Dapagliflozin Prevents Kidney Glycogen Accumulation and Improves Renal Proximal Tubule Cell Functions in a Mouse Model of Glycogen Storage Disease Type 1b

Abstract

Background: Mutations in SLC37A4, which encodes the intracellular glucose transporter G6PT, cause the rare glycogen storage disease type 1b (GSD1b). A long-term consequence of GSD1b is kidney failure, which requires KRT. The main protein markers of proximal tubule function, including NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, are downregulated as part of the disease phenotype.

Methods: We utilized an inducible mouse model of GSD1b, TM-G6PT^-/-, to show that glycogen accumulation plays a crucial role in altering proximal tubule morphology and function. To limit glucose entry into proximal tubule cells and thus to prevent glycogen accumulation, we administered an SGLT2-inhibitor, dapagliflozin, to TM-G6PT^-/- mice.

Results: In proximal tubule cells, G6PT suppression stimulates the upregulation and activity of hexokinase-I, which increases availability of the reabsorbed glucose for intracellular metabolism. Dapagliflozin prevented glycogen accumulation and improved kidney morphology by promoting a metabolic switch from glycogen synthesis toward lysis and by restoring expression levels of the main proximal tubule functional markers.

Conclusion: We provide proof of concept for the efficacy of dapagliflozin in preserving kidney function in GSD1b mice. Our findings could represent the basis for repurposing this drug to treat patients with GSD1b.

Keywords: NHE-3; Napi-2; SGLT-2 inhibitors; dapagliflozin; glycogen; glycogen storage disease 1b; hexokinase-1; proximal tubule.

Graphical abstract

Figure 1.

Renal phenotype in TM-G6PT^−/− mice. (A) Quantitative RT-PCR (RT-qPCR) expression of G6PT mRNA along the renal zone in wild-type C57BL/6J mice. Data are expressed as percentage of CTX/OSOM expression (n=3). ISOM (n=4) and IM (n=4). Statistics was performed by one-way ANOVA followed by Bonferroni post-test. (B) RT-qPCR expression of G6PT mRNA in control (ctr, gray dots) or TM-G6PT ^−/− mice (black dots) along the three renal zones (CTX/OSOM n=3+4; ISOM n=4+3; IM n=4+3, unpaired t test). (C) Urinary excretion of glucose and albumin over creatinine (ACR) (for glucose/creatinine n=4+8 and for ACR n=3+4; unpaired t test). (D) Urinary phosphate (Pi)/creatinine excretion and CC16/creatinine, as marker of low-molecular-weight proteinuria (Pi/creatinine n=5+14 and CC16/creatinine n=5+5; unpaired t test). I Representative images from renal cortex of Ctr and TM-G6PT^−/− mice stained with anti-NaPi2A antibody (upper panels, scale bar: 50 µm; lower panels, scale bar: 20 µm). (F) Immunoblotting from membrane fractions of whole kidney samples from Ctr and TM-G6PT^−/− mice reveals a downregulation of NaPi2A, NHE3, AQP1, and AQP2 in TM-G6PT^−/− mice (n=5+5, unpaired t test). All data are expressed as mean±SEM; ***P<0.001, **P<0.01, *P<0.05.

Figure 2.

PTs of TM-G6PT^−/− mice have large glycogen deposit and alteration in glucose transporters. (A) Cortical glycogen content is about six-fold larger in TM-G6PT^−/− (black dots) compared with control mice (Ctr, gray dots) (n=4+5, unpaired t test). (B) Representative electron microscopy images from renal cortex of control and TM-G6PT^−/− mice at low (upper panels, scale bar: 10 µm) and high (lower panels, scale bar: 1 µm) magnification. (C) Immunoblotting of membrane fraction of whole renal samples from control and TM-G6PT^−/− mice reveals a severe downregulation of both luminal glucose transporter, SGLT2 and basolateral glucose transporter, GLUT2 (n=5+5, unpaired t test). (D) Representative pictures from renal cortex of Ctr and TM-G6PT^−/− mice stained with anti-SGLT2 antibody (1:100 dilution; scale bar: 20 µm). All data are expressed as mean±SEM; **P<0.01, *P<0.05.

