近端腎小管XBP1缺失加劇缺血/再灌流誘導急性腎損傷到慢性腎臟病脂質累積

Abstract

慢性腎臟病 (chronic kidney disease, CKD)最終可能發展為終末期腎臟衰竭 (end-stage renal disease, ESRD)，其具有高盛行率以及高死亡率，並造成龐大的醫療負擔，在現今社會已成為全球迫切關注的公共衛生議題。近年許多的研究證據顯示，在急性腎臟損傷 (acute kidney injury, AKI)後，腎臟功能復原情形較差的病患可能是進展成CKD的潛在族群，因此驅使越來越多的研究著重於急性腎臟損傷至慢性腎臟病的機制探討。臨床上，纖維化為CKD不可逆的組織學表現，其中脂質累積以及脂肪酸β-氧化在腎臟病發展中扮演重要角色，特別是近端腎小管細胞，其對於能量需求極大，並以脂肪酸作為其主要能量來源。先前研究指出，當脂質代謝異常或粒線體功能障礙，都可能導致脂質累積，進而促進纖維化以及腎臟病的發展。根據本實驗室先前的研究成果，透過小鼠單側腎臟缺血再灌流損傷 (unilateral ischemia reperfusion injury, UIRI)，模擬急性腎臟損傷後腎臟纖維化進程的研究結果顯示，未折疊蛋白質反應作用因子X-box binding protein 1 (XBP1)的表現量與纖維化呈現負相關，我們進一步發現到UIRI後腎組織脂質累積增加，並伴隨調節脂肪酸合成轉錄因子：sterol regulatory element-binding protein 1 (SREBP-1)上升和調節脂肪酸β-氧化轉錄因子：peroxisome proliferator-activated receptor alpha (PPARα)下降。根據上述結果，本研究假設XBP1對於腎臟脂質累積和脂質代謝中扮演重要角色，首先，我們透過腎小管上皮細胞特異性XBP1基因剃除小鼠(XBP1 cKO)去驗證此推論，發現到在UIRI後XBP1 cKO 小鼠與wild-type 小鼠相比，其脂質累積加劇和SREBP-1, α-SMA表現顯著上升以及PPARα表現顯著下降的現象。此外，我們也證實在UIRI後處理fenofibrate的小鼠能夠維持PPARα表現，並能回復XBP1的表現以及減輕纖維化和脂質累積。接著在in vitro模式，我們透過人類腎臟近端腎小管上皮細胞 (HK-2)去探討XBP1缺乏下對脂質累積的影響，透過添加棕櫚酸 (Pa-BSA)以加強脂質累積的現象，並藉由Oil Red O染色觀察，發現到XBP1缺失下會造成脂質累積上升，也透過活細胞成像顯微鏡觀察到XBP1缺失下油滴數量增加。脂質代謝異常下會造成脂質累積，我們進一步觀察XBP1缺乏對於脂肪酸合成、脂肪酸攝取和脂肪酸β-氧化的影響，結果指出在XBP1缺乏下SREBP-1以及脂肪酸攝取膜蛋白：cluster of differentiation 36 (CD36) 表現顯著上升，而PPARα表現顯著下降。由於粒線體脂肪酸β-氧化主要由PPARα調控，因此粒線體功能障礙也是造成脂質累積的因素之一，我們接著去探討XBP1對於粒線體功能的影響，則結果發現到XBP1缺乏下，粒線體功能受到顯著的損害，我們進一步分析多種PPARα標的基因，發現到其中carnitine palmitoyltransferase-2 (CPT2) 表現上升和acyl-CoA dehydrogenase family member 11 (Acad11)表現下降。在先前文獻已指出PPARα在肝臟細胞中受到XBP1s調控，我們進一步去證明XBP1在腎小管細胞中調控PPARα的機制，透過大量表現XBP1s和XBP1u，發現到PPARα表現上升，進而透過Luciferase assay發現XBP1s會和PPARα promoter相互作用。本研究發現在腎小管上皮細胞中，XBP1可能作為調控脂質代謝以及粒線體功能的重要角色，缺乏XBP1下會造成脂質代謝異常及線粒體功能障礙，並加劇脂質累積，最後促使纖維化的進展。

