A kidney-hypothalamus axis promotes compensatory glucose production in response to glycosuria

  1. Tumininu S. Faniyan
  2. Xinyi Zhang
  3. Donald A. Morgan
  4. Jorge Robles
  5. Siresha Bathina
  6. Paul S. Brookes
  7. Kamal Rahmouni
  8. Rachel J. Perry
  9. Kavaljit H. Chhabra

  • Curated by eLife

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    eLife assessment

    The study presents valuable findings on compensatory mechanisms in response to glycosuria. The evidence supporting the claims is solid, although a causal relationship is somewhat uncertain and the addition of a more clinically relevant model would have strengthened the findings. The work will be of interest to diabetes investigators.

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Abstract

The kidneys facilitate energy conservation through reabsorption of nutrients including glucose. Almost all of the filtered blood glucose is reabsorbed by the kidneys. Loss of glucose in urine (glycosuria) is offset by an increase in endogenous glucose production to maintain normal energy supply in the body. How the body senses this glucose loss and consequently enhances glucose production is unclear. Using renal Glut2 knockout mice, we demonstrate that elevated glycosuria activates the hypothalamic-pituitary-adrenal axis, which in turn drives endogenous glucose production. This phenotype was attenuated by selective afferent renal denervation, indicating the involvement of the afferent nerves in promoting the compensatory increase in glucose production. In addition, through plasma proteomics analyses we observed that acute phase proteins - which are usually involved in body’s defense mechanisms against a threat – were the top candidates which were either upregulated or downregulated in renal Glut2 KO mice. Overall, afferent renal nerves contribute to promoting endogenous glucose production in response to elevated glycosuria and loss of glucose in urine is sensed as a biological threat in mice. These findings may be useful in improving efficiency of drugs like SGLT2 inhibitors that are intended to treat hyperglycemia by enhancing glycosuria, but are met with a compensatory increase in endogenous glucose production.

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  1. Version published to 10.7554/elife.91540.2 on eLife
  2. Version published to 10.7554/elife.91540 on eLife
  3. eLife

    Author Response

    The following is the authors’ response to the current reviews.

    Response to Reviewer 1:

    • We agree with the reviewer’s overall assessment of this manuscript.

    • Because multiple secreted proteins are changed between the control and experimental groups, some of them could be causal and others corelative in the context of enhancing compensatory glucose production in response to elevated glycosuria. Through future studies we will determine the causal factors that trigger the increase in glucose production.

    • Yes, we will correct the typographical errors in a revised version of this manuscript.

    Response to Reviewer 2:

    • We agree with reviewer on their comment about potential sex differences we may have missed in this study. Therefore, we will include this limitation in discussion section of a revised manuscript.

    • The reviewer’s statement ‘The methods of that publication indicate that all experiments were completed within 14 days of inducing the Glut2 knockout’ is incorrect. In the referred publication, we had explicitly mentioned in methods that ‘All of the experiments, except those using a diet-induced obesity mouse model or noted otherwise, were completed within 14 days of inducing the Glut2 deficiency.’ Please see figures 5h-l and 6 in that previous publication, which demonstrate that all the experiments were not completed within 14 days of inducing renal Glut2 deficiency. Per the reviewer’s advice, in the present manuscript we will include the timeline of the experiments (which in some cases is 4 months beyond inducing glycosuria) with all the figure legends. In addition, for a separate project (which is unpublished) we have measured glycosuria up to 1 year after inducing renal Glut2 deficiency. Therefore, the glycosuria observed in the renal Glut2 KO mice is not temporary.

    • In our previous response to the reviewer, we had already mentioned which control group was used in this study. Please see our response to the second reviewer’s point 3. As mentioned to the reviewer, we had used Glut2-loxp/loxp mice as the control group, which is also described multiple times in the figure legends of our previous paper that reported the phenotype of renal Glut2 KO mice and is cited in this manuscript so we don’t have to repeat the same information. Per the reviewer’s advice, we will also include the information in a revised version of this manuscript.

