姜黄素缓解草酸钙结晶肾损伤的机制研究

目的:观察姜黄素在草酸钙结晶性肾损伤过程中的作用,并初步探讨其可能的机制。方法:在乙醛酸盐腹腔注射诱导C57BL/6J小鼠和一水草酸钙(COM)刺激人肾小管上皮细胞(HK-2)的体内外草酸钙结晶肾损伤模型上,分别加用姜黄素进行干预,应用冯库萨染色观察肾组织草酸钙结晶沉积情况,应用qRT-PCR及Western-blot、免疫组化检测上皮细胞间充质转分化(epithelial-mesenchymal transition,EMT)标志物E-cadherin、N-cadherin和Vimentin在mRNA和蛋白质水平的表达情况。结果:(1)冯库萨染色显示姜黄素显著减少小鼠模型肾草酸钙结晶的沉积。(2)肾组织免疫组化和HK-2细胞qRT-PCR和Western-blot结果显示草酸钙结晶显著降低肾小管上皮细胞的上皮标志物E-cadherin表达和上调间质标志物N-cadherin及Vimentin表达,应用姜黄素干预后明显抑制以上改变。结论:姜黄素可能通过抑制草酸钙结晶所致肾小管上皮细胞EMT改变,从而缓解草酸钙结晶所致肾损伤。     

组蛋白去乙酰化酶抑制剂减轻草酸钙结晶肾损伤的实验研究

目的：观察组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid,SAHA）对草酸钙结晶模型小鼠肾结晶沉积的作用,初步探讨 SAHA 对减轻草酸钙结晶肾损伤的机制。方法：24只 ICR 雄性小鼠随机分为4组,即空白对照组（A 组）和实验组（B、C、D 组）;B 组：50 mg/ kg 的生理盐水（NS）＋100 mg/ kg 乙醛酸盐,C 组：50 mg/ kg 的DMSO ＋100 mg/ kg 乙醛酸盐,D 组：50 mg/ kg 的 SAHA ＋100 mg/ kg 乙醛酸盐;实验组给予乙醛酸盐6 h 前分别予以 NS、DMSO、SAHA 50 mg/ kg 等量腹腔注射,7 d 后处死全部小鼠。检测各组小鼠肾组织钙含量水平,丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽还原酶（GSH）的含量或活力,并且在光镜下观察各组小鼠肾组织切片中草酸钙结晶的分布,免疫组化及其半定量分析比较骨桥蛋白（OPN）、CD44分子在肾脏中的表达情况。结果：与 B、C 组相比,D 组小鼠的肾组织钙含量和MDA 含量显著下降（P 均〈0．05）,肾组织切片冯库萨染色显示草酸钙结晶沉积明显减少（P 〈0．05）,OPN 与 CD44表达水平也明显下降（P 〈0．05）。而 B 组与 C 组肾结晶形成的差异无统计学意义。结论：研究结果首次证实 SAHA 对减少小鼠草酸钙肾结晶形成有预防作用,而且其机制可能与抑制氧化应激和降低 OPN、CD44表达水平有关。     

复方金钱草减轻小鼠草酸钙结晶肾损伤的实验研究

目的：探讨复方金钱草对小鼠草酸钙结晶肾损伤的作用及可能的作用机制.方法：24只C57 BL/6小鼠按随机数字表法随机分为对照组（A组）、单纯结晶组（B组）、复方金钱草干预组（C组）,每组8只.B组：给予100 mg/kg乙醛酸盐;C组：给予100 mg/kg乙醛酸盐＋20 ml/kg复方金钱草浸膏;A组：给予100 mg/kg的生理盐水,在连续给药7 d后,检测各组小鼠血生化指标,肾组织钙含量水平,光镜下观察各组小鼠肾组织切片中草酸钙结晶体分布及组织病理改变,免疫组化及免疫荧光染色对TRPV5、Calbindin-D28进行定位分析,Western Blot法检测各组肾组织中TRPV5、Calbindin-D28k的含量.结果：与单纯结晶组相比,复方金钱草干预组小鼠血清尿素氮（BUN）及肌酐（Scr）水平、肾组织钙含量显著下降（P〈0.05）,肾组织草酸钙结晶沉积及肾脏病理组织损伤均显著减轻（P〈0.05）,但肾脏TRPV5及Calbindin-D28k表达量均强于单纯结晶组（P〈0.05）.结论：复方金钱草可能通过上调肾脏TRPV5及Calbindin-D28k表达量从而减少了肾小管内钙盐结晶体的形成,起到了保护肾脏的作用.     

