High glucose-induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model

Abstract

Background: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis.

Methods: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-β1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively.

Results: Only the combination of high D-glucose and TGF-β1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this rise was significantly reduced by both SGLT2 and MEK inhibition.

Conclusions: We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.

Keywords: Dapagliflozin; Diabetic Kidney Disease; Fibrosis; Glucose; Glucose Transporters; Smad3.

Figure 1. Effects of high glucose and TGF-β1 stimulation on PTECs that express CCN2

(A and B) Image of Western blot immunostained for secreted CCN2 protein in PTECs under various treatments and the ‘Mini-PROTEAN TGX Stain-Free’ gel used for normalisation purposes. (C) Data points for secreted CCN2 (∼36/38 kDa) abundance normalised for total protein. The Mini-PROTEAN TGX stain-free gels used for normalisation contain a trihalocompound that automatically produces fluorescence when covalently crosslinked to protein tryptophan residues. After the proteins are separated by electrophoresis, crosslinking is carried out by exposing the gel to UV light. This is the band density normalisation process used for all subsequent experiments. D-Glu+TGF-β1 treatment significantly up-regulated CCN2 secretion. *P<0.05 compared with all other treatments, n=3. Black circles ● = D-Glu, black triangles ▲ = D-Glu+TGF-β1, black stars ★ = control, black diamonds ♦ = control + TGF-β1, black hexagons = L-Glu, black squares ▓ = L-Glu + TGF-β1. Error bars indicate SD. Quantification of all proteins in these figures were a result of the light generated by the detection antibody indexed to total protein loaded in each sample. Control = 7 mM D-glucose, Control + TGF-β1 = 7 mM D-glucose + 0.75 ng/ml TGF-β1, D-Glu = 7 mM D-glucose + 18 mM D-glucose, L-Glu = 7 mM D-glucose + 18 mM L-glucose, D-Glu + TGF-β1 = 7 mM D-glucose + 18 mM D-glucose + 0.75 ng/ml TGF-β1, L-Glu + TGF-β1 = 7 mM D-glucose + 18 mM L-glucose + 0.75 ng/ml TGF-β1. This legend key is applicable to all subsequent figures.

Figure 2. Effects of Dapagliflozin (dapa) on up-regulated CCN2 mRNA and protein in PTECs

(A and B) Image of Western blot immunostained for secreted CCN2 protein in PTECs under various treatments. (C) Data points for secreted CCN2 (36–38 kDa) abundance normalised for total protein as mentioned above in Figure 1. Dapagliflozin at all three concentrations significantly attenuated CCN2 protein down to control levels; ††P<0.01 compared with D-Glu + TGF-β1, †††P<0.001 compared with D-Glu+TGF-β1, n=5. Black hexagons = control, upward black triangles ▲ = D-Glu+TGF-β1, white triangles Δ = D-Glu+TGF-β1+0.1 nM dapa, white circles ○ = D-Glu+TGF-β1+1 nM dapa, white squares □ = D-Glu+TGF-β1+ 10 nM dapa. (D) Effects of 0.1, 1 and 10 nM dapagliflozin on up-regulated CCN2 mRNA (ΔCt of CCN2 to GAPDH) in PTECs. Dapagliflozin at 1 and 10 nM significantly attenuated CCN2 relative mRNA abundance to control levels. †P<0.05 compared with D-Glu+TGF-β1. Black X X = control, black cross + = D-Glu+TGF-β1, white hexagons = D-Glu+TGF-β1+0.1 nM dapa, white diamonds ◊ = D-Glu+TGF-β1+1 nM dapa, white circles ○ = D-glu+TGF-β1+ 10 nM dapa, n=12. Error bars indicate SD. Quantification of all proteins in these figures were a result of the light generated by the detection antibody indexed to total protein loaded in each sample. Control = 7 mM D-glucose, Control + TGF-β1 = 7 mM D-glucose + 0.75 ng/ml TGF-β1, D-Glu = 7 mM D-glucose + 18 mM D-glucose, L-Glu = 7 mM D-glucose + 18 mM L-glucose, D-Glu+TGF-β1 = 7 mM D-glucose + 18 mM D-glucose + 0.75 ng/ml TGF-β1, L-Glu + TGF-β1 = 7 mM D-glucose + 18 mM L-glucose + 0.75 ng/ml TGF-β1. This legend key is applicable to all subsequent figures.

