应用MTS方法检测生物人工肾小管的细胞活性

目的:探讨应用MTS方法在生物人工肾小管(RAD)构建过程中测定血液滤器中肾小管上皮细胞活性的可行性.方法:采用人肾近曲小管上皮细胞株(HK2)体外培养,然后按不同浓度植入6孔板,应用MTS方法测定490 nm吸光度(OD)值,判断其测定值与细胞数量之间的关系.然后将培养的HK2细胞植入FH66血液滤过器内腔继续培养构建RAD,在培养过程中每隔3 d用MTS方法测定一次OD值,并与无细胞植入的空白滤器作对照.结果:HK2细胞数在105～106范围内,与OD值呈正相关关系,相关系数(r)为0.985,P<0.01.各时间点RAD组MTS测定均值与对照组相比均有统计学意义(P<0.01,n＝5).RAD组MTS测定值随培养时间的延长而增加,提示RAD在培养过程中细胞增殖.结论:MTS方法能较好地反映RAD内的细胞活性,能在RAD培养过程中作为一种简便有效的手段监测滤器内的细胞活性.     

Reactive Oxygen Species Independent Cytotoxicity Induced by Radiocontrast Agents in Tubular Cells (LLC-PK1 and MDCK)

Purpose. Radiocontrast agents (RAs) cause renal tubular damage by hemodynamic imbalance, which could cause hypoxic stimulus and direct cytotoxicity. However, reactive oxygen species (ROS) could be an important factor in RAs' direct cytotoxicity. This study investigated the involvement of ROS in deleterious effects produced by RAs on normoxic and hypoxic renal tubular cells. Materials and Methods. LLC-PK1 and MDCK were exposed to diatrizoate and ioxaglate in normoxic and hypoxic conditions. Apoptotic and necrotic cell death were assessed by acridine orange/ethidium bromide and annexin V methods. Hydrogen peroxide, superoxide anion, and malondialdehyde levels were analyzed by, respectively, 2′,7′-dichlorofluorescein, luminal, and thiobarbituric acid. Antioxidant agents were used to prevent cellular RAs damage. Results. Diatrizoate and ioxaglate decreased cellular viability in both cells, and this effect was enhanced by hypoxic conditions. Diatrizoate induced more injury than ioxaglate to both cell lines. LLC-PK1 underwent necrosis, while MDCK cells underwent apoptosis when exposed to diatrizoate. These results could not be attributed to an increase in osmolality. RAs did not increase hydrogen peroxide, superoxide anion or malondialdehyde levels in both cells. Additionally, N-acetyl-L-cysteine (NAC), ascorbic acid, α-tocopherol, glutathione, β-carotene, allopurinol, cimetidine, and citric acid did not protect cells against RAs damage. Surprising, NAC increased the cellular damage induced by ioxaglate in the both cell lines. Conclusion. The present study shows that RAs induce damage in cultured tubular cells, especially in hypoxic conditions. ROS were not involved in the observed RAs' cytotoxicity, and NAC increased ioxaglate-induced tubular damage.     

生物人工肾小管对β_2微球蛋白重吸收功能的研究

目的：探讨应用人肾近曲小管上皮细胞株（HK2）构建的生物人工肾小管辅助装置（RAD）体外对β2微球蛋白（β2-MG）的清除作用及机制。方法：A组以HK2体外培养后植入F60血液滤过器内腔构建的RAD后用含β2-MG的置换液体外模拟液体滤过,B组以上述条件构建的RAD用不含β2-MG的置换液模拟液体滤过,C组用含β2-MG的置换液对无细胞植入的F60滤器模拟液体滤过,对各组测定不同时间点的β2-MG滤过率,并应用免疫荧光法观察滤过后A、B组HK2细胞Megalin与β2-MG的蛋白表达。结果：A组β2-MG滤过率在各时间点均明显高于C组（P〈0.05）;C组β2-MG滤过率在第4h较同组前期各时间点明显降低（P〈0.05）。免疫荧光显示A组Megalin与β2-MG同步表达,B组则仅见Megalin表达。结论：以HK2细胞构建的RAD可显著提高β2-MG的滤过率,其功能在4小时内可稳定维持,作用机制可能与Megalin的内吞作用相关。     

