• Journal of Acupuncture Research
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• v.19 no.4
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• pp.190-199
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• 2002

Objective : This study was performed to determine if Scutellaria balicalensis Georgi extract (SbGE) prevents oxidant-induced membrane transport dysfunction in renal tubular cells. Methods : Membrane transport function was estimated by measuring $Na^+$-dependent inorganic phosphate transport in opossum kidney (OK) cells. $H_2O_2$ inhibited phosphate transport in a dose-dependent manner. Results : The inhibitory effect of $H_2O_2$ was significantly prevented SbGE over concentration range of 0.005-0.05%. $H_2O_2$ caused ATP depletion, which was prevented by SbGE. $H_2O_2$ induced the loss of mitochondrial function as evidenced by decreased MTT reduction and its effect was prevented by SbGE. The $H_2O_2$-induced inhibition of phosphate transport was not affected by a potent antioxidant DPPD, but the inhibition was prevented by an iron chelator deferoxamine, suggesting that $H_2O_2$ inhibits $Na^+$-dependent phosphate transport via an iron-dependent nonperoxidative mechanism in renal tubular cells. Conclusion : These data suggest that SbGE may exert the protective effect against oxidant-induced membrane transport dysfunction by a mechanism similar to iron chelators in renal epithelial cells. However, furher studies should be carried out to find the active ingredient(s) of SbGE that exerts the protective effect.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.2
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• pp.83-92
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• 2009

This study was undertaken to examine the effect of oxidants on membrane transport function in renal epithelial cells. Hydrogen peroxide ($H_2O_2$) was used as a model oxidant and the membrane transport function was evaluated by measuring $Na^+$-dependent phosphate ($Na^+$-Pi) uptake in opossum kidney (OK) cells. $H_2O_2$ inhibited $Na^+$-Pi uptake in a dose-dependent manner. The oxidant also caused loss of cell viability in a dose-dependent fashion. However, the extent of inhibition of the uptake was larger than that in cell viability. $H_2O_2$ inhibited $Na^+$-dependent uptake without any effect on $Na^+$-independent uptake. $H_2O_2$-induced inhibition of $Na^+$-Pi uptake was prevented completely by catalase, dimethylthiourea, and deferoxamine, suggesting involvement of hydroxyl radical generated by an iron-dependent mechanism. In contrast, antioxidants Trolox, N,N'-diphenyl-p-phenylenediamine, and butylated hydroxyanisole did not affect the $H_2O_2$ inhibition. Kinetic analysis indicated that $H_2O_2$ decreased Vmax of $Na^+$-Pi uptake with no change in the Km value. Phosphonoformic acid binding assay did not show any difference between control and $H_2O_2$-treated cells. $H_2O_2$ also did not cause degradation of $Na^+$-Pi transporter protein. Reduction in $Na^+$-Pi uptake by $H_2O_2$ was associated with ATP depletion and direct inhibition of $Na^+$-$K^+$-ATPase activity. These results indicate that the effect of $H_2O_2$ on membrane transport function in OK cells is associated with reduction in functional $Na^+$-pump activity. In addition, the inhibitory effect of $H_2O_2$ was not associated with lipid peroxidation.

• The Journal of Internal Korean Medicine
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• v.25 no.4
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• pp.75-85
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• 2004

목적 : 黃芩(황금)과 黃芩(황금)의 주요 flavonoid 성분인 baicalein이 신장세뇨관 상피세포에서 산화제에 의한 apoptosis에 미치는 효과를 살펴보고자 한다. 방법 : 신장세뇨관 상피세포주인 opossum kidney (OK) 세포를 유기산화제인 t-butylhydroperoxide (tBHP)에 노출시켜 apoptosis를 일으킨 후 관련된 변화를 관찰하였다. 결과 : tBHP는 농도에 의존하여 apoptosis를 유발시켰는데, 이러한 효과는 黃芩(황금)과 baicalein에 의해 농도 의존적으로 방지 되었다. tBHP에 의한 OK 세포사는 항산화제인 Trolox와 N-acetylcysteine에 의해 방지 되었다. tBHP는 mitogen-activated protein kinase의 subfamily인 extracellular signal-regulated kinase (ERK)를 활성화시켰다. ERK 억제제인 PD98059와 U0126은 tBHP에 의한 세포 사망을 방지하였다. tBHP에 의한 ERK 활성화는 U0126에 의해 억제되었으나 黃芩(황금)과 baicalein에 의해서는 영향을 받지 않았다. 철착염제인 deferoxamine은 tBHP에 의한 세포 사망과 ERK 활성화를 방지하였다. tBHP에 의한 세포 사망은 casopase 억제제인 BOD-U-FMK와 zDEVD-FMK에 의해 방지되었다. 결론 : 黃芩(황금)은 산화제에 의한 세포 사망을 방지하는데, 이는 kinase 억제, 항산화제 역할 및 철착염제의 작용에 기인하지 않았다. 黃芩(황금)의 이러한 효과는 산화제에 의관 신부전 예방 및 치료제로 개발하는데 이용될 수 있는 가능성을 보였다.