    • We request the reviewer to look at figure 1, showing an increase in glucose production in renal Glut2 KO mice and figure 3, which demonstrates that an afferent renal denervation reduces blood glucose levels by 50%. The afferent renal denervation (ablation of afferent renal nerves) does reduce blood glucose levels in renal Glut2 KO mice. Therefore, the use of the word ‘promote’ in the title is accurate and appropriate to reflect the role of the afferent renal nerves in contributing to about 50% increase in blood glucose levels in renal Glut2 KO mice. Regarding the reviewer's comment on changes in Crh gene expression, please look at figure 3. Ablation of renal afferent nerves decreases hypothalamic Crh gene expression and other mediators of the HPA axis by 50%. Therefore, the afferent renal nerves do contribute to regulating blood glucose levels, at least in part, by the HPA axis (which is widely known to change blood glucose levels). The use of words such as ‘required’ or ‘necessary’ in the title may have indicated causal role or could have been misleading here; therefore we have purposely used ‘promote’ in the title to accurately reflect the findings of this study.

    • Because we observed an increase in hepatic glucose production in renal Glut2 KO mice (Fig. 1) - which was reduced by 50% after selective afferent renal denervation (Fig. 3) - in the graphical abstract we are suggesting a neural connection between the kidney-brain-liver or an endocrine factor(s) to account for these changes in blood glucose levels as also described in the discussion section. We can include a question mark ‘?’ in the graphical abstract to show that further studies are need to validate these proposed mechanisms; however, we cannot just remove the arrow as advised by the reviewer.

    • Per the reviewer’s advice, in the methods we will include the dilutions used for each assay.

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Recommendations For The Authors):

    It would be helpful to the reader to specify in Figure 1a-c whether data were directly measured or calculated.

    We have now clarified this in method section of the revised manuscript. The glucose production was directly measured and then fractional contribution of the tissues was calculated from the former data. We have also included a reference research paper to further clarify the method.

    The methods section would be strengthened by clarifying the order in which experiments were performed, the age of the mice at each time point, and whether different cohorts were used for different techniques.

    We have included additional details in the method section with proper citations. For in-depth protocols we have cited our previous publications.

    It would be helpful to explain or provide a reference for how the post-mortem background activity measurement was performed.

    We have included this explanation in the revised manuscript.

    Similarly, details regarding the collection of blood for ACTH and corticosterone measurement are needed for the reader to evaluate whether the results are confounded by stress at the time of collection.

    We have added these details in the method section.

    I recommend stating, if accurate, that you used mixed-sex groups because your previous study found no sex differences in the phenotype of renal Glut2 KO mice.

    Yes, we have included these details in the revised manuscript.

    Sentence 239 is difficult to follow. Also, line 287 contains a contraction.

    We have revised the sentence per the reviewer’s advice.

    A graphical abstract would be helpful, bearing in mind conclusive vs suggestive findings.

    Yes, we have included the graphical abstract with the revised manuscript.

    Reviewer #2 (Recommendations For The Authors):

    Minor Comments to the Authors

    (1) The Methods also need to specify more of the critical details of the ELISAs, including the dilution factors used, and whether the values reported are dilution-corrected. Also, there is no description of how insulin was measured.

    We have included these details in the method section. The assay dilutions were performed per manufacturers’ instructions.

    (2) The Methods do not sufficiently describe how Crh mRNA was quantified in the hypothalamus. Presumably, they examined only the paraventricular nucleus? How many sections were used for in situ hybridization? How were the brains processed? What thickness of section was used? When were the brains collected?

    We have included these details in the method section and cited our previous publications for in-depth protocols. Some of the information is also available in the figure legends.

    (3) The number of mice that were used for plasma proteomics is not indicated.

    The number of mice is indicated using individual symbols or points presented on the bar graphs.

