α-酮戊二酸盐受体1与肾小管功能相互关系的探讨
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摘要
目的和意义：α-酮戊二酸（盐）受体1（Oxoglutarate Receptor1, OXGR1）曾是多年来引人关注的孤儿GPCR（orphan G protein-coupled receptor, oGPCR）之一。2004年，α-酮戊二酸盐（Alpha-Ketoglutarate,AKG）被确认为OXGR1的内源性配基，而且该受体在肾小管中高表达。迄今为止，尚无有关OXGR1生理功能和病理意义的研究报道。
糖尿病（Diabetes Mellitus, DM）是影响人类健康的第三严重疾病；糖尿病肾病（Diabetic Nephropathy, DN）是糖尿病最严重的并发症之一，也是终末期肾病主要原因。传统观点认为，肾小球及血管的改变在DN的病理生理学中起着重要作用，而往往忽视高血糖也可对肾脏的肾小管系统造成原发性损伤。新的观点认为，肾小管在DN及糖尿病中的作用，并非是一种继发性表现，而是具有其特有的作用机制，并且在病变早期就起作用。在糖尿病发展过程中，肾小球与肾小管之间存在着相互作用：一方面肾小球滤过率对肾小管产生影响，该作用称为球管平衡；另一方面存在着肾小管-肾小球反馈系统，起着稳定和交互性调节的作用。因此，有关肾小管病变在DN发病中重要作用的研究备受关注。
基于肾小管在DN中的重要作用以及OXGR1在肾小管中高表达，而且AKG是糖代谢的重要中间产物，研究OXGR1在DN中作用，可为探索DN的损伤机制提供新的思路，对糖尿病及DN的防治具有重要的理论和现实意义，这正是本研究工作的新意所在。OXGR1在肾肿瘤的表达也值得探索。
全氟烷酸类化合物(perfluoroalkyl acids, PFAAs)是指所有碳原子上的氢原子均被氟原子取代的一类化合物，其典型代表为全氟辛酸（Perfluorooctanoic acid,PFOA），它也是其它多种全氟化合物的重要前体之一。PFAAs广泛存在于人类的生活环境中，具有生物蓄积性并对人体有害。新近的研究证实，血浆中的PFOA及PFOS水平与高尿酸血症、慢性肾病具有相关性，但其机制尚未明确。另外，该类化合物的代谢存在种属差异，有人推测这是与肾小管上皮细胞的膜蛋白表达差异有关。
材料和方法：1、体外原代培养大鼠肾小管上皮细胞的相关研究：（1）采用雄性Wistar大鼠20只，无菌取双肾，剪取薄层肾皮质，至1mm3大小。以2g/L的Ⅰ型胶原酶消化，经80目和170目细胞筛过滤，短时培养后更换培养瓶。使用添加胰岛素-转铁蛋白-硒（Insulin-Transferrin-Selenium, ITS）营养添加剂的DMEM/F12培养液进行原代大鼠肾小管上皮细胞培养。（2）在96孔板中接种原代大鼠肾小管上皮细胞，在HBSS分别添加10或40μMAKG，同时设溶剂对照组（0μMAKG），孵育20min后吸除HBSS，测量每孔的荧光强度，观察其对不同浓度(1、2或4μM)的荧光素（Fluorescein, FL）吸收能力的影响。（3）将原代大鼠肾小管上皮细胞接种于Transwell小室中，在小室下部分别加入含1μM FL的PBS或含1μM FL及50μM AKG的PBS，在小室上部加入PBS，孵育5min后测量小室上部PBS的荧光强度，观察不同浓度的AKG对Transwell小室下部1μM FL转运的影响。（4）将大鼠肾小管悬液分别加入含1μM FL的孵育液或含1μM FL及50μMAKG的孵育液，孵育饱和后清洗、涂片，观察录像，测量荧光强度并对比。2、体内糖尿病模型的有关研究：将大鼠按65mg/kg剂量一次性腹腔注射STZ毁损胰岛制成糖尿病模型。于16W及32W取大鼠肾脏，制成蜡块、切片、HE染色，显微镜下观察病理变化。免疫组化染色观察Vimentin在DM肾脏表达的变化。免疫荧光标记观察OXGR1及Vimentin在DM肾脏表达变化及两者之间的关系。3、体外培养人源肾小管细胞系HK-2的相关研究：在6孔板中接种HK-2细胞，分别加入含0、5、15或30mM葡萄糖的1640培养液，72h后提取总mRNA，反转录为cDNA，PCR扩增后进行凝胶电泳，比较相对表达量。4、AKG和全氟烷酸协同作用于HK-2细胞的研究：将HK-2细胞接种于六孔板，观察不同浓度PFOA对HK-2细胞OXGR1mRNA表达的影响。应用MTT法，检测300μM PFOA和不同浓度的AKG对HK-2细胞活性的影响。应用Annexin V-FITC细胞凋亡检测试剂盒，检测300μMPFOA和不同浓度的AKG对HK-2细胞凋亡的影响。5、免疫组化方法标记OXGR1在原发肾脏肿瘤组织芯片中的表达，观察OXGR1在不同类型肾脏肿瘤之间的表达差异。
