血镁水平与糖尿病肾病

为探讨糖尿病肾病 (DN)患者血镁水平 ,以 1 5 9例 2型糖尿病 (T2DM)患者为研究对象 ,用xylidylblue比色法测定了其正常白蛋白尿期、微量白蛋白尿期 (早期DN)、临床DN及晚期DN血镁水平 ,与 2 0例正常对照组相比较 ;同时将 1 1 0例DN患者分为肾功能不全代偿组、失代偿组和肾功能衰竭组 ,将其血镁水平与 1 0 2例慢性肾小球肾炎 (CGN)患者相比较 ,组间比较采用t检验 ;对 79例T2DM正常白蛋白尿期及微量白蛋白尿期患者尿白蛋白排泄率 (UAER)与血镁水平进行了等级相关分析。结果表明 ,T2DM正常白蛋白尿期、早期DN及临床DN血镁水平降低 ,晚期DN血镁水平升高 ,各组与正常对照组相比存在显著性差异 (P <0 0 5或P <0 0 1 ) ;T2DMDN及CGN肾功能不全代偿期、失代偿期及肾功能衰竭期血镁水平渐升高 ,且肾功能处于同一期的DN和CGN相比 ,前者血镁水平均较后者显著降低 (P <0 0 1或P <0 0 5 ) ;T2DM正常白蛋白尿期和微量白蛋白尿期血镁水平与UAER呈负相关 (r=0 5 47,P <0 0 1 )。提示T2DM患者肾功能正常时存在低镁倾向 ,但晚期DN血镁水平升高 ;随着肾功能不全进展 ,DN和CGN患者血镁水平升高 ,但前者血镁水平仍较后者低 ,镁代谢紊乱与DM及其并发症的相互关系有待进一步研究 ;血镁水平测定可否作为DN的早期诊断指标亦     

Urinary d‐lactate levels reflect renal function in aristolochic acid‐induced nephropathy in mice

Urinary d ‐lactate is highly correlated to diabetic nephropathy – a progressive kidney disease in renal glomeruli. In this study, we used a C3H/3e mouse model to investigate the relationship between urinary d ‐lactate and aristolochic acid nephropathy where the glomerular structure is not affected. The nephropathy was induced using intravenous injections of aristolochic acid at a dosage of 10 mg/kg per day for 5 days and was characterized biochemically and histologically. The urinary excretions of proteins, N‐acetyl‐β‐d ‐glucosaminidase and serum creatinine were determined and connected to histological conventional findings. Urinary d ‐lactate was analyzed using column‐switching high‐performance liquid chromatography with fluorescence detection. The results showed a remarkable increase of urinary markers, including of urinary proteins and N‐acetyl‐β‐d ‐glucosaminidase, and the histological examination confirmed a diagnosis of acute tubule necrosis. The ratio of d ‐lactate to creatinine in the urine of aristolochic acid‐treated mice was approximately 36 times greater than that of the mice in the control group (p < 0.05). The ratios for the two groups of mice were 311.00 ± 71.70 and 8.60 ± 1.80 µmol/mmol creatinine, respectively. These data confirm in vivo that urinary d ‐lactate reflects renal injury conditions in aristolochic acid‐treated mice and may be a marker for the assessment of nephropathy. Copyright © 2013 John Wiley & Sons, Ltd.     

Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. An elevated fatty acid plasma concentration leads to podocyte injury and DN progression. This study aimed to identify and characterize cellular mechanisms of natural compounds that inhibit palmitic acid (PA)–induced human podocyte injury. By screening 355 natural compounds using a cell viability assay, 3-hydroxyterphenyllin (3-HT) and candidusin A (CDA), isolated from the marine-derived fungus Aspergillus candidus PSU-AMF169, were found to protect against PA-induced podocyte injury, with half-maximal inhibitory concentrations (IC₅₀) of ~16 and ~18 µM, respectively. Flow cytometry revealed that 3-HT and CDA suppressed PA-induced podocyte apoptosis. Importantly, CDA significantly prevented PA-induced podocyte barrier impairment as determined by 70 kDa dextran flux. Reactive oxygen species (ROS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) direct scavenging assays indicated that both compounds exerted an anti-oxidative effect via direct free radical–scavenging activity. Moreover, 3-HT and CDA upregulated the anti-apoptotic Bcl2 protein. In conclusion, 3-HT and CDA represent fungus-derived bioactive compounds that have a novel protective effect on PA-induced human podocyte apoptosis via mechanisms involving free radical scavenging and Bcl2 upregulation.     

