促紅血球生成素在缺血再灌流急性腎損傷模型小鼠中的效用與可能的作用機轉

Abstract

促紅血球生成素(erythropoietin, EPO)已普遍用來治療慢性腎臟病患者的貧血。自約西元2000年始便已有研究指出，在急性腎損傷的動物模型中，給予EPO的治療，能夠降低腎臟的損傷程度。但是關於EPO的詳細作用機轉，以及EPO與其受體之間的互動關係，甚至是受體在動物體內的表現情形等，卻遲遲未能有所定論。多數研究報告顯示刺激紅血球生成藥物(erythropoiesis-stimulating agents, ESA)對腎臟損傷有保護效用，但臨床使用顯示，ESA亦有其副作用，例如：提高心血管疾病、高血壓以及腫瘤形成的風險等，若能釐清ESA的作用機制，並找出最適當的投藥時機，或與EPO類似但具更專一性的組織保護分子，或許將能使ESA的效用發揮至最大，並期盼能對急性腎損傷的病患有所幫助。 本實驗選用八至十二週大的C57BL/6公鼠，施予右側腎臟摘除二週後緊接著左側腎臟缺血再灌流手術，做為本實驗的急性腎損傷小鼠模型，並採用不同的EPO投藥劑量與投藥時間點 (術前/術後) 來比較是否會造成療效差異。 另外，先前已有相關研究指出，EPO與不同的受體作用後，可產生兩種不同效果：若與EPOR-EPOR同型二聚體受體結合，會促進紅血球生成；若與EPOR-CSF2Rβ異型二聚體受體結合，則具有組織保護的效果。已知成年期的EPO絕大多數由腎臟血管周邊細胞製造產生，但關於EPO受體在腎臟中的表現與其功能，仍具相當大的爭議，因此成為本研究課題的一部分。 最後，延續林老師與本實驗室先前的研究主題，已知巨噬細胞在小鼠體內具備了不同的表現型，主要可分別為與發炎反應相關的M1型，將於損傷初期大量表現，以及與細胞修復再生相關的M2型，於損傷後期較為活躍，並可根據其細胞激素與趨化因子的表現情形，來辨別此兩種不同的表現型。在實驗室先前的研究中已發現，在小鼠單側輸尿管結紮的慢性腎病變模型中，給予EPO有抑制腎臟纖維化的效果，且應與EPO抑制了巨噬細胞的活化機制有關。因此，在本實驗中欲探究，在小鼠的缺血再灌流性急性腎損傷模型中，給予EPO是否亦能調控巨噬細胞，進而影響急性腎損傷與修復。 本實驗結果顯示，無論是在IRI術前或術後給予EPO，皆未能達到顯著降低腎臟損傷或促進腎功能恢復之療效，而僅提升了部分曾被指出具有促進組織修復功能的基因表現量，例如：Ccl17與Arg-1。另一方面，實驗中亦偵測了兩種EPO受體的表現情形，發現CSF2Rβ在腎臟組織中的表現遠高於EPOR，且CSF2Rβ在腎臟巨噬細胞中的表現會隨著IRI病程的進展而顯著增加，暗示了接下來的實驗可以就腎臟巨噬細胞的CSF2Rβ表現與EPO是否能藉此來調節腎臟巨噬細胞做更進一步的探討。 為了瞭解EPO是否能藉由巨噬細胞上的CSF2Rβ 受體來對其進行調控，我們未來將使用基因轉殖鼠來針對巨噬細胞上的CSF2Rβ 受體做剔除，並加以觀察EPO是否會對此基因轉殖鼠的IRI情形造成影響。若能釐清EPO與巨噬細胞之間的互動關係，或許能藉此找到對於急性腎損傷或是慢性腎臟病患者更佳的治療方針，增加病患福祉。

Erythropoietin (EPO), a kind of the erythropoiesis-stimulating agents (ESAs), is used commonly to correct anemia of chronic kidney disease (CKD) patients. Since the last decade, many studies have shown the protective role of EPO in animal models of acute kidney injury (AKI). However, the precise mechanism of EPO for tissue protection is still not fully understood. Moreover, evidence has shown that EPO administration is accompanied with side effects in clinical practice. For instance, EPO treatment may raise the risk of cardiovascular diseases, hypertension and tumor formation. Nowadays, scientists make efforts to clarify the functioning mechanisms of EPO, to define the most effective dosages or to find the alternative EPO-like molecules with higher specificity than EPO, in order to maximize the benefits of EPO and avoid side effects. In my experiments, AKI was induced by right nephrectomy followed by ischemia-reperfusion injury (IRI) surgery on left kidney 2 weeks later in 8-to-12-week-old male C57BL/6 mice. EPO was injected subcutaneously at different doses before or after IRI surgery to study whether it can prevent kidney injury or affect the repair and regeneration after kidney injury. On the other hand, it is still not clear about the interaction between EPO and its receptors, which might be EPOR-EPOR homodimer for erythropoiesis or EPOR-CSF2R heterodimer for tissue protection. In adults, EPO is produced mainly from the kidney pericytes, but the expression of EPO receptors in the kidney has not been confirmed. As a result, to detect the EPO receptors in the kidney is one of my goals in this study. In previous studies, Dr. Lin showed that macrophages can differentiate into two subtypes in vivo, which are so-called pro-inflammatory M1 macrophages and pro-reparative M2 macrophages. M1 macrophages will be dramatically increased in the early phase of injury and promote inflammation responses, whereas M2 macrophages are more active during the late phase and are associated with tissue repair and regeneration. Preliminary data in our lab suggested that EPO might ameliorate renal fibrosis in mice induced by unilateral ureteral obstruction (UUO) through inhibiting both subtypes of macrophages. Therefore, the effects of EPO on IRI kidney macrophages was investigated in this study. In present study, our data showed that recombinant human erythropoietin (rHuEPO) administration before or after IRI did not provide beneficial effects on reducing renal injury or promoting functional recovery, though it increased the expression of Ccl17 and Arg-1, which are factors that promote tissue repair and regeneration . On the other hand, the expression of CSF2Rβ was found in kidney and was significantly increased in kidney macrophages after IRI whereas EPOR expression in kidney was low. In an attempt to clarify whether rHuEPO regulates macrophages through CSF2Rβ receptor, we will use the genetic mouse model to specifically knock out CSF2Rβ on kidney macrophages and then evaluate the effects of rHuEPO on renal IRI. The final goal is to delineate the interaction between rHuEPO and macrophages, which might be the potential target cells of rHuEPO in kidney, hoping to find a better strategy of rHuEPO treatment on AKI or CKD patients.