Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This multicenter case-control study investigated risk factors for EPO hyporesponsiveness in 784 hemodialysis patients with anemia across six Latin American dialysis centers. Hyporesponsiveness was defined as requiring ≥200 IU/kg/week of EPO for ≥3 months to maintain hemoglobin levels of 10-12 g/dL, identifying 123 hyporesponsive cases (15.7%) and 661 responsive controls. Multivariate logistic regression revealed independent risk factors, including female sex, age <50 years, low serum albumin, high ferritin, low transferrin saturation, high parathyroid hormone, and use of RAS blockers. The study suggests these factors could guide personalized anemia management, though diabetes showed an inverse association in bivariate analysis that did not persist in the multivariate model. I have some comments and suggestions for this study.

1. The threshold of ≥200 IU/kg/week for hyporesponsiveness does not account for the route of EPO administration (intravenous vs subcutaneous), which can influence efficacy, nor does it consider individual variations in hemoglobin targets beyond the 10-12 g/dL range.

2. The counterintuitive association of younger age (<50 years) with hyporesponsiveness lacks a clear mechanistic explanation, and the study does not adequately explore this anomaly. Besides, the inverse association between diabetes and hyporesponsiveness seen in the bivariate analysis (though non-significant in the multivariate model) is also unexpected, given diabetes is often linked to more severe anemia.

3. The study relies heavily on ferritin as an indicator of inflammation and functional iron deficiency. While high ferritin is suggestive, the lack of systematic measurement of other specific inflammatory markers like hsCRP or cytokines (IL-6) limits a deeper understanding of the role of inflammation.

4. The dichotomization of continuous variables (like age, BMI, albumin, ferritin, TSAT, and PTH) based on specific cutoffs can lead to loss of information and may be somewhat arbitrary, even if based on literature or observed distributions.

5. Doses and routes of i.v. Iron, active vitamin D or calcimimetics, which directly influence ferritin, TSAT and PTH, are not quantified, inviting residual confounding.

6. A minor comment. The unit of PTH level is different. There are μg/mL and pg/mL. Please clarify.

Author Response

Response to Reviewer Comments

1. Summary

Thank you very much for taking the time to review our manuscript. We appreciate your thoughtful comments and constructive feedback, which have helped us improve the quality of our paper. Please find our detailed responses below and the corresponding revisions highlighted in the re-submitted files.

2. Response to Comments and Suggestions

Comment 1:

The threshold of ≥200 IU/kg/week for hyporesponsiveness does not account for the route of EPO administration (intravenous vs subcutaneous), which can influence efficacy, nor does it consider individual variations in hemoglobin targets beyond the 10-12 g/dL range.

Response 1: Thank you for this important observation. We agree that the route of administration can influence EPO efficacy. We have addressed this limitation on page 6, lines 151-157, where we clarify that all patients in our study received intravenous EPO administration during hemodialysis sessions, ensuring standardization of the delivery method. Regarding hemoglobin targets, we selected the 10-12 g/dL range based on current clinical guidelines for anemia management in CKD patients. We have further explained our rationale for this target range in the Methods section (page 4, lines 121-126) and acknowledge in the Discussion (page 12, lines 445-448) that individual variations in optimal hemoglobin targets could influence the classification of hyporesponsiveness. We have also added this consideration to our Limitations section.

Comment 2:

The counterintuitive association of younger age (<50 years) with hyporesponsiveness lacks a clear mechanistic explanation, and the study does not adequately explore this anomaly. Besides, the inverse association between diabetes and hyporesponsiveness seen in the bivariate analysis (though non-significant in the multivariate model) is also unexpected, given diabetes is often linked to more severe anemia.

Response 2: We appreciate this valuable insight. We have expanded our discussion of the unexpected association between younger age and EPO hyporesponsiveness on page 9, lines 316-324. We now propose several potential mechanisms, including age-related differences in hematopoietic stem cell function, inflammatory profiles, and hormonal factors that may differ between Latin American populations and other studied cohorts. Regarding the inverse association with diabetes, we have added a dedicated subsection (4.7, page 11, lines 390-404) that explores possible explanations for this finding, including differences in BMI, nephron-specific effects of diabetic nephropathy on EPO-producing cells, and treatment patterns. We acknowledge that both observations warrant further investigation with larger sample sizes and specific mechanistic studies.

