A genetic and physiological model of renal dysfunction in Lowe syndrome

  1. Navyashree A Ramesh
  2. Vaishali Kataria
  3. Indra Sara Lama
  4. Rajan Thakur
  5. Avishek Ghosh
  6. Sanjeev Sharma
  7. Aishwarya Venugopal
  8. Anil Vasudevan
  9. Raghu Padinjat

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Abstract

Lowe syndrome (LS) is an X-linked recessive genetic disorder characterized by renal dysfunction, neurodevelopmental defects, and cataract. The affected gene, OCRL encodes for a polyphosphoinositide 5-phosphatase. OCRL is localized to multiple sub-cellular locations in the endolysosomal system and defects in these organelles have been described in human cells depleted of OCRL. However, the relationship of the endolysosomal defects in OCRL depleted cells to the altered physiology of kidney cells of LS patients has not been completely determined. Here we model the kidney phenotypes of LS using a Drosophila nephrocyte model. Using this model system, we demonstrate that OCRL plays a cell-autonomous role in nephrocyte function. Deletion of the only OCRL ortholog in Drosophila ( dOCRL ) leads to cell-autonomous defects in larval nephrocyte structure and function. Null mutants of dOCRL ( dOCRL KO ) show defects in the endolysosomal system of larval nephrocytes that are associated with physiological defects in nephrocyte function. These defects could be rescued by reconstitution with a human OCRL transgene but not with a phosphatase dead version or a human LS patient derived mutation. Overall, this work provides a model system to understand the mechanisms by which the sub-cellular changes from loss of OCRL leads to defects in kidney function in human patients.

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    Reply to the reviewers

    Response to reviewer comments on Ramesh et.al and Revision plan

    Reviewer #1 (Evidence, reproducibility, and clarity (Required)):

    In this study the Drosophila orthologue of OCRL, the gene mutated in Lowe syndrome, is knocked out and effects upon whole organism physiology and upon the specific function of nephrocytes, the equivalent of the vertebrate kidney, are analysed. The authors report decreased viability of KO animals, in agreement with previous work, and go on to show that nephrocytes are defective in clearance of material from the hemolymph (equivalent of blood). This is accompanied by altered PIP2 and PI4P levels and perturbed endolysosmal organelles. Nephrocyte-specific KO indicates these changes are cell autonomous. Importantly, the phenotypes can be rescued by re-expression of dOCRL, and the human OCRL also rescues, but not when containing mutations that abrogate lipid phosphatase activity or seen in a human Lowe syndrome patient.

    The results are clear and convincing and indicate that the Drosophila OCRL KOs (global and nephrocyte-specific) are good models for understanding OCRL function in the kidney. The findings nicely recapitulate what has been shown in human cell lines and previously published zebrafish and mouse models. In that sense the findings are not unexpected and there is some lack of novelty. Nevertheless, the results here, showing the modelling of OCRL in flies, is important to publish. The fly model also offers certain advantages for future studies e.g. ease of genetics and lack of redundancy, which should prove valuable for such investigations. The paper serves as a very solid framework going forwards.

    We thank the reviewer for their positive assessment of our manuscript. We would like to reiterate the novel aspects of our study:

    • Lowe syndrome has three key clinical features: brain defect, renal dysfunction, and congenital cataract. Our work is a multiscale analysis of Lowe syndrome in a genetically tractable model organism, Drosophila including analysis of whole animal physiology, renal physiology, and the sub-cellular changes in Drosophila larval nephrocytes. As *Drosophila *nephrocytes are considered a good model of human renal function, we feel that our study lays the foundation for many future investigations of the renal aspects of the Lowe syndrome phenotype. Prior to this work, there was no Drosophila model of the renal phenotypes in Lowe syndrome.
    • As the reviewer correctly points out, the cell biological defects we describe in *Drosophila *OCRL knockout nephrocytes largely overlaps with that reported in multiple model systems including patient samples, human kidney cell lines, zebrafish larvae and a previous study in Drosophila We feel this is an important strength of this paper as this model can then work overlapping with other existing models. This is important since the Drosophila model is the only one with a single gene encoding for the ocrl/Inpp5b subfamily of 5’-phosphatases (in contrast to humans, mouse, and zebrafish) thus avoiding the complications arising from genetic redundancy.
    • Lastly, apart from a couple of studies from the Aguilar lab (done in cell lines), we believe that ours is the first study to look at patient derived mutations in an intact animal model. I only have a few suggestions for improving the manuscript, listed below:

    1.) The referencing is quite minimal and more relevant references should be cited. An obvious one is Del Signore et al describing KO of OCRL in flies, and there are others on OCRL on endocytosis that were not cited e.g. Erdmann et al, Nandez et al, Choudhury et al.

    There are almost 35 manuscripts on the cellular phenotypes of OCRL, many of them reporting cellular defects in various cell types and model system; indeed, there are 6 papers that mention Drosophila OCRL. It is hard to cite them all. Nevertheless, we will take on board the reviewer’s comment positively and try to cite several more. The paper of Signore et.al on *Drosophila *OCRL was omitted in error and will be included in the revision.

    2.) The figure panels should be presented in the right order in the text, which matches their numbering in the figures.

