了解RXR-SerpinB2-TGM2軸線在腎小管細胞衰老的作用

Abstract

人類平均壽命的延長使老化成為多數人會面對的問題，腎臟作為人體重要的代謝和排毒器官，其老化過程不僅直接影響到個體健康，還是許多年齡相關疾病的關鍵因素，例如慢性腎病 (chronic kidney disease, CKD)、第2型糖尿病和高血壓等，再者CKD還會加劇全身性器官的老化。因此了解腎臟老化與損傷的機制並設法延緩或改善具有重要意義。 已知隨著細胞分裂，端粒不斷縮短會引起DNA損傷反應 (DNA damage response, DDR)，誘導轉錄調節因子p53蛋白活化，促使下游基因Serpinb2和p21的表達增加。p21蛋白（也稱為CDKN1a）可結合並抑制細胞週期依賴性激酶 (cyclin-dependent kinase, CDK)的酵素活性，而SerpinB2會透過活化轉麩醯胺酸酶2 (transglutaminase 2, TGM2)的脫胺作用來穩定p21蛋白的結構，進而導致細胞週期停滯和細胞衰老。在過往的實驗發現，無論是老化的腎小管細胞，還是老年小鼠（24月齡）或是損傷小鼠模型的腎臟，SerpinB2的基因表達均顯著高於年輕的對照組。此外，給予小鼠類視網醇X受體 (retinoid X receptors, RXR)活化劑bexarotene的實驗中，觀察到其腎臟中另一個細胞週期抑制劑p16蛋白（也稱為CDKN2a或p16INK4a）的表現增加，意指RXR路徑與調節細胞週期、腎臟老化有關。故本研究是為了探討RXR-SerpinB2-TGM2訊號軸在腎小管細胞衰老中的作用。 由免疫螢光 (immunofluorescence, IF)染色確認RXR分布於小鼠的腎小管上皮細胞，故使用小鼠初代腎小管上皮細胞 (primary tubular epithelial cells, PTECs)來進行後續實驗。在觀察複製型衰老 (replicative senescence)的群體倍增 (population doubling, PD)實驗中，無法看到PTECs的自然老化，生長曲線持續上升，但給了RXR活化劑bexarotene一段時間後細胞就會出現生長停滯，生長曲線逐漸平緩；若給RXR抑制劑HX531時細胞則持續分裂，甚至比對照組上升得更快，不會出現生長停滯，表示活化與抑制RXR途徑會影響PTECs的生長。進一步看PTECs給予bexarotene和HX531後的基因和蛋白質表現變化，Tgm2基因的表達隨著給bexarotene顯著上升，給HX531則顯著下降，而p16、p21、p53以及部分衰老相關分泌表型 (senescence associated secretory phenotype, SASP)基因的表現在給bexarotene或HX531後與對照組皆無差異；在蛋白質表現的分析中，可發現p21、SerpinB2和TGM2在給予bexarotene時表現量顯著上升，而給予HX531時p21、SerpinB2和TGM2的表現量顯著下降，p16、p53的蛋白表現則在給bexarotene或HX531後與對照組無差異。以上代表在PTECs中，RXR對SerpinB2-TGM2軸線的基因和蛋白表現具有調控作用，但不影響促進發炎或纖維化的細胞激素表達。 在細胞週期分析實驗中，發現給bexarotene會使細胞週期停滯在G0/G1，傷口癒合實驗得知bexarotene作用讓PTECs生長與爬行較慢。同時，有加bexarotene的組別其衰老相關的β-半乳糖苷酶 (senescence-associated β-galactosidase, SA-β-gal)表現量上升且可被TGM2的活性抑制劑cystamine抑制，TGM2活性檢測，驗證了給bexarotene使TGM2活性增加，且同樣可受cystamine抑制。活化RXR會影響PTECs的衰老表型增加，而透過抑制TGM2活性，可以反轉活化RXR的作用效果。另外，用免疫螢光染色發現TGM2表現在小鼠的腎小管上皮細胞與間質，且在小鼠急性腎損傷 (Acute kidney injury, AKI)模型之腎臟組織的表現量會增多，而SerpinB2則表現在小鼠的腎小管上皮細胞及刷狀緣 (brush border)。未來我們將利用原位雜交技術確定表達Tgm2的腎細胞，並探討一種腎細胞分泌的TGM2是否可能影響另一種細胞。計劃利用基因轉殖技術在小鼠中進行Tgm2全身性及專一性剔除，觀察基因剔除對壽命的影響，同時驗證細胞層面觀察到的實驗結果，並給予AKI模型的小鼠bexarotene或HX531，看活化、抑制RXR對於腎臟損傷與修復的影響，將有助於深入理解RXR-SerpinB2-TGM2軸線在腎臟老化與損傷過程中的具體作用，並為針對這一途徑的治療策略開發提供實驗基礎。

