Review Reports

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript by Y. Wang et al., describes the role of voltage-gated potassium channel (Kv1.3) gene Kcma3 (encoding Kv1.3) in the regulation of hypertension. The authors indicated that Kcma3 gene-knockout (KO) mice exhibited elevated blood pressure (BP) and renal fibrosis. Transcriptomic analysis showed that renin-angiotensin-aldosterone system (RAAS) was upregulated in both Kcma3 KO mice. Also, the Kv1.3 inhibitor-treated cells showed the upregulation of RAAS. The authors concluded that Ca²⁺-dependent AKT-PKA-CREB signaling pathway seems to activate RAAS. The current study presents some interesting information; however, in the present form this manuscript is descriptive and preliminary in nature. There seems to be a fishing expedition strategy, which has been utilized to support the conclusions. However, some additional concerns on this manuscript remain as follows:

1. BP analysis is an important aspect of the present studies. The differences in BP between the groups is very minimal and seem to be insignificant. The disturbing issue is that there are not significant differences between systolic BP (SBP) and mean arterial pressure (MAP) analyses. At the same time the diastolic BP (DBP) is also seem to be very close among the groups. In control Bl/6 mice the SBP normally ranges between 100-110 mmHg, the increase in BP is very minimal in these exponential animal models. The statistical significance shown seems to be unrealistic here.
2. The authors have measured renin levels by Western blot and immune-fluorescence analyses and briefly renin activation in plasma by ELISA. A major concern of the current studies is the lack of measurement of renin activity, particularly renin concentration in these mouse models. Without data on renin concentrations the present study is very weak and poses uncertainly. Largely, the protein levels do not indicate the appropriate activation of renin and the optimum levels of formation of angiotensin(s), which are responsible for increasing BP.
3. The measurements of just plasma angiotensin II (ANG II) and aldosterone do not confirm the credible RAAS regulatory mechanisms.
4. To provide concrete evidence of the involvement of AKT-PKA-CREB pathway, the chromatin immunoprecipitation and transcriptional studies are critical. The current study lacks the fundamental and mechanistic basis to support the conclusions.
5. The hematoxylin & eosin and Mason’s trichome staining (Figure 2) and immunofluorescence staining (Figure 4) are of poor quality; these should have been substantially improved.
6. All Western blot cropped gels are overexposed. These blots should be supported by full length raw gel blots with appropriate ladder marker proteins. The authenticity of these data are of major concerns and reliability in the supplement materials.
7. The microarray gene expression data technology is almost obsolete, and it provides many misleading analytical information. The authors need to use single cell RNA sequencing (scRNAseq) with ATAC multimode analysis.
8. The statistical analyses utilized here are week, which cannot be accomplished by using the parametric analysis, including student’s “t” test and ANOVAs. These should be accessed by normality tests.
9. There are numerous grammatical, errors and sentence and syntax structures, which should be edited and corrected throughout the manuscript.

 

Author Response

Please see the attachment.

   

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors attempt to link Kv1.3 with hypertension by using whole animal and in vitro approach. They observe elevated blood pressure in animals with modified kcna3 status. The authors perform histological examinations, immunofluorescence, transcriptomic studies and pathway enrichment studies to attempt to elucidate the mechanism through which kcna3 influences blood pressure.

Major Comments

The global kcna3 knockout status is not very convincing since RNA transcripts and western blots still show the presence of large amounts of the channel. More genotype validation data are needed. 

Bar graphs are all missing labels.

The data are overinterpreted. The text at the end of every section needs to be toned down to be less declarative. 

Suggested edits/queries

Line 64: Full form, what does it encode?

Section 2.1 line 86 etc.: Did western blots confirm that this gene was indeed knocked out in these animals?

Section 3.1 line 281-283: 
Since Kcna3 modulation of RAAS has not yet been established at this stage of the study, this statement is misleading and not needed.

