埃他卡林肾脏保护作用药理学特征的研究
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摘要
导致肾脏损伤的因素复杂,危险因子众多。自然灾害、战争或恐怖袭击带来的挤压伤、烧伤、失血性休克、感染、中毒以及军事特殊作业环境如高原低氧、高温或高寒均可造成肾脏损伤;同时肾脏损伤也是多种心脑血管疾病、代谢性疾病如高血压、糖尿病、痛风等的严重并发症和伴发疾患;此外,多种药物及其中间代谢产物都可导致肾脏损伤。肾脏损伤作为一种可被多种因素诱发,并与多种疾病伴发,严重危害健康的疾患。因此,寻找新作用机制、有效、安全、且具有广泛临床性、适用性的抗肾脏损伤药物具有重要的实用意义和应用价值。
盐酸埃他卡林(iptakalim hydrochloride, Ipt)为新结构类型的ATP敏感性钾通道(KATP)开放剂(KCO),它对SUR2B/Kir6.1型KATP通道的开放作用强,具有一定的亚型选择性,对SUR2A/Kir6.2型作用较弱,对SUR1/Kir6.1型无激活作用[4]。本实验室的系列研究结果表明:埃他卡林对高血压所致肾脏损伤、缺血再灌注肾脏损伤及高尿酸血症所致肾脏损伤具有强效保护作用,其药理学机制与其抗高血压作用和内皮细胞保护作用密切相关[1,2,3]。其内皮细胞保护作用的分子途径与其下调大鼠血浆内皮素(ET-1)水平病理性增高;促进血管内皮细胞合成分泌一氧化氮(NO),抑制由尿酸(UA)诱导的单核细胞趋化蛋白-1(MCP-1)、细胞间粘附因子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1) mRNA表达上调等有关[1,2,3]。关于埃他卡林对于肾脏重要细胞是否具有直接的保护作用尚不清楚;关于油酸、内毒素、二甘醇其它致病因素所致肾脏损害,埃他卡林是否具有改善作用也非常值得探讨。
本实验在前期研究工作的基础上,首先在肾脏损伤致病因素尿酸、油酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞损伤的模型上,研究埃他卡林对肾脏重要细胞的保护作用,并与KCO类药物纳他卡林(Nat)和二氮嗪(Dia)的药理作用进行对比;结合KATP拮抗剂格列苯脲(Gli)和线粒体KATP拮抗剂5-羟基葵酸(5-HD)的拮抗实验,分析埃他卡林对肾脏重要细胞保护作用的药理学特征。在整体动物模型上,进一步评价埃他卡林抗其它致病因素如油酸、内毒素、二甘醇肾脏损的作用。本研究将为埃他卡林发展成为治疗肾脏损伤的药物、探讨其临床适应症提供实验依据。
第一部分:埃他卡林对肾脏重要细胞的保护作用及其药理学特征的研究
一、尿酸对肾脏重要细胞的损伤及埃他卡林保护作用的药理学特征
利用体外培养细胞和细胞存活率(MTT法)测定方法,研究了埃他卡林抗尿酸所致人肾小管上皮细胞、大鼠肾小球系膜细胞、人肾小球内皮细胞损伤作用的药理学特征。
1.埃他卡林对尿酸所致人肾小管上皮细胞损伤的影响
尿酸1200mg/l对体外培养人肾小管上皮细胞具有明显的损伤作用,细胞存活率66.0%±11.3%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,其中在10μM浓度下,细胞存活率提高到76.0%±8.1%,在100μM浓度下,细胞存活率提高到76.7%±8.1%;埃他卡林保护作用与KATP SUR2B/Kir6.1选择性开放剂纳他卡林相当,优于二氮嗪,该作用可被KATP拮抗剂格列苯脲和线粒体KATP阻断剂5-羟基葵酸所拮抗。
2.埃他卡林对尿酸所致大鼠肾小球系膜细胞损伤的影响
尿酸1200mg/l对体外培养大鼠肾小球系膜细胞具有明显的损伤作用,细胞存活率81.4%±6.6%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10、100μM时,细胞存活率分别增加至86.1%±7.3%、87.2%±10.4%、89.6%±8.1%、87.9%±8.3%,埃他卡林保护作用与纳他卡林及二氮嗪相当,也可被格列苯脲、5-羟基葵酸所拮抗。
3.埃他卡林对尿酸所致人肾小球内皮细胞损伤的影响
尿酸1200mg/l对体外培养人肾小球内皮细胞具有明显的损伤作用,细胞存活率73.1%±6.1%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10、100μM时,细胞存活率分别增加至85.0%±7.8%、87.5%±7.8%、90.5%±8.4%、90.6%±5.3%,埃他卡林保护作用与纳他卡林及二氮嗪相当;也可被格列苯脲、5-羟基葵酸所拮抗。
对比分析以上结果发现:1.尿酸对肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞具有明显的损伤作用。埃他卡林可减轻尿酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞的损伤,其中对肾小球系膜细胞、肾小球内皮细胞的保护作用强于对肾小管上皮细胞的保护作用。2.埃他卡林抗尿酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞损伤作用,与KATP SUR2B/Kir6.1选择性开放剂纳他卡林相当,二者对肾小管上皮细胞的保护作用均优于二氮嗪。二氮嗪在10μM浓度下对尿酸所致肾小管上皮细胞无保护作用。3.埃他卡林和纳他卡林均可减轻尿酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞的损伤,该保护作用可被KATP拮抗剂格列苯脲和线粒体KATP阻断剂5-羟基葵酸所拮抗,表明此保护作用与细胞膜KATP SUR2B/Kir6.1激活有关,且也可能与其激活线粒体KATP相关,但尚需进一步研究。
二、油酸对肾脏重要细胞的损伤及埃他卡林保护作用的药理学特征
利用体外培养细胞和细胞存活率(MTT法)测定方法,观察了埃他卡林抗油酸所致人肾小管上皮细胞、大鼠肾小球系膜细胞、人肾小球内皮细胞损伤作用的药理学特征。
1.埃他卡林对油酸所致人肾小管上皮细胞损伤的影响
油酸1.25μl/ml对体外培养人肾小管上皮细胞具有明显的损伤作用,细胞存活率23.3%±4.5%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10、100μM时,细胞存活率分别增加至28.6%±3.9%、30.9%±4.9%、35.2%±4.5%、36.4%±4.7%,埃他卡林保护作用弱于纳他卡林;也可被格列苯脲所拮抗。
2.埃他卡林对油酸所致大鼠肾小球系膜细胞损伤的影响
油酸1.25μl/ml对体外培养大鼠肾小球系膜细胞具有明显的损伤作用,细胞存活率39.4%±19.2%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为1、10、100μM时,细胞存活率分别增加至49.1%±15.2%,53.9%±25.2%,47.7%±9.8%,埃他卡林保护作用弱于纳他卡林;也可被格列苯脲所拮抗。
3.埃他卡林对是油酸所致人肾小球内皮细胞损伤的影响
油酸1.25μl/ml对体外培养人肾小球内皮细胞具有明显的损伤作用,细胞存活率9.1%±6.5%。埃他卡林在0.01-10μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10μM时,细胞存活率分别增加至11.7%±9.1%,11.6%±8.5%,11.9%±7.5%。埃他卡林保护作用弱于纳他卡林;该作用可被格列苯脲所拮抗。