    This will be corrected where needed.

    3.) Better description is required in a few places in the text so the reader can follow the experiments. For example, what cells are shown in figure 2? How were the PIP probes expressed? Is the imaging in vivo or ex vivo? In Fig 4, how ere the ex vivo experiments performed?

    As already indicated in the figure legend, the cells shown in fig 2 are pericardial nephrocytes and this has been specifically stated at the beginning of the results at line 131. We will now also explicitly state in the fig legend that pericardial nephrocytes are being shown.

    To measure the levels of PIP2 at the plasma membrane of pericardial nephrocytes we used the well-established PIP2 reporter, the PH domain of PLCδ tagged to mCherry (UAS PH-PLCδ::mCherry). These reporter probes were expressed in pericardial nephrocytes using Dot-Gal4. We dissected the nephrocytes from larvae and performed live imaging to measure the PIP2 levels. The intensity of these probes at the plasma membrane in the nephrocytes corresponds to the levels of the PIP2. The same strategy was used to measure the levels of PI4P, the probes for PI4P- P4M tagged to GFP were generated in our lab and previously published in Balakrishnan et al., J.Cell.Sci 2018- PMID: 29980590 and Basu et.al Dev.Biol, 2020- PMID: 32194035.

    For mbsa and dextran uptake assays, these maybe considered as ex-vivo experiments. They have been described in detail in the materials and methods.

    4.) The microscopy images in Figure 4 are too dark__.__

    We will redo these images in grayscale to resolve this issue.

    5.) Figure S2A needs some sort of schematic so the reader can understand what is being shown.

    We will include in this manuscript a schematic showing the scheme used to generate the crispr deletion mutant. This has already been published in Trivedi et.al eLife 2020.

    __ __6.) In Fig S2G the PIP2 distribution looks different in the nKO compared to the total KO- more on the PM. Is this a consistent result and what is the explanation if so?

    We believe the reviewer is referring to Fig S2E as there is no Fig S2G. Yes, the reviewer is correct in noting that the levels of PIP2 at the plasma membrane are higher in the nephroKO compared to the germline KO. We believe that the reason for the higher levels of PIP2 in the Nephrocyte specific ko is that this is an acute depletion of OCRL whereas in the germline mutant, over time, adaptation through other mechanisms may have partly restored PIP2 levels. Acute depletion offers limited scope for compensation.

    __ __7.) In Fig 7 the expression of phosphatase dead OCRL is barely detectable. This makes the functional data difficult to interpret with any certainty. The authors need to be more circumspect in their description of this data and change the writing accordingly.

    It is not uncommon for kinase and phosphatase dead mutant proteins to be expressed at lower levels than their wild type counterpart; this has been reported many times in the literature. However, we will look through our collection of independent transgenic lines and try to find a line where the phosphatase dead mutant expresses at levels as close to the wild type protein as possible.

    __

    __Reviewer #1 (Significance (Required)):

    The results are clear and convincing and indicate that the Drosophila OCRL KOs (global and nephrocyte-specific) are good models for understanding OCRL function in the kidney. The findings nicely recapitulate what has been shown in human cell lines and previously published zebrafish and mouse models. In that sense the findings are not unexpected and there is some lack of novelty. Nevertheless, the results here, showing the modelling of OCRL in flies, is important to publish. The fly model also offers certain advantages for future studies e.g. ease of genetics and lack of redundancy, which should prove valuable for such investigations. The paper serves as a very solid framework going forwards.

    We thank the reviewer for their positive assessment of our manuscript. We would like to reiterate the novel aspects of our study:

    • Lowe syndrome has three key clinical features: brain defect, renal dysfunction, and congenital cataract. Our work is a multiscale analysis of Lowe syndrome in a genetically tractable model organism, Drosophila including analysis of whole animal physiology, renal physiology, and the sub-cellular changes in Drosophila larval nephrocytes. As *Drosophila *nephrocytes are considered a good model of human renal function, we feel that our study lays the foundation for many future investigations of the renal aspects of the Lowe syndrome phenotype. Prior to this work, there was no Drosophila model of the renal phenotypes in Lowe syndrome.
    • As the reviewer correctly points out, the cell biological defects we describe in *Drosophila *OCRL knockout nephrocytes largely overlaps with that reported in multiple model systems including patient samples, human kidney cell lines, zebrafish larvae and a previous study in Drosophila We feel this is an important strength of this paper as this model can then work overlapping with other existing models. This is important since the Drosophila model is the only one with a single gene encoding for the ocrl/Inpp5b subfamily of 5’-phosphatases (in contrast to humans, mouse, and zebrafish) thus avoiding the complications arising from genetic redundancy.
    • Lastly, apart from a couple of studies from the Aguilar lab (done in cell lines), we believe that ours is the first study to look at patient derived mutations in an intact animal model.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The researchers have generated an OCRL knockout Drosophila model and successfully used it to model the kidney dysfunction phenotypes of the rare genetic condition Lowe syndrome. They demonstrate endolysosomal phenotypes consistent with observations reported in other model systems, and illustrate that these translate to disrupted endocytic uptake, and clearing of ingested silver nitrate. In addition, there was a significant effect on growth and development of larvae. Phenotypes could be rescued by expression of human WT OCRL, but not by expression of a patient derived mutant version.

