第二型血管張力素會增加大鼠腎臟間隔細胞上多巴胺D2及D4受器mRNA的表現

Abstract

背景與目的: 糖尿病是導致腎衰竭之主因。糖尿病腎病變在早期階段會有腎絲球擴大，腎絲球過濾率增加，及尿蛋白排出增加。其中腎絲球擴大相信是第二型血管張力素刺激導致。腎絲球間隔細胞的增生及轉型是糖尿病腎病變腎功能惡化的主因。間隔細胞在腎絲球內具有平滑肌細胞、纖維母細胞、及巨噬細胞的三重身份，也是血管活性物質之作用處, 這其中包括了血管收縮劑(如第二型血管張力素)及血管鬆弛劑(如多巴胺)。在過去糖尿病大鼠的動物研究中，發現腎臟內多巴胺的合成會增加，從而活化了腎臟內的多巴胺系統，而成為糖尿病腎絲球過濾率增加的主因。此外藉由過去糖尿病大鼠的研究，也可發現糖尿病腎病變腎功能之惡化和多巴胺受器有很大的關係。多巴胺受器是一種G蛋白耦合性受體，會透過MAPKs的傳遞路徑影響了細胞之複製、分化、及存活。經由以上線索， 是否在高糖或高第二型血管張力素的作用下，大鼠腎臟間隔細胞上多巴胺D2-like 接受器(D2 接受器或D4 接受器) mRNA的表現會有所改變，是值得探討的問題。 方法: 本研究首先嘗試建立穩定的大鼠腎臟間隔細胞初級培養株的培養環境及條件，並經由反轉錄聚合酵素連鎖反應檢驗大鼠腎臟間隔細胞上有多巴胺D2-like接受器(D2接受器及D4接受器) mRNA的存在，並具有MCP-1的表現。接下來分別使用第二型血管張力素或高糖刺激大鼠腎臟間隔細胞，於不同的時間點下，檢驗多巴胺D2接受器、多巴胺D4接受器、及MCP-1 mRNA的表現差異。研究最後階段使用多巴胺D2和D4的促進劑和抑制劑，探討對大鼠腎臟間隔細胞MCP-1 mRNA的表現。 結果: 吾人發現在加入10-6M （或是1 μM）第二型血管張力素後12小時，多巴胺D2受器mRNA及多巴胺D4受器mRNA皆有明顯增加14倍的表現量。10-6M第二型血管張力素刺激後8小時, 也會明顯增加MCP-1 mRNA為2.8倍的表現量。另外雖然高糖(30nM)也會增加大鼠腎臟間隔細胞造成多巴胺D2和D4受器、及MCP-1 mRNA的表現量，但應為高滲透壓所導致之結果。使用多巴胺D2或D4的促進劑或抑制劑對於大鼠腎臟間隔細胞，於各個時間點定量MCP-1 mRNA，並未發現有明顯之變化。 結論: 本研究發現大鼠腎臟間隔細胞表現多巴胺D2及D4受器 mRNA的表現，建立了多巴胺接受器於大鼠腎臟間隔細胞 mRNA表現的細胞研究模式。進一步發現第二型血管張力素會刺激大鼠腎臟間隔細胞多巴胺D2及D4接受器及MCP-1 mRNA的表現。D2或D4受器是否會調控經由其他發炎激素，有待進一步的研究。

Aim: Diabetes mellitus is the main cause for renal failure. In the early stage of diabetic nephropathy, there are mesangial hypertrophy, glomerular hyperfiltration, and albuminuria. The early changes in mesangial cell are believed to be related to the deterioration of the renal function. Mesangial cell is a kind of smooth muscle cell, a kind of fibroblast, and also a kind of macrophage. There are target sites of vasoactive agents on it, including vasoconstrictors (ex. angiotensin II) and vasodilators (ex. dopamine). By the experimental diabetes rat model, the production of dopamine in the kidney increases, this may activate the dopaminergic system in the kidney, and causes glomerular hyperfiltration. In diabetes rat model it is also said that there is relationship between dopamine D2-like receptors and glomerular function. Dopamine receptor is a kind of G protein-coupled receptors, which may influence the cell proliferation, differentiation, and survival by intra-cellular MAPK pathway. This study was designed to find out: under the high glucose or angiotensin II stimulation as diabetes status, the mesangial cell may change the mRNA expression of dopamine D2-like receptors (either dopamine D2 receptor or dopamine D4 receptor). Methods: This study set up an adequate culturing environment and condition for the primary culture rat mesangial cell. Furthermore, the existence of dopamine D2 and D4 receptors together with MCP-1 mRNA expression in primary culture rat mesangial cell were proved by RT-PCR. Then, the angiotensin II or high glucose was added in the cell culture environment to see the different mRNA expression of dopamine D2 receptor, dopamine D4 receptor, and MCP-1 in separate time points. Finally, the dopamine D2 or D4 agonist and antagonist were added respectively to the culture environment in order to see the changes of MCP-1 mRNA expression in the primary culture rat mesangial cell. Results: After the incubation of cells for 12 hours in 10-6M angiotensin II, the dopamine D2 receptor and dopamine D4 receptor mRNA expression increased. Additionally, the mRNA expression of MCP-1 increased apparently after 8 hours of the effect of angiotensin II. 30nM high glucose in the culture environment also increased the mRNA expression of dopamine D2 receptor, dopamine D4 receptor, and MCP-1, but these should be the effect of osmolarity. No apparent changes of mRNA expression in MCP-1 was found under the effect of dopamine D2 or D4 agonist and antagonist. Conclusions: This study found the mRNA existence of dopamine D2 and D4 receptors in primary culture rat mesangial cell, and this study also established a well-performed cellular phenotype model with adequate culturing environments and conditions. Furthermore, this study demonstrated that angiotensin II increased the mRNA expression of dopamine D2、D4 receptors and MCP-1. Further study is necessary for the underlying mechanism and their relationships to the MAPKs pathway.