对比分析以上结果发现:1.油酸对肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞具有明显的损伤作用。埃他卡林可减轻油酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞的损伤,其中对肾小管上皮细胞和肾小球内皮细胞的保护作用强于对肾小球系膜细胞的保护作用。2.埃他卡林抗油酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞损伤作用均弱于纳他卡林。3.埃他卡林和纳他卡林抗油酸所致肾小管上皮细胞、肾小球系膜细胞和内皮细胞损伤的作用均与其激活KATP及其亚型SUR2B/Kir6.1相关。4.埃他卡林抗油酸所致肾小管上皮细胞和肾小球内皮细胞损伤作用的有效浓度低于抗尿酸所致损伤的有效浓度;油酸所致肾脏细胞损伤的程度过重,即使在埃他卡林的保护下,肾脏细胞存活率依然很低。
第二部分:埃他卡林对其它致病因素所致肾脏损伤的影响
本研究发现埃他卡林对肾脏重要细胞包括肾小管上皮细胞、肾小球系膜细胞和内皮细胞具有直接的保护作用,以前研究发现埃他卡林对高血压所致肾脏损伤、缺血再灌注肾脏损伤及高尿酸血症所致肾损伤具有强效保护作用,在此基础上,本研究进一步探索埃他卡林对其它致病因素所致肾脏损伤的影响,为埃他卡林临床应用提供实验依据。
一、埃他卡林对内毒素休克所致肾脏损伤的影响
2%醋酸铅0.25ml/只,使动物致敏,随后注入内毒素1μg(0.1ml/100g)4h后导致内毒素性休克[5,6],血清Cr水平44.44±9.64μmol/l,较正常组21.48±1.55μmol/l显著增高,BUN水平12.65±10.92mmol/l,较正常组10.03±1.44 mmol/l显著增高。给予埃他卡林1、3和9mg/kg预防给药,其中高剂量9mg/kg组大鼠血清Cr水平21.48±1.55μmol/l 1,BUN水平10.3±0.41 mmol/l较模型组显著降低,内毒素休克所致肾脏病理变化也明显减轻。
二、埃他卡林对油酸所致肾脏损伤的影响
大鼠左肾动脉注射0.15ml/kg[7],24h后血清尿素氮水平由正常基础值7.27±0.60mmol/l显著升高到13.89±4.37mmol/l,血清肌酐水平也由正常对照组的15.78±1.96μmol/l显著升高到30.56±8.23μmol/l,埃他卡林对油酸所致肾损伤大鼠肾功能作用不明;在此实验条件下,油酸所致肾脏损伤的病理特征为肾小球毛细血管腔变窄,内皮细胞明显肿胀,坏死,球囊腔减小,基膜增厚,部分破坏,埃他卡林对油酸所致肾脏病理学损伤也无显著影响。
三、埃他卡林对二甘醇所致肾脏损伤的影响
二甘醇中毒模型组10g/kg:血清Cr水平16.71±2.27μmol/l,较正常组14.14±1.75μmol/l显著增高,给予埃他卡林1、3和9mg/kg治疗,其中高剂量9mg/kg组小鼠血清Cr水平14.54±1.62μmol/l,较模型组16.71±2.27μmol/l显著降低。在内毒素性休克、油酸、二甘醇所致肾脏损伤的模型上,埃他卡林在1、3、9mg/kg剂量下,评价其肾脏保护作用,结果表明:1.内毒素性休克大鼠模型上血清肌酐和尿素氮水平显著性升高,肾脏病理变化显示肾小球弥漫性血管内凝血所致微血栓、肾小管上皮变性和管腔内蛋白管型形成。埃他卡林1,3,9mg/kg预防给药后,剂量增加至9mg/kg时,才可显著减轻肾脏病理性损伤。2.肾动脉注射油酸导致肾脏损伤,血清肌酐和尿素氮水平显著性升高。肾小球毛细血管腔变窄,内皮细胞明显肿胀,坏死,球囊腔减小,基膜增厚,部分破坏。埃他卡林1,3,9mg/kg预防给药后,对肾脏损伤无显著性改善作用。3.小鼠腹腔注射二甘醇诱发肾脏损伤模型上,血清肌酐水平显著性增高。埃他卡林1,3,9mg/kg治疗给药后,剂量增加至9mg/kg时,可使血清肌酐水平恢复至正常水平,提示埃他卡林具有改善二甘醇所致肾功能的作用。
结合本研究室系列研究的结果分析,埃他卡林对高血压所致肾脏损伤、缺血再灌注肾脏损伤及高尿酸血症所致肾脏损伤具有强效保护作用,但对其他致病因素所致肾脏损伤的保护作用并非理想。其中,1.在内毒素所致肾脏损伤模型上,埃他卡林剂量增至9mg/kg时才呈保护作用,这可能还与其低血压的不良影响有关联2.在油酸所致肾脏损伤模型上,体外细胞实验证实埃他卡林对肾脏重要细胞的保护作用,但整体模型上未证实其肾脏保护作用,这可能与油酸所致肾脏细胞损伤严重,而埃他卡林保护作用程度不足相关3.在二甘醇所致肾脏损伤的模型上,埃他卡林剂量增至9mg/kg时才具有肾脏的保护作用,这可能与二甘醇所致肾损伤模型药理学特征表现为肾小管损伤严重,而埃他卡林对肾小管上皮细胞的保护作用较弱,对肾小球系膜细胞和内皮细胞作用较强有关联。
综上所述,本研究第一次证实了埃他卡林对尿酸所致肾脏重要细胞损伤具有直接保护作用,且存在一定的细胞差异性,其中对肾小球内皮细胞、肾小球系膜细胞的保护作用较强,对肾小管上皮细胞保护作用较弱。埃他卡林对肾脏重要细胞的保护作用与其激活细胞膜KATP亚型SUR2B/Kir6.1有关。
结合前期实验室研究发现和本实验结果,埃他卡林适用于高尿酸血症(或痛风)伴高血压且有肾脏损伤的患者,该类患者是埃他卡林的强临床适应症;其药理学途径包括:调控血压、保护血管内皮细胞和保护肾脏细胞。埃他卡林还适用于防治高血压性肾脏损伤和缺血再灌注肾脏损伤,但并不适合于内毒素性休克、挤压综合症肾脏损伤的防治;埃他卡林可否用于二甘醇所致肾损伤值得进一步研究。
本研究为埃他卡林发展成为治疗肾脏损伤的药物、确定其临床适应症提供了实验依据;同时也为抗肾脏损伤药物治疗新靶标的研究提供了实验线索。
Kidney injury is brought about by a lot of risk factors which include crush injury, burns and hemorrhagic shock, infection natural disasters, accidents,intoxation,war or terrorist attacks and military special environment such as high altitude hypoxia, heat or cold , and the kidney injury is also a serious complication or disease accompanied with many cardiovascular and cerebrovascular vascular diseases, metabolic diseases such as hypertension, diabetes, gout, etc. In addition, many drugs and their metabolites can lead to kidney damage. As matter of fact, kidney injury that is harmful to health could be induce by a variety of factors and associated with many other diseases. For this reason, the research of developing the new mechanism, the safe, the effective anti- injuries of kidney for clinical therapy is very important and has a great practical significance.
