Review Reports

Round 1

Reviewer 1 Report

The present study is an attempt by the authors to detect early changes in renal function in patients with stages 1-3 hypertension (ESC/ESH) with and without past COVID infection .   The authors  examine urinary microalbumin, nephrin, Creatinine, Cystatin -C, eGFR , markers of vascular injury and connective tissue injury (VEGF-A, and TGFbeta, and aldosterone).  The authors also exam renal reserve after protein loading, and intrarenal vascular indices by doppler.   They subsequently treated the patients hypetension using standard guideline based antihypertensive therapy and re-evaluated renal function, the urinary markers of renal injury, renalreserve and intrarenal vascular indices by doppler.   The authors conclude among other things that nephrin is the most sensitive early marker of renal injury, and  that guideline directed antihypertensive therapy improves all renal markers in HTN stages 1 and 2 but not significantly in hypertension stage 3.   

This is an interesting and ambitious study and although this reviewer has multiple comments and critiques the authors should be encouraged to revise this manuscript and resubmit.

MAJOR:

1. Diabetes is defined by fasting blood sugar, not HgbA1c or use of diabetic meds.
2. though purportedly excluded from enrollment, diabetics were probably included as the HTN 3 had FBS with SD >7 which is elevated . 
3. Nephrinuria methodology is problematic.   No "normal" is provided therefore to conclude that nephrin levels are "elevated" 1.4 fold suggests that you know what normal is.  The data presented provides neither usual range of sensitivity for your test, or usual range of normal.  Other reports of nephrin levels usually report in ng/ml... your data is presented in pg/ml and as such your values are barely within detection range and well within what is considered normal in other reports. 
4. In addition even  though the first morning urine sampel is presumed to be concentrated, as a general rule, measurement of sbustances in urine should be corrected to urine creatinine concentration in order to standarize the values and avoid variations due to changes in urine osmolarity. 
5. Define the HTN groups 1-3 ( ESC/ESH)
6. line 193 - what is meant by "early in the disease"
7. lines 204-206.  it is stated that serum cystatin C and egFR were significantly different between groups on ly from stage II, whileserum creatinine was significantly different only in stages II and III,  Both are significantly different in HTN 2 and 3 between covid and Non-Covid. 
8. you state that "nephrinuria is the only biomarker that is significantly i creasedin post-Covid 19 patients at Hypertension stage 1, when traditional functional markers are still normal"   this is not correct as microabluinuria is sinificantly different in HTN 1 in covid vs. non covid. 
9. It states in lines 259-260 that the authors are assessing the early renal impact of SARS CV@ infection in patients with pre-existing hypertension.  All we know from the description of the participants is that they had HTN on study enrollments and SARS CoV2 at some point more than 3 months prior to enrollment.  Unless you have specific information to the contrary , you don't know that the HTN antedated the SARS. 
10. The authors state but do not present in tables or figures the results of response to antihypertensive therpay in stage II or II in covid or non covid patients.
11. in the Conclusion it states that nephrinuria outperforms conventional markers.  As Stated aboe, the earliest biomarker of renal injury is microalbuminuria and it does not appear that nephrinuria outperforms microalbuminuria  from the data presented.
12. Please present the post reatment changes in nephrin in talbe form for the different HTN 1-3 w/wo covid.  this should be presented not just stated. 
13. correct duplication of glomerular hyperfiltration in line 296-297
14. You need to present the doppler data and renal resistive indices by group and post treatment in RESULTS Anot just present it in the discussion 

SEE ABOVE. 

Author Response

Response to Reviewer #1

We sincerely thank the reviewer for the careful reading of our manuscript and for the constructive and detailed comments. We appreciate the reviewer’s positive evaluation of the clinical relevance and ambition of the study. All comments were carefully considered, and the manuscript has been substantially revised accordingly. All revised text has been highlighted in yellow in the revised manuscript.

Comment 1:
Diabetes is defined by fasting blood sugar, not HbA1c or use of diabetic medication. Some patients in HTN stage III may have had undiagnosed diabetes.

Response:
Thank you for this important comment. We revised the Methods and Limitations sections to clarify the diabetes exclusion criteria. Patients with known diabetes mellitus, those taking glucose lowering drugs or those with fasting plasma glucose ≥7.0 mmol/L at enrolment were excluded. We also noted that HbA1c was not measured and that some patients with previously unrecognized dysglycemia may have been included, especially in stage III. In addition, fasting glucose values in Table 1 were carefully rechecked and corrected to ensure consistency with the exclusion criteria.