Figure 3.

In vitro silencing of G6PT leads to upregulation and increase activity of HK-1. (A) RT-qPCR expression of G6PT mRNA in TKPTS cells transfected with control siRNA (Ctr; gray dots) or three different siRNA interfering with the G6PT mRNA, namely A, B, and C. (A) RT-qPCR expression of G6PT mRNA and (B) HK-I (mRNA G6PT/Gapdh n=9+8+8+9 and mRNA HK1/Gapdh n=9+9+9+8). (C) Immunoblotting of cell lysate probed with an anti–HK-I antibody (n=3 per group). (D) HK-I activity from cell lysate (n=3 per group). For all of the panels statistics was performed with one-way ANOVA followed by Bonferroni post-test. All data are expressed as mean±SEM; ***P<0.001, **P<0.01, *P<0.05.

Figure 4.

Dapagliflozin prevents glycogen accumulation by facilitating its catabolism. (A) Protocol for dapagliflozin and TM administration. (B) Dapagliflozin prevents glycogen accumulation in renal cortex of TM-G6PT^−/− mice (n=4+6; unpaired t test). (C) Immunoblotting from total fraction of whole renal samples from control (Ctr) and TM-G6PT^−/− mice treated and untreated with dapagliflozin (n=3+4+4; one-way ANOVA). Dapagliflozin inhibits GYS by favoring its inactive S641-phosphorylated form and stimulates the activity of PYGL by favoring the expression of its active S15-phosphorylated form. All data are expressed as mean±SEM; ***P<0.001, **P<0.01, *P<0.05. The single asterisk (*) is for comparison Ctr versus TM-G6PT^−/− mice; the pound symbol (#) is for comparison versus untreated TM-G6PT^−/− mice.

Figure 5.

Dapagliflozin improves PT morphology and reduced urinary phosphate excretion. (A) Representative pictures of hematoxylin and eosin–stained renal section and their equivalent version edited with the Color Threshold option of Fiji software from control (Ctr) and, TM-G6PT^−/− mice treated with and without dapagliflozin (scale bar: 100 µm). The parenchyma was labeled with red color and quantified over the total surface area. (B) The amount of renal cortical parenchyma free of vacuoles increases substantially in TM-G6PT^−/− mice after treatment (n=3+3+4; one-way ANOVA). (C) Urinary phosphate (Pi)/creatinine excretion and CC16/creatinine, as marker of low-molecular-weight proteinuria from TM-G6PT^−/− mice treated with or without dapagliflozin (Pi/creatinine n=5+6 and CC16/creatinine n=4+5; unpaired t test). All data are expressed as mean±SEM; **P<0.01, *P<0.05. The single asterisk (*) is for comparison Ctr versus TM-G6PT^−/− mice; the pound symbol (#) is for comparison versus untreated TM-G6PT^−/− mice.

Figure 6.

Dapagliflozin ameliorates the expression of proteins markers of PT function. (A) Immunoblotting from membrane fraction of whole kidney samples from Ctr or TM-G6PT^−/− mice treated with or without dapagliflozin (n=3+4+4 respectively; one-way ANOVA). Dapagliflozin improves the expression of functional markers of PT such as NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, but also of distal tubule as AQP2. Representative pictures of renal cortex from Ctr or TM-G6PT^−/− mice treated with or without Dapa stained with (B) anti-NaPi2A (red) (1:300 dilution) and Lotus Tetragonolobus Lectin (LTL) (green) (1:400 dilution); scale bar: 10 µm; white asterisk indicates intracellular retention of NaPI2A or (C) anti-SGLT2 antibody (green) (1:100 dilution). All data are expressed as mean±SEM; ***P< 0.001, **P<0.01, *P<0.05. The single asterisk (*) is for comparison Ctr versus TM-G6PT^−/− mice; the pound symbol (#) is for comparison versus untreated TM-G6PT^−/− mice.