Iptakalim is a new ATP-sensitive potassium (KATP) channel opener. Iptakalim was more effective in activating Kir6.1/SUR2B channels, and less effective in activating Kir6.2/SUR2A, but not effective in activating Kir6.2/SUR1. Based on previous findings, iptakalim have protective effects against renal injuries induced by hypertension, ischemia reperfusion and hyperuricemia. Its pharmacological protective mechanism is supposed to be related with the role of anti-hypertension and the protective effects on endothelial cells. Molecular pathways of this protective effects on endothelial cells were involved in preventing pathological elevation of plasma endothelin-1(ET-1) , enhancing NO production in the endothelial cells and remarkably suppressed ICAM-1, VCAM-1, the MCP-1 gene over-expression.
Basing the foundation of the earlier work, we first observed the effects of iptakalim on damaged renal tubular epithelial cells, mesangial cells, and renal glomerulus endothelial cells induced by many risk factors for renal such as uric acid and oleic acid, and then compared with the effects of KCOs included natakalim and diaoxide. We also observed the inhibition of glibenclamide and 5-hydroxydecanoic acid to iptakalim, in order to analyses of the pharmacological characteristic of iptakalim protective effects on important renal cells. Finally we evaluated the effects of Iptakalim in anti- injuries of kidney in models induced by other pathogenic factors (eg.oleic acid, endotoxin and DEG). We would provide the experiment evidence for iptakalim developing into the anti-injuries of kidney drug and explore the clinical indication of Iptakalim through this research.
Part 1. The protective effects and pharmacological characteristics of iptakalim on important renal cells
1. The pharmacological characteristics of iptakalim protective effects against the damage of important renal cells induced by high uric acid The effects of iptakalim on the important renal cells damaged by uric acid were investigated by MTT method , and its pharmacological characteristics were further explored
1.1 Iptakalim protected against renal tubular epithelial cells damage induced by high uric acid
After incubation of cultured HK-2 cells with uric acid at the concentration of 1200mg/l for 24 h , decreased the cell survival rates( 66.0%±11.3%) was found. Pretreatment of HK-2 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 10μM and 100μM, the cell survival rates were 76.0%±8.1% and 76.7%±8.1%. The protective effect of iptakalim was consistent with natakalim and better than diazoxide, which could be reduced by both glibenclamide and 5-hydroxydecanoic acid at 10μM. 1.2 Iptakalim protected against mesangial cells damage induced by high uric acid
After incubation of cultured HBZY-1 cells with uric acid at the concentration of 1200mg/l for 24h , decreased the cell survival rates(81.4%±6.6%) was found. Pretreatment of HBZY-1 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-100μM, the cell survival rates were 86.1%±7.3%, 87.2%±10.4%, 89.6%±8.1%, 87.9%±8.3%, respectively. The protective effect of iptakalim was consistent with natakalim and diazoxide, which could be abolished by both glibenclamide and 5-hydroxydecanoic acid at 10μM.
1.3 Iptakalim protected against renal glomerulus endothelial cells damage induced by high uric acid
After incubation of cultured HRGEC cells with uric acid at the concentration of 1200mg/l for 24h , decreased the cell survival rates(73.1%±6.1%) was found. Pretreatment of HRGEC cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-100μM, the cell survival rates were 85.0%±7.8%, 87.5%±7.8%, 90.5%±8.4%, 90.6%±5.3%,respectively.The protective effect of iptakalim was consistent with natakalim and diazoxide, which could be abolished by both glibenclamide and 5-hydroxydecanoic acid at 10μM.
These results suggested that uric acid induce significant damage in tubular epithelial cells, mesangial cells and renal glomerulus endothelial cells. And iptakalim had definite but selective protective effects on these cells. The protective effects on mesangial cells and renal glomerulus endothelial cells were better than the effects on renal tubular epithelial cells. The protective effects of iptakalim against renal cells damage induced by uric acid was similar to the effect of natakalim which is a selective KATP channel opener on SUR2B/Kir6.1 subtypes. The protective effects of both drugs were better than diazoxide which had no protective effects on tubular epithelial cells damaged by uric acid. The protective effects of iptakalim and natakalim could be reduced by glibenclamide and 5-hydroxydecanoic acid at the concentration of 10μM. This finding showed that the protective effects of iptakalim and natakalim were related to activation of SUR2B/Kir6.1 subtypes of KATP on cell membrane and mitochondrion, which deserved further investigation.
2. The pharmacological characteristics of iptakalim protective effects against the damage of important renal cells induced by oleic acid
The effects of iptakalim on the important renal cells damaged by uric acid were investigated by MTT method , and its pharmacological characteristics were further explored
2.1 Iptakalim protected against renal tubular epithelial cells damage induced by oleic acid
After incubation of cultured HK-2 cells with oleic acid at the concentration of 1.25μl/ml for 24 h , decreased the cell survival rates(23.3%±4.5%) was found. Pretreatment of HK-2 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-100μM, the cell survival rates were 28.6%±3.9%, 30.9%±4.9%, 35.2%±4.5%, 36.4%±4.7%, respectively. The protective effect of iptakalim was consistent with natakalim, which could be reduced by glibenclamide at 10μM.
2.2 Iptakalim protected against mesangial cells damage induced by oleic acid After incubation of cultured HBZY-1 cells with oleic acid at the concentration of 1.25μl/ml for 24h , decreased the cell survival rates(39.4%±19.2%.) was found. Pretreatment of HBZY-1 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 1-100μM, the cell survival rates were 49.1%±15.2%, 53.9%±25.2%, 47.7%±9.8%, respectively. The protective effect of iptakalim was not as good as natakalim, which could be reduced by glibenclamide at 10μM.
