血管增滲酶遲緩激肽系統在腎臟疾病角色的探討

The Role of Kallikrein-Bradykinin SysTem in Kidney Disease

姜文智(Wen-Chih Chiang)

指導教授 ： 蔡敦仁

共同指導教授 ： 林琬琬(Wan-Wan Lin)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01537

摘要

腎衰竭是臨床上常見的併發症，會造成電解質不平衡、體液過多及尿毒症，引發更嚴重的併發症，使得致病率及死亡率增加。根據腎衰竭發展的快慢，一般會將腎衰竭區分為急性腎衰竭及慢性腎衰竭。急性腎衰竭的發生率在心血管手術可達1~15%，中等度敗血症19%，嚴重敗血症23%，敗血症合併休克且血液培養陽性時達51%。在這些急性腎衰竭發展過程中，缺氧(ischemia)扮演了很重要的角色；但是，急性腎衰竭的產生往往是缺氧及其他疾病引起反應交互作用的結果；例如敗血症發生時，發炎反應會與缺氧共同造成急性腎衰竭。這使得急性腎衰竭的診斷及治療更加的複雜及困難。慢性腎衰竭也是重要的疾病，特別是在台灣。2004年在台灣，末期腎病變需要洗腎的發生率為每百萬人有375人，盛行率為每百萬人有1706人，高居全世界的前2位。糖尿病腎病變及慢性腎絲球腎炎則是慢性腎臟疾病及導致末期腎病變的主要原因。腎衰竭不僅造個人健康上的損害，更形成家庭及社會的結構及經濟上的負擔，因此，了解腎臟病的致病機轉、預防及治療腎臟疾病是很重要的課題。
血管增滲酶遲緩激肽(kallikrein-bradykinin)系統是腎臟重要的旁泌素/細胞激素系統，此系統的組成包含血管增滲酶、激肽原(kininogen)、激肽(kinin)及遲緩激肽B1及B2受體。血管增滲酶會將激肽原分解成激肽，激肽與遲緩激肽B2受體結合後會激發二次傳遞物質的產生(如nitric oxide/cGMP及protaglandin)，引發許多生理反應，如血管舒張、發炎、利鈉及水腫。在腎臟，遲緩激肽可以增加血液灌流，因此，有可能在急性缺氧後的再灌流期增加血液的供應，或改善慢性腎臟病的腎臟缺氧狀況。另外，遲緩激肽促進細胞增生的作用也有可能有助於腎臟在受到傷害後細胞的存活及組織的修復。這些作用可能有助於減緩腎臟疾病產生的傷害。但是，遲緩激肽的發炎作用則可能有害於急性及慢性腎臟疾病。因此，吾人預期血管增滲酶遲緩激肽系統在急性及慢性腎臟疾病過程中會產生作用，影響腎臟疾病的進行。對於急性腎臟疾病，吾人將利用缺氧再灌流腎臟傷害(ischemic-reperfusion renal injury)動物模式，檢視血管增滲酶遲緩激肽系統在急性腎臟疾病的角色。對於慢性腎臟疾病，因為在主要的慢性腎臟疾病(如糖尿病腎病變及慢性腎絲球腎炎)中，目前沒有很好的動物模式可供研究，吾人將直接進行臨床研究，先探討尿液中血管增滲酶與腎功能及腎臟傷害程度的關係來著手，來導引出更進一步研究的方向。
許多研究顯示：活化血管增滲酶遲緩激肽系統對心臟的缺氧性病變有保護的作用。Schoelkens等人是第一個報導遲緩激肽對心臟的保護作用，另外，以血管增滲酶基因治療大白鼠也可以減緩腦及心臟的缺氧再灌流傷害。但是，早期投予遲緩激肽B2受體拮抗劑卻有助於肺臟、肝臟及腸的缺氧再灌流傷害。在腎臟，目前尚未有關於血管增滲酶遲緩激肽系統在腎臟缺氧再灌流傷害角色的研究。
急性缺氧腎病變(不論是否有合併有再灌流傷害)發生後，腎小管細胞的brush boder會產生融合現象，近端小管細胞產生壞死及凋亡(特別是近端小管的S3節段)，死去之細胞脫落至管腔中與蛋白形成柱狀體，基底膜並因而露出，組織切片中可以看到區狀壞死；假如，缺氧的時間更長，細胞壞死還會擴展至皮質，造成皮質的組織壞死。此外，腎臟還會有間質水腫及發炎細胞的浸潤，但是，腎絲球是不會被影響的。因為缺氧的傷害，血管內皮細胞會喪失分泌一氧化氮的能力，血管收縮物質(如endothelin 1)卻又大量產生，導致血管在再灌流期的持續收縮，使腎臟在缺氧(ischemia)傷害後仍處於持續的缺氧狀態，產生更多的組織傷害。遲緩激肽具有血管舒張的作用，或許可以改善腎臟在再灌流期的血液灌流，因此，有可能有助於減少缺氧再灌流的傷害。
在細胞的層次而言，缺氧發生後，細胞的ATP會被消耗殆盡並被轉換為ADP及AMP，AMP被進一步代謝為hypoxanthine，hypoxanthine的堆積會刺激reactive oxygen species (ROS)的產生。在再灌流期時，xathine oxidase會被活化，活化的xathine oxidase會將hypoxathine轉化為xathine，並產生hydrogen peroxide及superoxide等自由基。產生的自由基會藉由破壞DNA、氧化細胞膜及胞器膜上的脂質、直接活化引發細胞凋亡的基因及蛋白而導致細胞凋亡；自由基還會與鈣離子偕同作用活化粒腺體內的phospholipase A2，進而促進發炎反應，使缺氧再灌流的傷害更加嚴重。在粒腺體，產生的自由基則會打開permeability transition pore，使得粒腺體腫脹、粒腺體膜電位及粒腺體內的電子傳遞鏈氧化磷酸化功能喪失，促使更多自由基產生；粒腺體內的cytochrome c也會被釋放至細胞質中，並活化caspase，導致細胞壞死及凋亡。自由基的過度產生及粒腺體內的鈣離子堆積都是促使粒腺體permeability transition pore打開的重要因子。