黄芪对糖尿病造影剂肾损害的保护作用及机理研究

目的 :探讨黄芪对糖尿病 (DM)造影剂 (CM)肾损害的保护作用及其作用机理。方法 :建立单侧肾切除糖尿病肾病模型 ,比较静脉注入 76 %泛影葡胺 (10mg/kg)前2 4h黄芪灌胃和生理盐水灌胃的大鼠血肌酐、内生肌酐清除率和血浆ET - 1变化情况 ,以及肾脏ET - 1、ET受体A、B(ETR -A、ETR -B)表达情况。结果 :糖尿病大鼠在黄芪灌胃后禁水 2 4h,注射 76 %泛影葡胺 36h后 ,大鼠内生肌酐清除率下降幅度与生理盐水灌胃组相比有统计学意义 (P <0 .0 5 ) ,血浆ET - 1浓度无统计学意义。黄芪灌胃组与生理盐水灌胃组相比 ,肾小管间质ET - 1、ETR -A、ETR -B表达明显低。结论 :黄芪对糖尿病造影剂肾损害的发生有一定的预防作用 ,这种预防作用可能与抑制肾脏局部内皮素系统作用有关。     

1-Methylhydantoin Cytotoxicity on Renal Proximal Tubular Cells in Vitro

1-Methylhydantoin is produced by bacterial creatinine deaminase in the intestinal tract of uremic patients and retaken up into the body. The present study was designed to explore the toxic effect of 1-methylhydantoin on renal proximal tubular cells in vitro. HK-2 (Human renal proximal tubular cell line) was used as the subject. The cell viability was assessed by MTT assay. The cytotoxicity of 1-methylhydantoin to HK-2 was determined by NAG release test. Apoptosis of cultured HK-2 was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33258. Cells were exposed to 1-methylhydantoin (0.25mMol/L, 0.5mMol/L, or 1mMol/L), or creatinine (1mMol/L) for 24 h. 1-methylhydantoin induced a significant (p < 0.01) dose-dependent loss of cell viability. 1-methylhydantoin–treated HK-2 displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated that 1-methylhydantoin induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by flow cytometry, increased significantly (p < 0.01) after 1-methylhydantoin (0.5mMol/L ) for 24 h. The results of the present study clearly demonstrate that both 1-methylhydantoin and creatinine are toxic for proximal tubular cells but that the damage resulting from the 1-methylhydantoin is more severe.     

下调miR-153促进高糖诱导的肾小管上皮细胞-间充质转化

目的：探讨miR-153调控高糖诱导肾小管上皮细胞（HK2）-间充质转化（EMT）的机制,为研究糖尿病肾病（DN）间质纤维化机制提供新思路。方法：采用生物信息学方法预测调控Snail的候选miRNAs;在db/db小鼠肾组织中验证所有候选miRNAs的表达并分别与Snail做pearson相关性分析,筛选出与Snail负相关最显著的miRNA;以高糖诱导的HK2细胞为载体,转染该miRNA mimics 72 h后,采用real-time PCR、western blot方法,观察该miRNAs、Snail以及E-cadherin的表达改变。结果：调控Snail的候选miRNAs共7个,分别是：miR-153、miR-30e、miR-30d、miR-30b、miR-384-5p、miR-30a、miR-30c;与db/m相比,miR-153、miR-30d及miR-30e在db/db小鼠肾皮质中表达显著下调（P〈0.05）,其中miR-153与Snail表达水平负相关最显著（r=-0.501 56）;高糖诱导HK2细胞下调miR-153,肾小管上皮细胞标志蛋白E-cadherin表达减少;过表达miR-153抑制HK2细胞表达Snail,而E-cadherin水平增加。结论：MiR-153可能通过靶基因Snail参与调控高糖诱导的HK2细胞EMT。     