    Major comments:

    The experiments are generally well performed, logical and support the conclusions made by the authors. It would be nice to observe whether there is actin accumulation on the perturbed endosomal compartments described in Figure 4 as this is a common feature observed in other kidney model systems of the disease, although that is not an essential observation for the story outlined in the paper.

    Thanks for the comment. We will attempt to do this subject to the availability of suitable fluorophore combinations.

    The methods outlined are clear. N numbers and statistical results however are more opaquely reported. Although the number of replicates is mentioned in the material and methods, they are not mentioned in the figure legends, and at least for the silver nitrate uptake experiment, the N number reported does not seem to match the data points on the bar graph - the material and methods reports the experiment was done three times in triplicate, but there are only two individual data points on the bar graph itself. It is thus unclear what they represent. The colours are also not annotated.

    This will be mentioned clearly in both the figure legends and the materials & methods.

    With the phosphoinositide binding domain expression in Fig. 2, panel A image for dOCRL KO looks to be an outlier rather than a picture representing the mean.

    Overall, N numbers should be added to all figure legends, specifying X of cells assessed from Y number of pupae. In terms of the statistical analysis, exact p-values should be reported. It should be indicated where any relevant comparisons made were not significant. In places the authors have done so, but not consistently. In particular, it is unclear whether the differences in Figure 7D were statistically tested - no p values are reported in the figure legend and no comparisons are indicated in the figure itself.

    This will be done in the revised manuscript.__ __ In Figure 7B, it looks like hOCRL PD is barely expressed so it is hard to interpret the lack of rescue shown in panels C and D

    It is not uncommon for kinase and phosphatase dead mutant proteins to be expressed at lower levels than their wild type counterpart; this has been reported many times in the literature. However, will look through our collection of independent transgenic lines and try to find a line where the phosphatase dead mutant expresses at levels as close to the wild type protein as possible.

    Minor comments

    The length of scale bars needs reporting in the figure legend (or on the figures themselves)

    We will include the scale bars in the figure legend__ __ In figure 2A the cell in the control image is a substantially different shape to the other cells indicated in the figure: I assume this is just natural variation and bears no functional significance?

    This is natural variation. Even in a single wild type larva, one typically sees variation in the shape of individual pericardial nephrocytes.

    I was confused by what the difference between Sup Fig 2F vs Figure 6A was - is this reporting identical data for Control and Nephrocyte specific KO but just once on a log scale and once not (and in the supplemental with the addition of the whole organism knock-out)?

    These are not identical data plotted using two different scales, rather separate data.

    Were the authors surprised that the according to the data the nephrocyte specific knock-out elevated PI(4,5)P2 levels more than the whole organism knock-out?

    Yes, the reviewer is correct in noting that the levels of PIP2 at the plasma membrane are higher in the nephroKO compared to the germline KO. We believe that the reason for the higher levels of PIP2 in the Nephrocyte specific ko is that this is an acute depletion of OCRL whereas in the germline mutant adaptation through other mechanisms may have partly restored PIP2 levels over time. Acute depletion offers limited scope for compensation.

    __ __ For figures 6B-D and 7C-D representative examples of the images used to generate the data shown in the graphs should be added at least as a supplemental figure.

    This will be provided

    Line 196. Need to cite Sup Fig 1E-F in text Line 214 Need to cite Sup Fig 1I-J in text

    We will include it

    The figure legend for Figure 7 makes reference to a "Figure 7E" which is not present in the manuscript.

    This will be corrected.

    __Reviewer #2 (Significance (Required)):____ __ This paper describes a fly model that links nephrocyte physiology with molecular mechanism of rare disease significance. The paper characterises nephrocyte function by silver nitrate clearance and clathrin and bulk uptake pathways and links them to phosphoinositide lipid levels. Biosensor expression is used alongside lipid mass spectrometry measurements. The paper goes on to measure the effect of re-expression of the human gene and patient mutations. The paper reinforces existing understanding of the physiological and molecular basis of the human kidney disease.

    The nephrocyte phenotype mirrors the proximal tubule kidney phenotype observed in a variety of other models, such as the mouse model. Previous work in Drosophila and in other models needs setting out more thoroughly in the introduction and the advantages of the current work made more obvious. Drosophila has the added advantage of being more genetically tractable as a model than for example the mouse model, and so the similarity of behaviour between the two makes this model useful for the field. However it comes across in the text that this is the first use of Drosophila to examine OCRL when this is not the case. The authors are missing some key references to other work to place their study in context. This is not the first Drosophila model of Lowe syndrome. The authors do mention a study by El Kadhi and colleagues (2011) in passing, however a study from Del Signore and colleagues (2017: PMID 29028801) is missing, as is Mondin et al 2019 PMID: 31118240). Whilst Del Signore et al primarily concerns hemocytes, rather than nephrocytes, several comparable observations were made to the submitted work. The Del Signore paper reports several disruptions to the endolysosomal system in hemocytes, which would be consistent with the observations here in nephrocytes, and it also reports the larval lethality after the 3rd instar stage, again consistent with this study. The authors need to set out how is this paper different to what has previously been done in fly.

