Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome

  1. Mengyuan Ge
  2. Judith Molina
  3. Jin-Ju Kim
  4. Shamroop K Mallela
  5. Anis Ahmad
  6. Javier Varona Santos
  7. Hassan Al-Ali
  8. Alla Mitrofanova
  9. Kumar Sharma
  10. Flavia Fontanesi
  11. Sandra Merscher
  12. Alessia Fornoni  Is a corresponding author
  1. Katz Family Division of Nephrology and Hypertension, Department of Medicine, University of Miami Miller School of Medicine, United States
  2. Peggy and Harold Katz Family Drug Discovery Center, University of Miami Miller School of Medicine, United States
  3. Department of Radiation Oncology, University of Miami Miller School of Medicine, United States
  4. Center for Precision Medicine, School of Medicine, University of Texas Health San Antonio, United States
  5. Department of Biochemistry and Molecular Biology, University of Miami, United States
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Sodium-glucose cotransporter-2 (SGLT2) protein is expressed in the human kidney cortex and in cultured human and mouse podocytes.

(A) Immunohistochemistry staining of human kidney cortex for SGLT2 (left panel, scale bar: 50 μm; right panel, scale bar: 25 μm). (B) Western blot images demonstrating SGLT2 expression in cultured human podocytes (hPodo). Mouse liver lysate (mLiver), HepG2 liver cancer cells, and kidney lysate from Sglt2-/- mouse (Sglt2-/- mKidney) were used as the negative controls. HK2 proximal tubular cells were used as the positive control. (C,D) Western blot images (C) and quantification (D) demonstrating SGLT2 expression in mouse proximal tubular cells (Tubu) and podocytes (Podo) established from wild-type (WT) and Alport (AS) mice (n=3). (E) Sglt2 mRNA expression in WT and AS podocytes and tubular cells (n=3). (F) Representative confocal images of kidney cortices of AS mice (scale bars: 25 μm) stained with DAPI (blue), Synaptopodin (SYNPO, green) and SGLT2 (red). Yellow represents the co-localization of SYNPO and SGLT2. (D), (E), Two-tailed Student’s t-test. *p<0.5.

Figure 1—figure supplement 1
Podocyte-specific marker Synaptopodin (SYNPO) and tubule-specific marker Aquaporin 1 (AQP1) were confirmed in podocyte and tubular cell lines, respectively.
Figure 1—figure supplement 2
Single-cell transcriptomics indicates the expression of Sglt2 in podocyte.

Pod: podocyte; MC: mesangial cell; EC: endothelial cell; PT: proximal tubule; LH(AL/DL): loop of Henle ascending loop/descending loop; DCT: distal convoluted tubule; CNT: connecting tubule; CD-PC: collecting duct-principal cell; IC-A/B: intercalated cell type A/B; MΦ: macrophage.

Figure 2 with 1 supplement
Treatment of Alport syndrome (AS) podocytes with empagliflozin reduces lipid droplet (LD) accumulation and apoptosis.

(A–D) Immortalized podocytes and tubular cells of wild-type (WT) and AS mice treated with empagliflozin (Empa) or vehicle for 48 hr. (A,C) Bar graph analysis showing cytotoxicity normalized to viability, then compared to WT (n=3). (B,D) Bar graph analysis showing apoptosis normalized to viability, then compared to WT (n=3). (E,F) LD accumulation in tubular cells (E) and podocytes (F) was measured by Nile red staining. Bar graph analysis showing the quantification of the number of LDs per cell (n=3). (G) Correlation analyses between the LD accumulation and apoptosis in podocytes (n=12). (H) Representative images of Nile red staining demonstrate increased LD numbers (Nile red: green) in AS podocytes (Cell mask blue: blue; DAPI: red) compared to WT podocytes, which is reduced by Empa treatment. (A–F), Two-tailed Student’s t-test. (G), Pearson’s correlation coefficient. *p<0.5, **p<0.01.

Figure 2—figure supplement 1
Bar graph analysis of glucose content in Alport syndrome (AS) podocyte with or without empagliflozin (empa) treatment (n=3).
Figure 3 with 1 supplement
Empagliflozin inhibits the utilization of pyruvate as a metabolic substrate in Alport syndrome (AS) podocytes.

