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

Reviewer #1 (Public review):

Summary:

In this manuscript, the role of the insulin receptor and the insulin growth factor receptor was investigated in podocytes. Mice, where both receptors were deleted, developed glomerular dysfunction and developed proteinuria and glomerulrosclerosis over several months. Because of concerns about incomplete KO, the authors generated and studied 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 of cell death, the authors performed global proteomics and found that spliceosome proteins were downregulated. They confirmed this directly by using long-read sequencing. These results suggest a novel role for insulin and IGF1R signaling in RNA splicing in podocytes.

This is primarily a descriptive study and no technical concerns are raised. The mechanism of how insulin and IGF1 signaling regulates splicing is not directly addressed but implicates potentially the phosphorylation downstream of these receptors. In the revised manuscript, it is shown that the mouse KO is incomplete potentially explaining the slow onset of renal insufficiency. Direct measurement of GFR and serial serum creatinines might also enhance our understanding of progression of disease, proteinuria is a strong sign of renal injury. An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful but may be masked by defects in other spliceosome genes. As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on.

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. 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.

Latest comments:

The new reviewer raised two major points, whether the KO effect on splicing is specific to IGF1 and whether the interpretation could be developmental rather than due to splicing. The reviewer raises some important issues but the evidence to suggest that this is specific is data in the literature that IR/IGF signaling is already known to regulate splicing and that splicing defects were not detected in other models that they have analyzed. I agree with the reviewer (and authors) that the incomplete floxing of the genes is a major complication. The point that there could be a developmental defect with mice being born with fewer podocytes and perhaps the authors should caveat this point. The fact that they mice are born with normal function, that renal function can be maintained with up to 80% loss of podocytes suggest that they are likely born with a good number of podocytes and the dysfunction that occurs at 6 months is due to a process, induced by the loss of IR/IGF signaling that is detrimental to the podocyte.

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, bars/superimposed dot-plots.

Methods are generally well described.

Comments on previous version:

Coward and colleagues have done an excellent job of responding to all the reviewer comments.

Reviewer #4 (Public review):

Summary and background:

This report entitled "The insulin/IGF axis is critically important (for) controlling gene transcription in the podocyte" from Hurcombe et al is based on a mouse double knockdown of the IR and IGF1R and a parallel cultured mouse podocyte model. Insulin/IGF signaling system in mammals evolved as three gene reduplicated peptides (insulin, IGF-1, and IGF-2) and their two receptors IR and IGF1R that cross-react to variable extents with the peptides, are ubiquitously expressed, and signal through parallel pathways. The major downstream effect of insulin is to regulate glucose uptake and metabolism, while that of the IGF pathways is to regulate growth and cell cycling in part through mTORC1. The GH-IGF-1-IGF1R pathway regulates post-natal growth. IGF-2 signaling is thought to play a major role in regulating intrauterine growth and development, although IGF-2 is also present at high levels in post-natal life. Thus, one would anticipate that reducing IR/IGF1R signaling in any cell would slow growth and cell cycling by reducing growth factor and metabolic mTORC1-mediated and other processes including the splicing of RNA for protein synthesis.

Comments on revised version:

The second sentence of the Summary reads "This study sought to elucidate the compound role of the insulin/IGF1 axis in podocytes using transgenic mice and cell culture models deficient in both receptors." The study design and rationale for the proteosome analysis described is predicated on the finding that podocyte-specific knockdown of the IR/IGF-1R in mice is associated with development of proteinuria and reduced eGFR by 20months of life. Since the IR/IGF-1R are critically required for normal development and growth of all cells and organs, the obvious explanation for the observation would be that the model system results in defective podocyte development and deployment (caused by reduced IR/IGF-1) that, in turn, causes subsequent development of proteinuria and glomerulosclerosis (that may be much less dependent on a normal level of IR/IGF-1R expression). Thus, the experimental design does not allow a distinction between podocyte development and steady state function which are different biologic processes. The data provided does not examine podocyte status immediately after birth to confirm that podocyte number and size and structure is normal in mice that subsequently develop proteinuria and glomerulosclerosis. The response to the reviewer suggests that since this would require additional mice it has not been undertaken in order to reduce animal usage. This is not a valid argument, particularly when the investigators have not even used state-of-the-art methods to measure podocyte number, size and density in adult mice, key parameters that would be required to interpret their data. Counting podocyte nuclear number in glomerular cross-sections is simply an inadequate method, even if it is used and reported in other journals, and particularly where the examples given to justify its use can hardly be viewed as representing first rate science.

