Dapagliflozin improves endothelial integrity and hemodynamics in endotoxin treated mice through an apolipoprotein M dependent pathway
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eLife Assessment
SGLT2 inhibitors (SGLT2i) have assumed important roles in reducing cardiovascular risk, particularly in those with diabetes. It has become appreciated that its protective effects are likely beyond their ability to lower blood sugar levels. This research presents a novel approach to studying the SGLT2i mechanism of action which is yet to be fully elucidated.
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Abstract
Rationale
Sodium-glucose co-transporter inhibitors (SGLT2i) are under active clinical investigation in patients with acute inflammatory conditions, based on their clinical cardio-and nephroprotective effects, and a pre-clinical study that demonstrated SGLT2i improve renal outcomes and survival in a lipopolysaccharide (LPS) model. However, a unified mechanism that explains how SGLT2i could prevent hemodynamic consequences of inflammatory conditions has not been described. Apolipoprotein M (ApoM) is inversely associated with mortality in inflammatory conditions and improves cardiac function in endotoxin-treated mice via sphingosine-1-phosphate (S1P) signaling.
Objective
Test the hypothesis that pre-treatment with SGLT2i dapagliflozin (Dapa) improves hemodynamics in endotoxin-treated mice via the ApoM/S1P pathway.
Methods and Results
Mice with diet-induced obesity were gavaged with vehicle or Dapa for 4 days prior to LPS (10 mg/kg, IP). We found that mice receiving Dapa restored circulating ApoM levels, likely by increasing expression of the multi-ligand protein receptor megalin in the proximal tubules. Dapa attenuated LPS-induced reductions in cardiac dysfunction including reductions in ejection fraction, cardiac index, and coronary sinus area as well as vascular permeability as ascertained by intravital microscopy. Using both ApoM transgenic and knockout mice and S1P receptor inhibitors, we show that the ApoM/S1P pathway is important for the beneficial effects of Dapa in the LPS model.
Conclusions
In the setting of acute inflammation, our data suggest that SGLT2i maintains levels of megalin, leading to preservation of ApoM, which in turn promotes endothelial barrier integrity and improves hemodynamics. Our studies suggest a novel mechanism by which SGLT2i can preserve intravascular volume in the acute inflammatory setting.
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Author response:
Reviewer #1 (Public Review):
The authors examined the hypothesis that plasma ApoM, which carries sphingosine-1-phosphate (S1P) and activates vascular S1P receptors to inhibit vascular leakage, is modulated by SGLT2 inhibitors (SGLTi) during endotoxemia. They also propose that this mechanism is mediated by SGLTi regulation of LRP2/ megalin in the kidney and that this mechanism is critical for endotoxin-induced vascular leak and myocardial dysfunction. The hypothesis is novel and potentially exciting. However, the author's experiments lack critical controls, lack rigor in multiple aspects, and overall does not support the conclusions.
Thank you for these comments. We have now directly addressed this hypothesis by using proximal tubule-specific inducible megalin/Lrp2 knockout mice, which remains an innovative …
Author response:
Reviewer #1 (Public Review):
The authors examined the hypothesis that plasma ApoM, which carries sphingosine-1-phosphate (S1P) and activates vascular S1P receptors to inhibit vascular leakage, is modulated by SGLT2 inhibitors (SGLTi) during endotoxemia. They also propose that this mechanism is mediated by SGLTi regulation of LRP2/ megalin in the kidney and that this mechanism is critical for endotoxin-induced vascular leak and myocardial dysfunction. The hypothesis is novel and potentially exciting. However, the author's experiments lack critical controls, lack rigor in multiple aspects, and overall does not support the conclusions.
Thank you for these comments. We have now directly addressed this hypothesis by using proximal tubule-specific inducible megalin/Lrp2 knockout mice, which remains an innovative hypothesis about how SGLT2i can reduce vascular leak.
