腸源性尿毒癥毒素硫酸對甲酚和硫酸吲哚酚的研究進展

·綜述·

腸源性尿毒癥毒素硫酸對甲酚和硫酸吲哚酚的研究進展

成云曹學森鄒建洲

(復旦大學附屬中山醫(yī)院腎內科，上海市腎病與透析研究所， 上海200032)

關鍵詞

Research Development of Enterogenous Uremic Toxins: P-cresyl Sulfate and Indoxyl SulfateCHENGYunCAOXuesenZOUJianzhou

DepartmentofNephrology，ZhongshanHospital,FudanUniversity，Shanghai200032，China

尿毒癥毒素是指終末期腎病(end-stage renal disease，ESRD)時不能經尿液清除、潴留在體內且有毒性作用的物質。據(jù)歐洲尿毒癥毒素協(xié)作組(EUTox)統(tǒng)計，至2011年4月已發(fā)現(xiàn)160種尿毒癥毒素[1]。尿毒癥毒素根據(jù)其理化性質可分為3類。(1)不能與蛋白質結合的水溶性小分子物質：相對分子質量通常小于500，較易經血液透析清除，如尿素、肌酐；(2)蛋白質結合物質：大多數(shù)相對分子質量較小，很難通過血液透析清除，如硫酸對甲酚(p-cresyl sulfate，PCS)、硫酸吲哚酚(indoxyl sulfate，IS)；(3)中分子物質：相對分子質量通常大于500，常規(guī)血液透析效果不理想，如甲狀旁腺素、β2微球蛋白。尿毒癥毒素根據(jù)其來源分類也可分為3類，(1)內源性代謝產物：自身代謝產生，如非對稱性二甲基精氨酸(asymmetric dimethylarginine,ADMA)[2]；(2)微生物代謝產物：主要是腸道菌群代謝物質，如吲哚類、酚類；(3)外源性攝入物質：如草酸鹽[3]。研究[4- 5]表明，慢性腎臟病(chronic kidney disease，CKD)患者腸道菌群的種類和數(shù)量與健康人群顯著不同，其毒性產物與CKD及其并發(fā)癥的進展密切相關。其中，PCS和IS是當前研究最多的腸源性尿毒癥毒素，本文對其研究進展作一綜述。

1PCS、IS的產生和代謝

PCS主要在腸道產生，相對分子質量188，與血漿白蛋白結合率為94%[6]。腸道厭氧菌將食物中的苯丙氨酸和酪氨酸轉變?yōu)?-羥基苯乙酸。4-羥基苯乙酸脫羧為對甲酚，大部分對甲酚經腸道黏膜吸收，在腸道上皮細胞磺基轉移酶的作用下轉化為PCS[7]。PCS主要通過腎小管基底膜側的有機陰離子轉運體(organic anion transporter，OAT)分泌到腎小管管腔，經尿液排出[8]。

IS主要在腸道產生，相對分子質量251[9]，蛋白結合率達90%以上。食物中的色氨酸經大腸埃希菌分解產生吲哚，吲哚經門靜脈進入肝臟經羥化、硫酸化，最終生成IS。IS主要通過腎小管OAT分泌、排泄[10]。