实验结果：（1）优化的大鼠肾小管上皮细胞原代培养方法有效。添加ITS营养辅助剂可使原代细胞的传代7~9代，而缺乏ITS可导致原代细胞在传代后出现凋亡。（2）原代培养的肾小管上皮细胞具有吸收有机阴离子荧光素（FL）的能力。AKG（10或40μM）可明显促进细胞对FL（2或4μM）的吸收（P<0.05）；但当FL的浓度为1μM时，仅40μM AKG可明显促进FL的吸收（P<0.05）。（3）Transwell小室转运实验结果：与0μMAKG的对照组相比，50μMAKG对肾小管上皮细胞将FL转运至Transwell小室上部有更明显的促进作用（P<0.05）。（4）原代培养的肾小管细胞在FL饱和后可逐渐排出FL，与0μMAKG的对照组相比，50μMAKG组排出FL的速度明显加快，在8min及16min时有非常明显的差异（P<0.01），在24min时有明显差异（P<0.05）。（5）STZ注射大鼠成功诱发糖尿病，出现糖尿病肾病后，病理切片观察证实出现相应糖尿病肾病的病理表现，与新的国际糖尿病肾病病理分型标准相印证。在糖尿病肾脏改变中，炎症反应、间质纤维化以及肾小管萎缩等病变都以肾小管周围区域为著，而非以肾小球为中心，证实DM中肾小管病变具有独立性。免疫荧光标记证实OXGR1阳性区域主要位于肾小管的外缘区域，并在肾小管细胞之间也可见到阳性区域，这说明OXGR1主要位于肾小管细胞的底侧面表达。双重免疫荧光标记显示随着DM病程的加重，OXGR1阳性区域逐渐增强，同时Vimentin阳性区域逐渐增多，且OXGR1与Vimentin重叠的区域增多。肾小管上皮细胞Vimentin增多，提示肾小管出现了上皮-间充质转分化。肾小管上皮细胞的OXGR1表达增多，可能会导致肾小管的重吸收、转运功能出现障碍。（6）对照组培养液中不含葡萄糖，而含15mM或30mM葡萄糖培养液可明显增加HK-2细胞的OXGR1mRNA的表达（P<0.01），而含5mM葡萄糖培养液则与对照组无显著性差异（P>0.05）。（7）PFOA可上调HK-2细胞中OXGR1的mRNA表达。同时，AKG与PFOA协同作用可诱导HK-2细胞出现凋亡以及抑制其增殖活性（P<0.05）。（8）免疫组化标记证实， OXGR1蛋白表达在不同类型的肾脏肿瘤之间具有差异，肾母细胞瘤最弱，透明细胞性肾细胞癌和乳头状肾细胞癌较强，集合管癌最强；差异具有统计学意义（p<0.01）；但是透明细胞性肾细胞癌与乳头状肾细胞癌两组的OXGR1表达差异则没有统计学意义（p>0.05）。
结论：本研究应用多种可行的体内、外模型，初步探索了OXGR1在肾小管上皮细胞中的生理功能及病理意义。（1）建立并优化了大鼠原代肾小管上皮细胞培养策略，为更加准确地模拟研究肾小管吸收、转运功能奠定了基础。（2）首次证实，生理浓度相当的α-酮戊二酸盐可影响原代大鼠肾小管上皮细胞有机阴离子的转运功能，主要表现为对离子转运速度的调节，即可明显提高有机阴离子的转运速度；α-酮戊二酸盐的这种功能可能通过其受体OXGR1起作用。（3）本研究建立的大鼠DN模型，与新的国际糖尿病肾病病理分型相印证。（4）首次通过体内、外模型证实，高糖可促进肾小管上皮细胞OXGR1的表达；随着DM病程的进展，肾小管上皮细胞EMT加重，且OXGR1高表达与EMT存在着一定的关系；OXGR1在DM发生发展中的作用可能是与其调节肾小管功能有关，通过影响有机阴离子转运来实现。（5）首次经细胞实验证实，PFOA可上调HK-2细胞中OXGR1的mRNA表达；而AKG与PFOA共同作用可抑制HK-2细胞的增殖活性及诱导细胞凋亡；这种作用可能通过OXGR1起作用。（6）OXGR1在不同类型的肾脏肿瘤中表达不同，可作为集合管癌的标志物，用于原发肾脏肿瘤的鉴别诊断。本研究有关OXGR1在肾小管功能方面的原创性探索，为进一步研究OXGR1与相关致病因子的关系，深化对肾脏疾病的理解，开发新的治疗手段，提供了基础和可能方向。
Objective：Oxoglutarate Receptor1(OXGR1) was one of interesting orphanGPCRs for years. A2004study found that the endogenous ligand of OXGR1isα-ketoglutarate (AKG), and the receptors were highly expressed in the renal tubules.However, since the ligand of OXGR1was clarified, few literature has stated thephysiological functions and pathological effects of the receptor, so this field is stillblank.
