Associations of Transforming Growth Factor-β (TGF-β) with Chronic Kidney Disease Progression in Patients Attending a Tertiary Hospital in Johannesburg, South Africa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Past studies have indicated that TGF-β isoforms may exert both fibrotic and protective effects during CKD progression. The present study aimed to explore the causal relationship between TGF-β isoforms and CKD in the Black population, and the authors concluded that baseline levels of TGF-β isoforms were not predictive of disease progression. This work addresses a gap because, while TGF-β isoforms are known to play roles in renal fibrosis and inflammation, limited clinical data exist on their predictive utility for CKD progression, especially in African populations. While the negative findings limit the immediate clinical applicability of TGF-β isoforms as biomarkers, the study still makes a valuable contribution by clarifying their limitations in this population. While this is a notable contribution, several issues warrant further clarification and consideration:

1.Prior studies indicate that TGF-β isoforms may act as mediators of fibrosis in CKD. To rigorously test this hypothesis, a mediator/moderator approach may be more appropriate than logistic regression. The authors can present the pros and cons of using the current regression model in their study.

2.As most participants were in CKD stages 3–4, it remains unclear whether baseline TGF-β isoform levels reflect consequences of advanced CKD at enrollment rather than determinants of progression. This issue should be clarified.

3.The findings reveal that baseline serum TGF-β1 was lower in progressors, in contrast with prior studies (Refs. 38, 39), which reported higher TGF-β1 levels and associations with lower eGFR. Similarly, the role of TGF-β3 as potentially antifibrotic and renoprotective (Refs. 52, 54) diverges from the current results. The authors should provide further discussion to reconcile these inconsistencies.

4.A large proportion of urine samples had undetectable TGF-β isoform levels, limiting the statistical power. Also, the logistic regression models are limited by relatively few covariates and a modest sample size, which may introduce bias. This limitation should be more explicitly addressed.

5.Overall, the references are appropriate and include key studies on TGF-β biology, CKD biomarkers, and population-specific data. However, some recent reviews and meta-analyses on biomarker discovery in CKD could be added to strengthen the contextualization and underscore why TGF-β isoforms were prioritized.

6.The tables are comprehensive but could be reconstructed for easier reading.

Author Response

Dear Sir/ Madame,

Point-by-point Response TO EDITOR’S AND Reviewers’ COMMENTS

The authors would like to thank the Biomedicines journal and the reviewers for taking time and effort to review our manuscript submission and provide us with insightful and valuable feedback; we appreciate your comments, recommendations and queries.

Our detailed point-by-point response (in bolded bullet points) to each editorial point raised by the editors and reviewer during their review of the manuscript submission is below. 

All amendments and changes to the manuscript in response to the reviewer comments are highlighted in the manuscript. 

Below are the reviewers’ comments followed by bullet point replies in bolded text.

Reviewer 1:

Past studies have indicated that TGF-β isoforms may exert both fibrotic and protective effects during CKD progression. The present study aimed to explore the causal relationship between TGF-β isoforms and CKD in the Black population, and the authors concluded that baseline levels of TGF-β isoforms were not predictive of disease progression. This work addresses a gap because, while TGF-β isoforms are known to play roles in renal fibrosis and inflammation, limited clinical data exist on their predictive utility for CKD progression, especially in African populations. While the negative findings limit the immediate clinical applicability of TGF-β isoforms as biomarkers, the study still makes a valuable contribution by clarifying their limitations in this population. While this is a notable contribution, several issues warrant further clarification and consideration:

1. Prior studies indicate that TGF-β isoforms may act as mediators of fibrosis in CKD. To rigorously test this hypothesis, a mediator/moderator approach may be more appropriate than logistic regression. The authors can present the pros and cons of using the current regression model in their study.

Thank you for this comment. 

• It is true that studies have suggested that TGF-B isoforms may be mediators of fibrosis in CKD.  In our study, we have examined whether TGF-B isoform levels can be used as a predictor of CKD progression using liner regression with CKD progression as our dependent variable. 
• If the TGF-B isoforms are in fact mediators of this process rather than direct effectors, we would still expect to see an effect by the mediator on the dependent variable (i.e. an effect by the TGF-B isoforms on CKD progression), which is what we have looked for in our regression analysis. It is possible that there is an unknown independent variable, that we have not examined, that causes the CKD progression via the TGF-B isoforms. 
• However without knowing what this independent variable is, we are unable to test such a model.  Deducing this independent variable is beyond the scope of this study.  We have added the following line to our limitations section to reflect this: “It has been suggested that the TGF-B isoforms are either partial or complete mediators of CKD fibrosis and inflammation leading to a loss of kidney function. 
• Without knowing the initial variable that affects CKD progression by means of the TGF-B isoforms however, we are not able to assess this hypothesis.”