Line 291: These are WT mice and not a hypertension model. Since the gene was CRISPRd out, it should follow that RNA transcripts and subsequent protein translation should also be nearly undetectable. This is not new information. It is unclear why the authors keep mentioning the levels of a gene that has presumably been knocked out and interpreting data based on these findings

Line 292: Please explain how you got to this conclusion. This figure appears to be a knock out validation figure to show that it was successful and then experimentally elucidate whether these mice are hypertensive etc in the rest of the study.

FiG1A: Typo correct Chang to change

Figure 1 D, E: If this was a global knockout, why was half the fraction in the KO heart still preserved? It might be more appropriate to refer to this as a knockdown and NOT a knockout,

Figure 1 : Heart IF images needed

Figure 1 C legend needs to mention that C, D, E, F are in mice

Line 320, provide full forms before acronyms

Figure 2F : histology images need to be high resolution. Hard to look at in the current format.

Figure 4B, labels for bar graph are missing

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

General: This paper reports on a very comprehensive and detailed study describing the molecular pathway by which a decrease or absent Kv1.3 channel in kidney cells leads to the activation of renin release leading to increased activity of the intrarenal Renin-Angiotensin System and to the development of hypertension. The paper is well organized and clearly written. The results are presented in a clear logical manner and presented in detail. The use of several models that support the hypothesis strengthens the study. However, there are some overstatements that need to be deleted or corrected and some important issues.                          Specific comments: 1) The second sentence in the introduction should be deleted as there are many studies that have reported on the molecular pathways linking Kv1.3 channels to mechanisms of hypertension including renin. 2)  Missing from the study are in vivo functional measurements of renal hemodynamic function or GFR or of changes in Urinary electrolyte would have provided in vivo relevance. There was a chance to do functional measurements when the mice were anesthetized for the direct arterial pressure measurements. 3) While the results are presented in a comprehensive manner, there are so many panels in each of the figures that the individual graphs are difficult to read and follow. 4) Please check section 3.2 starting with lines 312 discussing the staining, you refer to Fig. 2D,E on line315. but Fig 2F shows the H&E and Masson's staining. Also, you refer to FigF,G on line 318, but should be G and H. 5) Section 3.5-It is surprising that you only used a CD cell line for the renin expressing studies when most of the renin is produced by JG cells. You acknowledge this later but suggest that most of the renal renin is from CD cells. Actually, much of the renal interstitial renin is from JG cells. The available data do not support the notion of a dual compartment of 2 distinct renin pools unless you men tubular versus interstitial renin. Do you have any data from JG cells? 6) I suggest deleting the first sentence in the discussion as it is not likely to be correct; there are many studies that have focused on the role of Kv1.3 in various aspects of blood pressure and cardiovascular homeostasis. 7) Finally, it seems to me that the increased intracellular Calcium that occurs in vascular cells of the knockout or blocked mice could also directly contribute to increased vascular tone and increased vascular resistance which would synergize with the volume retention and contribute to the hypertension. It is not correct to attribute all the hypertension that resulted solely to a volume dependent mechanism. These are all issues that are worthy of further discussion.    

 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have provided partial responses to the comments from the previous review; however, the manuscript has not been sufficiently revised to address the major concerns raised previously. In its current form, the manuscript remains largely descriptive and does not incorporate the specific revisions and mechanistic insights requested in the previous review. The revised manuscript should include the suggested experimental approaches listed below and clearly demonstrate how the new data supports the conclusions.

1. The major weaknesses identified earlier remain unresolved. To strengthen the scientific impact of the study, the authors should address these limitations by performing the additional new experiments and analyses previously recommended.
2. The determination of renin concentration is critical, which is a true measure of the actual renin activity, and without this data the manuscript is weak and erroneous to support the conclusion presented in the current manuscript.
3. Bulk RNA sequencing data is almost outdated. The authors need to perform single cell RNA sequencing and possibly with multiome including ATAC analysis.
4. The microarray experimental analysis is absolutely obsolete and should be deleted from the current manuscript.
5. AKT-PKA-CREB pathway needs to be revisited by incorporating the chromatin immunoprecipitation and transcriptional studies to make any realistic conclusions.
6. The immunofluorescence studies seem to reflect artifactual results, which need to be revised and improved.
7. The blood pressure studies are weak and largely without significant differences. It is unclear to state resting BP in these experiments, which should be clearly described.
8. All Western blot cropped gel bands need to be supported by full lengths raw gel with ladder marker proteins. Without the supporting data all Western blots cropped gel bands are highly questionable in this manuscript.