Comment 2:
Nephrinuria methodology is problematic. No normal range, assay sensitivity, or explanation of units was provided.

Response:
Thank you for this helpful comment. The methodology section for nephrinuria was significantly extended. We included the manufacturer of the ELISA kit, country, detection range of the assay, and the minimum detectable concentration. We also clarified why nephrin was reported in pg/mL and explained that all measured values were above the assay detection threshold and within the linear range of the assay. We explained that the non-COVID stage I group was not an external normal reference group but rather an internal comparator group for urinary nephrin, as there is currently no universally accepted clinical reference range for urinary nephrin.

Comment 3:
Urinary measurements should be corrected to urine creatinine concentration.

Response:
Thank you. We agree this is an important methodological consideration. We explained that first morning urine samples were used to minimize urine concentration and hydration variability. We also included this in the Limitations section and recommended that future studies report nephrin-to-creatinine ratios for standardization and inter-study comparison.

Comment 4:
Please define hypertension stages I–III according to ESC/ESH.

Response:
Thank you. The ESC/ESH hypertension stage definitions were added to Section 2.2 of the Methods.

Comment 5:
The phrase “early in the disease” was unclear.

Response:
The wording was revised for better clarity and specificity.

Comment 6:
The interpretation of cystatin-C and eGFR differences was unclear.

Response:
Thank you for reporting this problem. The Results section was modified to explicitly indicate that the difference between cystatin-C and eGFR was significant from stage II, while the difference between serum creatinine was significant from stage II and III.

Comment 7:
Microalbuminuria was also significantly different in HTN stage I; therefore nephrinuria was not the only altered marker.

Response:
We agree and appreciate this important correction by the reviewer. The manuscript was revised accordingly. We no longer state that nephrinuria was the only altered biomarker. We now report that nephrinuria exhibited a greater relative difference between groups at stage I, but that conventional renal functional markers were within the normal range.

Comment 8:
The manuscript cannot confirm that hypertension preceded SARS-CoV-2 infection.

Response:
Thank you for this important point. We have carefully revised the manuscript and changed the wording from “pre-existing hypertension” to more cautious language, “patients with established hypertension at enrollment.”

Comment 9:
The results of response to antihypertensive therapy were not adequately presented.

Response:
This is a good observation. A new table was added showing pre and post treatment values of systolic blood pressure, nephrinuria, microalbuminuria, TGF-β1 and aldosterone in all stages of hypertension in both post-COVID-19 and non-COVID-19 groups.

Comment 10:
The conclusion overstated that nephrinuria outperformed conventional markers.

Response:
Thank you. The conclusion was revised and moderated. The revised text now describes nephrinuria as a potential early marker associated with podocyte injury rather than a definitive superior biomarker.

Comment 11:
Please present post-treatment nephrinuria changes in table form.

Response:
Thank you. These data have been added to the newly added treatment-response table (Table 4).

Comment 12:
Please correct duplicated wording regarding glomerular hyperfiltration.

Response:
The duplicated wording was corrected in the revised manuscript.

Comment 13:
Doppler data and renal resistive indices should be presented in the Results section.

Response:
Thank you for this valuable suggestion. Table 5 was added to show the renal resistive index (RI) and pulsatility index (PI) values obtained from Doppler before and after treatment in all the study groups.

We are grateful to the reviewer for the thoughtful comments and suggestions that greatly enhanced the scientific quality, clarity, and methodological transparency of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript is a prospective comparative cohort study evaluating whether urinary nephrinuria can serve as an early biomarker of renal injury in hypertensive patients with a history of COVID-19. The topic is clinically interesting because it integrates post-COVID-19 status, hypertension, podocyte injury, nephrinuria, renal functional reserve, and renal hemodynamic parameters. The approach of attempting to detect renal injury at an earlier stage than conventional renal function markers in hypertensive patients is meaningful. In particular, the comparison between post-COVID-19 and non-COVID-19 groups according to hypertension stage, together with the assessment of nephrinuria, microalbuminuria, TGF-β1, aldosterone, and VEGF-A, represents a distinctive feature of this study.

