The insulin / IGF axis is critically important controlling gene transcription in the podocyte

Reviewer #1 (Public review):

Summary:

In this manuscript, the roles of the insulin receptor and the insulin growth factor receptor were investigated in podocytes. Mice in which both receptors were deleted developed glomerular dysfunction and developed proteinuria and glomerulrosclerosis over several months. Because of concerns about incomplete KO, the authors generated podocyte cell lines where both receptors were deleted. Loss of both receptors was highly deleterious with greater than 50% cell death. To elucidate the mechanism, the authors performed global proteomics and find that spliceosome proteins are down-regulated. They confirm this by using long-range sequencing. These results suggest a novel role for these pathways in podocytes.

This is primarily a descriptive study. The mechanism of how insulin and IGF1 signaling are linked to the spliceosome is not addressed and the phenotype of the mice is only superficially explored. The main issues are that the completeness of the mouse KO is never assessed nor is the completeness of the KO in cell lines. The absence of this data is a significant weakness. The mouse experiments would be improved if the serum creatinines were measured to provide some idea about the severity of the kidney injury. An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful. If this didn't rescue the phenotype, an explanation in the text would suffice. As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on. Lastly, in the cell line experiments, the authors should discuss the caveats associated with studying the 50% of the cells that survive vs the ones that died.

Significance:

With the GLP1 agonists providing renal protection, there is great interest in understanding the role of insulin and other incretins in kidney cell biology. It is already known that Insulin and IGFR signaling play important roles in other cells of the kidney, therefore, there is great interest in understanding these pathways in podocytes. The major advance is that these two pathways appear to have a role in RNA metabolism, the major limitations are the lack of information regarding the completeness of the KO's. If, for example, they can determine that in the mice, the KO is complete, that the GFR is relatively normal, then the phenotype they describe is relatively mild.

Comments on revision plan:

I agree with the suggested experiments especially, the experiments to examine whether insulin/IGF1 signaling have effects on splicing proteins. An alternative experiment would be to ask whether rescue of IR or IGF1R would ameliorate the splicing effects.

Reviewer #2 (Public review):

Summary:

In this manuscript, submitted to Review Commons (journal agnostic), Coward and colleagues report on the role of insulin/IGF axis in podocyte gene transcription. They knocked out both the insulin and IGFR1 mice. Dual KO mice manifested a severe phenotype, with albuminuria, glomerulosclerosis, renal failure and death at 4-24 weeks.

Long read RNA sequencing was used to assess splicing events. Podocyte transcripts manifesting intron retention were identified. Dual knock-out podocytes manifested more transcripts with intron retention (18%) compared wild-type controls (18%), with an overlap between experiments of ~30%.

Transcript productivity was also assessed using FLAIR-mark-intron-retention software. Intron retention w seen in 18% of ciDKO podocyte transcripts compared to 14% of wild-type podocyte transcripts (P=0.004), with an overlap between experiments of ~30% (indicating the variability of results with this method). Interestingly, ciDKO podocytes showed downregulation of proteins involved in spliceosome function and RNA processing, as suggested by LC/MS and confirmed by Western blot.

Pladienolide (a spliceosome inhibitor) was cytotoxic to HeLa cells and to mouse podocytes but no toxicity was seen in murine glomerular endothelial cells.

The manuscript is generally clear and well-written. Mouse work was approved in advance. The four figures are generally well-designed, with bars/superimposed dot-plots.

Methods are generally well described. It would be helpful to say that tissue scoring was performed by an investigator masked to sample identity.

Specific comments:

(1) Data are presented as mean/SEM. In general, mean/SD or median/IQR are preferred to allow the reader to evaluate the spread of the data. There may be exceptions where only SEM is reasonable.

(2) It would be useful to for the reader to be told the number of over-lapping genes (with similar expression between mouse groups) and the results of a statistical test comparing WT and KO mice. The overlap of intron retention events between experimental repeats was about 30% in both knock-out podocytes. This seems low and I am curious to know whether this is typical for typical for this method; a reference could be helpful.

(3) Please explain "adjusted p value of 0.01." It is not clear how was it adjusted. The number of differentially-expressed proteins between the two cell types was 4842.

Comments on revision plan:

The authors suggest additional experiments that should address my concerns and probably the other reviewers' concerns.

I encourage the authors to proceed with their proposed experiments and revisions.

