联合检测血清NGAL、KIM-1和SCr可有效提高慢性肾病分期诊断

为了探讨中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)和肾损伤分子-1 (kidney injury molecule-1, KIM-1)以及血肌酐(serum creatinine, SCr)联合检测对慢性肾病(chronic kidney disease, CKD)的早期诊断价值,本研究收集260例肾病患者和85例健康体检者,检测其血清NGAL、KIM-1和SCr水平。依据肾功能分级标准,CKD患者分为CKD 1期(53例),CKD 2期(68例),CKD 3期(71例),CKD 4期(46例)和CKD 5期(22例),并分析以上指标在各组间的含量差异,及其联合测定对CKD早期的敏感性。与健康对照组相比较,CKD 1期、CKD 2期、3期、4期和5期患者的NGAL、KIM-1水平均明显升高(p<0.001)。血清SCr含量在CKD 3期、4期和5期组较健康对照组显著增加(p<0.001)。以上3项指标均随着CKD严重程度增加而升高。各组指标阳性率分析显示,3项联合检测阳性率高于单项检测阳性率。ROC曲线分析NGAL、KIM-1、SCr对CKD诊断的AUC值F分别是为0.824、0.805、0.856。相关性分析结果显示,GFR和NGAL、KIM-1、SCr相关系数分别是r=-0.784、-0.756、-0.728 (p<0.05)。NGAL与KIM-1、SCr的相关系数分别是r=0.932、0.764 (p<0.05);KIM-1与SCr的相关系数r分别是0.791 (p<0.05)。本研究初步得出结论:血清NGAL、Kim-1可作为CKD早期诊断的重要指标,联合检测血清NGAL、Kim-1、SCr可有效提高CKD早期肾损伤诊断的敏感度,对CKD的分期诊断和治疗具有极其重要的临床价值。     

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.     

Therapeutic effects of CXCR4+ subpopulation of transgene‐free induced cardiosphere‐derived cells on experimental myocardial infarction

ObjectivesMyocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor‐like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus‐based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene‐free approaches.Materials and MethodsFour transgene‐free approaches for somatic reprogramming, including episome, minicircle, self‐replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations.ResultsThe self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C‐X‐C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach.ConclusionThus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.     

Can we reduce oxidative stress with liver transplantation?

BackgroundThe aim of this study was to determine the levels of lipid peroxidation (MDA) and antioxidants such as reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) in the blood serum of patients with cirrhosis and liver transplantation.MethodsIn this study, serum malondialdehyde acid (MDA) levels, superoxide dismutase (SOD), reduced glutathione (GSH), and catalase (CAT) activities were measured spectrophotometrically and compared to the results of the healthy control group.ResultsSOD, CAT and GSH activities were significantly decreased in the patient groups compared to the healthy control group (p<0.05). MDA levels were significantly higher in the patient group compared to the healthy control group (p <0.05).ConclusionsIn conclusion, this study demonstrated that oxidative stress may play an important role in the development of liver cirrhosis and in liver transplantation. This study is the first one to show how MDA, SOD, CAT and GSH levels change in liver cirrhosis and liver transplantation, while further studies are essential to investigate antioxidant enzymes and oxidative stress status in patients with cirrhosis and liver transplantation.     

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer''s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.     

Effects of dietary vitamin E and selenium on arginase activity in the liver, kidneys, and heart of rats treated with high doses of glucocorticoid

The effects of dietary intake of vitamin E and selenium on arginase activity in the liver, kidneys, and heart of rats treated with high doses of prednisolone were investigated. Rats were divided into five groups. Groups 3, 4, and 5 received a daily supplement in their drinking water of vitamin E, Se, and a combination of vitamin E and Se, respectively, for 30 days. For 3 days subsequently, the control group (group 1) was given a placebo, and the remaining four groups were injected intramuscularly with prednisolone. The tissue samples were collected from each group at 4, 8, 12, 24, and 48 h after the last administration of prednisolone. In the group treated with prednisolone alone, arginase activity in the liver was found to have increased at all the time periods, whereas it had decreased significantly in the heart at 48 h. Arginase activity in the kidneys was not affected by prednisolone. Compared to the control and prednisolone groups, arginase activity in the kidneys and heart of the vitamin E- and Se-supplemented groups was found to be significantly increased at all time periods, however, no difference was seen in the combination group. Arginase activity in the liver of the vitamin E-supplemented group was found to have decreased at all time periods, however, in the Se group compared to the prednisolone group it had reduced at 24 and 48 h only. In the combination group compared to the prednisolone group, liver arginase activity increased constantly up to 12 h returning to normal values at 48 h. Vitamin E and Se in combination may prevent the changes in arginase activity in various tissues caused by prednisolone.     

Failure to Reject an Allografted Tumor after Elimination of Macrophages in Mice

After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (MΦ)-rich, non-T, non-NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the MΦs in the rejection of allografted Meth A cells and characterized the MΦs in comparison with other well-known MΦs. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the MΦ-rich population. In addition, the lymphocyte-rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the MΦ-rich or the T cell-depleted MΦ-rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of MΦs by in vivo application of dichloromethylene diphosphonate-containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of MΦ is the effector cell essential for the rejection. In contrast to other well-known MΦs, the cytotoxic activity against Meth A cells was cell-to-cell contact dependent and soluble factor (e.g., NO and TNF-α) independent. Moreover, the cytotoxic activity of the MΦs (H-2^b) against ⁵¹Cr-labeled Meth A (H-2^d) cells was inhibited by the addition of unlabeled H-2^d, but not H-2^a, H-2^k or H-2^b, lymphoblasts as well as Meth A cells, implying the specific interaction of the MΦs with H-2^d cells.