Tamoxifen對缺血再灌流的急性腎損傷之影響

Abstract

急性腎損傷(Acute kidney injury, AKI)被定義為在數小時或數天內的急性腎臟功能下降，在醫療日益發達的現代，AKI的發生率以及致死率依舊居高不下，且伴隨著醫療的高支出，目前對於AKI還沒有有效的藥物能進行治療或預防，因此極需找出針對AKI有效的治療以及預防方法。 Tamoxifen (TAM)是常見的乳癌用藥，除了用於乳癌治療上，TAM也被用來活化基因轉殖鼠Cre重組酶的活性，我們發現給予C57BL/6J小鼠TAM後可以降低缺血再灌流所造成急性腎損傷的損傷程度，我們在小鼠8週大時將左側腎臟切除，並於兩週後進行腎臟缺血再灌流手術(IRI)，發現以管餵的方式在缺血再灌流前連續給予三天TAM後可以降低老鼠的腎損傷程度，給予TAM的組別血液中BUN及creatinine較低，並且在Periodic Acid-Schiff (PAS)染色的結果中腎臟損傷程度也較低，在Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL)染色的結果上，我們也發現TAM可以降低缺血再灌流後細胞凋亡的數量，透過酵素結合免疫分析法(Enzyme-Linked ImmunoSorbent Assay;ELISA)，我們發現在IRI後24小時，Neutrophil gelatinase-associated lipocalin (NGAL)的表現在給予olive oil與TAM的組別間沒有差異，但在48小時則是給予TAM的組別顯著低於給予olive oil的組別，Kidney injury molecule (KIM-1) 的表現不論在IRI後24小時或是48小時，皆是給予TAM的組別表現較低。此外，單純連續管餵TAM三天後且未進行IRI的小鼠血液中NGAL蛋白以及腎臟組織中的NGAL基因表現皆顯著上升，腎臟組織中的KIM-1基因表現也有增加，查詢相關文獻發現目前有部分研究顯示NGAL對於急性腎損傷有保護的作用，也有研究探討過KIM-1有助於急性腎損傷後的修復。我們進一步發現單純連續給予TAM且未進行IRI的小鼠在肝臟以及腎小管細胞中NGAL基因表現會增加，而嗜中性白血球NGAL的基因表現則沒有改變，此外buffy coat以及腎小管細胞的KIM-1基因表現也是增加的。另外我們也發現給予TAM可使IRI後腎臟中促發炎相關激素Tnfa、Il1b基因表現下降，而Egf基因以及粒線體功能相關基因Ppargc1a表現增加。除此之外，給予TAM後也在IRI前增加了腎臟中ki67基因的表現。 總結來說，小鼠術前三天連續給予TAM後降低了缺血再灌流的急性腎損傷，且單純給予TAM且尚未進行缺血再灌流手術就會使小鼠血液、腎臟及肝臟中的NGAL增加，未來須進一步透過NGAL基因剔除的小鼠進行實驗來證實TAM是否藉由NGAL的增加來減低急性腎損傷的嚴重程度，另外關於給予TAM後會使缺血再灌流手術小鼠腎臟粒線體功能相關基因Ppargc1a顯著增加的部分，也將進一步研究TAM是否也會藉由影響粒線體的功能來改善急性腎損傷。

Acute kidney injury (AKI) is defined as a sudden renal function lost within a few hours or a few days. In recent days, the mortality and the incidence of AKI are still high and contribute to a lot of burden of medical costs. There is no promising drug for AKI treatment or prevention currently. Thus, it is imperative to find effective therapeutics for AKI. Tamoxifen (TAM) is a medication used to treat breast cancer. Besides the anticancer effect, tamoxifen used to induce Cre recombinases for gene activation in mice. Here we demonstrated that TAM attenuated renal ischemia reperfusion injury in C57BL/6J mice. We performed right nephrectomy on 8-week-old C57BL/6J male mice and induced the ischemia-reperfusion injury in the left kidney 2 weeks later. These mice treated with TAM by oral gavage daily for three consecutive days before ischemia-reperfusion injury had lower injury severity. TAM treatment lowered plasma BUN and creatinine. Periodic Acid-Schiff (PAS) stain also showed that TAM treated mice had lower renal injury level. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining showed that TAM treated mice had lower apoptotic cells expression in renal tissues after ischemia-reperfusion injury. According to the results of Enzyme-Linked ImmunoSorbent Assay (ELISA), we found that plasma Neutrophil gelatinase-associated lipocalin (NGAL) expression had no difference between TAM treatment and olive oil treatment in 24 hours after ischemia-reperfusion injury. However, TAM treatment had lower plasma NGAL expression in 48 hours after ischemia-reperfusion injury. TAM treatment had lower expression of Kidney injury molecule (KIM-1) in both 24 and 48 hours after ischemia-reperfusion injury. In addition, our data showed that the plasma level of NGAL and gene expression of NGAL and KIM-1 in whole kidney of TAM treated mice were both elevated before ischemia-reperfusion injury, which were the plausible protective effect of TAM. Because several researchers have discussed the effect of NGAL on ischemia-reperfusion injury protection, and the role of KIM-1 signaling in renal tubule cells repair has been studied as well. According to the results, liver and renal tubule cells but not neutrophil increased NGAL secretion after TAM treatment. Moreover, buffy coat and renel tubule cells increased KIM-1 secretion. On the other hand, TAM treatment reduced inflammatory cytokines Tnfa、Il1b gene expression while induced Egf and mitochondria function related gene Ppargc1a expression in whole kidney after ischemia-reperfusion injury. TAM treatment also increased ki67 gene expression in whole kidney before ischemia-repurfusion injury. In conclusion, TAM treatment lowered acute renal ischemia reperfusion injury. On the other hand, administration of TAM without renal injury increased the NGAL plasma level and its expression in kidney and liver. In the future study, we will use NGAL gene knockout mice to confirm whether the renoprotective effect of TAM is contributed from the increase of NGAL for acute kidney injury. Regarding increased expression of mitochondria function related gene Ppargc1a after TAM treatment, we will also arrange further study to clarify if TAM has effect on mitochondira to reduce AKI.