    We apologise for missing out on citing the work of Signore e.al 2017. This will be done in the revised version.

    The discussion lacks sufficient detail on the work done in the humanised mouse model too (Festa et al , 2019). This study is mentioned in passing in the introduction, but needs fuller discussion compared to the fly model and mammalian cell culture and zebrafish larval models that the authors discuss.

    We will present a comparative discussion of Festa e.al 2019 in the revised version.

    The reviewer expertise is in cell biology of OCRL. Nephrocyte physiology and detailed fly issues are outside reviewer expertise.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary:

    This paper describes a function for the Lowe Syndrome phosphoinositide phosphatase OCRL in Drosophila kidney-like nephrocytes. The authors replicate previous findings that Drosophila ocrl null mutants are larval/pupal lethal, and further show that these null mutants fail to clear heavy metal from their nephrocytes. As previously shown in many cell types including in Drosophila, they find that ocrl mutant nephrocytes exhibit endocytic, endolysosomal, and autophagy defects. These defects are rescued by human OCRL but not an enzymatically inactive version or a patient mutation. Overall, this paper validates Drosophila as a model to explore the endocytic/endolysosomal basis of kidney defects in Lowe Syndrome.

    Major comments:

    For Figure 2E, ____please do not refer to a non-significant difference____ as a "trend". Trend is a statistical term that refers to patterns found in time series datasets. Datasets below the defined threshold of statistical significance are simply "not significantly different". Overall this figure shows a negative result (no change in PIP2 levels in whole animals, and no effect of rescue), and should be described as such. It is not surprising that whole animal PIP2 levels are unaltered in OCRL mutants as there are other phosphatases such as synaptojanin that may be more important in abundant cell types.

    Thank you, we will correct this.

    The dot-GAL4 driver used for CRISPR of OCRL is not nephrocyte-specific. It also expresses in salivary glands, lymph glands, and weakly in hemocytes (PMID 12324942). It is therefore possible that some of the phenotypes arise from non-cell-autonomous functions, notably hemocyte activation and systemic inflammatory responses as previously reported for Drosophila ocrl mutants (PMID 29028801). The conclusions about cell autonomy of the phenotype should either be softened, or additional experiments should be done with complementary drivers.

    We propose to carry out key nephrocyte phenotypes such as dextran uptake using other Gal4 lines such as AB1 Gal4, Hml Gal4 to rule contributions from salivary glands, lymph gland and hemocytes to the phenotypes seen with Dot-GAL4. We will also check phenotypes with Sns-Gal4 which is also a nephrocyte specific GAL4. This will be included in the revised manuscript.

    The very low expression level of the human phosphatase-dead mutant makes it impossible to assess if rescue is due to the mutation or simply to lack of protein. Do similarly low expression levels of the wild type protein rescue?

    It is not uncommon for kinase and phosphatase dead mutant proteins to be expressed at lower levels than their wild type counterpart; this has been reported many times in the literature. However, will look through our collection of independent transgenic lines and try to find a line where the phosphatase dead mutant expresses at levels as close to the wild type protein as possible.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    __Additional information is required for image analysis methods to enable replication: __It's not clear what the authors mean by "estimating the ratio of plasma membrane/cytoplasmic fluorescence" (p5 line 132). Why estimating and not measuring? If measured (as suggested by the graphs), details of the image analysis method (eg definition of plasma membrane and cytoplasmic ROI) must be described in such a way that they could be replicated. The only method currently provided is "Raw data of imaging were processed and analyzed using Fiji ImageJ,"

    Philosophically, all measurements are at some level an estimate; any measurement is the best estimate of what is under consideration, limited by the technical features of the measurement method being used. If the reviewer insists, we agree to change the word “estimating” to “measuring”. The Padinjat lab has published multiple times on the best possible way of estimating phosphoinositide levels at membranes including plasma membrane levels of PIP2 and PI4P. These methods consider various important factors such as the level of expression of the probe, the size of the cells being measured, method of imaging, plane of the cell being imaged, etc. These methods have been previously published in multiple peer-reviewed papers and described in detail in those studies including imaging parameters, sampling methods and data analysis approaches (Sharma et.al Cell.Reports 2019; PMID: 31091438-Star Methods and Basu et.al Dev.Biol 2020 PMID: 32194035). In this study we have used these methods. In view of the reviewer’s comments, we will cite these papers (one is already cited) and include them in the legends of the relevant figures.

    __ __For immunofluorescence, the authors state that mean fluorescence intensity of "EEA-1 and Rab-7 staining was quantified after background subtraction from the maximum projections of the stacks and normalized to the area of nephrocytes." Please detail how background was identified and subtracted.