The SHR as a Small Animal Model for Radiocontrast Renal Failure. Relation of Nephrotoxicity to Animal's Age,Gender, Strain,and Dose of Radiocontrast

The male spontaneously hypertensive rat (SHR), as it ages, suffers many of the renal and cardiovascular complications that are recognized in humans as risk factors for radiocontrast (RC) agent induced renal failure (RF). Knowledge of this led us to test this strain of rats as a small animal model for RC-induced renal failure (RC-RF). Functional studies demonstrated a significant fall in GFR in the recovery period after RC administration. In addition, histopathologic evaluation of the kidneys was done in this study. Our results are based on assigning separate scale values to the histopathological evaluation of the (a) glomeruli, (b) tubules, (c) interstitium, and (d) arteries and arterioles of the kidneys. Saline (S) was administered to one group and the RC agent Hypaque-76 (diatrizoate meglumine sodium) to paired groups of 5-, 8-, 10-, 12-, and 14-month-old male SHR. The results indicated that younger animals (5 and 8 months old) were resistant to the nephrotoxic effects of the RC, but developed susceptibility at 10 months of age, when spontaneous renal pathology became manifest. Both spontaneous renal pathology and RC-induced renal damage (RC-RD) increased as the animals aged. In addition, when the administered dose of RC was repeated after a short interval of only 6 h, the degree of RC-RD increased greatly. In parallel control studies of the influence of gender and strain on the response to RC in 12-month-old rats, neither hypertensive female SHR nor male normotensive Wistar-Kyoto (WKY) rats demonstrated significant spontaneous renal pathology or the marked susceptibility to RC nephrotoxicity shown by their male SHR counterparts.

This small animal model for RC-RD, the mature male SHR, has the distinct advantage that risk factors for RC-RD, similar to those characterized in humans for RC-RF, develop spontaneously without requiring any special treatment or surgical intervention.     