Diabetes is the third most serious illness; diabetic nephropathy (DN) is the mostserious complications of diabetes, and the main cause of the end-stage kidney disease.In the traditional view, the glomerular and vascular changes play an important role inthe pathophysiology of diabetic nephropathy, but the fact that the high blood sugarcan also cause primary damage to the kidney tubular system is often ignored. The roleof the renal tubules in diabetic nephropathy and diabetes is not secondary, but it hasits unique mechanism and works early in the diseases. In the development of diabetes,there is glomerular and tubular interaction: on the one hand, the glomerular filtrationrate have an impact on renal tubular system, which is called glomerulotubular balance;on the other hand, there is also a tubular-glomerular feedback system, stabilizing andinteractively modulating function. Therefore, the researches of effect of renal tubuleson DN are widely concerned.
Based on the important role of renal tubules in the DN, the high expression ofOXGR1in the renal tubules, and the fact that AKG is an important intermediateproduct of glucose metabolism, the research of the role of OXGR1in DN mayprovide new views to explore the mechanisms of injury in the DN and diabetes. Itmay has important theoretical and practical significance in DN prevention andtreatment. Expression of OXGR1in renal tumors is also very interesting.
Perfluoroalkyl acids(PFAAs) refer to a class of compounds, whose all carbonatoms on the hydrogen atoms are substituted by fluorine atoms. PerfluorooctaneSulfonate (PFOS) and perfluorooctanoic acid (PFOA) are typical PFAAs, and they arealso important precursors of a variety of other perfluorinated compounds. PFAAswidely present in the human living environment, accumulate in bodies and areharmful to humans. Recent studies have confirmed that PFOS and PFOA levels inplasma is relevant to hyperuricemia and chronic kidney disease, but its mechanism is not clear. In addition, the metabolism of these compounds is different among species,it may be due to expression differences of membrane receptors on the tubularepithelium.
Materials and methods:1Primary culture of renal tubular epithelial cells:(1)Kidneys of20male Wistar rats were taken sterilely, and renal cortices were cut intosmall pieces of of1mm3. The cortex pieces were digested in2g/L type I collagenasesolution and sieved through an80mesh and a170mesh. The renal tubule suspensionwere moved into new flasks after short-term culture. DMEM/F12medium containingITS was added for rat renal proximal tubular epithelial cell primary culture.(2)Primary rat renal tubular epithelial cells were cultivated in96-well plates.10or40μMAKG was added into HBSS. After incubation for20min, HBSS was suctioned, thefluorescence of each well was measured to observe the absorptive capacity of renaltubular cells in the different concentrations (1,2or4μM) of fluorescein.(3) After theprimary rat renal tubular epithelial cells were cultivated in the Transwell inserts, PBScontaining1μM FL or PBS containing1μM FL and50μM AKG were added into thelower part of the inserts and PBS was added in the upper part of the inserts. Afterincubation for5min, the fluorescence of PBS in the inserts were measured to observethe effect of different concentrations of AKG on transportation of1μM FL.(4) IMwith1μM FL, or IM with1μM FL and50μM AKG was added into the rat renaltubule suspension, washing after incubation saturation, smearing, observing recording,and the fluorescence was measured.2. The in vivo model of diabetic kidneys: the celldamage of pancreatic islets of rats was induced by a single intraperitoneal injection of65mg/kg STZ to make diabetic model. With the extension of the course, the ratsdeveloped diabetic nephropathy.16W and32W later, rat kidneys were collected tomake paraffin blocks and sections. Pathological changes were observed under themicroscope. Vimentin expression in DN kidneys were examined byimmunohistochemical staining. The relationship between changes of expression ofOXGR1and Vimentin in DN kidneys were observed by immunofluorescence.3. Invitro studies of cultured human kidney cell line HK-2: HK-2cells were cultivated in6-well plates, and1640medium containing0mM,15mM or30mM glucose wereadded.72hours later, total mRNA was obtained; reverse transcription PCR and gelelectrophoresis were performed. The gray value were measured by ImageJ to comparethe relative expression levels.4. HK-2cells were seeded in six-well plates and treatedwith different concentrations of PFOA to observe OXGR1mRNA expression of HK-2cells. MTT assay was used to detect the effect of300μM PFOA and differentconcentrations of AKG on HK-2cells viability. Using Annexin V-FITC apoptosisdetection kit, the effects of300μM PFOA and different concentrations of AKG onapoptosis of HK-2cells were detected.5. The expression of OXGR1on primary kidney tumor tissue microarray was detected by immunohistochemistry, to observeOXGR1expression differences among the different types of kidney tumors.