SAHA对草酸钙肾结晶小鼠肾功能的影响

目的:建立草酸钙肾结晶小鼠模型,探讨辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对模型小鼠肾功能的影响。方法:将24只C57BL/6雄性小鼠随机分为4组:空白对照组(无任何处理)、模型组(NS组)和干预组(DMSO、SAHA/DMSO组);NS组:50 mg/kg的生理盐水(NS)+100 mg/kg乙醛酸盐;DMSO组:50 mg/kg的DMSO+100 mg/kg乙醛酸盐;SAHA组:50 mg/kg的SAHA/DMSO+100 mg/kg乙醛酸盐;实验组分别预先予以NS、DMSO、SAHA 50 mg/kg等量腹腔注射,6 h后三组均给予100 mg/kg的乙醛酸盐腹腔注射,连续给药7 d后处死全部小鼠。在光镜下观察各组小鼠肾组织切片中草酸钙结晶的分布,比较钙盐沉积百分比;检测各组小鼠的血肌酐(血Scr)、血尿素氮(BUN),尿损伤因子1(尿KIM-1),尿钙/尿肌酐比值;检测肾组织的过氧化氢酶(CAT)、维生素E(VE)的含量或活力;并且对肾组织切片TUNEL染色及其半定量分析,比较肾小管凋亡细胞的表达情况。结果:(1)SAHA减少草酸钙肾结晶的形成;(2)SAHA减少模型小鼠的血肌酐、血尿素氮生成,降低尿KIM-1水平;(3)SAHA调节氧化应激水平;(4)SAHA组小鼠的肾小管细胞凋亡减少。结论:本项研究结果证实SAHA可改善草酸钙结晶小鼠的肾功能,减少肾小管细胞凋亡。     

糖肾方改善单侧输尿管梗阻小鼠肾间质纤维化的机制研究

目的:观察糖肾方对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的作用,并初步探讨其机制。方法:45只雄性C57BL/6小鼠随机分为假手术组(Sham)、UUO模型组(UUO)、糖肾方组(TSF);预先给药7 d,除Sham组外其余各组行UUO手术,术后7 d取肾组织进行HE染色及Masson染色观察肾组织病理改变,免疫组化染色检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(Collagen typeⅠ,ColⅠ)和Ⅲ型胶原(Collagen typeⅢ,ColⅢ)表达,Western Blot法检测转化生长因子-β_1(transforming growth factor-β_1,TGF-β_1)、E-钙连素(E-cadherin)和波形蛋白(vimentin)表达。结果:与Sham组比,UUO组小鼠肾小管间质损伤评分及纤维化面积百分比均显著升高;与UUO组比,TSF组肾小管间质损伤评分及纤维化面积百分比均显著降低。免疫组化与Western blot结果显示,与Sham组比,UUO组小鼠肾组织TGF-β_1及ColⅠ、ColⅢ表达水平显著升高;肾小管上皮细胞间质转分化(epithelial-mesenchymal transition,EMT)标志性分子α-SMA和vimentin蛋白表达水平明显升高,E-cadherin蛋白表达水平明显降低,TSF可显著逆转UUO小鼠肾组织上述蛋白表达。结论:糖肾方可改善UUO小鼠肾间质纤维化,其机制与其抑制TGF-β_1,减少EMT发生有关。     

草酸钙结晶-肾小管细胞损伤机制研究进展

草酸钙是肾结石中最常见的化学成分。肾钙盐结晶是草酸钙结石形成的关键步骤之一,而近年来研究发现肾钙盐结晶形成与肾小管上皮细胞损伤密切相关。本文就草酸钙结石形成和肾小管上皮细胞损伤相互作用机制方面进行综述。     