  4. eLife

    eLife assessment

    The study presents valuable findings on compensatory mechanisms in response to glycosuria. The evidence supporting the claims is solid, although a causal relationship is somewhat uncertain and the addition of a more clinically relevant model would have strengthened the findings. The work will be of interest to diabetes investigators.

  5. eLife

    Reviewer #1 (Public Review):

    Summary:

    In this study, Faniyan and colleagues build on their recent finding that renal Glut2 knockout mice display normal fasting blood glucose levels despite massive glucosuria. Renal Glut2 knockout mice were found to exhibit increased endogenous glucose production along with decreased hepatic metabolites associated with glucose metabolism. Crh mRNA levels were higher in the hypothalamus while circulating ACTH and corticosterone was elevated in this model. While these mice were able to maintain normal fasting glucose levels, ablating afferent renal signals to the brain resulted in substantially lower blood glucose levels compared to wildtype mice. In addition, the higher CRH and higher corticosterone levels of the knockout mice were lost following this denervation. Finally, acute phase proteins were altered, plasma Gpx3 was lower, and major urinary protein MUP18 and its gene expression were higher in renal Glut2 knockout mice. Overall, the main conclusion that afferent signaling from the kidney is required for renal glut2 dependent increases in endogenous glucose production is well supported by these findings.

    Strengths:

    An important strength of the paper is the novelty of the identification of kidney to brain communication as being important for glucose homeostasis. Previous studies had focused on other functions of the kidney modulated by or modulating brain function. This work is likely to promote interest in CNS pathways that respond to afferent renal signals and the response of the HPA axis to glucosuria. Additional strengths of this paper stem from the use of incisive techniques. Specifically, the authors use isotope enabled measurement of endogenous glucose production by GC-MS/MS, capsaicin ablation of afferent renal nerves, and multifiber recording from the renal nerve. The authors also paid excellent attention to rigor in the design and performance of these studies. For example, they used appropriate surgical controls, confirmed denervation through renal pelvic CGRP measurement, and avoided the confounding effects of nerve regrowth over time. These factors strengthen confidence in their results. Finally, humans with glucose transporter mutations and those being treated with SGLT2 inhibitors show a compensatory increase in endogenous glucose production. Therefore, this study strengthens the case for using renal Glut2 knockout mice as a model for understanding the physiology of these patients.

    Weaknesses:

    A few weaknesses exist. Most concerns relate to the interpretation of this study's findings. The authors state that loss of glucose in urine is sensed as a biological threat based on the HPA axis activation seen in this mouse model. This interpretation is understandable but speculative. Importantly, whether stress hormones mediate the increase in endogenous glucose production in this model and in humans with altered glucose transporter function remains to be demonstrated conclusively. For example, the paper found several other circulating and local factors that could be causal. This model is also unable to shed light on how elevated stress hormones might interact with insulin resistance, which is known to increase endogenous glucose production. That issue is of substantial clinical relevance for patients with T2D and metabolic disease. Finally, while findings from the Glut2 knockout mice are of scientific interest, it should be noted that the Glut2 receptor is critical to the function of pancreatic islets and as such is not a good candidate for pharmacological targeting

  6. eLife

    Reviewer #2 (Public Review):

    Summary:

    The authors previously generated renal Glut2 knockout mice, which have high levels of glycosuria but normal fasting glucose. They use this as an opportunity to investigate how compensatory mechanisms are engaged in response to glycosuria. They show that renal and hepatic glucose production, but not metabolism, is elevated in renal Glut2 male mice. They show that renal Glut2 male mice have elevated Crh mRNA in the hypothalamus, and elevated plasma levels of ACTH and corticosterone. They also show that temporary denervation of renal nerves leads to a decrease in fasting and fed blood glucose levels in female renal Glut2 mice, but not control mice. Finally, they perform plasma proteomics in male mice to identify plasma proteins with a greater than 25% (up or down) between the knockouts and controls.

    Strengths:

    The question that is trying to be addressed is clinically important: enhancing glycosuria is a current treatment for diabetes, but is limited in efficacy because of compensatory increases in glucose production.