鈣離子在缺氧再灌流腎臟傷害中也扮演了重要的角色。缺氧會使細胞的ATP耗盡，細胞膜上的鈉-鉀幫浦因而喪失功能，導致細胞膜電位喪失，voltage-gated鈣離子通道因此會被打開，細胞外鈣離子進入細胞內，提升細胞內游離鈣離子濃度。ATP耗盡也會使內質網汲取鈣離子的能力減低，進一步使細胞內游離鈣離子濃度增加。細胞內游離鈣離子濃度增加會活化portease、phospholipase及細胞骨幹(cytoskeleton)的破壞，導致細胞死亡。同時，粒腺體也會汲取細胞內增加的游離鈣離子，permeability transition pore因此被打開，造成粒腺體傷害及細胞死亡。許多研究也顯示：阻斷鈣離子通道或加入鈣離子螫合劑可以減緩缺氧再灌流腎臟傷害。遲緩激肽在一般細胞培養狀態下可以增加細胞內鈣離子濃度及自由基的產生，因此，也有可能在細胞或腎臟缺氧狀態下，加強缺氧引發的細胞內鈣離子增加及自由基的產生，加重腎臟缺氧再灌流的傷害。
吾人因此假設：血管增滲酶遲緩激肽系統在腎臟缺氧再灌流傷害中，會影響缺氧再灌流造成的傷害，有可能是減少傷害，也有可能是加重傷害。為證實吾人假設，在動物實驗，吾人以皮下給予組織型血管增滲酶蛋白以活化遲緩激肽受體，或給予遲緩激肽受體拮抗劑抑制遲緩激肽作用，在缺氧再灌流傷害後，檢視腎功能、病理變化、自由基的產生、發炎反應及細胞凋亡。在細胞實驗，吾人以ATP去除來模擬細胞缺氧(ischemia)狀態，檢視遲緩激肽對細胞去除引發之細胞死亡、自由基產生、粒腺體傷害、鈣離子濃度變化及凋亡途徑的活化，並嘗試解析其訊息傳導途徑。
實驗顯示：缺氧再灌流發生48小時後，對照組大白鼠之血清肌酸酐及尿素氮値上升，腎臟鈉離子排除率(FeNa)增加，給予血管增滲酶治療的大白鼠腎功能惡化的現象更加明顯，但若同時給予遲緩激肽B2受體拮抗劑，則會將血管增滲酶的作用抵消，恢復至與對照組相同的程度。同時投予遲緩激肽B1受體拮抗劑拮抗遲緩激肽B1受體的作用或PBS之治療以補充可能流失的體液，都沒有辦法抵消血管增滲酶的傷害加成作用。吾人也測量了腎動脈在缺氧再灌流發生1小時後的血流量，缺氧再灌流傷害本身的確會減少再灌流期的腎臟血流量，但是血管增滲酶的治療並不影響其血流量變化。同時，吾人也測量了缺氧再灌流發生1小時後的血壓，實驗顯示各組間之血壓並無明顯差異。這些結果顯示：活化遲緩激肽B2受體會加強腎臟的缺氧再灌流傷害，而且，這些作用並不是經由改變體液容積、腎臟血液灌流或血壓所產生的。
吾人進一步檢視缺氧再灌流後的腎小管壞死及發炎反應，類似的情況也可以在此實驗看到：活化遲緩激肽B2受體會加強腎臟缺氧再灌流傷害引起的腎小管壞死、單核球/中性球的浸潤及monocyte chemoattractant protein 1/tumor necrosis factor-alpha(MCP-1/TNF-alpha)基因及蛋白的表現。吾人並以即時監測化學冷光的方式，了解各組大白鼠在缺氧期及再灌流期前3.5小時自由基產生的狀況。在缺氧一開始，腎臟自由基便開始產生並逐漸增加，當再灌流期血流重建的的一剎那，會有自由基的大量產生，並持續到測量結束。活化遲緩激肽B2受體會加強腎臟缺氧期及再灌流期的自由基產生，測量組織的hydrogen peroxide、游離malondialdehyde及氧化/還原型glutothione也證實活化遲緩激肽B2受體會加強腎臟缺氧再灌流傷害引起氧化壓力(oxidative stress)。以TUNEL及anti-caspase-cleased fragment (p85) poly (ADP-ribose) polymerase (PARP)免疫組織染色檢視腎臟組織內的凋亡細胞，也發現自由基產生較多的血管增滲酶治療組其細胞凋亡的情形也較對照組明顯。這些結果證實：活化遲緩激肽B2受體會加強腎臟缺氧再灌流傷害是經由加強自由基產生的結果。
組織在受到缺氧再灌流傷害後，自由基來自於發炎細胞或組織所構成的細胞。鄭等人發現腎臟的近端腎小管細胞是腎臟缺氧再灌流傷害後自由基主要產生的部位，這個部位與腎小管壞死的主要部位是相同的。所以，活化遲緩激肽B2受體加強腎臟自由基的產生也可能發生在近端腎小管細胞。過去的研究證實遲緩激肽在心臟細胞及血管平滑肌細胞一般培養狀態下，可以增加細胞自由基的產生，因此，吾人也推測：遲緩激肽B2受體活化後，也會加強近端腎小管細胞在缺氧或ATP去除引起的自由基產生，因而有更多的粒腺體傷害及細胞凋亡。
