Title: Dapagliflozin improves endothelial integrity and hemodynamics in endotoxin 1 treated mice through an apolipoprotein M dependent pathway

25 Rationale: Sodium-glucose co-transporter inhibitors (SGLT2i) are under active clinical 26 investigation in patients with acute inflammatory conditions, based on their clinical cardio- and 27 nephroprotective effects, and a pre-clinical study that demonstrated SGLT2i improve renal 28 outcomes and survival in a lipopolysaccharide (LPS) model. However, a unified mechanism that 29 explains how SGLT2i could prevent hemodynamic consequences of inflammatory conditions 30 has not been described. Apolipoprotein M (ApoM) is inversely associated with mortality in 31 inflammatory conditions and improves cardiac function in endotoxin-treated mice via 32 sphingosine-1-phosphate (S1P) signaling. 33 Objective: Test the hypothesis that pre-treatment with SGLT2i dapagliflozin (Dapa) improves 34 hemodynamics in endotoxin-treated mice via the ApoM/S1P pathway. 35 Methods and Results: Mice with diet-induced obesity were gavaged with vehicle or Dapa for 4 36 days prior to LPS (10 mg/kg, IP). We found that mice receiving Dapa restored circulating ApoM 37 levels, likely by increasing expression of the multi-ligand protein receptor megalin in the 38 proximal tubules. Dapa attenuated LPS-induced reductions in cardiac dysfunction including 39 reductions in ejection fraction, cardiac index, and coronary sinus area as well as vascular 40 permeability as ascertained by intravital microscopy. Using both ApoM transgenic and knockout 41 mice and S1P receptor inhibitors, we show that the ApoM/S1P pathway is important for the 42 beneficial effects of Dapa in the LPS model. 43 Conclusions: In the setting of acute inflammation, our data suggest that SGLT2i maintains 44 levels of megalin, leading to preservation of ApoM, which in turn promotes endothelial barrier 45 integrity and improves hemodynamics. Our studies suggest a novel mechanism by which 46 SGLT2i can preserve intravascular volume in the acute inflammatory setting. barrier integrity and intravascular volume. We hypothesized that Dapa attenuates 243 cardiac dysfunction in LPS-treated mice by reducing vascular inflammation and maintaining 244 endothelial barrier integrity, thus preserving intravascular volume. To test this, we investigated Study conception: CVR, ZG, JO, RE, MK, AJ. Study design: CVR, ZG, TK, KNM, AK, CW, JO, 339 RE, JS, MK, CC, JC, AJ. Data acquisition: CVR, ZG, TK, KNM, MO, AD, AG, LH, AK, CW, JN. Data interpretation: CVR, ZG, TK, KNM, MO, AD, AK, CW, JO, RE, MK, CC, JC, AJ.


Introduction 49
Sodium-glucose co-transporter inhibitors (SGLT2i) such as dapagliflozin (Dapa) have 50 well-described clinical benefits in diabetes, heart failure (HF) and chronic kidney disease (1-5). 51 Interestingly, a recent post-hoc analysis of the Dapagliflozin in Patients with Chronic Kidney 52 Disease trial demonstrated reduced infectious death in patients treated with SGLT2i (6), findings 53 that are concordant with preclinical studies suggesting that SGLT2i can improve survival and 54 renal function in a lipopolysaccharide (LPS) murine model (7). This recent post hoc analysis 55 suggests the hypothesis that SGLT2i may reduce infectious death, but as in HF and kidney 56 disease, the downstream mechanism by which SGLT2i leads to these improved outcomes 57 remains enigmatic, particularly with respect to the integrative physiology. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint

Intravital Microscopy 148
Ly6G-Cre LoxP-STOP-TdTomato mice on chow diet were treated with vehicle or 149 VPC23019 (0.75 mg/kg body weight (BW) IP daily) and gavaged with vehicle or dapagliflozin 150 (1.25 mg/kg) for 4 days before IP injection of LPS (7.5 mg/kg). Twenty-four hours after LPS 151 challenge, intravital microscopy was performed as we described (27). FITC-conjugated dextran 152 (1 mg/g) was infused into a jugular venous cannulus. Neutrophil recruitment and vascular leak 153 were monitored in an area of 0.02 mm 2 (number/field/5 minutes) in the inflamed cremaster 154 venules with a diameter of 25-40 μm. The number of neutrophils that visibly roll over the 155 inflamed endothelium over 5 minutes were counted by an observer blinded to the treatment 156 groups and unfamiliar with the study hypotheses. Adherent neutrophils were defined as 157 neutrophils that were stationary for more than 30 seconds or crawled on the inflamed 158 endothelium but did not roll over. Vascular leak was calculated by subtraction of the median 159 fluorescence intensities of FITC-conjugated dextran inside of blood vessels from those of FITC-160 conjugated dextran in the field of view. The median extravascular FITC intensity per unit time 161 (across the first 5 randomly selected vessels) and the per vessel area under the curve were 162 calculated. In independent experiments, WT littermate controls, Apom KO , Apom TG (along with 163 their respective littermates) mice on chow diet were gavaged with vehicle or dapagliflozin for 4 164 days prior to LPS injection. Vascular leak and neutrophil recruitment were monitored by injection 165 of FITC-conjugated dextran and Alexa Fluor 647-conjugated anti-Ly-6G antibodies (0.1 μg/g), 166 respectively. Fluorescence and bright-field images were recorded using a Zeiss Axio examiner 167 Z1 microscope system with a Yokogawa confocal spinning disk (CSU-W1) equipped with four 168 stack laser system (405 nm, 488 nm, 561 nm, and 637 nm wave lengths). Images were 169 collected with a high-speed, high-resolution camera (2304 x 2304 pixel format, ORCA-Fusion 170 BT sCMOS, Hamamatsu). Data were analyzed using SlideBook, version 6.0 (Intelligent Imaging 171 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint Innovations). Each curve is the median extravascular FITC-Dextran intensity across the 172 quantified vessels (n = 5 vessels per mouse, 3-5 mice per group). 173