(A, B) Bar graph analysis of endogenous and substrate-driven oxygen consumption rates in wild-type (WT) and Alport (AS) tubular cells (A) and podocytes (B) treated with or without empagliflozin (E) (n=3). The sequential addition of permeabilizing agent and substrates was labeled in the figure. (C) Pyruvate dehydrogenase (PDH) activity was measured by a colorimetric assay in protein extracts from AS podocytes, normalized to protein concentration (n=3). Two-tailed Student’s t-test, *p<0.5. FA: octanoylcarnitine; ML: malate-low concentration; MH: malate-high concentration; P: pyruvate; G: glutamate.

Figure 3—figure supplement 1
Empagliflozin inhibits NADH-linked oxygen consumption rate in Alport syndrome (AS) podocytes.

(A, B) Bar graph analysis of endogenous and substrate-driven oxygen consumption rates in wild-type (WT) and Alport (AS) tubular cells (A) and podocytes (B) treated with or without empagliflozin (E) (n=3). The sequential addition of permeabilizing agent and substrates was labeled in the figure. (C) Bar graph analysis of the relative rate of extracellular acidification in AS podocyte after empagliflozin treatment (n=3). Two-tailed Student’s t-test, *p<0.5, ***p< 0.001. MH: malate-high concentration; P: pyruvate; G: glutamate.

Sglt2 knockdown reduces lipotoxicity in Alport syndrome (AS) podocytes.

(A, B) Western blot images (A) and quantification (B) of Sodium-glucose cotransporter-2 (SGLT2) protein in AS podocytes transfected with Sglt2 siRNA (siSglt2) or nontargeting siRNA (siCtrl) for 72 hr. GAPDH was used as a sample loading control (n=3). (C,D) Bar graph analysis showing cytotoxicity (C) and apoptosis (D) normalized to viability (n=3) in siCtrl and siSGLT2 AS podocytes, with or without the treatment of empagliflozin (E), then compared with siCtrl. (E,F) Western blot images (E) and quantification (F) of CPT1A protein in siCtrl and siSglt2 AS podocytes, with or without the treatment of empagliflozin (n=3). (B), Two-tailed Student’s t-test, (C), (D), (F), One-Way ANOVA followed by Holm-Sidak’s multiple comparisons. *p < 0.05, ***p< 0.001.

Empa improves the survival of Alport syndrome (AS) mice.

(A) Survival curve (n=4–5) of AS mice fed empagliflozin-supplemented (E) chow versus placebo diet starting at 4 weeks of age, compared to age-matched wild-type (WT) control mice. (B) Glycemia levels of WT and AS mice fed placebo diet and AS mice fed empagliflozin chow (n=3–8). (B), AS vs AS +E: Two-tailed Student’s t-test.

Empagliflozin improves renal function in a mouse model of Alport syndrome.

(A) Urinary albumin-to-creatinine ratio (ACR) in WT and Alport syndrome (AS) mice fed with placebo, empagliflozin (E), ramipril (R), or the combination of empagliflozin and ramipril (E+R). Urines were collected at the time of sacrifice (n = 7–8). (B) Bar graph analysis of body weights of mice from all experimental groups. (C,D) Bar graph analysis of blood urea nitrogen (BUN) (C) and creatinine (D) levels of mice from all experimental groups (n = 7–8). (E) Representative images of Periodic acid-Schiff (PAS) staining and bar graph analysis showing the mesangial expansion score of kidney cortices sections (scale bar: 50 μm; n = 7–8). (F) Representative Picrosirius red staining and bar graph analysis showing the quantification of fibrosis in kidney cortices sections (scale bar: 100 μm; n=7–8). (G) Representative images of kidney cortices stained with WT1 (green) to detect podocytes and DAPI (blue) to reveal nuclei and bar graph quantification of the average number of WT1-positive podocytes per glomerulus (scale bar: 25 μm, n = 7–8). One-Way ANOVA followed by Holm-Sidak’s multiple comparisons. *p < 0.05, **Pp< 0.01, ***p< 0.001.

Empagliflozin prevents renal lipid accumulation in experimental Alport syndrome.