If the absence of studies that would answer the above questions, the investigators should add a sentence to the Discussion dealing with study limitations as follows. "The study design does not allow us to determine whether the primary effect of reduced IR/IGF-1R expression on the phenotype is during in utero and post-natal podocyte development and deployment, during periods of rapid growth when IGF-1 levels are highest, in steady state adult podocytes, or under all of the above conditions".

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, the role of the insulin receptor and the insulin growth factor receptor was investigated in podocytes. Mice, where both receptors were deleted, developed glomerular dysfunction and developed proteinuria and glomerulrosclerosis over several months. Because of concerns about incomplete KO, the authors generated and studied 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 of cell death, the authors performed global proteomics and found that spliceosome proteins were downregulated. They confirmed this directly by using long-read sequencing. These results suggest a novel role for insulin and IGF1R signaling in RNA splicing in podocytes.

This is primarily a descriptive study and no technical concerns are raised. The mechanism of how insulin and IGF1 signaling regulates splicing is not directly addressed but implicates potentially the phosphorylation downstream of these receptors. In the revised manuscript, it is shown that the mouse KO is incomplete potentially explaining the slow onset of renal insufficiency. Direct measurement of GFR and serial serum creatinines might also enhance our understanding of progression of disease, proteinuria is a strong sign of renal injury. An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful but may be masked by defects in other spliceosome genes. As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on.

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. 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.

Comments on revised version:

I'm satisfied with the revised manuscript and the responses to my previous concerns.

Thank you.

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, bars/superimposed dot-plots.

Methods are generally well described.

Comments on revised version:

Coward and colleagues have done an excellent job of responding to all the reviewer comments.

Thank you.

Reviewer #4 (Public review):

Summary and background:

This report entitled "The insulin/IGF axis is critically important (for) controlling gene transcription in the podocyte" from Hurcombe et al is based on a mouse double knockdown of the IR and IGF1R and a parallel cultured mouse podocyte model. Insulin/IGF signaling system in mammals evolved as three gene reduplicated peptides (insulin, IGF-1, and IGF-2) and their two receptors IR and IGF1R that cross-react to variable extents with the peptides, are ubiquitously expressed, and signal through parallel pathways. The major downstream effect of insulin is to regulate glucose uptake and metabolism, while that of the IGF pathways is to regulate growth and cell cycling in part through mTORC1. The GH-IGF-1-IGF1R pathway regulates post-natal growth. IGF-2 signaling is thought to play a major role in regulating intrauterine growth and development, although IGF-2 is also present at high levels in post-natal life. Thus, one would anticipate that reducing IR/IGF1R signaling in any cell would slow growth and cell cycling by reducing growth factor and metabolic mTORC1-mediated and other processes including the splicing of RNA for protein synthesis.

Thank you for this new extra review and assessing our paper with new suggestions (we addressed the previous suggestions to the satisfaction of other reviewers). Of note -regarding this introduction – the podocyte is a terminally differentiated cell and may have unique responses to insulin / IGF as it is accepted it does not generally proliferate (hence we consider understanding the actions of insulin / IGF and their receptors to be of interest). Indeed, we have recently shown a contrasting effect of IGF signalling in the podocyte. Partial suppression of the IGF1 receptor is beneficial in contrast to near complete suppression that results in mitochondrial dysfunction (PMID:38706850).

Mouse IR/IGF1R double knockdown model:

A double knockdown mouse model was generated by interbreeding mice with different genetic backgrounds carrying floxed sites for IR and IGF-1R to produce mixed background offspring with both floxed IR and IGF-1R genes. These mice were crossed so that the podocin promoter driven-Cre (that comes on at about embryonic day 12 bas podocytes are developing) would delete IR and IGF-1R genes. Since podocin is believed to be an absolutely podocyte-specific protein, this podocin promoter this is predicted to specifically knock down the IR and IGF1R genes only in podocytes. The weight and growth of double KO offspring was not different from controls, but some proportion of the double knockdown mice subsequently developed proteinuria by 6 months and 20% died, although no specific data is provided to identify the cause of the deaths since eGFR was not decreased. Surviving mice were evaluated at 6 months of age. The efficacy of knockdown was not demonstrated in the mouse model itself, although a temperature-sensitive cell line developed from these double knockdown mice showed that expression of IR and IGF-1R proteins in the Cre-treated cell line were both reduced by about 50% (no statistical analysis of this result provided).