Cubilin Is Essential for Albumin Reabsorption in the Renal Proximal Tubule

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the “specific” cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.The renal handling of plasma proteins involves ultrafiltration in the glomerulus followed by tubular reabsorption. As a result of the essentially size-selective properties of the glomerular filter, the primary urine contains proteins of low molecular weight (<60 kD) such as vitamin D-binding protein (DBP) or free retinol-binding protein (RBP),¹ whereas larger proteins are excluded. Albumin, the single most abundant plasma protein, is partially filtered, and the reported amount present in the glomerular ultrafiltrate varies from 1 to 50 μg/ml.² Ultrafiltered protein, whatever the total amount in the lumen of the initial proximal tubule may be under physiologic conditions, is reabsorbed because normal urine is virtually protein free. Reabsorption takes place in the proximal tubule via receptor-mediated endocytosis, which, at present, is the only documented process for tubular protein clearance. Two receptors, physically and physiologically associated, have been identified.¹ Megalin is a large transmembrane protein (approximately 600 kD) that belongs to the LDL receptor family. Cubilin,³ also known as the intrinsic factor cobalamin receptor,^4,5 is a peripheral membrane protein (approximately 460 kD).³ Megalin binds cubilin with high affinity and may contribute to the internalization of cubilin-ligand complexes. Cubilin also binds amnionless (AMN),^6,7 a 50-kD transmembrane protein that is required for its membrane expression and may permit internalization. Most proteins potentially present in the glomerular ultrafiltrate, and all of those that have been specifically studied have been identified as ligands of megalin, cubilin, or both. This is in particular the case for the most abundant, albumin, which binds both megalin and cubilin.¹The functional relevance of cubilin for tubular uptake of proteins relies on observations made in patients with Imerslund-Grasbeck syndrome (I-GS; also known as megaloblastic anemia 1, OMIM No. 261100) caused by inheritable cubilin or AMN gene defects.^8–11 Functional cubilin deficiency resulting from inappropriate membrane insertion^6,12 and/or synthesis of a truncated form of cubilin¹³ is associated with urine excretion of cubilin ligands such as albumin, transferrin, or apoA-I. Similar observations are made in a model of I-GS in dogs.^6,12 On the other hand, the functional relevance of megalin relies on observations made in mice. Megalin-deficient mice^14–17 excrete megalin ligands (RBP, DBP, cathepsin B, and albumin) as well as cubilin-specific ligands (transferrin and apoA-I). The latter finding has been tentatively related to the fact that megalin is essential for the internalization of cubilin-ligand complexes. Several questions remain unanswered. For instance can apoA-I (or other cubilin ligands), which does not bind megalin, be reabsorbed in the absence of cubilin? We do not know either whether megalin and cubilin function in parallel or as an integrated system for albumin reabsorption. To evaluate the respective roles of cubilin and megalin, it is necessary to compare the effects of cubilin and megalin deficiency in the same system (mouse or man) as well as to analyze the combined effects of simultaneous cubilin and megalin deficiency. This has not yet been achieved because interference with cubilin function early in utero results in early embryonic lethality^18,19 related, at least in part, to dysfunction of extra embryonic tissues. We report here an efficient conditional inactivation system on the basis of loxP and the use of MORE mice that express the Cre recombinase in the whole epiblast but not in extra embryonic tissues.²⁰ The inactivation of cubilin in the kidney results in a dramatic decrease of albumin and intrinsic factor vitamin B12 (IF-B12) complex reabsorption but, unexpectedly, does not affect the reabsorption of several other cubilin ligands.     

In Vitro Differentiation of MSC into Cells with a Renal Tubular Epithelial-Like Phenotype

Bone marrow mesenchymal stem (stromal) cells (MSCs) are shown to differentiate into different renal lineages in in vivo injury models. Nevertheless, the in vitro differentiation of MSCs into a renal tubular epithelial lineage has not been investigated. We hypothesize that the injured renal epithelial cells express renotypic factors that may influence the differentiation of MSCs into a renal tubular epithelial lineage. MSCs were cocultured for up to seven days with injured or uninjured murine cortical tubular renal epithelial cells (MCTs), which are separated by a physical barrier; following the coculture, MSCs were examined for the expression of two renal tubular epithelial-specific markers, kidney-specific cadherin (Ksp-cadherin) and aquaporin-1 (AQP1). MSCs differentiated into a tubular epithelial-like phenotype, as shown by the appearance of Ksp-cadherin and AQP1 by day 7 when cocultured with injured MCTs. Further, MSCs showed tubulogenic characteristics when cocultured in a three-dimensional matrix. Nonetheless, MSCs cultured with the conditioned medium from injured MCTs, cocultured with ureteric bud cells, or treated with nephrogenic factors did not differentiate into renal epithelial cells. Based on our findings, we conclude that MSCs can differentiate into a renal epithelial lineage independent of cell fusion when cocultured with injured renal cells.     