    Weaknesses:

    (1) Although I appreciate that the initial characterization of the mice in another publication showed that both males and females have glycosuria, this does not mean that both sexes have the same mechanisms giving rise to glycosuria. There are many examples of sex differences in HPA activation in response to threat, for example. There is an unfounded assumption here that males and females have the same underlying mechanisms of glycosuria that undermines the significance of the findings.

    (2) The authors state that they induced the Glut2 knockout with taxomifen as in their previous publication. The methods of that publication indicate that all experiments were completed within 14 days of inducing the Glut2 knockout. This means that the last dose of tamoxifen was delivered 14 days prior to the experimental endpoint of each experiment. This seems like an important experimental constraint that should be discussed in this manuscript. Is the glycosuria that follows Glut2 knockout only a temporary change? If so, then the long-term change in glycosuria that follows SGLT2 inhibition in humans might not be best modelled by this knockout. Please specify when the surgeries to implant a jugular catheter or ablate the renal nerves performed relative to the Glut2 knockout in the Methods.

    (3) I am still unclear what group was used for controls. Are these wild-type mice who receive tamoxifen? Are they KspCadCreERT2;Glut2loxP/loxP mice who do not receive tamoxifen? This is important and needs to be specified.

    (4) The authors should report some additional control measures for the renal denervation that could also impact blood glucose and perhaps some of their other measures. The control measures, which one would like to see unimpacted by renal denervation, include body weights, food consumption and water intake, and glycosuria itself.

    (5) The graphical abstract shows a causal link between the hypothalamus and the liver that is unsupported by any of the current findings. That arrow should be removed or a question mark should be added next to the arrow.

    (6) Though the authors have toned down their language implying a causal link between the HPA measures and compensatory elevation of blood glucose in the face of glycosuria, the title still implies this causal link. It is still the case that their data do not support causation. There are many potential ways to establish a causal link but those experiments are not performed here. The renal afferents are correlated with Crh content of the PVN, but nothing has been done to show that the Crh content is important for elevating blood glucose. In light of this, the title should be toned down. Perhaps something like "Renal nerves maintain blood glucose production and elevated HPA activity in response to glycosuria". The link between HPA and glucose is not shown in this paper.

  7. Version published to 10.1101/2023.09.01.555894v2 on bioRxiv
  8. Version published to 10.7554/elife.91540.1 on eLife
  9. eLife

    Author Response

    We appreciate the reviewers’ and editors’ advice on further improving this manuscript. We have provided point by point responses to the reviewers’ comments mentioned below. A revised version of this manuscript will be uploaded within a few weeks.

    Authors’ response to Reviewer 1 comments:

    • We appreciate the reviewer’s time in highlighting the strengths and weaknesses of this manuscript.

    • Per the reviewer’s advice, we will provide further description of the methods in a revised version of this manuscript.

    • The interpretation about the biological threat in response to elevated glycosuria in renal Glut2 KO mice is based on our observation that these mice exhibit changes in acute phase proteins measured using plasma proteomics. We will further discuss this in a revised version of this manuscript.

    • We acknowledge that this manuscript provides a resource for future mechanistic studies. Because multiple secreted proteins are changed between the control and experimental groups, some of them could be causal and others corelative in the context of enhancing compensatory glucose production in response to elevated glycosuria. Through future studies we will determine the causal proteins that trigger the increase in glucose production and identify the tissues that secrete these proteins.

    • We have shown previously (Cordeiro et al., Diabetologia 2022) that renal Glut2 deficiency doesn’t change insulin sensitivity (i.e. renal Glut2 KO mice don’t exhibit insulin resistance despite the activation of the HPA axis). It is likely that the massive glycosuria in renal Glut2 KO mice may overcome or mask the phenotype of insulin resistance potentially induced by an increase in the stress hormones.

