Sodium-Glucose Cotransporter Inhibition Preserves Apolipoprotein M During Acute Inflammation in Mice and Humans

Abstract

Background: Sodium-glucose cotransporter inhibitors (SGLT2is) reduce inflammation and maintain vascular integrity. Apolipoprotein M (ApoM) is crucial for vascular integrity via sphingosine-1-phosphate (S1P) signaling and is inversely linked with mortality in sepsis and COVID-19.

Objectives: The authors tested if SGLT2i (dapagliflozin [Dapa]) increases ApoM in mice using lipopolysaccharide (LPS) and in humans with COVID-19.

Methods: Diet-induced obese mice (n = 14-15/group), proximal tubule-specific knockout of the multiligand protein receptor Lrp2 (Lrp2^KO) mice (n = 5-8/group), Ly6G-Cre LoxP-STOP-TdTomato mice (n = 3-5/group), Apom^KO mice (n = 3-5/group), and Apom^TG mice (n = 3-5/group) were randomized to receive either vehicle or Dapa (1.25 mg/kg daily) for 4 days before LPS (10 mg/kg IP). Outcomes included ApoM protein levels (Western and enzyme-linked immunosorbent assay) and intravital microscopy to assess endothelial leak and neutrophil behavior. Plasma samples from ACTIV-4a participants (standard of care, n = 37; standard of care + SGLT2i, n = 15) were analyzed for circulating ApoM by enzyme-linked immunosorbent assay. Statistical analyses included two-way analysis of variance for mice and t-test or Mann-Whitney test for humans.

Results: Dapa restored circulating ApoM levels in LPS-treated mice (0.017 vs 0.035 [a.u./μL], P = 0.0489) and increased ApoM levels in patients randomized to SGLT2i (0.5240 vs 0.6537 [μM], P = 0.0101). LRP2 knockout blocked Dapa's effect on ApoM. In vitro, Dapa stimulated Lrp2-dependent uptake of ApoM-GFP. Dapa attenuated vascular leak induced by LPS in an ApoM-dependent manner.

Conclusions: SGLT2i maintains Lrp2 levels, preserving ApoM and promoting endothelial barrier integrity in acute inflammation, indicating a novel protective mechanism of SGLT2i through ApoM preservation.

Keywords: LRP2; SGLT2; apolipoprotein M; endothelial vascular integrity.

Graphical abstract

Figure 1

Dapagliflozin Preserves Circulating Apolipoprotein M in LPS-Treated Mice (A) Diet-induced obesity mice were randomized to vehicle or Dapa (1.25 mg/kg gavage daily for 4 days) before saline or LPS (10 mg/kg IP) and euthanized the next day. (B) Representative images of immunoblots for murine ApoM from plasma with quantification in (C) (n = 14-15/group); (D) plasma ApoM level by ELISA (n = 6-10/group). (E) Representative fluorescence images were obtained from HK2 cells, which were grown on 8-chamber slides for 24 hours, thereafter, exposed to Dapa (6 nM) or vehicle for 16 hours, followed by addition of saline or LPS (0.1 μg/mL) as well as ApoM-GFP (24,000 arb units) for further 6 hours (scale bar = 20 μm). (F) Quantification of mean fluorescence intensity in (H) (n = 6-8/group). All data are presented as mean ± SD or median ± IQR. Each dot represents 1 mouse. Two-way ANOVA with Sidak's correction for multiple comparisons in panels (C, D, and F). ANOVA = analysis of variance; ApoM = apolipoprotein M; ApoM-GFP = green fluorescent protein labeled human ApoM; DAPI = 4’,6-diamidino-2-phenylindole; ELISA = enzyme-linked immunosorbent assay; LPS = lipopolysaccharide.

Figure 2

Dapagliflozin Preserves Circulating Apolipoprotein M in Human With COVID-19 (A and B) Circulating ApoM in patients randomized to SOC (n = 37) vs SGLT2i (n = 15) who had banked plasma available at day 0 and day 3. (C) Percentage change in ApoM on day 3 vs day 0 in patients randomized to standard of care vs SGLT2i. All data are presented as mean ± SD or median ± IQR. Each dot represents 1 person. Student's t-test for in panels (A and B), and Mann-Whitney test in C. ApoM = apolipoprotein M; SGLT2i = sodium-glucose co-transporter 2 inhibitor; SOC = standard of care.

Figure 3

Lipopolysaccharide Reduces Apolipoprotein M Protein and mRNA Abundance in Liver and Kidney Diet-induced obesity mice were randomized to vehicle or Dapa (1.25 mg/kg gavaged daily for 4 days) before saline or LPS (10 mg/kg IP) and euthanized the next day. (A) Representative images of immunoblots for ApoM from hepatic protein isolates with quantification in (B) and hepatic ApoM mRNA abundance in (C) (n = 3-7/group); (D) representative images of immunoblots for kidney ApoM with quantification in (E) and renal ApoM mRNA abundance in (F) (n = 3-7/group). (G) Representative images of immunoblots for hepatic LDLR and quantification in (H) (n = 4/group). β-Actin serves as loading control for (A, B, D, E, G, and H). All data are presented as mean ± SD or median ± IQR. Each dot represents 1 mouse. Two-way ANOVA with Sidak's correction for multiple comparisons in panels (B, C, E, F, and H). ANOVA = analysis of variance; ApoM = apolipoprotein M; LDLR = low-density lipoprotein receptor; LPS = lipopolysaccharide.