In the knockout mice, proteinuria was significantly increased by 6 months, but not at earlier time points. Histologic analysis showed proteinaceous casts, glomerulosclerosis and interstitial fibrosis. Podocyte number was stated to be reduced by about 30% in double knockdown mice, although the method by which this was evaluated seems to have been by counting WT1 positive nuclei in glomerular cross-sections, an approach that is well-known not to be a reliable way of assessing true podocyte number. No information is provided about podocyte size, density or glomerular volume.

Comment: If IR/IGF1R deletion plays a significant role in normal podocyte function sufficient to cause proteinuria and glomerulosclerosis then the effect of reduced IR and IGF1R protein expression on podocyte function would have been expected to produce a phenotype before 6 months. A more likely scenario to explain the overall result is that deleting the IR and IGF1R genes at about embryonic day12 impacted podocyte development to a variable extent such that some mice developed fewer podocytes per glomerulus than other mice. As mice grow and their glomeruli and glomerular capillary area increases, those mice with fewer podocytes would not be able to completely cover the filtration surface with foot processes and would develop proteinuria and glomerulosclerosis. If reduced podocyte number per glomerulus is the proximate cause of the observed proteinuria, then modulation of the body and kidney growth rate by calorie restriction to slow growth (lower circulating IGF-1 levels) would be expected to be protective, while a high protein high calorie diet (higher circulating IGF-1 levels) or uni-nephrectomy to increase kidney growth rate would be expected to enhance proteinuria and glomerulosclerosis.

Thank you for these comments. In response to them:

(1) WT1 as a marker of podocyte number. We agree may not be the most accurate way of precisely measuring podocyte number but is widely accepted in the field (PMID:33655004 / PMID:38542564) and we think convincingly shows fewer podocytes at 6-months.

(2) Podocyte size and density was not measured. This was not the focus of the paper and the histology obviously showed a significant phenotype in several mice (Figs 1D-F). Of note we did objectively assess a glomeruloscleorosis index (Fig 1D). We took the approach to understand mechanism through non-biased proteomics and phospho-proteomics of conditionally immortalised podocytes in which we had convincingly knocked down the insulin and IGF1 receptors (Figure 2)

(3) You did not study the mice earlier to ascertain the developmental phenotype. We concede we did not do this but there was no significant proteinuria detected early in the mice so elected not to increase mouse numbers by studying them then (which we consider good practice for reduction, replacement and refinement). We suspect there would have been subtle changes in those mice that had significantly reduced simultaneous IR and IGF1R knockdown. It was precisely because of this that we generated a conditionally immortalised podocyte cell line with robust simultaneous knock-down of both receptors.

(4) You did not show significant insulin and IGF1 receptor knockdown in the conditionally immortalised cell line (reviewer states it was 50%). We clearly knocked both receptors down (insulin and IGF1R) in the podocyte line by >80% which was highly statistically significant (p<0.00001). Figure 2A. We agree this was crucial (and we made the cell line because of the variability in the mouse model).

The model as used may be more representative of a variable degree of podocyte depletion than an effect of impaired IR/IGF1R signaling. Therefore, although the phenotype may be ultimately attributable to the IR/IGF1R gene deletions the proteinuria and glomerulosclerotic phenotype itself was probably a consequence of defective podocyte development. Examining podocyte number, size, density and glomerular volume at earlier time points (4 weeks) would help to answer this question. Therefore, a more appropriate title would be "The insulin/IGF axis is critically important (for) normal podocyte development and deployment". In this context the effect of the knockdowns on splicing would make more sense.

Please see our response (above). We think our final conclusion that in the podocyte the insulin/IGF axis is important for spliceosome activity and control is valid. This is due to our findings (both total and phospho proteomics results) and considering recent other papers showing this axis can rapidly phosphorylate a variety of spliceosome proteins in different cell types (PMID:39939313 / PMID:32888406). All discussed in detail in the manuscript).

Cell culture studies. A cell line was generated using a temperature sensitive SV40 system that has been previously reported from this laboratory. A detailed analysis is provided to show that double knockout cells exhibited abnormal spliceosome activity. This forms the basis for the conclusion that "The insulin/IGF axis is critically important (for) controlling gene transcription in the podocyte". There are several concerns that weaken this conclusion.