吾人以去除葡萄糖等受質及加入antimycin A方式模擬細胞缺氧(ischemia)，在NRK-52E近端腎小管細胞株進行實驗。吾人證實：活化遲緩激肽B2受體會加強ATP去除引發之細胞死亡，這可由細胞LDH的釋放、sub G0/G1細胞的比率、Hoechest染色凋亡細胞的計算及Annexin binding assay的分析得知。以螢光染色劑及流式細胞儀分析，吾人也發現活化遲緩激肽B2受體會加強ATP去除引發之自由基產生(包含hydrogen peroxide及superoxide)及粒腺體膜電位喪失。粒腺體膜電位喪失會導致粒腺體傷害及引發相關的細胞凋亡活化途徑。在遲緩激肽治療的細胞也發現有更多的cytochrome c釋放至細胞質中，活化的PARP及caspase 9也比對照組多。這些結果說明了活化遲緩激肽B2受體會加強ATP去除引起的自由基產生，自由基產生越多，所受到的粒腺體傷害及細胞凋亡也越明顯。在吾人hypoxia的實驗中也發現：活化遲緩激肽B2受體會加強hypoxia引起的細胞凋亡。從吾人的動物及細胞實驗可以證實：活化遲緩激肽B2受體加強腎臟缺氧再灌流傷害是經由加強自由基產生所引起的。
除了自由基之外，鈣離子也是打開粒腺體permeability transition pore的重要因子，並在腎臟缺氧再灌流傷害中扮演重要角色。遲緩激肽已知可以在一般細胞培養狀態下增加細胞內鈣離子，不僅如此，吾人發現活化遲緩激肽B2受體也會加強ATP去除引起的細胞內及粒腺體鈣離子增加，即使是細胞已接受ATP去除1.5小時之後，加入遲緩激肽仍然可以再增加細胞內鈣離子濃度。更多的鈣離子將與自由基偕同打開粒腺體permeability transition pore，導致粒腺體更嚴重的膜電位喪失，粒腺體腫脹及破裂，更多的cytochrome c釋放至細胞質，活化caspase 9，導致更多的細胞凋亡。這個結果說明了鈣離子在遲緩激肽加強ATP去除引起細胞傷害的關連性。
使細胞內游離鈣離子增加有三個途徑：1)從細胞內的儲存處所(如內質網)釋放出來。2)由細胞外經voltage-gated鈣離子通道進入細胞內。3)由細胞外經receptor mediated鈣離子通道進入細胞內。鈣離子自內質網釋放出來需要inositol 1,4,5-triphosphate(IP3)的刺激，活化遲緩激肽B2受體會活化phospholipase C (PLC)，造成細胞膜上phosphoinositide的水解，並釋放出IP3。吾人在細胞實驗中投予PLC的抑制劑(U73122)，發現PLC抑制劑可以拮抗遲緩激肽加強ATP去除引起的細胞內及粒腺體內鈣離子增加，至與對照組細胞相同的程度，此結果顯示PLC-IP3訊息傳導途徑在遲緩激肽加強ATP去除引起細胞內鈣離子增加的角色。而且，遲緩激肽加強ATP去除引起自由基增加及粒腺體膜電位喪失的作用也會被PLC抑制劑所拮抗，這些結果證實：遲緩激肽加強ATP去除引起的細胞傷害是經由PLC訊息傳導途徑活化的。但是，遲緩激肽在一般細胞培養狀態下，也可經由voltage-gated及receptor-mediated鈣離子通道增加細胞內的鈣離子濃度，因此，遲緩激肽也有可能經由這兩種通道加強ATP去除引起的細胞內鈣離子增加、自由基增加及細胞傷害，這需要吾人進一步實驗加以釐清。但由吾人實驗知道，阻斷PLC傳導途徑可以將遲緩激肽的作用幾乎完全抵消，所以此兩種通道在遲緩激肽的加強ATP去除引起的傷害中，角色可能不重要或必須經由PLC訊息傳導途徑活化方能作用。
吾人並無法由以上的實驗得知，遲緩激肽是否會經由粒腺體不相關的途徑來加強ATP去除引起的細胞傷害。內質網的壓力(endoplasmic reticulum stress)已知可以在缺氧再灌流傷害中引發細胞凋亡，遲緩激肽會加強ATP去除引起細胞內鈣離子的增加、自由基的產生及鈣離子自內質網的釋放，這些都會形成內質網壓力，內質網壓力的存在會活化caspase 12、c-Jun NH2-terminal knase訊息傳導途徑及growth arrest and DNA damage-inducible gene 153的表現，導致細胞進入凋亡。欲了解此一途徑的角色需要進一步實驗來探討。
另外一個活化遲緩激肽B2受體加強腎臟缺氧再灌流傷害的可能機轉為經由phospholipase A2(PLA2)的活化。許多實驗顯示:細胞質內、粒腺體及微小體(microsome)的PLA2在腎臟缺氧再灌流傷害後會被活化，在單獨分離出的腎臟及缺氧再灌流腎傷害，自由基也可與鈣離子偕同活化粒腺體的PLA2；遲緩激肽本身也會經由增加細胞內鈣離子活化PLA2，因此，活化遲緩激肽B2受體有可能會加強缺氧再灌流引起的PLA2活化。PLA2活化產生的脂質發炎物質是發炎反應的重要引發因子，而發炎反應是缺氧再灌流組織傷害的原因之一。遲緩激肽引發的更多PLA2活化有可能加強缺氧再灌流的腎臟傷害。