Rapid determination of aristolochic acids I and II in herbal products and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs) are a mixture of structural-related compounds, in which aristolochic acid I (AA I) and aristolochic acid II (AA II) are reported to be correlated with Aristolochic acid nephropathy (AAN). In this work, a rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to determine AA I and AA II in herbal products and biological fluids. By using gradient elution with a mobile phase composed of a mixture of 10 mM ammonium formate buffer (pH 3.0) and acetonitrile, AAs could be determined within 10 min. Under optimum UHPLC-MS/MS conditions, the limit of detections was 0.14 and 0.26 ng mL⁻¹ for AA I and AA II, respectively. Run-to-run repeatability and intermediate precision of peak area for AA I and AA II were less than 5.74% relative standard deviation (RSD). Accuracy was tested by spiking 10, 100 and 1000 ng mL⁻¹ in rat serum and the recoveries were within 76.5-92.9%. Matrix effects were within 78.8-127.7%. The developed method was successfully applied to determine AA I and AA II in several herbal products and to investigate their pharmacokinetic behavior in female Wister rats. The result shows that the developed UHPLC-MS/MS method is efficient, sensitive, and accurate for the determination of AA I and AA II in herbal products and biological samples.     

Phenolic Compounds from Mori Cortex Ameliorate Sodium Oleate-Induced Epithelial–Mesenchymal Transition and Fibrosis in NRK-52e Cells through CD36

Lipid deposition in the kidney can cause serious damage to the kidney, and there is an obvious epithelial–mesenchymal transition (EMT) and fibrosis in the late stage. To investigate the interventional effects and mechanisms of phenolic compounds from Mori Cortex on the EMT and fibrosis induced by sodium oleate-induced lipid deposition in renal tubular epithelial cells (NRK-52e cells), and the role played by CD36 in the adjustment process, NRK-52e cells induced by 200 μmol/L sodium oleate were given 10 μmoL/L moracin-P-2″-O-β-d-glucopyranoside (Y-1), moracin-P-3′-O-β-d-glucopyranoside (Y-2), moracin-P-3′-O-α-l-arabinopyranoside (Y-3), and moracin-P-3′-O-[β-glucopyranoside-(1→2)arabinopyranoside] (Y-4), and Oil Red O staining was used to detect lipid deposition. A Western blot was used to detect lipid deposition-related protein CD36, inflammation-related protein (p-NF-κB-P65, NF-κB-P65, IL-1β), oxidative stress-related protein (NOX1, Nrf2, Keap1), EMT-related proteins (CD31, α-SMA), and fibrosis-related proteins (TGF-β, ZEB1, Snail1). A qRT-PCR test detected inflammation, EMT, and fibrosis-related gene mRNA levels. The TNF-α levels were detected by ELISA, and the colorimetric method was used to detects SOD and MDA levels. The ROS was measured by flow cytometry. A high-content imaging analysis system was applied to observe EMT and fibrosis-related proteins. At the same time, the experiment silenced CD36 and compared the difference between before and after drug treatment, then used molecular docking technology to predict the potential binding site of the active compounds with CD36. The research results show that sodium oleate can induce lipid deposition, inflammation, oxidative stress, and fibrosis in NRK-52e cells. Y-1 and Y-2 could significantly ameliorate the damage caused by sodium oleate, and Y-2 had a better ameliorating effect, while there was no significant change in Y-3 or Y-4. The amelioration effect of Y-1 and Y-2 disappeared after silencing CD36. Molecular docking technology showed that the Y-1 and Y-2 had hydrogen bonds to CD36 and that, compared with Y-1, Y-2 requires less binding energy. In summary, moracin-P-2″-O-β-d-glucopyranoside and moracin-P-3′-O-β-d-glucopyranoside from Mori Cortex ameliorated lipid deposition, EMT, and fibrosis induced by sodium oleate in NRK-52e cells through CD36.     