    • In this manuscript, our major goal was to determine how elevated glycosuria leads to an increase in compensatory glucose production. We are not suggesting renal Glut2 as a therapeutic in this manuscript (that was already demonstrated in our previously published manuscript, Cordeiro et al., Diabetologia 2022).

    Authors’ response to Reviewer 2 comments:

    1. Renal Glut2 KO mice didn’t exhibit sex differences for the variables reported in our previous manuscript (Cordeiro et al., Diabetologia 2022). Therefore, in the present manuscript we decided to use male or female mice depending on their availability for each reported experiment. Per the reviewer’s advice, we will describe these details including age and sexes in each figure legend.

    2. For the method description, we have cited previous publications and mentioned ‘as described previously’. Based on the reviewer’s suggestion we will further describe the methods in detail to clarify the reviewer’s concerns. In addition, we will include age and sexes in the legends of each figure.

    3. For littermate controls, we had used Glut2loxp/loxp mice (which are like WT controls as described in Cordeiro et al., Diabetologia 2022) that were injected with tamoxifen exactly in the same way as the experimental mice. Het mice for Cre were not used as controls because they would have confounded the results as pointed out by the reviewer.

    4. Because elevated HPA activity is known to increase blood glucose levels, we suggested ‘the HPA axis may…..’. Given the nature of this manuscript, we agree the secreted proteins identified using plasma proteomics could contribute to enhanced glucose production directly or through secondary mechanisms. Afferent renal denervation using capsaicin reduced blood glucose levels concomitant with the suppression of the HPA axis in renal Glut2 KO mice. Based on these findings we speculated that the HPA axis may be partly responsible for increasing glucose production in renal Glut2 KO mice.

    We had considered using CRF antagonist and glucocorticoid receptor antagonists to determine the causal role of the HPA axis in contributing to the increase in glucose production in renal Glut2 KO mice. However, these drugs activate compensatory mechanisms including changes in insulin sensitivity. Therefore, use of these drugs would further confound the results instead of providing a clarity on the causal role of the HPA axis in enhancing glucose production in renal Glut2 KO mice.

    1. We understand the reviewer’s concerns whether the results reported here are translatable to humans. Please note that expression of SGLT2 is not kidney-specific; therefore, pleiotropic effects of SGLT2 inhibition in tissues other than the kidney cannot be excluded in animal models and humans. In contrast, the mouse model reported in this manuscript is kidney-specific Glut2 KO mice. Therefore, phenotype produced in renal Glut2 KO mice cannot be directly compared with that produced after SGLT2 inhibition. It may be too early to speculate whether the results reported in this manuscript are translatable to humans.

    In the referred research papers by the reviewer, the authors have used either models of different types of diabetes or included individuals with diabetes in their study. Notedly, diabetes itself affects the HPA axis independently of SGLT2 or GLUT2 inhibition. Therefore, it may not be appropriate to compare results obtained from animals or individuals with diabetes with that reported in this manuscript from renal Glut2 KO mice.

    1. Yes, we are currently performing mechanistic studies including assessment of mitochondrial function in renal Glut2 KO mice to determine whether and how the kidneys sense loss of glucose in urine.

    2. We apologize for the lack of methods description. We will provide additional method details in a revised version of this manuscript. All the assays were performed as per manufacturer’s instructions. Aliquots of the same samples were used for analyses of the hormones and for consistency across different assays.

  10. eLife

    eLife assessment

    This study presents a useful characterization of mechanisms underlying glycosuria-mediated increase in compensatory glucose production in Glut2 knockout mice. The strength of support is incomplete but the data represent a starting point for further studies regarding the role of the HPA axis and acute phase proteins in regulating blood glucose during glycosuria.