吾人的實驗結果:早期活化遲緩激肽B2受體會加強腎臟缺氧再灌流的傷害，這與其他作者發現遲緩激肽在腦及心臟有保護作用的結果不同，原因尚不明瞭。其中可能的原因為:血管增腎酶遲緩激肽系統在缺氧再灌流不同時期可能有不同的作用。在Xia等人的實驗中，血管增滲酶基因是在再灌流開始後才給予的，等到腦細胞攝取基因，分泌出蛋白，產生遲緩激肽，再與受體結合，發揮保護腦細胞的作用至少已經是許多小時以後的事了。而其它作者在缺氧再灌流發生之前即給予遲緩激肽B2受體拮抗劑，發現可以減緩腸、肝及肺的缺氧再灌流傷害。在早期遲緩激肽可能發揮其發炎之作用，而在晚期則可促進細胞再生，因此可在缺氧再灌流傷害不同時期顯現不同的效果。另外的解釋是:心臟及腦有側支循環(collateral circulaltion)，在其中一條血管阻塞後，遲緩激肽還可通過血管的擴張作用增加側支循環的血液供應，改善缺氧的程度。但腎臟只有一條腎動脈供應血流，血管阻塞後，遲緩激肽無法增加側支循環，而只能在早期產生發炎反應，增加傷害。
臨床上，血漿型的血管增滲酶在敗血症時會被活化，導致全身血管的擴張造成血壓降低，並引起發炎反應；研究顯示:發炎反應與缺氧是導致敗血症相關急性腎傷害產生的重要原因。組織型血管增滲酶在動物及人類的小腸發炎疾病會被釋放出來，在呼吸器引發的肺臟傷害，肺臟分泌物也有較高濃度的血管增滲酶。因此，腎臟的組織型血管增滲酶也有可能被敗血症引發的發炎反應刺激而釋放出來，並產生作用。既然活化遲緩激肽B2受體會加強腎臟缺氧再灌流傷害，被敗血症刺激釋放的血管增滲酶可能會與缺氧偕同引發敗血性急性腎臟傷害。為了證實這項假說，吾人必需研究敗血症發生時，腎臟的血管增腎酶遲緩激肽系統是否被活化，以及其活性是否與敗血症相關的急性腎臟傷害發生率有相關性。
吾人也嘗試對血管增滲酶遲緩激肽系統在慢性腎臟病變的角色做研究。尿液中組織型血管增滲酶的排出可反應腎臟內血管增滲酶的合成及活化，評估慢性腎臟病患者尿液中血管增滲酶的排出可能有助於了解血管增滲酶遲緩激肽系統在慢性腎臟病變的角色。因此，吾人進行了臨床研究，探討尿液組織型血管增滲酶排出與腎功能或蛋白尿程度的關係。以multiple regression分析，吾人發現：尿液中血管增滲酶的排出率與腎功能有正相關，但與蛋白尿程度沒有關係；追蹤12個月後，腎功能惡化也伴隨著尿液血管增滲酶排出率的減少。血管增滲酶是由腎小管排出的，腎功能減少也代表著腎元減少，尿液血管增滲酶的排出也隨者減少。吾人的研究結果顯示：尿液血管增滲酶的排出可能可作為腎功能變化的指標。
有趣的是，研究發現血管增滲酶與尿液或血液的MCP-1濃度成正相關，分析顯示：此相關並不是因為管增滲酶與MCP-1分別與腎功能有正相關所產生的。尿液中的MCP-1排出越多代表腎臟發炎的活性越高，吾人的研究結果顯示：當腎臟有發炎傷害時，血管增滲酶會被釋放至尿液中。這個發現與過去在腸及肺的研究吻合，在動物及人類的腸發炎性疾病或呼吸器相關的肺臟傷害，都可以看到血管增滲酶的釋出。但是，吾人只在一個時間點看見其相關性，系列的追蹤尿液中血管增滲酶與MCP-1的相關性，才能證實其真正的關係。假如，尿液中血管增滲酶確實與腎臟傷害的活性有相關性，測量尿液中的血管增滲酶將有助於了解腎臟內慢性疾病的進行；但是，這樣的臨床應用還必須進一步研究尿中血管增滲酶與腎臟傷害活性的關聯性，特別是與病理檢查發炎細胞浸潤程度作關聯探討，才能確認其應用價值。
血管增滲酶釋放出來後，有可能會分解激肽元，產生遲緩激肽，引發發炎反應。但也有報導，遲緩激肽可以經由抑制PAI-1的表現，減緩腎臟的纖維化。發炎及纖維化都是慢性腎臟疾病重要的反應過程，因此，血管增滲酶遲緩激肽系統很有可能影響慢性腎臟病變的進行，究竟是好的影響或是壞的影響，目前尚不清楚。為解答這樣的問題，吾人將進行動物實驗，藉由活化或抑制遲緩激肽受體，在慢性腎臟疾病模式中，檢視其對腎臟發炎及纖維化的影響，相信更能確認血管增滲酶遲緩激肽系統在慢性腎臟病變的角色。
總之，早期活化遲緩激肽B2受體會在缺氧期及再灌流期，經由加強自由基產生的途徑，加強缺氧再灌流的腎臟傷害；活化遲緩激肽B2受體會經由活化PLC訊息傳導途徑，加強ATP去除引起的細胞內及粒腺體內的鈣離子增加、自由基產生、粒腺體傷害及細胞的凋亡。但還必須從臨床上證實此研究結果的重要性，並探討其它可能的訊息傳導途徑。在慢性腎臟疾病，尿液血管增滲酶的排出與腎功能及發炎細胞激素呈正相關，這樣結果顯示:尿液的血管增滲酶也許是慢性腎臟病腎臟傷害進行的指標。但有關於血管增滲酶遲緩激肽系統在慢性腎臟病的角色，目前還不了解，需要更多的研究來釐清其真相。

