Journal: Journal of the American Society of Nephrology

Article Title: Empagliflozin Inhibits Proximal Tubule NHE3 Activity, Preserves GFR, and Restores Euvolemia in Nondiabetic Rats with Induced Heart Failure

doi: 10.1681/asn.2020071029

Figure Lengend Snippet: Figure 3. SGLT2 is overexpressed in the PT of nondiabetic HF rats. (A) Representative immunohistochemical staining of SGLT2 in the PT of sham and HF rats treated with empagliflozin (EMPA) or untreated. Scale bar: 50 mm. (B) Representative immunoblots from SDS-PAGE of renal cortical proteins isolated from sham and HF rats treated with EMPA or untreated and probed with antibodies against SGLT2 and b-actin. (C) Graphic representation of the relative levels of SGLT2 protein abundance in the renal cortex of the four groups of rats. (D) Graphic representation of the relative mRNA expression of SGLT2 in the renal cortex of sham and HF rats treated with EMPA or untreated. The levels of SGLT2 mRNA were measured using quantitative PCR, and GAPDH (glyceraldehyde 3-phosphate dehydroge- nase) was used as an internal control. The values represent individual measurements and the means 6 SEM (Supplemental Material). *P50.01 versus sham; **P50.006 versus sham; ***P,0.001 versus sham; †††P,0.001 versus sham 1 EMPA; ##P50.008 versus HF; ###P,0.001 versus HF.

Article Snippet: The separated proteins were transferred from the gel to a polyvinylidene difluoride membrane (Immobilon-P; Merck Millipore, Bedford, MA) at 350 mA for 8–10 hours at 4 C with a TE 62 Transfer Cooled Unit (GE HealthCare, Piscataway, NJ) and stained with Ponceau S. Then, the polyvinylidene difluoride membranes were incubated with blocking solution (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 hour and overnight (4 C) with specific primary antibodies: a polyclonal antibody against SGLT2 (1:1000, NBP1–92384; Novus Biologicals, Centennial, CO); a mousemAb againstNHE3, clone 3H3 (1:1000; PeterAronson, Yale University, New Haven, CT)22; a phosphospecific mAb against NHE3, PS552-NHE3, clone 14D522 (1:1000; Santa Cruz Biotech, Dallas, TX); or a polyclonal antibody against b-actin (1:5000, ab8227; Abcam).

Techniques: Immunohistochemical staining, Staining, Western Blot, SDS Page, Isolation, Quantitative Proteomics, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: Redefining the Role of ADAM17 in Renal Proximal Tubular Cells and Its Implications in an Obese Mouse Model of Pre-Diabetes

doi: 10.3390/ijms222313093

Figure Lengend Snippet: Influence of HFD and proximal tubular Adam17 deletion on SGLT2 protein expression. Quantification of the SGLT2 immunostaining and representative images depicting SGLT2 staining from all experimental groups. 200× magnification, scale bar 50 μm. Data are expressed as mean ± SD. Abbreviations: SD, standard diet; HFD, high-fat diet; Adam17WT , wild type; Adam17KO , knockout. * p < 0.05 HFD vs. SD, $ p < 0.05 KO vs. WT.

Article Snippet: Endogenous peroxidase was blocked by 3% H 2 O 2 in TBS1X incubation for 15 min. Non-specific interactions blocking was done by 1% BSA and 3% goat serum for 1 h. Then, sections were incubated with anti-WT-1 antibody (1:1000; sc192, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Galectin-3 antibody (1:1000; 126701, Biolegend, San Diego, CA, USA), anti-F4/80 antibody (1:500; 400501, Biolegend, San Diego, CA, USA), or anti-SGLT2 antibody (1:200; 24654-1-AP, Proteintech, Manchester, UK) at 4 °C overnight.

Techniques: Expressing, Immunostaining, Staining, Knock-Out

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Overview of study design and phenotypes. A , Overall discovery strategy. Wild-type (WT) and hyperglycemic type 1 diabetic Akita mice were put on a high-fat, high-carbohydrate Western diet for 4 weeks, followed by SGLT2i (sodium-glucose cotransporter 2 inhibitor), dapagliflozin (10 mg/kg of Western diet), or vehicle (repelleted Western diet) for 1 week. B , Organs and body fluids were snap-frozen and subjected to multi-omics analysis using mass spectrometry. SGLT2 interactome was determined from kidney. C , Main findings. D , Overview of functional follow-up studies and key findings. GDF15 indicates growth/differentiation factor 15; RBC, red blood cell; and WAT, white adipose tissue.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Western Blot, Biomarker Discovery, Mass Spectrometry, Functional Assay

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Integrated proteome/interactome analysis indicates broad remodeling of kidney metabolite transport by SGLT2i. A , Volcano plot quantification of proteomics analysis of kidney cortex. Overlapping significant proteins (false discovery rate <0.05) between wild-type (WT) and Akita mice upon SGLT2i (sodium-glucose cotransporter 2 inhibitor) treatment are labeled with their gene name. B , Enrichment of Gene Ontology Biological Process (GOBP) terms in WT kidney cortex (kidney cortex proteome was used as background). C , Nephron localization of altered transmembrane transporters of the solute carrier group (SLC). D , Mass spectrometry–based interactome analysis of mouse SGLT2i. SGLT2 interacts with membrane transporters that mediate the reabsorption or secretion of sodium, amino acids, urate, protons, anions, and multiple other compounds. E , Human tissue atlas of selected putative human SGLT2 interactors (source: The Human Proteome Atlas; https://www.proteinatlas.org ).

