SDS Electrophoresis on Gradient Polyacrylamide Gels as a Semiquantitative Tool for the Evaluation of Proteinuria.

Proteinuria is an important sign of kidney diseases. Different protein patterns in urine associated with glomerular, tubular and overload proteinuria may be differentiated using the immunochemical detection of indicator proteins or via urinary proteins electrophoresis. Our aim was to characterize sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using commercially available 4-20% gradient gels as a method to detect and differentiate proteinuria. Our laboratory-based study used excess urine samples collected for routine diagnostic purposes from adult patients of a tertiary-care hospital, including patients with albumin/creatinine < 30 mg/g and patients with dipstick proteinuria. The limit of albumin detection was estimated to be 3 mg/L. In 93 samples with albumin/creatinine < 30 mg/g, an albumin fraction was detected in 87% of samples with a minimum albumin concentration of 2.11 mg/L. The separation of 300 urine samples of patients with proteinuria revealed distinct protein patterns differentiated using the molecular weights of the detected proteins: glomerular (albumin and higher molecular weights) and two types of tubular proteinuria ("upper" ≥20 kDa and "lower" with lower molecular weights). These patterns were associated with different values of the glomerular filtration rate (median 66, 71 and 31 mL/min/1.72 m2, respectively, p = 0.004) and different proportions of multiple myeloma and nephrological diagnoses. As confirmed using tandem mass spectrometry and western blot, the SDS-PAGE protein fractions contained indicator proteins including immunoglobulin G, transferrin (glomerular proteinuria), α1-microglobulin, retinol-binding protein, neutrophil gelatinase-associated lipocalin, cystatin C, and ß2-microglobulin (tubular), immunoglobulin light chain, myoglobin, and lysozyme (overflow). SDS-PAGE separation of urine proteins on commercially available 4-20% gradient gels is a reliable technique to diagnose proteinuria and differentiate between its main clinically relevant types.

Acute Phase Proteins and Vitamin D Seasonal Variation in End-Stage Renal Disease Patients.

End-stage renal disease (ESRD) patients are vulnerable to vitamin D deficiency due to impaired renal hydroxylation, low dietary intake and inadequate sun exposure. Vitamin D plays a role in innate and adaptive immunity and its seasonal variation has been linked to mortality. ESRD is associated with inadequate removal of pro-inflammatory cytokines regulating acute phase protein (APP) synthesis. Our aim was to look for associations between lifestyle factors, diet, and vitamin D seasonal variation and their relationship with selected APPs and calcium-phosphate metabolism. The study included 59 ESRD patients treated with maintenance hemodialysis. A 24-hour dietary recall was conducted in the post-summer (November 2018, PS) and post-winter (February/March 2019, PW) period, and blood was collected for the measurements of serum total vitamin D, α1-acid glycoprotein (AGP), C-reactive protein (CRP), albumin, prealbumin (PRE), parathormone, calcium and phosphate. A self-constructed questionnaire gathered information on vitamin D supplementation, sun exposure and physical activity. Higher caloric intake was observed PW compared PS. Less than 15% of participants met the dietary recommendations for energy, protein, fiber, vitamin D and magnesium intake. Vitamin D supplementation was associated with higher serum vitamin D regardless of season. AGP, PRE, albumin, and vitamin D presented seasonal changes (higher values PS). In patients with serum vitamin D below 25 ng/mL, vitamin D seasonal change correlated with CRP and prealbumin change. Phosphate and Ca × P correlated positively with AGP. A low vitamin D serum level could impact the inflammatory process; however, more studies are needed to confirm the relationship.

Does Beta-Trace Protein (BTP) Outperform Cystatin C as a Diagnostic Marker of Acute Kidney Injury Complicating the Early Phase of Acute Pancreatitis?