Results:(1) Primary culture procedure of rat renal proximal tubule epithelialcells was optimized. ITS could help primary cells passage for7-9generations, and thelack of ITS could lead to apoptosis of primary cells.(2)The primary cultured renaltubular cells had the ability to absorb the organic anion, fluorescein. AKGsignificantly promoted the FL absorption (p<0.05).(3) Transwell insert transportationexperiment: Compared with the control group of0μM AKG,50μM AKG promotedthe transportation of renal proximal tubule cells of FL to the upper Transwell chambersignificantly (P <0.05).(4)Primary cultured renal proximal tubule cells could excreteFL gradually. Compared with the control group of0μM AKG,50μM AKG groupexcreted FL very significantly faster in the8min and16min (P <0.01), significantlyfaster in24min (P <0.05).(5) Injection of STZ into rats induced diabetes successfully.Diabetic nephropathy developed and the typical pathological features of diabeticnephropathy were confirmed by histological observations, according to the newinternational diabetic nephropathy pathological standard. Inflammation, interstitialfibrosis and tubular atrophy lesions in diabetic nephropathy, were usually peritubular,rather than glomerular centered, suggesting that the DN tubular lesions were primary.Immunofluorescence labeling confirmed that OXGR1positive areas were mainlylocated in the outer edge the renal tubules and there were also positive region betweenthe renal tubular cells, which meaned the expression OXGR1mainly located in thebasolateral side of renal tubular cells. With double immunofluorescence labeling,OXGR1positive areas gradually increased in the process of DN, while Vimentinpositive areas gradually increased too, and OXGR1and Vimentin overlapping regionincreased. The Vimentin positive areas in the renal tubular epithelial cells promptedrenal epithelial-mesenchymal transdifferentiation. The increased OXGR1expressionin renal tubular epithelial cells, may lead to dysfunction renal tubular reabsorption.(6)Compared with the control group with non-glucose medium,15mM or30mM glucosemedium significantly increased the OXGR1mRNA expression of HK-2cells (P<0.01),5mM glucose medium did not promote the expression compared with thecontrol group (P>0.05).(7) mRNA expression of OXGR1in HK-2cells wasupregulated by PFOA. The combination of AKG and PFOA could induce apoptosisand necrosis of HK-2cells (P <0.05).(8) OXGR1expression in Wilms' tumors wasthe weakest, expression in collecting duct carcinomas was the strongest, andexpression in clear cell renal cell carcinomas and papillary renal cell carcinomas wasstrong; OXGR1expressions in the different tumors were significantly different (p<0.01). However, OXGR1expression were not significantly different betweenCCRCC and PRCC(p>0.05).
Conclusions: Many practical models in vivo and in vitro were applied toinvestigate the physiological and pathological functions of OXGR1on the renaltubular epithelial cells.(1) The procedure of primary culture of rat tubular epithelialcells was established and optimized, in order to simulate the tubular absorption ofkidney more accurately.(2) The fact that α-ketoglutarate in physiologicalconcentration could affect organic anion transport function of primary rat kidneyproximal tubule epithelial cells is demonstrated for the first time, mainly regulatingtransporting speed; this function may be OXGR1dependant.(3) DN model in ratswas established, corroborating with the new international diabetic nephropathypathological standard.(4) It was confirmed, by in vivo and in vitro model for the firsttime, that high glucose can promote the OXGR1expression of renal tubular epithelialcells; EMT of renal tubular epithelial cells aggravated with the progression of DN,and there was a certain relationship between OXGR1high expression and EMT; therole of OXGR1in the development of DN may stated in the modulation of renaltubular function relating to its influence of organic anion transporter.(5) It wasdemonstrated by cell experiment for the first time that PFOA can upregulate themRNA expression of HK-2cells OXGR1; the combination of AKG and PFOA couldinhibit the proliferation and induce apoptosis of HK-2cells.(6) OXGR1expression isdifferent among different types of kidney tumors, so it may be used as a marker ofcollecting duct carcinoma, especially for the differential diagnosis of primary renaltumors. The original findings on the effect of OXGR1on the functions of renaltubular cells in our study provide basis and possible directions for the further researchof relationship between OXGR1and related pathogens, deep understanding of renaldiseases, and the development of new treatments.