Reviewer #2 (Public Review):
Apolipoprotein M (ApoM) is a plasma carrier for the vascular protective lipid mediator sphingosine 1-phospate (S1P). The plasma levels of S1P and its chaperones ApoM and albumin rapidly decline in patients with severe sepsis, but the mechanisms for such reductions and their consequences for cardiovascular health remain elusive. In this study, Ripoll and colleagues demonstrate that the sodium-glucose co-transporter inhibitor dapagliflozin (Dapa) can preserve serum ApoM levels as well as cardiac function after LPS treatment of mice with diet-induced obesity. They further provide data to suggest that Dapa preserves serum ApoM by increasing megalin-mediated reabsorption of ApoM in renal proximal tubules and that ApoM improves vascular integrity in LPS treated mice. These observations put forward a potential therapeutic approach to sustain vascular protective S1P signaling that could be relevant to other conditions of systemic inflammation where plasma levels of S1P decrease. However, although the authors are careful with their statements, the study falls short of directly implicating megalin in ApoM reabsorption and of ApoM/S1P depletion in LPS-induced cardiac dysfunction and the protective effects of Dapa.
The observations reported in this study are exciting and potentially of broad interest. The paper is well written and concise, and the statements made are mostly supported by the data presented. However, the mechanism proposed and implied is mostly based on circumstantial evidence, and the paper could be substantially improved by directly addressing the role of megalin in ApoM reabsorption and serum ApoM and S1P levels and the importance of ApoM for the preservation for cardiac function during endotoxemia. Some observations that are not necessarily in line with the model proposed should also be discussed.
The authors show that Dapa preserves serum ApoM and cardiac function in LPS-treated obese mice. However, the evidence they provide to suggest that ApoM may be implicated in the protective effect of Dapa on cardiac function is indirect. Direct evidence could be sought by addressing the effect of Dapa on cardiac function in LPS treated ApoM deficient and littermate control mice (with DIO if necessary).
The authors also suggest that higher ApoM levels in mice treated with Dapa and LPS reflect increased megalin-mediated ApoM reabsorption and that this preserves S1PR signaling. This could be addressed more directly by assessing the clearance of labelled ApoM, by addressing the impact of megalin inhibition or deficiency on ApoM clearance in this context, and by measuring S1P as well as ApoM in serum samples.
Methods: More details should be provided in the manuscript for how ApoM deficient and transgenic mice were generated, on sex and strain background, and on whether or not littermate controls were used. For intravital microscopy, more precision is needed on how vessel borders were outland and if this was done with or without regard for FITC-dextran. Please also specify the type of vessel chosen and considerations made with regard to blood flow and patency of the vessels analyzed. For statistical analyses, data from each mouse should be pooled before performing statistical comparisons. The criteria used for choice of test should be outlined as different statistical tests are used for similar datasets. For all data, please be consistent in the use of post-tests and in the presentation of comparisons. In other words, if the authors choose to only display test results for groups that are significantly different, this should be done in all cases. And if comparisons are made between all groups, this should be done in all cases for similar sets of data.
Thank you for these comments. We have now tested the direct role of Lrp2 with respect to SGLT2i in vivo and in vitro, and our study now shows that Lrp2 is required for the effect of dapagliflozin on ApoM. ApoM deficient and transgenic mice were previously described and published by our group (PMID: 37034289) and others (PMID: 24318881), and littermate controls were used throughout our manuscript. We agree that the effect on cardiac function is likely indirect in these models, and as yet we do not have the tools in the LPS model to separate potential endothelial protective vs cardiac effects. In addition, since the ApoM knockout has multiple abnormalities that include hypertension, secondary cardiac hypertrophy, and an adipose/browning phenotype, all of which may influence its response to Dapa in terms of cardiac function, these studies will be challenging to perform and will require additional models that are beyond the scope of this manuscript.
For intravital microscopy, vessel borders were outlined blindly without regard for FITC-dextran. We believe it is important to show multiple blood vessels per mouse since, as the reviewer points out, there is quite a bit of vessel heterogeneity. These tests were performed in the collaborator’s laboratory, and data analysis was blinded, and the collaborator was unaware of the study hypothesis at the time the measurements were performed and analyzed. They have previously reported this is a valid method to show cremaster vessel permeability (PMID: 26839042).