Reviewer #3 (Public review):

Summary:

These investigators have previously shown important roles for either insulin receptor (IR) or insulin-like growth factor receptor (IGF1R) in glomerular podocyte function. They now have studied mice with deletion of both receptors and find significant podocyte dysfunction. They then made a podocyte cell line with inducible deletion of both receptors and find abnormalities in transcriptional efficiency with decreased expression of spliceosome proteins and increased transcripts with impaired splicing or premature termination.

The studies appear to be performed well and the manuscript is clearly written.

There are a number of potential issues and questions with these studies.

(1) For the in vivo studies, the only information given is for mice at 24 weeks of age. There needs to be a full time course of when the albuminuria was first seen and the rate of development. Also, GFR was not measured. Since the podocin-Cre utilized was not inducible, there should be a determination of whether there was a developmental defect in glomeruli or podocytes. Were there any differences in wither prenatal post natal development or number of glomeruli?

(2) Although the in vitro studies are of interest, there are no studies to determine if this is the underlying mechanism for the in vivo abnormalities seen in the mice. Cultured podocytes may not necessarily reflect what is occurring in podocytes in vivo.

(3) Given that both receptors are deleted in the podocyte cell line, it is not clear if the spliceosome defect requires deletion of both receptors or if there is redundancy in the effect. The studies need to be repeated in podocyte cell lines with either IR or IGFR single deletions.

(4) There are no studies investigating signaling mechanisms mediating the spliceosome abnormalities.

Comments on revision plan:

I do not have any changes from my prior review. I applaud the authors for developing a plan to address the questions and concerns raised in my prior review.

Author response:

Evidence reducibility and clarity

Reviewer 1:

In this manuscript, the role of the insulin receptor and the insulin growth factor receptor was investigated in podocytes. Mice, were both receptors were deleted, developed glomerular dysfunction and developed proteinuria and glomerulosclerosis over several months. Because of concerns about incomplete KO, the authors generated podocyte cell lines where both receptors were deleted. Loss of both receptors was highly deleterious with greater than 50% cell death. To elucidate the mechanism, the authors performed global proteomics and find that spliceosome proteins are downregulated. They confirm this by using long-range sequencing. These results suggest a novel role for these pathways in podocytes.

Thank you

This is primarily a descriptive study and no technical concerns are raised. The mechanism of how insulin and IGF1 signaling are linked to the spiceosome is not addresed.

We do not think the paper is descriptive as we used non-biased phospho and total proteomics in the DKO cells to uncover the alterations in the spliceosome (that have not been previously described) that were detrimental. However, we are happy to look further into the underlying mechanism.

We would propose:

(1) Stimulating/inhibiting insulin/IGF signalling pathways in the Wild-type and DKO knockout cells and check expression levels and/or phosphorylation status of splice factors (including those in Figure 3E) and those revealed by phospho-proteomic data; a variety of inhibitors of insulin/IGF1 pathways could also be used along the pathways that are shown in Fig 2.

(2) Looking at the RNaseq data bioinformatically in more detail – the introns/exons that move up or down are targets of the splice factors involved; most splice factors binding sequences are known, so it should be possible to ask bioinformatically – from the sequences around the splice sites of the exons and introns that move in the DKO, which splice factors binding sites are seen most frequently? To uncover splice factors/RNA-binding proteins (RBPs) that are involved in the insulin signaling we will use a software named MATT which was specifically designed to look for RNA-binding motifs (PMID 30010778). In brief, using the long-sequencing data, we will test 250 nt sequences flanking the splice sites of all regulated splicing events (intronic and exonic) against all RNA- binding proteins in the CISBP-RNA database (PMID 23846655) using MATT. This will result in a list of RBPs potentially involved in the insulin signaling. We will validate these by activating insulin signaling (similar to Figures 2 B,C) and probe whether the RBPs are activated (e.g. phosphorylated or change in expression) or we will manipulate expression of the candidate RBPs and measure how they affect the insulin signaling.

(3) Examining the phospho and total proteomic data for IGF1R and Insulin receptor knockout alone podocytes (which we have already generated) and analysing these in more detail and include this data set to elucidate the relative importance of both receptors to spliceosome function.

The phenotype of the mouse is only superficially addressed. The main issues are that the completeness of the mouse KO is never assessed nor is the completeness of the KO in cell lines. The absence of this data is a significant weakness.

