枸橼酸钾干预下肾结石大鼠肾组织内Tamm-Horsfall 蛋白的表达

有研究结果显示Tamm-Horsfall 蛋白(THP)是一种与尿路结石形成密切相关的大分子有机物[1-2].我们通过观察枸橼酸钾干预下肾结石大鼠肾组织内THP 的表达,了解构橼酸钾对THP表达的影响,探讨枸橼酸钾抑制结石形成的机制及枸橼酸钾与THP的关系.     

大鼠低亲和力钠依赖二羧酸转运蛋白对枸橼酸及草酸转运电生理特性的研究

目的 探讨大鼠低亲和力钠依赖二羧酸转运蛋白(SDCT1)对枸橼酸及草酸的转运特点。方法克隆出大鼠SDCT1全长cDNA基因。应用爪蟾卵母细胞异源性表达SDCT1，并使用双电极电压钳法记录通道电流。通过改变灌流液中枸橼酸、草酸的浓度，以及两种底物转运时钠离子的浓度及pH值，对其特性进行了研究。结果SDCT1对枸橼酸及草酸的转运均为底物浓度及钠离子依赖。pH值变化显著影响2者的转运。但低pH对草酸的转运影响要远大于枸橼酸。结论在临床实践中，虽然升高尿液pH值可以抑制枸橼酸的重吸收，发挥其抑制钙盐沉积的功能。但过度地碱化尿液也会使草酸的重吸收受到抑制，使草酸大量存留在尿中而导致结行。保持适当的尿酸碱度是必要的。     

尿凝血酶原片断1在肾结石模型大鼠肾组织的表达及其意义

目的　研究尿液中尿凝血酶原片段 1(UPTF1)的来源和UPTF1在肾结石模型大鼠肾组织的表达 ,探讨尿结石形成对肾组织UPTF1表达的影响及其在尿结石形成中的意义。方法用乙二醇和 1α 羟基维生素D3 灌胃制作大鼠肾草酸钙结石模型。采用半定量逆转录聚合酶链反应(RT PCR)检测UPTF1mRNA在结石模型大鼠和正常对照大鼠肾组织中的表达及水平变化。结果　偏光显微镜下结石模型大鼠肾乳头和肾皮质内布满草酸钙晶体 ,肾钙含量、2 4h尿草酸和尿钙分泌量分别为 13 8.3 9mg/g、82 .89μmol和97.3 5 μmol;对照组肾钙含量、2 4h尿草酸和尿钙分泌量分别为 1.5 4mg/g、2 4.2 2 μmol和3 .14 μmol,组间差异均有统计学意义(P <0 .0 1)。UPTF1mRNA在所有大鼠肾组织和肝组织中都有表达 ,但在正常大鼠和肾结石大鼠肾组织中的相对表达量分别为 1.73± 0 .2 5、1.86± 0 .19,两组差异无统计学意义意义 (P >0 .0 5 )。结论　尿液中的UPTF1来源于大鼠肾组织的生物合成 ,可能是草酸钙结石形成的生理性抑制因子 ,从而可以借助实验动物模型为研究UPTF1在尿结石形成中的作用提供了依据。     

草酸钙结石形成对肾组织bikunin和ⅠαⅠ表达的影响

目的研究肾草酸钙结石形成对肾组织bikunin基因和间α胰蛋白酶抑制物(ⅠαⅠ)蛋白表达的影响,探讨bikunin在尿结石形成中的意义.方珐诱导实验性大鼠肾草酸钙结石模型,收集结石大鼠、正常大鼠、临床肾结石和非结石患者每组各8例的肾组织标本.采用免疫组化、逆转录聚合酶链反应(RT-PCR)和计算机图像分析技术分别检测所有大鼠和人肾组织中bikuninmRNA和ⅠαⅠ蛋白的表达水平.结果正常大鼠和非结石患者肾组织均存在bikunin mRNA和IαI蛋白的表达.肾草酸钙结石形成后,结石大鼠肾组织ⅠαⅠ蛋白的灰度值和bikunin mRNA的相对表达水平分别为198.43士15.17、0.70士0.14;肾结石患者肾组织IαI蛋白的灰度值和bikunin mRNA的相对表达水平分别为263.25士17.41、1.27±0.13,分别和对照组相比,均显著增加(P＜0.05).结论Bikunin作为构成IαI的轻链结构,在肾草酸钙结石形成后,bikunin mRNA的表达迅速增强,提示机体通过肾脏合成更多的bikunin来抑制肾草酸钙结石的形成.     

