SGLT2 inhibitor dapagliflozin limits podocyte damage in proteinuric nondiabetic nephropathy - PubMed (original) (raw)
SGLT2 inhibitor dapagliflozin limits podocyte damage in proteinuric nondiabetic nephropathy
Paola Cassis et al. JCI Insight. 2018.
Abstract
Sodium-glucose cotransporter 2 (SGLT2) inhibitors have pleiotropic properties beyond blood glucose-lowering effects and modify important nonglycemic pathways, leading to end-organ protection. SGLT2 inhibitors display renoprotective effects in diabetic kidney disease, which creates a rationale for testing the therapeutic potential of this drug class in nondiabetic chronic kidney disease. Here, we have shown that dapagliflozin provided glomerular protection in mice with protein-overload proteinuria induced by bovine serum albumin (BSA), to a similar extent as an ACE inhibitor used as standard therapy for comparison. Dapagliflozin limited proteinuria, glomerular lesions, and podocyte dysfunction and loss. We provide the observation that SGLT2 was expressed in podocytes and upregulated after BSA injections. Through in vitro studies with cultured podocytes loaded with albumin we have identified what we believe to be a novel mechanism of action for SGLT2 inhibitor that directly targets podocytes and relies on the maintenance of actin cytoskeleton architecture. Whether SGLT2 inhibitors represent a possible future therapeutic option for some patients with proteinuric glomerular disease who do not have as yet an effective treatment will require ad hoc clinical studies.
Keywords: Chronic kidney disease; Nephrology; Protein traffic.
Conflict of interest statement
Conflict of interest: The authors received funding from AstraZeneca Lab Italia.
Figures
Figure 1. Dapagliflozin induces glycosuria and natriuresis, and limits proteinuria in mice with protein overload.
(A–D) Mice were placed in metabolic cages and 24-hour urine was collected for evaluation of glucose excretion (A, n = 8–12), diuresis (B, n = 8–12), sodium excretion (C, n = 8–12), and urinary protein to creatinine ratio (D, n = 8–12) on day 23 after starting BSA injections. Data are the mean ± SEM and were analyzed by ANOVA with Tukey’s post hoc test. DAPA, dapagliflozin; ACEi, ACE inhibitor.
Figure 2. Dapagliflozin limits glomerular structural lesions and inflammation in mice with protein overload.
(A) Periodic acid–Schiff–stained images of representative glomeruli from control mice (n = 5) and in BSA-mice treated with vehicle, dapagliflozin (DAPA), or ACE inhibitor (ACEi) (n = 8 each group). The overall glomerular damage score was calculated by summing mesangial matrix expansion (arrow), glomerular capillary dilation (asterisks), and adhesions of the glomerular tuft to the Bowman’s capsule (arrowheads). Scale bars: 20 μm. (B) Glomerular and interstitial accumulation of Mac-2–positive monocytes/macrophages in control mice (n = 5) and in BSA-mice treated with vehicle, dapagliflozin, or ACE inhibitor (n = 8 each group). Data are the mean ± SEM and were analyzed by ANOVA with Tukey’s post hoc test.
Figure 3. Dapagliflozin limits ultrastructural podocyte damage in mice with protein overload.
Representative electron micrographs of glomeruli from control mouse and BSA-mice treated with vehicle, dapagliflozin (DAPA), or ACE inhibitor (ACEi). Focal areas of podocyte damage with effacement of foot processes are indicated by arrowheads in a mouse treated with BSA + vehicle. Scale bars: 2,000 nm.
Figure 4. Effects of dapagliflozin on nephrin expression in mice with protein overload.
Representative images and quantification showing glomerular nephrin expression (red) in control mice (n = 5) and in BSA-mice treated with vehicle, dapagliflozin (DAPA), or ACE inhibitor (ACEi) (n = 8 each group), evaluated on day 23 after starting BSA. Scale bars: 20 μm. Data are the mean ± SEM and were analyzed by ANOVA with Tukey’s post hoc test.
Figure 5. Dapagliflozin limits podocyte depletion in mice with protein-overload proteinuria.