Acute pancreatitis (AP) belongs to the commonest acute gastrointestinal conditions requiring hospitalization. Acute kidney injury (AKI) often complicates moderately severe and severe AP, leading to increased mortality. Among the laboratory markers proposed for early diagnosis of AKI, few have been studied in AP, including cystatin C and neutrophil gelatinase-associated lipocalin (NGAL). Beta-trace protein (BTP), a low-molecular-weight glycoprotein proposed as an early marker of decreased glomerular filtration, has never been studied in AP. We investigated the diagnostic usefulness of serum BTP for early diagnosis of AKI complicating AP in comparison to previously studied markers. BTP was measured in serum samples collected over the first three days of hospital stay from 73 adult patients admitted within 24 h of mild to severe AP. Thirteen patients (18%) developed AKI in the early phase of AP. Serum BTP was higher in patients who developed AKI, starting from the first day of hospitalization. Strong correlations were observed between BTP and serum cystatin C but not serum or urine NGAL. On admission, BTP positively correlated with endothelial dysfunction. The diagnostic usefulness of BTP for AKI was similar to cystatin C and lower than NGAL. Increased BTP is an early predictor of AKI complicating AP. However, it does not outperform cystatin C or NGAL.

Redox Balance Correlates with Nutritional Status among Patients with End-Stage Renal Disease Treated with Maintenance Hemodialysis.

Over 50% of end-stage renal disease (ESRD) patients die of cardiovascular disease. ESRD patients treated with maintenance hemodialysis are repeatedly exposed to oxidative stress. The aim of the study was to find the relationship between lifestyle factors, nutritional status, calcium-phosphate metabolism, and selected redox parameters such as glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), uric acid (UA), and total antioxidant capacity expressed as ferric reducing antioxidant power (FRAP). The study included 97 ESRD hemodialysis patients and 42 controls with no renal disease. Patients were asked to complete a questionnaire which gathered information on their physical activity, hours of sleep, smoking, and frequency of fruit and vegetable intake; the blood samples were then drawn before the midweek dialysis session. The ESRD patients had lower levels of GR, GPx, and SOD activity, a lower level of FRAP, and a higher UA concentration than the control group. The FRAP value decreased with age (ρ = -0.32, p = 0.001); smokers had a significantly lower SOD activity in comparison to nonsmokers (p = 0.03). In the ESRD patients, FRAP and UA correlated with both albumin (ρ = 0.26, p = 0.011; ρ = 0.41, p = 0.006, respectively) and prealbumin (ρ = 0.34, p ≤ 0.001; ρ = 0.28, p = 0.006, respectively), whereas UA, GR, GPx, and SOD correlated with calcium, UA, GR, and GPx with phosphate level. Based on the findings, there are weak associations between nutritional status and selected redox parameters in hemodialyzed patients. Further studies are needed to establish if diet modifications and adequate nutritional status can positively impact the antioxidant capacity in this group of patients.

Markers of Glomerular and Tubular Damage in the Early Stage of Kidney Disease in Type 2 Diabetic Patients.

Diabetic kidney disease develops in half of genetically predisposed patients with type 2 diabetes (T2DM). Early diagnosis of kidney damage and nephroprotective treatment are the ways of preventing the disease progression. Our aim was to evaluate selected laboratory markers of glomerular and tubular damage in T2DM patients with early stages of chronic kidney disease (G1/G2, A1/A2) for their associations with A2 albuminuria and early decline in the estimated glomerular filtration rate (eGFR). Among 80 T2DM patients with median eGFR of 92.4 ml/min/1.73 m2 and median urinary albumin to creatinine ratio (uACR) of 4.69 mg/g, 19 had uACR > 30 mg/g (A2). Higher serum cystatin C, serum and urine neutrophil gelatinase associated lipocalin (NGAL), urine kidney injury molecule 1 (KIM-1), detectable urine transferrin and IgG, and lower serum uromodulin significantly predicted A2 albuminuria, urine KIM-1/creatinine ratio, and IgG being the best predictors. Albuminuria, urine NGAL/creatinine, and IgG correlated with diabetes duration. Albuminuria, urine NGAL, transferrin, IgG, and uromodulin correlated with diabetes control. In a subgroup of 29 patients, retrospective data were available on changes in eGFR and uACR over one year. Decline in eGFR was observed in 17 patients and increase in uACR in 10 patients. Serum and urine NGAL correlated with eGFR changes. Higher urine NGAL, KIM-1/creatinine ratio, and detectable IgG were significantly associated with the increase in uACR. Widely available markers, serum cystatin C, urine IgG, transferrin, and NGAL, may help in early assessment of kidney disease in T2DM patients; however, large prospective studies are needed to confirm the conclusion.

