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Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury

View Article: PubMed Central - PubMed

ABSTRACT

Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney.

No MeSH data available.


The role of autophagy in AGEs-induced mesangial cell apoptosis.Mesangial cells were transfected with single Atg5 siRNA [(MSS247019; siRNA (1), (A-a)] or mixture of Atg5 siRNAs [(MSS247019, MSS247020, and MSS247021; siRNA (2), (A-b)] or single Atg5 siRNA [(MSS247020; siRNA (3), (B-a)] or scramble control before AGEs treatment. Cells were treated with BSA (160 μg/ml) or AGEs (160 μg/ml) for 24 h. The protein levels of Atg5, p62, LC3, phospho-eIF2α, CHOP, and cleaved caspase-3 were determined by Western blot (A). The percentages of apoptotic cells were determined by PI-Annexin V staining (B). Data are presented as mean ± SEM of three independent experiments performed in duplicates (A) or triplicates (B). *P < 0.05 as compared to BSA-treated group or AGEs-treated group.
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f8: The role of autophagy in AGEs-induced mesangial cell apoptosis.Mesangial cells were transfected with single Atg5 siRNA [(MSS247019; siRNA (1), (A-a)] or mixture of Atg5 siRNAs [(MSS247019, MSS247020, and MSS247021; siRNA (2), (A-b)] or single Atg5 siRNA [(MSS247020; siRNA (3), (B-a)] or scramble control before AGEs treatment. Cells were treated with BSA (160 μg/ml) or AGEs (160 μg/ml) for 24 h. The protein levels of Atg5, p62, LC3, phospho-eIF2α, CHOP, and cleaved caspase-3 were determined by Western blot (A). The percentages of apoptotic cells were determined by PI-Annexin V staining (B). Data are presented as mean ± SEM of three independent experiments performed in duplicates (A) or triplicates (B). *P < 0.05 as compared to BSA-treated group or AGEs-treated group.

Mentions: To clarify the role of autophagy in AGEs-induced mesangial cell apoptosis, cells were transfected with scramble or Atg5 siRNA for 6 h before AGEs treatment. Transfection with Atg5 siRNA (MSS247019) significantly reduced Atg5 protein expression and LC3 cleavage and p62 protein degradation (Fig. 8Aa). To confirm the effect of Atg5 knockdown, a single Atg5 siRNA (MSS247020) and a mixture of three Atg5 siRNAs (MSS247019, MSS247020, and MSS247021) were used. As shown in Fig. 8Ab, Atg5 siRNAs (MSS247020 and mixture) could also reduce Atg5 protein expression and LC3 cleavage and p62 protein degradation. These results indicate that Atg5 siRNA transfection inhibits AGEs-induced autophagy. Moreover, the AGEs-induced cleavage of caspase-3 was significantly enhanced by Atg5 siRNAs transfection (Fig. 8Aa,b). Similarly, transfection of Atg5 siRNAs significantly enhanced AGEs-induced cell apoptosis (Fig. 8Ba,b). These results may not due to the off-target effect of the Atg5 knockdown. However, transfection of Atg5 siRNA did not alter the protein expression of CHOP and the phosprylation of eIF2α in AGEs-treated mesangial cells (Fig. 8Aa). These results suggest that autophagy may play a protective role in AGEs-induced mesangial cell apoptosis. ER stress is capable of interfering with the function of autophagy in mesangial cells.


Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury
The role of autophagy in AGEs-induced mesangial cell apoptosis.Mesangial cells were transfected with single Atg5 siRNA [(MSS247019; siRNA (1), (A-a)] or mixture of Atg5 siRNAs [(MSS247019, MSS247020, and MSS247021; siRNA (2), (A-b)] or single Atg5 siRNA [(MSS247020; siRNA (3), (B-a)] or scramble control before AGEs treatment. Cells were treated with BSA (160 μg/ml) or AGEs (160 μg/ml) for 24 h. The protein levels of Atg5, p62, LC3, phospho-eIF2α, CHOP, and cleaved caspase-3 were determined by Western blot (A). The percentages of apoptotic cells were determined by PI-Annexin V staining (B). Data are presented as mean ± SEM of three independent experiments performed in duplicates (A) or triplicates (B). *P < 0.05 as compared to BSA-treated group or AGEs-treated group.
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f8: The role of autophagy in AGEs-induced mesangial cell apoptosis.Mesangial cells were transfected with single Atg5 siRNA [(MSS247019; siRNA (1), (A-a)] or mixture of Atg5 siRNAs [(MSS247019, MSS247020, and MSS247021; siRNA (2), (A-b)] or single Atg5 siRNA [(MSS247020; siRNA (3), (B-a)] or scramble control before AGEs treatment. Cells were treated with BSA (160 μg/ml) or AGEs (160 μg/ml) for 24 h. The protein levels of Atg5, p62, LC3, phospho-eIF2α, CHOP, and cleaved caspase-3 were determined by Western blot (A). The percentages of apoptotic cells were determined by PI-Annexin V staining (B). Data are presented as mean ± SEM of three independent experiments performed in duplicates (A) or triplicates (B). *P < 0.05 as compared to BSA-treated group or AGEs-treated group.
Mentions: To clarify the role of autophagy in AGEs-induced mesangial cell apoptosis, cells were transfected with scramble or Atg5 siRNA for 6 h before AGEs treatment. Transfection with Atg5 siRNA (MSS247019) significantly reduced Atg5 protein expression and LC3 cleavage and p62 protein degradation (Fig. 8Aa). To confirm the effect of Atg5 knockdown, a single Atg5 siRNA (MSS247020) and a mixture of three Atg5 siRNAs (MSS247019, MSS247020, and MSS247021) were used. As shown in Fig. 8Ab, Atg5 siRNAs (MSS247020 and mixture) could also reduce Atg5 protein expression and LC3 cleavage and p62 protein degradation. These results indicate that Atg5 siRNA transfection inhibits AGEs-induced autophagy. Moreover, the AGEs-induced cleavage of caspase-3 was significantly enhanced by Atg5 siRNAs transfection (Fig. 8Aa,b). Similarly, transfection of Atg5 siRNAs significantly enhanced AGEs-induced cell apoptosis (Fig. 8Ba,b). These results may not due to the off-target effect of the Atg5 knockdown. However, transfection of Atg5 siRNA did not alter the protein expression of CHOP and the phosprylation of eIF2α in AGEs-treated mesangial cells (Fig. 8Aa). These results suggest that autophagy may play a protective role in AGEs-induced mesangial cell apoptosis. ER stress is capable of interfering with the function of autophagy in mesangial cells.

View Article: PubMed Central - PubMed

ABSTRACT

Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2&alpha; and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney.

No MeSH data available.