蔡佳盈　沈世忠　綜述　萬(wàn)建新　審校

近年的研究表明血管緊張素Ⅱ(Ang Ⅱ)通過(guò)其AT1受體(AT1R)導(dǎo)活性氧(ROS)的產(chǎn)生[1，2]，活化瞬時(shí)受體電位通道蛋白6(TRPC6)并增強(qiáng)TRPC6的核轉(zhuǎn)錄，上調(diào)及活化的TRPC6通道可增加Ca2+內(nèi)流并介導(dǎo)足細(xì)胞肌動(dòng)蛋白骨架的重排致足突融合、參與足細(xì)胞損傷及凋亡的發(fā)生、發(fā)展。本文分別闡述足細(xì)胞TRPC6通道、Ang Ⅱ上調(diào)及活化TRPC6通道、上調(diào)及活化TRPC6通道在Ang Ⅱ致足細(xì)胞損傷及凋亡中的作用。

足細(xì)胞TRPC6通道

經(jīng)典型TRPC是存在于哺乳類(lèi)動(dòng)物細(xì)胞膜上的一類(lèi)重要的非選擇性陽(yáng)離子通道。該家族總共有7個(gè)亞型， TRPC為同源或異源四聚體蛋白，含有6個(gè)跨膜亞單位，一些TRPC(包括TRPC6)其細(xì)胞內(nèi)氨基端和羧基端可結(jié)合鈣調(diào)素。G蛋白偶聯(lián)受體(GPCR)及受體酪氨酸激酶活化后，參與細(xì)胞內(nèi)Ca2+濃度的調(diào)控。在TRP家族中，TRPC6亞型被認(rèn)為是選擇性最強(qiáng)的通道蛋白，通過(guò)介導(dǎo)Ca2+內(nèi)流而發(fā)揮生理作用[2]。在腎臟生理進(jìn)程中，該通道涉及腎小管Ca2+和Mg2+的重吸收，滲透壓調(diào)節(jié)等。足細(xì)胞表達(dá)的TRPC6可相互結(jié)合在胞膜形成同型或異型四聚體離子通道。2005年，Winn等[3]首次發(fā)現(xiàn)在家族遺傳性局灶節(jié)段性腎小球硬化(FSGS)患者中存在TRPC6基因突變，并且TRPC5和TRPC6被認(rèn)為是足細(xì)胞中的Ca2+內(nèi)流通道，而TRPC6對(duì)Ca2+的通透是無(wú)限的，因此TRPC6介導(dǎo)的Ca2+內(nèi)流活化下游信號(hào)分子也是充足的，故可理解為信號(hào)整合及放大器。TRPC6亦屬于足細(xì)胞裂孔隔膜蛋白，免疫共沉淀法顯示TRPC6和裂孔隔膜蛋白nephrin及podocin緊密聯(lián)系，提示TRPC6可能涉及裂孔隔膜的信號(hào)傳導(dǎo)[4,5]。

Ang Ⅱ通過(guò)AT1R介導(dǎo)足細(xì)胞損傷

已證實(shí)大部分腎臟細(xì)胞，包括足細(xì)胞、系膜細(xì)胞及腎小管上皮細(xì)胞可產(chǎn)生Ang Ⅱ[6,7]，腎素-血管緊張素系統(tǒng)(RAS)在調(diào)節(jié)心腎功能發(fā)揮重要作用，但是，過(guò)多RAS活化將促進(jìn)炎癥和氧化應(yīng)激，導(dǎo)致腎小球?yàn)V過(guò)屏障損害和蛋白尿[8]。關(guān)于Ang Ⅱ可直接損害足細(xì)胞不斷得到認(rèn)可[6]。Ang Ⅱ作為腎臟病進(jìn)展的危險(xiǎn)因素，可誘導(dǎo)產(chǎn)生大量非血流動(dòng)力影響，如產(chǎn)生氧自由基、細(xì)胞因子，并可刺激細(xì)胞凋亡、增生和肥大[9]。Ang Ⅱ可影響裂孔隔膜復(fù)合物基因的表達(dá)，如nephrin和podocin、TRPC6等。很多作用主要是通過(guò)AT1R介導(dǎo)的[10]，早在2004年Hoffmann等[11]在轉(zhuǎn)基因小鼠研究中證實(shí)，足細(xì)胞AT1R過(guò)表達(dá)足以導(dǎo)致蛋白尿和FSGS。AT1R通過(guò)與異源三聚體G蛋白結(jié)合激活不同第二信使介導(dǎo)的轉(zhuǎn)導(dǎo)通路[12]。

