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Jeong, Chung, Jung, Doh, Kim, Park, Park, Kim, Ha, Kim, Lee, and Ko: The Effect of High Glucose and TGF-β on the Cellular Injury in Cultured Glomerular Epithelial Cells
Original Article
The Journal of Korean Diabetes Association

The Journal of Korean Diabetes Association 2006; 30(4): 254-263.

Published online: 31 July 2006

DOI: https://doi.org/10.4093/jkda.2006.30.4.254

The Effect of High Glucose and TGF-β on the Cellular Injury in Cultured Glomerular Epithelial Cells

Gui Hwa Jeong2, Sung Chang Chung1, Eui Dal Jung2, Yun Jeong Doh2, Hee Kyoung Kim2, Soon Hong Park2, In Hae Park2, Jung Guk Kim2, Sung Woo Ha2, Bo Wan Kim2, In Kyu Lee2, Cheol Woo Ko3

1Donggang medical center, Woolsan, Korea.

2Department of Internal Medicine, Kyungpook National University, School of Medicine, Daegu, Korea.

3Department of Pediatric, Kyungpook National University, School of Medicine, Daegu, Korea.

Received 21 March 2006       Accepted 25 July 2006

Copyright © 2006 Korean Diabetes Association

Abstract

Background

The glomerulus is a complex physiological structure, as well as selective filtration barrier in the control of renal blood flow and blood pressure. Glomerular epithelial cells may play an important role in development of diabetic nephropathy. Apoptosis of the glomerular epithelial cells are characterized by disappearance of a selective filtration barrier. TGF-Β is a key factor in the development of diabetic nephropathy because of its effects on the accumulation of extracellular matrix and mesangial cell proliferation. We examined whether the high glucose and TGF-Β induce the apoptosis in cultured rat glomerular epithelial cells.

Methods

Glomerular epithelial cells were cultured from rat glomeruli and conditioned with different concentration of TGF-Β or high-glucose. We measured apoptosis of cultured rat glomerular epithelial cell conditioning with different concentration of TGF-Β or high-glucose by using DNA electrophoresis.

Results

High glucose (25 mM) induced apoptosis of cultured rat glomerular epithelial cells compared to controls. TGF-Β also induced cell death of cultured rat glomerular epithelial cells in dose dependent manner.

Conclusion

These results suggest that high glucose and TGF-Β-induced cell death of glomerular epithelial cell may play an important role in diabetic nephropathy and proteinuria. Pathway of apoptosis or cell death by high glucose and TGF-Β must be investigated in the glomerular epithelial cells.

Keywords: Apoptosis, Diabetic nephropathy, Glomerular epithelial cell, High-glucose, TGF-β

Figures and Tables

Fig. 1
Cultured renal epithelial cells were exposed to normal glucose (5.5 mM), high glucose (25 mM), normal glucose (5.5 mM) plus TGF-β (1 ug/mL), high glucose (25 mM) plus TGF-β (1 ug/mL). (A) DAPI stain in renal epithelial cells treated with high glucose and TGF-β. Apoptotic DNA fregmentation of cells treated with high glucose and TGF-β were increased in DAPI stain. (B) The mean ± S.D. of the three different experiments expressed as number of apoptotic cells per normal cells are shown in the graph.
*p < 0.05 versus controls.
jkda-30-254-g001
Fig. 2
High glucose and TGF-β induce apoptosis in renal epithelial cells.
A. Cells were exposed to normal glucose (5.5 mM), normal glucose (5.5 mM) plus TGF-β (1 ng/mL), high glucose (25 mM), high glucose (25 mM) plus TGF-β (1 ng/mL) for 24 hr after which nuclei were stained by PI stain and analysed by flow cytometry. The mean ± S.D. of the three different experiments expressed as number of apoptotic cells per total cells are shown in the graph.
*p < 0.05 versus controls compared.
B. Cells were exposed to normal glucose (5.5 mM), high glucose (25 mM) plus TGF-β (1 ng/mL) for 24, 48, 72 hr were stained by PI stain and analysed by flow cytometry. The mean ± S.D. of the three different experiments expressed as number of apoptotic cells per total cells are shown in the graph.
*p < 0.05 versus controls.
jkda-30-254-g002
Fig. 3
High glucose induced apoptosis in renal epithelial cells.
After cells were exposed to starvation state for 24 h, they were exposed to normal glucose (5.5 mM), normal glucose (5.5 mM) plus mannitol (19.5 mM), high glucose (25 mM) for 24 hr. A. Renal epithelial cells were stained by TUNEL stain. B. Three types of bars (white, gray, black) indicate percentage of apoptotic cells per total number. The mean ± S.D. of the three different experiments expressed as number of apoptotic cells per total cells are shown in the graph.
*p < 0.05 versus controls compared.
jkda-30-254-g003
Fig. 4
A. Effects of glucose concentration (5.5 mM, 25 mM) on caspase-3 activity. Time course of caspase 3-activity of renal epithelial cells induced by high glucose (25 mM). The level of caspase-3 activity in high glucose (25 mM) was significantly increased compared with the control cells at 24, 48, 72 hr. B. Effects of TGF-β treatment (1 ng/mL) on caspase-3 activity. Caspase-3 activity was increased in the cells treated with normal glucose (5 mM) plus TGF-β (1 ng/mL), high glucose (25 mM), high glucose plus TGH-β (1 ng/mL) at 24 hr. At 48 hr, caspase-3 activity was increased in group treated with high glucose.
*p < 0.05 compared wth normal glucose (5.5 mM).
jkda-30-254-g004
Fig. 5
Western blot assay about p21 Cip, p27 Kip of renal epithelial cells 12, 24, 48, 72 hours after cell lysis with IPH lysis buffer. A. Western blot shows p21 Cip level and p27 Kip level after treatment of high glucose. B. p21 Cip and p27 Kip concentration were measured at 12, 24, 48, 72 hr after normal (5.5 mM), high (25 mM) glucose treatment.
jkda-30-254-g005

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