Statistical analyses 174
Sample size was estimated based on literature review of murine SGLT2i studies (7, 28). 175 Data were considered non-normally distributed if either the Shapiro-Wilk or Kolmogorov-176 Smirnov tests were statistically significant (P < 0.05). Data are presented as mean ± SEM or 177 median ± 95% CI and analyzed by GraphPad Prism 9.0. Statistical significance among multiple 178 groups was analyzed by analysis of variance (ANOVA) and multiple testing corrections as 179 specified in each figure legend. In all of the analyses, a value of P < 0.05 was considered 180 statistically significant. 181

Study approval 182
All murine studies were approved by the Institutional Animal Care and Usage Committee To test our hypothesis that Dapa preserves circulating ApoM in acute inflammation, we 191 utilized a model of diet-induced obesity (DIO) generated by feeding mice 60% high-fat high-192 calorie diet from 6 to 16 weeks of age. We performed studies in DIO mice, because it is a well- was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint mg/kg daily), prior to saline or LPS injection (10 mg/kg IP) on Day 4, followed by in vivo 196 phenotyping (see below) and euthanasia on Day 5. LPS administration induced hypothermia, 197 weight loss, and transient hypoglycemia, none of which was influenced by Dapa 198 (Supplementary Figure 1). 199 In DIO mice, Dapa pre-treatment significantly attenuated LPS-induced decreases in 200 circulating ApoM (Figure 1A, B). In contrast, Dapa did not have a significant effect on serum 201 albumin concentrations ( Figure 1C). We thus investigated how Dapa might attenuate LPS-202 induced reductions in ApoM. The liver, and to a lesser extent the kidney, are the main sites of 203 ApoM production (18). We found that while LPS reduces both hepatic ApoM protein abundance 204 and renal ApoM protein and mRNA abundance, none of these were affected by Dapa ( Figure  205 1D-I). Since we did not find considerable change in ApoM mRNA and protein abundance in 206 either the liver or the kidney, we sought to examine other potential mechanisms by which Dapa 207 might preserve circulating ApoM. 208 Hepatic ApoM clearance is dependent on low-density lipoprotein receptor (LDLR) uptake 209 (33); however, LPS suppressed LDLR equally in both vehicle and Dapa-treated mice ( Figure  210  antibody showed that LPS-treated mice that received Dapa had higher levels of Lrp2 219 immunofluorescence compared to controls (Figure 2C, D). These findings suggest that Dapa 220 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022.