2. As most participants were in CKD stages 3–4, it remains unclear whether baseline TGF-β isoform levels reflect consequences of advanced CKD at enrollment rather than determinants of progression. This issue should be clarified.

        Thank you for the comment

• A total of 275 (88.1%) of the study patients were in early stage CKD (CKD stages 1- 3) at baseline.

3. The findings reveal that baseline serum TGF-β1 was lower in progressors, in contrast with prior studies (Refs. 38, 39), which reported higher TGF-β1 levels and associations with lower eGFR. Similarly, the role of TGF-β3 as potentially antifibrotic and renoprotective (Refs. 52, 54) diverges from the current results. The authors should provide further discussion to reconcile these inconsistencies. Thank you for the comment

• Further discussion has been provided as advised

4. A large proportion of urine samples had undetectable TGF-β isoform levels, limiting the statistical power. Also, the logistic regression models are limited by relatively few covariates and a modest sample size, which may introduce bias. This limitation should be more explicitly addressed.

Thank you for the comment

• This limitation has been addressed as advised.

5. Overall, the references are appropriate and include key studies on TGF-β biology, CKD biomarkers, and population-specific data. However, some recent reviews and meta-analyses on biomarker discovery in CKD could be added to strengthen the contextualization and underscore why TGF-β isoforms were prioritized.

Thank you for the comment

• Recent reviews and meta-analyses on biomarker discovery in CKD have been added as advised.

6. The tables are comprehensive but could be reconstructed for easier reading.

Thank you for the comment

• We advise that the tables could not be reconstructed and we ask the reviewers/editors for any suggestions on how we could adjust these for ease.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments

1. Need to include most recent prevalence data (e.g., WHO or Global Burden of Disease reports). Furthermore, although the authors highlight the higher prevalence of CKD in low- and middle-income countries, they do not provide Africa-specific or South Africa-specific epidemiological data, which would be particularly relevant since the study was conducted in Johannesburg. Including these details would strengthen the contextual relevance of the introduction.
2. Lines 57–60: The role of TGF-β in fibrosis and inflammation is mentioned, but the underlying molecular pathways are not discussed. Even a brief overview of these pathways would strengthen the biological rationale for investigating TGF-β isoforms as potential predictors of CKD progression.
3. A total of 164 patients were excluded, and most of them (110) had uncontrolled hypertension. Since uncontrolled hypertension and diabetes are very common in real CKD patients, leaving them out makes the study less representative of the real-world situation. The authors should explain why these patients were excluded and discuss whether this could affect how useful the biomarkers are in higher-risk patients.
4. Most of the enrolled patients were already in advanced CKD (stages 3–4). This skewed distribution limits the ability to assess the predictive value of TGF-β isoforms in early CKD, where early detection is most clinically useful. The authors should acknowledge and discuss this limitation.
5. The use of the Kish and Leslie formula for sample size determination is noted, but the exact calculation steps are not provided.
6. The follow-up duration of only 2 years is relatively short for a slow-progressing disease such as CKD. Important cases of mild or gradual progression may have been missed, which could underestimate the predictive value of the biomarkers. The authors should acknowledge this limitation.
7. Need to specify how long samples were stored before assay or whether multiple freeze–thaw cycles occurred. Since these factors can significantly influence biomarker stability, additional details should be provided.
8. The conclusion section should be made more informative to clearly reflect the complete findings of the study. This will help readers better understand the significance and context of research.
9. A separate Discussion section should be included in the manuscript. In this section, need to compare your findings with previous similar studies, if available. Discussing similarities or differences with earlier research will help place your results in context and provide more depth to your study. This will also strengthen the manuscript and make your conclusions more comprehensive for readers.
10. The current title, “Do Transforming Growth Factor-β (TGF-β) Isoforms Predict Chronic Kidney Disease Progression?”, reads more like a heading or a research question. For better clarity and impact, it is recommended to modify the title to more clearly reflect the key findings or scope of the study.

Author Response

Dear Sir/ Madame,

Point-by-point Response TO EDITOR’S AND Reviewers’ COMMENTS

The authors would like to thank the Biomedicines journal and the reviewers for taking time and effort to review our manuscript submission and provide us with insightful and valuable feedback; we appreciate your comments, recommendations and queries.

