Review Reports - Oxidative Stress and Endothelial Dysfunction in Patients with Hypertensive Nephropathy: Role of the Mediterranean Diet

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an important and clinically relevant topic by exploring the interplay between oxidative stress, endothelial dysfunction, intrarenal vascular resistance, and adherence to the Mediterranean diet in patients with essential hypertension and hypertensive nephropathy. The study is timely, as it integrates vascular, biochemical, and nutritional aspects of chronic kidney disease progression, using renal resistive index as a surrogate of renal microvascular damage. Overall, the work is well structured, the rationale is sound, and the findings are generally consistent with current pathophysiological concepts. Nevertheless, several aspects of study design, data analysis, and presentation could be improved to strengthen the scientific rigor, clarity, and interpretability of the results.

The observational and cross-sectional nature of the study should be more explicitly emphasized throughout the manuscript, particularly in the Abstract and Discussion, to avoid any implicit suggestion of causality. Although this limitation is acknowledged, some interpretations still verge on causal language, especially regarding the role of oxidative stress and Mediterranean diet adherence in modulating RRI. A more cautious phrasing that consistently frames the findings as associations would improve conceptual clarity and align conclusions with the study design.
The potential confounding effect of age, pharmacological treatments, and comorbidities on RRI and oxidative stress markers warrants deeper consideration. Patients in the higher RRI group were older and more frequently treated with beta-blockers and statins, which may independently influence endothelial function and oxidative stress. Multivariable regression analyses adjusting for these variables would substantially strengthen the robustness of the associations reported and reduce the risk of residual confounding.
The rationale for dichotomizing patients according to the 75th percentile of RRI, in addition to using the established clinical cut-off of 0.7, should be better justified. While percentile-based stratification is acceptable, it may reduce comparability with other studies. Clarifying whether results are consistent across both approaches, or presenting sensitivity analyses using RRI as a continuous variable, would enhance methodological transparency.
The assessment of Mediterranean diet adherence relies on a self-reported questionnaire administered at a single time point, which may not adequately capture long-term dietary patterns. This limitation should be discussed in greater depth, particularly in light of the modest correlations observed. Additionally, reporting the distribution of PREDIMED scores and adherence categories would help readers better contextualize the strength and clinical relevance of the dietary associations.
The biological interpretation of the differential behavior of oxidative stress markers deserves further refinement. While the discussion appropriately highlights sNOX2-dp as a sensitive marker of NOX2 activation, the lack of significant associations with NO and H₂O₂ could be explored more critically, considering issues such as biological variability, assay limitations, and the temporal dynamics of these biomarkers. This would prevent overemphasis on a single marker and provide a more balanced redox profile interpretation.
The manuscript would benefit from a clearer articulation of its clinical implications. While RRI and oxidative stress biomarkers are proposed as tools for refined risk stratification, practical guidance on how these measures could be integrated into routine clinical practice is limited. Expanding this aspect, even in a speculative manner, and outlining concrete future research directions, such as longitudinal or interventional dietary studies, would enhance the translational impact of the work.

Author Response

Answer: Thank you for your kind comment

________________________________________________________________________________

Nevertheless, several aspects of study design, data analysis, and presentation could be improved to strengthen the scientific rigor, clarity, and interpretability of the results.

The observational and cross-sectional nature of the study should be more explicitly emphasized throughout the manuscript, particularly in the Abstract and Discussion, to avoid any implicit suggestion of causality. Although this limitation is acknowledged, some interpretations still verge on causal language, especially regarding the role of oxidative stress and Mediterranean diet adherence in modulating RRI. A more cautious phrasing that consistently frames the findings as associations would improve conceptual clarity and align conclusions with the study design.

Answer: Thank you for your comment. We modified the manuscript and the abstract according to your comment.

________________________________________________________________________________

The potential confounding effect of age, pharmacological treatments, and comorbidities on RRI and oxidative stress markers warrants deeper consideration. Patients in the higher RRI group were older and more frequently treated with beta-blockers and statins, which may independently influence endothelial function and oxidative stress. Multivariable regression analyses adjusting for these variables would substantially strengthen the robustness of the associations reported and reduce the risk of residual confounding.

Answer: Thank you for your helpful comment. We modified Methods and Results according to your comment and we added a multivariable linear regression analysis adjusted for age, sex, beta blockers and statin use as suggested, adding also a further table (Table 2).

In Methods

“Finally, we performed a multivariable stepwise linear regression analysis evaluating the association between RRI and sNOX2-dp, NO and H2O2, respectively, adjusting for age, sex and concomitant use of beta blockers and statins that could influence OxS biomarkers.”

In Results

“Furthermore, we performed a multivariable stepwise linear regression analysis (Table 2) to evaluate the association between RRI and Ox stress biomarkers adjusting for potential confounding factors as age, sex and use of beta-blockers and statins. We found a significant direct association between RRI and sNOX2-dp and H₂O₂ serum levels and an inverse association between RRI and NO serum levels (Table 2).”

