非经典型蛋白激酶C在尿蛋白诱导肾小管上皮细胞转分化中的作用  

The Role of Atypical PKC Isoenzymes on the Transdifferentiation of Renal Tubular Epithelial Cells Induced by Urinary Proteins

作　　者：安宁 罗玉婷 李亚宁 吴洪銮 杨陈 潘庆军 刘华锋 

机构地区：[1]广东医科大学附属医院肾脏疾病研究所,湛江524001

出　　处：《中国中西医结合肾病杂志》2017年第9期771-775,I0002-I0004,共8页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：国家自然科学基金资助项目(No.30971370);广东省自然科学基金资助项目(No.8152402301000014);湛江市科技计划项目(No.2015A06009)

摘　　要：目的:探讨非经典型PKC同工酶(PKC-ζ和PKC-ι)在尿蛋白诱导的肾小管上皮细胞(RTECs)转分化中的作用。方法:首先免疫组化法检测PKC-ζ和PKC-ι在人正常及不同类型肾小球疾病患者肾组织的表达并分析其表达量的变化。接着应用不同浓度的尿蛋白(0 mg/ml、0.5 mg/ml、1.0 mg/ml、2.0 mg/ml、4.0 mg/ml及8.0 mg/ml)处理体外培养的HK-2细胞,应用免疫细胞化学法检测HK-2细胞E-cadherin和α-SMA的表达以观察HK-2细胞转分化情况,选择诱导HK-2细胞转分化适宜的尿蛋白浓度进行后续试验。将HK-2细胞分对照组和尿蛋白组,分别给予无血清培养基和尿蛋白(4.0 mg/ml)刺激,分别用免疫荧光及Western Blot法检测各组细胞内PKC-ζ和PKC-ι在细胞膜及细胞浆的活化易位情况。结果:(1)两种非经典型PKC同工酶PKC-ζ和PKC-ι在正常和肾小球疾病肾组织肾小球和RTECs均有表达,在肾间质均无表达,表达量不尽相同。PKC-ζ在肾小球疾病肾组织肾小球中的表达量均较正常肾组织显著减少(P<0.05),在MCD和FSGS的RTECs中的表达量较正常肾组织显著减少(P<0.05)。PKC-ι在FSGS的肾小球和RTECs中的表达量均显著减少(P<0.05),在Ig AN肾组织RTECs中表达显著增高(P<0.05),在肾小球表达无明显差别。(2)不同浓度的尿蛋白刺激HK-2细胞48 h后,当尿蛋白的浓度逐渐升高时,HK-2细胞随之转变为类似成纤维细胞形态,细胞E-cadherin表达下调及α-SMA表达上调。尿蛋白浓度为4.0 mg/ml诱导HK-2细胞转分化最明显。(3)尿蛋白诱导HK-2细胞转分化时,PKC-ζ和PKC-ι无明显活化易位。结论:两种非经典型PKC同工酶均在人类肾组织表达,提示PKC-ζ和PKC-ι均参与肾脏的生理过程,但PKC-ζ和PKC-ι在尿蛋白诱导的肾小管上皮细胞转分化中并未发挥相关作用。Objective： To investigate the role of atypical PKC isoenzymes involved in the transdifferentiation of renal tubular epithelial cells（ RTECs） induced by urinary proteins. Methods： 50 cases of patients with biopsy-proven glomerular disease were collected in the Affiliated Hospital of Guangdong Medical University,including 10 patients with minimal change disease,10 patients with membranous nephropathy,10 patients with focal segmental glomerulosclerosis,10 patients with Ig A nephropathy,and 10 patients with lupus nephritis,respectively. 6 cases of normal kidney specimens,far away from tumor margin and proven to be normal renal tissue by pathology examination were obtained from patients with renal cell carcinoma after unilateral nephrectomy. The expression of PKC-ζ and PKC-ι were detected in those tissues by immunohistochemistry. In order to determine the optimal concentration of urine protein to induce epithelial – mesenchymal transdifferentiation,HK-2 cells were stimulated with different concentrations（ 0,0. 5,1. 0,2. 0,4. 0,and 8. 0 mg/ml） of urinary proteins. The expression level of E-cadherin and α-SMA in cultured HK-2 cells were assessed by immunocytochemistry. The localization of two a PKCs in membrane/cytoplasm was detected by laser confocal microscopy and Western Blot in HK-2 cells with or without 4 mg/ml urinary protein stimulation. Results： PKC-ζ and PKC-ι were expressed in glomeruli and RTECs in normal human renal tissues as well as in renal tissues from pateints with glomerular diseases. None of them were detected in renal interstitium. The distribution of atypical PKCs in glomerular disease renal tissues was similar to that in normal tissues,but the levers of expression of atypical PKCs were different. The PKC-ζ staining was lower both in glomeruli and RTECs of renal tissues from patients with FSGS and LN than those in normal renal tissues（ P〈 0. 05）. The expression of PKC-ι was higher in RTECs of renal tissues originated from patients with Ig AN than that in normal renal tis

关 键 词：PKC-ζ PKC-ι 尿蛋白 肾小管上皮细胞 转分化 

分 类 号：R692[医药卫生—泌尿科学]