PONE-D-23-40891Dach1 is essential for maintaining normal mature podocytesPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: I Don't Know

Reviewer #4: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a well-designed and interesting article regarding podocyte pathophisiology. I have several concerns which should be addressed adequately.

1. It is nice to examine some interactions between Dach1 and WT1 expression in the podocyte.

2. How about the expression of DACH1 is in renal biopsy specimens from patients with WT1 mutations?

3. The authors examined the implication of DACH1 in in vivo murine models. Were the results obtained same as those of in a cultured human podocyte?

4. The implication of the results obtained for future perspective particulary in clinical applications should be mentioned.

5. When podocytes injured, was the loss of DACA1 expression firster than that of the other sturauctual molecules?

Reviewer #2: Matsusaka et al present a manuscript describing the renal, specifically the glomerular, phenotype of several DACH1 knockout animals. DACH1 has previously been described by this group and others as a gene expressed highly in glomerular podocytes as well as other renal cells and non-renal tissues, with loss of DACH1 expression in injured glomeruli. Global DACH1 knockout has been reported to confer perinatal lethality by an unclear mechanism; renal hypoplasia was reported by one group, though its connection to lethality was unclear. Cau et al previously reported that podocyte specific knockout animals are normal into adulthood but are highly susceptible to glomerular stress, including diabetes. The current group, using a previously unreported knockout construct, report slightly smaller kidneys in the global DACH1 knockout, but no loss of glomerular number or architecture. Animals with a podocyte-specific Cre driven knockout show overall poor Cre-mediated excision in podocytes, and a variable glomerular phenotype with albuminuria and glomerular scerlosis. Global DACH1 knockout during adulthood also leads to only incomplete DACH1 loss in glomeruli, and a mild renal phenotype. Overall, the results are of interest to the glomerular disease research community, but the results are difficult to fully interpret given their presentation, and the analysis is rather superficial and limited. Clearer presentation of the results already at hand is needed, though additional data generation would be of benefit.

Major concerns:

1. Histologic analysis is provided for 11 podocyte-specific knockout mice. However, per methods, these span in age from 8 weeks to 7-8 months. The age of control mice is not provided. Given the wide span in ages and the authors’ report that albuminuria worsens with age, combining all age groups for analysis is no appropriate. As the authors report that 7 experimental animals were of age 7-8 months, it would seem reasonable to limit histologic analysis to this group, comparing them to similarly aged control mice. Sex-specific data also need to be provided, as there are many instances of sex-specific differences in susceptibility to glomerular injury.

2. The degree of DACH1 excision in podocytes of the NPHS1-Cre and the ROSA-Cre mice is difficult to gauge from the available images and data provided by the authors. It would be helpful to provide some quantitative analysis, or co-immunofluorescence staining with WT1, of a young (perhaps 1mo old) animal to better gauge the efficacy of Cre-mediated excision. An analysis of total podocyte number might also be informative, as one could postulate that once DACH1 is lost in podocytes, these particular cells rapidly die and are therefore not seen in IHC analysis.

3. It would be helpful to provide experimental details for the tamoxifen-mediated DACH1 knockout experiments in the methods, or at the beginning of the results section related to these experiments. Specifically, please specify the age at which Cre-induction occurred, the time between tamoxifen administration and urine and histological analysis, and the sex of the animals.

4. The authors conclude that the lack of staining of several distal nephron markers is indicative of no suggestion of a critical role for DACH1 in tubular cells. This seems a premature conclusion. Urine concentrating ability, assessment of serum electrolyte and acid-base parameters would seem to be useful in better addressing whether there are functional effects on the distal nephron. But even here, it would be possible to postulate indirect effects from other organ systems affected by global DACH knockout. It may be best to temper the language of the conclusion, and just state that distal nephron marker expression was not affected in these animals.

5. Addition of some mechanistic insight into what role DACH1 is playing in the maintenance of podocytes and their function would really add to the manuscript. While not absolutely necessary, without mechanistic data, the descriptive analysis of the renal pathology needs to be enhanced. This could include glomerular injury scores across time and between sexes, analysis of podocyte structure by ultrastructure beyond the single SEM image shown for the NPHS1-Cre;DACH1 flox mice.

Minor:

There is mention of performing a renal biopsy on a mouse in the results section. This is not a routine procedure performed on mice in this reviewer’s experience; please provide details in the methods section. In addition, it would seem that a renal biopsy/partial nephrectomy in a mouse might lead to proteinuria and/or FSGS lesions afterwards, or at least act as a potential second hit. It is not clear that providing histology from this animal (supplemental figure 2) is helpful in assessing the development of pathology in the tamoxifen induced knockout animals. More granular data presentations, as noted above, would be more useful overall.

Reviewer #3: In this manuscript Tanaka and colleagues analyze the phenotypic effects of global, tissue-specific and temporal deletion of Dach1 expression in mice. They find that ubiquitous deletion leads to perinatal death consistent with two previous reports but, in contrast to one previous study, they find relatively normal podocyte and kidney development and conclude that Dach1 is not essential for podocyte precursor differentiation. More strikingly, in adult mice they find that podocyte specific KO of Dach1 (as well as ubiquitous inducible deletion in adult) leads to mice with progressive albuminuria and an FSGS-like syndrome while previous studies have suggested virtually no baseline phenotype in similar KOs (although the latter mice do show a potent susceptibility to injury induced FSGS-like disease). They speculate that these differences could reflect differences in the architecture of the constructs used to generate the KOs and highlight the need for further studies to elucidate the precise reasons for these discrepancies.