(A) Representative Oil Red-O (ORO) images of stained kidney cortices sections (scale bar: 20 μm) and bar graph quantification of the number of glomeruli with lipid droplets (LD) in ORO-stained slides (n = 7–8). (B–D) Bar graph analysis of triglyceride TG, (B), total cholesterol TC, (C), and cholesterol ester CE, (D) contents in kidney cortices. Values are normalized to protein concentrations (n = 6–8). (E–G) Correlation analyses between the CE content of kidney cortices and albumin-to-creatinine ratio (ACR), blood urea nitrogen (BUN), or creatinine (n = 27, 31, 31). (H–J) Correlation analyses between the TG content of kidney cortices and ACR, BUN, or creatinine (n = 29, 29, 29). (A–D), One-Way ANOVA followed by Holm-Sidak’s multiple comparisons. (E–J), Pearson’s correlation coefficient. *p< 0.05, **p< 0.01, ***p< 0.001.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (M. musculus)immortalized podocytesThis paper;
PMID:33340991
Cell line established and maintained in Fornoni lab
Cell line (M. musculus)immortalized tubular cellsThis paperCell line established and maintained in Fornoni lab
Genetic reagent (M. musculus)CBA/CaxC57BL/10-H-2Kb-tsA58Charles River;
(PMID:1711218)
Genetic reagent (M. musculus)129-Col4a3tm1Dec/JJackson LaboratoryStrain# 002908
RRID:IMSR_JAX:002908
Antibodyanti-WT1 (rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-192
RRID:AB_632611
1:300 (IF)
Antibodyanti-SGLT2 (mouse monoclonal)Santa Cruz BiotechnologyCat# sc-21537
RRID:AB_2814658
1:100 (IHC)
1:500 (WB)
Antibodyanti-SGLT2
(rabbit polyclonal)
BiCell scientificCat# 20802
RRID:AB_2935905
1:100 (IF)
Antibodyanti-SYNAPTOPODIN (goat polyclonal)Santa Cruz BiotechnologyCat# sc-21537
RRID:AB_2201166
1:300 (IF)
1:1,000 (WB)
Antibodyanti-AQP1 (rabbit polyclonal)ProteintechCat# 20333–1-AP
RRID:AB_10666159
1:2,000 (WB)
Antibodyanti-CPT1A
(mouse monoclonal)
AbcamCat# ab128568
RRID:AB_11141632
1:1,000 (WB)
Antibodyanti-GAPDH
(mouse monoclonal)
Sigma-AldrichCat# CB1001
RRID:AB_2107426
1:10,000 (WB)
Sequence-based reagentSglt2_FThis paperPCR primersATGGAGCAACACGTAGAGGC
Sequence-based reagentSglt2_RThis paperPCR primersATGACCAGCAGGAAATAGGCA
Sequence-based reagentGapdh_FThis paperPCR primersCCTGGAGAAACCTGCCAAGTATG
Sequence-based reagentGapdh_RThis paperPCR primersGGTCCTCAGTGTAGCCCAAGATG
Sequence-based reagentsiRNA: Sglt2Santa Cruz BiotechnologyCat# sc-6154020 nM
Sequence-based reagentsiRNA: nontargetin controlThermo ScientificCat# 439084320 nM
Commercial kitApoTox-Glo Triplex assayPromegaCat# G6320
Commercial kitAmplex Red Cholesterol AssayThermo ScientificCat# A12216
Commercial kitTriglyceride Colorimetric AssayCaymanCat# 10010303
Chemical compound, drugEmpagliflozin (BI 10773)SelleckchemCat# S8022500 nM
Software, algorithmGraphpad PrismGraphpad softwareSCR_002798

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Mengyuan Ge
  2. Judith Molina
  3. Jin-Ju Kim
  4. Shamroop K Mallela
  5. Anis Ahmad
  6. Javier Varona Santos
  7. Hassan Al-Ali
  8. Alla Mitrofanova
  9. Kumar Sharma
  10. Flavia Fontanesi
  11. Sandra Merscher
  12. Alessia Fornoni
(2023)
Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome
eLife 12:e83353.
https://doi.org/10.7554/eLife.83353