2PCS、IS的腎臟毒性

PCS可通過促進腎臟纖維化加快腎臟病進展、腎功能下降[11]。PCS主要通過以下機制促進腎臟纖維化：(1)PCS可顯著增加腎組織腎素、血管緊張素Ⅱ1型受體(AT1R)表達，激活腎素-血管緊張素-醛固酮系統(tǒng)(renin-angiotensin-aldosterone system，RAAS)，進而促進腎間質成纖維細胞的增殖與分化，加重腎組織纖維化[12]；(2)PCS具有促炎作用，可促進腎間質單核細胞/巨噬細胞浸潤[11]，上調促炎因子表達[13]，引起腎間質纖維化；(3)體外實驗證實，PCS可促進小鼠近端腎小管上皮細胞炎性相關基因的表達，如轉化生長因子-β(transforming growth factor-β，TGF-β)、白介素-6(interleukin-6，IL-6)等[14]，而TGF-β可促進腎小管間質纖維化[15]，IL-6可通過誘導腎臟纖維化相關基因及內皮素-1基因的表達加速CKD的進展[16]；(4)Klotho基因可編碼一種參與成纖維細胞生長因子受體構成的跨膜蛋白，這種跨膜蛋白可延緩腎臟纖維化進程，發(fā)揮腎臟保護作用[17-18]，而PCS通過促進DNA甲基轉移酶表達，使Klotho基因超甲基化，進而抑制Klotho基因表達[19]，使Klotho基因產物的腎臟保護作用下降或消失，促進腎臟纖維化，加速腎臟病進展。

IS促進腎臟纖維化的機制主要有：(1)IS促進腎小管上皮細胞活性氧簇(reactive oxygen species，ROS)的產生，激活核轉錄因子κB(nuclear factor-κB，NF-κB)、p53、 細胞外信號調節(jié)激酶(extracellular signal-regulated kinase，ERK)等調節(jié)因子，使單核細胞趨化蛋白-1(monocyte chemotactic protein-1，MCP-1)、細胞間黏附分子-1(intercellular adhesion molecule-1，ICAM-1)的表達上調，引起單核細胞/巨噬細胞在小管間質聚集，進而促進腎臟纖維化[20-21]；(2)IS使腎組織腎素、血管緊張素原、AT1R表達增加，AT2R表達減少，進而通過激活RAAS及促進TGF-β表達，使腎間質細胞向成纖維細胞轉化，引起腎臟纖維化[22]；(3)IS也可通過促進Klotho基因超甲基化而促進腎臟纖維化[19]。

3PCS、IS的心血管毒性

大量研究[7，23-24]證實，PCS水平與CKD患者心血管疾病的發(fā)生及全因死亡獨立相關。Schepers等[13]研究發(fā)現(xiàn)，PCS可誘導白細胞產生自由基，進而引起ESRD患者的血管損傷。Watanabe等[25]研究發(fā)現(xiàn)，PCS可使人臍靜脈內皮細胞及人主動脈平滑肌細胞內NADPH氧化酶(NAPDH oxidase，NOX)的表達顯著增加，促進細胞內產生ROS，進而損害血管內皮細胞及平滑肌細胞。Han等[26]研究發(fā)現(xiàn)，PCS可通過增強NOX活性、增加ROS，促進心肌細胞凋亡。

研究[27]顯示，IS可提高ESRD患者全因死亡率及心血管事件發(fā)病率，其機制主要有以下兩方面。(1)IS可促進血管損傷，研究[28]發(fā)現(xiàn)，IS可致循環(huán)中內皮損傷標志物內皮微粒(endothelial microparticles，EMPs)產生增加，提示其有致血管內皮損傷作用。IS引起內皮損傷主要是通過促氧化應激作用實現(xiàn)的。IS可促進NOX活化、使內皮細胞產生的ROS增多[29]，升高的ROS可通過激活NF-κB，增加MCP-1及ICAM-1的表達[30]，導致血管內皮損傷。此外，IS可以通過激活絲裂原活化蛋白激酶(mitogen-activated protein kinase ，MAPK)途徑促進血管平滑肌細胞(vascular smooth muscle cell ，VSMC)增殖[31]；并可通過促進骨母細胞特異性蛋白表達增加而加重動脈鈣化、使動脈壁增厚[32]。近年來研究[33]證實，IS可促進大鼠主動脈細胞衰老相關蛋白，如p16INK4a、p21WAF1/CIP1的表達，提示IS有加速動脈衰老作用。(2)IS可加速心肌損傷，研究[34]發(fā)現(xiàn)，IS可通過促氧化應激、削弱抗氧化屏障作用促進心肌纖維化及心肌細胞肥大。此外，IS可通過抑制單磷酸腺苷活化蛋白激酶/解偶聯(lián)蛋白2(AMP-activated protein kinase/uncoupling protein 2，AMPK/UCP2)途徑促進心肌肥大[35]。

