Empagliflozin protects the kidney by reducing toxic ALB (albumin) exposure and preventing autophagic stagnation in proximal tubules

Abstract

The renoprotective effects of SLC5A2/SGLT2 (solute carrier 5 (sodium/glucose cotransporter), member 2) inhibitors have recently been demonstrated in non-diabetic chronic kidney disease (CKD), even without overt albuminuria. However, the mechanism underlying this renoprotection is largely unclear. We investigated the renoprotective mechanisms of the SLC5A2 inhibitor empagliflozin with a focus on ALB (albumin) reabsorption and macroautophagy/autophagy in proximal tubules using wild-type or drug-inducible lrp2/Megalin or atg5 knockout mice with high-fat diet (HFD)-induced obesity or 5/6 nephrectomy that elevated intraglomerular pressure without overt albuminuria. Empagliflozin treatment of HFD-fed mice reduced several hallmarks of lipotoxicity in the proximal tubules, such as phospholipid accumulation in the lysosome, inflammation and fibrosis. Empagliflozin, which decreases intraglomerular pressure, not only reduced the HFD-induced increase in ALB reabsorption via LRP2 in the proximal tubules (i.e. total nephron ALB filtration), as assessed by urinary ALB excretion caused by genetic ablation of Lrp2, but also ameliorated the HFD-induced imbalance in circulating ALB-bound fatty acids. Empagliflozin alleviated the HFD-induced increase in autophagic demand and successfully prevented autophagic stagnation in the proximal tubules. Similarly, empagliflozin decreased ALB exposure and autophagic demand in 5/6 nephrectomized mice. Finally, empagliflozin reduced HFD-induced vulnerability to ischemia-reperfusion injury, whereas LRP2 blockade and atg5 ablation separately diminished this effect. Our findings indicate that empagliflozin reduces ALB exposure and prevents autophagic stagnation in the proximal tubules even without overt albuminuria. Autophagy improvement may be critical for the renoprotection mediated by SLC5A2 inhibition.

Keywords: Albuminuria; LRP2/Megalin; SLC5A2/SGLT2 inhibitors; autophagic stagnation; lipotoxicity; lysosome.

Figure 1.

Empagliflozin reduces an HFD-induced phospholipid accumulation in the PTEC lysosomes. (A and B) Images of PAS staining, LAMP1 immunostaining, toluidine blue staining, and nile red staining of the kidney cortical regions in ND-fed, HFD-fed, and empagliflozin (EMPA)-treated HFD-fed mice. Sections were coimmunostained for LRP2/MEGALIN, a marker of proximal tubules (blue) (A, middle) and counterstained with DAPI (blue) (B). (C and D) The vacuole scores from PAS staining images (C) and the relative sizes and number (per proximal tubule) of nile red-positive puncta (D) are shown. The data in (D) were counted in at least 10 high-power fields (×600) (each high-power field contains 10–15 proximal tubules). (E) Electron micrographs of kidneys from the HFD-fed and EMPA-treated HFD-fed mice showing lamellar structures in lysosomes (arrow) (n = 3 in each group). Bars: 50 μm (A), 20 μm (B), 5 μm (E). Data are provided as means ± SE. Statistically significant differences (*p < 0.01) are indicated. All images are representative of multiple experiments.

Figure 2.

Empagliflozin alleviates kidney injury in HFD-induced CKD mice. (A) Images of COL1A1 immunostaining, Masson’s trichrome staining, and ADGRE1/F4/80 immunostaining of kidney cortical regions in ND-fed, HFD-fed, and EMPA-treated HFD-fed mice (n = 6–7 in each group). COL1A1- or ADGRE1/F4/80-positive areas were quantified in at least 10 high-power fields (×200). (B) Plasma cystatin C levels of the mice in ND-fed, HFD-fed, and EMPA-treated HFD-fed mice. (C) Images of DT and 4HNE immunostaining of kidney cortical regions in ND-fed, HFD-fed, and EMPA-treated HFD-fed mice. (D) Western blots of DT and 4HNE in the kidneys of ND-fed, HFD-fed, and EMPA-treated HFD-fed mice. Bars: 100 μm (A, C). Data are provided as means ± SE. Statistically significant differences (*p < 0.01, #P < 0.05) are indicated. All images are representative of multiple experiments.

Figure 3.

Empagliflozin alleviates the HFD-induced increase in total nephron ALB filtration and tubular ALB reabsorption. (A) The evaluation of SNGFR of ND-fed, HFD-fed, and EMPA-treated HFD-fed wild-type mice (n = 8–12 in each group). (B) The daily urinary ALB excretion of ND-fed, HFD-fed, and EMPA-treated HFD-fed iLrp2KO mice before tamoxifen and after tamoxifen (n = 5–7 in each group). Total nephron ALB filtration is estimated by urinary ALB excretion in iLrp2KO mice given tamoxifen. (C) The estimated tubular ALB reabsorption. Subtraction of ALB excretion before tamoxifen from that after tamoxifen corresponds to estimated tubular ALB reabsorption (n = 6–7 in each group). Data are provided as means ± SE. Statistically significant differences (*P < 0.01, #P < 0.05, N.S. not significant) are indicated.