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

All points have been satisfactorily resolved.

Author Response

We sincerely thank the reviewer for taking the time to re-evaluate our revised manuscript. We are pleased that all points have been satisfactorily resolved. We appreciate the reviewer’s constructive input throughout the review process.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Thank you for your detailed responses to the concerns and issues rised nd the suggestions made. 

Author Response

We thank the reviewer for the positive feedback and for acknowledging our detailed responses. We appreciate the reviewer’s time and valuable suggestions, which have helped us improve the quality of this work.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

      Wang et al. investigated the role of the Kv1.3 channel in blood pressure hypothesizing that loss of Kv1.3 could lead to an increase in blood pressure.  To assess this tenet, KV1.3 KO mouse model was generated that showed a marked reduction of the channel in hart, liver and kidney.  Using telemetry, Kv1.3 KO mice exhibited higher systolic, diastolic and mean blood pressure that associated with an increase in circulating renin, angiotensin and aldosterone.  Increased renin expression was detected in tubular areas of the kidney that localized with aquaporin 2, a marker of the collecting duct.  Authors also demonstrate a PKA and Akt activated pathway associated with higher phosphorylated CREB in in the kidney of Kv1.3 KO mice   Finally, the study demonstrates a Kv1.3 pathway regulating PKA, Akt, CREB and renin in the M1 collecting duct cells that supports the mouse studies.   

      While the study addresses an important question on renin regulation and hypertension, as well as providing substantial datasets, several significant methodological and mechanistic concerns limit interpretation which are detailed below of the review.  Collectively, these issues raise concerns about data validity, mechanistic interpretation, and overall conclusions.

Major concerns: 

1. The premise of the study is the Kv1.3 deletion increase intracellular Ca2+ levels and kinase associated pathways including PKA, Akt and CREB.  However, activation of these pathways would lead to the downregulation of juxtaglomerular renin which is secreted into the circulation to activate the angiotensin pathway.  The author show an increase in collected duct renin but these cell secrete renin into the tubular fluid which are excreted in urine, but not into the circulation.   Therefore, the reviewer doesn’t understand how there can be an increase in circulating renin  in this study.  Indeed, an increase in blood pressure should inhibit renin release into the circulation through activation of renal baroreceptors.  

2. In addition to circulating renin, the authors demonstrate an increase in angiotensin. However, what angiotensin are measuring as there are many “angiotensins”?    Moreover, no information is provided in the paper as to what and how the “angiotensin” was measured.   Finally, serum is not appropriate to measure angiotensin levels as its metabolized rapidly in samples without addition of strong inhibitors. 

3. No details are provided on the measurement of renin or aldosterone in the study.   

4. The authors show that Kv1.3 is not different between WKY and SHR but there appears to trend for higher Kv1.3 in WKY and only 4 animals were measured. Authors should increase the N in their study as well as more specifically assess Kv1.3 distribution. 

5. How does an increase in CD renin increase blood pressure in their Kv1.3 KO mice since CD renin is secreted into the tubular fluid/urine and not into the circulation.

6. For the authors to claim that Kv1.3 activates the renin-angiotensin system (RAS) leading to hypertension, authors must show that administration of a renin inhibitor and/or an AT1 receptor blocker (ARB) administered separately abolishes the increase in blood pressure in the Kv1.3 KO mice to confirm that RAS activation is the hypertensive mechanism.

7. The blood pressure increases from 12 to 14 in the adult Kv1.3 KO mice. Why does this increase occur over a short period of time in the adult transgenic mice?