氯沙坦含药血清对肾小管上皮-间充质转分化的抑制作用

目的:肾小管上皮细胞-间充质转分化(epithelial-to-mesenchymal transition,EMT)是肾小管间质纤维化(tubulointerstitial fibrosis,TIF)的关键发病机制,而肾小管间质纤维化是慢性肾脏病进展为终末期肾病(end-stage renal dis-ease,ESRD)的重要共同通路。血管紧张素ⅡAT1受体阻断剂氯沙坦对于延缓慢性肾脏病进展有一定的作用,但其能否抑制肾小管上皮细胞-间充质转分化继而抑制肾间质纤维化尚不清楚。本实验通过体外TGF-β1诱导HK-2细胞向间充质细胞转分化,观察氯沙坦对肾小管上皮细胞-间充质转分化的抑制作用及其可能机制。方法:在体外使用TGF-β1诱导HK-2细胞表型改变并给予氯沙坦大鼠含药血清干预。氯沙坦大鼠含药血清按照既定的操作程序获取。HK-2细胞行E-cadher-in,Vimentin,β-catenin和ZEB1免疫荧光染色及Western blot分析。结果:TGF-β1诱导肾小管上皮细胞HK-2转化为间充质细胞,细胞形态由卵圆形变为长梭形,上皮标志物E-cadherin表达下调,间充质标志物Vimentin表达上调,上皮细胞-间充质转分化相关分子β-catenin在胞浆、胞核的积聚增多以及ZEB1表达增强;氯沙坦大鼠含药血清能够部分抑制TGF-β1诱导的HK-2转化为间充质表型,并维持HK-2细胞的上皮表型,抑制E-cadherin的表达下调和Vimentin的表达上调;同时抑制β-catenin在胞浆、胞核的积聚以及ZEB1的表达。结论:研究结果提示氯沙坦可抑制体外的肾小管上皮细胞-间充质转分化,其机制可能与氯沙坦抑制β-catenin/ZEB1通路有关。     

丹参多酚酸盐对实验性小鼠草酸钙结晶生成的影响

目的 观察中药丹参多酚酸盐对小鼠肾脏草酸钙结晶生成的干预效果,并探讨其可能的作用机制.方法 24只8周龄雄性C56BL/6小鼠根据丹参多酚酸盐干预与否以及干预浓度（80mg/kg、160 mg/kg）进行分组,每组8只.各组小鼠均给予腹腔注射乙醛酸盐（100 mg/kg）造模,丹参多酚酸盐干预组在乙醛酸盐诱导结晶肾损伤基础上加用不同浓度的丹参多酚酸盐注射液,均为1次/d,连续给药7d后,收集肾脏组织标本.光镜下观察各组小鼠肾组织中草酸钙结晶生成情况;检测肾组织中钙含量、脂质过氧化丙二醛（malondialdehyde,MDA）、谷胱甘肽（glutathione,GSH）、过氧化氢酶（catalase,CAT）及超氧化物歧化酶（superoxide dismutase,SOD）活性.结果 与模型组相比,给予丹参多酚酸盐注射液后的小鼠肾组织SOD、GSH和CAT显著升高,而MDA和肾组织钙含量显著下降（P＜0.05）,但丹参多酚酸盐两种浓度间未见明显的量效关系.结论 丹参多酚酸盐能有效的抑制由乙醛酸盐诱导的小鼠草酸钙结晶的生成.     

肾小管上皮细胞转分化与肾小管间质纤维化

肾小管上皮细胞转分化（EMT）是肾间质纤维化（R1F）的重要发病机制之一，TGF—β1等细胞因子可促进EMT从而促进RIF形成，而HGF、BMP-7可抑制EMT而减轻肾纤维化。本文重点介绍EMT在肾间质纤维化中的病理学意义、分子机制及治疗干预的研究。     

