Investigations On The Pharmacological Characteristics Of Renal Protective Actions Of Iptakalim

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 cells1. 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 explored1.1 Iptakalim protected against renal tubular epithelial cells damage induced by high uric acidAfter 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 acidAfter 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 acidAfter 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 acidThe effects of iptakalim on the important renal cells damaged by uric acid were investigated by MTT method , and its pharmacological characteristics were further explored2.1 Iptakalim protected against renal tubular epithelial cells damage induced by oleic acidAfter 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 acidAfter 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 factorsThe 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 LPSIn 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 acidIn 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 DEGIn 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.