We have updated our methods section and updated the figure legends to clearly indicate the statistical analyses we used. For 2 group comparison we used student’s t-test, and for multiple groups one-way ANOVA with Sidak's correction for multiple comparisons was used throughout the paper when the data are normally distributed, and Kruskal-Wallis was used when the data are not normally distributed.
Reviewer #3 (Public Review):
The authors have performed well designed experiments that elucidate the protective role of Dapa in sepsis model of LPS. This model shows that Dapa works, in part, by increasing expression of the receptor LRP2 in the kidney, that maintains circulating ApoM levels. ApoM binds to S1P which then interacts with the S1P receptor stimulating cardiac function, epithelial and endothelial barrier function, thereby maintaining intravascular volume and cardiac output in the setting of severe inflammation. The authors used many experimental models, including transgenic mice, as well as several rigorous and reproducible techniques to measure the relevant parameters of cardiac, renal, vascular, and immune function. Furthermore, they employ a useful inhibitor of S1P function to show pharmacologically the essential role for this agonist in most but not all the benefits of Dapa. A strength of the paper is the identification of the pathway responsible for the cardioprotective effects of SGLT2is that may yield additional therapeutic targets. There are some weaknesses in the paper, such as, studying only male mice, as well as providing a power analysis to justify the number of animals used throughout their experimentation. Overall, the paper should have a significant impact on the scientific community because the SGLT2i drugs are likely to find many uses in inflammatory diseases and metabolic diseases. This paper provides support for an important mechanism by which they work in conditions of severe sepsis and hemodynamic compromise.
Thank you for these comments.
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eLife Assessment
SGLT2 inhibitors (SGLT2i) have assumed important roles in reducing cardiovascular risk, particularly in those with diabetes. It has become appreciated that its protective effects are likely beyond their ability to lower blood sugar levels. This research presents a novel approach to studying the SGLT2i mechanism of action which is yet to be fully elucidated.
-
Reviewer #1 (Public Review):
The authors examined the hypothesis that plasma ApoM, which carries sphingosine-1-phosphate (S1P) and activates vascular S1P receptors to inhibit vascular leakage, is modulated by SGLT2 inhibitors (SGLTi) during endotoxemia. They also propose that this mechanism is mediated by SGLTi regulation of LRP2/ megalin in the kidney and that this mechanism is critical for endotoxin-induced vascular leak and myocardial dysfunction. The hypothesis is novel and potentially exciting. However, the author's experiments lack critical controls, lack rigor in multiple aspects, and overall does not support the conclusions.
-
Reviewer #2 (Public Review):
Apolipoprotein M (ApoM) is a plasma carrier for the vascular protective lipid mediator sphingosine 1-phospate (S1P). The plasma levels of S1P and its chaperones ApoM and albumin rapidly decline in patients with severe sepsis, but the mechanisms for such reductions and their consequences for cardiovascular health remain elusive. In this study, Ripoll and colleagues demonstrate that the sodium-glucose co-transporter inhibitor dapagliflizin (Dapa) can preserve serum ApoM levels as well as cardiac function after LPS treatment of mice with diet-induced obesity. They further provide data to suggest that Dapa preserves serum ApoM by increasing megalin-mediated reabsorption of ApoM in renal proximal tubules and that ApoM improves vascular integrity in LPS treated mice. These observations put forward a potential …
Reviewer #2 (Public Review):
Apolipoprotein M (ApoM) is a plasma carrier for the vascular protective lipid mediator sphingosine 1-phospate (S1P). The plasma levels of S1P and its chaperones ApoM and albumin rapidly decline in patients with severe sepsis, but the mechanisms for such reductions and their consequences for cardiovascular health remain elusive. In this study, Ripoll and colleagues demonstrate that the sodium-glucose co-transporter inhibitor dapagliflizin (Dapa) can preserve serum ApoM levels as well as cardiac function after LPS treatment of mice with diet-induced obesity. They further provide data to suggest that Dapa preserves serum ApoM by increasing megalin-mediated reabsorption of ApoM in renal proximal tubules and that ApoM improves vascular integrity in LPS treated mice. These observations put forward a potential therapeutic approach to sustain vascular protective S1P signaling that could be relevant to other conditions of systemic inflammation where plasma levels of S1P decrease. However, although the authors are careful with their statements, the study falls short of directly implicating megalin in ApoM reabsorption and of ApoM/S1P depletion in LPS-induced cardiac dysfunction and the protective effects of Dapa.