We apologise for not making clear but we did assess the level of receptor knockdown in the animal and cell models. The in vivo model showed variable and non-complete levels of insulin receptor and IGF1 receptor podocyte knock down (shown in supplementary figure 1B). This is why we made the in vitro floxed podocyte cell lines in which we could robustly knockdown both the insulin receptor and IGF1 receptor (shown in Figure 2A)

The mouse experiments would be improved if the serum creatinines were measured to provide some idea how severe the kidney injury is.

We can address this:

We have further urinary Albumin:creatinine ratio (uACR) data at 12, 16 and 20 weeks. We also have more blood tests of renal function that can be added. There is variability in creatinine levels which is not uncommon in transgenic mouse models (probably partly due to variability in receptor knock down with cre-lox system). This is part of rationale of developing the robust double receptor knockout cell models where we knocked out both receptors by >80%.

An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful. If this didn't work, an explanation in the text would suffice.

We would consider over express SF3BF4 in the Wild type and DKO cells and assess the effects on spliceosome if deemed necessary. However, we think it is unlikely to rescue the phenotype as so many other spliceosome components are downregulated in the DKO cells.

As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on.

We have some detail on this and can add to the manuscript. However it is not extensive as not a major driver of this work.

Lastly, the authors should caveat the cell experiments by discussing the ramifications of studying the 50% of the cells that survive vs the ones that died.

Thank you, we appreciate this and this was the rationale behind cells being studied after 2 days differentiation before significant cell loss in order to avoid the issue of studying the 50% of cells that survive.

Reviewer 2:

In this manuscript, submitted to Review Commons (journal agnostic), Coward and colleagues report on the role of insulin/IGF axis in podocyte gene transcription. They knocked out both the insulin and IGFR1 mice. Dual KO mice manifested a severe phenotype, with albuminuria, glomerulosclerosis, renal failure and death at 4-24 weeks.

Long read RNA sequencing was used to assess splicing events. Podocyte transcripts manifesting intron retention were identified. Dual knock-out podocytes manifested more transcripts with intron retention (18%) compared wild-type controls (18%), with an overlap between experiments of ~30%.

Transcript productivity was also assessed using FLAIR-mark-intron-retention software. Intron retention w seen in 18% of ciDKO podocyte transcripts compared to 14% of wild-type podocyte transcripts (P=0.004), with an overlap between experiments of ~30% (indicating the variability of results with this method). Interestingly, ciDKO podocytes showed downregulation of proteins involved in spliceosome function and RNA processing, as suggested by LC/MS and confirmed by Western blot.

Pladienolide (a spliceosome inhibitor) was cytotoxic to HeLa cells and to mouse podocytes but no toxicity was seen in murine glomerular endothelial cells.
Specific comments.

The manuscript is generally clear and well-written. Mouse work was approved in advance. The six figures are generally well-designed, bars/superimposed dot-plots.

Thank you

Evaluation.

Methods are generally well described. It would be helpful to say that tissue scoring was performed by an investigator masked to sample identity.

We did this and will add this information to the methods/figure legend.

Specific comments.

(1) Data are presented as mean/SEM. In general, mean/SD or median/IQR are preferred to allow the reader to evaluate the spread of the data. There may be exceptions where only SEM is reasonable.

Graphs can be changed to SD rather than SEM.

(2) It would be useful to for the reader to be told the number of over-lapping genes (with similar expression between mouse groups) and the results of a statistical test comparing WT and KO mice. The overlap of intron retention events between experimental repeats was about 30% in both knock-out podocytes. This seems low and I am curious to know whether this is typical for typical for this method; a reference could be helpful.

This is an excellent question. We had 30% overlap as the parameters used for analysis were very stringent. We suspect we could get more than 30% by being less stringent, which still be considered as similar events if requested. Our methods were based on FLAIR analysis (PMID: 32188845)

(3) Please explain "adjusted p value of 0.01." It is not clear how was it adjusted. The number of differentially-expressed proteins between the two cell types was 4842.

We used the Benjamini-Hochberg method to adjust our data. We think the reviewer is referring to the transcriptomic data and not the proteomic data.

Minor comments

Page numbers in the text would help the reviewer communicate more effectively with the author.