關鍵字

血管增滲酶遲緩激肽缺氧再灌流 ； 慢性腎臟疾病 ； 自由基 ； 細胞凋亡 ； 鈣離子 ； 細胞激素

並列摘要

Renal failure is a common complication seen in clinical practice. Renal failure usually results in electrolyte imbalance, fluid overload and uremia which cause further complications leading to higher morbidity or mortality. Renal failure is divided into acute reanl failure (ARF) and chronic renal failure (CRF) according to the developmentory rate. The incidence of ARF is 1~15% in cardiovascular surgery, 19 % in moderate sepsis, 23 % in severe sepsis, and 51% in septic shock when blood cultures are positive. In these events ischemia plays an important role in the pathogenesis of shock-related acute renal injury. However, the development of ARF is sometimes the result of synergistic effect of ischemia with other intra-renal response subsequent to disease process, such as inflammation in septic ARF, thus further complicates the diagnosis and treatment of ischemic ARF. CRF is also an important disease in Taiwan. The prevalence and incidence of end stage renal disease (most coming from CRF) under dialysis is 1706 and 375/million respectively which are the top two in the world. Diabetes nephropathy and chronic glomerulonephritis are the two major diseases that lead to CRF therefore end stage renal disease. Renal failure not only hurt the personal health but also put a great impact on the family and society.
The kallikrein-kinin system is an important cytokine/paracrine system in kidney. This system includes kallikrein, kininogen, kinin, and bradykinin B2 and B1 receptors. Kallikrein cleaves kininogen substrate to release vasoactive kinin peptide via limited proteolysis. Binding of intact kinin to the B2 receptor activates secondary messengers, such as nitric oxide (NO)/cGMP and prostacyclin, and triggers many biological effects, such as vasodilation, inflammation, natriuresis, and edema formation. In kidney, bradykinin could increase renal perfusion which may improve the renal blood supply after acute ischemic renal injury or attenuate ischemia in chronic renal disease (CKD), which is found in many CKD includning chronic glomerulonephritis. The anti-hypertensive effect may be helpful in attenuating the disease progression in CKD. Besides, the cellular proliferative effect may also help the cell survival and renal repair process after reanl injury either in acute kidney injury (AKI) or CKD. However, the inflammatory effect may be detrimental to AKI and CKD, since inflammation is a very important factor in reanl damage.
We expect that kallikrein-bradykinin system will exert their action and influence the disease process in AKI and CKD. For the study of AKI, we apply acute ischemic reperfusion (I/R) renal injury as animal model and study the role of this system in I/R injury. For CKD, we conduct clinical study to explore the role of kallikrein-bradykinin system since there is lack of good animal model of major CKD, diabetes nephropahty and chronic glomerulonephritis. It is also difficult to obtain renal tissue or to measure the bradykinin level in urine. So, we first study the relationship between urinary kallikrein and renal function or the status of reanl injury to find out any clue of the role of kallikrein-bradykinin system in CKD.
The kallikrein-kinin system contributes to the protection of ischemic heart. Schoelkens et al. were the first to report the cardioprotective effects of bradykinin. Kallikrein gene delivery has also been observed to attenuate ischemic stroke and acute myocardial ischemia/reperfusion (I/R) injury. Conversely, several studies have shown bradykinin plays a deleterious role in brain, heart and other organs, such as lung, liver and intestine after I/R injury. Yet there is still no study evaluating the role of kallikrein-bradykinin system in I/R renal injury.
After the ischemic with/without perfusion injury, there will be effacement of the brush boder and necrosis/apoptosis of proximal tubular cells. The dead cells will slough from tubular lumen which will combine with luminal protein and form cast in the lumen. The basement membrane is denuded. Patchy necrosis is seen in outer medulla, especially in the S3 segment of proximal tubule. If the ischemia is more severe, tubular necrosis will extend to proximal tubule in the cortex. There is edema and inflammatory infiltration in the interstitium. However, the glomerulus is unaffected. Due to the ischemic insult, the endothelium is injured which impair the release the endothelium-derived nitric oxide. In contrast, the vasoconstrictors, such as endothelin-1, are released. Both mechanisms impair the renal reperfusion resulting in persistent ischemia in reperfusion phase, further extend renal injury. Bradykinin may improve the renal perfusion in the reperfusion phase of I/R injury. This effect may be beneficial in I/R renal injury.
After ischemia cellular ATP is degraded into ADP and AMP. The AMP is further degraded into hypoxanthine and the accumulation of hypoxanthine will stimulate the generation of reactive oxygen species (ROS). During the reperfusion phase, xanthine oxidase is activated and transforms hypoxanthine into xanthine with the by-product formation of hydrogen peroxide and superoxide. ROS can induce apoptosis by damaging DNA, oxidizing membrane lipids, and/or directly activating the expression of the genes/proteins responsible for apoptosis. The generated ROS also act synergistically with calcium to activate mitochondrial phospholipase A2 enhancing further inflammatory response and renal damage in renal I/R injury. In mitochondria, the generated ROS opens the permeability transition pore (PTP) which induces swelling of mitochondria, collapse of mitochondrial membrane potential, and uncoupling of mitochondrial oxidative phosphorylation which produces more ROS. Cytochrome c is therefore released into cytosole leading to cell necrosis and apoptosis. The overproduction of ROS and accumulation of mitochondrial Ca2+ have been proposed to be the main triggers of the PTP opening.
Calcium also plays an important role in ischemic reperfusion (I/R) renal injury. Ischemia causes the loss of ATP which will result in the impairment of Na+-K+ pump in cell membrane leading to loss of membrane potential and opening of voltage-gated Ca2+ channel, therefore influx of extracellular Ca2+ and elevation of intracellular Ca2+. Besides, ATP depletion also impairs the re-uptake of Ca2+ by endoplasmic reticulum, further enhances the intracellular Ca2+ accumulation. Elevation of intracellular Ca2+ will activate protease, phospholipase and degradation of cytoskeleton leading to cell death. Mitochondria takes intracellular excess Ca2+ and initiate the opening of PTP leading to mitochondrial damage and related apoptotic pathway. Blocking the Ca2+ channel or using Ca2+ chelator attenuates I/R renal injury. Bradykinin is known to enhance the intracellular free Ca2+ accumulation, mitochondria Ca2+ uptake and mitochondrial ROS generation in cell culture. So it may enhance the Ca2+ accumulation and ROS generation in kidney during I/R renal injury leading to more cell damage.
Therefore, we hypothesize that kallikrein-bradykinin system could play a role in I/R renal injury, either beneficial or detrimental. We conducted animal and cell culture studies to evaluate the role of kallikrein-bradykinin system in acute I/R renal injury. We activated the tissue kallikrein-kinin system by administration of rat tissue kallikrein protein in a rat model of renal I/R injury and examined the effects on renal function, pathology, ROS generation, apoptosis and inflammatory response. In cell culture studies, we use ATP depletion model in renal proximal tubular cells to simulate ischemia. The effect of bradykinin on cellular death, ROS generation, mitochondrial damage, and apoptotic pathway activation induced by ATP depletion was examined. Possible signaling pathway was also approached. The effect of bradykinin on cell death was further examined in hypoxic study. These results provided insights into the role of the kallikrein-kinin system in acute I/R renal injury.