________________________________________________________________________________

The rationale for dichotomizing patients according to the 75th percentile of RRI, in addition to using the established clinical cut-off of 0.7, should be better justified. While percentile-based stratification is acceptable, it may reduce comparability with other studies. Clarifying whether results are consistent across both approaches, or presenting sensitivity analyses using RRI as a continuous variable, would enhance methodological transparency.

Answer: Thank you for your comment and we are sorry that the presentation of the results is not clear. We analyzed RRI as continuous variable in correlation and multivariable linear regression analysis. The use of percentiles is only descriptive of the cohort population. We analyzed RRI as continuous variable and describe the cohort population according to a previous study performed (Geraci et al. Choroidal thickness is associated with renal hemodynamics in essential hypertension, PMID: 31945274, DOI: 10.1111/jch.13777). To clarify the manuscript, we removed the descriptive analysis of RRI cut-off 0.7.

________________________________________________________________________________

The assessment of Mediterranean diet adherence relies on a self-reported questionnaire administered at a single time point, which may not adequately capture long-term dietary patterns. This limitation should be discussed in greater depth, particularly in light of the modest correlations observed. Additionally, reporting the distribution of PREDIMED scores and adherence categories would help readers better contextualize the strength and clinical relevance of the dietary associations.

Answer: Thank You for you suggestions. When we talk about limitations in the discussion section we add “dietary adherence was evaluated using a self-reported questionnaire collected at a single time point. This approach may be influenced by recall and social desirability biases and may not accurately capture long-term habitual dietary patterns or changes over time. Consequently, some degree of exposure misclassification cannot be excluded and may have biased associations toward the null, potentially contributing to the modest correlations observed. Future studies should consider repeated dietary assessments and/or complementary methods (e.g., dietary records and objective nutritional biomarkers) to better characterize long-term adherence” (page 10, 265, 272)

We reported the distribution of PREDIMED scores and adherence categories in Table 1 according to < or ≥75^th percentile of RRI as suggested, please see Table 1.

________________________________________________________________________________

The biological interpretation of the differential behavior of oxidative stress markers deserves further refinement. While the discussion appropriately highlights sNOX2-dp as a sensitive marker of NOX2 activation, the lack of significant associations with NO and H₂O₂ could be explored more critically, considering issues such as biological variability, assay limitations, and the temporal dynamics of these biomarkers. This would prevent overemphasis on a single marker and provide a more balanced redox profile interpretation.

Answer: Thank you for your comment. We add “Although sNOX2-dp is considered a sensitive marker of NOX2 activation, the absence of significant associations with NO and H₂O₂ should be interpreted cautiously. NO-related measures are characterized by substantial biological variability and are highly influenced by diet, renal function, inflammation, and pre-analytical factors (e.g., processing time and storage conditions), which may increase measurement noise in cross-sectional settings. Similarly, H₂O₂ levels may be affected by assay-related limitations and by its rapid turnover within antioxidant systems, resulting in transient fluctuations that a single measurement may fail to capture. Therefore, the differential behavior of these biomarkers likely reflects both biological kinetics and methodological constraints, and our results should be viewed as a composite redox profile rather than evidence driven by a single marker” in Discussion (page 9, 232, 242)

________________________________________________________________________________

The manuscript would benefit from a clearer articulation of its clinical implications. While RRI and oxidative stress biomarkers are proposed as tools for refined risk stratification, practical guidance on how these measures could be integrated into routine clinical practice is limited. Expanding this aspect, even in a speculative manner, and outlining concrete future research directions, such as longitudinal or interventional dietary studies, would enhance the translational impact of the work.

Answer: thank you for your comment. We add “From a clinical standpoint, RRI can be obtained during routine renal Doppler ultrasonography with minimal additional burden and may help identify hypertensive CKD patients with higher intrarenal vascular resistance who could benefit from closer follow-up and intensified risk-factor control. Oxidative stress and endothelial biomarkers may complement RRI by providing a mechanistic ‘redox–vascular’ profile, particularly in patients with discordant clinical risk (e.g., relatively preserved eGFR but elevated vascular risk features). At present, these biomarkers should be regarded as investigational; however, they may inform hypothesis generation and patient selection for lifestyle or dietary interventions. Future research should include longitudinal studies assessing whether RRI and redox biomarkers predict renal and cardiovascular outcomes, as well as interventional trials evaluating whether structured Mediterranean diet programs can improve biomarker trajectories, RRI, and the rate of eGFR decline. Additional work is needed to establish assay reproducibility, clinically meaningful thresholds, and cost-effectiveness before routine implementation” in Discussion (page 9, 243, 256).

________________________________________________________________________________

Reviewer 2 Report

Comments and Suggestions for Authors

This observational study investigates associations between renal resistive index (RRI), oxidative stress/endothelial dysfunction markers, and Mediterranean diet (MD) adherence in 99 hypertensive nephropathy patients. The topic is timely and clinically relevant, with novel integration of RRI and sNOX2-dp in this context. However, methodological limitations, inconsistencies, and overinterpretation weaken the conclusion:

1)This is a single-center, cross-sectional analysis (n=99) precludes causality claims about MD "modulating" pathways or "slowing CKD progression." Many correlations (e.g., RRI-MD: p=0.06; MD-H2O2/NO: nonsignificant) lack power; multivariable adjustment for confounders (age, drugs, dyslipidemia) is absent.