Chronic kidney disease (CKD) progression to end-stage renal disease (ESRD), which causes the high prevalence and mortality and huge economic burden. Nowadays, it has turned into the public health issues of global concern. In recent years, there is a lot of evidence that patients with poor ability to recover after acute kidney injury (AKI) may be the potential group of CKD. Thus, prompting more and more studies focused on mechanism investigation of AKI to CKD transition. Clinically, fibrosis is an irreversible histological manifestation of CKD. However, the lipid accumulation and fatty acid β-oxidation play a crucial role in the development of renal disease, which is especially for proximal tubular cells (PTCs). Owing to the high-energy demand, and they use fatty acid as the major energy source. Previous studies have showed that the aberrant lipid metabolism or mitochondrial dysfunction cause the lipid accumulation, which further promoted the progression of fibrosis and renal disease. In our previous study, we mimicked the process of renal fibrosis after acute kidney injury using the mice model of unilateral ischemia reperfusion injury (UIRI). We observed the expression of the unfolded protein response effector, X-box binding protein 1 (XBP1) was negatively correlated with the degree of post-AKI fibrosis. In addition, we further found the gradual increase of lipid accumulation in renal tissue after UIRI, and accompanied with the increase of fatty acid synthesis regulated transcription factor-sterol regulatory element-binding protein 1 (SREBP-1) and the decrease of fatty acid β-oxidation regulated transcription factor-peroxisome proliferator-activated receptor alpha (PPARα). On the basis of above results, this study hypothesized that XBP1 may play an important role in lipid accumulation and lipid metabolism of renal. Then, we examined this suppose using renal tubular epithelial specific XBP1 knockout (XBP1 cKO) mice. We observed the lipid accumulation was aggravated, the expression of SREBP-1 was significantly increased, and the expression of PPARα was significantly decreased in XBP1 cKO mice after UIRI. Moreover, we confirmed that fenofibrate-treated mice maintain the expression of PPARα after UIRI. Simultaneously, the expression of XBP1 were recovered, and ameliorated the fibrosis and lipid accumulation. In the in vitro model, we first investigated the impact of XBP1 deficiency on the lipid accumulation using human renal proximal tubule epithelial cells (HK-2). We added the palmitate (Pa-BSA) to enhance the lipid accumulation and observed with Oil Red O staining. The data showed XBP1 deficiency promoted the lipid accumulation. Also, we observed the XBP1 deficiency increased the number of lipid droplets by using Nanolive 3D cell explorer. We further examined the impact of XBP1 deficiency on fatty acid synthesis, fatty acid uptake and fatty acid β-oxidation. The results showed that the expression of SREBP-1 and fatty acid uptake membrane protein—cluster of differentiation 36 (CD36) were significantly increased, whereas the expression of PPARα was significantly decreased in the XBP1 deficiency. Moreover, the fatty acid β-oxidation is mainly regulated by PPARα and plays a role in mitochondria. Therefore, the mitochondrial dysfunction is also one of the cause that result in the lipid accumulation. We further explored the effect of XBP1 on mitochondrial function. The data showed that XBP1 deficiency was markedly inhibited the mitochondrial function. We further analyzed a variety of PPARα target genes. The XBP1 deficiency were up-regulated the expression of carnitine palmitoyltransferase-2 (CPT2), whereas down-regulated the expression of acyl-CoA dehydrogenase family member 11 (Acad11). The previous study have indicated that PPARα is regulated by XBP1s in liver cells. Thus, we further elucidated the potential mechanisms of XBP1-regulated PPARα. Through the overexpression of XBP1s and XBP1u, we found that the expression of PPARα was increased. We further used the luciferase assay to find that XBP1s could interact with PPARα promoter. To summary our results, XBP1 play an important role in regulating lipid metabolism and mitochondrial function in PTCs. The lack of XBP1 will cause aberrant lipid metabolism and mitochondrial dysfunction, resulting in lipid accumulation, and eventually promote the progression of fibrosis.