Malnutrition, Inflammation, Atherosclerosis Syndrome (MIA) and Diet Recommendations among End-Stage Renal Disease Patients Treated with Maintenance Hemodialysis.

Malnutrition-inflammation-atherosclerosis syndrome is one of the causes of increased mortality in chronic kidney disease (CKD). The aim of the study was to assess the inflammation and nutritional status of patients in end-stage kidney disease treated with maintenance hemodialysis. The study included a group of 98 hemodialyzed patients with stage 5 CKD (38 women and 60 men). Albumin, prealbumin (PRE), and C-reactive protein (CRP) were measured in serum samples collected before mid-week dialysis. Fruit and vegetables frequency intakes were assessed with a questionnaire. CRP was above the reference limit of 5 mg/L in 53% of patients. Moreover, the Glasgow Prognostic Score (GPS) indicated the co-occurrence of inflammation and protein calorie malnutrition in 11% of patients, and the presence of either inflammation or malnutrition in 25%. The questionnaire revealed that hemodialyzed patients frequently exclude fruit and vegetables from their diets. Nearly 43% of the interviewed patients declared frequently eating vegetables, and 35% declared frequently eating fruit, a few times per week or less. The most frequently selected fruit and vegetables had a low antioxidant capacity. The strict dietary restrictions in CKD are difficult to fulfill, and if strictly followed, may lead to protein-calorie malnutrition.

Serum pregnancy-associated plasma protein A correlates with inflammation and malnutrition in patients treated with maintenance hemodialysis.

Advanced chronic kidney disease (CKD) leads to complications such as anemia, electrolyte abnormalities, bone and mineral disorder, and malnutrition-inflammation-atherosclerosis (MIA) syndrome, that result in high cardiovascu- lar mortality. One of the biomarkers associated with inflammation and cardiovascular events is pregnancy-associated plasma protein A (PAPP-A). The aim of the study was to measure serum PAPP-A in hemodialyzed CKD patients, and to investigate its correlations with the laboratory markers of the complications. We enrolled 78 consecutive stable adult CKD patients treated with maintenance hemodialysis for median period of 60 months. PAPP-A concentrations were measured with by electrochemiluminescence immunoassay. Average serum PAPP-A in hemodialyzed patients was almost two times higher than the upper reference limit. It positively correlated with N-terminal pro-brain natriuretic peptide (NT-proBNP), serum sodium, and the markers of inflammation and malnutrition. In conclusion, serum PAPP-A seems a useful biomarker associated with cardiovascular dysfunction, inflammatory state and malnutrition in hemodialysis patients.

Dynamics of reactive oxygen species generation in the presence of copper(II)-histidine complex and cysteine.

Histidine-copper(II) complex (Cu-His2) is a form of bound copper necessary for cellular copper uptake. Due to the high affinity of histidine to copper(II) ions, the binding of copper(II) by histidine is considered a substantial part of plasma antioxidative defense. Also cysteine plays a role in the antioxidative system. However, we show here that in the presence of oxygen the histidine-copper(II) complex plus cysteine produces reactive oxygen species (ROS). Cysteine concentration was assayed using a thiol specific silver-mercury electrode. Hydrogen peroxide was assayed amperometrically using platinum electrode. ROS formation was followed by chemiluminescence of luminol-fluoresceine-enhanced system. Addition of cysteine to Cu-His2 solution at pH 7.4 in the presence of atmospheric oxygen initiates the synthesis of H2O2 and generation of ROS, which manifests as a burst of chemiluminescence. The reaction has two stages; in the first stage, cysteine is utilized for the synthesis of an unstable intermediary product which becomes a substrate for ROS formation. Anaerobic conditions inhibit ROS formation. Increased cysteine concentration enhances the lag phase of the oxidative burst without influencing the amount of ROS. The synthesis of ROS (measured by chemiluminescence) is proportional to the concentration of Cu-His2 employed. ROS production can be repetitively initiated by further additions of cysteine to the reaction medium. The study suggests that Cu-His2 catalyzes cysteine-dependent reduction of oxygen to superoxide employing an intermediary cysteine-copper(I) complex and enabling Fenton reaction with copper and hydrogen peroxide produced as a secondary product. In effect, Cu-His2 with cysteine may be a source of ROS in biological media.

