Studies On Mechanisms And Actions Of Renal Vascular Endothelium Injury In Contrast Media-induced Nephropathy

Contrast media-induced nephropathy (CMN) is an important cause of hospital acquired renal failure. Large doses of iodinated water soluble contrast media (CM) are often administered during interventional cardiovascular procedures. The patients undergoing these examinations are usually suffering from specific diseases (such as hypercholesterolemia, hypertension, diabetic nephropathy, renal failure and so on) and vulnerable to develop into CMN, which can negatively affect the state of illness and even increase mortality. Therefore, preventing CMN effectively will ensure interventional cardiovascular procedures in safety. However, the mechanisms of renal injury induced by CM remain to be clarified. It is very important to deploy more investigations on the pathogenesis of CMN at cellular and molecular levels, in order to find preventive methods for clinical instructions.It has been suggested that the factors of CMN relate to two major causes: one is homodynamic changes of renal blood flow, which leads to anoxia and ischemia of the renal medulla, and the other is direct cytotoxic effects of contrast media on renal cells. These two aspects may also affect each other and consequently accelerate the development of CMN. Since CM could induce acute renal failure, many scholars focused their studies on the toxic actions of CM on tubular epithelial cells and tubular function. But speculating from the two major causes mentioned above, we think that the effects of CM on renal vascular endothelium may also possess an important role in the development of CMN. First, CM can directly contact with and produce toxic actions on renal vascular endothelium, and may even induce endothelial cells apoptosis or necrosis. Secondly, it has been shown that a renal vasoconstriction appeared following intravascular administration. It's well known that severe renal vasoconstriction would cause tubular anoxia and ischemia, and on the other hand, renal vasoconstriction must be related to the functional changes of endothelial cells. Therefore, the effects of CM on renal vascular endothelium should also be deeply investigated, and which may reveal the potential role of cell-biological and functional changes of renal vascular endothelium in CMN and bring a breakthrough for fully understanding the mechanisms of CMN.In the present study, in order to explore the toxic actions of CM on renal vascular endothelium and its mechanisms, by using the methods of radioimmunoassay, tetrazolium dye (MTT), western blot, reverse transcription polymerase chain reaction (RT-PCR) and fluorescent staining, we designed and carried out the following experiments:â‘ Analysis of effects of different types and doses of CM on the functional changes and cellular injury of rat renal artery endothelium;â‘¡Investigation of the influences of CM on cell viability and apoptosis in rat glomerular endothelial cells cultured in vitro;â‘¢Exploration of the mechanisms of glomerular endothelial cells injury induced by CM. Results and conclusions are summarized as follows:1. Both high and low osmolar CM affected the function of renal artery endothelium by increasing the release of endothelin-1 (ET-1) with dose dependent manner. Since they had no influence on the secretion of nitric oxide (NO), the proportion of NO/ET-1 was unbalanced and which would consequently induce renal artery constriction. The significant action was observed at the dose of 30μl/ml of Diatrizoate (high osmolar CM, HOCM), and 60μl/ml of Ioversol (low osmolar CM, LOCM). It is suggested that the effect of HOCM is stronger than LOCM on the functional changes of renal artery endothelium.2. The release of lactatedehydrogenase (LDH) increased significantly from rat renal artery endothelium after being treated with 90μl/ml of Diatrizoate for 4 hours. A slight, but nonsignificant increased release of LDH induced by Ioversol was observed. There was a notable difference of LDH release between the Diatrizoate and Ioversol groups at the concentration of 90μl/ml. It indicates that the injury of renal artery endothelium induced by Diatrizoate is more severe than Ioversol, especially at high concentrations.3. After being incubated with 90μl/ml of Ioversol for 1hours, the release of ET-1 but not NO increased in renal artery endothelium from both normal and atherosclerotic rats. However, there was no significant difference in the increased release of ET-1 between the two groups. When being incubated with 90μl/ml of Ioversol for 4 hours, the release of LDH from renal artery endothelium in atherosclerotic rats increased significantly, while it did not change obviously in normal rats. Therefore, our findings suggest that CM induce more severe injury in atherosclerotic renal artery endothelium.4. A significant decrease of cell viability was detected not only in the glomerular endothelial cells treated with Ioversol, but also in the cells treated with iso-osmolatic mannitol. However, the extent of viability suppression was much higher in Ioversol group. In addition, the release of LDH increased significantly in glomerular endothelial cells after 24 hours exposure to Ioversol but not mannitol. It implies that the cytotoxicity of Ioversol could not be simply explained by hyperosmolality, and it may be related to some other reasons.5. Further investigations showed that the viability of rat glomerular endothelial cells was inhibited by Ioversol in a dose- and time-dependent manner. After exposure of glomerular endothelial cells to 90μl/ml of Ioversol for 24 hours, except for the viability suppression, cell apoptosis was also detected by morphological observation and DNA agarose gel electrophoresis, as well as Hoechst33342/PI staining. It reflects that even non-ionic LOCM can significantly affect the viability of glomerular endothelial cells and induce apoptosis at high concentrations. Glomerular endothelial cells apoptosis would further influence the function of renal vascular endothelium, and which may contribute to the renal vasoconstriction as well as tubular ischemia and anoxia.6. Following treatment with 90μl/ml of Ioversol for 24 hours, the expression of Bcl-2 mRNA was down-regulated, and on the contrary, the expression of Bax mRNA was up-regulated. Hence the ratio of Bcl-2 mRNA to Bax mRNA increased significantly. Additionally, the protein level of caspase-3 cleaved fragments was elevated by increasing the dose of Ioversol. Compared with control group, the number of cells positively stained with Annexin V/PI was increased significantly in Ioversol group. But after being anticipated with caspase-3 inhibitor, the proportion of apoptotic cells markedly declined. Therefore, the activation of caspase-3 following the increase of Bax/Bcl-2 may be closely related to glomerular endothelial cells apoptosis induced by CM.7. After glomerular endothelial cells incubated with 90μl/ml of Ioversol for 24 hours, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and the protein level of cytochrome-C increased markedly, but there was no significant change in the content of glutathione peroxides (GSH-PX). It hints that the mechanism of glomerular endothelial cells apoptosis induced by CM may be mostly related to the disruption of calcium homeostasis and mitochondrial injury.In conclusion, the present study demonstrates that CM can affect the function of renal vascular endothelium. Large doses of LOCM can markedly inhibit glomerular endothelial cells viability and even induce apoptosis. The disruption of calcium homeostasis and mitochondrial injury maybe contribute to glomerular endothelial cell injury induced by CM.