this important comment. Dose for LOXL2 inhibitor was determined from our previous animal study on idiopathic pulmonary fibrosis (IPF) [1]. In the previous IPF mice model using C57B/6 mice, LOXL2 inhibitor doses ranging from 1, 10, and 30mg/kg were administered for 14 days with significant anti-fibrotic effects shown in 10mg/kg and 30mg/kg groups. However, 30 mg/kg group showed somewhat increased body weight loss into 2 weeks of the study. We therefore elected to use 10 mg/kg dose for our experiments that lasts up to 4 weeks.

We acknowledge the reviewer’s concern regarding the relatively small sample size and the absence of a formal power analysis. The sample size of n=4-5 per group employed in this study is consistent with previously published studies using CsA-induced kidney fibrosis models in rodents, in which similar group sizes (n=4-6) have been commonly used and have yielded statistically significant and reproducible results [2]. Importantly, despite the modest sample size, our study demonstrated statistically significant differences across multiple key outcome measures, suggesting that the study was adequately powered to detect biologically meaningful differences. The consistency of these findings further supports the reliability of our conclusions.

We acknowledge, however, that the absence of a formal, a priori power calculation represents a limitation of the current study, and we have added the following statement to the Discussion section of the revised manuscript: “Second, the relatively small sample size in each experimental group (n = 4–5) may limit the statistical power of our findings.”

 

Reference

1. Shim DH, Kim MJ, Chung HW, Kim MN, Sohn MH, Lee S, et al. Targeting Lysyl Oxidase-like 2: A Therapeutic Strategy for Idiopathic Pulmonary Fibrosis with a Novel Indolizine Derivative. Pharmaceutics. 2026;18(5):554. PubMed PMID: doi:10.3390/pharmaceutics18050554.
2. Stangenberg, S., Saad, S., Schilter, H.C. et al. Lysyl oxidase-like 2 inhibition ameliorates glomerulosclerosis and albuminuria in diabetic nephropathy. Sci Rep 8, 9423 (2018). https://doi.org/10.1038/s41598-018-27462-6

 

Comment 3: The statistical methods (ANOVA with Tukey post hoc test and t-test) are appropriate, but there is no mention of normality testing or variance homogeneity. Exact p-values are not consistently reported; reliance on significance markers (*, #) limits interpretability.

Answer: We thank the reviewer for this careful and constructive comment. We confirm that prior to all statistical analyses, data distribution was assessed using the Shapiro-Wilk test for normality, and homogeneity of variance was evaluated using Levene's test. All datasets met the assumptions of normality and equal variance, justifying the use of one-way ANOVA with Tukey's post hoc test for multiple comparisons and unpaired Student's t-test for two-group comparisons. These details have been added to the Statistical Analysis section of the revised manuscript. “Data are expressed as mean ± standard deviation. Prior to statistical analysis, normality of data distribution was confirmed using the Shapiro-Wilk test, and homogeneity of variance was assessed using Levene's test. The one-way ANOVA test with post hoc Tukey comparison was used for multiple group comparison between the control and treatment.  Student’s t-test was further used to assess the difference between two groups. P <0.05 was considered statistically significant, and all statistical analyses were performed using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA)”

Regarding the reporting of exact p-values, we agree with the reviewer that reliance solely on significance markers (*, #) limits the interpretability and transparency of the results. In the revised manuscript, we have replaced all significance markers with exact p-values. Significance markers have been retained in figures for visual clarity, but exact p-values are now reported in the Results section of the revised manuscript. However, given the large number of comparisons across multiple figures, reporting all exact p-values within the main text would compromise the readability and flow of the manuscript. Therefore, we have compiled comprehensive statistical summaries — including mean ± SD and exact p-values for all pairwise comparisons — into Supplementary Tables, each corresponding to the respective figures in the manuscript. We believe this approach balances scientific rigor with readability, and we hope this adequately addresses the reviewer's concern.

 

Comment 4: Figures lack sufficient quantitative detail. For example Western blot results should include full-length blots or uncropped images. Densitometric analysis methods are not described in detail. Units and scales in some graphs are unclear or insufficiently labeled (e.g., Figure 2 panels).

Answer: We thank the reviewer for these detailed comments. Full-length, uncropped Western blot images for all reported proteins have been included as Supplementary Figures (uploaded in non-published material). The original blot images were cropped solely for clarity of presentation, and the uncropped versions confirm the specificity of each band without any image manipulation.

In addition, densitometric quantification of Western blot bands was performed using ImageJ software (version 1.54, NIH, Bethesda, MD, USA). Band intensities were normalized to the corresponding loading control (β-actin) within each membrane. Results are expressed as relative protein expression normalized to the control group (set to 1.0). These details have been

added to the Materials and Methods section of the revised manuscript.

Regarding units and axis labels in figures, we have carefully reviewed all figure panels and corrected any unclear or missing labels in the revised manuscript.

 

Comment 5: The authors conclude that LOXL2 inhibition may be a therapeutic strategy; however, translational relevance is overstated. The discussion does not adequately address conflicting literature where LOXL2 inhibition showed limited or variable effects in different models. Potential off-target effects or safety concerns of LOXL2 inhibition are not discussed.

