Regulation of the Actin Cytoskeleton in Podocytes

Round 1

Reviewer 1 Report

Review of the manuscript “Regulation of the Actin Cytoskeleton in Podocytes” written by Judith Blaine and James Dylewski: A great amount of work has been devoted to write this review considering the results of 207 studies. I agree that it would have been impossible to include all studies performed on this topic. However, at least one recent study has been overlooked, that seems to me of basic importance when reviewing the regulation of the actin cytoskeleton of podocytes. Suleiman and Miner (JCI Insight 2017) have shown that podocyte foot processes do not contain myosin, thus do not have contractile abilities. This lack uncovers a deficiency of this review. The mechanical challenges to podocytes, that underlie the regulation of the actin cytoskeleton have not been defined. The authors talk of “enormous hemodynamic stresses” (line 29), which one?, “local changes in the microenvironment” (31), which one?, “are under significant tensile and shear stress forces”(240) from where do they come from?, “is subjected to a wide area of hemodynamic forces”(321), which one? This review reports, mostly correctly, the cell biological mechanisms that underlie the regulation of the actin cytoskeleton, but it does not relate these regulations to the challenges that have provoked them.
Other points: (i) Line 28; “wrap tightly around glomerular endothelial capillaries”. Podocytes never wrap around a capillary, they only cover part of their surface. (ii) Line 99 ff: the mechanical relevance of the slit diaphragm concerning the interconnecting foot processes is lacking

Author Response

We appreciate the reviewer's thoughtful comments that have helped to strengthen this manuscript.

Below please find a point-by-point response to the reviewer's comments.

1)  "However, at least one recent study has been overlooked, that seems to me of basic importance when reviewing the regulation of the actin cytoskeleton of podocytes. Suleiman and Miner (JCI Insight 2017) have shown that podocyte foot processes do not contain myosin, thus do not have contractile abilities. This lack uncovers a deficiency of this review"

We appreciate the reviewer directing us to this important paper and have included this study and an explanation of its importance in the manuscript (lines 84-90).

2) "The mechanical challenges to podocytes, that underlie the regulation of the actin cytoskeleton have not been defined. The authors talk of “enormous hemodynamic stresses” (line 29), which one?, “local changes in the microenvironment” (31), which one?, “are under significant tensile and shear stress forces”(240) from where do they come from?, “is subjected to a wide area of hemodynamic forces”(321), which one? This review reports, mostly correctly, the cell biological mechanisms that underlie the regulation of the actin cytoskeleton, but it does not relate these regulations to the challenges that have provoked them. "

We have included sentences in the Introduction explaining the hemodynamic forces (tensile and shear stresses, lines 29-34) that podocytes are subject to as well as a new section on hemodynamic forces affecting podocytes (lines 464-479) and how these relate to changes in the podocyte actin cytoskeleton. We have removed the words "local changes in the microenvironment"). 

3)"Other points: (i) Line 28; “wrap tightly around glomerular endothelial capillaries”. Podocytes never wrap around a capillary, they only cover part of their surface. (ii) Line 99 ff: the mechanical relevance of the slit diaphragm concerning the interconnecting foot processes is lacking"

We have changed line 28 to read "Podocytes are terminally differentiated cells that form the outer layer of the GFB .."

We have added the following to the section on the slit diaphragm (section 2.3):

"The slit diaphragm (SD) is a size-selective barrier between two podocyte foot processes that prevents filtration of large macromolecules into the ultrafiltrate.  The SD is also subject to significant shear stress from glomerular filtrate flow [23] (see section 3.1 below)" (lines 110-112) We also describe the importance of contractile elements within the podocyte in maintaining slit diaphragm structure and function (lines 84-90).

Reviewer 2 Report

The authors have submitted a review of actin cytoskeleton regulation in podocytes. As one of the final barriers to urinary protein loss and a complex signaling platform, understanding podocyte biology is crucial in the quest for the development of novel therapeutics. This topic is of interest to the field but the manuscript requires modification.

Some suggestions/questions:

1. Section headings should be revised to be more concise as many parts of the manuscript have overlapping and repetitive content. The content in sections 3.3, 3.4 and 3.5 should be integrated into the relevant earlier parts of the manuscript. No point for example in discussing a gene mutation in one section and then later describing the mechanism by which the mutation causes actin cytoskeletal injury. Likewise, not ideal to separate focal adhesion abnormalities from the initial description of focal adhesions.
2. The section on integrins should be expanded given the large body of work on integrin-based regulation of podocyte focal adhesion dynamics
3. Page 8: “Drugs” deserves in own section. It’s very incomplete to only mention cyclosporine and Rituximab as the only podocyte-acting drugs when many other such as ACEi/ARB and steroids and many other agents in development are relevant. There are also other agents like dasatinib that disrupt the podocyte actin cytoskeleton.
4. The introduction references hemodynamic forces that cause podocyte injury but these are not covered. There is a significant body of literature describing podocyte shear stress and downstream consequences

Author Response

We thank the reviewer for the thoughtful comments that have helped to strengthen this manuscript.

Below please find a point-to-point response to the reviewer's comments:

1. "Section headings should be revised to be more concise as many parts of the manuscript have overlapping and repetitive content. The content in sections 3.3, 3.4 and 3.5 should be integrated into the relevant earlier parts of the manuscript. No point for example in discussing a gene mutation in one section and then later describing the mechanism by which the mutation causes actin cytoskeletal injury. Likewise, not ideal to separate focal adhesion abnormalities from the initial description of focal adhesions."This is an excellent suggestion. We have moved the content in sections 3.3, 3.4 and 3.5 to the parts of the manuscript describing the slit diaphragm or focal adhesions and created new sections 2.6 and 2.8.
2. "The section on integrins should be expanded given the large body of work on integrin-based regulation of podocyte focal adhesion dynamics"The section on integrins has been expanded (please see lines 376-398).
3. "Page 8: “Drugs” deserves in own section. It’s very incomplete to only mention cyclosporine and Rituximab as the only podocyte-acting drugs when many other such as ACEi/ARB and steroids and many other agents in development are relevant. There are also other agents like dasatinib that disrupt the podocyte actin cytoskeleton."Section 3.3 "Drugs" has been expanded to include RAS inhibitors, glucocorticoids, abatacept and dasatanib (lines 510-547).
4. "The introduction references hemodynamic forces that cause podocyte injury but these are not covered. There is a significant body of literature describing podocyte shear stress and downstream consequences"We have added a section on hemodynamic factors (section 3.1, lines 464-479).

Round 2

Reviewer 2 Report

The authors have addressed prior concerns raised