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

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

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.

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.