Dapagliflozin Attenuates Renal Tubulointerstitial Fibrosis Associated With Type 1 Diabetes by Regulating STAT1/TGFβ1 Signaling

Abstract

Tubulointerstitial fibrosis (TIF) plays an important role in the progression of renal fibrosis in diabetic nephropathy (DN). Accumulating evidence supports a crucial inhibitory effect of dapagliflozin, a SGLT2 inhibitor, on TIF, but the underlying mechanisms remain largely unknown. This study aimed to shed light on the efficacy of dapagliflozin in reducing TIF as well as its possible impact on renal function. TIF in human kidney biopsies obtained from patients with DN was quantified by histopathological staining. In vitro, HK-2 cells were incubated in high glucose with dapagliflozin or fludarabine, and epithelial-mesenchymal transition (EMT) was determined. In vivo experiments were performed in streptozotocin (STZ)-induced type 1 diabetic mice treated with dapagliflozin by gavage for 16 weeks, after which specific functional characteristics and TIF were analyzed. In both DN patients and diabetic mice, fibronectin and Col IV, as well as STAT1 protein in the kidneys were increased as compared with controls. Dapagliflozin significantly decreased blood glucose, and renal STAT1 and TGF-β1 expression in mice. Furthermore, dapagliflozin improved renal function, and attenuated diabetes-induced TIF. In HK-2 cells, dapagliflozin, and fludarabine directly decreased aberrant STAT1 expression and reversed high glucose-induced downregulation of E-cadherin and α-SMA induction. Thus, the results demonstrate that dapagliflozin not only improves hyperglycemia but also slows down the progression of diabetes-associated renal TIF by improving hyperglycemia-induced activation of the STAT1/TGF-β1 pathway.

Keywords: STAT1; TGF-β1; dapagliflozin; epithelial mesenchymal transition; tubulointerstitial fibrosis.

Figure 1

The expressions of STAT1, TGFβ1, FN, and Col IV in non-diabetic adjacent normal kidney tissues (NC), and renal biopsy sections from DN patients. (A) Protein expression levels of STAT1, TGFβ1, FN, and Col IV were quantified by western blot analysis. (B) mRNA expressions levels of STAT1, TGFβ1, FN, and Col IV were quantified by qRT-PCR. *P < 0.05 vs. NC.

Figure 2

Histopathological examination of non-diabetic adjacent normal kidney tissues (NC) and renal biopsy sections from DN patients. Representative images of (A) HE staining, (B) PAS staining, (C) Picrosirius red staining, and (D) Masson's trichrome staining for assessing TIF. Immunohistochemistry staining of (E) FN, (F) Col IV, and (G) STAT1 in human kidney sections. Original magnification, 100–200x. Quantitative analysis of (H) PAS staining, (I) Picrosirius red staining, (J) Masson's trichrome staining, (K) relative FN staining intensity, (L) relative Col IV staining intensity, and (M) relative STAT1 staining intensity. All data are expressed as means ± SEM, n = 10 (*P < 0.05 vs. NC).

Figure 3

Effect of DAPA on renal production of fibrotic markers in diabetic mice. (A) Representative western blots for STAT1, TGF-β1, Col IV, and FN in renal cortical lysates. β-actin was used as internal control. (B) Density ratio of STAT1 to GAPDH mRNA expressions. (C–F) Normalized quantification of western blot data in diabetic mice at different times. *P < 0.05 vs. NC; **P < 0.05 vs. DM-8W; * ^&P < 0.05 vs. DM-12W; ^#P < 0.05 vs. DM-16W. Values are means ± SEM. n = 3.

Figure 4

DAPA prevents renal TIF in diabetic mice. Representative images of immunofluorescence staining for Col IV and FN in tubular areas of diabetic mice. Original magnification, 200×.

Figure 5

Effect of DAPA on extracellular matrix (ECM) protein accumulation and renal TIF. (A–C) IHC examination of kidney sections for FN, Col IV, and STAT1. Original magnification, 200×. (D–F) Quantification of FN, Col IV, and STAT1 staining. *P < 0.05 vs. NC; ^#P < 0.05 vs. DM-12W; ^&P < 0.05 vs. DM-16W. Values are means ± SEM.

Figure 6

Effect of DAPA on renal TIF. (A–D) Representative photomicrographs of H&E, PAS, Picrosirius red, and Masson's trichrome staining. (E–G) Quantification of PAS, Picrosirius red and Masson's trichrome staining. *P < 0.05 vs. NC; ^#P < 0.05 vs. DM-12W; ^&P < 0.05 vs. DM-16W. Values are mean ± SEM.

Figure 7

Effects of DAPA and Flu on the expression of EMT-related proteins in HK-2 cells. (A) Representative western blots illustrating STAT1, TGF-β1, E-cadherin, α-SMA, and β-actin protein expression in HK2 cells. (B–E) Quantification of the western blot data in HK-2 cells. Statistical analysis was performed with one-way ANOVA. Results are expressed as the mean ± SEM. *P < 0.05 vs. NC, ^#P < 0.05 vs. HG, ^#&P < 0.05 vs. HG+DAPA-1.