Role of follistatin-like 1 (FSTL1) in kidney fibrosis

Ph.D.Kidney fibrosis is a final common pathway of progressive chronic kidney disease and is characterized by the increase of interstitial fibroblasts and myofibroblasts, and the excessive production and accumulation of extracellular matrix (ECM) proteins, such as collagens and fibronectins, within the kidney. The Wnt family of ligands controls a variety of cellular activities. Wnt/β-catenin signalling in the kidney is silent in normal adults, but it is reactivated in tubular cells, pericytes, fibroblasts and macrophages in injured kidneys. Wnt/β-catenin signaling has been found to promote renal fibrosis by facilitating partial tubular epithelial-mesenchymal transition (EMT)/dedifferentiation, and promoting myofibroblast differentiation, proliferation and function. Follistatin-like 1 (FSTL1) is a secreted glycoprotein and comprises a follistatin domain and an extracellular calcium-binding (EC) domain. The functions of FSTL1 have been studied in heart injury, lung development and fibrosis, arthritis, and wound healing. However, the role of FSTL1 in kidney is largely unknown. In the present study, we showed that FSTL1 expression in the kidney was dramatically increased 1, 3, and 7 days after unilateral ureteral obstruction (UUO), while it was not changed by bilateral ischemia/reperfusion (I/R) and cisplatin nephrotoxicity in mice. Immunofluorescent staining showed that FSTL1 was expressed in fibroblasts in the kidneys with UUO. Overexpression of FSTL1 in vivo aggravated renal fibrosis after UUO, as indicated by increased expression of α-SMA, fibronectin and collagen I. Immunoprecipitation assay (IP) demonstrated that FSTL1 interacted with various Wnt ligands in vitro, both caonical and non-canonical, including Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt8a, Wnt8b, Wnt9a, and Wnt10b, which are concurrently increased in fibrotic kidneys after UUO. In addition, FSTL1 further upregulated the β-catenin responsive 8× TOPFlash luciferase reporter activity induced by canonical Wnt ligands in mIMCD3, HEK293T, and NRK-49F cells. Overexpression of FSTL1 in vivo also promoted the activation of Wnt/β-catenin signaling as shown by increased expression of active β-catenin, β-catenin, phospho-GSK3β, and cyclin D1 in UUO mice. Taken together, these results suggest FSTL1 promotes kidney fibrosis by amplifying Wnt/β-catenin signaling.腎纖維化是進行性慢性腎病的最終途徑，其特點是間質成纖維細胞和肌成纖維細胞的增加，細胞外基質蛋白（ECM）如膠原蛋白和纖連蛋白的過量產生和積累。Wnt配體家族可以控制多種細胞活動。腎臟中的Wnt/β-catenin信號通路在正常成人腎臟中是沉默的，但它在受損腎臟中的腎小管細胞，周細胞，成纖維細胞和巨噬細胞中被重新激活。以前的研究已發現Wnt/β-catenin信號通路通過促進腎小管上皮-間質轉化（EMT）/去分化，促進肌成纖維細胞分化，增值和功能来促進腎纖維化。卵泡抑素樣蛋白1 （FSTL1）是一種分泌型糖蛋白，包含卵泡抑素結構域和細胞外鈣結合（EC）結構域。FSTL1的功能已經在心臟損傷，肺發育和纖維化，關節炎和傷口愈合中研究過。然而，FSTL1在腎臟中的作用在很大程度上是未知的。在本研究中，我們發現單側輸尿管結扎（UUO）後1,3和7天腎臟中FSTL1的表達量顯著增加，但是雙側缺血再灌注（I/R）和順鉑導致的腎毒性腎病中FSTL1的表達量沒有改變。免疫熒光染色結果顯示FSTL1在UUO小鼠腎臟中的成纖維細胞中表達。體內過表達FSTL1加重了UUO引起的腎纖維化，表現為α-SMA、纖連蛋白和I型膠原蛋白的表達增加。免疫沉澱試驗（IP）證明FSTL1在可以與多種經典和非經典Wnt配體相互作用，包括Wnt1、 Wnt2、 Wnt3a、Wnt4、 Wnt5a、 Wnt7a、 Wnt8a、 Wnt8b、 Wnt9a和Wnt10b，這些配體都是在UUO 小鼠中上升的配體。另外，在mIMCD3, HEK293T和NRK-49F 細胞中FSTL1進一步上調了由Wnt配體誘導的β-catenin介導的8× TOPFlash 熒光素酶報告基因的活性。體內過表達FSLT1也促進Wnt/β-catenin信號通路的激活，提高UUO小鼠腎臟中β-catenin、 active β-catenin、phospho-GSK3β和cyclin D1的表達量。總之，這些結果表明FSTL1可以通過增強Wnt/β-catenin信號通路的活性來促進腎纖維化。Zhang, Yu.Thesis Ph.D. Chinese University of Hong Kong 2019.Includes bibliographical references (leaves 141-157).Abstracts also in Chinese.Title from PDF title page (viewed on 23, December, 2020)