2.3 Iptakalim protected against renal glomerulus endothelial cells damage induced by high uric acid
After incubation of cultured HRGEC cells with oleic acid at the concentration of 1.25μl/ml for 24 h , decreased the cell survival rates(9.1%±6.5%) was found.
Pretreatment of HRGEC cells with 0.01-10μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-10μM, the cell survival rates were 11.7%±9.1%, 11.6%±8.5%, 11.9%±7.5%,respectively. The protective effect of iptakalim was not as good as natakalim, which could be reduced by both glibenclamide at 10μM.
These results suggested that oleic acid induce significant damage in tubular epithelial cells, mesangial cells and renal glomerulus endothelial cells. And Iptakalim had definite but selective protective effects on these cells. The protective effects on renal tubular epithelial cells and renal glomerulus endothelial cells were better than the effects on mesangial cells. The protective effects of iptakalim against renal cells damage induced by oleic acid was not as good as the effect of natakalim which is a selective KATP channel opener on SUR2B/Kir6.1 subtypes. The protective effects of iptakalim and natakalim could be reduced by glibenclamide at the concentration of 10 uM. This finding showed that the protective effects of iptakalim and natakalim were related to activation of SUR2B/Kir6.1 subtypes of KATP on cell membrane .The effective concentration at which iptakalim protect against injuries of mesangial cells and renal glomerulus endothelial cells induced by uric acid is not as good as the one at which iptakalim protect against injuries of mesangial cells and renal glomerulus endothelial cells induced by oleic acid. In renal glomerulus endothelial cells, the level of the protective effects of iptakalim and natakalim against injury induced by uric acid is better than anti-oleic acid.
Part 2. The effects of iptakalim on renal injuries induced by other pathogenic factors
The result showed that iptakalim has direct protective effects on important renal cells .The previous finding suggested that iptakalim protects against hypertensive renal damage and renal injury induced by ischemia-reperfusion and by hyperuricemia. On the basis of these findings, the research will further investigate the effect of iptakalim on renal injury induced by other pathogenic factor such as LPS, oleic acidand, DEG. These investigations will provide experimental evidence for clinical indication.
1.The effects of iptakalim on renal injuries induced by LPS
In rats with the shock induced 1μg(0.1ml/100g) of LPS, significantly increased serum levels of Cr and BUN were found ,which proved renal function. The serum levels of Cr in rats changed from 21.48±1.55μmol/l to44.44±9.64μmol/l. The serum levels of BUN in rats changed from 10.03±1.44 mmol/l to12.65±10.92mmol/l. Rats with pretreatment of iptakalim at the doses of 9 mg/kg showed improved renal dysfunction and pathological changes in renal tissue. The result may be related to the role of iptakalim anti-hypertension.
2.The effects of iptakalim on renal injuries induced by oleic acid
In Rats with the renal injuries induced 0.15ml/kg of oleic acid, significantly increased serum levels of Cr and BUN were found, which proved renal function. The serum levels of Cr in rats changed from 15.78±1.96μmol/l to 30.56±8.23μmol/L. The serum levels of BUN in rats changed from 7.27±0.60mmol/l to13.89±4.37mmol/l. Iptakalim has no effect on damaged renal function and the morphological changes derived from rats with treatment of oleic acid . These result was not consistent with the result of cellular model, renal injuries induced by oleic acid was too Serious to be improved by iptakalim .
3.The effects of iptakalim on renal injuries induced by DEG
In Rats with the renal injuries induced 10g/kg of DEG, significantly increased serum levels of Cr were found, which proved renal function. The serum levels of Cr changed from 14.14±1.75μmol/L to 16.71±2.27μmol/L. Rats with pretreatment of iptakalim at the doses of 9 mg/kg showed improved renal dysfunction and pathological changes in renal tissue. The serum levels of Cr changed from 16.71±2.27μmol/L to 14.54±1.62μmol/L. Iptakalim at the doses of 9 mg/kg has a certain protective effects against renal dysfunction induced by DEG which caused severe renal tubular injury . This may be due to the protective effects of iptakalim on mesangial cells and renal glomerulus endothelial cells better than effects on renal tubular epithelial cells.
In summary, we first confirmed the direct protective effects of iptakalim on important renal cells, which had cellular selectivity. The effects of iptakalim on mesangial cells, and renal glomerulus endothelial cells were more protective than renal tubular epithelial cells. The protective effects of iptakalim were related to activation of SUR2B/Kir6.1 subtypes of KATP on cell membrane and mitochondrion, which deserved further investigation. On the basis of our previous findings, we realized that the protective effects of iptakalim against renal damage were associated with preventing hypertension and retarding the pathogenesis of endothelial dysfunction and direct effects on renal cells. Iptakalim is suitable for use in hypertensive individuals with hyperuricemia and renal injury, and also suitable for hypertensive renal injury and ischemia reperfusion renal injury, but not fit for individuals of renal damage caused by endotoxin shock, crush sydrome. The protective effects of iptakalim against renal damaged by DEG needed further investigating. We would provide the experiment evidence for iptakalim developing into the anti-injuries of kidney drug and explore the clinical indication of iptakalim through this research, meanwhile provide the experiment clues for investigating strategy for the treatment of renal damage.
