何華妮
(海南省瓊海市人民醫(yī)院 腎內(nèi)科,海南 瓊海 571400)
腎組織內(nèi)皮素受體A、B在老年糖尿病腎病發(fā)展中的作用研究
何華妮
(海南省瓊海市人民醫(yī)院 腎內(nèi)科,海南 瓊海 571400)
目的探討腎組織中內(nèi)皮素受體A、B(ETAR、ETBR)表達(dá)變化在老年糖尿病腎病發(fā)展中的作用。方法選取2015年1月-2016年6月該院收治的糖尿病腎病老年患者34例,根據(jù)糖尿病腎病病理分型分為:糖尿病腎?、窦墸―1組10例),Ⅱ級(D2組12例),Ⅲ級(D3組12例);選取同期在該院行腎腫瘤切除術(shù)后遠(yuǎn)離腎腫瘤的正常組織為對照組(10例)。免疫組織化學(xué)檢測ETAR、ETBR的表達(dá)量;免疫熒光雙染法定位ETAR、ETBR在腎組織的分布部位。收集生化檢測指標(biāo),計(jì)算腎小球硬化率,進(jìn)行相關(guān)性分析。結(jié)果D3組尿蛋白水平高于D2和D1組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);D3組腎小球?yàn)V過率(GFR)和白蛋白(Alb)與D1組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05);D3組和D2組腎小球硬化率均高于D1組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。糖尿病腎病各組ETAR、ETBR表達(dá)量均高于對照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);對照組ETAR表達(dá)量少于ETBR,隨著病情加重糖尿病腎病患者腎組織中ETAR表達(dá)量逐漸增加,組間比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05);D2組ETBR表達(dá)量最高,與D1組和D3組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05);ETAR/ETBR在各組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。ETAR在腎小管近端和遠(yuǎn)端均存在表達(dá),ETBR主要表達(dá)于腎小管近端。糖尿病腎病患者腎組織中ETAR表達(dá)量與病程、尿蛋白水平、腎小球硬化率(ROG)呈正相關(guān);ETBR表達(dá)量與病程呈負(fù)相關(guān);ETAR/ETBR比值與病程、尿蛋白水平及ROG呈正相關(guān),與Alb、GFR呈負(fù)相關(guān)。結(jié)論老年糖尿病患者腎組織中ETAR、ETBR表達(dá)量增加,ETAR/ETBR值倒置與病情發(fā)展呈正相關(guān)。
糖尿病腎?。荒I組織;內(nèi)皮素受體;老年人
Abstract:ObjectiveTo investigate the endothelin receptor A,B (ETAR,ETBR)expression in renal tissue of the development in elder diabetic nephropathy.MethodsFrom January 2015 to June 2016,34 elderly patients with diabetic nephropathy in our hospital were divided into 3 groups according to pathological type:10 cases of group D1(12 cases),grade D2 group(12 cases),grade D3 group.Patients underwent renal tumor resection from renal tumors in our hospital as the control group (10 cases).The expressions of ETAR and ETBR were detected by immunohistochemistry.The location of ETAR and ETBR in renal tissue was detected by immunofluorescence double staining method.Biochemical detection indexes were collected to calculate the rate of glomerular sclerosis,and the correlation was analyzed.ResultsThe urine protein level of D3 group was significantly higher than that of D2 and D1 group,the difference was statistically significant (P<0.05).Glomerular filtration rate(GFR)and albumin(Alb)had significant difference between group D3 and D1(P<0.05).D3 group and D2 group glomerular sclerosis rate were significantly higher than that in D1 group,thedifference had statistical difference (P<0.05).The expression of ETAR and ETBR in diabetic nephropathy group were significantly higher than the control group,the difference was statistically significant(P<0.05).The expression of ETAR was less than ETBR in the control group,with the aggravation of increased expression of ETAR in renal tissue of diabetic nephropathy patients,the differences between groups were statistically significant(P<0.05).The expression of ETBR in D2 group was the highest,and no statistically significant differences between D1 group and D3 group (P>0.05).ETAR/ETBR in each group had statistically significant difference(P<0.05).ETAR was expressed in proximal and distal renal proximal tubule.ETBR was mainly expressed in proximal tubule.The expression of ETAR in renal tissue of patients with diabetic nephropathy and duration,urinary protein level and ROG were positively correlated.The expression of ETBR was negatively related to the amount and duration.ETAR/ETBR ratio and the course of disease,urinary protein level,ROG were positively correlated,negatively correlated with Alb,GFR.ConclusionsThe expression of ETAR and ETBR in the renal tissues of the elderly patients with diabetes mellitus are significantly increased,and the inversion of ETAR/ETBR value is positively correlated with the development of the disease.
