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Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury.

Yilmaz TU, Yazihan N, Dalgic A, Kaya EE, Salman B, Kocak M, Akcil E - Indian J. Med. Res. (2015)

Bottom Line: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury.EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (p <0.01) whereas glibenclamide decreased ( p<0.05) HIF-1 α mRNA expression.Glibenclamide significantly ( p<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Department of General Surgery, Kocaeli University, Kocaeli, Turkey.

ABSTRACT

Background & objectives: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP ) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions.

Methods: Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by Western blot.

Results: Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (p <0.01) whereas glibenclamide decreased ( p<0.05) HIF-1 α mRNA expression. Glibenclamide significantly ( p<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group.

Interpretation & conclusions: Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic/normal conditions.

No MeSH data available.


Related in: MedlinePlus

Effects of different concentrations (1,5,10,50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in hypoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).
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Figure 4: Effects of different concentrations (1,5,10,50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in hypoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).

Mentions: Determination of apoptosis: The apoptosis levels in the proximal renal tubule cells were determined by measuring the caspase-3 activity levels. In cell culture media, while cell proliferation continued, caspase activation led to apoptotic cell death. In normoxic conditions, apoptosis decreased with increased EPO amount over time (P<0.05). Similarly, treatment with Dia decreased the apoptosis over time in a dose-dependent manner (P<0.05). Glibenclamide treatment significantly increased apoptosis at 48 h as compared with the control group (P<0.05). Also, addition of Gli to EPO significantly increased the apoptosis when compared with the EPO 10 group (Fig. 3). Hypoxia significantly (P<0.01) increased the apoptosis at 48 h when compared with 2 h. EPO treatment decreased the caspase-3 levels when compared with the control group (P<0.01). Similarly, treatment with Dia decreased the apoptosis within time in a dose-dependent manner (P<0.01). Glibenclamide treatment increased the caspase-3 levels at 48 h when compared with the control group. Also, treatment with Gli significantly increased the caspase-3 levels over time (P<0.01). Addition of Gli to EPO significantly increased the apoptosis when compared with the EPO 10 group in hypoxic conditions (P<0.01) (Fig. 4).


Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury.

Yilmaz TU, Yazihan N, Dalgic A, Kaya EE, Salman B, Kocak M, Akcil E - Indian J. Med. Res. (2015)

Effects of different concentrations (1,5,10,50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in hypoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525406&req=5

Figure 4: Effects of different concentrations (1,5,10,50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in hypoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).
Mentions: Determination of apoptosis: The apoptosis levels in the proximal renal tubule cells were determined by measuring the caspase-3 activity levels. In cell culture media, while cell proliferation continued, caspase activation led to apoptotic cell death. In normoxic conditions, apoptosis decreased with increased EPO amount over time (P<0.05). Similarly, treatment with Dia decreased the apoptosis over time in a dose-dependent manner (P<0.05). Glibenclamide treatment significantly increased apoptosis at 48 h as compared with the control group (P<0.05). Also, addition of Gli to EPO significantly increased the apoptosis when compared with the EPO 10 group (Fig. 3). Hypoxia significantly (P<0.01) increased the apoptosis at 48 h when compared with 2 h. EPO treatment decreased the caspase-3 levels when compared with the control group (P<0.01). Similarly, treatment with Dia decreased the apoptosis within time in a dose-dependent manner (P<0.01). Glibenclamide treatment increased the caspase-3 levels at 48 h when compared with the control group. Also, treatment with Gli significantly increased the caspase-3 levels over time (P<0.01). Addition of Gli to EPO significantly increased the apoptosis when compared with the EPO 10 group in hypoxic conditions (P<0.01) (Fig. 4).

Bottom Line: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury.EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (p <0.01) whereas glibenclamide decreased ( p<0.05) HIF-1 α mRNA expression.Glibenclamide significantly ( p<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Department of General Surgery, Kocaeli University, Kocaeli, Turkey.

ABSTRACT

Background & objectives: Erythropoietin (EPO) has cytoprotective and anti-apoptotic effects in pathological conditions, including hypoxia and ischaemia-reperfusion injury. One of the targets to protect against injury is ATP-dependent potassium (KATP ) channels. These channels could be involved in EPO induced ischaemic preconditoning like a protective effect. We evaluated the cell cytoprotective effects of EPO in relation to KATP channel activation in the renal tubular cell culture model under hypoxic/normoxic conditions.

Methods: Dose and time dependent effects of EPO, KATP channel blocker glibenclamide and KATP channel opener diazoxide on cellular proliferation were evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] under normoxic and hypoxic conditions in human renal proximal tubular cell line (CRL-2830). Evaluation of the dose and time dependent effects of EPO, glibenclamide and diazoxide on apoptosis was done by caspase-3 activity levels. Hypoxia inducible factor-1 alpha (HIF-1 α) mRNA levels were measured by semi-quantative reverse transcription polymerase chain reaction (RT)-PCR. Kir 6.1 protein expresion was evalutaed by Western blot.

Results: Glibenclamide treatment decreased the number of living cells in a time and dose dependent manner, whereas EPO and diazoxide treatments increased. Glibenclamide (100 μM) treatment significantly blocked the anti-apoptotic effects of EPO (10 IU/ml) under both normoxic and hypoxic conditions. EPO (10 IU/ml) and diazoxide (100 μM) treatments significantly increased (p <0.01) whereas glibenclamide decreased ( p<0.05) HIF-1 α mRNA expression. Glibenclamide significantly ( p<0.01) decreased EPO induced HIF-1 α mRNA expression when compared with the EPO alone group.

Interpretation & conclusions: Our results showed that the cell proliferative, cytoprotective and anti-apoptotic effects of EPO were associated with KATP channels in the renal tubular cell culture model under hypoxic/normal conditions.

No MeSH data available.


Related in: MedlinePlus