草酸钙结石形成对肾组织bikunin和IαⅠ表达的影响

目的研究肾草酸钙结石形成对肾组织bikunin基因和间α胰蛋白酶抑制物(ⅠαⅠ)蛋白表达的影响,探讨bikunin在尿结石形成中的意义.方珐诱导实验性大鼠肾草酸钙结石模型,收集结石大鼠、正常大鼠、临床肾结石和非结石患者每组各8例的肾组织标本.采用免疫组化、逆转录聚合酶链反应(RT-PCR)和计算机图像分析技术分别检测所有大鼠和人肾组织中bikuninmRNA和ⅠαⅠ蛋白的表达水平.结果正常大鼠和非结石患者肾组织均存在bikunin mRNA和IαI蛋白的表达.肾草酸钙结石形成后,结石大鼠肾组织ⅠαⅠ蛋白的灰度值和bikunin mRNA的相对表达水平分别为198.43士15.17、0.70士0.14;肾结石患者肾组织IαI蛋白的灰度值和bikunin mRNA的相对表达水平分别为263.25士17.41、1.27±0.13,分别和对照组相比,均显著增加(P＜0.05).结论Bikunin作为构成IαI的轻链结构,在肾草酸钙结石形成后,bikunin mRNA的表达迅速增强,提示机体通过肾脏合成更多的bikunin来抑制肾草酸钙结石的形成.     

单味中药金钱草、石韦、车前子对肾结石模型大鼠的预防作用

目的：观察单味中药金钱草、石韦、车前子对肾结石的预防作用。方法：90只大鼠随机分为5组,采用1.25%乙二醇和1%氯化铵制备大鼠肾结石模型,用上述3种单味中药的免煎剂型分别给各实验组大鼠肾结石模型灌胃,对照组用枸橼酸钾,4周后观察尿和肾组织中草酸钙结晶的形成情况。结果：单味中药金钱草组、石韦组、车前子组大鼠肾内草酸钙结晶明显少于模型组,而与西药枸橼酸钾组相当。金钱草组、石韦组、车前子组大鼠尿中草酸钙结晶的排泄均明显多于模型组。结论：单味中药金钱草、石韦、车前子对预防大鼠肾结石的形成有确切效果,并与西药枸橼酸钾相当,其作用可能主要是通过增加尿中草酸钙结晶的排泄而达到的。     

白藜芦醇介导Nrf2/ARE信号通路对大鼠草酸钙肾结石模型的抗氧化损伤作用

目的探讨白藜芦醇(Res)在大鼠草酸钙肾结石模型中的抗氧化作用。方法将24只SD大鼠随机分为对照组、结石模型组和结石模型+Res干预组,每组8只。结石组给予自由饮水并用乙二醇+氯化铵溶液灌胃(2mL/d)法制备草酸钙结石模型,干预组在结石模型基础上加用Res灌胃(每日200mg/kg)。28d造模完成后采集肾组织标本,用HE染色法比较各组肾脏病理改变及草酸钙结晶的沉积情况并检测肾组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽(GSH)含量;RT-PCR法检测核因子E2相关因子2(Nrf2)mRNA表达水平;Western blot法检测Nrf2蛋白表达水平。结果 (1)肾组织病理结果:肾组织与对照组比较,结石模型组大鼠肾组织HE染色切片中可见肾小管的病理性改变并大量结晶形成(P0.05);与结石模型组比较,结石模型+Res干预组肾小管扩张不明显,炎细胞浸润以及管腔内草酸钙晶体明显减少(P0.05)。(2)肾组织氧化应激物水平:与对照组比较,结石模型组SOD、CAT和GSH含量降低,MDA含量升高(P0.05);与结石模型组比较,结石模型+Res干预组SOD、CAT和GSH含量显著升高,MDA含量显著降低(P0.05)。(3)肾组织Nrf2蛋白水平:与对照组比较,结石模型组Nrf2mRNA和Nrf2蛋白表达量降低(P0.05);与结石模型组比较,结石模型组+Res干预组Nrf2mRNA和蛋白表达量明显升高(P0.05)。结论 Res通过其抗氧化作用抑制/延缓了草酸钙晶体对大鼠肾小管上皮细胞的损伤及粘附,此作用可能与Nrf2/ARE信号通路有关。     