8. The blood pressure data should be analyzed by a 2-way ANOVA with time and species as the two variables.

9. Renal renin is typically expressed as two bands by Western at 50-55 and 35-37 as both prorenin and renin, respectively; however, the authors detect a singe band of 45 kDa in kidney and M-1 cells.   What is this band – prorenin or renin? 

10. Authors should demonstrate that renin release/secretion is increased in the M-1 cells in addition to changes in cellular expression following various treatments.

   

 

 

Author Response

Please see the attachment.

   

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates an interesting and potentially important mechanism linking Kcna3/Kv1.3 deficiency with renin upregulation, RAAS activation, hypertension and renal injury. The study combines animal experiments, transcriptomic analysis, blood pressure measurements, histology, western blotting, immunofluorescence and pharmacological inhibition in renal cells. Overall, the topic is relevant for the field of hypertension, renal biology and ion channel signalling, and the proposed Ca²⁺-dependent AKT–PKA–CREB axis is biologically plausible and potentially novel.

1. The main strength of the study is the integrated experimental design. The authors do not rely on only one type of data, but combine in vivo findings in Kcna3 knockout mice with transcriptomic and in vitro pharmacological evidence. This gives the manuscript a stronger mechanistic structure. The finding that Kcna3 deficiency is associated with increased blood pressure, renal fibrosis, RAAS activation and Renin upregulation is interesting and deserves attention.
2. The authors propose that loss of Kv1.3 impairs K⁺ efflux, causes membrane depolarization, increases intracellular Ca²⁺, and then activates AKT, PKA and CREB signalling, leading to Renin upregulation. This pathway is attractive, but the manuscript would benefit from a clearer explanation of how each step was directly demonstrated and which steps remain inferred. In particular, the authors should more carefully separate confirmed experimental results from proposed mechanistic interpretation.
3. The cellular source of Renin should be discussed more carefully. The immunofluorescence data suggest that Renin is increased mainly in tubular regions and partially overlaps with Aqp2-positive collecting duct areas. This is an important observation, but it also raises questions. Since renin is classically linked to juxtaglomerular cells, the authors should discuss more clearly why collecting duct-derived Renin is relevant here, how strong the evidence is, and whether additional markers or cell-specific approaches would be needed in future studies.
4. The study would be stronger with additional in vivo validation of the signalling pathway. The in vitro inhibitor experiments with BAPTA-AM and H89 support the involvement of Ca²⁺ and PKA signalling. However, the same pathway is not directly inhibited or rescued in vivo. This does not weaken the whole manuscript, but it should be presented as a limitation. The authors may also consider adding, if available, in vivo or ex vivo validation of Ca²⁺/PKA/CREB signalling in kidney tissue.
5. There are several formatting and consistency issues that should be addressed. For example, section numbering appears inconsistent in the Results section, where subsections are labelled as 2.2, 2.3, 2.4 and 2.5 instead of 3.2, 3.3, 3.4 and 3.5. Some headings also need capitalization correction, such as “measurement of Tissue Total Calcium Content” and “Rna Extraction and Quantitative Real-Time Pcr”. The authors should also check spacing errors, figure references, statistical descriptions, and consistency in gene/protein naming.

Comments on the Quality of English Language

The manuscript is generally readable, and the scientific message can be traced. The English needs cleaning up before publication. Many expressions need polishing to make them more natural and precise, some sentences are repetitive, and some transitions are mechanical. We recommend some moderate language editing by a fluent scientific English speaker, especially in the Abstract, Discussion and figure legends.

Author Response

Please see the attachment.

   

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The current manuscript presents compelling evidence linking Kv1.3 channel dysfunction to hypertension through calcium-dependent signaling pathways. The research is innovative and clinically relevant, combining transcriptomics with mouse models and pharmacological studies. While the foundational work is solid, the reviewer identified gaps in study design and data interpretation requiring attention before publication. Addressing these concerns will strengthen your manuscript's impact.