糖尿病（Diabetes Mellitus, DM）是影响人类健康的第三严重疾病；糖尿病肾病（Diabetic Nephropathy, DN）是糖尿病最严重的并发症之一，也是终末期肾病主要原因。传统观点认为，肾小球及血管的改变在DN的病理生理学中起着重要作用，而往往忽视高血糖也可对肾脏的肾小管系统造成原发性损伤。新的观点认为，肾小管在DN及糖尿病中的作用，并非是一种继发性表现，而是具有其特有的作用机制，并且在病变早期就起作用。在糖尿病发展过程中，肾小球与肾小管之间存在着相互作用：一方面肾小球滤过率对肾小管产生影响，该作用称为球管平衡；另一方面存在着肾小管-肾小球反馈系统，起着稳定和交互性调节的作用。因此，有关肾小管病变在DN发病中重要作用的研究备受关注。
基于肾小管在DN中的重要作用以及OXGR1在肾小管中高表达，而且AKG是糖代谢的重要中间产物，研究OXGR1在DN中作用，可为探索DN的损伤机制提供新的思路，对糖尿病及DN的防治具有重要的理论和现实意义，这正是本研究工作的新意所在。OXGR1在肾肿瘤的表达也值得探索。
全氟烷酸类化合物(perfluoroalkyl acids, PFAAs)是指所有碳原子上的氢原子均被氟原子取代的一类化合物，其典型代表为全氟辛酸（Perfluorooctanoic acid,PFOA），它也是其它多种全氟化合物的重要前体之一。PFAAs广泛存在于人类的生活环境中，具有生物蓄积性并对人体有害。新近的研究证实，血浆中的PFOA及PFOS水平与高尿酸血症、慢性肾病具有相关性，但其机制尚未明确。另外，该类化合物的代谢存在种属差异，有人推测这是与肾小管上皮细胞的膜蛋白表达差异有关。
材料和方法：1、体外原代培养大鼠肾小管上皮细胞的相关研究：（1）采用雄性Wistar大鼠20只，无菌取双肾，剪取薄层肾皮质，至1mm3大小。以2g/L的Ⅰ型胶原酶消化，经80目和170目细胞筛过滤，短时培养后更换培养瓶。使用添加胰岛素-转铁蛋白-硒（Insulin-Transferrin-Selenium, ITS）营养添加剂的DMEM/F12培养液进行原代大鼠肾小管上皮细胞培养。（2）在96孔板中接种原代大鼠肾小管上皮细胞，在HBSS分别添加10或40μMAKG，同时设溶剂对照组（0μMAKG），孵育20min后吸除HBSS，测量每孔的荧光强度，观察其对不同浓度(1、2或4μM)的荧光素（Fluorescein, FL）吸收能力的影响。（3）将原代大鼠肾小管上皮细胞接种于Transwell小室中，在小室下部分别加入含1μM FL的PBS或含1μM FL及50μM AKG的PBS，在小室上部加入PBS，孵育5min后测量小室上部PBS的荧光强度，观察不同浓度的AKG对Transwell小室下部1μM FL转运的影响。（4）将大鼠肾小管悬液分别加入含1μM FL的孵育液或含1μM FL及50μMAKG的孵育液，孵育饱和后清洗、涂片，观察录像，测量荧光强度并对比。2、体内糖尿病模型的有关研究：将大鼠按65mg/kg剂量一次性腹腔注射STZ毁损胰岛制成糖尿病模型。于16W及32W取大鼠肾脏，制成蜡块、切片、HE染色，显微镜下观察病理变化。免疫组化染色观察Vimentin在DM肾脏表达的变化。免疫荧光标记观察OXGR1及Vimentin在DM肾脏表达变化及两者之间的关系。3、体外培养人源肾小管细胞系HK-2的相关研究：在6孔板中接种HK-2细胞，分别加入含0、5、15或30mM葡萄糖的1640培养液，72h后提取总mRNA，反转录为cDNA，PCR扩增后进行凝胶电泳，比较相对表达量。4、AKG和全氟烷酸协同作用于HK-2细胞的研究：将HK-2细胞接种于六孔板，观察不同浓度PFOA对HK-2细胞OXGR1mRNA表达的影响。应用MTT法，检测300μM PFOA和不同浓度的AKG对HK-2细胞活性的影响。应用Annexin V-FITC细胞凋亡检测试剂盒，检测300μMPFOA和不同浓度的AKG对HK-2细胞凋亡的影响。5、免疫组化方法标记OXGR1在原发肾脏肿瘤组织芯片中的表达，观察OXGR1在不同类型肾脏肿瘤之间的表达差异。