Comment 3:

The study relies heavily on ferritin as an indicator of inflammation and functional iron deficiency. While high ferritin is suggestive, the lack of systematic measurement of other specific inflammatory markers like hsCRP or cytokines (IL-6) limits a deeper understanding of the role of inflammation.

Response 3: We agree with this observation. We have enhanced our discussion of inflammatory markers on page 10, lines 358-363, where we now highlight that our study did include hs-CRP measurements, which were significantly higher in the hyporesponsive group. However, we acknowledge the limitation of not measuring specific pro-inflammatory cytokines such as IL-6, TNF-α, and others that might provide more comprehensive insights into the inflammatory mechanisms underlying EPO hyporesponsiveness. We have added this limitation to our discussion section (page 12, lines 436-438) and suggested that future studies should incorporate more extensive inflammatory marker panels.

Comment 4:

The dichotomization of continuous variables (like age, BMI, albumin, ferritin, TSAT, and PTH) based on specific cutoffs can lead to loss of information and may be somewhat arbitrary, even if based on literature or observed distributions.

Response 4: Thank you for highlighting this methodological concern. We have expanded our explanation of the rationale for dichotomizing continuous variables in the Methods section (page 5, lines 174-178). While we acknowledge that this approach may result in some loss of information, we selected clinically relevant thresholds based on established literature and clinical practice guidelines. We have added a note in the Limitations section (page 12, lines 442-444) acknowledging this limitation and suggesting that future studies might consider alternative analytical approaches such as spline functions or categorical variables with multiple levels to better capture the potentially non-linear relationships between these parameters and EPO responsiveness.

Comment 5:

Doses and routes of i.v. Iron, active vitamin D or calcimimetics, which directly influence ferritin, TSAT and PTH, are not quantified, inviting residual confounding.

Response 5: This is an excellent point. We have acknowledged this limitation more explicitly in our discussion section (page 12, lines 439-441). We now clarify that although we recorded the use of these medications as categorical variables, we did not systematically collect data on specific doses, duration, or timing of iron supplementation, vitamin D analogs, or calcimimetics relative to laboratory measurements. This could indeed introduce residual confounding, as these treatments directly influence the parameters we identified as risk factors. We have added this as an important consideration for future studies, which should incorporate more detailed treatment data to better adjust for these potential confounders.

Comment 6:

A minor comment. The unit of PTH level is different. There are μg/mL and pg/mL. Please clarify.

Response 6: Thank you for catching this inconsistency. We have corrected all references to PTH units to consistently use pg/mL throughout the manuscript, which is the standard unit for reporting intact PTH levels in clinical practice. The corrections appear on pages 7 (lines 240-242), 8 (lines 271-272), 9 (lines 289-290), and 10 (lines 365-367).

3. Additional clarifications

We appreciate the reviewer's thorough assessment of our manuscript. The comments have helped us identify important areas for improvement and clarification. We have addressed all points raised and believe these revisions have significantly strengthened our paper. We have also performed an additional review of the entire manuscript to ensure consistency in units, terminology, and clarity of presentation.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study by Tulcanaza et al is a multicenter case-control study which analyzed data from 784 hemodialysis patients with regards to hyporesponsiveness to erythropoietin. As the mechanisms underlying EPO hyporesponsiveness are not fully understood, this study is of clinical interest. The authors identified some predictors of EPO hyporesponsiveness which may help to identify patients at risk.

Here are some comments which may help to improve the manuscript:

• Figure 1. Typically, inclusion / exclusion criteria are applied as a first step in patient selection. Why was this applied at a later stage? Are these really exclusion / inclusion criteria, as the Figure states that these are risk factors. This figure should rater be a flow chart showing the inclusion flow. It is also not clear was does exposed / unexposed mean here. This figure and description should be improved.
• Figure 1: “Source: own elaboration” should be removed.
• Figure 1: The number of patients in the different boxes and the number of patients excluded from the study should be given at each step.
• Line 107: Region/County information is missing.
• Methods: “Cases (n=123) were defined as patients requiring ≥200 IU/kg/week of EPO alfa for a continuous period of ≥3 months to maintain hemoglobin levels between 10-12 g/dL, consistent with established definitions in the literature (23, 24)”. What is with patients above 12g/dL?
• Line 112: What is the Hb target for the controls, also 10-12g/dL?
• Line 157-159: Why didn’t the authors use continuous variables but defined thresholds, which reduce the level of information?
• Table 1 should be closer to the text when then results are presented (section 3.1). The same with Table 4 (section 3.4).
• Line 219: “Laboratory parameters showed several significant differences between groups.” Should be rather “Several laboratory parameters showed significant …”
• How were the parameters for the multivariate analysis selected?
• Are the identified independent predictors really independent or are they just maybe reflecting e.g. higher inflammation?