  11. eLife

    Reviewer #1 (Public Review):

    Summary:
    In this study, Faniyan and colleagues build on their recent finding that renal Glut2 knockout mice display normal fasting blood glucose levels despite massive glucosuria. Renal Glut2 knockout mice were found to exhibit increased endogenous glucose production along with decreased hepatic metabolites associated with glucose metabolism. Crh mRNA levels were higher in the hypothalamus while circulating ACTH and corticosterone were elevated in this model. While these mice were able to maintain normal fasting glucose levels, ablating afferent renal signals to the brain resulted in substantially lower blood glucose levels compared to wildtype mice. In addition, the higher CRH and higher corticosterone levels of the knockout mice were lost following this denervation. Finally, acute phase proteins were altered, plasma Gpx3 was lower, and major urinary protein MUP18 and its gene expression were higher in renal Glut2 knockout mice. Overall, the main conclusion that afferent signaling from the kidney is required for renal glut2 dependent increases in endogenous glucose production is well supported by these findings.

    Strengths:
    An important strength of the paper is the novelty of the identification of kidney-to-brain communication as being important for glucose homeostasis. Previous studies had focused on other functions of the kidney modulated by or modulating brain activity. This work is likely to promote interest in CNS pathways that respond to afferent renal signals and the response of the HPA axis to glucosuria. Additional strengths of this paper stem from the use of incisive techniques. Specifically, the authors use isotope-enabled measurement of endogenous glucose production by GC-MS/MS, capsaicin ablation of afferent renal nerves, and multifiber recording from the renal nerve. The authors also paid excellent attention to rigor in the design and performance of these studies. For example, they used appropriate surgical controls, confirmed denervation through renal pelvic CGRP measurement, and avoided the confounding effects of nerve regrowth over time. These factors strengthen confidence in their results. Finally, humans with glucose transporter mutations and those being treated with SGLT2 inhibitors show a compensatory increase in endogenous glucose production. Therefore, this study strengthens the case for using renal Glut2 knockout mice as a model for understanding the physiology of these patients.

    Weaknesses:
    A few weaknesses exist. Clarification of some aspects of the experimental design would improve the manuscript. However, most concerns relate to the interpretation of this study's findings. The authors state that loss of glucose in urine is sensed as a biological threat based on the HPA axis activation seen in this mouse model. This interpretation is understandable but speculative. Importantly, whether stress hormones mediate the increase in endogenous glucose production in this model and in humans with altered glucose transporter function remains to be demonstrated conclusively. For example, the paper found several other circulating and local factors that could be causal. In addition, the approach used in these studies cannot definitively determine whether renal glucose production or only hepatic glucose production was altered. This model is also unable to shed light on how elevated stress hormones might interact with insulin resistance, which is known to increase endogenous glucose production. That issue is of substantial clinical relevance for patients with T2D and metabolic disease. Finally, while findings from the Glut2 knockout mice are of scientific interest, it should be noted that the Glut2 receptor is critical to the function of pancreatic islets and as such is not a good candidate for pharmacological targeting.

  12. eLife

    Reviewer #2 (Public Review):

    Summary:
    The authors previously generated renal Glut2 knockout mice, which have high levels of glycosuria but normal fasting glucose. They use this as an opportunity to investigate how compensatory mechanisms are engaged in response to glycosuria. They show that renal and hepatic glucose production, but not metabolism, is elevated in renal Glut2 male mice. They show that renal Glut2 male mice have elevated Crh mRNA in the hypothalamus and elevated plasma levels of ACTH and corticosterone. They also show that temporary denervation of renal nerves leads to a decrease in fasting and fed blood glucose levels in female renal Glut2 mice, but not control mice. Finally, they perform plasma proteomics in male mice to identify plasma proteins with a greater than 25% (up or down) between the knockouts and controls.

    Strengths:
    The question that is trying to be addressed is clinically important: enhancing glycosuria is a current treatment for diabetes, but is limited in efficacy because of compensatory increases in glucose production.
    Also, the mouse line used is an inducible knockout, thus minimizing the impact of compensatory mechanisms engaged in early development.