急性缺血再灌注小鼠肾损伤修复过程中Wnt-β-catenin信号的表达

目的 观察急性肾损伤（AKI）后恢复过程中肾组织内Wnt-β-catenin信号分子表达的分布及变化, 探讨Wnt-β-catenin信号通路在AKI修复过程中的作用。 方法 (1)应用BAT-gal转基因报告鼠建立急性肾缺血再灌注损伤（IRI）模型, 在IRI后1 d断尾取血,并于IRI后第2和7天处死报告鼠取血并收集肾脏标本。肾组织切片行PAS染色观察肾病理改变及X-gal-LTL(近曲小管标记物)、X-gal-NKCC2（髓袢标记物）、X-gal-DBA（集合管标记物）免疫荧光组织化学双重染色,观察Wnt-β-catenin信号的分布、表达与变化。(2)应用野生型小鼠建立IRI模型,于IRI后第2、5、7天处死小鼠获取肾脏标本用Western 印迹方法检测Wnt4及其共同受体低密度脂蛋白受体相关蛋白6（Lrp6）的表达水平。 结果 IRI小鼠肾损伤后第2天,PAS结果显示损伤肾脏的肾小管上皮细胞排列紊乱,可见明显的肾小管上皮细胞脱落及基底膜裸露,管腔内可见脱落的细胞和细胞碎片;7 d时小鼠肾小管上皮细胞损伤面积较2 d时明显缩小。X-gal染色显示第2天损伤的肾脏皮质和外髓质层中可见少数肾小管出现Wnt信号的表达,间质中Wnt信号的反应较高,第7天与第2天比较,Wnt信号表达明显增强。Western印迹结果显示,Wnt4于IRI后2 d增高,第5、7天亦较第2天明显增高;其共同受体磷酸化Lrp6代表激活的Wnt-β-catenin信号,在IRI 2 d后明显增高,而在无损伤成鼠肾组织中未检测到。免疫双染提示主要是近曲小管和髓袢的上皮细胞对Wnt信号通路应答。 结论 急性肾损伤时Wnt-β-catenin信号通路被激活,可能参与了肾小管上皮细胞的修复机制。     

Expression of inter-alpha inhibitor related proteins in kidneys and urine of hyperoxaluric rats

PURPOSE: To investigate the involvement of the inter-alpha inhibitor family of proteins in calcium oxalate stone formation we determined immunohistochemical distribution in the kidneys and excretion in the urine of these proteins in normal and hyperoxaluric rats. Various members of the family have been shown to inhibit the formation and retention of calcium oxalate crystals in the kidneys. MATERIALS AND METHODS: Hyperoxaluria was induced in male Sprague-Dawley rats by administering 0.75% ethylene glycol. The inter-alpha inhibitor family consists of inter-alpha inhibitor, pre-alpha inhibitor, the so-called heavy chains H1, H2 and H3, and the light chain bikunin. Antibodies against these molecules were used to localize various proteins in rat kidneys by immunohistochemical techniques. Urine was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis to determine the expression of various members of the inter-alpha inhibitor family. RESULTS: In normal kidneys staining for inter-alpha inhibitor and other members of the family was mostly limited to the proximal tubules and generally to their luminal contents. Eight weeks after the induction of hyperoxaluria various sections of renal tubules stained positive for inter-alpha inhibitor, bikunin and H3. Positive staining was observed in the tubular lumina as well as in the cytoplasm of epithelial cells. Crystal associated material was heavily stained. Western blot analysis recognized 7 protein bands in the urine. The urinary expression of H1, H3 and pre-alpha-inhibitor was significantly increased. CONCLUSIONS: Apparently hyperoxaluria and renal calcium oxalate crystal deposition result in the increased expression of crystallization inhibitors, such as inter-alpha-inhibitor related proteins, in the kidneys and urine. Results indicate that kidneys respond to nephrolithic challenges by producing proteins that inhibit crystal formation and retention.     

Effect of oxalate on the growth of renal tubular epithelial cells

PURPOSE: Until recently, oxalate was considered merely as a major component of calcium oxalate stones, forming crystals in the lumen of the renal tubules. However, new evidence suggests that oxalate is not only a major constituent of calcium oxalate stones but also has effect on renal tubular epithelial cells, affecting the pathogenesis of nephrolithiasis. We tried to elucidate the effect of oxalate on the growth of renal tubular epithelial cells of different species and locations and also to interpret the possible role of the oxalate in the pathogenesis of urolithiasis. MATERIALS AND METHODS: Porcine proximal renal tubular epithelial cells (LLC-PK1) and canine distal renal tubular epithelial cells (MDCK) were incubated with different concentrations of oxalate, and the effect of oxalate on the growth of the cells was assessed by methylthiazoletetrazolium assay. RESULTS: Growth of the renal tubular epithelial cells was inhibited with increasing concentrations of oxalate in both proximal and distal lines. CONCLUSION: Oxalate may cause stone formation by affecting the growth of renal tubular epithelial cells as well as by providing a constituent of calcium oxalate stones.     

