Inactivation of TSC1 promotes epithelial-mesenchymal transition of renal tubular epithelial cells in mouse diabetic nephropathy

Abstract

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic nephropathy (DN). However, the molecular mechanisms of EMT remain largely unknown. Tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling, and the loss of TSC1 or TSC2 function leads to a spectrum of diseases that underlie abnormalities in cell growth, proliferation, differentiation, and migration. In this study, we investigated the effects of TSC1 on high glucose (HG)-induced EMT of human proximal tubular epithelial HK-2 cells in vitro and renal fibrosis in TSC1^-/- and db/db mice. We found that the exposure of HK-2 cells to HG (30 mM) time-dependently decreased TSC1 expression, increased the phosphorylation of mTORC1, P70S6K, and 4E-BP-1, and promoted cell migration, resulting in EMT. Transfection of the cells with TSC1 mimic significantly ameliorated HG-induced EMT of HK-2 cells. The tubules-specific TSC1 knockout mice (TSC1^-/-) displayed a significant decline in renal function. TSC1^-/- mice, similar to db/db mice, showed greatly activated mTORC1 signaling and EMT process in the renal cortex and exacerbated renal fibrosis. Overexpression of TSC1 through LV-TSC1 transfection significantly alleviated the progression of EMT and renal fibrosis in the renal cortex of db/db mice. Taken together, our results suggest that TSC1 plays a key role in mediating HG-induced EMT, and inhibition of TSC1-regulated mTORC1 signaling may be a potential approach to prevent renal fibrosis in DN.

Keywords: TSC1−/− mice; db/db mice; diabetic nephropathy; epithelial–mesenchymal transition; human proximal tubular epithelial cell line HK-2; rapamycin; renal fibrosis; tuberous sclerosis complex 1.

Fig. 1

The time course change of TSC1 expression and downstream molecules phosphorylation with high-glucose stimulation. Data are presented as the mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, compared with NG

Fig. 2

Effect of high glucose on TSC1 signaling in HK-2 cells. a Effects of HG, TSC1, and rapamycin stimulation on the changes of HK-2 cells morphology (magnification, ×400). b The expression of TSC1, mTOR, p70S6K, and 4E-BP1 in HK-2 cells through Western blotting. c Statistical analysis of TSC1 expression and relative phosphorylation levels of mTOR, p70S6K, and 4E-BP1 in HK-2 cells. NG: cells treated with normal glucose 5.56 mM; HG: cells treated with high glucose 30 mM; HG+TSC1 vehicle: cells treated with high glucose +TSC1 vehicle plasmids for 48 h; HG+TSC1 mimic: cells treated with high glucose +TSC1 mimic plasmids for 48 h. HG+Rap: cells treated with high glucose +20 nM rapamycin. Cells were starved for 24 h and treated with normal glucose, high glucose and rapamycin for 72 h. Data are presented as the mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, compared with NG; ^#P < 0.05, ^##P < 0.01, compared with HG. Bar = 50 μm

Fig. 3

Effect of TSC signaling activation on high glucose-induced EMT in HK-2 cells. a Longer invaded distances in HK-2 cells transfected with TSC1 mimic plasmids and treated with rapamycin by wound-healing assay. b, c Expression of ZO-1, E-cadherin, vimentin, α-SMA in HK-2 cells. d, e Expression of snail and twist in HK-2 cells. f The mRNA levels of snail and twist in high glucose-cultured HK-2 cells by qRT-PCR. Data are presented as the mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, compared with NG; ^#P < 0.05, ^##P < 0.01, compared with HG

Fig. 4

Distribution and expressions of ZO-1 and α-SMA in HK-2 cells by immunofluorescence. Bar = 25 μm

Fig. 5

Effect of TSC1 silencing on mTOR signaling of normal glucose-cultured HK-2 cells. a Effects of TSC1 shRNA stimulation on the changes of HK-2 cells morphology (magnification, ×400). b The expression of TSC1, p-mTOR, mTOR, p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1 in HK-2 cells through Western blotting. c Statistical analysis of TSC1 expression and relative phosphorylation levels of mTOR, p70S6K, and 4E-BP1 in HK-2 cells. Vehicle: cells treated with normal glucose 5.56 mM + TSC1 vehicle plasmids for 48 h; TSC1 shRNA: cells treated with normal glucose 5.56 mM + TSC1 shRNA plasmids for 48 h. Data are presented as the mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, compared with Vehicle. Bar = 50 μm

Fig. 6

Effect of TSC1 silencing on EMT of normal glucose-cultured HK-2 cells. a Invaded distances in HK-2 cells transfected with TSC1 shRNA plasmids by wound-healing assay. b Expression of ZO-1, E-cadherin, Vimentin, α-SMA in HK-2 cells. c The protein and mRNA levels of two transcription factors (Snail and Twist) in normal glucose-cultured HK-2 cells by qRT-PCR. d Distribution and expressions of ZO-1 and α-SMA in HK-2 cells by immunofluorescence (bar = 25 μm). Vehicle: cells treated with normal glucose 5.56 mM + TSC1 vehicle plasmids for 48 h; TSC1 shRNA: cells treated with normal glucose 5.56 mM+TSC1 shRNA plasmids for 48 h. Data are presented as the mean ± SEM, n = 3. ^*P < 0.05, ^**P < 0.01, compared with Vehicle

Fig. 7

The origin and identification of TSC1^−/− mice. a Strategy for generating mice with tubules-specific deletion of TSC1. b Genotyping of the mice by PCR analysis of genomic DNA. c Expression of TSC1^−/− mice by immunofluorescence (bar = 20 μm). LTL: fluorescein-labeled Lotus Tetragonolobus Lectin. LTL is specifically expressed in the tubules of kidney

Fig. 8

Effect of TSC1 on kidney interstitial fibrosis. a Masson staining of renal cortex sections of mice. b Sirius Red staining of renal cortex sections of mice. c Collagen IV expression in renal cortex of mice through immunohistochemistry. d Laminin expression in renal cortex of mice through immunohistochemistry. Images a, b, c, and d are representative staining images (magnification, ×40). Data are presented as the mean ± SEM, n = 6. ^**P < 0.01, compared with control; ^#P < 0.05, ^##P < 0.01, compared with TSC1^−/−; ^ΔΔP < 0.01, compared with db/m mice; ^▲P < 0.05, compared with db/db mice

Fig. 9

Effect of TSC1 on mTORC1 signaling in TSC1^−/− mice and db/db mice. The expression of phosphorylation of TSC1, mTORC1, p70S6K, and 4E-BP1 levels in renal cortex of mice. Data are presented as the mean ± SEM, n = 6. ^*P < 0.05, ^**P< 0.01, compared with control;    ^##P < 0.01, compared with TSC1^−/−; ^ΔΔP < 0.01, compared with db/m mice; ^▲P < 0.05, compared with db/db mice

Fig. 10

Effect of TSC1 on EMT in TSC1^−/− mice and db/db mice. a Expression of ZO-1, E-cadherin, Vimentin, and α-SMA in renal cortex of mice. b Expression of Snail and Twist in renal cortex of mice. Data are presented as the mean ± SEM, n = 6. ^*P < 0.05, ^**P < 0.01, compared with control; ^#P < 0.05, ^##P < 0.01, compared with TSC1^−/−; ^ΔΔP < 0.01, compared with db/m mice; ^▲P < 0.05, compared with db/db mice