盐酸埃他卡林(iptakalim hydrochloride, Ipt)为新结构类型的ATP敏感性钾通道(KATP)开放剂(KCO),它对SUR2B/Kir6.1型KATP通道的开放作用强,具有一定的亚型选择性,对SUR2A/Kir6.2型作用较弱,对SUR1/Kir6.1型无激活作用[4]。本实验室的系列研究结果表明:埃他卡林对高血压所致肾脏损伤、缺血再灌注肾脏损伤及高尿酸血症所致肾脏损伤具有强效保护作用,其药理学机制与其抗高血压作用和内皮细胞保护作用密切相关[1,2,3]。其内皮细胞保护作用的分子途径与其下调大鼠血浆内皮素(ET-1)水平病理性增高;促进血管内皮细胞合成分泌一氧化氮(NO),抑制由尿酸(UA)诱导的单核细胞趋化蛋白-1(MCP-1)、细胞间粘附因子-1(ICAM-1)、血管细胞粘附分子-1(VCAM-1) mRNA表达上调等有关[1,2,3]。关于埃他卡林对于肾脏重要细胞是否具有直接的保护作用尚不清楚;关于油酸、内毒素、二甘醇其它致病因素所致肾脏损害,埃他卡林是否具有改善作用也非常值得探讨。
本实验在前期研究工作的基础上,首先在肾脏损伤致病因素尿酸、油酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞损伤的模型上,研究埃他卡林对肾脏重要细胞的保护作用,并与KCO类药物纳他卡林(Nat)和二氮嗪(Dia)的药理作用进行对比;结合KATP拮抗剂格列苯脲(Gli)和线粒体KATP拮抗剂5-羟基葵酸(5-HD)的拮抗实验,分析埃他卡林对肾脏重要细胞保护作用的药理学特征。在整体动物模型上,进一步评价埃他卡林抗其它致病因素如油酸、内毒素、二甘醇肾脏损的作用。本研究将为埃他卡林发展成为治疗肾脏损伤的药物、探讨其临床适应症提供实验依据。
第一部分:埃他卡林对肾脏重要细胞的保护作用及其药理学特征的研究
一、尿酸对肾脏重要细胞的损伤及埃他卡林保护作用的药理学特征
利用体外培养细胞和细胞存活率(MTT法)测定方法,研究了埃他卡林抗尿酸所致人肾小管上皮细胞、大鼠肾小球系膜细胞、人肾小球内皮细胞损伤作用的药理学特征。
1.埃他卡林对尿酸所致人肾小管上皮细胞损伤的影响
尿酸1200mg/l对体外培养人肾小管上皮细胞具有明显的损伤作用,细胞存活率66.0%±11.3%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,其中在10μM浓度下,细胞存活率提高到76.0%±8.1%,在100μM浓度下,细胞存活率提高到76.7%±8.1%;埃他卡林保护作用与KATP SUR2B/Kir6.1选择性开放剂纳他卡林相当,优于二氮嗪,该作用可被KATP拮抗剂格列苯脲和线粒体KATP阻断剂5-羟基葵酸所拮抗。
2.埃他卡林对尿酸所致大鼠肾小球系膜细胞损伤的影响
尿酸1200mg/l对体外培养大鼠肾小球系膜细胞具有明显的损伤作用,细胞存活率81.4%±6.6%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10、100μM时,细胞存活率分别增加至86.1%±7.3%、87.2%±10.4%、89.6%±8.1%、87.9%±8.3%,埃他卡林保护作用与纳他卡林及二氮嗪相当,也可被格列苯脲、5-羟基葵酸所拮抗。
3.埃他卡林对尿酸所致人肾小球内皮细胞损伤的影响
尿酸1200mg/l对体外培养人肾小球内皮细胞具有明显的损伤作用,细胞存活率73.1%±6.1%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10、100μM时,细胞存活率分别增加至85.0%±7.8%、87.5%±7.8%、90.5%±8.4%、90.6%±5.3%,埃他卡林保护作用与纳他卡林及二氮嗪相当;也可被格列苯脲、5-羟基葵酸所拮抗。
对比分析以上结果发现:1.尿酸对肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞具有明显的损伤作用。埃他卡林可减轻尿酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞的损伤,其中对肾小球系膜细胞、肾小球内皮细胞的保护作用强于对肾小管上皮细胞的保护作用。2.埃他卡林抗尿酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞损伤作用,与KATP SUR2B/Kir6.1选择性开放剂纳他卡林相当,二者对肾小管上皮细胞的保护作用均优于二氮嗪。二氮嗪在10μM浓度下对尿酸所致肾小管上皮细胞无保护作用。3.埃他卡林和纳他卡林均可减轻尿酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞的损伤,该保护作用可被KATP拮抗剂格列苯脲和线粒体KATP阻断剂5-羟基葵酸所拮抗,表明此保护作用与细胞膜KATP SUR2B/Kir6.1激活有关,且也可能与其激活线粒体KATP相关,但尚需进一步研究。
二、油酸对肾脏重要细胞的损伤及埃他卡林保护作用的药理学特征
利用体外培养细胞和细胞存活率(MTT法)测定方法,观察了埃他卡林抗油酸所致人肾小管上皮细胞、大鼠肾小球系膜细胞、人肾小球内皮细胞损伤作用的药理学特征。
1.埃他卡林对油酸所致人肾小管上皮细胞损伤的影响
油酸1.25μl/ml对体外培养人肾小管上皮细胞具有明显的损伤作用,细胞存活率23.3%±4.5%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10、100μM时,细胞存活率分别增加至28.6%±3.9%、30.9%±4.9%、35.2%±4.5%、36.4%±4.7%,埃他卡林保护作用弱于纳他卡林;也可被格列苯脲所拮抗。
2.埃他卡林对油酸所致大鼠肾小球系膜细胞损伤的影响
油酸1.25μl/ml对体外培养大鼠肾小球系膜细胞具有明显的损伤作用,细胞存活率39.4%±19.2%。埃他卡林在0.01-100μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为1、10、100μM时,细胞存活率分别增加至49.1%±15.2%,53.9%±25.2%,47.7%±9.8%,埃他卡林保护作用弱于纳他卡林;也可被格列苯脲所拮抗。
3.埃他卡林对是油酸所致人肾小球内皮细胞损伤的影响
油酸1.25μl/ml对体外培养人肾小球内皮细胞具有明显的损伤作用,细胞存活率9.1%±6.5%。埃他卡林在0.01-10μM浓度范围内,可浓度依赖性地提高细胞存活率,浓度为0.1、1、10μM时,细胞存活率分别增加至11.7%±9.1%,11.6%±8.5%,11.9%±7.5%。埃他卡林保护作用弱于纳他卡林;该作用可被格列苯脲所拮抗。
对比分析以上结果发现:1.油酸对肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞具有明显的损伤作用。埃他卡林可减轻油酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞的损伤,其中对肾小管上皮细胞和肾小球内皮细胞的保护作用强于对肾小球系膜细胞的保护作用。2.埃他卡林抗油酸所致肾小管上皮细胞、肾小球系膜细胞、肾小球内皮细胞损伤作用均弱于纳他卡林。3.埃他卡林和纳他卡林抗油酸所致肾小管上皮细胞、肾小球系膜细胞和内皮细胞损伤的作用均与其激活KATP及其亚型SUR2B/Kir6.1相关。4.埃他卡林抗油酸所致肾小管上皮细胞和肾小球内皮细胞损伤作用的有效浓度低于抗尿酸所致损伤的有效浓度;油酸所致肾脏细胞损伤的程度过重,即使在埃他卡林的保护下,肾脏细胞存活率依然很低。
第二部分:埃他卡林对其它致病因素所致肾脏损伤的影响
本研究发现埃他卡林对肾脏重要细胞包括肾小管上皮细胞、肾小球系膜细胞和内皮细胞具有直接的保护作用,以前研究发现埃他卡林对高血压所致肾脏损伤、缺血再灌注肾脏损伤及高尿酸血症所致肾损伤具有强效保护作用,在此基础上,本研究进一步探索埃他卡林对其它致病因素所致肾脏损伤的影响,为埃他卡林临床应用提供实验依据。
一、埃他卡林对内毒素休克所致肾脏损伤的影响