    Weaknesses:

    1. Though the Methods specify that both male and female mice were used, it appears each experiment was performed only on one sex, rather than each experiment being performed on both sexes. For example, renal denervation was performed only on females, whereas all other experiments were performed exclusively on males. This makes it impossible to examine whether there are sex differences in any measures.

    2. This study appears to use an inducible Glut2 knockout with tamoxifen, yet nothing describes when the tamoxifen was delivered relative to the experimental manipulations. Was the knockout performed in young animals? In adult animals? This is important both for the ability of readers to repeat the experiment, but also to interpret the results in light of potential compensatory changes (if the knockout was performed at an early age, for example).

    3. In Methods, please clarify whether littermate controls were WT, het, or both. If het mice were used as controls, this is potentially problematic.

    4. Conclusions like "the HPA axis may contribute to the compensatory increase in glucose production in renal Glut2 knockout mice" (line 215) are premature. All that is shown is that renal Glut2 male mice have elevated HPA activity. There are no experiments establishing causation. For example, the authors could administer a CRF antagonist or a glucocorticoid receptor antagonist in this mouse line, and examine whether this impacts blood glucose. This was not done.

    5. If elevated glycosuria drives HPA activity, one would expect to see elevated HPA activity in humans who take SGLT2 inhibitors. Yet, this does not seem to be the case (Higashikawa et al, 2021; see also Perry et al, 2021 for rodent example). This raises the question of whether the glycosuria observed in the mouse line here is relevant to any human conditions. The relevance of the mechanisms proposed here would be much more convincing if a second model of glycosuria was used here (for example, inducing diabetes in mice and treating with SGLT2 inhibitors). Without these types of experiments, any relevance to human conditions is highly speculative and should be reserved for the Discussion. What the authors are studying here is one mechanism for maintaining blood glucose when glycosuria is induced by a genetic knockout.

    6. The experiment examining the impact of renal denervation is nice but incomplete. For example, what is the relevance to the hepatic glucose production that was reported? It is interesting that the renal denervation normalized the elevated HPA activity in Glut2 female mice, but it is not clear how this signaling would alter HPA activity.

    7. The Methods need to describe the plasma collection procedure for both ELISA and plasma proteomic experiments. What time of day were samples collected? Were samples collected when animals were euthanized from other experiments after experimental manipulations, or in animals without other experimentation?

    8. In general, the links between the disparate mechanisms (signals in the plasma, changes in renal activity, changes in HPA activity) are weak. There are more experiments needed to establish a direct kidney-hypothalamus axis. If renal activity elevates blood glucose in the face of glycosuria, why are there no differences in renal activity between control and Glut2 knockout mice? If the blood glucose levels are regulated by renal activity, it must be the sensitivity to the renal activity that differs between control and knockout mice - perhaps this should be investigated. If one stimulates afferent renal nerves, can one drive HPA activation and elevate blood glucose? How are these measures related to the plasma proteins identified? Without these links, this study is descriptive and correlational.

  13. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8431146.

    This review reflects comments and contributions from Marina Schernthanner, Femi Arogundade and Pablo Ranea-Robles. Review synthesized by Jonny Coates.

    The study leverages the phenotype presented by the renal Glut2 KO mice (glycosuria with normal glycemia) to investigate how the body senses this glucose loss and the mechanisms behind metabolic homeostasis processes that lead to enhanced glucose production so glycemia remains stable. The use of a genetically modified mouse model with renal Glut2 knockout provides a controlled system for studying the specific role of renal glucose transporters in glucose homeostasis. The study involves various methods, including measurements of glucose production, metabolomics, gene expression related to the hypothalamic-pituitary-adrenal axis, afferent renal nerve ablation, and analysis of secreted proteins. The authors point to a kidney/hypothalamus axis and suggest the involvement of different acute phase proteins in this homeostatic response. The limitations of the study are acknowledged, and further research is suggested to delve deeper into the role of secretory proteins and the specific source of endogenous glucose production after afferent renal denervation. The manuscript is well written and the results are potentially of interest. The study's findings have potential implications for the field of diabetes treatment, as they suggest a mechanism that may explain why SGLT2 inhibitors don't achieve their full potential in lowering blood glucose levels. However, we think that some of the conclusions are merely based on descriptive assessments of changes occurring in the renal Glut2 KO mice. There are a lack of details in the reporting of some of the results and, in particular, in the discussion section, that would also require a bit more explanation from the authors. Right now, it could be hard for the reader to place this research in context. We have summarized our comments below