马兜铃酸促进肾小管上皮细胞转分化的发生及温阳活血方干预作用研究

目的：探讨马兜铃酸（aristolochic acid,AA）在大鼠近端肾小管上皮细胞（NRK-52E）转分化中的可能作用,及温阳活血方拮抗转分化机制。方法：（1）MTT法检测不同浓度温阳活血方含药血清对NRK-52E细胞增殖的影响。（2）免疫细胞化学法检测不同浓度AA（0、5、10、20、40μg/ml）对NRK-52E细胞表达α-平滑肌肌动蛋白的影响。（3）ELISA法检测AA刺激下Col-Ⅰ的表达及温阳活血方对其调控作用。（4）Real-Time PCR法检测温阳活血方对TGF-β1mRNA、ET-1mRNA、VEGF mRNA等肾间质纤维化相关因子的调控作用。结果：（1）AA5μg/ml、AA10μg/ml、AA20μg/ml、AA40μg/ml均可刺激NRK-52E表达α-平滑肌肌动蛋白,AA10μg/ml浓度下α-平滑肌肌动蛋白表达较强。（2）AA刺激NRK-52E细胞12h、24h、48h后,Col-Ⅰ的表达水平随着时间的延长而增强。（3）马兜铃酸刺激NRK-52E细胞24h后,TGF-β1mRNA表达上调（P〈0.05）,VEGF mRNA表达显著下调（P〈0.01）,ET-1mRNA的表达显著上调（P〈0.01）,Col-Ⅰ表达水平显著高于正常对照组（P〈0.01）,温阳活血方干预下,TGF-β1mRNA表达下调（P〈0.05）,VEGF mRNA表达显著上调（P〈0.01）,ET-1mRNA有下调趋势,Col-Ⅰ表达水平显著下调。结论：（1）马兜铃酸可促进肾小管上皮细胞转分化的发生。（2）温阳活血方具有拮抗肾小管上皮细胞转分化作用。     

Comparison of Thymoglobulin and Grafalon as Induction Agents in Renal Transplantation: A Prospective Study

BackgroundInduction immunosuppression is used to reduce the incidence of acute rejection and prevent delayed graft function. The 2 rabbit anti-thymocyte globulins- thymoglobulin and Grafalon (ATG Fresenius) have been commonly used for induction immunosuppression and treatment of acute rejection in solid organ transplantation. There are very few studies comparing the efficacy and side effects of both the anti-thymocyte globulins therefore this prospective study comparing the 2 types of anti-thymocyte globulins would be of clinical interest.Patients and MethodsThis prospective single center study was conducted at Rabindranath Tagore International Institute of Cardiac Sciences, Kolkata, India from April 2019 to June 2020. Sixty-two ABO-compatible renal transplant recipients were included in the study. They were divided in 2 groups of 31 patients each. One group received thymoglobulin (3 mg/kg) and the second group received Grafalon (6 mg/kg). All patients were followed up for 12 months and the 2 groups were compared for incidence of rejections, infections, graft function, patient survival, and graft survival.ResultsThere was no significant difference in the incidence of rejections, infective episodes, graft function, posttransplant diabetes mellitus, graft survival and patient survival in thymoglobulin or Grafalon groups. The hematological parameters were similar in both groups at 7 days, 1 month, and 6 months of follow-up. The absolute lymphocyte count was significantly lower in the thymoglobulin group at 12 months posttransplant.ConclusionsThymoglobulin and Grafalon were found to be equivalent in terms of safety and efficacy in short term, with no difference in rejections, infections, graft survival, or patient survival.     