2%醋酸铅0.25ml/只,使动物致敏,随后注入内毒素1μg(0.1ml/100g)4h后导致内毒素性休克[5,6],血清Cr水平44.44±9.64μmol/l,较正常组21.48±1.55μmol/l显著增高,BUN水平12.65±10.92mmol/l,较正常组10.03±1.44 mmol/l显著增高。给予埃他卡林1、3和9mg/kg预防给药,其中高剂量9mg/kg组大鼠血清Cr水平21.48±1.55μmol/l 1,BUN水平10.3±0.41 mmol/l较模型组显著降低,内毒素休克所致肾脏病理变化也明显减轻。
二、埃他卡林对油酸所致肾脏损伤的影响
大鼠左肾动脉注射0.15ml/kg[7],24h后血清尿素氮水平由正常基础值7.27±0.60mmol/l显著升高到13.89±4.37mmol/l,血清肌酐水平也由正常对照组的15.78±1.96μmol/l显著升高到30.56±8.23μmol/l,埃他卡林对油酸所致肾损伤大鼠肾功能作用不明;在此实验条件下,油酸所致肾脏损伤的病理特征为肾小球毛细血管腔变窄,内皮细胞明显肿胀,坏死,球囊腔减小,基膜增厚,部分破坏,埃他卡林对油酸所致肾脏病理学损伤也无显著影响。
三、埃他卡林对二甘醇所致肾脏损伤的影响
二甘醇中毒模型组10g/kg:血清Cr水平16.71±2.27μmol/l,较正常组14.14±1.75μmol/l显著增高,给予埃他卡林1、3和9mg/kg治疗,其中高剂量9mg/kg组小鼠血清Cr水平14.54±1.62μmol/l,较模型组16.71±2.27μmol/l显著降低。在内毒素性休克、油酸、二甘醇所致肾脏损伤的模型上,埃他卡林在1、3、9mg/kg剂量下,评价其肾脏保护作用,结果表明:1.内毒素性休克大鼠模型上血清肌酐和尿素氮水平显著性升高,肾脏病理变化显示肾小球弥漫性血管内凝血所致微血栓、肾小管上皮变性和管腔内蛋白管型形成。埃他卡林1,3,9mg/kg预防给药后,剂量增加至9mg/kg时,才可显著减轻肾脏病理性损伤。2.肾动脉注射油酸导致肾脏损伤,血清肌酐和尿素氮水平显著性升高。肾小球毛细血管腔变窄,内皮细胞明显肿胀,坏死,球囊腔减小,基膜增厚,部分破坏。埃他卡林1,3,9mg/kg预防给药后,对肾脏损伤无显著性改善作用。3.小鼠腹腔注射二甘醇诱发肾脏损伤模型上,血清肌酐水平显著性增高。埃他卡林1,3,9mg/kg治疗给药后,剂量增加至9mg/kg时,可使血清肌酐水平恢复至正常水平,提示埃他卡林具有改善二甘醇所致肾功能的作用。
结合本研究室系列研究的结果分析,埃他卡林对高血压所致肾脏损伤、缺血再灌注肾脏损伤及高尿酸血症所致肾脏损伤具有强效保护作用,但对其他致病因素所致肾脏损伤的保护作用并非理想。其中,1.在内毒素所致肾脏损伤模型上,埃他卡林剂量增至9mg/kg时才呈保护作用,这可能还与其低血压的不良影响有关联2.在油酸所致肾脏损伤模型上,体外细胞实验证实埃他卡林对肾脏重要细胞的保护作用,但整体模型上未证实其肾脏保护作用,这可能与油酸所致肾脏细胞损伤严重,而埃他卡林保护作用程度不足相关3.在二甘醇所致肾脏损伤的模型上,埃他卡林剂量增至9mg/kg时才具有肾脏的保护作用,这可能与二甘醇所致肾损伤模型药理学特征表现为肾小管损伤严重,而埃他卡林对肾小管上皮细胞的保护作用较弱,对肾小球系膜细胞和内皮细胞作用较强有关联。
综上所述,本研究第一次证实了埃他卡林对尿酸所致肾脏重要细胞损伤具有直接保护作用,且存在一定的细胞差异性,其中对肾小球内皮细胞、肾小球系膜细胞的保护作用较强,对肾小管上皮细胞保护作用较弱。埃他卡林对肾脏重要细胞的保护作用与其激活细胞膜KATP亚型SUR2B/Kir6.1有关。
结合前期实验室研究发现和本实验结果,埃他卡林适用于高尿酸血症(或痛风)伴高血压且有肾脏损伤的患者,该类患者是埃他卡林的强临床适应症;其药理学途径包括:调控血压、保护血管内皮细胞和保护肾脏细胞。埃他卡林还适用于防治高血压性肾脏损伤和缺血再灌注肾脏损伤,但并不适合于内毒素性休克、挤压综合症肾脏损伤的防治;埃他卡林可否用于二甘醇所致肾损伤值得进一步研究。
本研究为埃他卡林发展成为治疗肾脏损伤的药物、确定其临床适应症提供了实验依据;同时也为抗肾脏损伤药物治疗新靶标的研究提供了实验线索。
Kidney injury is brought about by a lot of risk factors which include crush injury, burns and hemorrhagic shock, infection natural disasters, accidents,intoxation,war or terrorist attacks and military special environment such as high altitude hypoxia, heat or cold , and the kidney injury is also a serious complication or disease accompanied with many cardiovascular and cerebrovascular vascular diseases, metabolic diseases such as hypertension, diabetes, gout, etc. In addition, many drugs and their metabolites can lead to kidney damage. As matter of fact, kidney injury that is harmful to health could be induce by a variety of factors and associated with many other diseases. For this reason, the research of developing the new mechanism, the safe, the effective anti- injuries of kidney for clinical therapy is very important and has a great practical significance.
Iptakalim is a new ATP-sensitive potassium (KATP) channel opener. Iptakalim was more effective in activating Kir6.1/SUR2B channels, and less effective in activating Kir6.2/SUR2A, but not effective in activating Kir6.2/SUR1. Based on previous findings, iptakalim have protective effects against renal injuries induced by hypertension, ischemia reperfusion and hyperuricemia. Its pharmacological protective mechanism is supposed to be related with the role of anti-hypertension and the protective effects on endothelial cells. Molecular pathways of this protective effects on endothelial cells were involved in preventing pathological elevation of plasma endothelin-1(ET-1) , enhancing NO production in the endothelial cells and remarkably suppressed ICAM-1, VCAM-1, the MCP-1 gene over-expression.