Dapa attenuates vascular leak and neutrophil transendothelial migration 241
One mechanism that exacerbates cardiac loading and function in sepsis is loss of 242 endothelial barrier integrity and intravascular volume. We hypothesized that Dapa attenuates 243 cardiac dysfunction in LPS-treated mice by reducing vascular inflammation and maintaining 244 endothelial barrier integrity, thus preserving intravascular volume. To test this, we investigated 245 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. S1P signaling, we tested the combined effect of Dapa and S1PRi. We found that compared with 253 vehicle, S1PRi significantly increased vascular leak, and that treatment with S1PRi blocked the 254 effect of Dapa on FITC-Dextran extravasation ( Figure 4A, B and Supplementary Figure 3). 255 Pre-treatment with Dapa increased the number of rolling neutrophils, and decreased the number 256 of adherent neutrophils (Figure 4C, D); nonetheless, S1PRi blocked the effects of Dapa on 257 rolling neutrophils, but not adherent neutrophils, implying that Dapa in part affects neutrophil 258 recruitment in a S1PR-dependent manner. Importantly, we did not observe any consistent 259 effects of Dapa on S1P receptor expression (Supplementary Figure 4), or significant increases 260 in the number of circulating neutrophils (Supplementary Figure 5). These results indicate that 261 Dapa prevents vascular leak induced by LPS, an effect dependent on S1PR signaling. 262 Given that Dapa prevented reductions in circulating ApoM in LPS-treated DIO mice, we 263 sought to determine whether ApoM mediated the effects of Dapa on endothelial integrity and 264 neutrophils. To test this, we utilized both ApoM knockout mice (Apom KO ), which exhibit 50% 265 reductions in plasma S1P (9), and mice with hepatocyte-specific overexpression of human 266 APOM (Apom TG ), which results in 3-5 fold increase in plasma ApoM and S1P (14). While 267 Apom KO mice exhibited increased vascular leak compared to littermate controls following LPS, 268 Dapa did not significantly reduce FITC-Dextran extravasation in Apom KO mice (Figure 4E and 269 Supplementary Figure 6). Compared to control mice, Apom KO mice did not affect neutrophil 270 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint rolling and adhesion on the inflamed endothelium. Dapa pretreatment did not affect neutrophil 271 rolling and adhesion in Apom KO mice (Figure 4F, G). In contrast, IVM demonstrated that 272 Apom TG mice exhibit reduced vascular leak (Figure 4H and Supplementary Figure 7), fewer 273 rolling neutrophils (Figure 4I), and more adherent neutrophils (Figure 4J), but Dapa 274 pretreatment did not significantly alter any of these parameters in Apom TG mice. These results 275 suggest that Dapa reduces LPS-induced endothelial leak via Apom-S1P signaling, but that the 276 effect of Dapa on neutrophil recruitment may only be partially dependent on ApoM/S1P. suggest that SGLT2i acutely reduce excess extravascular volume (41). We identify a 290 mechanism by which SGLT2 inhibition in the proximal tubule preserves levels of ApoM, via 291 increased re-uptake, contributing to preserved endothelial barrier integrity, therefore providing a 292 potential explanation for how SGLT2i could acutely reduce excess extravascular volume 293 compared to other diuretics. Finally, our results are of potential translational relevance given two 294 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made where ApoM is also inversely associated with clinical severity (20, 21). 296 One of the main findings of our study is that SGLT2i preserved cardiac preload, as 297 evidenced by preserved end-diastolic volume index, coronary sinus area, and an increased right  Our work must be considered in the context of its limitations. First, we utilize an LPS 318 model in both high-fat fed and chow-fed animals. We utilize lean animals in our IVM 319 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022.  ApoM mRNA abundance in (I) (n = 3-7). All data are presented as mean ± SEM. *P < 0.05, **P 504 < 0.01, ***P < 0.001, ****P < 0.0001. Each dot represents one mouse. One-way ANOVA with 505 Sidak's correction for multiple comparisons in panels (B), (C), (E), (F), (H) and (I). 506 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  prior to LPS (10 mg/kg IP). A) Representative Western blot for LDL receptor and quantification 511 in (B) (n = 4). C) Immunofluorescence staining for megalin/Lrp2 (Green) and DAPI in kidney 512 frozen sections with quantification of mean fluorescence intensity in (D) (n = 5-11). Scale bar = 513 50 μm. E) Representative fluorescence images were obtained from HK2 cells which were grown 514 on 8-chamber slides for 24 hours, thereafter, exposed to Dapaliflozin (6 nM) or vehicle for 16 515 hours, followed by addition of LPS (0.1 µg/ml) or saline as well as ApoM-GFP (2100 arb units) 516 for further 6 hours. Scale bar = 20 μm. F) Quantification of mean fluorescence intensity in (E) (n 517 = 6-8). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 518 0.0001. Each dot represents one mouse or one biological replicate (individual wells). One-way 519 ANOVA with Sidak's correction for multiple comparisons in panels (B), (D) and (F). 520 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  treated with the S1P receptor 1 and 3 inhibitor (S1PRi, 0.75 mg/kg IP daily), simultaneously 531 gavaged with dapagliflozin or vehicle for 4 days before saline or LPS (10 mg/kg IP) followed the 532 next day by echocardiography and quantitative assessment of ejection fraction, cardiac index, 533 and coronary sinus area, respectively (n = 4-6). All data are presented as mean ± SEM or 534 median ± 95% CI. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot represents one 535 mouse. One-way ANOVA with Sidak's correction for multiple comparisons in panels (B-E) and 536 (I-K), Kruskal-Wallis test with Dunn's correction for multiple comparisons in panels (F) and (H). 537 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint for assessment of extravascular FITC-Dextran and rolling and adherent neutrophils. All 553 experiments quantified vessels (> 4 per mouse, n = 3-5 mice per group. Each dot represents 554 one vessel. All data are presented as mean ± SEM or median ± 95% CI. *P < 0.05, **P < 0.01, 555 ***P < 0.001, ****P < 0.0001. One way ANOVA with Sidak's correction for multiple comparisons 556 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted April 28, 2022. ; https://doi.org/10.1101/2022.04.27.489709 doi: bioRxiv preprint