Glyoxylate induces renal tubular cell injury and microstructural changes in experimental mouse

Crystal formation in mice could not be induced either by the administration of ethylene glycol or by glycolate. To clarify the reasons for the difference among these oxalate precursors in mice, we studied renal tubular epithelial injury by immunohistochemical staining of oxidative stress and observing microstructures. Daily intra-abdominal injection of saline solution [10 ml/(kg day)], ethylene glycol[(48.3 mmol/(kg day)], glycolate [1.31 mmol/(kg day)], and glyoxylate [1.35 mmol/(kg day)] into C57BL/6 male mice (8 weeks) was performed for 7 days. Immunohistochemical staining of superoxide dismutase (SOD) and malondialdehyde (MDA), and transmission electron microscopy (TEM) of renal tubular epithelial cells were performed to observe oxidative stress and morphological changes, respectively. Decreased SOD and increased MDA were shown only in glyoxylate-treated mouse kidneys. The TEM study with glyoxylate-treated mouse kidneys demonstrated that the internal structure of mitochondria in renal tubular cells underwent destruction and vacuolization, and microvilli density decreased. These changes in renal tubular cells were located in the crystal-forming area. However, such changes were not detected in the other groups. Each precursor of oxalate induces different changes in renal epithelial cells regarding oxidative stress and the microstructural changes. It is suggested that calcium oxalate crystal formation requires cell injury and morphological changes of renal epithelial tubular cells induced by glyoxylate administration in the mouse kidney.     

Membrane-associated crystallization of calcium oxalatein vitro

Summary Incubation of proximal tubular brush border membrane in a metastable calcium oxalate solution of low supersaturation resulted in the equimolar depletion of calcium and oxalate and the formation of monoclinic calcium oxalate crystals. We propose that membrane fragments from sloughed epithelial cells of the nephron can similarly induce crystallization in urine that is metastable for calcium oxalate.     

高血糖在高草酸尿环境下促进肾草酸钙结石形成的机制研究

目的通过细胞及动物实验,探究高血糖促进草酸钙结石形成的机制。方法八周龄Wistar雄性大鼠(共40只)随机分为4组:阴性对照组、高草酸尿模型组,高血糖模型组,高血糖合并高草酸尿模型组;每组10只。采用q RT-PCR检测在含有不同浓度葡萄糖培养基下HK2细胞中OPN、MCP-1、Cbfa1、BMP-2 m RNA的表达;酶联免疫吸附测定(ELISA)检测OPN与MCP-1在培养基中的浓度;钙盐染色检测各组大鼠肾组织中草酸钙结石晶体;细胞凋亡试验检测各组大鼠肾组织中肾小管上皮细胞凋亡。免疫组织化学染色法(IHC)检测OPN在各组大鼠肾组织中的表达;ELISA检测MCP-1在各组大鼠24小时尿液中的含量。结果高浓度葡萄糖环境中,HK-2细胞的OPN与MCP-1表达上调。高血糖合并高草酸尿模型组肾组织中有大量草酸钙沉积,大量肾小管上皮细胞凋亡,OPN表达显著增加,其尿液中MCP-1总量明显增加,与其他组相比有统计学差异(P0.05)。结论高血糖能促使肾小管上皮细胞炎症趋化因子OPN、MCP-1表达增加。在高草酸尿环境中,高血糖能促进肾小管上皮细胞凋亡及草酸钙结石形成。高血糖可能是通过炎症趋化因子加重局部炎症反应,促进肾小管上皮细胞凋亡及草酸钙结石形成。     

Role of inter-α-inhibitor and its related proteins in experimentally induced calcium oxalate urolithiasis. Localization of proteins and expression of bikunin gene in the rat kidney

Summary Our earlier studies indicated that members of the inter-α-inhibitor (IαI) family of glycoproteins may play an important role in urolithiasis. Indeed bikunin, the light chain of IαI is a potent inhibitor of calcium oxalate crystallization. In order to understand this role, the distribution of IαI and its related proteins, as well as the expression of bikunin, were studied in normal and nephrolithic rats. In normal rats, IαI immunoreactivity was located mainly in proximal tubules. However, in nephrolithic rats, in addition to proximal tubules, the staining was intensively extended to tubules in the corticomedullary junction. Furthermore, by using polymerase chain reaction technique, we demonstrated that gene encoding for bikunin was activated in kidneys of nephrolithic rats. We have previously demonstrated increased staining for osteopontin in association with calcium oxalate crystal deposition in rat kidneys. Others have shown an increase in osteopontin production by renal epithelial cells on exposure to calcium oxalate crystals. Based on these observations we conclude that kidney cells possess an auto-defense system against calcium oxalate crystallization and stone formation in which members of the IαI family may be closely involved. Received: 18 February 1998 / Accepted: 9 July 1998