Basing the foundation of the earlier work, we first observed the effects of iptakalim on damaged renal tubular epithelial cells, mesangial cells, and renal glomerulus endothelial cells induced by many risk factors for renal such as uric acid and oleic acid, and then compared with the effects of KCOs included natakalim and diaoxide. We also observed the inhibition of glibenclamide and 5-hydroxydecanoic acid to iptakalim, in order to analyses of the pharmacological characteristic of iptakalim protective effects on important renal cells. Finally we evaluated the effects of Iptakalim in anti- injuries of kidney in models induced by other pathogenic factors (eg.oleic acid, endotoxin and DEG). We would provide the experiment evidence for iptakalim developing into the anti-injuries of kidney drug and explore the clinical indication of Iptakalim through this research.
Part 1. The protective effects and pharmacological characteristics of iptakalim on important renal cells
1. The pharmacological characteristics of iptakalim protective effects against the damage of important renal cells induced by high uric acid The effects of iptakalim on the important renal cells damaged by uric acid were investigated by MTT method , and its pharmacological characteristics were further explored
1.1 Iptakalim protected against renal tubular epithelial cells damage induced by high uric acid
After incubation of cultured HK-2 cells with uric acid at the concentration of 1200mg/l for 24 h , decreased the cell survival rates( 66.0%±11.3%) was found. Pretreatment of HK-2 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 10μM and 100μM, the cell survival rates were 76.0%±8.1% and 76.7%±8.1%. The protective effect of iptakalim was consistent with natakalim and better than diazoxide, which could be reduced by both glibenclamide and 5-hydroxydecanoic acid at 10μM. 1.2 Iptakalim protected against mesangial cells damage induced by high uric acid
After incubation of cultured HBZY-1 cells with uric acid at the concentration of 1200mg/l for 24h , decreased the cell survival rates(81.4%±6.6%) was found. Pretreatment of HBZY-1 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-100μM, the cell survival rates were 86.1%±7.3%, 87.2%±10.4%, 89.6%±8.1%, 87.9%±8.3%, respectively. The protective effect of iptakalim was consistent with natakalim and diazoxide, which could be abolished by both glibenclamide and 5-hydroxydecanoic acid at 10μM.
1.3 Iptakalim protected against renal glomerulus endothelial cells damage induced by high uric acid
After incubation of cultured HRGEC cells with uric acid at the concentration of 1200mg/l for 24h , decreased the cell survival rates(73.1%±6.1%) was found. Pretreatment of HRGEC cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-100μM, the cell survival rates were 85.0%±7.8%, 87.5%±7.8%, 90.5%±8.4%, 90.6%±5.3%,respectively.The protective effect of iptakalim was consistent with natakalim and diazoxide, which could be abolished by both glibenclamide and 5-hydroxydecanoic acid at 10μM.
These results suggested that uric acid induce significant damage in tubular epithelial cells, mesangial cells and renal glomerulus endothelial cells. And iptakalim had definite but selective protective effects on these cells. The protective effects on mesangial cells and renal glomerulus endothelial cells were better than the effects on renal tubular epithelial cells. The protective effects of iptakalim against renal cells damage induced by uric acid was similar to the effect of natakalim which is a selective KATP channel opener on SUR2B/Kir6.1 subtypes. The protective effects of both drugs were better than diazoxide which had no protective effects on tubular epithelial cells damaged by uric acid. The protective effects of iptakalim and natakalim could be reduced by glibenclamide and 5-hydroxydecanoic acid at the concentration of 10μM. This finding showed that the protective effects of iptakalim and natakalim were related to activation of SUR2B/Kir6.1 subtypes of KATP on cell membrane and mitochondrion, which deserved further investigation.
2. The pharmacological characteristics of iptakalim protective effects against the damage of important renal cells induced by oleic acid
The effects of iptakalim on the important renal cells damaged by uric acid were investigated by MTT method , and its pharmacological characteristics were further explored
2.1 Iptakalim protected against renal tubular epithelial cells damage induced by oleic acid
After incubation of cultured HK-2 cells with oleic acid at the concentration of 1.25μl/ml for 24 h , decreased the cell survival rates(23.3%±4.5%) was found. Pretreatment of HK-2 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-100μM, the cell survival rates were 28.6%±3.9%, 30.9%±4.9%, 35.2%±4.5%, 36.4%±4.7%, respectively. The protective effect of iptakalim was consistent with natakalim, which could be reduced by glibenclamide at 10μM.
2.2 Iptakalim protected against mesangial cells damage induced by oleic acid After incubation of cultured HBZY-1 cells with oleic acid at the concentration of 1.25μl/ml for 24h , decreased the cell survival rates(39.4%±19.2%.) was found. Pretreatment of HBZY-1 cells with 0.01-100μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 1-100μM, the cell survival rates were 49.1%±15.2%, 53.9%±25.2%, 47.7%±9.8%, respectively. The protective effect of iptakalim was not as good as natakalim, which could be reduced by glibenclamide at 10μM.
2.3 Iptakalim protected against renal glomerulus endothelial cells damage induced by high uric acid
After incubation of cultured HRGEC cells with oleic acid at the concentration of 1.25μl/ml for 24 h , decreased the cell survival rates(9.1%±6.5%) was found.
Pretreatment of HRGEC cells with 0.01-10μM iptakalim for 24 h, the cell survival rates increased with the concentration. At the concentration of iptakalim in 0.1-10μM, the cell survival rates were 11.7%±9.1%, 11.6%±8.5%, 11.9%±7.5%,respectively. The protective effect of iptakalim was not as good as natakalim, which could be reduced by both glibenclamide at 10μM.
These results suggested that oleic acid induce significant damage in tubular epithelial cells, mesangial cells and renal glomerulus endothelial cells. And Iptakalim had definite but selective protective effects on these cells. The protective effects on renal tubular epithelial cells and renal glomerulus endothelial cells were better than the effects on mesangial cells. The protective effects of iptakalim against renal cells damage induced by oleic acid was not as good as the effect of natakalim which is a selective KATP channel opener on SUR2B/Kir6.1 subtypes. The protective effects of iptakalim and natakalim could be reduced by glibenclamide at the concentration of 10 uM. This finding showed that the protective effects of iptakalim and natakalim were related to activation of SUR2B/Kir6.1 subtypes of KATP on cell membrane .The effective concentration at which iptakalim protect against injuries of mesangial cells and renal glomerulus endothelial cells induced by uric acid is not as good as the one at which iptakalim protect against injuries of mesangial cells and renal glomerulus endothelial cells induced by oleic acid. In renal glomerulus endothelial cells, the level of the protective effects of iptakalim and natakalim against injury induced by uric acid is better than anti-oleic acid.