Author Response

Response to Reviewer Comments

1. Summary

Thank you very much for taking the time to review our manuscript. Your thoughtful comments will help us improve the clarity and scientific rigor of our paper. Please find our detailed responses below and the corresponding revisions in the re-submitted files.

2. Point-by-point response to Comments and Suggestions

Comment 1: Figure 1 issues (flow chart, "exposed/unexposed," and source statement)

Response 1: Thank you for this constructive feedback. We agree that Figure 1 requires significant revision. We have redesigned it as a proper patient flow diagram following STROBE guidelines for case-control studies. The new figure clearly shows:

1. Initial population (n=932 hemodialysis patients)
2. Application of exclusion criteria with specific numbers excluded at each step (148 patients excluded: 52 hospitalized within 3 months, 37 with active malignancy/hematological disorders, 29 with recent blood transfusions, 30 with incomplete records)
3. Final study population (n=784)
4. Division into cases (n=123) and controls (n=661)

We have removed the inappropriate terms "exposed/unexposed" and the "Source: own elaboration" statement. The revised figure now accurately reflects the patient selection process rather than describing risk factors.

Comment 2: Regional information missing

Response 2: We have added the regional information in line 107 (now reads: "...examining factors associated with erythropoietin (EPO) hyporesponsiveness in chronic kidney disease (CKD) patients on hemodialysis in Ecuador, South America."). This information is also provided in the Methods section where we specify that the study was conducted across six dialysis centers in Ecuador.

Comment 3: Hemoglobin targets for cases and controls

Response 3: Thank you for highlighting this important point. We have clarified that both cases and controls had the same target hemoglobin range of 10-12 g/dL. We have revised the text in lines 121-126 to explicitly state: "Cases (n=123) were defined as patients requiring ≥200 IU/kg/week of EPO alfa for a continuous period of ≥3 months to maintain hemoglobin levels between 10-12 g/dL. Controls (n=661) were defined as patients responsive to lower doses of EPO while maintaining the same target hemoglobin levels of 10-12 g/dL." Patients with hemoglobin levels above 12 g/dL were not included in the hyporesponsive group as per our definition, as these patients would not meet the criteria of requiring high EPO doses to maintain target hemoglobin levels.

Comment 4: Use of thresholds for continuous variables

Response 4: We appreciate this methodological observation. We have expanded our explanation in lines 174-180: "Continuous variables were dichotomized based on clinically relevant thresholds established in the literature and clinical practice guidelines to facilitate clinical interpretation and application of findings. While we acknowledge that this approach may result in some information loss compared to treating variables as continuous, it allows for clear identification of practical clinical cutoffs that can guide treatment decisions. In sensitivity analyses not shown in the main text, we also analyzed these variables in their continuous form, which yielded consistent directions of associations but was less interpretable for clinical application."

Comment 5: Table placement

Response 5: We agree with this suggestion and have repositioned Tables 1 and 3 to be closer to their respective text sections (3.1 and 3.4) in the final manuscript layout.

Comment 6: Writing style in line 219

Response 6: We have revised this sentence as suggested: "Several laboratory parameters showed significant differences between groups."

Comment 7: Selection of parameters for multivariate analysis

Response 7: We have clarified our approach to multivariate model building in the statistical analysis section (lines 172-174): "Variables with p<0.10 in the bivariate analyses were included in the initial multivariate model. The final model was built using a backward elimination approach (likelihood ratio test), retaining variables with p<0.05 and those considered clinically relevant regardless of statistical significance."