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Labeling, Mass Spectrometry, Membrane

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Untargeted metabolome analysis reveals SGLT2i-induced alterations in amino acid and organic anion metabolism. A , Overview of untargeted metabolomics results in wild-type (WT) and diabetic Akita mice. Significant metabolites from >30 000 quantified features with breakdown of altered metabolites by organ and genotype. B , The metabolite pool is sensitive to changes in production as well as kidney secretion and reabsorption. C , Illustration of interorgan communication by linking solute carrier group (SLCs) downregulated in the kidney to enhanced urinary metabolites and depleted metabolites in other organs. D , SGLT2 (sodium-glucose cotransporter 2)–dependent changes in plasma:urine ratios indicate increased urinary excretion of glucose and glucose metabolites, and less secretion of substrates of the kidney organic anion transporters (see text for details). Urine metabolites were normalized to creatinine. E , Immunoblot analysis of OAT1 (organic anion transporter 1) in SGLT2i-treated WT mice shows reduction of OAT1 (Slc22a6; normalized to b-actin; 2-sided t test), consistent with proteomics results. F , Targeted analysis of microbiota-derived organic anions (uremic toxins) in the plasma of WT and diabetic Akita mice. RBC indicates red blood cell.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Clinical Proteomics, Western Blot, Derivative Assay

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Metaproteomics analysis demonstrates reshaping and attenuation of the microbiome fermentation of amino acids to uremic toxins by SGLT2i. A , Taxonomic analysis of metaproteome of gut microbiota with SGLT2i (sodium-glucose cotransporter 2 inhibitor) on family level. B , Cumulative histogram analysis of cresol- and phenol-producing bacteria species on metaproteome level, as well as tryptophan-metabolizing bacteria. C , Murine fecal metabolomics results of key metabolites. D , Targeted metabolomics of human fecal microbiota fermentation (48 hours) in the presence or absence of SGLT2i dapagliflozin (100 nM). Metabolites with >0.5-fold change (48 hours fermentation versus control) and P <0.05 between both conditions are labeled with an open circle (3 donors, with 3 fermentations each). IAA indicates indoleacrylic acid; and IL, indolelactate.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Metaproteomics, Bacteria, Control, Labeling

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Using Sglt2 KO mice to probe for metabolic off-target effects of SGLT2i. A , Study design. B , Quantification of glucosuria. C , Quantification of plasma:urine ratios in dapagliflozin-treated mice and Sglt2 knockout (KO) mice as compared with untreated wild-type (WT) mice. D , Rank of changes in plasma:urine ratios with SGLT2i (sodium-glucose cotransporter 2 inhibitor; all P <0.05). E , Analysis of off-target effects on mouse plasma, defined as significant changes in the presence of SGLT2i in Sglt2 KO mice. F , Quantification of p-cresol sulfate abundance in response to SGLT2i in both WT and Sglt2 KO mice. B and F , 2-way ANOVA to probe for a significant effect of Sglt2 genotype ( Sglt2 ), dapagliflozin (dapa), or the interaction between the 2 factors ( P interaction ). If the interaction was statistically significant, then a pairwise multiple comparison procedure (Holm-Sidak method) identified the significant effects. * P <0.05 versus vehicle; # P <0.05 versus WT.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Clinical Proteomics, Knock-Out, Comparison

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: SGLT2i-induced reduction in uremic toxins translates to humans. A and D , Design of decompensated heart failure (HF) cohort and randomized controlled trial in patients with diabetes. B and C , Decompensated HF: heatmaps of SGLT2i (sodium-glucose cotransporter 2 inhibitor)–induced log2 fold changes for comparisons at discharge ( B ) and between admission and discharge ( C ). D , Randomized control trial of patients with diabetes: heatmaps of SGLT2i-induced log2 fold changes for SGLT2i versus placebo ( E ) and treatment versus baseline ( F ). SGLT2i treatment reduces uremic toxins originating from aromatic amino acid fermentation.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Control