Figure 4

Proximal Tubule-Specific Knockout or Inhibition of Lrp2 Attenuates the Effects of Dapagliflozin on ApoM Maintenance (A) Representative immunofluorescence staining images for Lrp2 (green) and DAPI (blue) in kidney frozen sections from Lrp2^KO and littermate controls (scale bar = 50 μm). (B) Representative immunofluorescence staining images for Lrp2 (green) and DAPI (blue) in kidney frozen sections from Lrp2^KO and littermate controls randomized to vehicle or Dapa treatment (1.25 mg/kg daily for 4 days) and treated with LPS (10 mg/kg IP), with (C) quantification of LRP2 MFI, shown as fold change of Dapa to vehicle (n = 6-7/group; scale bar = 50 μm). (D) Representative images of immunoblots for ApoM from paired plasma with quantification in (E) from Lrp2^KO and littermate control mice randomized to vehicle or Dapa (1.25 mg/kg gavage daily for 4 days) before saline or LPS (10 mg/kg IP) and euthanized the next day (n = 5-8/group). (F) MFI of ApoM-GFP in HK2 cells which were grown on 8-chamber slides for 24 hours and then treated with 1 μM LRP inhibitor for 1 hour prior to ApoM-GFP (24,000 arb units) addition (n = 3). (G) MFI of ApoM-GFP in above HK2 cells setting with additional treatment including exposure to Dapa (6 nM) or vehicle for 16 hours and followed by addition of LPS (0.1 μg/mL) or saline prior to ApoM-GFP addition (n = 3/group). All data are presented as mean ± SD or median ± IQR. Each dot represents 1 mouse or 1 biological replicate (individual wells). Student's t-test in panels (C and F). Two-way ANOVA with Sidak's correction for multiple comparisons in panels (E and G). ANOVA = analysis of variance; ApoM = apolipoprotein M; DAPI = 4’,6-diamidino-2-phenylindole; LPS = lipopolysaccharide; LRP2, low-density lipoprotein receptor-related protein 2; MFI = mean fluorescence intensity.

Figure 5

Dapagliflozin Attenuates LPS-Induced Endothelial Leak in an S1P and ApoM-Dependent Manner Ly6G-Cre-TdTomato mice on chow diet were treated with control or S1PRi (0.75 mg/kg IP daily) and gavaged with vehicle or Dapa (1.25 mg/kg daily for 4 days) before LPS (7.5 mg/kg IP). Intravital microscopy was performed. (A) Representative images of cremaster venules, FITC-Dextran (green), neutrophils (pseudo color blue), white dotted lines indicate vessel border, and red arrows highlighting areas of extravascular FITC-Dextran (scale bar = 10 μm); (B) AUC per vessel of extravascular FITC-Dextran; (C) The numbers of rolling and (D) adherent neutrophils observed in 5 minutes; (E-G) Littermate controls (wild type) gavaged with vehicle for 4 days then treated with LPS compared to Apom^KO mice fed a chow diet and treated with vehicle or dapagliflozin gavage for 4 days prior to LPS injection. Intravital microscopy was performed to assess extravascular FITC-Dextran and rolling, and adherent neutrophils; (H-J) Littermate control nontransgenic mice (ntg) vs Apom^TG mice on chow diet were gavaged with vehicle or Dapa (1.25 mg/kg daily for 4 days) prior to LPS injection and intravital microscopy for assessment of extravascular FITC-Dextran and rolling and adherent neutrophils. All experiments quantified vessels >4 per mouse (n = 3-5 mice per group). All data are presented as min to max in box plot. One way ANOVA with Sidak's correction for multiple comparisons in panels (E-G); Two-way ANOVA with Sidak's correction for multiple comparisons in panels (B-D) and (H-J). ANOVA = analysis of variance; AUC = area under the curve; FITC = fluorescein isothiocyanate; LPS = lipopolysaccharide.

Central Illustration

Dapagliflozin Preserves Apolipoprotein M During Acute Inflammation in Mice and Humans In humans from the ACTIV-4a trial, randomization to sodium-glucose co-transporter inhibitors increased circulating ApoM levels. In mice, dapagliflozin pretreatment attenuated loss of the scavenger receptor LRP2 and ApoM, defending against lipopolysaccharide-induced disruption of endothelial barrier integrity. ApoM = apolipoprotein M; LRP2 = low-density lipoprotein receptor-related protein 2; SGLT2i = sodium-glucose co-transporter 2 inhibitor; SOC = standard of care.