夹竹桃麻素对高草酸尿症大鼠肾脏氧化应激损伤的保护作用

目的 观察NADPH氧化酶特异抑制剂夹竹桃麻素（apocynin）对高草酸尿症大鼠肾脏氧化应激（OS）损伤的保护作用。 方法 自由饮用含有0.8%乙二醇的水4周建立高草酸尿症SD大鼠模型。大鼠按随机数字表法分为4个组：空白组、高草酸尿症组、apocynin干预组、apocynin对照组。后两组给予apocynin（0.2 g&#8226;kg-1&#8226;d-1）灌胃,对照组给予正常饮水。4周后检测大鼠肾脏OS 指标（尿H2O2和8-异前列腺素）,以及Ccr及肾脏/体质量比值。免疫组化观察NADPH氧化酶亚基p47phox在肾脏中的表达位置。RT-PCR和免疫印迹法分别检测肾组织NADPH氧化酶亚基p47phox、gp91phox、Nox-1 mRNA以及p47phox蛋白的表达水平。 结果 p47phox在各组肾脏中均有广泛的表达,包括肾皮质区、内髓区、外髓区等。与空白组比较,高草酸尿症组大鼠尿H2O2和8-异前列腺素水平显著升高,Ccr降低,肾脏/体质量比值增高（均P < 0.05）;肾脏p47phox、gp91phox和Nox-1 的mRNA表达均显著增加（均P ＜ 0.05）, p47phox蛋白表达也增多（P ＜ 0.01）。apocynin干预治疗可抑制肾脏p47phox、Nox-1 mRNA及p47phox蛋白的表达,但gp91phox mRNA表达未明显减少,而大鼠尿H2O2和8-异前列腺素水平下降,Ccr增加,肾脏/体质量比值减少,但仍高于对照组水平。 结论 NADPH氧化酶是高草酸尿症诱导大鼠肾脏OS损伤过程中活性氧形成的来源之一。使用apocynin抑制NADPH氧化酶活性可部分减轻肾脏的OS损伤程度,保护肾功能。     

酒石酸钾预防草酸钙肾结石形成的研究

体外实验证明酒石酸钾对一水草酸钙晶体的生长和聚集有抑制作用。动物实验发现酒石酸钾能够降低鼠肾组织中钙及草酸的沉积。２７例患者口服酒石酸钾期间，２４小时尿钙、磷和草酸明显下降，尿枸橼酸和尿ＰＨ显著上升，实验证明，酒石酸钾能够抑制草酸钙肾结石的形成，是一种有希望的防石药物。     

来氟米特对糖尿病大鼠肾脏Podocalyxin表达的影响

目的：研究来氟米特对糖尿病大鼠肾脏Podocalyxin蛋白表达的影响。方法：48只雄性Wistar大鼠随机分为正常对照组、糖尿病肾病模型组、来氟米特干预组、氯沙坦干预组,每组12只。腹腔注射STZ建立糖尿病肾病大鼠模型,给予药物干预。观察各组大鼠血糖、24h尿微量白蛋白量的变化,8周、12周末各组大鼠血尿素氮（BUN）、肌酐（Scr）、肾脏组织病理变化及肾组织Podocalyxin表达水平。结果：8周、12周末模型组、来氟米特干预组、氯沙坦干预组24h尿微量白蛋白定量较正常对照组显著增高（P〈0.05）,肾组织病理损伤明显,大鼠Podocalyxin蛋白表达均显著低于正常对照组（P〈0.05）。8周、12周末干预组24h尿微量白蛋白定量、BUN、Scr水平显著低于同期模型组（P〈0.05）,肾组织病理损伤较模型组明显减轻,大鼠肾组织Podocalyxin蛋白表达显著高于同期模型组（P〈0.05）。结论：来氟米特能够上调糖尿病大鼠肾组织Podocalyxin表达水平,对糖尿病肾脏损伤有保护作用。     