实验结果：（1）优化的大鼠肾小管上皮细胞原代培养方法有效。添加ITS营养辅助剂可使原代细胞的传代7~9代，而缺乏ITS可导致原代细胞在传代后出现凋亡。（2）原代培养的肾小管上皮细胞具有吸收有机阴离子荧光素（FL）的能力。AKG（10或40μM）可明显促进细胞对FL（2或4μM）的吸收（P<0.05）；但当FL的浓度为1μM时，仅40μM AKG可明显促进FL的吸收（P<0.05）。（3）Transwell小室转运实验结果：与0μMAKG的对照组相比，50μMAKG对肾小管上皮细胞将FL转运至Transwell小室上部有更明显的促进作用（P<0.05）。（4）原代培养的肾小管细胞在FL饱和后可逐渐排出FL，与0μMAKG的对照组相比，50μMAKG组排出FL的速度明显加快，在8min及16min时有非常明显的差异（P<0.01），在24min时有明显差异（P<0.05）。（5）STZ注射大鼠成功诱发糖尿病，出现糖尿病肾病后，病理切片观察证实出现相应糖尿病肾病的病理表现，与新的国际糖尿病肾病病理分型标准相印证。在糖尿病肾脏改变中，炎症反应、间质纤维化以及肾小管萎缩等病变都以肾小管周围区域为著，而非以肾小球为中心，证实DM中肾小管病变具有独立性。免疫荧光标记证实OXGR1阳性区域主要位于肾小管的外缘区域，并在肾小管细胞之间也可见到阳性区域，这说明OXGR1主要位于肾小管细胞的底侧面表达。双重免疫荧光标记显示随着DM病程的加重，OXGR1阳性区域逐渐增强，同时Vimentin阳性区域逐渐增多，且OXGR1与Vimentin重叠的区域增多。肾小管上皮细胞Vimentin增多，提示肾小管出现了上皮-间充质转分化。肾小管上皮细胞的OXGR1表达增多，可能会导致肾小管的重吸收、转运功能出现障碍。（6）对照组培养液中不含葡萄糖，而含15mM或30mM葡萄糖培养液可明显增加HK-2细胞的OXGR1mRNA的表达（P<0.01），而含5mM葡萄糖培养液则与对照组无显著性差异（P>0.05）。（7）PFOA可上调HK-2细胞中OXGR1的mRNA表达。同时，AKG与PFOA协同作用可诱导HK-2细胞出现凋亡以及抑制其增殖活性（P<0.05）。（8）免疫组化标记证实， OXGR1蛋白表达在不同类型的肾脏肿瘤之间具有差异，肾母细胞瘤最弱，透明细胞性肾细胞癌和乳头状肾细胞癌较强，集合管癌最强；差异具有统计学意义（p<0.01）；但是透明细胞性肾细胞癌与乳头状肾细胞癌两组的OXGR1表达差异则没有统计学意义（p>0.05）。
结论：本研究应用多种可行的体内、外模型，初步探索了OXGR1在肾小管上皮细胞中的生理功能及病理意义。（1）建立并优化了大鼠原代肾小管上皮细胞培养策略，为更加准确地模拟研究肾小管吸收、转运功能奠定了基础。（2）首次证实，生理浓度相当的α-酮戊二酸盐可影响原代大鼠肾小管上皮细胞有机阴离子的转运功能，主要表现为对离子转运速度的调节，即可明显提高有机阴离子的转运速度；α-酮戊二酸盐的这种功能可能通过其受体OXGR1起作用。（3）本研究建立的大鼠DN模型，与新的国际糖尿病肾病病理分型相印证。（4）首次通过体内、外模型证实，高糖可促进肾小管上皮细胞OXGR1的表达；随着DM病程的进展，肾小管上皮细胞EMT加重，且OXGR1高表达与EMT存在着一定的关系；OXGR1在DM发生发展中的作用可能是与其调节肾小管功能有关，通过影响有机阴离子转运来实现。（5）首次经细胞实验证实，PFOA可上调HK-2细胞中OXGR1的mRNA表达；而AKG与PFOA共同作用可抑制HK-2细胞的增殖活性及诱导细胞凋亡；这种作用可能通过OXGR1起作用。（6）OXGR1在不同类型的肾脏肿瘤中表达不同，可作为集合管癌的标志物，用于原发肾脏肿瘤的鉴别诊断。本研究有关OXGR1在肾小管功能方面的原创性探索，为进一步研究OXGR1与相关致病因子的关系，深化对肾脏疾病的理解，开发新的治疗手段，提供了基础和可能方向。
Objective：Oxoglutarate Receptor1(OXGR1) was one of interesting orphanGPCRs for years. A2004study found that the endogenous ligand of OXGR1isα-ketoglutarate (AKG), and the receptors were highly expressed in the renal tubules.However, since the ligand of OXGR1was clarified, few literature has stated thephysiological functions and pathological effects of the receptor, so this field is stillblank.
