探討CASK在腎臟缺血-再灌流損傷的角色

Abstract

腎臟是體內清除廢物、調節電解質、平衡體液的重要器官。在台灣腎病人口逐年增加，而腎病也是全球十大主要死因之一。過濾血液是腎臟的重要功能，腎小球在這種功能中扮演重要的角色。系膜細胞佔腎小球細胞的三分之一，它已被發現與腎臟纖維化具有高度相關。鈣/鈣調蛋白依賴性絲氨酸激酶（CASK）首先在腦中被發現，而後在小腸，結腸和腎臟也被報導具有高度表達。目前已有許多與CASK相關的疾病被檢測出來。在過去文獻中發現，全身性剔除CASK 基因的小鼠，在出生後數小時內便會死亡。同時也有報導指出，在CASK 基因表現低下的小鼠，其腎臟的發育會受到阻礙。然而，CASK如何參與腎臟疾病現在仍不清楚。因此，本篇研究試圖找出CASK在缺血/再灌注損傷誘導的腎損傷中的作用及其可能的機制。結果我們發現在缺血/再灌流的小鼠腎臟中，CASK蛋白量降低，然而腎小球系膜細胞株（Mes13）在缺氧後則表現量增加。 經過實驗發現，在過度表達CASK的Mes13細胞給予缺氧的刺激後，其發炎因子IL-1β、IL-6，腎損傷指標LCN2的表現量，相較於CASK 表達量正常的組別來的低。在Mes13細胞個別給予HIF-1α、ERK、或p38抑制劑時，可以減少缺氧所導致的CASK 蛋白表現量上升。同時，我們也發現CASK蛋白表現下降時，能加速Mes13的爬行速度，反之則減少其移動能力。總而言之，CASK腎臟之表現量在腎損傷過程中會受到調控，並參與損傷後的反應如炎症及纖維化，但其背後機制需要進行更詳細的研究。

Kidney is an important organ to clear the waste in our body, regulate the electrolytes, and balance the fluid. Kidney disease is the top ten leading cause of death worldwide, especially in Taiwan. Filtering the blood is an important function of kidney, and glomeruli play a major role in this function. Mesangial cells take one-third population of the cells in glomeruli, and have been found highly associated to renal fibrosis. Calcium/calmodulin-dependent serine kinase (CASK) is highly expressed in brain, small intestine, colon, and kidney. Previously it was reported that the development of kidney is delayed in CASK knockdown mice. However, how CASK involves in kidney diseases is still unclear now. Our research is trying to figure out the role of CASK in ischemia/reperfusion injury (IRI) in kidney and the possible mechanisms. We applied hypoxia condition as an environment mimicking the consequence of acute kidney injury (AKI). Here, we found that CASK protein level in kidney is decreased in IRI animal model after operation for 1 and 14 days, which represent the AKI and chronic kidney disease (CKD) states, respectively. Besides increasing renal injury markers (LCN2, Cyr61) and inflammatory cytokines (IL-6, IL-1, p65) at both disease states, expression levels of fibrotic markers (-SMA, periostin, elastin) were also enhanced in CKD state. In contrast, we found the increased CASK expression in mesangial Mes13 cells and renal tubular epithelial NRK-52E cells after hypoxia stimulation. Although hypoxia-induced cell death in Mes13 cells was not changed by CASK silencing, increased protein expressions of HIF-1, knocking down CASK inhibited HO-1 and PDGFβ and p38 activation. Meanwhile gene expression of LCN2 and IL-1 were enhanced in CASK silenced Mes13 cells. In contrast, overexpressing CASK in Mes13 cells lead to the inhibition of hypoxia-induced LCN2, IL-1, IL-6 and TNF gene expression. Of note, KC7F2 (a HIF-1α inhibitor), U0126 (an ERK inhibitor) and SB202190 (a p38 inhibitor) can reduce the elevation of CASK after hypoxia. In wound healing assay, our data revealed the increased and decreased cell migration in Mes13 cells of CASK silencing and overexpression, respectively. In summary, CASK expression in kidney is regulated upon facing the hypoxia and reperfusion stresses, and CASK might play a protective role in kidney disease by attenuating the inflammation and fibrosis.