Keywords:diabetic nephropathy;renal tissue;endothelin receptor;elder
糖尿病腎病多發(fā)于老年人群,是導(dǎo)致糖尿病患者死亡的主要并發(fā)癥之一[1]。研究發(fā)現(xiàn),糖尿病腎病患者血漿和尿液中內(nèi)皮素1(endothelin-1,ET-1)含量增加并參與了病情的發(fā)展,ET-1主要通過內(nèi)皮素受體 A(endothelin type a receptor,ETAR)、內(nèi)皮素受體 B(endothelin type b receptor,ETBR)2種受體亞型發(fā)揮作用[2-3]。相關(guān)研究[4]顯示,正常人腎臟ETAR與ETBR的比值為1∶2,但糖尿病腎病患者腎組織ETAR和ETBR的變化仍處在動(dòng)物實(shí)驗(yàn)層面,且結(jié)論存在爭議[5-7]。本研究通過觀察不同糖尿病腎病時(shí)期老年患者腎組織中ETAR和ETBR表達(dá)變化,探討其與老年糖尿病發(fā)展的關(guān)系。
1.1 一般資料
選取2015年1月-2016年6月本院收治的糖尿病腎病老年患者34例。腎活檢前均告知患者必要性和危險(xiǎn)性,簽署知情同意書;排除合并繼發(fā)性腎臟病變、心力衰竭及嚴(yán)重感染的患者。其中,男性25例,女性 11例;年齡 60~79歲,平均(64.91±9.35)歲。根據(jù)美國腎病學(xué)會2010年頒布的糖尿病腎病病理分型標(biāo)準(zhǔn),分為糖尿病腎病Ⅰ級(D1組10例),Ⅱ級(D2組12例),Ⅲ級(D3組12例)。選取同期在本院行腎腫瘤切除術(shù)后距腎腫瘤>3 cm的組織,蘇木精 - 伊紅染色法(hematoxylin-eosin staining,HE)染色后顯微鏡下觀察為形態(tài)正常的腎組織為對照組(10例)。其中,男性7例,女性3例;年齡60~76歲,平均(66.08±11.70)歲。糖尿病腎病患者與對照組患者之間性別構(gòu)成、年齡比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。
1.2 方法
1.2.1 腎病理檢查 入選患者進(jìn)行腎組織光學(xué)顯微鏡、免疫熒光及電子顯微鏡檢查,其中,光學(xué)顯微鏡檢查包括:HE染色;PAS染色法(periodic acid-schiff stain,PAS);正六胺銀染色法 (aeriodic acid-silver methe-namine,PASM-Masson) 染色;Masson 三色染色。計(jì)算腎小球硬化率(Glomerular sclerosis rate,ROG)。經(jīng)本科與超聲科協(xié)作進(jìn)行腎活檢組織穿刺,將標(biāo)本初步處理后送上級醫(yī)院或廣州金域醫(yī)學(xué)檢驗(yàn)中心進(jìn)一步檢查及處理。
1.2.2 免疫組織化學(xué)檢測 取腎組織石蠟包塊,二甲苯脫蠟,酒精梯度脫水,3%過氧化氫37℃孵育15min,磷酸鹽緩沖液沖洗3次;修復(fù)抗原,羊血清工作液封閉20 min,分別滴加ETAR、ETBR多克隆抗體(上海研謹(jǐn)生物科技有限公司,1∶1 000稀釋),4℃冷藏過夜,磷酸鹽緩沖液沖洗3次,滴加生物素標(biāo)記的二抗,37℃孵育60 min,磷酸鹽緩沖液洗凈;滴加辣根過氧化物酶(horse radish peroxidase,HRP)標(biāo)記的鏈霉素卵白素工作液,37℃孵育60 min,二氨基聯(lián)苯胺(Diaminobenzidine,DAB)染色,蒸餾水洗凈后蘇木素復(fù)染,脫水、透明、干燥及封片,高倍光鏡視野下觀察胞質(zhì)為棕黃色的為陽性,進(jìn)行半定量分析;采用Image pro plus 6.0軟件測定ETAR、ETBR表達(dá)量。
1.2.3 免疫熒光雙染法 腎組織制作2 μm冷凍切片,采用直接免疫熒光法和間接免疫熒光法,共聚焦顯微鏡下觀察染色結(jié)果。近端和遠(yuǎn)端腎小管的蛋白標(biāo)志物分別為LTL、THP(上海玉博生物科技有限公司),集合管蛋白標(biāo)志物為DBA(上海浩然生物技術(shù)有限公司),按試劑盒說明書進(jìn)行操作。