Diabetes is the third most serious illness; diabetic nephropathy (DN) is the mostserious complications of diabetes, and the main cause of the end-stage kidney disease.In the traditional view, the glomerular and vascular changes play an important role inthe pathophysiology of diabetic nephropathy, but the fact that the high blood sugarcan also cause primary damage to the kidney tubular system is often ignored. The roleof the renal tubules in diabetic nephropathy and diabetes is not secondary, but it hasits unique mechanism and works early in the diseases. In the development of diabetes,there is glomerular and tubular interaction: on the one hand, the glomerular filtrationrate have an impact on renal tubular system, which is called glomerulotubular balance;on the other hand, there is also a tubular-glomerular feedback system, stabilizing andinteractively modulating function. Therefore, the researches of effect of renal tubuleson DN are widely concerned.
Based on the important role of renal tubules in the DN, the high expression ofOXGR1in the renal tubules, and the fact that AKG is an important intermediateproduct of glucose metabolism, the research of the role of OXGR1in DN mayprovide new views to explore the mechanisms of injury in the DN and diabetes. Itmay has important theoretical and practical significance in DN prevention andtreatment. Expression of OXGR1in renal tumors is also very interesting.
Perfluoroalkyl acids(PFAAs) refer to a class of compounds, whose all carbonatoms on the hydrogen atoms are substituted by fluorine atoms. PerfluorooctaneSulfonate (PFOS) and perfluorooctanoic acid (PFOA) are typical PFAAs, and they arealso important precursors of a variety of other perfluorinated compounds. PFAAswidely present in the human living environment, accumulate in bodies and areharmful to humans. Recent studies have confirmed that PFOS and PFOA levels inplasma is relevant to hyperuricemia and chronic kidney disease, but its mechanism is not clear. In addition, the metabolism of these compounds is different among species,it may be due to expression differences of membrane receptors on the tubularepithelium.
Materials and methods:1Primary culture of renal tubular epithelial cells:(1)Kidneys of20male Wistar rats were taken sterilely, and renal cortices were cut intosmall pieces of of1mm3. The cortex pieces were digested in2g/L type I collagenasesolution and sieved through an80mesh and a170mesh. The renal tubule suspensionwere moved into new flasks after short-term culture. DMEM/F12medium containingITS was added for rat renal proximal tubular epithelial cell primary culture.(2)Primary rat renal tubular epithelial cells were cultivated in96-well plates.10or40μMAKG was added into HBSS. After incubation for20min, HBSS was suctioned, thefluorescence of each well was measured to observe the absorptive capacity of renaltubular cells in the different concentrations (1,2or4μM) of fluorescein.(3) After theprimary rat renal tubular epithelial cells were cultivated in the Transwell inserts, PBScontaining1μM FL or PBS containing1μM FL and50μM AKG were added into thelower part of the inserts and PBS was added in the upper part of the inserts. Afterincubation for5min, the fluorescence of PBS in the inserts were measured to observethe effect of different concentrations of AKG on transportation of1μM FL.(4) IMwith1μM FL, or IM with1μM FL and50μM AKG was added into the rat renaltubule suspension, washing after incubation saturation, smearing, observing recording,and the fluorescence was measured.2. The in vivo model of diabetic kidneys: the celldamage of pancreatic islets of rats was induced by a single intraperitoneal injection of65mg/kg STZ to make diabetic model. With the extension of the course, the ratsdeveloped diabetic nephropathy.16W and32W later, rat kidneys were collected tomake paraffin blocks and sections. Pathological changes were observed under themicroscope. Vimentin expression in DN kidneys were examined byimmunohistochemical staining. The relationship between changes of expression ofOXGR1and Vimentin in DN kidneys were observed by immunofluorescence.3. Invitro studies of cultured human kidney cell line HK-2: HK-2cells were cultivated in6-well plates, and1640medium containing0mM,15mM or30mM glucose wereadded.72hours later, total mRNA was obtained; reverse transcription PCR and gelelectrophoresis were performed. The gray value were measured by ImageJ to comparethe relative expression levels.4. HK-2cells were seeded in six-well plates and treatedwith different concentrations of PFOA to observe OXGR1mRNA expression of HK-2cells. MTT assay was used to detect the effect of300μM PFOA and differentconcentrations of AKG on HK-2cells viability. Using Annexin V-FITC apoptosisdetection kit, the effects of300μM PFOA and different concentrations of AKG onapoptosis of HK-2cells were detected.5. The expression of OXGR1on primary kidney tumor tissue microarray was detected by immunohistochemistry, to observeOXGR1expression differences among the different types of kidney tumors.