We will do this

Reviewer 3:

These investigators have previously shown important roles for either insulin receptor (IR) or insulin-like growth factor receptor (IGF1R) in glomerular podocyte function. They now have studied mice with deletion of both receptors and find significant podocyte dysfunction. They then made a podocyte cell line with inducible deletion of both receptors and find abnormalities in transcriptional efficiency with decreased expression of spliceosome proteins and increased transcripts with impaired splicing or premature termination.

The studies appear to be performed well and the manuscript is clearly written.

Thank you

Referees cross-commenting

I am in agreement with Reviewer 1 that the studies are overly descriptive and do not provide sufficient mechanism and the lack of more investigation of the in vivo model is a significant weakness.

Please see our responses to reviewer 1 above.

Significance

Reviewer 1:

With the GLP1 agonists providing renal protection, there is great interest in understanding the role of insulin and other incretins in kidney cell biology. It is already known that Insulin and IGFR signaling play important roles in other cells of the kidney. So, there is great interest in understanding these pathways in podocytes. The major advance is that these two pathways appear to have a role in RNA metabolism, the major limitations are the lack of information regarding the completeness of the KO's. If, for example, they can determine that in the mice, the KO is complete, that the GFR is relatively normal, then the phenotype they describe is relatively mild.

Thank you. The receptor KO in the mice is unlikely to be complete (Please see comments above and Supplementary Figure 1b). There are many examples of KO models targeting other tissues showing that complete KO of these receptors seems difficult to achieve , particularly in reference to the IGF1 receptor. In the brain (which is also terminally differentiated cells PMID:28595357 (barely 50% iof IGF1R knockdown was achieved in the target cells). Ovarian granulosa cells PMID:28407051 -several tissue specific drivers tried but couldn't achieve any better than 80%. The paper states that 10% of IGF1R is sufficient for function in these cells so they conclude that their knockdown animals are probably still responding to IGF1. Finally, in our recent IGF1R podocyte knockdown model we found Cre levels were important for excision of a single floxed gene (PMID: 38706850) hence we were not surprised that trying to excise two floxed genes (insulin receptor and IGF1 receptor) was challenging. This is the rationale for making the double receptor knockout cell lines to understand process / biology in more detail.

Reviewer 2:

The manuscript is generally clear and well-written. Mouse work was approved in advance. The figures are generally well-designed, bars/superimposed dot-plots.

Evaluation.

Methods are generally well described. It would be helpful to say that tissue scoring was performed by an investigator masked to sample identity.

Thank you we will do this.

Reviewer 3:

There are a number of potential issues and questions with these studies.

(1) For the in vivo studies, the only information given is for mice at 24 weeks of age. There needs to be a full time course of when the albuminuria was first seen and the rate of development. Also, GFR was not measured. Since the podocin-Cre utilized was not inducible, there should be a determination of whether there was a developmental defect in glomeruli or podocytes. Were there any differences in wither prenatal post natal development or number of glomeruli?

Thank you we will add in further phenotyping data. We do not think there was a major developmental phenotype as albuminuria did not become significantly different until several months of age. We could have used a doxycycline inducible model but we know the excision efficiency is much less than the podocin-cre driven model SUPP FIGURE 1. This would likely give a very mild (if any) phenotype and not reveal the biology adequately.

(2) Although the in vitro studies are of interest, there are no studies to determine if this is the underlying mechanism for the in vivo abnormalities seen in the mice. Cultured podocytes may not necessarily reflect what is occurring in podocytes in vivo.

Thank you for this we are happy to employ Immunohistochemistry (IHC) and immunofluorescence (IF) using spliceosome antibodies on tissue sections from DKO and control mice to examine spliceosome changes. However, as the DKO results in podocyte loss, there may not be that many DKO podocytes still present in the tissue sections. This will be taken into consideration.

(3) Given that both receptors are deleted in the podocyte cell line, it is not clear if the spliceosome defect requires deletion of both receptors or if there is redundancy in the effect. The studies need to be repeated in podocyte cell lines with either IR or IGFR single deletions.

Thank you. We have full total and phospho-proteomic data sets from single insulin receptor and IGF1 receptor knockout cell lines that we will investigate for this point.

(4) There are not studies investigating signaling mechanisms mediating the spliceosome abnormalities.

Thank you as outlined as above to reviewer 1 point 1 we are very happy to investigate insulin / IGF signalling pathways in more detail.