4PCS、IS的其他作用

近年研究提示，PCS可能與CKD相關的胰島素抵抗有關。PCS通過激活胰島素信號轉導通路中的ERK1/2誘導小鼠出現(xiàn)胰島素抵抗，使其脂肪含量減少，脂肪在肝臟及肌肉重新分布[36]。骨代謝方面，Tanaka等[37]研究發(fā)現(xiàn)，PCS通過激活c-Jun 氨基末端激酶( c-Jun N-terminal kinase，JNK) 和p38分裂原激活蛋白激酶(p38 mitogen activated protein kinases，p38MAPK)信號轉導途徑導致成骨細胞功能障礙，引起腎性骨病。

Kim等[38]研究發(fā)現(xiàn)，IS可通過抑制成骨細胞的分化、誘導成骨細胞凋亡，從而引起骨骼病變。此外，研究[39]發(fā)現(xiàn)，IS可導致體外培養(yǎng)的成骨細胞抵抗甲狀旁腺激素，從而導致腎性骨病的發(fā)生。

5PCS和IS的清除

PCS和IS均為蛋白質高親和力毒素，常規(guī)透析方法難以清除。Meert等[40]研究發(fā)現(xiàn)，透析中增加對流量也利于PCS及IS的清除。不同材質的透析膜對這兩種毒素的清除率無差異[41]。Meijers等[42]發(fā)現(xiàn)，血漿分離吸附技術對PCS的清除效果顯著優(yōu)于高通量透析，但血漿分離吸附技術成本高昂，目前無法在臨床推廣。

此外，由于PCS和IS主要由腸道產生，理論上可以通過改變腸道菌群降低PCS和IS的濃度，但目前尚無相關研究。目前研究較多的腸道吸附劑，如AST-120，Owada 等[43]的研究顯示，AST-120可清除部分腸源性毒素，并可延緩尿毒癥大鼠的腎功能惡化。

6展望

目前對PCS及IS 作用機制的了解已較深入，但是仍無有效的、適合臨床應用的清除PCS及IS的透析方式或藥物，需要進一步探索。

參考文獻

[ 1 ]Duranton F, Cohen G, De Smet R, et al. Normal and pathologic concentrations of uremic toxins[J]. J Am Soc Nephrol, 2012,23(7):1258-1270.

[ 2 ]Kielstein JT, Zoccali C. Asymmetric dimethylarginine: a cardiovascular risk factor and a uremic toxin coming of age?[J]. Am J Kidney Dis, 2005,46(2):186-202.

[ 3 ]Goldfarb DS, Modersitzki F, Asplin JR. A randomized, controlled trial of lactic acid bacteria for idiopathic hyperoxaluria[J]. Clin J Am Soc Nephrol, 2007,2(4):745-749.

[ 4 ]Vaziri ND, Wong J, Pahl M, et al. Chronic kidney disease alters intestinal microbial flora[J]. Kidney Int, 2013,83(2):308-315.

[ 5 ]Wu IW, Hsu KH, Lee CC, et al. p-Cresyl sulphate and indoxyl sulphate predict progression of chronic kidney disease[J]. Nephrol Dial Transplant, 2011,26(3):938-947.

[ 6 ]Niwa T. Update of uremic toxin research by mass spectrometry[J]. Mass Spectrom Rev, 2011,30(3):510-521.

[ 7 ]Bammens B, Evenepoel P, Keuleers H, et al. Free serum concentrations of the protein-bound retention solute p-cresol predict mortality in hemodialysis patients[J]. Kidney Int, 2006,69(6):1081-1087.

[ 8 ]Miyamoto Y, Watanabe H, Noguchi T, et al. Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney[J]. Nephrol Dial Transplant, 2011,26(8):2498-2502.