Results:(1) Primary culture procedure of rat renal proximal tubule epithelialcells was optimized. ITS could help primary cells passage for7-9generations, and thelack of ITS could lead to apoptosis of primary cells.(2)The primary cultured renaltubular cells had the ability to absorb the organic anion, fluorescein. AKGsignificantly promoted the FL absorption (p<0.05).(3) Transwell insert transportationexperiment: Compared with the control group of0μM AKG,50μM AKG promotedthe transportation of renal proximal tubule cells of FL to the upper Transwell chambersignificantly (P <0.05).(4)Primary cultured renal proximal tubule cells could excreteFL gradually. Compared with the control group of0μM AKG,50μM AKG groupexcreted FL very significantly faster in the8min and16min (P <0.01), significantlyfaster in24min (P <0.05).(5) Injection of STZ into rats induced diabetes successfully.Diabetic nephropathy developed and the typical pathological features of diabeticnephropathy were confirmed by histological observations, according to the newinternational diabetic nephropathy pathological standard. Inflammation, interstitialfibrosis and tubular atrophy lesions in diabetic nephropathy, were usually peritubular,rather than glomerular centered, suggesting that the DN tubular lesions were primary.Immunofluorescence labeling confirmed that OXGR1positive areas were mainlylocated in the outer edge the renal tubules and there were also positive region betweenthe renal tubular cells, which meaned the expression OXGR1mainly located in thebasolateral side of renal tubular cells. With double immunofluorescence labeling,OXGR1positive areas gradually increased in the process of DN, while Vimentinpositive areas gradually increased too, and OXGR1and Vimentin overlapping regionincreased. The Vimentin positive areas in the renal tubular epithelial cells promptedrenal epithelial-mesenchymal transdifferentiation. The increased OXGR1expressionin renal tubular epithelial cells, may lead to dysfunction renal tubular reabsorption.(6)Compared with the control group with non-glucose medium,15mM or30mM glucosemedium significantly increased the OXGR1mRNA expression of HK-2cells (P<0.01),5mM glucose medium did not promote the expression compared with thecontrol group (P>0.05).(7) mRNA expression of OXGR1in HK-2cells wasupregulated by PFOA. The combination of AKG and PFOA could induce apoptosisand necrosis of HK-2cells (P <0.05).(8) OXGR1expression in Wilms' tumors wasthe weakest, expression in collecting duct carcinomas was the strongest, andexpression in clear cell renal cell carcinomas and papillary renal cell carcinomas wasstrong; OXGR1expressions in the different tumors were significantly different (p<0.01). However, OXGR1expression were not significantly different betweenCCRCC and PRCC(p>0.05).
Conclusions: Many practical models in vivo and in vitro were applied toinvestigate the physiological and pathological functions of OXGR1on the renaltubular epithelial cells.(1) The procedure of primary culture of rat tubular epithelialcells was established and optimized, in order to simulate the tubular absorption ofkidney more accurately.(2) The fact that α-ketoglutarate in physiologicalconcentration could affect organic anion transport function of primary rat kidneyproximal tubule epithelial cells is demonstrated for the first time, mainly regulatingtransporting speed; this function may be OXGR1dependant.(3) DN model in ratswas established, corroborating with the new international diabetic nephropathypathological standard.(4) It was confirmed, by in vivo and in vitro model for the firsttime, that high glucose can promote the OXGR1expression of renal tubular epithelialcells; EMT of renal tubular epithelial cells aggravated with the progression of DN,and there was a certain relationship between OXGR1high expression and EMT; therole of OXGR1in the development of DN may stated in the modulation of renaltubular function relating to its influence of organic anion transporter.(5) It wasdemonstrated by cell experiment for the first time that PFOA can upregulate themRNA expression of HK-2cells OXGR1; the combination of AKG and PFOA couldinhibit the proliferation and induce apoptosis of HK-2cells.(6) OXGR1expression isdifferent among different types of kidney tumors, so it may be used as a marker ofcollecting duct carcinoma, especially for the differential diagnosis of primary renaltumors. The original findings on the effect of OXGR1on the functions of renaltubular cells in our study provide basis and possible directions for the further researchof relationship between OXGR1and related pathogens, deep understanding of renaldiseases, and the development of new treatments.
引文
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