    The steps were followed for the background subtraction in quantifying the MFI of EEA-1 and Rab7 staining:

    1. Open the Raw image in ImageJ Fiji.
    2. Make a maximum projections of all the stacks by selecting Image> stack>Z project>select maximum projection.
    3. Convert the Max Z projected image to HiLo mode by selecting LUT>HiLO.
    4. To subtract the background manually draw an ROI in the image on an area that is devoid of any nephrocytes by selecting the oval selection tool. Six such 6 ROIs were drawn in the background of the image.
    5. Now measure the MFI of these 6 background ROIs by selecting Analyze>Measure
    6. Copy the MFI of all these 6 backgrounds ROIs into the Excel file and calculate the average MFI of these backgrounds 7 Using this average value of the background MFI in ImageJ select Process>Math>Subtract>Enter the average MFI of the background>Click OK

    You can always preview the image with background subtraction. This image has been background subtracted. Post the above streps, we drew an ROI around the nephrocyte border and measured the MFI of the EEA-1/Rab7 staining.

    All the measurements with the ROIs have been stored in the server along with the Raw images__. __

    __ __ For Figure 3C, 6B, 7D it is not clear why the authors have used the categorical measurement of % of cells with red pixels rather than simply measuring the continuous variable of mean pixel intensity. Can more explanation be provided for this choice?

    Our goal here is quantifying the level of AgNO3 in nephrocytes. Since AgNO3 it is not a fluorochrome traditional methods of quantification used for fluorochromes are not applicable as one would encounter the problem of non-linearity and saturating images. Since it is difficult to assess the intensity values from the color brightfield images, we used the following method.

    The raw brightfield images are opened in FIJI and are converted to 8-bit images (Image>type>8-bit). The images are then inverted using edit>invert and further converted to 16 color pixel LUT (Image>Lookup table> 16 colours) which shows the distribution and intensity of AgNO3 in the following order from white corresponding to the high intensity of AgNO3 and black being the least intense.

    To validate our method, we tested it using Rab5-DN/Rab5-RNAi which shows no uptake of AgNO3 (previously published in PMID: PMC5429992). This experiment showed that our analysis works as expected. __ __

    • Are prior studies referenced appropriately?

    Referencing of prior studies is extremely inadequate, resulting in inflated claims of novelty. Comments can be found below in the significance section.

    We will revise the referencing (more details below).

    • Are the text and figures clear and accurate?

    Text and figures are ok.

    Reviewer #3 (Significance (Required)):

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

    The study provides a validated new system in which to study ocrl function in kidney-like cells in flies. There are a few technical and interpretation concerns that should be easily addressed. One main limitation of the study is that it does not provide new mechanistic or physiological insight into how OCRL regulates kidney function. A related major limitation is that the manuscript is not placed in its proper context in the field - these phenotypes have been previously observed in other animal models and also in other cell types in the fly, but the paper does not properly cite that previous literature.

    We respectfully reiterate that the title of our paper “A genetic and physiological model of renal dysfunction in Lowe syndrome”

    Further we would like to reiterate the last line of the abstract which typically sums up what the paper is about is as follows: “Overall, this work provides a model system to understand the mechanisms by which the sub-cellular changes from loss of OCRL leads to defects in kidney function in human patients.”

    Nowhere in the manuscript, neither title, abstract or elsewhere have we claimed to have provided new mechanistic or physiological insight into how OCRL regulates kidney function. However, this study is a very detailed and in-depth description of a model system to stud the renal manifestations of Lowe syndrome using the genetically tractable model system, Drosophila. It will be a solid foundation on which many labs can base future studies, both basic and applied in relation to Lowe syndrome.

    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

    Referencing of prior studies is extremely inadequate, and many of the claims of novelty are incorrect. The introduction asserts that only cellular studies have been conducted in kidney cells and animals, and that "the relationship of the endolysosomal defects in OCRL depleted cells to the altered physiology of kidney cells of LS patients has not been completely determined". Some of the cited papers (e.g. PMID 30590522) did characterize renal physiology at the level of proteinuria, very similar to the silver clearance described in this paper. Additional but important uncited papers that correlate cellular defects with kidney function include PMID 31676724 and 22680056. The authors should thoroughly acknowledge and reference the previous literature on animal and cellular models of kidney dysfunction upon loss of OCRL.

    There are almost 35 manuscripts on the cellular phenotypes of OCRL, many of them reporting cellular defects in various cell types and model system; indeed, there are 6 papers that mention Drosophila OCRL. It is hard to cite them all and, in some cases, reconcile findings between them. Nevertheless, we will take on board the reviewer’s comment positively and try to cite several more.

    It is also essential to cite published Drosophila in vivo OCRL literature (PMID 29028801), which is completely omitted. A naïve reader would miss that fly OCRL null mutants have previously been characterized in vivo, and that many of the reported findings are duplicated in this paper, including lethal phase, transgene rescue, and most of the cellular phenotypes (PIP2 levels, endocytic and endosomal defects, lysotracker, and autophagy defects, though in hemocytes rather than nephrocytes, and with some interesting differences that are worth pursuing, such as Rab7 levels). The paragraph on p 9 discussing comparison of Drosophila to other systems completely ignores these previous findings. Further, the current manuscript uses specific fly OCRL tools (antibodies and transgenes) from the previous paper without citation, and the reader would not know to look up how these tools were generated and validated. I have signed this review to note that the previous Drosophila work happens to have been from my group, but objectively any knowledgeable reviewer would recognize that it should have been cited and discussed in this paper. Overall it is a disservice to the field to claim novelty by failing to cite the relevant literature. The introduction and discussion should be extensively revised to put the work in its proper context.