[ 9 ]Dou L, Bertrand E, Cerini C, et al. The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation and wound repair[J]. Kidney Int, 2004,65(2):442-451.

[10]Deguchi T, Ohtsuki S, Otagiri M, et al. Major role of organic anion transporter 3 in the transport of indoxyl sulfate in the kidney[J]. Kidney Int, 2002,61(5):1760-1768.

[11]Lee SB, Kalluri R. Mechanistic connection between inflammation and fibrosis[J]. Kidney Int Suppl, 2010(119):S22-S26.

[12]Huang Y, Wongamorntham S, Kasting J, et al. Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin Ⅱ-independent mechanisms[J]. Kidney Int, 2006,69(1):105-113.

[13]Schepers E, Meert N, Glorieux G, et al. P-cresylsulphate, the main in vivo metabolite of p-cresol, activates leucocyte free radical production[J]. Nephrol Dial Transplant, 2007,22(2):592-596.

[14]Sun CY, Hsu HH, Wu MS. p-Cresol sulfate and indoxyl sulfate induce similar cellular inflammatory gene expressions in cultured proximal renal tubular cells[J]. Nephrol Dial Transplant, 2013,28(1):70-78.

[15]Wang L, Cao AL, Chi YF, et al. You-gui Pill ameliorates renal tubulointerstitial fibrosis via inhibition of TGF-beta/Smad signaling pathway[J]. J Ethnopharmacol, 2015,169:229-238.

[16]Zhang W, Wang W, Yu H, et al. Interleukin 6 underlies angiotensin Ⅱ-induced hypertension and chronic renal damage[J]. Hyp ertension, 2012,59(1):136-144.

[17]Barker SL, Pastor J, Carranza D, et al. The demonstration of alpha Klotho deficiency in human chronic kidney disease with a novel synthetic antibody[J]. Nephrol Dial Transplant, 2015,30(2):223-233.

[18]Haruna Y, Kashihara N, Satoh M, et al. Amelioration of progressive renal injury by genetic manipulation of Klotho gene[J]. Proc Natl Acad Sci USA, 2007,104(7):2331-2336.

[19]Sun CY, Chang SC, Wu MS. Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation[J]. Kidney Int, 2012,81(7):640-650.

[20]Shimizu H, Bolati D, Higashiyama Y, et al. Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-kappaB, p53, ERK, and JNK in proximal tubular cells[J]. Life Sci, 2012,90(13-14):525-530.

[21]Shimizu H, Yisireyili M, Higashiyama Y, et al. Indoxyl sulfate upregulates renal expression of ICAM-1 via production of ROS and activation of NF-kappaB and p53 in proximal tubular cells[J]. Life Sci, 2013,92(2):143-148.

[22]Sun CY, Chang SC, Wu MS. Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition[J]. PLoS One, 2012,7(3):e34026.

[23]Liabeuf S, Barreto DV, Barreto FC, et al. Free p-cresylsulphate is a predictor of mortality in patients at different stages of chronic kidney disease[J]. Nephrol Dial Transplant, 2010,25(4):1183-1191.

[24]Wu IW, Hsu KH, Hsu HJ, et al. Serum free p-cresyl sulfate levels predict cardiovascular and all-cause mortality in elderly hemodialysis patients--a prospective cohort study[J]. Nephrol Dial Transplant, 2012,27(3):1169-1175.

[25]Watanabe H, Miyamoto Y, Enoki Y, et al. p-Cresyl sulfate, a uremic toxin, causes vascular endothelial and smooth muscle cell damages by inducing oxidative stress[J]. Pharmacol Res Perspect, 2015,3(1):e92.

[26]Han H, Zhu J, Zhu Z, et al. p-Cresyl sulfate aggravates cardiac dysfunction associated with chronic kidney disease by enhancing apoptosis of cardiomyocytes[J]. J Am Heart Assoc, 2015,4(6):e001852.