Ang Ⅱ活化TRPC6并增強(qiáng)TRPC6的核轉(zhuǎn)錄

Ang Ⅱ活化下游TRPC6最近，電生理學(xué)詳細(xì)揭示了TRPC5和TRPC6作為Ang Ⅱ誘導(dǎo)非選擇性陽(yáng)離子傳導(dǎo)的下游離子通道[13]。TRPC5和TRPC6可被上游的受體活化，如GPCR，包括AT1R。Ang Ⅱ在多種酶[如黃嘌呤氧化酶、細(xì)胞色素P450、一氧化氮合成酶(NOS)、黃素氧化還原酶]參與下通過(guò)其AT1R介導(dǎo)ROS的產(chǎn)生[1]。在足細(xì)胞中，Ang Ⅱ與酶之間的聯(lián)系及上調(diào)信號(hào)分子調(diào)節(jié)還原型煙酰嘌呤二核苷酸磷酸(NADPH)氧化酶機(jī)制尚不完全清楚，可能有磷脂酶D(PLD)、蛋白激酸C(PKC)、非受體酪氨酸激酶(c-Src)、表皮生長(zhǎng)因子受體(EGFR)、磷脂酰肌醇-3激酶(PI3K)和Rac的參與。ROS為高活性分子，它可氧化脂質(zhì)、促進(jìn)內(nèi)皮NOS解偶聯(lián)從而減少NO的合成[14]，局部組織ROS還可將局部釋放的NO轉(zhuǎn)化成ONOO-,ONOO-極易與蛋白質(zhì)酪氨酸殘基作用生成3-硝基酪氨酸(3-NT)[14]，NO減少、ROS增多被認(rèn)為是脂質(zhì)、蛋白質(zhì)和DNA氧化損傷的主要原因。體內(nèi)實(shí)驗(yàn)證明，Ren2小鼠給予ROS清除劑、AT1R阻滯劑可降低腎組織內(nèi)NADPH活性，減少ROS的生成，減輕足突融合[8]。ROS的增加降低了NO的生物活性，NO是RAS作用于腎臟的關(guān)鍵調(diào)節(jié)分子[7]。NO能夠激發(fā)細(xì)胞內(nèi)多種信號(hào)轉(zhuǎn)導(dǎo)通路，刺激可溶性鳥(niǎo)苷環(huán)化酶催化細(xì)胞內(nèi)環(huán)磷酸鳥(niǎo)苷(cGMP)的合成，再由cGMP激活蛋白激酶G(PKG)調(diào)節(jié)局部和全身信號(hào)。PKG存在于多種組織細(xì)胞，是重要的信號(hào)分子NO的下游底物，能夠在多種水平調(diào)節(jié)Ca2+水平。故考慮足細(xì)胞是否存在NO-cGMP-PKG信號(hào)通路并通過(guò)該通路激活TPRC6通道，尚待進(jìn)一步證實(shí)。TRPC6還可被Ang Ⅱ活化的AT1R及其下游第二信使二酰苷油(DAG)所激活的蛋白激酶C活化[17]。

