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 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.

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.

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.