Answer: We thank the reviewer for this thoughtful and constructive comment. We acknowledge that the translational relevance of our findings was overstated in the original manuscript. In the revised manuscript, we have moderated the language throughout the Discussion and Conclusion sections. Specifically, strong claims such as "LOXL2 inhibition represents a promising therapeutic strategy" have been revised to “The present preclinical findings suggest that Compound #765-mediated LOXL2 inhibition may offer a potential therapeutic benefit in CsA-induced fibrosis, though further validation is warranted.”

             We have also expanded the Discussion to address the variable efficacy of LOXL2 inhibition across kidney disease models. While LOXL2 inhibition preserved kidney function and reduced fibrosis in Alport syndrome models [1], effects were more limited in diabetic nephropathy, where improvement was confined to glomerular fibrosis with minimal tubulointerstitial impact [2]. These discrepancies likely reflect differences in the site of injury, stage of fibrosis, and disease-specific pathological mechanisms. In CsA-induced nephropathy, tubulointerstitial fibrosis is the hallmark feature, and LOXL2 was the most highly upregulated LOX family member in our model (Figure 1), supporting the rationale for selective LOXL2 targeting. Although dual LOX/LOXL inhibition has been proposed as a broader approach [3], our findings suggest that selective LOXL2 inhibition is sufficient in the CsA nephropathy model, likely owing to the predominant role of LOXL2 in this pathological setting.

We also acknowledge that potential off-target effects and safety concerns were insufficiently addressed. LOXL2 contributes to ECM homeostasis and connective tissue integrity, and its inhibition may carry unintended consequences such as impaired wound healing, skeletal abnormalities, and vascular dysfunction. The possibility that Compound #765 may affect other LOX family members at higher concentrations also cannot be excluded. These points have been added to the limitations of the revised Discussion, and we agree that further preclinical toxicity and safety evaluations are necessary before any translational application.

 

Reference

1. Cosgrove D, Dufek B, Meehan DT, Delimont D, Hartnett M, Samuelson G, et al. Lysyl oxidase like-2 contributes to renal fibrosis in Col4α3/Alport mice. Kidney Int. 018;94(2):303-14. Epub 20180516. doi: 10.1016/j.kint.2018.02.024. PubMed PMID: 29759420; PubMed central PMCID: PMCPMC7523185.
2. Stangenberg S, Saad S, Schilter HC, Zaky A, Gill A, Pollock CA, et al. Lysyl oxidase-like 2 inhibition ameliorates glomerulosclerosis and albuminuria in diabetic nephropathy. Sci Rep. 2018;8(1):9423. Epub 20180621. doi: 10.1038/s41598-018-27462-6. PubMed PMID: 29930330; PubMed Central PMCID: PMCPMC6013429.
3. Saifi MA, Shaikh AS, Kaki VR, Godugu C. Disulfiram prevents collagen crosslinking and inhibits renal fibrosis by inhibiting lysyl oxidase enzymes. J Cell Physiol. 2022;237(5):2516-27. Epub 20220313. doi: 10.1002/jcp.30717. PubMed PMID: 35285015.

 

Comment 6: The study reports reduction in F4/80-positive cells and TUNEL-positive cells; however. quantification methods need clearer description. Additional markers (e.g., caspase activation, cytokine panels) would strengthen conclusions.

Answer: We thank the reviewer for this valuable comment. We have revised the Materials and Methods section to provide a more detailed description of the quantification methods for both F4/80 and TUNEL staining. Specifically, F4/80-positive cells were counted manually in 20 randomly selected high-power fields (×400 magnification) per section in a blinded manner, and results are expressed as the number of positive cells per high-power field (cells/HPF). TUNEL-positive cells were quantified as a percentage of total cells per HPF, also assessed in a blinded manner across 20 randomly selected fields.

We appreciate the reviewer's suggestion to include additional markers such as caspase activation and cytokine panels to strengthen our mechanistic conclusions. We acknowledge that these additional analyses would provide further insight into the specific apoptotic pathways and inflammatory mechanisms involved. However, we would like to emphasize that TUNEL staining is widely accepted as a validated and robust method for quantifying apoptotic cell death in kidney tissue sections, and has been extensively used as a primary readout in CsA-induced nephropathy models [1-3]. Moreover, concerning cytokine panels, we would like to highlight that our study already examined multiple inflammatory and fibrotic mediators that collectively constitute a targeted cytokine profile relevant to CsA-induced kidney fibrosis. Specifically, we assessed TGF-β1 protein expression by Western blot, TGF-β1 and MCP-1 mRNA levels by RT-qPCR, and macrophage infiltration by F4/80 immunohistochemistry. Furthermore, downstream signaling pathways including TGF-β/Smad3 and MAPK (p38 and ERK1/2) were also evaluated, providing additional mechanistic insight into the inflammatory and fibrotic processes. While we acknowledge that a broader cytokine panel (e.g., TNF-α, IL-6, IL-1β) would further strengthen our conclusions, these analyses were beyond the scope of the current study.