1.3 統(tǒng)計(jì)學(xué)方法
數(shù)據(jù)分析采用SPSS 14.0統(tǒng)計(jì)軟件,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(±s)表示,用方差分析,兩兩比較用t檢驗(yàn);回歸分析用Pearson法,P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 生化檢測結(jié)果和腎小球硬化率
經(jīng)單因素方差分析,各組尿蛋白、腎小球?yàn)V過率(glomerular filtration rate,GFR)、白蛋白(Albumin,Alb)、腎小球硬化率(Glomerular sclerosis rate,ROG)間差異有統(tǒng)計(jì)學(xué)意義(P<0.05),各組尿蛋白水平差異有統(tǒng)計(jì)學(xué)意義;進(jìn)一步兩兩比較顯示,D3組尿蛋白高于D2和D1組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);D3組GFR、Alb均低于D2和D1組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);D3組和D2組ROG高于D1組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。糖尿病腎病各組間糖化血紅蛋白(HbA1C)、血清肌酐(serum creatinine,Scr)水平比較差異無統(tǒng)計(jì)學(xué)意義P>0.05)。見表1。
2.2 免疫組織化學(xué)檢測結(jié)果
經(jīng)單因素方差分析,各組ETAR、ETBR表達(dá)量比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05),各組ETAR、ETBR表達(dá)量有差異;進(jìn)一步兩兩比較顯示,糖尿病腎病各組ETAR、ETBR表達(dá)量高于對照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);對照組ETAR表達(dá)量少于ETBR,隨著病情加重糖尿病腎病患者腎組織中ETAR表達(dá)量逐漸增加,組間比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05);D2組ETBR表達(dá)量最高,與D1組和D3組比較差異無統(tǒng)計(jì)學(xué)意義(P>0.05);ETAR/ETBR在各組比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見表2和圖1。
表1 各組患者生化檢測結(jié)果和腎小球硬化率比較 (±s)
表1 各組患者生化檢測結(jié)果和腎小球硬化率比較 (±s)
注:1)與 D1 組比較,P<0.05;2)與 D2 組比較,P<0.05
組別ROG/%對照組 0.04±0.02 4.21±0.52 115.62±3.78 39.96±6.15 59.71±10.05 0.50±0.11 D1 組 1.57±0.35 7.52±1.67 104.05±7.43 39.02±5.36 86.63±4.93 2.11±1.95 D2 組 2.51±0.83 7.28±1.31 88.19±22.59 33.38±8.09 83.85±6.79 13.80±5.161)D3 組 5.17±2.661)2) 7.60±1.69 68.27±26.131)2) 31.89±9.771) 93.05±6.19 18.63± 15.541)F值 4.068 0.826 8.339 6.350 0.995 10.985P值 0.035 0.195 0.005 0.018 0.082 0.001尿蛋白/(g/d)HbA1C/%GFR Alb/(g/L)Scr/(μmol/L)
2.3 糖尿病腎病患者免疫熒光雙染色結(jié)果
以LTL為近端腎小管標(biāo)志物,4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)為藍(lán)色熒光染料,Merge為藍(lán)色與綠色染料混合染色結(jié)果(見圖2)。ETAR在腎小管近端和遠(yuǎn)端存在表達(dá),ETBR主要表達(dá)于腎小管近端。