However, the current manuscript requires further improvement in several areas, including the reproducibility of the methods, the presentation of results, the distinction between results and interpretation, the specificity of the treatment protocol, and the strength of the clinical conclusions. Therefore, I believe that this study would be suitable for re-evaluation after the following issues have been addressed.

1. Although the Abstract states that nephrinuria and related biomarkers were increased in hypertensive patients after COVID-19, it is not immediately clear to the reader which comparison group these findings refer to. For example, briefly specifying that the comparison was made against non-COVID-19 patients at the same hypertension stage would improve the clarity of the Abstract.

2. It would enrich Section 2.1 if brief information were added regarding Bukhara State Medical Institute, such as its hospital capacity and its role in the local community or regional healthcare system.

3. In Section 2.3, biochemical tests were performed using serum samples. To improve transparency and reproducibility, please add the manufacturer and country of origin of the analytical equipment used, as well as information on the reagents. The same applies to the ELISA kits used in the study.

4. In Sections 2.5 and 3.4, the manuscript refers to treatment according to current guidelines. It would be helpful to briefly specify which current hypertension or nephroprotective treatment guidelines were followed.

5. In Section 3.2, the means and standard deviations for each group are presented. Please consider adding 95% confidence intervals to provide a clearer indication of the precision of the estimates.

6. Some sentences in the Results section appear to be interpretive and may be more appropriate for the Discussion section. For example, in lines 202–204, the statement “while nephrinuria was already significantly different in this same group, indicating that nephrinuria may enable earlier detection of podocyte injury” is interpretive. Similarly, in lines 230–232, the statement “suggesting that the convergence of profibrotic, endothelial and podocyte injury signals is enhanced in patients with a history of SARS-CoV-2 infection (Table 3, Figure 3)” would fit better in the Discussion. The same applies to Section 3.4, lines 250–257: “Nephrinuria, microalbuminuria, TGF-β1 and aldosterone concentrations were reduced following therapy with statistically significant improvements in stages I and II in both groups. In stage III patients, post-treatment changes were less pronounced and some markers did not change significantly, in line with structural changes at this stage and relative irreversibility of late-stage hypertensive nephropathy. Doppler-derived intrarenal velocities improved more consistently in non-COVID-19 patients than in post-COVID-19 patients at the same stage of hypertension: this suggests persistent microvascular dysfunction in post-COVID-19 patients.” The latter parts of these statements contain explanatory interpretations and could be moved to the Discussion section.

7. Please compare the present findings with studies from other countries or institutions in the Discussion section.

8. The claim that nephrinuria enables early CKD risk stratification should be moderated. The study did not assess long-term renal outcomes, such as incident CKD, sustained eGFR decline, or progression to overt kidney disease. The findings support nephrinuria as a candidate early biomarker, but its prognostic value requires validation in longitudinal outcome-based studies.

9. Additional limitations should also be mentioned. First, COVID-19 severity and the clinical status at the time of infection were not sufficiently reflected. Second, the type, dose, adherence, and prior use of antihypertensive medications were not clearly described, making it difficult to attribute the six-month treatment response to specific drugs or RAAS blockade. Third, the possibility of false-positive findings due to multiple comparisons and the small sample size should be acknowledged. Fourth, because no reference range or clinically meaningful threshold for nephrinuria was provided, its immediate clinical applicability remains limited.

 

 

Author Response

Response to Reviewer #2

We are grateful to the reviewer for his careful reading of our manuscript and for his constructive and thoughtful comments. The reviewer's appreciation of the clinical relevance and originality of the study is appreciated. Every comment was taken into account and the manuscript was revised accordingly. All revised text has been highlighted in yellow in the revised manuscript.

Comment 1:
The Abstract does not clearly specify which comparison group was used.

Response:
Thank you for this valuable suggestion. The Abstract was amended to state that comparisons were made between post-COVID-19 patients and non-COVID-19 patients in the same hypertension stage.

Comment 2:
Please provide brief information about Bukhara State Medical Institute and its role in the regional healthcare system.

Response:
Thank you. The section 2.1 was extended to provide a short description of Bukhara State Medical Institute, its status as a major academic medical institution and tertiary referral center for the Bukhara region and neighboring regions.

Comment 3:
Please add manufacturer and country information for the analytical equipment, reagents, and ELISA kits.

Response:
We appreciate this valuable suggestion. The manufacturer names, countries of origin, analyzer details and reagent information for all major biochemical and ELISA based measurements used in the study were added to section 2.3.