    The introduction and discussion will be revised accordingly.

    To summarize: previously it was known that defects in endosomal membrane traffic in kidney cells of "humanized" ocrl mice or of zebrafish correlated with defects in renal function. It was also known that Drosophila ocrl null mutants are larval/pupal lethal and that their blood cells exhibit endosomal trafficking defects similar to those shown in the current study. This paper shows for the first time that ocrl null mutants also have endosomal trafficking defects in kidney-like nephrocytes, and show defects in the physiological clearing functions of nephrocytes. Thus, this paper replicates the literature for ocrl function in other cell types in Drosophila and in other animal models, and provides a helpful new experimental system for future mechanistic or therapeutic tests of OCRL function in kidney-like cells. However, it does not provide a mechanistic advance into which of the many cellular phenotypes previously observed (and repeated here) lead to kidney dysfunction.

    We would respectfully reiterate the title of our paper “A genetic and physiological model of renal dysfunction in Lowe syndrome”

    Further we would like to reiterate the last line of the abstract which typically sums up what the paper is about: “Overall, this work provides a model system to understand the mechanisms by which the sub-cellular changes from loss of OCRL leads to defects in kidney function in human patients.”

    Nowhere in the manuscript, neither title, abstract or elsewhere have we claimed to have provided new mechanistic or physiological insight into how OCRL regulates kidney function.

    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

    This paper will be of interest to researchers studying Lowe Syndrome or membrane traffic in Drosophila nephrocytes.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This paper describes a function for the Lowe Syndrome phosphoinositide phosphatase OCRL in Drosophila kidney-like nephrocytes. The authors replicate previous findings that Drosophila ocrl null mutants are larval/pupal lethal, and further show that these null mutants fail to clear heavy metal from their nephrocytes. As previously shown in many cell types including in Drosophila, they find that ocrl mutant nephrocytes exhibit endocytic, endolysosomal, and autophagy defects. These defects are rescued by human OCRL but not an enzymatically inactive version or a patient mutation. Overall, this paper validates Drosophila as a model to explore the endocytic/endolysosomal basis of kidney defects in Lowe Syndrome.

    Major comments:

    For Figure 2E, please do not refer to a non-significant difference as a "trend". Trend is a statistical term that refers to patterns found in time series datasets. Datasets below the defined threshold of statistical significance are simply "not significantly different". Overall this figure shows a negative result (no change in PIP2 levels in whole animals, and no effect of rescue), and should be described as such. It is not surprising that whole animal PIP2 levels are unaltered in OCRL mutants as there are other phosphatases such as synaptojanin that may be more important in abundant cell types.

    The dot-GAL4 driver used for CRISPR of OCRL is not nephrocyte-specific. It also expresses in salivary glands, lymph glands, and weakly in hemocytes (PMID 12324942). It is therefore possible that some of the phenotypes arise from non-cell-autonomous functions, notably hemocyte activation and systemic inflammatory responses as previously reported for Drosophila ocrl mutants (PMID 29028801). The conclusions about cell autonomy of the phenotype should either be softened, or additional experiments should be done with complementary drivers.

    The very low expression level of the human phosphatase-dead mutant makes it impossible to assess if rescue is due to the mutation or simply to lack of protein. Do similarly low expression levels of the wild type protein rescue?

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Additional information is required for image analysis methods to enable replication: It's not clear what the authors mean by "estimating the ratio of plasma membrane/cytoplasmic fluorescence" (p5 line 132). Why estimating and not measuring? If measured (as suggested by the graphs), details of the image analysis method (eg definition of plasma membrane and cytoplasmic ROI) must be described in such a way that they could be replicated. The only method currently provided is "Raw data of imaging were processed and analyzed using Fiji ImageJ,"

    For immunofluorescence, the authors state that mean fluorescence intensity of "EEA-1 and Rab-7 staining was quantified after background subtraction from the maximum projections of the stacks and normalized to the area of nephrocytes." Please detail how background was identified and subtracted.

    For Figure 3C, 6B, 7D it is not clear why the authors have used the categorical measurement of % of cells with red pixels rather than simply measuring the continuous variable of mean pixel intensity. Can more explanation be provided for this choice?

    • Are prior studies referenced appropriately?

    Referencing of prior studies is extremely inadequate, resulting in inflated claims of novelty. Comments can be found below in the significance section.

    • Are the text and figures clear and accurate?

    Text and figures are ok.

    Significance

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

    The study provides a validated new system in which to study ocrl function in kidney-like cells in flies. There are a few technical and interpretation concerns that should be easily addressed. One main limitation of the study is that it does not provide new mechanistic or physiological insight into how OCRL regulates kidney function. A related major limitation is that the manuscript is not placed in its proper context in the field - these phenotypes have been previously observed in other animal models and also in other cell types in the fly, but the paper does not properly cite that previous literature.