2.4 相關(guān)性分析
單因素相關(guān)性分析顯示,糖尿病腎病患者腎組織中ETAR表達(dá)量與病程、尿蛋白水平及ROG呈正相關(guān);ETBR表達(dá)量與病程呈負(fù)相關(guān);ETAR/ETBR比值與病程和尿蛋白水平、ROG呈正相關(guān),與Alb、GFR呈負(fù)相關(guān)。見表3。
表2 各組腎小管ETAR和ETBR表達(dá)量 (±s)
表2 各組腎小管ETAR和ETBR表達(dá)量 (±s)
組別ETAR/ETBR對照組 1.24±0.09 2.89±0.10 0.36±0.15 D1 組 5.56±0.34 6.61±1.86 0.94±0.21 D2 組 8.63±1.39 7.05±1.91 1.37±0.11 D3 組 12.65±1.84 6.02±1.18 2.20±0.25F值 11.931 0.539 9.138P值 0.001 0.261 0.003 ETAR ETBR
圖1 各組腎小管ETAR和ETBR染色結(jié)果 (免疫組織化學(xué)×400)
圖2 糖尿病腎病患者腎小管ETAR和ETBR雙染色結(jié)果 (免疫熒光×400)
表3 ETAR、ETBR影響因素相關(guān)性分析
ET-1與ETAR結(jié)合后可誘導(dǎo)炎癥反應(yīng)、血管收縮及細(xì)胞增殖[8-10],與ETBR結(jié)合后可介導(dǎo)內(nèi)皮素濃度相關(guān)的NO釋放[11],促進(jìn)循環(huán)中ET-1清除和水鈉排泄,起到舒張血管、利尿降壓的作用[12-13]。本研究顯示,人正常腎組織中多表達(dá)于腎小管,腎小球中無表達(dá);糖尿病腎病腎組織中ETAR、ETBR在腎小管表達(dá)水平提升,腎小球中也有表達(dá)。免疫熒光雙染法顯示,腎組織中皮質(zhì)內(nèi)部集合管、近曲小管和遠(yuǎn)端小管中多分布ETAR;近端小管中多分布ETBR。本研究中收集人腎組織時(shí)多為腎皮質(zhì)部分,只有少量髓質(zhì),因此無法得到人腎臟髓質(zhì)和集合系統(tǒng)的分布情況。
本研究顯示,人正常腎組織中ETAR表達(dá)量低于ETBR,糖尿病腎病早期腎組織中ETAR和ETBR表達(dá)水平均提升,隨著病情進(jìn)展ETAR表達(dá)水平高于ETBR,在糖尿病腎?、蠹墪r(shí)ETAR與ETBR的比值達(dá)到2∶1。說明ET系統(tǒng)在糖尿病腎病發(fā)展中發(fā)揮作用,其機(jī)制可能與糖尿病腎病患者血管內(nèi)皮功能受損有關(guān)。當(dāng)腎臟局部ET-1分泌量增加時(shí),其相應(yīng)的2個(gè)受體含量也存在改變,在病情早期ETBR可競爭性抑制ETAR與ET-1結(jié)合,擴(kuò)張血管,保護(hù)腎組織;隨病情發(fā)展ETBR分泌不足,腎組織損傷加重。相關(guān)性研究顯示,ETAR表達(dá)水平與病程、尿蛋白量、ROG呈正相關(guān),ETBR與病程呈負(fù)相關(guān),與其他兩項(xiàng)無相關(guān)性,提示ETAR在糖尿病腎病進(jìn)展中發(fā)揮重要作用;ETAR與ETBR的比值異常與病程、ROG、尿蛋白量、GFR有相關(guān)性,說明在糖尿病腎病發(fā)展中的作用不可忽視。
綜上所述,糖尿病可激活內(nèi)皮素系統(tǒng),腎臟組織中持續(xù)高表達(dá)ETAR及ETAR/ETBR比值異常參與糖尿病腎病的發(fā)生、發(fā)展。本研究可為干預(yù)內(nèi)皮素途徑治療糖尿病腎病提供新的理論基礎(chǔ)。
[1]ZANATTA C M,VERIONESE F V,DASILVALORETO M,et al.Endothelin-1 and endothelin a receptor immunoreactivity is increased in patients with diabetic nephropathy[J].Renal Failure,2012,34(3):308-315.