Forty eight hours after I/R renal injury, the serum creatinine, blood urea nitrogen and excretion fraction of sodium increased in control rats. Administration of kallikrein protein further increased the impairment of renal function and this effect was blocked by co-treatment with bradykinin B2 receptor (B2R) antagonist (HOE140), but not bradykinin B1 receptor (B1R) antagonist (Lys-(des-Arg9- Leu8-bradykinin). Supplement with phosphate buffered saline could not reverse the detrimental effect of kallikrein. Administration with B2R or B1R antagonist only did not attenuate I/R renal injury. We also measured the renal artery blood flow one hour after I/R injury. Renal blood flow did decrease after I/R injury in control rats, but there was no difference between rats whether treated with kallikrein or not. The blood pressure 1 hour after reperfusion was also not different between each group. These results indicate that the activation of B2R is detrimental to I/R renal injury and this effect is not through enhanced natriuresis by bradykinin activation, change of renal blood flow or systemic blood pressure.
We further examined the degree of tubular necrosis and inflammatory response after I/R injury. Similar phenomenon was seen. Activation of B2R enhanced the tubular necrosis, macrophage/neutrophil infiltration, the gene/protein expression of MCP-1 and TNF-alpha in renal tissue. To explore the possible mechanism, we measured the oxidative stress between different groups. Using real-time chemiluminescence recording to measure superoxide production, we found that superoxide generation began in the beginning of ischemia and increased progressively. The initiation of blood re-flow in beginning of reperfusion stimulated a surge of superoxide production and this production sustained till 3.5 hours after I/R procedure. Activation of B2R aggravated the ROS generation in both ischemic and reperfusion phases. Measurement of tissue hydrogen peroxide, free malondialdehyde, reduced and oxidized glutathione also demonstrated the enhancement of oxidative stress by activation of B2R. The renal apoptotic cells, stained by TUNEL and anti-caspase-cleaved fragment (p85) of human poly (ADP-ribose) polymerase (PARP) antibody, were examined 4 hours after I/R injury from the same tissue from ROS measurement. The kallikrein treated rats, which had more ROS generation, also had higher degree of tubular cell apoptosis. Therefore, the in vivo study indicates that early activation of B2R aggravates I/R renal injury through enhancement of ROS generation in rats. This finding is compatible with the findings of Chien et al. (2001), in that the amount of ROS was positively correlated to the duration of ischemia and the degree of apoptosis.
The sources for ROS generation after I/R may come from inflammatory cells or the resident cells. In kidney Chien et al. (2001) demonstrated the major sources of ROS came from proximal tubular cells in early stage (within 4 h after ischemia) of renal I/R injury. The site of ROS generation was compatible with the site of tubular necrosis induced by I/R injury. It is probable the augmentation of ROS generation by bradykinin receptor activation also happened in proximal tubular cells after I/R injury. In cell culture, bradykinin increases mitochondria ROS generation in cardiomyocyte or vascular smooth muscle cell. Therefore, we hypothesize that bradykinin could enhance ROS generation, mitochondrial injury and cellular death induced by ischemic injury in renal tubular cells culture model.
Cell culture studies were conducted to uncover the underlying mechanism of the detrimental effect of bradykinin system in ischemic injury. Using substrate deprivation and treatment with antimycin A, cellular ATP was depleted to simulate ischemia in proximal tubular cell line, NRK52E. We found that activation of B2R aggravated the cellular death induced by ATP depletion, which were revealed by more LDH release, more subG0/G1 cells, more apoptotic body and Annexin V biding assay in bradykinin treated cells. Using sub G0/G1 evaluation, we further confirmed that bradykinin also enhanced the apoptosis induced by hypoxic stimuation for 9 hours. Using fluorescence dye and flow cytometry, we proved that activation of B2R enhanced the ROS generation (including superoxide and hydrogen peroxide) and loss of mitochondrial membrane potential induced by ATP depletion. Loss of mitochondrial membrane potential causes mitochondrial damage, therefore related apoptotic pathway activation. Our results revealed that there was more cytochrome c released into cytoplasma in bradykinin treated cells. The activated form of caspase 9 and PARP was more in bradykinin treated cells than in control cells. Renal tubular cell was known to be the major source of ROS in I/R renal injury and prolong ischemia was associated with more ROS generation and tubular damage. Our studies demonstrated that activation of B2R enhanced ROS generation induced by ATP depletion in renal tubular cells. With more ROS, more mitochondrial damage and cell death were observed in bradykinin treated cells when compared to cells subjected to ATP depletion only. This in vitro study complements the in vivo evidence that the enhancement of ischemic reperfusion renal injury by early B2R activation was through the enhancement of ROS generation.
In addition to ROS, calcium also facilitate the opening of mitochondrial membrane permeability transition pore and plays an important role in ischemic/reperfusion injury in vivo and in vitro. Bradykinin is known to enhance intracellular Ca2+ accumulation in cell culture. In this study, we demonstrated activation of B2R also enhanced the intracellular and mitochondrial Ca2+ accumulation induced by ATP depletion. Even in cells already subjected to ATP depletion for 1.5 hours, bradykinin also could increase intracellular Ca2+ concentration. More intracellular and mitochondrial Ca2+ act synergistically with ROS to open the mitochondrial membrane permeability transition pore leading to greater loss of mitochondrial membrane potential, therefore more severe mitochondrial swelling, and rupture of the mitochondrial membrane. Accompanying the mitochondrial damage, cytochrome c is released into cytosole, which cleaves procaspase 9 into the active form of caspase 9, thus conferring the apoptotic death signal. These more apoptotic events are seen in bradykinin treated cells. These results indicate the important role of Ca2+ in bradykinin mediated detrimental role in cellular death induced by ATP depletion.
There are three sources of calcium mobilization to elevate intracellular Ca2+: (1) Ca2+ release from internal stores, such as the endoplasmic reticulum; (2) influx through dihydropyridine sensitive (voltage-dependent) Ca2+ channels; (3) and dihydropyridine insensitive (receptor mediated) Ca2+ input from extracellular space. The release of Ca2+ from internal stores could be stimulated by increase of inositol 1,4,5-trisphosphate (IP3). Activation of bradykinin receptors is known to activate PLC resulting in phosphoinositide hydrolysis in the plasma membrane and generation of IP3 in different cell types. We applied PLC inhibitor (U73122) in cell culture studies and demonstrated that the enhancement of ATP depletion related intracellular and mitochondrial Ca2+ accumulation by bradykinin could be blocked by PLC inhibitor to the similar level as cells subjected to ATP depletion only. This indicates the possible role of PLC-IP3 signaling pathway in bradykinin enhanced Ca2+ accumulation induced by ATP depletion. The detrimental effect of bradykinin on generation of ROS, loss of mitochondrial membrane potential and apoptosis was also reversed by co-treatment with PLC inhibitor. These results indicate the detrimental effect of bradykinin on ATP depletion related cellular injury is through activation of PLC signaling pathway. However, bradykinin could also increase intracellular Ca2+ through the opening of voltage-dependent or receptor mediate Ca2+ channel. The possible involvement of other two pathways in bradykinin enhanced calcium accumulation and ROS generation induced by ATP depletion needs further studies to clarify. Our studies revealed that PLC inhibitor could almost blunted the dentrimental effect of bradykinin on ATP depletion related cell injury, so the voltage-dependent or receptor mediate Ca2+ channel may play a minor role or should be activated through PLC pathway activation.