    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

    Referencing of prior studies is extremely inadequate, and many of the claims of novelty are incorrect. The introduction asserts that only cellular studies have been conducted in kidney cells and animals, and that "the relationship of the endolysosomal defects in OCRL depleted cells to the altered physiology of kidney cells of LS patients has not been completely determined". Some of the cited papers (e.g. PMID 30590522) did characterize renal physiology at the level of proteinuria, very similar to the silver clearance described in this paper. Additional but important uncited papers that correlate cellular defects with kidney function include PMID 31676724 and 22680056. The authors should thoroughly acknowledge and reference the previous literature on animal and cellular models of kidney dysfunction upon loss of OCRL.

    It is also essential to cite published Drosophila in vivo OCRL literature (PMID 29028801), which is completely omitted. A naïve reader would miss that fly OCRL null mutants have previously been characterized in vivo, and that many of the reported findings are duplicated in this paper, including lethal phase, transgene rescue, and most of the cellular phenotypes (PIP2 levels, endocytic and endosomal defects, lysotracker, and autophagy defects, though in hemocytes rather than nephrocytes, and with some interesting differences that are worth pursuing, such as Rab7 levels). The paragraph on p 9 discussing comparison of Drosophila to other systems completely ignores these previous findings. Further, the current manuscript uses specific fly OCRL tools (antibodies and transgenes) from the previous paper without citation, and the reader would not know to look up how these tools were generated and validated. I have signed this review to note that the previous Drosophila work happens to have been from my group, but objectively any knowledgeable reviewer would recognize that it should have been cited and discussed in this paper. Overall it is a disservice to the field to claim novelty by failing to cite the relevant literature. The introduction and discussion should be extensively revised to put the work in its proper context.

    To summarize: previously it was known that defects in endosomal membrane traffic in kidney cells of "humanized" ocrl mice or of zebrafish correlated with defects in renal function. It was also known that Drosophila ocrl null mutants are larval/pupal lethal and that their blood cells exhibit endosomal trafficking defects similar to those shown in the current study. This paper shows for the first time that ocrl null mutants also have endosomal trafficking defects in kidney-like nephrocytes, and show defects in the physiological clearing functions of nephrocytes. Thus, this paper replicates the literature for ocrl function in other cell types in Drosophila and in other animal models, and provides a helpful new experimental system for future mechanistic or therapeutic tests of OCRL function in kidney-like cells. However, it does not provide a mechanistic advance into which of the many cellular phenotypes previously observed (and repeated here) lead to kidney dysfunction.

    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

    This paper will be of interest to researchers studying Lowe Syndrome or membrane traffic in Drosophila nephrocytes.

    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I am an expert in the cell biology of membrane traffic, Drosophila as a model system, and imaging and image analysis. Avital Rodal Professor of Biology Brandeis University

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The researchers have generated an OCRL knockout Drosophila model and successfully used it to model the kidney dysfunction phenotypes of the rare genetic condition Lowe syndrome. They demonstrate endolysosomal phenotypes consistent with observations reported in other model systems, and illustrate that these translate to disrupted endocytic uptake, and clearing of ingested silver nitrate. In addition, there was a significant effect on growth and development of larvae. Phenotypes could be rescued by expression of human WT OCRL, but not by expression of a patient derived mutant version.

    Major comments:

    The experiments are generally well performed, logical and support the conclusions made by the authors. It would be nice to observe whether there is actin accumulation on the perturbed endosomal compartments described in Figure 4 as this is a common feature observed in other kidney model systems of the disease, although that is not an essential observation for the story outlined in the paper.

    The methods outlined are clear. N numbers and statistical results however are more opaquely reported. Although the number of replicates is mentioned in the material and methods, they are not mentioned in the figure legends, and at least for the silver nitrate uptake experiment, the N number reported does not seem to match the data points on the bar graph - the material and methods reports the experiment was done three times in triplicate, but there are only two individual data points on the bar graph itself. It is thus unclear what they represent. The colours are also not annotated.

    With the phosphoinositide binding domain expression in Fig. 2, panel A image for dOCRL KO looks to be an outlier rather than a picture representing the mean.

    Overall, N numbers should be added to all figure legends, specifying X of cells assessed from Y number of pupae. In terms of the statistical analysis, exact p-values should be reported. It should be indicated where any relevant comparisons made were not significant. In places the authors have done so, but not consistently. In particular, it is unclear whether the differences in Figure 7D were statistically tested - no p values are reported in the figure legend and no comparisons are indicated in the figure itself.

    In Figure 7B, it looks like hOCRL PD is barely expressed so it is hard to interpret the lack of rescue shown in panels C and D.

    Minor comments

    The length of scale bars needs reporting in the figure legend (or on the figures themselves)

    In figure 2A the cell in the control image is a substantially different shape to the other cells indicated in the figure: I assume this is just natural variation and bears no functional significance?

    I was confused by what the difference between Sup Fig 2F vs Figure 6A was - is this reporting identical data for Control and Nephrocyte specific KO but just once on a log scale and once not (and in the supplemental with the addition of the whole organism knock-out)? Were the authors surprised that the according to the data the nephrocyte specific knock-out elevated PI(4,5)P2 levels more than the whole organism knock-out?

    For figures 6B-D and 7C-D representative examples of the images used to generate the data shown in the graphs should be added at least as a supplemental figure.