 

1. The Kv1.3-RAAS hypothesis is genuinely innovative. However, three gaps need attention: a) Functional vs. transcriptional disconnect: Expression unchanged in hypertension, yet you use complete knockout. Discuss what suppresses channel function without altering gene expression. b) Missing cardiac data: Despite Kv1.3 heart expression, you lack echocardiography or heart remodeling metrics—critical given systemic hypertension. c) Model limitations: Global knockout doesn't reflect human disease mechanisms.

 

2. Figure 2A-C: Blood pressure measurements start at weeks 12-14, missing critical early phases. Add baseline and earlier timepoints (weeks 4-8) to show progressive hypertension onset and strengthen disease trajectory characterization.

 

 

3. Figure 3I and Figure 5: The study focuses on collecting duct cells, but juxtaglomerular cells are the primary systemic renin source—a major interpretive gap. Perform in situ hybridization or targeted staining to distinguish JG versus CD renin contributions and contextualize collecting duct findings in your discussion. Clarifying which cell type drives hypertension is essential for mechanistic credibility.

 

Minor:

1. Figure 1: Correct "IIeart-Kv1.3" to "Heart-Kv1.3" in western blot label.
2. Figure 2: Verify scale bar accuracy in figure legend (Line 303). Confirm whether 50 μm or 100 μm matches image annotations.
3. Figure 3: Fix Y-axis typo in GSEA plots: change "signa_to_noise" to "signal_to_noise" (Figs 3E, 3F).
4. Line 134: Add missing space: "SBP and DBP were" (not "DBPwere").
5. Line 164: Fix header formatting: "2.5 Measurement of Tissue Total Calcium Content" (capitalization and spacing).
6. Line 179: Correct acronyms: "2.7 RNA Extraction and Quantitative Real-Time PCR (qPCR)" (not "Rna... Pcr").
7. Line 199: Fix antibody dilution typo: "1:1000 dilution" (remove extra characters).
8. Line 215: Correct numbering error. Section 2.8 appears twice; renumber sequentially.
9. Line 249: Bold/italicize "2.10 Statistical Analysis" to match other subheadings.
10. Lines 324, 326, 329: Use standard format: "Figure 3G, J" (not "Figure 3G and J").
11. Line 493: Add missing spaces: "National Science and Technology Major Project" (not "NationalScience").

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

     

      Wang et al. have provided a revised version of the study that investigated the role of the Kv1.3 channel in blood pressure hypothesizing that loss of Kv1.3 could lead to an increase in blood pressure. However, there remain major concerns with the study. 

Major concerns: 

1. The authors should refer to renin as collecting duct renin or CD renin as no evidence is provided that increased renin is derived from JG cells, the predominant source of renin in the body.
2. Again, authors fail to adequately explain how there can be an increase in circulating renin as the CD secretes renin into tubular fluid and excreted into the urine.
3. Again, authors fail to adequately explain why an increase in blood pressure doesn’t reduce circulating renin and Ang II since the kidney reduces JG renin release in response to higher blood pressure and increased Ang II.
4. Although the reviewer requested confirmation that Ang II contributes to hypertension, authors failed to provide data that RAAS blockade actually reduces blood pressure in their model.
5. Authors should clarify whether their renin ELISA measure prorenin as well.
6. The authors failed to appropriately analyze their blood pressure data as requested by the reviewer and should be analyzed by a 2-way ANOVA with time and species as the two variables, not a t test.
7. The telemetry recording should be extended over several weeks and show both daytime and nighttime blood pressures to better support chronic changes in pressure in their model.   
8. Authors failed to demonstrate that renin release/secretion is increased in the M-1 cells in addition to changes in cellular expression as requested by the reviewer.
9. Finally, the supplied gel images described by the authors as the original Western blot gels appear as cutouts of images rather than full length gels with corresponding mol wet markers. The reviewer has no confidence that these cut out images are original

Reviewer 2 Report

Comments and Suggestions for Authors

My comments have been addressed and the manuscript is significantly improved.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript seems much improved. The responses provide by the authors seem satisfactory.