Association of High Calcitriol Serum Levels and Its Hydroxylation Efficiency Ratio with Disease Risk in SLE Patients with Vitamin D Deficiency

Vitamin D (calcidiol) deficiency in systemic lupus erythematosus (SLE) is more frequent than in healthy subjects (HS); it is associated with clinical activity and damage in SLE. Although calcidiol is considered the best indicator of the vitamin D serum status, its deficiency could not reflect its hydroxylation efficiency ratio and calcitriol serum status. This study was aimed at assessing the association of calcidiol and calcitriol serum levels and its hydroxylation efficiency ratio with the risk to clinical and renal disease activities in SLE patients. A cross-sectional study was conducted in 308 SLE and HS women; calcidiol and calcitriol serum levels were evaluated by immunoassays. SLE patients showed lower calcidiol serum levels vs. HS (21.2 vs. 24.2 ng/mL; p < 0.001 ). Active SLE patients presented higher calcidiol/calcitriol ratio scores vs. inactive SLE patients (2.78 vs. 1.92 pg/ng; p = 0.02 ), and SLE patients with renal disease activity showed a pattern of calcidiol-deficient levels (19.5 vs. 25.3 ng/mL; p < 0.04 ) with higher calcitriol levels (47 pg/mL vs. 41.5 pg/mL; p = 0.02 ) and calcidiol/calcitriol ratio scores (2.13 vs. 1.54 pg/ng; p < 0.02 ) compared to SLE patients without renal disease activity. Calcidiol levels were negatively correlated with calcitriol levels ( r = − 0.26 ; p = 0.001 ) and urine proteins (mg/dL) ( r = − 0.39 ; p < 0.01 ). Regarding calcitriol levels, it was positively correlated with the blood lymphocyte count ( r = 0.30 ; p < 0.001 ) and negatively correlated with the glomerular filtration rate ( r = − 0.28 ; p = 0.001 ). Moreover, the calcitriol/calcidiol ratio was positively correlated with urine proteins ( r = 0.38 ; p < 0.01 ). The calcidiol deficiency ( OR = 2.27 ; 95% CI = 1.15 -4.49; p < 0.01 ), high calcitriol levels ( T 3 rd , OR = 4.19 , 95% CI = 2.23 -7.90; p < 0.001 ), and a high calcitriol/calcidiol ratio score ( T 3 rd , OR = 5.93 , 95% CI: 3.08-11.5; p < 0.001 ) were associated with the risk for SLE. In conclusion, a pattern of calcidiol deficiency with high calcitriol serum levels and a high vitamin D hydroxylation efficiency ratio was associated with disease risk in SLE patients.

Correlation between Apelin and Some Angiogenic Factors in the Pathogenesis of Preeclampsia: Apelin-13 as Novel Drug for Treating Preeclampsia and Its Physiological Effects on Placenta

Preeclampsia (PE) is one of the commonest causes for maternal and fetal morbidity and mortality. Imbalances of angiogenic factors, oxidative stress, and inflammatory response have a role in the pathogenesis of PE. Data regarding the circulating apelin level and its role in PE remains controversial. This study was formulated to assess the serum apelin level in PE, investigate its correlation with some inflammatory, oxidative stress, and angiogenic proteins in a nitric oxide synthase inhibitor; the N (gamma)-nitro-L-arginine methyl ester (L-NAME)-induced rat model of PE and determine whether apelin administration could protect against development of PE. 40 healthy adult female albino rats and 10 adult male albino rats were used in this study. The pregnant female rats were randomly divided into three groups: group 1 (normal pregnant group), group 2 (PE-induced group), injected subcutaneously with 75 mg L-NAME/kg bodyweight/day starting from day 9 to 20 of gestation, and group 3 (PE-induced group supplemented with apelin (PE + apelin)); PE induced as before and simultaneously subcutaneously injected with apelin-13 (6 × 10−8 mol/kg bodyweight/twice daily) beginning from day 6 to 20 of gestation. In all groups, blood pressure and urine protein were determined at gestation days (GD) 0, 10, and 18. Moreover, serum apelin, placental growth factor (PLGF), vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), interferon-gamma (IFN-γ), and interleukin-10 (IL-10) levels and serum superoxide dismutase enzyme (SOD) and catalase (CAT) activities of all groups were estimated at the end of experiment. Placental histopathological examination was also performed. PE-induced rats showed significantly decreased serum apelin levels. Moreover, they showed significantly increased blood pressures, urine proteins, sFlt-1, sEng, and IFN-γ (mean arterial blood pressure, urine proteins, sFlt-1, sEng, and IFN-γ showed significant negative correlations with serum apelin level), but it showed significantly decreased VEGF, PLGF, IL-10, SOD, and CAT (VEGF, PLGF, IL-10, and SOD showed significant positive correlations with serum apelin level). In contrast, exogenous apelin administration significantly ameliorated these parameters together with improvement in the placental histoarchitecture in the apelin-supplemented PE group. This study demonstrated the protective effects of apelin administration on the pathogenesis of PE.

Approval of the method of electrophoretic separation and identification of some urine proteins in rats with toxic nephropathy

The aim of the article. The aim was to test the method for determining certain protein markers in rat urine by electrophoretic separation followed by mass spectrometric identification for early diagnostic of nephrotoxicity. The objectives of the study included assessing the reproducibility of the protein separation method, comparing rats urine protein profiles normal and after gentamicin sulfate administration, as well as a biochemical and morphological study of kidney biopsy specimens. Materials and methods. Gentamicin sulfate (GS) was administered intramuscularly to rats of both sexes at a dose of 60 mg/kg b.w. for consequence 5 days. Daily urine was collected in the background, on the 3rd, 7th and 14th day from the start of the administration of GS in the metabolic cages. We measured the concentrations of total protein and creatinine in urine samples, the level of lipids in the homogenates of the kidneys. Electrophoretic separation of urine proteins was performed in the Mini-PROTEAN Tetra Vertical Electrophoresis. The gels were stained, and the stained zones were excised and enzymatic hydrolysis of the proteins was performed. Spectra of tryptic peptides were recorded on an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker, Germany). Proteins were identified in the Biotools program by accessing the Mascot database (matrixscience.com). Results. Assessment of reproducibility of the results of electrophoretic separation of rat urine proteins showed an acceptable value of the coefficient of variation (on average 8.03%) and the error of analysis (analysis = 14.53%). It that normally in rats of males the alpha-2-uroglobulin precursor dominates in urine and in females immunoglobulin G light chains dominate was show. Against the background of the administration of GS, leukocytes in pathological concentrations appear in the urine in rats of both sexes; in males, the proteomic profile has a strong intraspecific dispersion, including due to sperm contamination; in females, well identified zones of albumin, alpha-1-antitrypsin, aminopeptidase M, beta-2-microglobulin, and transferrin was appear. A biochemical study of kidney homogenates an increase in total cholesterol (males) and triacylglycerides (females) revealed. Pathomorphological changes were similar in male and female rats in the form of fatty degeneration of the proximal tubule nephrothelium and leukocyte infiltration of interstitium and confirmed changes in the spectrum of urine proteins. Conclusion. Based on the analysis of the experimental results it was found that the use of the technology of electrophoretic separation of proteins in PAGE under denaturing conditions followed by MALDI mass spectrometric identification (PAGE-MALDI-TOF/TOF) and densitometric determination of the percentage of protein fractions is applicable for the detection of nephrotoxicity. Due to gender differences in the protein spectrum it is preferable to examine the urine of female rats. Pathomorphological changes did not have gender differences.

The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.