Figure 3. Phosphorylation state of various signalling pathways upon treatment with high D-glucose and TGF-β1 in PTECs

(A–C) Image of western blots immunostained for cellular phospho-ERK protein in PTECs under various treatments and time points. (D–F) Data points for cellular phospho-ERK (∼40 kDa) abundance normalised for total protein as mentioned above in Figure 1. TGF-β1 induced ERK phosphorylation significantly, +/- high D-glucose for 60 min, while exclusive high D-glucose treatment phosphorylated ERK at 30 min; *P<0.05. (G–I) Image of Western blots immunostained for cellular phospho-Smad3 protein in PTECs under various treatments and time points; (G and H) Smad3 phosphorylated at serine 423/425; (I and J) Smad3 phosphorylated at serine 204. (J–L) Data points for cellular phospho-Smad3 (∼50 kDa) abundance normalised for total protein. TGF-β1 significantly phosphorylated Smad3, +/- high D-glucose from 15 min onwards; **P<0.01. (M–O) Image of Western blots immunostained for cellular phospho-serine 204 of Smad3 LR protein in PTECs under various treatments and time points. (P–R) Data points for cellular phospho-serine 204 of Smad3 LR protein (∼25 kDa) abundance normalised for total protein. D-Glu+TGF-β1 treatment at 30 min significantly phosphorylated serine 204 compared to L-Glu+TGF-β1 at 30 min; *P<0.05, n=3–4. Black squares ▓ = L-Glu+TGF-β1, black circles ● = D-Glu+TGF-β1, white circles ○ = D-Glu, white squares □ = L-Glu. Error bars indicate SD. Quantification of all phosphorylated proteins in these figures were a result of the light generated by the detection antibody indexed to total protein loaded in each sample. Control = 7 mM D-glucose, Control + TGF-β1 = 7 mM D-glucose + 0.75 ng/ml TGF-β1, D-Glu = 7 mM D-glucose + 18 mM D-glucose, L-Glu = 7 mM D-glucose + 18 mM L-glucose, D-Glu + TGF-β1 = 7 mM D-glucose + 18 mM D-glucose + 0.75 ng/ml TGF-β1, L-Glu + TGF-β1 = 7 mM D-glucose + 18 mM L-glucose + 0.75 ng/ml TGF-β1. This legend key is applicable to all subsequent figures.

Figure 4. Phosphorylation state of Smad3 serine 204 and ERK after U0126 and dapagliflozin treatment

(A) Image of Western blot immunostained for cellular phospho-ERK (∼40 kDa) protein in PTECs treated with dapagliflozin and (B) the corresponding data points for cellular phospho-ERK abundance normalised for total protein as mentioned above in Figure 1. Dapagliflozin significantly reduced the cellular content of phosphorylated ERK (∼40 kDa). **P<0.01 compared with D-Glu+TGF-β1+dapa 1 nM, n=3. (C) Image of Western blot immunostained for cellular phospho-serine 204 of Smad3 LR protein in PTECs treated with dapagliflozin and (D) the corresponding data points for cellular phospho-serine 204 of Smad3 LR abundance normalised for total protein. Dapagliflozin (1 nM) significantly reduced the cellular content of phosphorylated serine 204. *P<0.05 compared with D-Glu + TGF-β1 + dapa 1 nM, n=3. (E) Image of Western blots immunostained for cellular phospho-serine 204 of Smad3 LR protein in PTECs treated with U0126 and (F) the corresponding data points for cellular phospho-serine 204 of Smad3 LR (∼25kDa) abundance normalised for total protein. U0126 (10 μM) significantly reduced the cellular content of Smad3 phosphorylated serine 204 at 30 min. **P<0.05 compared to D-Glu + TGF-β1 + U0126, n=3. Black hexagons = L-Glu + TGF-β1, white hexagons = L-Glu + TGF-β1 + U0126, black circles ● = D-Glu + TGF-β1, white circles ○ = D-Glu + TGF-β1 + U0126, white squares □ = L-Glu + TGF-β1, white diamonds ◊ = D-Glu + TGF-β1 + dapa 1 nM. Quantification of all phosphorylated proteins in these figures were a result of the light generated by the detection antibody indexed to total protein loaded in each sample.

Figure 5. Schematic of proposed signalling mechanism: glucose mediated pro-fibrotic CCN2 induction

(A) Typically, the TGF-β1 type 2 receptor phosphorylates the cytoplasmic domain of the type 1 receptor, which then proceeds to phosphorylate Smad3 on the MH2 domain, (B) specifically at serine 423 and 425. The fully activated Smad complex is then translocated to the nucleus where it controls gene transcription. Under hyperglycaemic conditions, ERK is also activated and phosphorylates the serine 204 located on the linker region between the C (SSXS) and N terminal domain. This then increases TGF-β specific signalling of the complex, thereby potentiating transcriptional activity at the nucleus. Control = 7 mM D-glucose, Control + TGF-β1 = 7 mM D-glucose + 0.75 ng/ml TGF-β1, D-Glu = 7 mM D-glucose + 18 mM D-glucose, L-Glu = 7 mM D-glucose + 18 mM L-glucose, D-Glu + TGF-β1 = 7 mM D-glucose + 18 mM D-glucose + 0.75 ng/ml TGF-β1, L-Glu + TGF-β1 = 7 mM D-glucose + 18 mM L-glucose + 0.75 ng/ml TGF-β1. This legend key is applicable to all subsequent figures.