Figure 4.

Empagliflozin decreases proximal tubule exposure to albumin, especially in S3 segments. (A) Kidney cortical regions of mice injected with Alexa Fluor 555–conjugated BSA (red) were stained with LRP2/MEGALIN (green) and counterstained with DAPI (blue). (B) The number of dots positive for Alexa Fluor 555–conjugated BSA was counted in LRP2/MEGALIN-positive proximal tubules (n = 3 in each group). (C) Schematic illustration of LRP2/MEGALIN and SLC5A2/SGLT2 staining patterns in the S1–2 and S3 segments of the proximal tubules. (D) Kidney cortical regions of ND-fed, HFD-fed, and EMPA-treated HFD-fed mice injected with Alexa Fluor 555–conjugated BSA (red) were stained with LRP2/MEGALIN (white) and SLC5A2/SGLT2 (green), and counterstained with DAPI (blue). (E) The numbers of dots positive for Alexa Fluor 555–conjugated BSA dots were counted in SLC5A2/SGLT2-positive (S1–2 segments) and -negative (S3 segments) proximal tubules (n = 3 in each group). Bars: 50 μm (A, D). Data are provided as means ± SE. Statistically significant differences (*p < 0.01, # p< 0.05) are indicated. All images are representative of multiple experiments.

Figure 5.

Empagliflozin improves the metabolic status of HFD-fed mice. (A) Changes in body weight and blood glucose levels during the experimental period. (B) Images of HE staining and ADGRE1/F4/80 immunostaining of visceral white adipose tissue (n = 6 in each group). (C) The relative cell size and ADGRE1/F4/80-positive area were quantified in at least 5 high-power fields (×100). Bars: 100 μm (B). Data are provided as means ± SE. Statistically significant differences (*p < 0.01, #P < 0.05, N.S. not significant) are indicated. All images are representative of multiple experiments.

Figure 6.

Empagliflozin alleviates the HFD-induced increase in autophagic demand and prevents autophagic stagnation in the proximal tubules. (A) Kidney cortical regions of ND-fed, HFD-fed, and EMPA-treated HFD-fed (Atg5^F/F control and iAtg5KO) mice were immunostained for SQSTM1/p62 and ubiquitin after treatment with vehicle or tamoxifen 3 weeks before euthanasia (n = 4–5 in each group). Sections were coimmunostained for LRP2/MEGALIN, a marker of proximal tubules (blue). Magnified images from the tamoxifen-treated iAtg5KO mice are also presented. The number of SQSTM1/p62- or ubiquitin-positive dots was counted in at least 10 high-power fields (×400). (B) GFP-positive puncta formation was assessed in the proximal tubules of ND-fed, HFD-fed, and EMPA-treated HFD-fed GFP-MAP1LC3 transgenic mice that were either fed a ND or HFD diet or subjected to 24 h of starvation, with or without chloroquine (CQ) administration 6 h before euthanasia. Kidney sections were immunostained for LRP2/MEGALIN (red), and counterstained with DAPI (blue). The number of GFP-positive puncta per proximal tubule under each condition was counted in at least 10 high-power fields (original magnification, × 600) (each high-power field contained 10–15 proximal tubules). Bars: 10 μm (A, B). Data are provided as means ± SE. Statistically significant differences (*p < 0.01, N.S. not significant) are indicated. All images are representative of multiple experiments. CQ, chloroquine.

Figure 7.

Empagliflozin reduces HFD-induced vulnerability to IR injury in HFD-fed mice in an autophagy-dependent manner. (A, B) Images of PAS (A) and TUNEL (B) staining of kidney cortexes from ND-fed, HFD-fed, and EMPA-treated HFD-fed Atg5^F/F control and iAtg5KO mice 2 days after a unilateral IR injury (n = 4–7 in each group). (C) the PAS injury score is shown. (D)The number of TUNEL-positive PTECs was calculated in at least 10 high-power fields (×200). (E, F) The mRNA levels of Havcr1/Kim1 (E), and the representative images of immunostaining for HAVCR1/KIM1 (F) in ND-fed, HFD-fed, and EMPA-treated HFD-fed Atg5^F/F control and iAtg5KO mice 2 days after a unilateral IR injury. Bars: 100 μm (A, B, F). Data are provided as means ± SE. Statistically significant differences (*p < 0.01, #p < 0.05, N.S. not significant) are indicated. All images are representative of multiple experiments.

Figure 8.

Schematic illustration of this study. Increased glomerular pressure caused by HFD or 5/6 nephrectomy induces tubular reabsorption of toxic ALB via LRP2, leading to increased autophagic demand and stagnation of autophagy flux, which can increase vulnerability to IR injury. Empagliflozin reduces intraglomerular pressure and ameliorates metabolic dysfunctions, thereby improving the quantity and quality of filtered ALB and alleviating autophagic stagnation in PTECs. This prevention of autophagic stagnation can eventually improve the integrity of PTECs, thus leading to renoprotection.