2)Hypertensive nephropathy diagnosis relies on history/eGFR without biopsy or proteinuria exclusion evidence, risking misclassification amid strict exclusions (no diabetes, obesity, CVD). The authors should state it in the limitations section.

3)NO assayed indirectly as NOx (nitrite/nitrate), yet labeled "NO" throughout—misleading given NOx reflects multiple sources. sNOX2-dp/H2O2 correlations with RRI conflict between abstract (p<0.0018) and results figures.

4)PREDIMED questionnaire prone to recall bias; no validation against intake data. Trend-level RRI association (r=-0.18, p=0.06) overstated as supportive evidence.

5)Table 1 reports means/SD despite nonparametric tests used; the authors should clarify distributions. No correction for multiple comparisons in correlations.

6)The authors should revise for mistypos (e.g., "Sens Organs", "HO" vs H2O2 inconsistency); Figure 2 caption errors ("Nitric Perox-ide"). Abstract p-values mismatch results.

Author Response

Answer: Thank you for your kind comment.

________________________________________________________________________________

However, methodological limitations, inconsistencies, and overinterpretation weaken the conclusion:

Answer: Thank you for your comment. According to your corrections, we change in the Abstract “the Mediterranean diet (MD) has been associated with a more favorable oxidative and endothelial profile, although data linking diet to renal microcirculation in hypertensive nephropathy remain limited” in background and “… supporting a potential association between higher MD adherence and lower NOX2-related oxidative stress. These findings are hypothesis-generating and require confirmation in adequately powered longitudinal and interventional studies before any clinical inference on CKD progression can be made” in conclusions.

In the Discussion we add “Overall, our findings are consistent with a potential link between dietary pattern, systemic redox status, and renal vascular damage” (page 9, 232, 233) and “However, given the cross-sectional design, these associations should be interpreted as hypothesis-generating rather than causal. In particular, any mechanistic inference that MD ‘modulates’ oxidative pathways or influences CKD progression cannot be supported by the present dataset and warrants confirmation in prospective studies.” (page 9, 239, 243).

In Limitations paragraph of the discussion, we add and “Moreover, residual confounding cannot be excluded. Although we performed multivariable models for the association between RRI and biomarkers (adjusted for age, sex, and selected therapies), we could not comprehensively adjust all analyses for additional clinical covariates (e.g., dyslipidemia and other treatments) and lifestyle factors. Finally, the modest sample size may have limited statistical power to detect small-to-moderate associations, potentially contributing to trend-level or null findings” (page 10, 281, 285)

________________________________________________________________________________

2) Hypertensive nephropathy diagnosis relies on history/eGFR without biopsy or proteinuria exclusion evidence, risking misclassification amid strict exclusions (no diabetes, obesity, CVD). The authors should state it in the limitations section.

Answer: Thank you for your comment. In the Limitations paragraph of the discussion, we add “Second, hypertensive nephropathy was defined clinically in the absence of histological confirmation. While extensive exclusion criteria were applied to reduce etiologic heterogeneity, some degree of diagnostic misclassification cannot be excluded, particularly regarding the exclusion of alternative causes of CKD (including proteinuric and primary glomerular diseases) in a real-world setting” (page 10, 272, 277)” and in Methods we add “In the present study, kidney biopsy was not performed as part of the protocol; therefore, the diagnosis relied on clinical criteria and exclusion of alternative etiologies based on history, laboratory evaluation, and imaging” (page 11, 346, 348)

________________________________________________________________________________

Answer: Thank you for your comment. We correct the mistakes.

________________________________________________________________________________

4) PREDIMED questionnaire prone to recall bias; no validation against intake data. Trend-level RRI association (r=-0.18, p=0.06) overstated as supportive evidence.

Answer: Thank you for your comment. In the discussion we add “Notably, the association between MD adherence and RRI did not reach conventional statistical significance and should therefore be interpreted as a trend-level finding rather than supportive evidence of a diet–microcirculation effect” (page 8, 210, 213).

________________________________________________________________________________

5)Table 1 reports means/SD despite nonparametric tests used; the authors should clarify distributions. No correction for multiple comparisons in correlations.

Answer: Thank you for your comment, as reported in the Methods, we evaluated normal distribution of continuous variables by the Kolmogorov-Smirnov test, then we applied mean and standard deviation (SD) if they have a normal distribution or median and interquartile range (IQR) if the distribution is non-normal. Then we compare compared by Student’s t-test or Mann-Whitney U test, respectively. We checked and correct table 1 according to your comment. We also specified in the Methods that we performed Bonferroni test for correction of multiple comparison in correlations.

________________________________________________________________________________

6)The authors should revise for mistypos (e.g., "Sens Organs", "HO" vs H2O2 inconsistency); Figure 2 caption errors ("Nitric Perox-ide"). Abstract p-values mismatch results.

Answer: Thank you, we correct all the mistakes.

________________________________________________________________________________

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

accept with no further revision