Dapagliflozin Treatment Augments Bioactive Phosphatidylethanolamine Concentrations in Kidney Cortex Membrane Fractions of Hypertensive Diabetic db/db Mice and Alters the Density of Lipid Rafts in Mouse Proximal Tubule Cells

Abstract

In addition to inhibiting renal glucose reabsorption and allowing for glucose excretion, the sodium/glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin may be efficacious in treating various comorbidities associated with type 2 diabetes mellitus (T2DM). The molecular mechanisms by which dapagliflozin exerts its beneficial effects are largely unknown. We hypothesized dapagliflozin treatment in the diabetic kidney alters plasma membrane lipid composition, suppresses extracellular vesicle (EV) release from kidney cells, and disrupts lipid rafts in proximal tubule cells. In order to test this hypothesis, we treated diabetic db/db mice with dapagliflozin (N = 8) or vehicle (N = 8) and performed mass spectrometry-based lipidomics to investigate changes in the concentrations of membrane lipids in the kidney cortex. In addition, we isolated urinary EVs (uEVs) from urine samples collected during the active phase and the inactive phase of the mice and then probed for changes in membrane proteins enriched in the EVs. Multiple triacylglycerols (TAGs) were enriched in the kidney cortex membrane fractions of vehicle-treated diabetic db/db mice, while the levels of multiple phosphatidylethanolamines were significantly higher in similar mice treated with dapagliflozin. EV concentration and size were lesser in the urine samples collected during the inactive phase of dapagliflozin-treated diabetic mice. In cultured mouse proximal tubule cells treated with dapagliflozin, the lipid raft protein caveolin-1 shifted from less dense fractions to more dense sucrose density gradient fractions. Taken together, these results suggest dapagliflozin may regulate lipid-mediated signal transduction in the diabetic kidney.

Keywords: dapagliflozin; diabetes; kidney; lipid rafts; lipidomics.

Figure 1

Analysis of lipids in the kidneys of dapagliflozin- or vehicle-treated salt-loaded hypertensive diabetic db/db mice. (A) Volcano plot showing 34 lipids that are significantly down-regulated and 19 lipids that are significantly upregulated in the kidneys of diabetic db/db mice treated with dapagliflozin compared to vehicle. A 2.0-fold change threshold and a p-value threshold of 0.05 were applied. Data were normalized by the mean and then to the median in MetaboAnalyst with Pareto scaling. The points representing specific lipids on the left (value < 0) are expressed at a lower level, and those on the right (value > 0) are expressed at a higher level, in the dapagliflozin group compared to the vehicle group. (B) Heatmap with Euclidean distance measure, Ward clustering method showing the top 50 lipids, with each colored cell on the map corresponding to a concentration value in the data table.

Figure 1

Analysis of lipids in the kidneys of dapagliflozin- or vehicle-treated salt-loaded hypertensive diabetic db/db mice. (A) Volcano plot showing 34 lipids that are significantly down-regulated and 19 lipids that are significantly upregulated in the kidneys of diabetic db/db mice treated with dapagliflozin compared to vehicle. A 2.0-fold change threshold and a p-value threshold of 0.05 were applied. Data were normalized by the mean and then to the median in MetaboAnalyst with Pareto scaling. The points representing specific lipids on the left (value < 0) are expressed at a lower level, and those on the right (value > 0) are expressed at a higher level, in the dapagliflozin group compared to the vehicle group. (B) Heatmap with Euclidean distance measure, Ward clustering method showing the top 50 lipids, with each colored cell on the map corresponding to a concentration value in the data table.

Figure 2

Lipids significantly downregulated in the kidneys of the dapagliflozin-treated salt-loaded hypertensive diabetic db/db mice compared to the vehicle-treated db/db mice. (A) Plots of triacylglycerols (TAGs), (B) plots of phosphatidylethanolamines (PEs), (C) plots of phosphatidylglycerols (PGs), (D) plots of phosphatidylserines (PSs), and (E) plots of phosphatidylinositols (PIs) significantly different between the two groups. The concentration of each lipid is given in µmol/g. The concentration of each lipid was normalized to the median. The black (left bar) represents the dapagliflozin group, and the white (right bar) represents the vehicle group.

Figure 3

Lipids significantly upregulated in the kidneys of dapagliflozin-treated salt-loaded hypertensive diabetic db/db mice compared to the vehicle-treated db/db mice. (A) Plots of PEPs, PEOs, and PEs, (B) plots of lysophosphatidylethanolamines (LPEs), (C) plots of lysophosphatidylcholines (LPCs), (D) plots of phosphatidylinositols (PIs), and (E) plots of phosphatidylcholines (PCs). The concentration of each lipid is given in µmol/g. The concentration of each lipid was normalized to the median. The black (left bar) represents the dapagliflozin group, and the white (right bar) represents the vehicle group.

Figure 4

Characterization of uEVs isolated from the inactive and active phases of salt-loaded hypertensive diabetic db/db mice. (A) Representative nanoparticle tracking analysis plots of uEVs isolated from diabetic db/db mice treated with vehicle or dapagliflozin during the inactive or active phases. (B) Summary plots showing the comparison of uEV size between the two groups and two phases. ** represent a p-value of <0.01 (C) Summary plots showing the comparison of uEV concentration between the two groups and two phases. *** represent a p-value of <0.005 (D) Western blots of established uEV markers in each uEV sample from the two groups and phases. n = 4 uEV preparations from inactive-phase urine collections and the same number of uEV preparations from active-phase urine collections of n = 4 vehicle-treated db/db mice and n = 4 dapagliflozin-treated db/db mice. (E) Densitometric analysis of the immunoreactive bands in Panel D normalized to urinary albumin levels (uAlbumin).

Figure 5

Sucrose density gradient assays from mouse proximal tubule cells treated with dapagliflozin or vehicle. (A) Western blot analysis of lipid-raft- and non-lipid-raft-associated fractions from sucrose density gradient fractions of mouse proximal tubule cells treated with vehicle. (B) Western blot analysis of lipid-raft- and non-lipid-raft-associated fractions from sucrose density gradient fractions of mouse proximal tubule cells treated with dapagliflozin.

Figure 6

Schematic showing the effects of dapagliflozin treatment on membrane lipids in the kidneys of diabetic db/db mice. An increase in plasmalogen phosphatidylethanolamines in the form of PEPs and PEOs in the membranes of diabetic db/db kidneys after dapagliflozin treatment may alleviate the progression of diabetic kidney disease. ? represents putative effects of dapagliflozin treatment.