    Major comments:

    • The study mentions the use of male and female mice, but it's important to know the sample sizes for each experimental group and how gender might influence the results. Additionally, the authors should provide more details about the control groups and their matching criteria to ensure the validity of comparisons. Moreover, the exact genetic information for the knockout mice i.e. what is the CreER driver that makes it kidney-specific? Is missing. It is currently inconsistent in terms of sex and age of mice used for different experiments. 

    Minor comments:

    • While the metabolomics analysis is described, more information is needed about the biological significance of the changes observed in the metabolites. How do these changes relate to the compensatory glucose production, and are they causally linked?

    • The paper would benefit from improved organization and clarity, particularly in the results and discussion sections. 

    • Crh+ cells in control image of fig 2 are not clear. The authors could consider highlighting the are where these cells are present, or add an inset showing a zoomed image of some positive cells

    • How specific is the use of capsaicin to selectively suppress afferent renal nerve activity? Does this impact other neurons? Either citations or experimental data should be included here. 

    • The conditions of mice in Sup Fig. 1 are not clear and should be stated clearly in this part of the text and in the figure legend.

    • The study is transparent about its limitations and raises important questions for future research. This acknowledgment of limitations contributes to the scientific rigor of the work.

    • While control groups are mentioned, it's not clear how these controls were chosen or matched to the experimental group. Further information is needed on how these controls were used to make valid comparisons.

    • While the study describes the experimental procedures in detail, it's essential to provide information on how many times these experiments were replicated to assess the reproducibility of the results. This is especially crucial given the complex methods used.

    • Blocking the HPA axis and assessing responses in KO and WT mice would strengthen the data in Fig 2

    • Investigating or showing the levels of glucagon and adrenaline to delineate mechanisms of tissue-specific glucose production would further strengthen the data presented. 

    • Is it possible to measure glucose production under denervation conditions? That would support the conclusion if the increased glucose production is blunted 

    • Not everyone might be familiar with the abbreviation 2D-DIGE. Explaining this before first use would be beneficial. 

    • Supp fig 2 could be fused with Fig 4 to make the argument more convincing.

    • The authors state that "It is possible that afferent renal denervation in the present study attenuated only hepatic glucose production through the hypothalamus without affecting the compensatory increase in renal (local) glucose production". Addressing this would significantly strengthen the manuscript, particularly given that the title includes "hypothalamus-kidney axis". 

    Comments on reporting:

    • The paper mentions the use of statistical tests but lacks information on the specific statistical tests performed for each analysis. It's crucial to provide details on the tests used, assumptions made, and how p-values were adjusted for multiple comparisons, if applicable.

    Suggestions for future studies:

    • Extend the research to human subjects, particularly individuals with diabetes treated with SGLT2 inhibitors. Investigate whether similar mechanisms and pathways are at play in humans, and whether these findings have clinical relevance.

    • Investigate the specific roles of secreted proteins, such as acute phase proteins and major urinary proteins, in glucose regulation and potential interactions with the kidney-hypothalamus axis.

    • Explore how the kidney-hypothalamus axis integrates with other nervous system and endocrine signals involved in glucose regulation, such as insulin and glucagon.

    • Conduct in-depth studies on the impact of afferent renal nerve activity on glucose homeostasis and the signaling pathways involved. Investigate the role of sensory nerves in detecting glycosuria and triggering compensatory responses.

    Competing interests

    The author declares that they have no competing interests.

  14. Version published to 10.1101/2023.09.01.555894v1 on bioRxiv