• Journal of Acupuncture Research
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• v.21 no.3
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• pp.1-12
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• 2004

Objective: This study was undertaken to evaluate the role of lipid peroxidation in oxidant-induced apoptosis and effect of JS on the apoptosis in opossum kidney (OK) cells, an established renal proximal tubular cells. Methods : Exposure of cells to 0.1mM tBHP for 2hr did not induce apoptosis, but subsequent incubation in normal culture medium for 18hr after tBHP treatment induced apoptotic cell death which is dependent of tBHP concentration. Results : JS decreased tBHP-induced apoptotic cell death in a dose-dependent fashion and at concentrations higher than 0.01 mg/ml completely prevented the apoptosis. tBHP-induced apoptosis was prevented by the lipid soluble antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) and water-soluble antioxidant Trolox. tBHP increased lipid peroxidation, which was inhibited by JS and DPPD. tBHP-induced DNA damage was prevented by JS and DPPD. Conclusion : These results indicate that tBHP induces apoptosis through a lipid peroxidation-dependent mechanism and JS exerts the protective effect against the apoptosis by preventing peroxidation of membrane lipids.

• Journal of Acupuncture Research
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• v.18 no.1
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• pp.88-99
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• 2001

Objectives : This study was performed to determine if Orostachys japonicus A. Berger aquacupuncture (OjB) provides the protective effect against the loss of celi viability and DNA damage induced by oxidant in renal proximal tubular cells. Methods : The cell viability was evaluated by a MTT reduction assay and DNA damage was estimated by measuring double stranded DNA breaks in opossum kidney (OK) cells, an established proximal tubular cell line. Lipid peroxidation was determined by measuring malondialdehyde (MDA), a product of lipid peroxidation. Results : $H_2O_2$ increased the loss of cell viability in a time-dependent manner, which were prevented by 0.1% OjB. The protective effect of OjB was dose-dependent over concentration range of 0.05-0.5%. $H_2O_2$ caused ATP depletion and DNA damage, which were prevented by OjB and the hydrogen peroxide scavenger catalase. The loss of cell viability by $H_2O_2$ was not affected by the antioxidant DPPD, but lipid peroxidation by the oxidant was completely inhibited by DPPD. Conclusions : These data suggest that $H_2O_2$-induced death results from a lipid peroxidation-independent mechanism and the protective effect of OjB is not associated with its antioxidant activity.

• Journal of Acupuncture Research
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• v.17 no.3
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• pp.208-218
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• 2000

This study was undertaken to determine if Salviae Radix (SR) exerts protective effect against oxidant-induced inhibition of phosphate uptake in renal proximal tubular cells. Membrane transport function and cell death were evaluated by measuring phosphate uptake and trypan blue exclusion, respectively, in opossum kidney (OK) cells, an established proximal tubular cell line. $H_2O_2$ was used as a model oxidant. $H_2O_2$ inhibited the phosphate uptake in a dose-dependent manner over the concentration range of 0.1-0.5 mM. Similar fashion was observed in cell death. However, the phosphate uptake was more vulnerable to $H_2O_2$ than cell death, suggesting that $H_2O_2$-induced inhibition of phosphate uptake is not totally attributed to cell death. Decreasedphosphate uptake was associated with ATP depletion and inhibition of $Na^+$-pump activity as determined by direct inhibition of $N^+-K^+$-ATPase activity. When cells were treated with $H_2O_2$ in the presence of 0.05% SR, the inhibition of phosphate uptake and cell death induced by $H_2O_2$ was significantly attenuated. SR restored ATP depletion and decreased $Na^+-K^+$-ATPase activity, and this is likely responsible for the protective effect of SR on decreased phosphate uptake. The protective effect of SR was similar to the $H_2O_2$ scavenger catalase. SR reacts directly with $H_2O_2$ to reduce the effective concentration of the oxidant. The iron chelator deferoxamine prevented the inhibition of phosphate uptake and cell death induced by $H_2O_2$, suggesting that $H_2O_2$-induced cell injury is resulted from an iron-dependent mechanism. These results indicate that SR exerts the protective effect against $H_2O_2$-induced inhibition of phosphate uptake by reacting directly with $H_2O_2$ like the $H_2O_2$scavenger enzyme catalase, in OK cells. However, the underlying mechanism remains to be explored.