We do not know whether bradykinin receptor activation could enhance ROS generation and apoptosis induced by ATP depletion independent of mitochondrial injury. Endoplasmic reticulum (ER) stress is known to induce apoptosis in ischemic reperfusion injury. Bradykinin could enhance ROS generation, intracellular Ca2+ accumulation, release of Ca2+ from ER in cells subjected to ATP depletion. All these events could result in ER stress. Once ER stress is present, ER stress related apoptotic pathway, such as caspase 12 activation, c-Jun NH2-terminal kinase pathway activation and growth arrest and DNA damage–inducible gene 153 expression will be activated. Therefore, bradykinin may enhance cell death through enhancement of ER stress induced by ATP depletion. But this hypothesis needs further studies to clarify.
One of the other possible mechanisms in which activation of bradykinin B2 receptor aggravates I/R renal injury is through the activation of phospholipase A2 (PLA2). Several studies have shown that cytosolic, mitochondrial and microsomal subcellular fractions of PLA2 is activated after I/R injury. PLA2 could also be activated by increases in intracellular or extracellular Ca2+ elicited by the inflammatory mediator bradykinin. ROS has also been observed to act synergistically with calcium to activate mitochondrial PLA2 in isolated kidney and in vivo I/R renal injury. Therefore, it is probable that activation of PLA2 induced by ischemic reperfusion renal injury is further enhanced by activation of B2R. The activated PLA2 is critical to the production of proinflammatory lipid mediators, as the initiation of the inflammatory response. Inflammation is considered to be the major pathophysiological pathway of acute renal failure resulting from ischemia. More activation of PLA2 may induce more tissue injury in I/R renal injury with bradykinin system activation.
In contrast to the protective role of bradykinin system in heart and brain in some studies, our study did not reveal a beneficial role of early activation of bradykinin system in I/R renal injury. The reason for this discrepancy is unknown. One possible explanation is that bradykinin may play different role in different stage of I/R injury. In beneficial effect study of brain the activation of bradykinin indeed was initiated many hours after ischemia since Xia et al.(2004) administrated kallikrein gene after the initiation of reperfusion. The studies of detrimental effect of bradykinin in other organs were usually performed using early blocking of B2R strategy. In early stage bradykinin might exert the inflammatory response while in late stage bradykinin may facilitate cell proliferation. These effects put different impact on I/R injury. The other explanation is that heart and brain have collateral circulation that bradykinin could enhance collateral circulation through vasodilatory effect once one supply vessel is occluded. But there is only one artery to supply blood in kidney. Therefore, bradykinin may only exert the inflammatory effect in early stage of I/R renal injury.
In clinical practice, plasma kallikrein-bradykinin system is activated during sepsis leading to systemic vasodilation and enhances inflammatory response. Besides, recent studies have revealed that inflammatory response, in addition to ischemia, also played a role in the pathogenesis of septic ARF. Tissue kallikrein is found to be released during intestinal inflammation in animal, human inflammatory bowel disease and mechanical ventilation related lung injury. Therefore, it is possible that renal tissue kallikrein-bradykinin system is also released and activated by the stimulation of inflammation in sepsis. Since activation of B2R will aggravates I/R renal injury, kallikrein-bradykinin system may play a synergistic action with ischemia on the development of septic ARF. To test this hypothesis, we need to examine whether renal kallikrein-bradykinin system is activated during sepsis and study the correlation between the development of ARF and the activity of kallikrein-bradykinin system.
We also try to study the role or kallikrein-bradykinin system in CKD. Changes in the urinary excretion of kallikrein are predictive of the synthesis and activation of kallikrein within the kidney. Evaluation of urinary kallikrein excretion in patients with kidney disease may be helpful to clarify the role of kallikrein-kinin system in kidney disease. We conducted the clinical study to evaluate the relationship between urinary kallikrein and renal function or proteinuria. Using multiple regression analysis, we found that the urinary kallikrein excretion was positively correlated to reanl function but not the degree of proteinuria. After 12 months of follow up, the renal function deteriorated while the urinary kallikrein excretion also decreasesd. Kallikrein is excreted from renal tubule. As the renal injury progress there will be loss of the nephron number. So the urinary kallikrein excretion decreases. Our results indicate that urinarly kallikrein excretion may be an indicator of reanl function change.
Interestingly, the urinary kallikrein concentration is positively correlated to urinary MCP-1 concentration. Our analysis revealed that the correlation was not related to their relationship to renal function independently. The higher urinary MCP-1 may indicate the higher acitivity of inflammation in kidney. The result indicates that urinary kallikrein is excreted when renal inflammatory damage is present. This finding is compatible to the previous studies that kallikrein is excreted in animal and human inflammatory intestinal disease and in mechanimal lung injury. But the correlation is only found in one time point in our study. Serial follow up of the relationship between urinary kallikrein and MCP-1 is necessary to clarifly the true relationship. If urinary kallikrein did correlate to the activity of renal damage, measuring urinary kallikrein serially may be helpful in realizing the activity of disease process. But the application in clinical practice need further studies to clarify the relationship between urinary kallikrein excretion and status of renal injury, especially the correlation to the renal pathological findings.
After release kallikrein may exert its action, cleave kininogen into bradykinin and initiate the inflammatory reaction. But bradykinin is also reported to enhance matrix degradation through inhibition of PAI-1 expression. Inflammation and fibrosis are two important processes in CKD. Therefore, kallikrein-bradykinin system may have effect on renal disease progression. But whether the released kallikrein is beneficial or detrimental to CKD is unknown. To study the role of kallikrein-bradykinin system in CKD, we will conduct animal model study, by activating or blocking bradykinin receptor, to see the effect of bradykinin system on renal inflammation and fibrosis. These studies will more specifically demonstrate the role of bradykinin system in CKD.
In conclusion, early activation bradykinin B2 receptor has a deleterious effect on I/R renal injury through enhancement of ROS generation in both ischemic and reperfusion phases. The in vitro study reveals that the detrimental effect of B2R activation in cell injury induced by ATP depletion or hypoxia is through accumulation of intracellular Ca2+, generation of ROS, and mitochondrial damage by PLC pathway activation. However, the clinical implication and other possible signaling pathway of which B2R activation enhances I/R renal injury needs further studies to clarify. In CKD, urinary kallikrein excretion is positively correlated to renal function and urinary inflammatory cytokines. This indiates that urinary kallikrein may be a potential marker of renal damage in CKD. But the role of kallikrein-bradykinin system in CKD is still unclear and is to be studied.