Ang Ⅱ增強(qiáng)TRPC6的核轉(zhuǎn)錄Ang Ⅱ活化TRPC6并增強(qiáng)其核轉(zhuǎn)錄[18,19]。在心肌細(xì)胞中，Ca2+無(wú)疑被認(rèn)為是導(dǎo)致心臟肥大的原因之一，Ang Ⅱ活化TRPC6和Ca2+依賴(lài)鈣調(diào)磷酸酶、細(xì)胞活化T細(xì)胞核因子(NFAT)的正反饋環(huán)，導(dǎo)致TRPC6轉(zhuǎn)錄增加及心肌肥大[20]。而在小鼠足細(xì)胞中，Tom Nijenhuis等的研究也顯示Ang Ⅱ可誘導(dǎo)TRPC6表達(dá)和足細(xì)胞損傷，TRPC6通過(guò)PLC相關(guān)轉(zhuǎn)導(dǎo)信號(hào)被活化、鈣離子內(nèi)流、鈣離子依賴(lài)的鈣調(diào)磷酸酶活化進(jìn)一步導(dǎo)致NFAT核轉(zhuǎn)錄而增強(qiáng)TRPC6相關(guān)基因的表達(dá)，足細(xì)胞細(xì)胞膜TRPC6表達(dá)增多將導(dǎo)致這一正反饋調(diào)節(jié)。這正反饋調(diào)節(jié)導(dǎo)致鈣調(diào)磷酸酶的持續(xù)活化和TRPC6的進(jìn)一步表達(dá)[21]。該作者為了進(jìn)一步證實(shí)上述觀點(diǎn)，用血管緊張素Ⅱ受體拮抗劑(ARB)與血管緊張素轉(zhuǎn)換酶抑制劑(ACEI)處理Ang Ⅱ活化的足細(xì)胞后，Ang Ⅱ誘導(dǎo)的TRPC6過(guò)表達(dá)得到抑制。Wang等[22]發(fā)現(xiàn)TRPC6活化后介導(dǎo)Ca2+內(nèi)流增強(qiáng)了足細(xì)胞NADPH氧化酶活性，而NADPH來(lái)源的ROS可上調(diào)TRPC6的表達(dá)。此外，Zhang等[23]的研究中顯示，Ang Ⅱ處理足細(xì)胞后還可通過(guò)激活細(xì)胞外調(diào)節(jié)蛋白激酶(ERK)通路致核因子κB(NF-κB)核轉(zhuǎn)錄而上調(diào)膜表面TRPC6表達(dá)。Ang Ⅱ活化TRPC6并增強(qiáng)TRPC6的核轉(zhuǎn)錄，故TRPC6放大了Ang Ⅱ?qū)ψ慵?xì)胞的損傷(圖1)。

圖1　Ang Ⅱ活化TRPC6并增強(qiáng)其核轉(zhuǎn)錄示意圖

活化TRPC6介導(dǎo)足細(xì)胞足突融合

足細(xì)胞膜上信號(hào)通路調(diào)控足突融合越來(lái)越多證據(jù)顯示，腎小球上皮細(xì)胞(足細(xì)胞)損傷是形成蛋白尿的一個(gè)最重要機(jī)制[25-27]。以大量蛋白尿?yàn)楸憩F(xiàn)的腎小球疾病，包括FSGS[27]、微小病變(MCD)[28]和糖尿病腎病[29]，其中足細(xì)胞的損傷以足突融合為主要表現(xiàn)?；谶@些特殊的細(xì)胞生物學(xué)特點(diǎn)，足細(xì)胞及裂孔隔膜對(duì)維持腎小球?yàn)V過(guò)屏障的完整性具有重要作用[30]。近年來(lái)足細(xì)胞膜分子的作用越來(lái)越受到重視，它們將細(xì)胞外信號(hào)傳遞到細(xì)胞內(nèi)并影響細(xì)胞結(jié)構(gòu)，尤其是足突結(jié)構(gòu)。事實(shí)上，與感覺(jué)神經(jīng)元相似，足細(xì)胞的細(xì)胞體和主突的功能可能僅僅是向足突提供營(yíng)養(yǎng)。足細(xì)胞足突被功能性分為頂膜區(qū)、基膜區(qū)和裂孔隔膜區(qū)，這些區(qū)域細(xì)胞膜在結(jié)構(gòu)和功能上與足突的肌動(dòng)蛋白骨架緊密聯(lián)系，這些細(xì)胞膜區(qū)域任何部位改變將影響肌動(dòng)蛋白骨架，使其由平行可收縮的捆狀排列變成雜亂無(wú)序的網(wǎng)狀排列，肌動(dòng)蛋白骨架的重排致足突融合。這一過(guò)程受很多信號(hào)分子的調(diào)控，不僅僅局限于整合素的活化、GPCR的和生長(zhǎng)因子受體的活化的調(diào)控，還受Ca2+和內(nèi)流信號(hào)通路的調(diào)控。體內(nèi)研究證實(shí)，足突融合與蛋白尿的出現(xiàn)密切相關(guān)[31,32]，并可解釋足細(xì)胞的損傷可出現(xiàn)大量尿蛋白。