Comment 4:
Please specify which hypertension and nephroprotective treatment guidelines were followed.

Response:
Thank you. Sections 2.5 and 3.4 were updated to reflect treatment according to the ESH Guidelines for arterial hypertension management 2023 and KDIGO 2021 Clinical Practice Guideline for blood pressure management in chronic kidney disease.

Comment 5:
Please consider adding 95% confidence intervals.

Response:
Thank you for this suggestion. To aid interpretation and precision of the estimates, 95% confidence intervals were added for the key nephrinuria comparisons in Section 3.2.

Comment 6:
Some statements in the Results section are interpretive and would fit better in the Discussion.

Response:
We agree and appreciate this important observation. Some interpretive/explanatory statements were moved from the Results section to the Discussion section to help keep the objective findings separate from the interpretation/explanation.

Comment 7:
Please compare the findings with studies from other countries and institutions.

Response:
Thank you. The Discussion section was extended to compare with international studies and other post-COVID renal cohorts from other countries, such as those on podocyte injury, nephrinuria, endothelial dysfunction, and renal involvement in long COVID.

Comment 8:
The claim regarding early CKD risk stratification should be moderated.

Response:
This is a valuable comment, which we appreciate. The Conclusions and Clinical Implications sections were modified to temper the statements about prognostic value. The manuscript now states that nephrinuria is a potential or candidate early biomarker, and that its prognostic value needs to be confirmed in longitudinal outcome-based studies.

Comment 9:
Additional limitations should be acknowledged.

Response:
Thank you for this valuable suggestion. The Limitations section was significantly expanded. We added limitations related to:
(i) lack of stratification according to COVID-19 severity and clinical course,
(ii) incomplete characterization of prior antihypertensive treatment and medication adherence,
(iii) possible false-positive findings related to multiple comparisons and moderate sample size, and
(iv) the absence of a universally accepted clinical reference range or threshold for urinary nephrin.

We are grateful to the reviewer for the constructive comments and suggestions that greatly enhanced the methodological clarity, scientific rigor and clinical interpretation of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I would like to thank the authors for their careful revision and detailed responses to the reviewer’s comments. The revised manuscript has been substantially improved, particularly in terms of clarification of the study setting, laboratory methods, treatment guidelines, separation of results and interpretation, and expansion of the limitations.

I have only one remaining minor comment. The authors have added a 95% confidence interval for the key stage I nephrinuria comparison, which is helpful. However, most comparisons in Table 2 are still presented mainly as mean ± SD with p-values. I would encourage the authors to reconsider adding between-group mean differences with 95% confidence intervals more consistently, at least for the main nephrinuria comparisons across hypertension stages, and preferably for the key renal biomarkers in Table 2. This would improve the clarity, precision, and interpretability of the reported results.

Overall, the manuscript is much improved, and I recommend minor revision.

Author Response

Major comments

I would like to thank the authors for their careful revision and detailed responses to the reviewer’s comments. The revised manuscript has been substantially improved, particularly in terms of clarification of the study setting, laboratory methods, treatment guidelines, separation of results and interpretation, and expansion of the limitations.

Detailed comments

I have only one remaining minor comment. The authors have added a 95% confidence interval for the key stage I nephrinuria comparison, which is helpful. However, most comparisons in Table 2 are still presented mainly as mean ± SD with p-values. I would encourage the authors to reconsider adding between-group mean differences with 95% confidence intervals more consistently, at least for the main nephrinuria comparisons across hypertension stages, and preferably for the key renal biomarkers in Table 2. This would improve the clarity, precision, and interpretability of the reported results.

Overall, the manuscript is much improved, and I recommend minor revision.

Response:
We sincerely thank the reviewer for the positive evaluation of our revised manuscript and for the constructive final suggestion. In response to this suggestion, we have added between-group mean differences with 95% confidence intervals for the principal renal biomarkers (nephrinuria, microalbuminuria, creatinine, cystatin-C, and eGFR) across the various stages of hypertension in Table 2. We also included the corresponding text in the Results section to enhance clarity and interpretation of the effect sizes between the post-COVID-19 and non-COVID-19 groups.

We agree that the addition of mean differences and confidence intervals makes the results more precise and easier to interpret and enhances the statistical presentation of the manuscript. The reviewer's comments are appreciated and have contributed to further enhancing the quality and transparency of the study.

 

 

Author Response File: Author Response.pdf