 

Reference

1. Yang, C.W. Faulkner, G.R. Wahba, I.M. Christianson, T.A. Bagby, G.C. Jin, D.C., et al. Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice. J. Transplant. 2002, 2, 391–399.
2. Thomas, S.E, Andoh, T.F. Pichler, R.H. Shankland, S.J. Couser, W.G. Bennett, W.M. et al. Accelerated apoptosis characterizes cyclosporine-associated interstitial fibrosis. Kidney Int. 1998, 53, 897–908.
3. Shihab, F.S. Andoh, T.F. Tanner, A.M. Yi, H. Bennett, W.M. Expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis. Kidney Int. 1999, 56, 2147–2159.

 

Comment 7: Several grammatical errors and typographical issues are present throughout the manuscript (e.g., spacing, punctuation, inconsistent use of abbreviations). Terms such as “aĴenuated” appear with formatting issues. Some abbreviations are inconsistently defined (e.g., LOXL2i vs LOXL2 inhibitor). Ensure all abbreviations are defined at first use.

Answer: We thank the reviewer for the careful review of the manuscript. We have thoroughly revised the manuscript to correct grammatical and typographical errors, including spacing, punctuation, and formatting inconsistencies. In addition, abbreviations and terminology were carefully standardized throughout the manuscript.

 

Comment 8: Figure legends are generally descriptive but could be improved by including sample size (n), clarifying statistical comparisons, avoiding repetition. More detail is needed for antibody validation and sources, RT-qPCR efficiency and normalization strategy and criteria for histological scoring

Answer: We thank the reviewer for the careful review of the manuscript. Sample size and statistical comparison indicators are stated in all revised figure legends. In addition, all primary antibodies used in this study are commercially available and validated by the manufacturers. Detailed information has been added to the revised Materials and Methods section, including antibody sources, RT-qPCR normalization methods, and histological scoring criteria. We also clarified that gene expression levels were normalized to the housekeeping gene GAPDH and analyzed using the ΔΔCT method. Furthermore, the criteria used for semi-quantitative histological assessment and the number of analyzed fields/sections per sample have been described more clearly in the revised manuscript. Repetitive descriptions in the figure legends were minimized to improve readability.

 

Comment 9: In page 3–4 under results section, the increase in LOXL2 expression is clearly shown, but fold-change values should be explicitly stated rather than inferred from figures.

Answer: We thank the reviewer for the careful review of the manuscript. As recommended, fold-change values have been explicitly stated in the revised Results section. The sentence has been revised as follows: “Typically, among these LOX and LOXL families, a notable upregulation was observed in LOXL2, which showed a 2.87-fold increase compared to the control (mean difference = −1.87, 95% CI: −2.62 to −1.12, P < 0.001).”

Additionally, detailed numeric values for all LOX and LOXL family members, including mean ± SD, mean difference, 95% confidence interval, and P-values, are presented in Supplemental Table.

 

Comment 10: In page 5–6 under histology, quantification of fibrosis using MT and PSR staining is appropriate; however, inter-observer variability is not addressed.

Answer: We thank the reviewer for this important methodological comment. To address inter-observer variability, all histological sections were evaluated in a blinded manner by two independent observers, and any discrepancies in fibrosis quantification were resolved by consensus. In addition, fibrosis area was quantified using automated image analysis software (MetaMorph; Molecular Devices, San Jose, CA, USA), which further minimized subjective bias. These clarifications have been added to the Materials and Methods section of the revised manuscript.

 

Comment 11: In page 9–10 (signaling pathways), the reduction in p-p38, p-Smad3, and p-ERK1/2 is interesting, but upstream regulators are not explored.

Answer: We thank the reviewer for this insightful comment. We acknowledge that the upstream regulators linking LOXL2 inhibition to the observed reductions in p-Smad3, p-ERK1/2, and p-p38 were not directly investigated in the present study. Based on existing literature, LOXL2-mediated crosslinking of extracellular matrix components has been shown to activate integrin-dependent signaling cascades, which may converge on TGF-β/Smad and MAPK pathways [1, 2]. However, the precise upstream mechanisms — such as integrin engagement, focal adhesion kinase (FAK) activation, or receptor tyrosine kinase transactivation — through which LOXL2 inhibition modulates these downstream effectors remain to be elucidated. This has been acknowledged as a limitation in the revised Discussion, and future studies incorporating upstream pathway interrogation (e.g., FAK/Src signaling, integrin blocking, or transcriptomic profiling) are warranted to fully delineate the mechanistic axis.

 

Reference

1. Amendola PG, Reuten R, Erler JT. Interplay Between LOX Enzymes and Integrins in the Tumor Microenvironment. Cancers (Basel). 2019;11(5):729. doi: 10.3390/cancers11050729. PMID: 31130685; PMCID: PMC6562985.
2. Luo J, Wu Y, Zhu X, Wang S, Zhang X, Ning Z. LOXL2 silencing suppresses angiotensin II-induced cardiac hypertrophy through the EMT process and TGF-β1/Smad3/NF-κB pathway. Iran J Basic Med Sci. 2022;25(8):964-969. doi: 10.22038/IJBMS.2022.63338.13981. PMID: 36159334; PMCID: PMC9464345.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors replied to the comments.