Representative images and quantification of WT1-positive podocytes (red) expressed as number per glomerulus (A), podocyte density (B), and glomerular volume (C) in control mice (n = 6) and in BSA-mice treated with vehicle, dapagliflozin (DAPA), or ACE inhibitor (ACEi) (n = 8 each group), evaluated on day 23 after starting BSA. Scale bars: 20 μm. Data are the mean ± SEM and were analyzed by ANOVA with Tukey’s post hoc test.
Figure 6. Dapagliflozin ameliorates defective podocyte nestin expression in mice with protein overload.
Representative images and quantification showing glomerular nestin expression (green) in control mice (n = 5) and in BSA-mice treated with vehicle, dapagliflozin (DAPA), or ACE inhibitor (ACEi) (n = 7–8 each group), evaluated on day 23 after starting BSA. Scale bars: 20 μm. Data are the mean ± SEM and were analyzed by ANOVA with Tukey’s post hoc test.
Figure 7. SGLT2 is expressed in podocytes and upregulated after BSA injections.
Representative images of renal tissue from control mice and mice treated with BSA showing merged area (yellow) of costaining of glomerular SGLT2 expression (red) and nephrin (green) in podocytes. Boxes indicate area of enlarged images to the right. Nuclei and cell membranes are stained with DAPI (blue) and Cy5-labeled lectin (gray), respectively. Scale bars: 20 μm.
Figure 8. Human cultured podocytes express SGLT2 that is upregulated by albumin.
(A) Representative Western blot and densitometric analysis of SGLT2 protein in renal proximal tubular cells (RPTECs), used as positive control, and podocytes exposed to medium alone (control) or albumin (10 mg/ml, 6 hours). Actin was used as sample loading control. Data are the mean ± SEM (n = 4 samples) and were analyzed by Student’s t test. (B) SGLT2 mRNA expression evaluated by real-time qPCR analysis in podocytes exposed to medium alone (control) or albumin (10 mg/ml) for 3 and 6 hours. Hypoxanthine phosphoribosyltransferase 1 (HRPT1) was used as endogenous control. SGLT2 levels were normalized to HPRT1 levels and reported as fold change (FC) relative to the corresponding control. Data are the mean ± SEM (n = 6 samples) and were analyzed by ANOVA with Tukey’s post hoc test.
Figure 9. Effect of dapagliflozin on cytoskeletal remodeling in podocytes loaded with albumin.
Representative images and quantification of immunofluorescence staining for F-actin (red) (A), α-actinin-4 (red) (B), and β1-integrin (red) (C) in podocytes exposed to control medium (control) or albumin (10 mg/ml), in the presence or absence of dapagliflozin (DAPA, 10 nM) for 6 hours. Nuclei were counterstained with DAPI (blue). Asterisks indicate podocytes with F-actin cytoskeletal remodeling. Quantifications were performed in 15 fields per sample. Data are the mean ± SEM (n = 4–6 samples for F-actin, n = 3 samples for α-actinin-4, n = 5 samples for β1-integrin) and were analyzed by ANOVA with Tukey’s post hoc test. Original magnification, ×630.
Figure 10. NF-κB mediates albumin-induced SGLT2 mRNA expression in human cultured podocytes.
SGLT2 mRNA expression evaluated by real-time qPCR analysis in podocytes exposed to medium alone (control) or albumin (10 mg/ml) in the presence or absence of SN50 (10 μM) for 3 hours. Hypoxanthine phosphoribosyltransferase 1 (HRPT1) was used as endogenous control. SGLT2 levels were normalized to HPRT1 levels and reported as fold change relative to control. Data are the mean ± SEM (n = 4 samples) and were analyzed by ANOVA with Tukey’s post hoc test.
Figure 11. SGLT2 expression in renal biopsies of patients with idiopathic membranous nephropathy.
(A) Representative double immunofluorescence staining for SGLT2 (red) and nephrin (green) in normal control kidney (n = 3) and (B) in the renal biopsy of proteinuric patients with idiopathic membranous nephropathy (n = 4). DAPI (blue) stains nuclei. Scale bars: 20 μm.
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