In general I find these studies to well-performed and detailed but the conclusions are somewhat dissatisfying in that the authors have not been able to identify a clear reason for the discrepancies between their studies and the previous ones. While some of reported differences appear large and noteworthy, they may not be as significant as the authors conclude. I think they could consider and discuss a much larger range of potential causes for the discrepancy in results between their studies and those of other groups. For example:

1) One rather trivial explanation for the differences between the complete KO data could be strain differences. I note that the authors here have used ES cells from “C57BL/6J x 129S6/SvEvTac F1 mice” to make there KO and then sibling crossed these mice to generate homozygotes. This hybrid background, rather than the cited differences in genetic constructs, is a second possible cause for the discrepant results in the perinatal studies.

2) With regard to the phenotypes in the adult ubiquitous and podocyte-specific deletions, the authors identify what appears to be a more striking phenotype in that their mice exhibit clear evidence of an FSGS-like disease progression whereas previous groups observed normal phenotypes unless the mice were challenged pharmacologically. But this too may actually be a more subtle difference. For example, it is known that a mere 50% reduction in expression of the major podocyte sialomucin, podocalyxin, renders mice and humans highly susceptible to late onset FSGS-like syndromes (Refaeli I et al Sci. Reports 10: 9419, 2020, Barua M et al Kid Int. 85: 124, 2014, Lin FG et al Clin. Sci. 133: 9–21, 2018). This is one of many examples where a subtle change in protein expression can have major consequences on podocyte and kidney function. Accordingly, if in the current studies Dach1 deletion conferred susceptibility, but microbiome status, nutritional or other environmental influences that varied between studies altered the expression of other podocyte proteins, these very subtle differences between experimental conditions could account for what appear to be more major discrepancies between the studies. Minimally the authors should raise this as a possibility in their Discussion section.

Minor criticisms:

1) I was puzzled by thickened endothelial walls and the lack of obvious endothelia fenestrae in the TEM images in Fig 1. These should be readily apparent in glomerular endothelia. Is there an endothelial phenotype in the mice?

2) In Fig 2A there appears to be some evidence of weak deletion of the Dach1 targeted allele in the tail DNA of one of the samples. Is the NHPS1-Cre “leaky” and perhaps deleting in vascular endothelia or other cells in the tail?

3) The authors refer to inefficient deletion of Dach1 in podocytes based on residual expression in many of their mice. Can the authors be sure this inefficient deletion or does this mere reflect a high degree of protein stability that fails to disappear after gene deletion?

Reviewer #4: Tanaka et al. have conducted research on the function of Dach1 using various knockout (KO) mouse models. They have clearly elucidated the phenotypic differences among several types of Dach1 KO mice and highlighted the significance of Dach1. However, there are several concerns within this manuscript that need to be addressed.

Major

1. In Fig 1 Global Dach1 KO Mice, the authors report no significant differences in the expression of podocyte marker proteins between control and Dach1 KO mice in lines 171-173. However, there appear to be no images examining the expression of podocyte markers in this article. Given that the article's central theme is Dach1 and podocytes, the authors should include images that evaluate the podocyte markers.

2. The authors note that kidneys in global Dach1 KO mice were significantly smaller, yet the glomerular diameter and density were not reduced. However, they conclude that the absence of Dach1 causes kidney hypoplasia. Does this imply that Dach1 KO primarily affects tubules and collecting ducts formation without influencing the glomeruli? The mechanism behind this size difference should be explained in more detail.

3. In Fig 2 Nphs1-Cre/Dach1fl/fl mice, while Fig 2B shows the significance of albuminuria, the age of the mice evaluated is unclear.

4. Indeed, Fig 2C demonstrates the increased albuminuria in Nphs1-Cre/Dach1fl/fl mice as they age. However, it is unusual to show images of SDS-PAGE directly. Instead, the authors should present the quantified values of albuminuria by ELISA using a time-course figure, while the SDS-PAGE data could be included in a supplementary figure.

5. The results in Fig 2F are quite complex. Nphs1-Cre/Dach1fl/fl mice exhibited glomerulosclerosis and albuminuria, but the bottom figures of Fig 2F suggest that even preserved Dach1 expression might lead to glomerulosclerosis. Does this indicate that Dach1 expression is not directly associated with glomerulosclerosis? These results seem inconsistent with the authors' conclusion in this section.

6. In Fig 3 Inducible Dach1 KO Mice, similar to Fig 2B, the age of the mice evaluated in Fig 3A is unclear.

7. Also, like Fig 2F, Fig 3C suggests that Dach1 expression in podocytes does not influence podocyte survival. Unfortunately, these results do not support the authors' hypothesis.

8. In Disucssion, the detailed comparisons between the authors' data and previous studies are valuable. If possible, shorten the length of these comparisons.

9. The authors report that various types of Dach1 KO mice exhibit glomerulosclerosis and albuminuria. These findings are of significant interest and represent a novel contribution. However, some results seem inconsistent with the authors' hypothesis.

10. Both Table 1 and supplementary Table 1 are titled "Antibodies for Immunostaining." However, the distinction between these tables is unclear, even though they list different molecules. It is recommended to combine these tables into a single table, preferably as supplementary Table 1, for better clarity.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Dach1 is essential for maintaining normal mature podocytes

PONE-D-23-40891R1

Dear Dr. Matsusaka,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors addressed their original MS. The revised MS is addressed almost adequately. I have no further concerns.

Reviewer #3: (No Response)

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Reviewer #1: Yes: Hiroshi Tanaka

Reviewer #3: No

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