並列關鍵字

kallikrein ； bradykininvischemic reperfusion ； chronic kidney disease ； reactive oxygen species ； apoptosis ； calciumvcytokine

參考文獻

Agata J, Chao L, Chao J. Kallikrein gene delivery improves cardiac reserve and attenuates remodeling after myocardial infarction. Hypertension 2002, 40:653-659.

Ahluwalia A, Perretti M. Involvement of bradykinin B1-receptors in the polymorphonuclear leukocyte accumulation induced by IL-1 beta in vivo in the mouse. J Immunol 1996, 156:269-274.

Arakawa K, Takeyoshi I, Akao Y, Totsuka O, Matsumoto K, Morishita Y. Bradykinin B2 receptor antagonist FR173657 ameliorates small bowel ischemia-reperfusion injury in dogs. Dig Dis Sci 2005, 50:27-36.

Badr KF, Kelley VE, Rennke HG, Brenner BM. Roles for thromboxane A2 and leukotrienes in endotoxin-induced acute renal failure. Kidney Int 1986, 30:474-480.

Bailly V, Zhang Z, Meier W, Cate R, Sanicola M, Bonventre JV. Shedding of kidney injury molecule-1, a putative adhesion protein involved in renal regeneration. J Biol Chem 2002, 277:39739-39748.