Sodium dicarboxylate cotransporter-1 expression in renal tissues and its role in rat experimental nephrolithiasis

BACKGROUND: Nephrolithiasis is a common disease with a high recurrence rate; however, calcium stone pathogenesis remains unknown because of complex multiple factors. Hypocitraturia induced by citrate transport disturbance is known to be involved in nephrolithiasis development. Sodium dicarboxylate cotransporter (NaDC) mediates citrate uptake from the renal proximal tubule. However, the role of NaDC in nephrolithiasis is unclear. This study aimed to investigate NaDC-1 expression in rat renal proximal tubule epithelial cells and its relationship with experimental nephrolithiasis. METHODS: Male Wastar rats were divided into control, ethylene glycol (EG)-treated and potassium citrate-treated groups. Calcium oxalate (CaOx) crystal deposition and histological changes in the kidneys were examined with anatomical and light microscopes. The plasma and urinary biochemical parameters, such as citrate, oxalate etc, were analyzed by routine biochemical methods. NaDC-1 mRNA expression in kidneys was determined by northern blot analysis, the change in NaDC-1 protein abundance was detected by immunohistochemistry. RESULTS: It was found that NaDC-1 expression and its mRNA significantly increased in the EG group when compared with controls. Increased NaDC-1 expression was associated with a decline in urinary citrate excretion. Potassium citrate administration could significantly down-regulate NaDC-1 expression and its mRNA, and elevate urinary citrate content alleviate renal pathological changes and reduce nephrolithiasis occurrence. CONCLUSION: Increased NaDC-1 expression on the renal proximal tubule epithelial cells could play an important role in nephrolithiasis development, suggesting it could be a therapeutic target for the treatment of nephrolithiasis.     

Osteopontin knockdown in the kidneys of hyperoxaluric rats leads to reduction in renal calcium oxalate crystal deposition

Osteopontin (OPN) expression is increased in kidneys of rats with ethylene glycol (EG) induced hyperoxaluria and calcium oxalate (CaOx) nephrolithiasis. The aim of this study is to clarify the effect of OPN knockdown by in vivo transfection of OPN siRNA on deposition of CaOx crystals in the kidneys. Hyperoxaluria was induced in 6-week-old male Sprague–Dawley rats by administering 1.5 % EG in drinking water for 2 weeks. Four groups of six rats each were studied: Group A, untreated animals (tap water); Group B, administering 1.5 % EG; Group C, 1.5 % EG with in vivo transfection of OPN siRNA; Group D, 1.5 % EG with in vivo transfection of negative control siRNA. OPN siRNA transfections were performed on day 1 and 8 by renal sub-capsular injection. Rats were killed at day 15 and kidneys were removed. Extent of crystal deposition was determined by measuring renal calcium concentrations and counting renal crystal deposits. OPN siRNA transfection resulted in significant reduction in expression of OPN mRNA as well as protein in group C compared to group B. Reduction in OPN expression was associated with significant decrease in crystal deposition in group C compared to group B. Specific suppression of OPN mRNA expression in kidneys of hyperoxaluric rats leads to a decrease in OPN production and simultaneously inhibits renal crystal deposition.     

肾结石大鼠肾组织bikunin mRNA的表达

目的 :研究bikunin在实验性肾草酸钙结石大鼠肾组织的表达及意义。方法 :采用乙二醇和氯化铵诱导大鼠肾草酸钙结石模型形成 ,检测各组大鼠肾功能、肾组织Ca2 + 含量和草酸钙晶体沉积、尿生化指标 ,并用逆转录聚合酶链反应 (RT PCR)检测bikuninmRNA在肾组织的表达情况。结果 :模型组大鼠的血清Cr、BUN、肾Ca2 + 含量、2 4h尿Ca2 + 、草酸 (Ox)分泌量和肾组织bikuninmRNA的表达均明显高于正常组 (P <0 .0 5 )。结论 :高草酸尿和草酸钙结晶的沉积能促使大鼠肾脏通过合成更多的bikunin来抑制大鼠肾组织草酸钙晶体的形成。     