甘草酸对马兜铃酸致肾小管上皮细胞损害保护作用的初步研究

目的:探讨甘草酸(glycyrrhizin,GL)对马兜铃酸(aristolorchic acid,AA)肾损害的保护作用,观察GL是否可以减轻AA对培养的肾小管上皮细胞的损害.方法:5?S RPMI-1640培养肾小管上皮细胞(LLC-PK1),采用结晶紫染色法观察细胞增殖;荧光染色观察活细胞数目;酶动力学检测LDH活性,比色法检测NAG水平;透射电镜观察细胞超微结构.结果:(1)AA 40、80、160 μg/ml可明显抑制细胞增殖,结晶紫OD值显著减少,与无AA对照组比较(P<0.01).(2)GL在5、10、25 μg/ml时结晶紫OD值与无GL对照组比较(P>0.05);GL在50 μg/ml时P<0.05,100 μg/ml、200 μg/ml时P<0.01,提示大剂量GL可改善AA抑制细胞增殖的作用.(3)在AA 40 μg/ml作用下;随着时间延长,无GL组的细胞存活数目逐渐减少,而GL(100 μg/ml、200 μg/ml)作用组,活细胞数目明显增多,显示高浓度GL对细胞损伤有一定的保护作用.(4)GL在5、10、25 μg/ml时,LDH释放率、NAG酶水平与无GL对照组比较(P>0.05),GL在50 μg/ml时P<0.05,GL在100 μg/ml、200 μg/ml时LDH释放率、NAG酶明显减少,与无GL组比较(P<0.01).提示大剂量GL可减轻AA的细胞毒作用.(5)细胞超微结构观察:AA 40 μg/ml作用24 h后,细胞超微结构发生显著改变,以核变异最突出,出现核分叶、巨核、染色质浓染、核边集、核缺失、核膜卷曲增厚,线粒体肿胀等严重细胞损伤改变.GL 100 μg/ml、200 μg/ml组上述改变均较轻微.结论:GL可明显改善AA对肾小管上皮细胞增殖的抑制作用;改善细胞超微结构.     

Changes in Pericardial Calcification Due to Antiplatelet Agents: In Vitro Studies

To develop tissue valves for prolonged use in the cardiovascular system, the complicated process of surface induced calcification must be better understood. Calcification was examined for 60 days on glutaraldehyde treated bovine pericardium (GABP) and enzyme extracted tissues fixed in glutaraldehyde (GATBP) incubated in metastable solutions of calcium phosphate, and the roles of aspirin and persantine in conjunction with vitamins C, B, or E, gentamycin (antibiotic), or pentothal sodium (anesthetic) in the medium were examined. Further, the diffusion of calcium across the GATBP was evaluated using a diffusion cell with 2 compartments. Pericardial calcification as also observed using scanning electron microscopy (SEM) techniques. It seems that the examined antiplatelet agents can modify the pericardial surfaces and subsequently their mineralization processes (GATBP, 31.7 μg/mg tissue; in the presence of 5 mg% vitamin C, 13.1 μg/mg tissue; in 1.5 mg% aspirin, 17.2 μg/ mg tissue; and 1 mg% gentamycin, 14.8 μg/mg tissue) on exposure with the metastable calcium phosphate solution for 60 days. In addition, these agents may modify calcium transport and interfere with the adsorption at the surface, hence reducing calcium nodulation on GATBP. Scanning electron micrographs also revealed a reduction in calcium deposition on the pericardium due to these antiplatelet agents. It may be hypothesized that the influx of calcium on GATBP may be due to the cellular components or the involvement of plasma proteins like the fibrinogen molecule. The exact mechanism of these changes in the calcification of the pericardium are still unknown. From these in vitro findings, it appears that a combined vitamin therapy with low doses of aspirin may be beneficial for platelet suppression and thereby for prevention of thrombosis and calcification. However, more in vivo studies are needed to develop applications.     

Psychotherapeutic Agents in End‐Stage Renal Disease

Patients with end‐stage renal disease (ESRD) are often affected by many comorbid conditions, including mental health disorders. Psychiatric illness among patients with ESRD has been associated with increased risks for nonadherence, hospitalizations, suicide, and all‐cause mortality. We reviewed the pharmacokinetic data available with psychotherapeutic agents, focusing on physiologic data rather than specific dosing recommendations. Unfortunately data regarding the pharmacokinetics, efficacy, and safety of psychotherapeutic agents in ESRD remain rather limited. Of the agents available, it appears that the most data in this patient group were found with selective serotonin reuptake inhibitors and benzodiazepines. Given the small number of patients enrolled in many of the studies and the wide inter‐individual variability, it was difficult to interpret the significance of results in many instances. A number of agents, such as tricyclic antidepressants, were associated with adverse effects that would be imperative to avoid in patients with ESRD. Psychotherapeutic medications should be started at low doses and titrated carefully, while monitoring the efficacy and safety of each agent.