Comparison of high-sensitivity C-reactive protein serum assay results obtained using Dade-Behring BNII nephelometer and Ortho Vitros FS 5.1 clinical analyzer in respect of CRP-related risk assessment of chronic metabolic diseases.

BACKGROUND: Serum concentration of high sensitive C-reactive protein (hsCRP) can predict the risk of chronic metabolic and cardiovascular diseases but it is unclear whether turbidimetric high sensitive assays of CRP are adequate. METHODS: Concentrations of serum CRP in 126 samples of serum were measured with high-sensitivity methods using nephelometry (BN II Nephelometer) and turbidimetry (Ortho Vitros FS 5.1). RESULTS: For CRP concentrations measured by nephelometry and turbidimetry intra-assay CVs were 3.2 and 0.9% at mean CRP concentrations of 1.4 and 2.1 mg/l, inter-assay CVs for commercial controls were 3.1% and 3.6% at mean concentrations of 1.3 and 1.7 mg/l, and mean biases were 7.62% and 2.26%, respectively. Measurements were strongly, linearly correlated (r = 0.99; CRP vitros = 0.03 +1.03 CRP (BN II)). When disease risk was assessed by nephelometry and turbidimetry, results were similar. If the risk of disease was classified as moderate (1.0 < CRP < or = 3.0 mg/l) or high (CRP > 3.0 mg/l), the frequency of misclassified cases was only 2.3 and 2.1%, respectively. The classification agreement weighted kappa coefficient was 0.94 (95% C.I.: 0.89-0.98). CONCLUSIONS: turbidimetric high sensitive CRP assays can properly classify CRP-related prediction of chronic metabolic diseases with special consideration on cardiovascular risk.

Potentiometric determination of cysteine with thiol sensitive silver-mercury electrode.

A potentiometric procedure for cysteine thiol group concentration monitoring in media generating free radicals was developed using a thiol specific silver-mercury electrode. Electrolytic deposition of mercury on a silver wire and treatment with 20 mM cysteine in 0.5 M NaOH were used to produce the electrode. A silver-chloride electrode in saturated KCl was the reference. A glass capillary with 1 M KNO3 in 1% agarose gel was the liquid junction. The electrode responded to cysteine concentration in the range from 0.01 to 20 mM yielding a perfect linear relationship for the dependence of log [cysteine] versus electrode potential [mV], with b0 (constant) = -373.43 [mV], b1 (slope) = -53.82 and correlation coefficient r2 = 0.97. The electrode potential change per decade of cysteine concentration was 57 mV. The minimal measurable signal response was at a cysteine concentration of 0.01 mM. The signal CV amounted to 4-6% for cysteine concentrations of 0.01 to 0.05 mM and to less than 1% for cysteine concentrations of 0.5 to 20 mM. The response time ranged from about 100 s for cysteine concentrations of 0.01 to 0.1 mM to 30 s at higher cysteine concentrations. The standard curve reproducibility was the best at cysteine concentrations from 0.1 to 20 mM. In a reaction medium containing cysteine and copper(II)-histidine complex ([His-Cu]2+) solution in 55 mM phosphate buffer pH 7.4 the electrode adequately responded to changes in cysteine concentration. Beside cysteine, the silver-mercury electrode responded also to thiol groups of homocysteine and glutathione, however, the Nernst equation slope was about half of that for cysteine.