Dietary oxalate and calcium oxalate nephrolithiasis

PURPOSE: Patients with calcium oxalate kidney stones are advised to decrease the consumption of foods that contain oxalate. We hypothesized that a cutback in dietary oxalate would lead to a decrease in the urinary excretion of oxalate and decreased stone recurrence. We tested the hypothesis in an animal model of calcium oxalate nephrolithiasis. MATERIALS AND METHODS: Hydroxy-L-proline (5%), a precursor of oxalate found in collagenous foods, was given with rat chow to male Sprague-Dawley rats. After 42 days rats in group 1 continued on hydroxy-L-proline, while those in group 2 were given chow without added hydroxy-L-proline for the next 21 days. Food and water consumption as well as weight were monitored regularly. Once weekly urine was collected and analyzed for creatinine, calcium, oxalate, lactate dehydrogenase, 8-isoprostane and H(2)O(2). Urinary pH and crystalluria were monitored. Rats were sacrificed at 28, 42 and 63 days, respectively. Renal tissue was examined for crystal deposition by light microscopy. RESULTS: Rats receiving hydroxy-L-proline showed hyperoxaluria, calcium oxalate crystalluria and nephrolithiasis, and by day 42 all contained renal calcium oxalate crystal deposits. Urinary excretion of lactate dehydrogenase, 8-isoprostane and H(2)O(2) increased significantly. After hydroxy-L-proline was discontinued in group 2 there was a significant decrease in urinary oxalate, 8-isoprostane and H(2)O(2). Half of the group 2 rats appeared to be crystal-free. CONCLUSIONS: Dietary sources of oxalate can induce hyperoxaluria and crystal deposition in the kidneys with associated degradation in renal biology. Eliminating oxalate from the diet decreases not only urinary oxalate, but also calcium oxalate crystal deposits in the kidneys and improves their function.     

Characterization of Tamm-Horsfall protein in a rat nephrolithiasis model

PURPOSE: The role of Tamm-Horsfall protein in calcium oxalate stone formation is controversial. It is unclear whether Tamm-Horsfall protein has a role in crystallization. If it does, does it act as an inhibitor or promoter of crystallization? To elucidate the nature of its involvement we characterized Tamm-Horsfall protein in a rat model of calcium oxalate nephrolithiasis by in vivo and in vitro techniques. MATERIALS AND METHODS: Calcium oxalate nephrolithiasis was induced in male Sprague-Dawley rats. The amino acid and carbohydrate composition of Tamm-Horsfall protein from normal rats and those with nephrolithiasis was determined. The Tamm-Horsfall protein gene and protein expression in the kidneys were examined by in situ hybridization and immunohistochemistry. Furthermore, the interaction of Tamm-Horsfall protein and calcium oxalate crystals was assessed by an in vitro crystal aggregation assay. RESULTS: Tamm-Horsfall protein from rats with nephrolithiasis was biochemically similar to that from normal rats. Although Tamm-Horsfall protein was associated with crystal deposits in the renal papillae of rats with nephrolithiasis, Tamm-Horsfall protein messenger RNA expression in the kidneys remained unchanged. In each group Tamm-Horsfall protein inhibited calcium oxalate crystal aggregation by 47%, indicating no change in functional capabilities. CONCLUSIONS: The results of this study indicate that urinary excretion, and the biochemical nature and functional capabilities of Tamm-Horsfall protein remain unchanged during experimental calcium oxalate nephrolithiasis. Although staining for Tamm-Horsfall protein was evident in the papillae of rats with nephrolithiasis, the site of Tamm-Horsfall protein synthesis remained cells of the thick ascending limbs of the loop of Henle.     