• The Journal of Korean Medicine
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• v.22 no.3
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• pp.21-30
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• 2001

목적 : 이전 연구에서 단삼 추출액이 강력한 항산화 작용이 있음을 확인한 바 있어 단삼 추출액이 신장세뇨관 세포에서 oxidant에 의한 세포사망 및 DNA 손상을 방지하는 지를 조사하고 이러한 효과가 지질의 과산화를 억제하는 효과에 기인하는 지를 시험하였다. 방법 : 신장 근위세뇨관 세포 유래 세포주인 opossum kidney (OK)세포를 이용하여 세포 사망은 frypan blue exclusion방법을 이용하여 평가하였고, DNA손상 정도는 double stranded DNA의 파괴를 측정하여 평가하였다. Oxidant 약물 모델로는 $H_2O_2$를 사용하였다. 결과 : $H_2O_2$는 적용시 간과 농도에 비례하여 세포 사망을 유도하였다. 단삼 추출액은 0.05% 농도에서 $H_2O_2$에 의한 세포사망 및 DNA 손상을 방지하였다. 이러한 방지효과는 $H_2O_2$ 제거 효소인 catalase와 철 착염제인 deferoxamine에 의해서도 나타났다. 그러나 강력한 항산화제인 DPPD는 $H_2O_2$에 의한 세포 사망이나 DNA손상을 방지하지 못하였다. $H_2O_2$는 세포내 ATP 농도를 감소시켰으며. 이러한 감소는 poly (ADP-ribose) polymerase억제제인 3-aminobenzamide에 의해 방지되었으나 단삼 추출액에 의해서는 영향을 받지 않았다. 3-aminobenzamide는 $H_2O_2$에 의한 세포 사망을 방지하였다. $H_2O_2$는 지질의 과산화를 증가시켰으며, 이러한 변화는 단삼 추출액과 DPPD에 의해 방지되었다 결론 : OK 세포에서 $H_2O_2$에 의한 세포사망과 DNA 손상에는 지질의 과산화가 중요한 역할을 하지 않으며, 단삼 추출액의 $H_2O_2$에 의한 세포 사망과 DNA 손상 방지 효과는 항산화 작용이 아닌 다른 기전에 기인하는 것으로 사료된다.

• Korean Journal of Acupuncture
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• v.24 no.1
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• pp.171-187
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• 2007

Objectives : This study was performed to determine if Orostachys japonicus A. Berger herbal acupuncture (OjB) provides the protective effect against the loss of cell viability and DNA damage induced by oxidant in renal proximal tubular cells. Methods : The cell viability was evaluated by a MTT reduction assay and DNA damage was estimated by measuring double stranded DNA breaks in opossum kidney (OK) cells, an established proximal tubular cell line. Lipid peroxidation was determined by measuring malondialdehyde (MDA), a product of lipid peroxidation. Results : H2O2 increased the loss of cell viability in a time-dependent manner, which were prevented by 0.1% OjB. The protective effect of OjB was dose-dependent over concentration range of 0.05-0.5%. H2O2 caused ATP depletion and DNA damage, which were prevented by OjB and the hydrogen peroxide scavenger catalase. The loss of cell viability by H2O2 was not affected by the antioxidant DPPD, but lipid peroxidation by the oxidant was completely inhibited by DPPD. Generation of superoxide and H2O2 in neutrophils activated by phorbol-12,13-dibutyrate was inhibited by OjB in a dose-dependent manner. OjB inhibited generation of H2O2 in OK cells treated with antimycin A and exerted a direct H2O2 scavenging effect. Exposure of OK cells to 1 mM tBHP caused a significant depletion of glutathione which was prevented by OjB. OjB accelerated the recovery in cells cultured for 20 hr in normal medium without oxidant following oxidative stress. Conclusions : These results suggest that OjB exerts the protective effect against oxidant-induced cell injury and its protective effect was resulted from radical scavenging and antioxidant activities.

• Journal of Acupuncture Research
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• v.20 no.6
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• pp.128-139
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• 2003

Juglans sinensis Dode has been reported to have antioxidant activity. However, the effect of Juglans sinensis Dode aquacupuncture(JS) on reactive oxygen species(ROS)-induced alterations in membrane transport function in renal tubular cells. This study was performed to evaluate the effect of JS on the organic hydroperoxide t-butylhydroperoxide(tBHP)-induced inhibition of $Na^+$-dependent phosphate($Na^+$-Pi) uptake in opossum kidney (OK) cells, an established renal proximal epithelial cell line. tBHP inhibited $Na^+$-Pi uptake in a time-dependent manner. The inhibitory effect of tBHP was prevented by JS over concentration range of 0.05-1mg/100ml in a dose-dependent manner. Kinetic studies showed that tBHP caused an decrease in Vmax for $Na^+$-Pi uptake without any a significant change in Km. $Na^+$-dependent phosphonoformic acid binding, a irreversible inhibitor of renal $Na^+$-Pi uptake, was decreased by tBHP treatment. The reduction in Vmax and phosphonoformic acid binding by tBHP was prevented by JS. tBHP induced lipid peroxidation and its effect was completely inhibited by JS and antioxidant N,N'-diphenyl-p-phenylenediamine. These data suggest that the oxidant inhibits phosphate uptake by a reduction in the number of active carrier across the membrane. JS may prevent oxidant-induced inhibition of membrane transport function by a mechanism similar to antioxidants in renal epithelial cells. Although the precise constituents remain to be explored, JS may be employed as a useful candidate herb for drug development to prevent and treat oxidant-mediated renal failure.