Our detailed point-by-point response (in bolded bullet points) to each editorial point raised by the editors and reviewer during their review of the manuscript submission is below. 

All amendments and changes to the manuscript in response to the reviewer comments are highlighted in the manuscript. 

Below are the reviewers’ comments followed by bullet point replies in bolded text.

Reviewer 2:

1. Need to include most recent prevalence data (e.g., WHO or Global Burden of Disease reports). Furthermore, although the authors highlight the higher prevalence of CKD in low- and middle-income countries, they do not provide Africa-specific or South Africa-specific epidemiological data, which would be particularly relevant since the study was conducted in Johannesburg. Including these details would strengthen the contextual relevance of the introduction.

    Thank you for the comment

• Most recent prevalence data on CKD has been included as advised.

2. Lines 57–60: The role of TGF-β in fibrosis and inflammation is mentioned, but the underlying molecular pathways are not discussed. Even a brief overview of these pathways would strengthen the biological rationale for investigating TGF-β isoforms as potential predictors of CKD progression.

     Thank you for the comment

• The role of TGF-β in fibrosis and inflammation and the underlying molecular pathways have been discussed as advised.

3. A total of 164 patients were excluded, and most of them (110) had uncontrolled hypertension. Since uncontrolled hypertension and diabetes are very common in real CKD patients, leaving them out makes the study less representative of the real-world situation. The authors should explain why these patients were excluded and discuss whether this could affect how useful the biomarkers are in higher-risk patients.

     Thank you for the comment

• Studies have demonstrated that patients with uncontrolled hypertension and diabetes mellitus are associated with rapid CKD progression. Also, uncontrolled hypertension and diabetes mellitus are associated with increased levels of Transforming Growth Factor-beta 1 (TGF-β1). Despite controlled blood pressure and blood sugar patients still progress to ESRD. The study aimed to determine if Transforming Growth Factor-beta can predict CKD progression among these patients while excluding uncontrolled hypertension and uncontrolled diabetes as confounders. 

4. Most of the enrolled patients were already in advanced CKD (stages 3–4). This skewed distribution limits the ability to assess the predictive value of TGF-β isoforms in early CKD, where early detection is most clinically useful. The authors should acknowledge and discuss this limitation.

Thank you for the comment

• A total of 275 (88.1%) of the study patients were in early stage CKD (CKD stages 1- 3) at baseline. Thus most patients were in early stage CKD.

5. The use of the Kish and Leslie formula for sample size determination is noted, but the exact calculation steps are not provided.

Thank you for the comment

• The sample size was obtained using the Krejcie and Morgan formula (1970) for prospective studies and exact calculation steps are provided.

6. The follow-up duration of only 2 years is relatively short for a slow-progressing disease such as CKD. Important cases of mild or gradual progression may have been missed, which could underestimate the predictive value of the biomarkers. The authors should acknowledge this limitation.

Thank you for the comment

• The duration of only 2 years of follow up has been acknowledged as a limitation as advised.

7. Need to specify how long samples were stored before assay or whether multiple freeze–thaw cycles occurred. Since these factors can significantly influence biomarker stability, additional details should be provided.

Thank you for the comment

• Serum samples were stored at -80°C while urine samples were centrifuged at 4000 rpm for 20 minutes and the supernatant stored at -80°C. There were no multiple freeze – thaw cycles.

8. The conclusion section should be made more informative to clearly reflect the complete findings of the study. This will help readers better understand the significance and context of research.

Thank you for the comment

• The conclusion has been revised accordingly.

9. A separate Discussion section should be included in the manuscript. In this section, need to compare your findings with previous similar studies, if available. Discussing similarities or differences with earlier research will help place your results in context and provide more depth to your study. This will also strengthen the manuscript and make your conclusions more comprehensive for readers.

Thank you for the comment

• The discussion has been revised accordingly.

10. The current title, “Do Transforming Growth Factor-β (TGF-β) Isoforms Predict Chronic Kidney Disease Progression?”, reads more like a heading or a research question. For better clarity and impact, it is recommended to modify the title to more clearly reflect the key findings or scope of the study.

Thank you for the comment

• The title has been changed to reflect the key findings or scope of the study as recommended.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revision has improved the clarity and quality of the manuscript. I have no more comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed all my concern, therefore, this manuscript should be accepted for publication in the journal.