Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions

Sun Ha Lee, Tae Hyun Yoo, Bo Young Nam, Dong Ki Kim, Jin Ji Li, Dong Sub Jung, Seung Jae Kwak, Dong Ryeol Ryu, Seung Hyeok Han, Jung Eun Lee, Sung Jin Moon, Dae Suk Han, Shin Wook Kang

Research output: Contribution to journalArticle

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Abstract

Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10-7 M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.

Original languageEnglish
Pages (from-to)F1381-F1390
JournalAmerican Journal of Physiology - Renal Physiology
Volume297
Issue number5
DOIs
Publication statusPublished - 2009 Nov 1

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Podocytes
Aldosterone
Apoptosis
Cytochrome P-450 CYP11B2
Mineralocorticoid Receptors
Spironolactone
Diabetes Mellitus
Western Blotting
Diabetic Nephropathies
Staining and Labeling
Glucose
Experimental Diabetes Mellitus
DNA Nucleotidylexotransferase
Mannitol
Transferases
Proteinuria
Caspase 3
Radioimmunoassay
Sprague Dawley Rats
Real-Time Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Physiology
  • Urology

Cite this

Lee, Sun Ha ; Yoo, Tae Hyun ; Nam, Bo Young ; Kim, Dong Ki ; Li, Jin Ji ; Jung, Dong Sub ; Kwak, Seung Jae ; Ryu, Dong Ryeol ; Han, Seung Hyeok ; Lee, Jung Eun ; Moon, Sung Jin ; Han, Dae Suk ; Kang, Shin Wook. / Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions. In: American Journal of Physiology - Renal Physiology. 2009 ; Vol. 297, No. 5. pp. F1381-F1390.
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abstract = "Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10-7 M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.",
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Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions. / Lee, Sun Ha; Yoo, Tae Hyun; Nam, Bo Young; Kim, Dong Ki; Li, Jin Ji; Jung, Dong Sub; Kwak, Seung Jae; Ryu, Dong Ryeol; Han, Seung Hyeok; Lee, Jung Eun; Moon, Sung Jin; Han, Dae Suk; Kang, Shin Wook.

In: American Journal of Physiology - Renal Physiology, Vol. 297, No. 5, 01.11.2009, p. F1381-F1390.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions

AU - Lee, Sun Ha

AU - Yoo, Tae Hyun

AU - Nam, Bo Young

AU - Kim, Dong Ki

AU - Li, Jin Ji

AU - Jung, Dong Sub

AU - Kwak, Seung Jae

AU - Ryu, Dong Ryeol

AU - Han, Seung Hyeok

AU - Lee, Jung Eun

AU - Moon, Sung Jin

AU - Han, Dae Suk

AU - Kang, Shin Wook

PY - 2009/11/1

Y1 - 2009/11/1

N2 - Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10-7 M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.

AB - Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10-7 M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.

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