Comment 8: Independence of predictors

Response 8: This is an excellent point. We have added a paragraph in the Discussion section (page 10, lines 357-364) addressing the potential interrelationships among identified risk factors: "While our multivariate analysis identified several independent risk factors for EPO hyporesponsiveness, it is important to note that these factors may be biologically interrelated. For instance, high ferritin, low transferrin saturation, and low albumin often coexist in the context of malnutrition-inflammation complex syndrome in dialysis patients. Rather than representing truly independent mechanisms, these factors may reflect different aspects of underlying inflammatory processes. Our finding of significantly elevated hs-CRP levels in hyporesponsive patients supports this interpretation. Future studies incorporating path analysis or structural equation modeling could better elucidate these complex interrelationships."

3. Additional clarifications

We thank the reviewer for their thorough assessment, which has significantly improved the clarity and scientific rigor of our manuscript. We have conducted an additional review to ensure consistency throughout the paper and have revised the figures and text accordingly.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Congratulations on your manuscript addressing an important topic in nephrology. I have a few comments aimed at improving your paper; these will require only minor revisions:

1. Title: Include the designation “case–control” in the title, in accordance with the STROBE statement.
   E.g.: “Risk Factors Associated with Hyporesponsiveness to Erythropoietin in Chronic Kidney Disease Patients on Hemodialysis Who Present Anemia: A Multicenter Case–Control Study.”
2. Abstract: Add the total number of participants and the data-collection period.
3. Introduction: State your primary hypotheses explicitly, as recommended by the STROBE statement.
4. Methods: Provide the study’s prior registration number or reference the published study protocol, if one exists.
5. Abbreviations: Ensure consistency throughout the text. Once an abbreviation is defined, use only the abbreviation thereafter. For example, “erythropoietin (EPO)” on page 2, line 86, and “chronic kidney disease (CKD)” on line 87.
6. Figure 1: Replace the current graphic with a STROBE-compliant flow diagram (see https://www.strobe-statement.org/), showing the initial sample size, exclusions (with reasons), and final numbers of cases and controls.
7. Figure Style: Prepare the flow diagram in black-and-white or grayscale to improve clarity.
8. Control Selection: Specify how controls were chosen (e.g., random sampling, exact ratio).
9. Ethics Section: Add an ethics statement at the end of Methods detailing the information from section 2.1 (IRB approval, consent waiver, etc.).
10. Kt/V Calculation: Describe the method used to calculate Kt/V.
11. Font Consistency: Use a uniform font throughout. For example, in Table 1 “Obese (≥30.0 kg/m²)” appears in a different font, as does text in Table 3.
12. Reporting of Variables: Report results for all variables listed in Methods. For instance, “serum calcium and phosphorus levels” are mentioned but not shown in Table 2; the same applies to medications.
13. Units of PTH: Harmonize the units for parathyroid hormone between text and tables (either pg/mL or μg/mL).
14. Discussion of Population Differences: In the Introduction you note that “many existing studies have been conducted in North American, European, or East Asian populations, with limited data from Latin American patients…” but the Discussion does not explore these differences. Please analyze similarities and differences between your cohort and published literature, and reflect on this in your Conclusions.
15. Bibliography – DOIs: Check all DOIs, as several do not match the cited articles (e.g., reference 6’s DOI points to a book chapter rather than an article, and reference 7’s DOI is incorrect).
16. Bibliography – Non-existent Citations: Remove or correct citations that do not exist (e.g., reference 11).

Thank you for the opportunity to review your work. I look forward to seeing these revisions.

Author Response

Response to Reviewer Comments

1. Summary

Thank you very much for taking the time to review our manuscript and for your constructive feedback. We appreciate your recognition of the importance of our research topic in nephrology. We have carefully addressed each of your comments and made the corresponding revisions in the manuscript, as detailed below.

2. Point-by-point response to Comments and Suggestions

Comment 1: Title

Response 1: Thank you for this suggestion. We have revised the title to include the study design as recommended by STROBE guidelines: "Risk Factors Associated with Hyporesponsiveness To Erythropoietin in Chronic Kidney Disease Patients on Hemodialysis Who Present Anemia: A Multicenter Case-Control Study."

Comment 2: Abstract - participant numbers and data collection period

Response 2: We have updated the abstract to include the total number of participants (784) and the data collection period (January to December 2019) in the Methods section of the abstract.