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Effects of SGLT2i-modulated uremic toxin, p-cresol, on human engineered heart tissue. A , Summary of metabolite changes in response to SGLT2i (sodium-glucose cotransporter 2 inhibitor) in mouse, rat, and human. B , Human engineered heart tissue (EHT) was exposed to either DMSO (black) or 300 µM p-cresol (PCL; green) for 5 days. Bar charts show force (mN), and relaxation time from peak to 80% relaxation (RT 80%; seconds) in spontaneously contracting EHTs. C , Time course of spontaneous force 1 day before exposure to 3 mM PCL (−1; green) or DMSO (black) until 5 days of daily treatment. After 3 days of treatment, PCL was replaced by DMSO (green arrows) for another 48 hours’ incubation (n=16–22 per group, derived from 4 [300 µM] and 5 [3 mM] independent rounds of cardiac differentiation unpaired, parametric T test [** = P<0.01 and **** = P<0.0001]). D and E , Proteomic effects of incubation of EHT with p-cresol (300 µM) or other SGLT2i-modulated metabolites for 5 days. F , GDF15 (growth/differentiation factor 15) in patients with heart failure (HF) with new SGLT2i treatment (patients from Figure 6A). FDR indicates false discovery rate; HTN, hypertension; and WT, wild type.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Incubation, Derivative Assay

Journal: Circulation

Article Title: Metabolic Communication by SGLT2 Inhibition

doi: 10.1161/CIRCULATIONAHA.123.065517

Figure Lengend Snippet: Integrated views on the reconfiguration by SGLT2i of the early proximal tubule and systemic metabolic communication. A , Summary of findings and metabolic communication of SGLT2i (sodium-glucose cotransporter 2 inhibitor) in an early proximal tubule centric view in wild-type (WT) mice. Proteins with red lining were upregulated and proteins with blue lining were downregulated by SGLT2i. Proteins showing green filling have been shown to interact with SGLT2 in affinity purification analysis (see also Figure 2D). For further discussion, see text and Figure S8 . B , Summary of findings and metabolic communication of SGLT2i on a systemic level in WT mice. C , Chemical structures of SGLT2 inhibitors dapagliflozin and empagliflozin and p-cresol. ACAT3 indicates acetyl-coenzyme A acetyltransferase 3; AGE, advanced glycation end products; AMPK, 5′-adenosine monophosphate–activated protein kinase; CKD, chronic kidney disease; ER, endoplasmic reticulum; GLUT2, glucose transporter 2; HGD, homogentisate 1,2-dioxygenase; NHE3, Na + /H + exchanger 3; OAT1, organic anion transporter 1; PDZK1IP1, PDZK1 interacting protein 1; SLC, solute carrier; TMEM27, transmembrane protein 27; and WAT, white adipose tissue.

Article Snippet: The antibodies used were rabbit anti-murine SGLT2 (20802; BiCell), specificity for SGLT2 previously confirmed using Sglt2 KO mice, rabbit anti-human SGLT2 (142195; Cell Signaling; amino terminus targeted), and, for isotype control, rabbit immunoglobulin G (Sigma).

Techniques: Affinity Purification

Journal: International Journal of Molecular Sciences

Article Title: Ageing-Related Alterations in Renal Epithelial Glucose Transport

doi: 10.3390/ijms242216455

Figure Lengend Snippet: Protein expression of klotho, SGLT1, SGLT2, GLUT1 and GLUT2 in renal tissue of control, 6-month, 6-month with GYY4137 treatment (G6M), 12-month and 12-month with GYY4137 treatment (G12M) groups. * p < 0.05 vs. 3-month group, ## p < 0.05: 12-month group vs. G12-month group.

Article Snippet: After complete blocking with 5% skim milk, specific antibodies for molecules, such as SGLT1/2, GLUT1/2, and klotho, were applied (SGLT1/2: 1:1000, ABclonal, MA, USA; SGLT2: 1:1000, Santa Cruz Biotechnology, CA, USA; GLUT1: 1:25,000, Abcam, Cambridge, UK; GLUT2: 1:1000, Proteintech, IL, USA; klotho: 1:1000, Proteintech, IL, USA).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Ageing-Related Alterations in Renal Epithelial Glucose Transport

doi: 10.3390/ijms242216455

Figure Lengend Snippet: Protein expression of klotho, SGLT1, SGLT2, GLUT1 and GLUT2 in renal tissue of control, 6-month, 6-month with GYY4137 treatment (G6M), 12-month and 12-month with GYY4137 treatment (G12M) groups. * p < 0.05 vs. 3-month group, ## p < 0.05: 12-month group vs. G12-month group.

Article Snippet: After complete blocking with 5% skim milk, specific antibodies for molecules, such as SGLT1/2, GLUT1/2, and klotho, were applied (SGLT1/2: 1:1000, ABclonal, MA, USA; SGLT2: 1:1000, Santa Cruz Biotechnology, CA, USA; GLUT1: 1:25,000, Abcam, Cambridge, UK; GLUT2: 1:1000, Proteintech, IL, USA; klotho: 1:1000, Proteintech, IL, USA).

Techniques: Expressing, Control