The influence of sex hormones on renal osteopontin expression and urinary constituents in experimental urolithiasis

PURPOSE: To our knowledge the influence of sex hormones on urinary stone formation remains undetermined. We investigated the effect of castration on urinary lithogenic factors and renal osteopontin expression in rats previously treated with ethylene glycol. MATERIALS AND METHODS: Sprague-Dawley rats were divided normal males, castrated males, males with 2 weeks of 0.75% ethylene glycol treatment, castrated males with 2 weeks of 0.75% ethylene glycol treatment, normal females, castrated females, females with 2 weeks of 0.75% ethylene glycol treatment and castrated females with 2 weeks of 0.75% ethylene glycol treatment. We analyzed 24-hour urine samples for urinary constituents, such as calcium, oxalate, citrate, uric acid, phosphate, magnesium, sodium, potassium and creatinine. The kidneys were examined for osteopontin expression by Northern blot analysis and for crystal deposition by histological examination. RESULTS: In intact male rats calcium and citrate excretion decreased and oxalate excretion increased significantly after ethylene glycol treatment. Castrated male rats with ethylene glycol had greater calcium and less oxalate excretion than male intact rats with ethylene glycol. In intact female rats uric acid excretion decreased and only calcium excretion increased significantly after ethylene glycol treatment. Castrated female rats with ethylene glycol excreted significantly more oxalate and less calcium than intact female rats with ethylene glycol. Renal osteopontin expression was the same in male intact and castrated rats, and in female intact and castrated rats. In males with ethylene glycol expression was stronger in castrated than in intact rats. In females with ethylene glycol expression was weaker in castrated than in intact rats. No crystal deposits were found in the kidneys in any group. CONCLUSIONS: Testosterone appears to promote stone formation by suppressing osteopontin expression in the kidneys and increasing urinary oxalate excretion. Estrogen appears to inhibit stone formation by increasing osteopontin expression in the kidneys and decreasing urinary oxalate excretion.     

Prophylactic effects of quercetin and hyperoside in a calcium oxalate stone forming rat model

Quercetin and hyperoside (QH) are the two main constituents of the total flavone glycosides of Flos Abelmoschus manihot, which has been prescribed for treating chronic kidney disease for decades. This study aimed to investigate the effect of QH on calcium oxalate (CaOx) formation in ethylene glycol (EG)-fed rats. Rats were divided into three groups: an untreated stone-forming group, a QH-treated stone-forming group (20 mg/kg/day) and a potassium citrate-treated stone-forming group (potassium citrate was a worldwide-recognized calculi-prophylactic medicine). Ethylene glycol (0.5 %) was administered to the rats during the last week, and vitamin D3 was force-fed to induce hyperoxaluria and kidney calcium oxalate crystal deposition. 24 h urine samples were collected before and after inducing crystal deposits. Rats were killed and both kidneys were harvested after 3 weeks. Bisected kidneys were examined under a polarized light microscope for semi-quantification of the crystal-formation. The renal tissue superoxide dismutase and catalase levels were measured by Western blot. QH and potassium citrate have the ability to alkalinize urine. The number of crystal deposits decreased significantly in the QH-treated stone-forming group as compared to the other groups. Superoxide dismutase and catalase levels also increased significantly in the QH-treated stone-forming group, as compared with the untreated stone-forming group. QH administration has an inhibitory effect on the deposition of CaOx crystal in EG-fed rats and may be effective for preventing stone-forming disease.     

Effect of potassium depletion on urinary stone risk factors in Wistar rats

Various studies have suggested that potassium depletion leads to acidosis and hypocitraturia. In Northeastern Thailand, for example, mild hypokalemia and mild hyperoxaluria are observed in most stone formers. However, there are limited reports about the direct link between potassium depletion and the formation of urinary stones, most of which are calcium oxalate stones. Therefore, we studied the direct effect of potassium depletion on the risk factors for calcium oxalate stone formation. Seventy-two rats were fed a control diet or a potassium-deficient diet for 1, 2, or 3 weeks (n = 12 per group). Twenty-four-hour urine collection was done for the measurement of potassium, calcium, oxalate, glycolate, citrate, phosphorus, and magnesium. Lactate dehydrogenase activity was also measured in order to assess renal tubular damage, and kidneys were harvested for histological examination. Furthermore, urinary supersaturation of calcium oxalate was calculated. With potassium depletion, the urinary concentrations of potassium, citrate, magnesium, and phosphorus decreased rapidly. There was no detectable renal damage, renal calcium deposition, and no significant increase of urinary oxalate or calcium. However, the urinary supersaturation index of calcium oxalate increased significantly in rats with potassium depletion. These findings indicate that potassium deficiency may increase the risk of stone formation through enhanced supersaturation.