Low molecular weight proteins in urines from healthy subjects as well as diabetic,nephropathic and diabetic‐nephropathic patients: a MALDI study

Urine samples from healthy subjects as well as diabetic, nephropathic and diabetic‐nephropathic patients were analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry in order to establish evidence of some possible differences in the peptide profile related to the pathological states. Multivariate analysis suggested the possibility of a distinction among the considered groups of patients. Some differences have been found, in particular, in the relative abundances of three ions at m/z 1912, 1219 and 2049. For these reasons, further investigation was carried out by MALDI/TOF/TOF to determine the sequence of these peptides and, consequently, to individuate their possible origin. By this approach, the peptide at m/z 1912 was found to originate from uromodulin, and its lower expression in the case of nephropathy can be well related to the pathological condition. Ions at m/z 2049 and 1219 originate from the collagen α‐1(I) chain precursor and from the collagen α‐5 (IV) chain precursor, respectively, and, also in this case, their different expressions can be related to the pathologies under investigation. The obtained data seem to indicate that urine is an interesting biological fluid to investigate on the peptide profile and to obtain, consequently, information on the dismetabolism activated by specific pathologies. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation of herbs linked to "Chinese herb nephropathy" from the liquid chromatographic determination of aristolochic acids

A HPLC method was developed and applied to analyze aristolochic acids (AA-I and AA-II) in Chinese medicinal herbs. The herb samples were extracted by using ultrasonication with the extraction efficiency of better than 82%. Extracts were then filtered and injected onto a C18 column eluting under a gradient program using methanol and water-containing 0.5% acetic acid. The method with the detection limits of 1.33 ng for AA-I and 7.29 ng for AA-II per injection was successfully applied for the analysis of traditional Chinese medicine (TCM) and related products and differentiation of Chinese medicinal herbs that have previously been misused and caused toxicological effects. The developed protocol provided an example that analysis of selected component markers could serve for health security and quality control of TCM consumption.     

高效液相色谱法测定糖尿病肾病患者血液中的非蛋白氮

采用反相高效液相色谱法对糖尿病患者血液中与肾病相关的四种非蛋白氮代谢产物（肌酸、肌酐、尿素和尿酸）进行了测定。获得的优化条件为：采用C18反相色谱柱,在室温下,以含10mmol／L,pH6．86的KH2PO4和体积分数30％的甲醇水溶液为流动相,流速0．9mL／min,检测波长200nm。在优化的条件下。5min内可对病人血液中上述4种物质同时进行测定。方法具有较好的重现性（迁移时间和峰面积的RSD均为3％）和较高的灵敏度以及较宽的线性范围（肌酸：5—300μmol／L,肌酐：10—200μmol／L,尿素：1～30mmol／L,尿酸：10—1500μmol／L）,适用于临床上病人血液的日常分析和早期肾病的监测。     

IgA肾病免疫吸附剂的研究(Ⅱ)

通过1,4-丁二醇-二缩水甘油醚法活化琼脂糖载体,既起到了活化载体的作用,同时又由于该活化剂分子是长链结构而给载体提供了一定长度的手臂,有利于人免疫球蛋白G(IgG)大分子配基与载体的连接,用该方法制成的免疫吸附剂对高IgA肾病病人血清中IgA进行了吸附研究.通过体外实验条件的优化发现,最高可吸附60%的IgA,同CDI法比较(吸附率可达35%左右),该吸附剂具有较高的吸附效率.     

Development and validation of an LC–MS/MS method for simultaneous determination of remdesivir and its hydrolyzed metabolite and nucleoside,and its application in a pharmacokinetic study of normal and diabetic nephropathy mice

Remdesivir (RDV), a phosphoramidate prodrug, has broad-spectrum antiviral activity. It is the first antiviral drug approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Remdesivir is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, the phosphatase inhibitor PhosSTOP and carboxylesterase inhibitor 5,5′-dithiobis-2-nitrobenzoic acid were used to improve stability of RDV in mouse blood. We developed a rapid and sensitive LC–MS/MS method to simultaneously quantify RDV, RM-442 and RN in mouse blood. Chromatographic separation was achieved by gradient elution on an Acquity HSS T₃ column. The run time was 3.2 min. The linearity ranges of the analytes were 0.5–1,000 ng/ml for RDV and 5–10,000 ng/ml for both RM-442 and RN. The method had an acceptable precision (RSD < 8.4% for RDV, RSD < 10.7% for RM-442 and RSD < 7.2% for RN) and accuracy (91.0–106.3% for RDV, 92.5–98.6% for RM-442 and 87.5–98.4% for RN). This method was successfully applied to analyze RDV, RM-442 and RN in the blood of normal and diabetic nephropathy DBA/2 J mice after intravenous injection of RDV at 20 mg/kg. The area under the concentration–time curve of RN between the normal and diabetic nephropathy mice showed a significant difference (P < 0.01).