活化TRPC6介導(dǎo)的Ca2+內(nèi)流信號(hào)致足突細(xì)胞骨架重排Winn等[3]對(duì)TRPC6的研究發(fā)現(xiàn)，當(dāng)Ang Ⅱ使活化TRPC6介導(dǎo)的Ca2+內(nèi)流顯著增加時(shí)，足細(xì)胞內(nèi)細(xì)胞骨架的排列出現(xiàn)異常，可改變足細(xì)胞足突的收縮結(jié)構(gòu)。Ca2+內(nèi)流和細(xì)胞骨架主要調(diào)節(jié)分子RhoGTP酶活化之間存在緊密關(guān)系[33]，基于鈣離子和肌動(dòng)蛋白細(xì)胞骨架的重要作用，RhoGTP酶已成為目前很多足細(xì)胞研究的方向。在細(xì)胞水平，未受損的足突中RhoA活性占主導(dǎo)地位，而不穩(wěn)定、可伸縮的足突中，Cdc42/Rac1活性占主導(dǎo)。最近研究顯示，Synaptopodin是足細(xì)胞RhoGTP酶的重要調(diào)節(jié)因子，可抑制RhoGTP酶的泛素化和蛋白酶降解[34]，可通過(guò)促進(jìn)RhoA而抑制Cdc42/Rac1，因此可預(yù)防足突細(xì)胞骨架的重排。而活化的TRPC6通過(guò)Ca2+內(nèi)流活化鈣調(diào)磷酸酶并促進(jìn)Synaptopodin降解，故TRPC6可通過(guò)此途徑使足細(xì)胞骨架重排、足突融合。Rac1促進(jìn)鹽皮質(zhì)激素受體進(jìn)入足細(xì)胞核內(nèi)。Tian D研究顯示，TRPC6敲除將導(dǎo)致應(yīng)力纖維減少、Rac1活化、運(yùn)動(dòng)力增強(qiáng)，并可通過(guò)RhoA持續(xù)活化改善[13]。

活化TRPC6介導(dǎo)足細(xì)胞凋亡

Ang Ⅱ被認(rèn)為是誘導(dǎo)足細(xì)胞凋亡的始動(dòng)因素之一[35]，其誘導(dǎo)凋亡途徑尚未明確，而通過(guò)TRPC6介導(dǎo)足細(xì)胞凋亡途徑的研究則更為有限。Zhang H等體外實(shí)驗(yàn)證實(shí)，Ang Ⅱ可通過(guò)上調(diào)TRPC6，活化下游ERK及NF-κB的核移位，誘導(dǎo)足細(xì)胞凋亡[36]。Yang等[37]則在體外高糖環(huán)境下培養(yǎng)的小鼠足細(xì)胞研究中發(fā)現(xiàn)，高糖誘導(dǎo)ROS產(chǎn)生，ROS通過(guò)活化足細(xì)胞TRPC6介導(dǎo)Ca2+內(nèi)流增加，并通過(guò)Ca2+內(nèi)流激活RhoA/ROCK通路導(dǎo)致足細(xì)胞凋亡，敲除小鼠足細(xì)胞TRPC6基因，則足細(xì)胞凋亡明顯受到抑制。Ang Ⅱ也通過(guò)其AT1R介導(dǎo)ROS的產(chǎn)生[1]，故有理由推測(cè)，Ang Ⅱ是否也可通過(guò)活化TRPC6通道介導(dǎo)Ca2+內(nèi)流及RhoA/ROCK通路致足細(xì)胞凋亡，但國(guó)內(nèi)外尚缺乏相關(guān)文獻(xiàn)報(bào)道(圖2)。

圖2　TRPC6介導(dǎo)足細(xì)胞損傷、凋亡信號(hào)傳遞示意圖

綜上所述，通過(guò)對(duì)Ang Ⅱ誘導(dǎo)足細(xì)胞損傷的信號(hào)傳導(dǎo)機(jī)制和TRPC6通道的研究，我們發(fā)現(xiàn)足細(xì)胞內(nèi)AT1R活化、TRPC6通道、Ca2+信號(hào)通路、RhoGTP酶活化、足突細(xì)胞骨架的重排和足突融合之間的聯(lián)系。因此，AT1R阻滯劑、TRPC6通道可能成為非常有價(jià)值的藥物靶點(diǎn)。

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