    Line 196. Need to cite Sup Fig 1E-F in text

    Line 214 Need to cite Sup Fig 1I-J in text

    The figure legend for Figure 7 makes reference to a "Figure 7E" which is not present in the manuscript.

    Significance

    This paper describes a fly model that links nephrocyte physiology with molecular mechanism of rare disease significance. The paper characterises nephrocyte function by silver nitrate clearance and clathrin and bulk uptake pathways and links them to phosphoinositide lipid levels. Biosensor expression is used alongside lipid mass spectrometry measurements. The paper goes on to measure the effect of re-expression of the human gene and patient mutations. The paper reinforces existing understanding of the physiological and molecular basis of the human kidney disease.

    The nephrocyte phenotype mirrors the proximal tubule kidney phenotype observed in a variety of other models, such as the mouse model. Previous work in Drosophila and in other models needs setting out more thoroughly in the introduction and the advantages of the current work made more obvious. Drosophila has the added advantage of being more genetically tractable as a model than for example the mouse model, and so the similarity of behaviour between the two makes this model useful for the field.

    However it comes across in the text that this is the first use of Drosophila to examine OCRL when this is not the case. The authors are missing some key references to other work to place their study in context. This is not the first Drosophila model of Lowe syndrome. The authors do mention a study by El Kadhi and colleagues (2011) in passing, however a study from Del Signore and colleagues (2017: PMID 29028801) is missing, as is Mondin et al 2019 PMID: 31118240). Whilst Del Signore et al primarily concerns hemocytes, rather than nephrocytes, several comparable observations were made to the submitted work. The Del Signore paper reports several disruptions to the endolysosomal system in hemocytes, which would be consistent with the observations here in nephrocytes, and it also reports the larval lethality after the 3rd instar stage, again consistent with this study. The authors need to set out how is this paper different to what has previously been done in fly. The discussion lacks sufficient detail on the work done in the humanised mouse model too (Festa et al , 2019). This study is mentioned in passing in the introduction, but needs fuller discussion compared to the fly model and mammalian cell culture and zebrafish larval models that the authors discuss.

    The reviewer expertise is in cell biology of OCRL. Nephrocyte physiology and detailed fly issues are outside reviewer expertise.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this study the Drosophila orthologue of OCRL, the gene mutated in Lowe syndrome, is knocked out and effects upon whole organism physiology and upon the specific function of nephrocytes, the equivalent of the vertebrate kidney, are analysed. The authors report decreased viability of KO animals, in agreement with previous work, and go on to show that nephrocytes are defective in clearance of material from the hemolymph (equivalent of blood). This is accompanied by altered PIP2 and PI4P levels and perturbed endolysosmal organelles. Nephrocyte-specific KO indicates these changes are cell autonomous. Importantly, the phenotypes can be rescued by re-expression of dOCRL, and the human OCRL also rescues, but not when containing mutations that abrogate lipid phosphatase activity or seen in a human Lowe syndrome patient.

    The results are clear and convincing and indicate that the Drosophila OCRL KOs (global and nephrocyte-specific) are good models for understanding OCRL function in the kidney. The findings nicely recapitulate what has been shown in human cell lines and previously published zebrafish and mouse models. In that sense the findings are not unexpected and there is some lack of novelty. Nevertheless, the results here, showing the modelling of OCRL in flies, is important to publish. The fly model also offers certain advantages for future studies e.g. ease of genetics and lack of redundancy, which should prove valuable for such investigations. The paper serves as a very solid framework going forwards.

    I only have a few suggestions for improving the manuscript, listed below:

    1. The referencing is quite minimal and more relevant references should be cited. An obvious one is Del Signore et al describing KO of OCRL in flies, and there are others on OCRL on endocytosis that were not cited e.g. Erdmann et al, Nandez et al, Choudhury et al.
    2. The figure panels should be presented in the right order in the text, which matches their numbering in the figures.
    3. Better description is required in a few places in the text so the reader can follow the experiments. For example, what cells are shown in figure 2? How were the PIP probes expressed? Is the imaging in vivo or ex vivo? In Fig 4, how ere the ex vivo experiments performed?
    4. The microscopy images in Figure 4 are too dark.
    5. Figure S2A needs some sort of schematic so the reader can understand what is being shown.
    6. In Fig S2G the PIP2 distribution looks different in the nKO compared to the total KO- more on the PM. Is this a consistent result and what is the explanation if so?
    7. In Fig 7 the expression of phosphatase dead OCRL is barely detectable. This makes the functional data difficult to interpret with any certainty. The authors need to be more circumspect in their description of this data and change the writing accordingly.

    Significance

    The results are clear and convincing and indicate that the Drosophila OCRL KOs (global and nephrocyte-specific) are good models for understanding OCRL function in the kidney. The findings nicely recapitulate what has been shown in human cell lines and previously published zebrafish and mouse models. In that sense the findings are not unexpected and there is some lack of novelty. Nevertheless, the results here, showing the modelling of OCRL in flies, is important to publish. The fly model also offers certain advantages for future studies e.g. ease of genetics and lack of redundancy, which should prove valuable for such investigations. The paper serves as a very solid framework going forwards.

  5. Version published to 10.1101/2024.01.15.575703v1 on bioRxiv