The increase in human life expectancy has made aging a common issue faced by many. As a crucial metabolic and detoxifying organ, the kidney plays a significant role in individual health. It is a key factor in numerous age-related diseases such as chronic kidney disease (CKD), type 2 diabetes, and hypertension. Moreover, CKD can exacerbate the aging of systemic organs. Thus, it is important to understand the mechanisms of renal aging and injury and find ways to slow or improve them. Telomere attrition with each cell division initiates a DNA damage response (DDR), activating the transcription regulator p53, which enhances Serpinb2 and p21 gene expressions. The p21 protein inhibits the activity of cyclin-dependent kinases (CDKs), and SerpinB2 stabilizes p21 through the action of transglutaminase 2 (TGM2), leading to cell cycle arrest and senescence. Previous experiments have found that whether in senescent renal tubular cells, aged mice (24 months old), or an injury mouse model, the gene expression of SerpinB2 is significantly higher than in young control groups. Furthermore, in experiments in which mice were given the retinoid X receptor (RXR) activator bexarotene, an increase in the expression of another cell cycle inhibitor protein p16 was observed, indicating that the RXR pathway is involved in regulating the cell cycle and kidney aging. Therefore, this study aims to explore the role of the RXR-SerpinB2-TGM2 signaling axis in renal tubular cell senescence. Immunofluorescence staining confirmed the distribution of RXR in the renal tubular epithelial cells of mice, so primary tubular epithelial cells (PTECs) were used for subsequent experiments. In the population doubling (PD) experiments for observing replicative senescence, the natural aging of PTECs was not observed, and the growth curve continued to rise. However, after a period of treatment with the RXR activator bexarotene, cell growth stalled, and the growth curve gradually flattened. When the RXR inhibitor HX531 was administered, the cells continued to divide, even increasing faster than the control group without showing growth arrest. This indicates that activating and inhibiting the RXR pathway affects the aging and growth of PTECs, respectively. Further experiments revealed that bexarotene and HX531 increased and decreased the expression of Tgm2 gene in PTECs, respectively. Meanwhile, the gene expression of cell cycle regulators including p16, p21, p53, and senescence-associated secretory phenotype (SASP) including IL1β, MMP3, and TGFβ1 remained unchanged compared to the control group after treatment with bexarotene or HX531. However, the protein expression of p21, SerpinB2, and TGM2 significantly increased with bexarotene treatment, while they decreased significantly with HX531 treatment. The protein expressions of p16 and p53 remained unchanged compared to the control group after treatment with bexarotene or HX531. This indicates that RXR activation upregulates the protein expression of SerpinB2-TGM2-p21 axis in PTECs but does not affect the expression of cytokines that promote inflammation or fibrosis. In cell cycle analysis experiments, bexarotene was found to cause cell cycle arrest at G0/G1 phase and fewer cells entering S phase. Wound healing experiments revealed that bexarotene treatment slowed the growth and migration of PTECs. Additionally, the group treated with bexarotene exhibited an increase in senescence-associated β-galactosidase (SA-β-gal) expression, which could be inhibited by the TGM2 inhibitor cystamine. TGM2 activity assay confirmed that bexarotene increased TGM2 activity, which could also be inhibited by cystamine. These data indicate that RXR activation promotes the aging phenotype of PTECs; however, TGM2 inhibition by cystamine can reverse the effects of RXR activation. We found that TGM2 is expressed in the renal tubular epithelial cells and interstitium of mice by immunofluorescence staining. Its expression increases in kidney tissues of acute kidney injury (AKI) mouse models, while SerpinB2 is expressed in the renal tubular epithelial cells and brush border of mice. We will first clarify Tgm2-expressing kidney cells by in situ hybridization and the possibility of TGM2 secretion from one kidney cell type affecting the other cell type. Plans include using transgenic mice for systemic and specific knockout of Tgm2 to observe the impact of gene knockout on lifespan and renal function during aging and diseases, while also verifying the experimental results observed at the cellular level. Administering bexarotene or HX531 to mice in AKI models to see the effects of activating and inhibiting RXR on kidney injury and repair will help deepen the understanding of the specific role of the RXR-SerpinB2-TGM2 axis in renal aging and diseases. This will provide an experimental basis for the development of therapeutic strategies targeting this pathway.