Comment 3: Introduction - explicit hypotheses

Response 3: We have revised the introduction to explicitly state our primary hypothesis on page 2, lines 82-84: "We hypothesize that multiple factors, including demographic characteristics, nutritional status, iron parameters, and comorbidities, independently contribute to EPO hyporesponsiveness in Latin American hemodialysis patients."

Comment 4: Methods - study registration

Response 4: As this was a retrospective study that did not require registration, we have added a statement in the Methods section clarifying this: "This retrospective study was not registered in a clinical trials registry as it did not involve any intervention or prospective assignments."

Comment 5: Abbreviations consistency

Response 5: We have conducted a thorough review of abbreviation usage throughout the manuscript. Once defined, we consistently use the abbreviation thereafter. We have specifically corrected "erythropoietin (EPO)" and "chronic kidney disease (CKD)" to use only the abbreviations after first definition.

Comment 6 & 7: Figure 1 - STROBE-compliant flow diagram

Response 6 & 7: We have completely redesigned Figure 1 according to STROBE guidelines for case-control studies. The new diagram clearly shows:

• Initial population (932 hemodialysis patients)
• Exclusion process with specific reasons and numbers (148 patients excluded)
• Final study population (784 patients)
• Division into cases (123) and controls (661) The flow diagram has been prepared in grayscale with a clean, professional design for optimal clarity in both print and digital formats.

Comment 8: Control selection

Response 8: We have added information on control selection on page 4, lines 125-126: "Controls were selected from the same centers as cases, using all eligible patients who met the inclusion criteria and did not meet the definition of hyporesponsiveness, resulting in a control-to-case ratio of approximately 5:1."

Comment 9: Ethics section

Response 9: We have added a dedicated Ethics section at the end of the Methods section (page 5, lines 187-190): "Ethics: The study protocol was approved by the Institutional Review Board of all participating centers. As this was a retrospective study using anonymized medical records, the requirement for individual patient informed consent was waived. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines."

Comment 10: Kt/V calculation

Response 10: We have added details on Kt/V calculation on page 4, lines 142-143: "Dialysis adequacy (Kt/V) was calculated using the single-pool Daugirdas second-generation formula: Kt/V = -ln(R - 0.008 × t) + (4 - 3.5 × R) × UF/W, where R is the post/pre-dialysis BUN ratio, t is dialysis session length in hours, UF is ultrafiltration volume in liters, and W is post-dialysis weight in kg."

Comment 11: Font consistency

Response 11: We have standardized the font throughout the manuscript, ensuring consistency in all tables, particularly Table 1 and Table 3.

Comment 12: Reporting of all variables

Response 12: We have added calcium and phosphorus values to Table 2, along with information about phosphate binder and vitamin D analog use. We have ensured that all variables mentioned in the Methods section are reported in the Results section and corresponding tables.

Comment 13: Units of PTH

Response 13: We have standardized all references to parathyroid hormone to consistently use pg/mL as the unit of measurement throughout both text and tables.

Comment 14: Discussion of population differences

Response 14: We have added a new subsection (4.8) in the Discussion entitled "Comparison with Other Populations" (page 11, lines 406-424), where we analyze similarities and differences between our findings and those from North American, European, and Asian populations. We discuss consistent factors across populations (hypoalbuminemia, functional iron deficiency, secondary hyperparathyroidism) and highlight population-specific differences, particularly regarding the association of younger age with hyporesponsiveness and the lower prevalence of diabetes in our hyporesponsive cohort.

Comment 15: Bibliography - DOIs

Response 15: We have reviewed and corrected all DOIs in the references section. Specifically, we have fixed references 6 and 7, ensuring that all DOIs correctly link to their respective articles.

Comment 16: Non-existent citations

Response 16: We have verified all references and have corrected or removed non-existent citations, including reference 11. The reference list has been updated accordingly.

3. Additional clarifications

We have conducted a thorough review of the entire manuscript to ensure consistency in terminology, units, and formatting. The revisions have improved the clarity and scientific rigor of our work while adhering to STROBE guidelines for reporting case-control studies. We believe these changes have significantly enhanced the quality of our manuscript.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded accordingly. I have no further comments.