The observations reported in this study are exciting and potentially of broad interest. The paper is well written and concise, and the statements made are mostly supported by the data presented. However, the mechanism proposed and implied is mostly based on circumstantial evidence, and the paper could be substantially improved by directly addressing the role of megalin in ApoM reabsorption and serum ApoM and S1P levels and the importance of ApoM for the preservation for cardiac function during endotoxemia. Some observations that are not necessarily in line with the model proposed should also be discussed.
The authors show that Dapa preserves serum ApoM and cardiac function in LPS-treated obese mice. However, the evidence they provide to suggest that ApoM may be implicated in the protective effect of Dapa on cardiac function is indirect. Direct evidence could be sought by addressing the effect of Dapa on cardiac function in LPS treated ApoM deficient and littermate control mice (with DIO if necessary).
The authors also suggest that higher ApoM levels in mice treated with Dapa and LPS reflect increased megalin-mediated ApoM reabsorption and that this preserves S1PR signaling. This could be addressed more directly by assessing the clearance of labelled ApoM, by addressing the impact of megalin inhibition or deficiency on ApoM clearance in this context, and by measuring S1P as well as ApoM in serum samples.
Methods: More details should be provided in the manuscript for how ApoM deficient and transgenic mice were generated, on sex and strain background, and on whether or not littermate controls were used. For intravital microscopy, more precision is needed on how vessel borders were outland and if this was done with or without regard for FITC-dextran. Please also specify the type of vessel chosen and considerations made with regard to blood flow and patency of the vessels analyzed. For statistical analyses, data from each mouse should be pooled before performing statistical comparisons. The criteria used for choice of test should be outlined as different statistical tests are used for similar datasets. For all data, please be consistent in the use of post-tests and in the presentation of comparisons. In other words, if the authors choose to only display test results for groups that are significantly different, this should be done in all cases. And if comparisons are made between all groups, this should be done in all cases for similar sets of data.
-
Reviewer #3 (Public Review):
The authors have performed well designed experiments that elucidate the protective role of Dapa in sepsis model of LPS. This model shows that Dapa works, in part, by increasing expression of the receptor LRP2 in the kidney, that maintains circulating ApoM levels. ApoM binds to S1P which then interacts with the S1P receptor stimulating cardiac function, epithelial and endothelial barrier function, thereby maintaining intravascular volume and cardiac output in the setting of severe inflammation. The authors used many experimental models, including transgenic mice, as well as several rigorous and reproducible techniques to measure the relevant parameters of cardiac, renal, vascular, and immune function. Furthermore, they employ a useful inhibitor of S1P function to show pharmacologically the essential role for …
Reviewer #3 (Public Review):
The authors have performed well designed experiments that elucidate the protective role of Dapa in sepsis model of LPS. This model shows that Dapa works, in part, by increasing expression of the receptor LRP2 in the kidney, that maintains circulating ApoM levels. ApoM binds to S1P which then interacts with the S1P receptor stimulating cardiac function, epithelial and endothelial barrier function, thereby maintaining intravascular volume and cardiac output in the setting of severe inflammation. The authors used many experimental models, including transgenic mice, as well as several rigorous and reproducible techniques to measure the relevant parameters of cardiac, renal, vascular, and immune function. Furthermore, they employ a useful inhibitor of S1P function to show pharmacologically the essential role for this agonist in most but not all the benefits of Dapa. A strength of the paper is the identification of the pathway responsible for the cardioprotective effects of SGLT2is that may yield additional therapeutic targets. There are some weaknesses in the paper, such as, studying only male mice, as well as providing a power analysis to justify the number of animals used throughout their experimentation. Overall, the paper should have a significant impact on the scientific community because the SGLT2i drugs are likely to find many uses in inflammatory diseases and metabolic diseases. This paper provides support for an important mechanism by which they work in conditions of severe sepsis and hemodynamic compromise.
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