(1) In the double knockdown cell culture system about 30% of cells were "lost" by 3 days and about 70% of cells were "lost" by 5days. The studies were done at the 3 day time point. It is not clear whether "lost" cells were in the process of dying, stress-induced detachment, or just growing more slowly than control due to reduced IR and IGF-1R signaling. These processes could have impacted splicing in a non-specific way independent of IR/IGF1R signaling itself.

(2) Can a single cell line derived from the double floxed mice be relied on to provide an unbiased picture of the effect of deleting IR and IGF-1R? Presumably, the transfection and selection process will select for cells that survive thereby including unknown biases, possibly related to spliceosome function. Is a single cell line adequate? These investigators have extensive experience with this type of analysis, but this question is not addressed in the discussion.

(3) To determine whether the effect is specific to reduced IR/IGFR signaling the deletion of IR and IGF-1R could be corrected by transfecting full length IR and IGF-1R cDNAs into the cells to restore normal IR/IGF1R signaling. If transfected cells with intact IR and IGF-1R expression and activity returns spliceosome activity to normal this would be evidence that receptors themselves play some role in spliceosome activity, as opposed to the downstream effect on growth limitation/stress on the cells.

(4) Other ways of testing whether the splicing effect is specifically due to reduced IR/IGF-1R signaling would be to (a) block IR and IGF1R receptors using available inhibitors, (b) remove or reduce insulin, IGF-1 and IGF-2 levels in the culture medium, (c) use low glucose and amino acid culture medium to slow growth rate independent of receptor function, (d) or block intra-cellular signaling via the IR and IGF-1R receptors through mTORC1 inhibition using rapamycin or other signaling targets.

(5) It would be useful to determine whether the cultured cells stressed in other ways (e.g. ischemia, toxins, etc.) also results in the same splicing abnormalities.

Point 1. 70% cell loss was observed at day 7 (not day 5). We found approximately 20% loss at day 3. We opted to go for this early date hypothesising the key detrimental processes would be clear then. This 3 day time point also ensures there has been enough time to allow for the expression of Cre recombinase, receptor gene excision and degradation of existing endogenous IR/IGF1R following lentiviral transduction. Interestingly we did not find a major “death or apoptosis” signal in our data then but agree it should be considered. We think this is a specific pathway as we have examined several other conditionally immortalised detrimental podocyte cell line previously using proteomics with a much more severe phenotype of cell death (E.g. podocyte GSK3 alpha/beta knockdown) and we detected NO spliceosome signal (PMID:30679422). Furthermore, there are now other podocyte proteomics “stress” studies that have been published in which there is proteinuria and significant cell loss / death that also do not show spliceosome dysfunction. These include studying the detailed proteosomal signature of podocytes stressed with Doxorubicin and Lipopolysaccharide endotoxin LPS in mice (PMID:32047005) and bradykinin stimulation of rat podocytes (PMID:32518694).

Point 2. Yes, we think it is valuable and reproducible. We generated a podocyte cell line from insulin receptor and IGF1 receptor homozygous floxed cells. Hence there is no selection bias in the cells when generating the line as both receptors are effectively intact. We then temporally “knocked down” the receptors with extrinsic lentiviral Cre.

Importantly we validated our cell line findings both back in the cells (with Western blotting) and in our transgenic receptor knockdown mice and found evidence of spliceosomal dysregulation (Figure 3E and 3F). Also as discussed above the spliceosome has been identified in other models in the insulin/IGF pathway.

Point 3. We don’t think the experiment of knocking down the receptors and then reconstituting them would prove this hypothesis. This is because if splicing abnormality was due to generalised cell dysfunction (which we do not think is the case in this situation) then putting the receptors back may simply restore cell health and the spliceosomal function (e.g. it does not prove it is via the receptors). Secondly, the process of transduction with multiple lentiviruses may be inherently stressful to the cell and there may be a high level of extrinsic receptor inserted which may also be confounding/detrimental. Finally, as discussed there are now several lines of evidence describing insulin / IGF signalling to spliceosomal proteins which we consider important (discussed in the paper in detail).

Point 4. We think modulating the receptors using the Cre-lox approach is the cleanest approach (with fewer off-target effects) to interrogate the insulin / IGF axis. It allows us to differentiate the cells by thermo-switching (which is crucial for this terminally differentiated cell) and then robustly knocking down both receptors simultaneously to investigate mechanism. We agree these supplementary approaches may give some extra information if their limitations (eg off target effects of inhibitors) are also taken into consideration.

Point 5. They do not. Please see response to point 1 above regarding GSK3, Doxorubicin, LPS and bradykinin challenge.