[2]WATSON A M,LI J,SCHUMACHER C,et al.The endothelin receptor antagonist avosentan ameliorates nephropathy and atherosclerosis in diabetic apolipoprotein E knockout mice[J].Diabetologia,2010,53(1):192-203.
[3]ANDRESS D L,COLL B,PRITCHETT Y,et al.Clinical efficacy of the selective endothelin A receptor antagonist,atrasentan,in patients with diabetes and chronic kidney disease (CKD)[J].Life Sciences,2012,91(13/14):739-742.
[4]YUAN W M,LI Y,WANG J,et al.Endothelin-receptor antagonists for diabetic nephropathy:a meta-analysis[J].Nephrology,2015,20(7):459-466.
[5]SALEH M A,POLLOCK J S,POLLOCK D M,et al.Distinct actions of endothelin A selective versus combined endothelin A/B receptor antagonists in early diabetic kidney disease[J].Journal of Pharmacology&Experimental Therapeutics,2011,338(1):263-270.
[6]RITZ E,WENZEL R R.Endothelin antagonist as add-on treatment for proteinuria in diabetic nephropathy:is there light at the end ofthe tunnel[J].Journalofthe American Society of Nephrology Jasn,2011,22(4):593-595.
[7]KOHAN D E,BARTON M.Endothelin and endothelin antagonists in chronic kidney disease[J].Kidney International,2014,86(5):896-904.
[8]HU C,CONG X D,DAI D Z,et al.Argirein alleviates diabetic nephropathy through attenuating NADPH oxidase,Cx43,and PERK in renaltissue[J].Naunyn-Schmiedeberg'sArchivesof Pharmacology,2011,383(3):309-319.
[9]GRAY S P,JANDELEIT-DAHM K.The pathobiology of diabetic vascular complications-cardiovascular and kidney disease[J].Journal of Molecular Medicine,2014,92(5):441-452.
[10]ABDEL-RAHMAN E M,SAADULLA L,REEVES W B,et al.Therapeutic modalities in diabetic nephropathy:standard and emerging approaches[J].Journal of General Internal Medicine,2012,27(4):458-468.
[11]MANN J F,GREEN D,JAMERSON K,et al.Avosentan for overt diabetic nephropathy[J].Journal of the American Society of Nephrology Jasn,2010,21(3):527-535.
[12]TURGUT F,BOLTON W K.Potential new therapeutic agents for diabetic kidney disease[J].American Journal of Kidney Diseases,2010,55(5):928-940.
[13]REICHETZEDER C,TSUPRYKOV O,HOCHER B,et al.Endothelin receptor antagonists in clinical research-Lessons learned from preclinical and clinical kidney studies[J].Life Sciences,2014,118(2):141-148.
ETAR and ETBR receptor subtypes in development of diabetic nephropathy in elderly
Hua-ni He
(Department nephrology,Qionghai People's Hospital,Qionghai,Hainan 571400,China)
R323.6
A
10.3969/j.issn.1005-8982.2017.15.009
1005-8982(2017)15-0042-05
2016-12-26