Part 2. The effects of iptakalim on renal injuries induced by other pathogenic factors
The result showed that iptakalim has direct protective effects on important renal cells .The previous finding suggested that iptakalim protects against hypertensive renal damage and renal injury induced by ischemia-reperfusion and by hyperuricemia. On the basis of these findings, the research will further investigate the effect of iptakalim on renal injury induced by other pathogenic factor such as LPS, oleic acidand, DEG. These investigations will provide experimental evidence for clinical indication.
1.The effects of iptakalim on renal injuries induced by LPS
In rats with the shock induced 1μg(0.1ml/100g) of LPS, significantly increased serum levels of Cr and BUN were found ,which proved renal function. The serum levels of Cr in rats changed from 21.48±1.55μmol/l to44.44±9.64μmol/l. The serum levels of BUN in rats changed from 10.03±1.44 mmol/l to12.65±10.92mmol/l. Rats with pretreatment of iptakalim at the doses of 9 mg/kg showed improved renal dysfunction and pathological changes in renal tissue. The result may be related to the role of iptakalim anti-hypertension.
2.The effects of iptakalim on renal injuries induced by oleic acid
In Rats with the renal injuries induced 0.15ml/kg of oleic acid, significantly increased serum levels of Cr and BUN were found, which proved renal function. The serum levels of Cr in rats changed from 15.78±1.96μmol/l to 30.56±8.23μmol/L. The serum levels of BUN in rats changed from 7.27±0.60mmol/l to13.89±4.37mmol/l. Iptakalim has no effect on damaged renal function and the morphological changes derived from rats with treatment of oleic acid . These result was not consistent with the result of cellular model, renal injuries induced by oleic acid was too Serious to be improved by iptakalim .
3.The effects of iptakalim on renal injuries induced by DEG
In Rats with the renal injuries induced 10g/kg of DEG, significantly increased serum levels of Cr were found, which proved renal function. The serum levels of Cr changed from 14.14±1.75μmol/L to 16.71±2.27μmol/L. Rats with pretreatment of iptakalim at the doses of 9 mg/kg showed improved renal dysfunction and pathological changes in renal tissue. The serum levels of Cr changed from 16.71±2.27μmol/L to 14.54±1.62μmol/L. Iptakalim at the doses of 9 mg/kg has a certain protective effects against renal dysfunction induced by DEG which caused severe renal tubular injury . This may be due to the protective effects of iptakalim on mesangial cells and renal glomerulus endothelial cells better than effects on renal tubular epithelial cells.
In summary, we first confirmed the direct protective effects of iptakalim on important renal cells, which had cellular selectivity. The effects of iptakalim on mesangial cells, and renal glomerulus endothelial cells were more protective than renal tubular epithelial cells. The protective effects of iptakalim were related to activation of SUR2B/Kir6.1 subtypes of KATP on cell membrane and mitochondrion, which deserved further investigation. On the basis of our previous findings, we realized that the protective effects of iptakalim against renal damage were associated with preventing hypertension and retarding the pathogenesis of endothelial dysfunction and direct effects on renal cells. Iptakalim is suitable for use in hypertensive individuals with hyperuricemia and renal injury, and also suitable for hypertensive renal injury and ischemia reperfusion renal injury, but not fit for individuals of renal damage caused by endotoxin shock, crush sydrome. The protective effects of iptakalim against renal damaged by DEG needed further investigating. We would provide the experiment evidence for iptakalim developing into the anti-injuries of kidney drug and explore the clinical indication of iptakalim through this research, meanwhile provide the experiment clues for investigating strategy for the treatment of renal damage.
引文
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12. Chao-Liang Long, Xiu-Chuan Qin, Zhi-yuan Pan,et al. Activation of ATP-sensitive potassium channels protects vascular endothelial cells from hypertension and renal injury induced by hyperuricemia[J]. Journal of Hypertension, 2008, 26:2326–38
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10. Kanellis J, Watanabe S, Li JH et al. Uric acid stimulates monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein kinase and cyclooxygenase-2[J]. Hypertension,2003,41: 1287–93.
11.Khosla UM, Zharikov S, Finch JL et al. Hyperuricemia induces endothelial dysfunction[J]. Kidney Int, 2005, 67: 1739–42.
12. Chao-Liang Long, Xiu-Chuan Qin, Zhi-yuan Pan,et al. Activation of ATP-sensitive potassium channels protects vascular endothelial cells from hypertension and renal injury induced by hyperuricemia[J]. Journal of Hypertension, 2008, 26:2326–38
13. Mount DB, Kwon CY, Zandi-Nejad K. Renal urate transport[J]. Rheum Dis Clin North Am, 2006, 32:313-31, vi.
14. Waring WS, Webb DJ, Maxwell SR. Uric acid as a risk factor for cardiovascular disease[J]. Q J Med, 2000a ,93:707-713.
15. Bakhiya A,Balm A,Burckhardt G,et a1,Human organic anion transporter3 (hOAT3)can operate as an exchanger and mediate secretory urate flux[J].Cell Physiol Biochem, 2003, l3:249-56.
16. Netea MG,Kullberg BJ,Block WL,et a1.The role of hypemricemia in the increased cytokine production after lipopolysaccharide challenge in neutropenic mice[J].Blood, 1997, 89:577-82.
17. Watanabe S,Kang DH,Feng L.Uric acid,hominoid evolution,and the pathogenesis of salt-sensitivity[J]. Hypertension, 2002, 40 (3):355-60.
18. Mazzali M,Kanellls J,Han L,et a1.Hypernricemia induces a primary arteriolopathy in rats by a blood pressure independent mechanism[J]. Am J Physiol Renal Physiol, 2002, 282:991-7.
19. Kang D,Nakagawa T,Feng L,et a1.A role for uric acid in renal progression[J]. J Am Soc Nephrol, 2002, 13:2888-97.
20. Xue H, Zhang YL, Liu GS,et al . A new ATP-sensitive potassium channel opener protects the kidney from hypertensive damage in spontaneously hypertensive rats[J]. J Pharmacol Exp Ther, 2005,315(2):501-9.
21吕昌迎,龙超良,高俊钰等.埃他卡林对大鼠肾脏缺血再灌注损伤的保护作用国际药学研究杂志2008年第02期
22. Mazzali,M,Hughes J,Kim YG,,et al.Elevated uric acid increases blood pressure in the rat by a novel crystal-independent mechanism.Hypertension.2001,38:1101-1106
23. Sanchez-lozada LG,Tapia E,Avila-casado C,et al.Mild hyperuricemia induces glomerular hypertension in normal rats.Am J Physiol Renal Physiol.2002,283:F1105-F1110