[27]Melamed ML, Plantinga L, Shafi T, et al. Retained organic solutes, patient characteristics and all-cause and cardiovascular mortality in hemodialysis: results from the retained organic solutes and clinical outcomes (ROSCO) investigators[J]. BMC Nephrol, 2013,14:134.

[28]Faure V, Dou L, Sabatier F, et al. Elevation of circulating endothelial microparticles in patients with chronic renal failure[J]. J Thromb Haemost, 2006,4(3):566-573.

[29]Dou L, Jourde-Chiche N, Faure V, et al. The uremic solute indoxyl sulfate induces oxidative stress in endothelial cells[J]. J Thromb Haemost, 2007,5(6):1302-1308.

[30]Tumur Z, Shimizu H, Enomoto A, et al. Indoxyl sulfate upregulates expression of ICAM-1 and MCP-1 by oxidative stress-induced NF-kappaB activation[J]. Am J Nephrol, 2010,31(5):435-441.

[31]Yamamoto H, Tsuruoka S, Ioka T, et al. Indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells[J]. Kidney Int, 2006,69(10):1780-1785.

[32]Adijiang A, Goto S, Uramoto S, et al. Indoxyl sulphate promotes aortic calcification with expression of osteoblast-specific proteins in hypertensive rats[J]. Nephrol Dial Transplant, 2008,23(6):1892-1901.

[33]Adijiang A, Higuchi Y, Nishijima F, et al. Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats[J]. Biochem Biophys Res Commun, 2010,399(4):637-641.

[34]Yisireyili M, Shimizu H, Saito S, et al. Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats[J]. Life Sci, 2013,92(24-26):1180-1185.

[35]Yang K, Xu X, Nie L, et al. Indoxyl sulfate induces oxidative stress and hypertrophy in cardiomyocytes by inhibiting the AMPK/UCP2 signaling pathway[J]. Toxicol Lett, 2015,234(2):110-119.

[36]Koppe L, Pillon NJ, Vella RE, et al. p-Cresyl sulfate promotes insulin resistance associated with CKD[J]. J Am Soc Nephrol, 2013,24(1):88-99.

[37]Tanaka H, Iwasaki Y, Yamato H, et al. p-Cresyl sulfate induces osteoblast dysfunction through activating JNK and p38 MAPK pathways[J]. Bone, 2013,56(2):347-354.

[38]Kim YH, Kwak KA, Gil HW, et al. Indoxyl sulfate promotes apoptosis in cultured osteoblast cells[J]. BMC Pharmacol Toxicol, 2013,14:60.

[39]Nii-Kono T, Iwasaki Y, Uchida M, et al. Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells[J]. Kidney Int, 2007,71(8):738-743.

[40]Meert N, Eloot S, Schepers E, et al. Comparison of removal capacity of two consecutive generations of high-flux dialysers during different treatment modalities[J]. Nephrol Dial Transplant, 2011,26(8):2624-2630.

[41]Ficheux A, Gayrard N, Szwarc I, et al. The use of SDS-PAGE scanning of spent dialysate to assess uraemic toxin removal by dialysis[J]. Nephrol Dial Transplant, 2011,26(7):2281-2289.

[42]Meijers BK, Weber V, Bammens B, et al. Removal of the uremic retention solute p-cresol using fractionated plasma separation and adsorption[J]. Artif Organs, 2008,32(3):214-219.

[43]Owada S, Maeba T, Sugano Y, et al. Spherical carbon adsorbent (AST-120) protects deterioration of renal function in chronic kidney disease rats through inhibition of reactive oxygen species production from mitochondria and reduction of serum lipid peroxidation[J]. Nephron Exp Nephrol, 2010,115(4):e101-e111.

中圖分類號R692.5

文獻標識碼A

通訊作者鄒建洲，E-mail：jianzzou@163.com

基金項目：上海市科學技術委員會基金項目(編號：15DZ0503402)