Aleksandra Wyczalkowska-Tomasik

Comparison of Two Methods for Determination of NGAL Levels in Urine: ELISA and CMIA

Neutrophil gelatinase‐associated lipocalin (NGAL) is a new useful biomarker for the early diagnosis of acute kidney injury. The aim of the study was to compare two analytical methods for measurement of urinary NGAL: enzyme‐linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA).

Cathepsin B and L activity in the serum during the human aging process: Cathepsin B and L in aging

Cathepsin B and L are active in several physiological processes that occur in the human body. Their synthesis and activity is detectable in several tissues and processes, including skin hyperkeratosis, insemination, and embryonic development. Proteolytic enzymes are very important cellular constituents and have been the subject of much research. The enzymes are in the limelight of scientists first of all as ECM degradation factors. Most publications describe proteolytic enzymes as tumor and metastasis factors. These proteases also have a central function as apoptosis factors. The aim of this study was to evaluate correlations between age and the enzymatic activities of the proteolytic enzymes cathepsin B and L together. This research was conducted using blood samples provided by healthy people (n=60, 30 men and 30 women). Subjects were subdivided into groups with respect to age: young (about 20 years old, n=20), middle age (about 50 years old, n=20), and elderly (about 80 years old, n=20). Fluorometric assay was used to determine proteolytic enzyme activity. Cathepsin B and L activity was measured as a total activity. The activity of cathepsin B and L significantly increases with age.

Age-dependent increase in serum levels of indoxyl sulphate and p-cresol sulphate is not related to their precursors: Tryptophan and tyrosine.

Retention of indoxyl sulphate and p‐cresol sulphate is associated with many diseases. The aim of the present study was to examine serum levels of indoxyl sulphate and p‐cresol sulphate, the dynamics of their changes according to age, and their precursors.

Repeated restraint stress produces acute and chronic changes in hemodynamic parameters in rats

Abstract Noninvasive hemodynamic measurements in rats require placing animals in restrainers. To minimize restraint stress-induced artifacts several habituation protocols have been proposed, however, the results are inconclusive. Here, we evaluated if a four-week habituation is superior to a shorter habituation, or no habituation. This is the first study comparing different habituation protocols with the use of four-week continuous telemetry measurements. We did the experiments on male, 16-week old, Sprague–Dawley rats. Continuous recordings of mean arterial blood pressure (MABP) and heart rate (HR) were made before and during habituation protocols. Rats were subjected either to control (four weeks of restraint-free recordings, n = 5) or two-week (seven restraints, n = 6) or four-week (14 restraints, n = 6) restraint sessions. The restraint protocols included placement of rats in the middle of the dark phase into plastic restrainers as used for tail-cuff measurements. Restraint lasted for 60 min, and was repeated every second day. Each restraint significantly increased MABP (by 15–25 mmHg) and HR (by 40–120 beats/min). Exposure to the restraint protocols decreased diurnal variation in MABP. There was no hemodynamic adaptation to repeated restraint, and no significant difference in hemodynamic response to restraint among controls, the two-week and the four-week groups. In conclusion, our study indicates that measurements in restrained rats are not likely being made without stress-induced changes in MABP. Moreover, in hemodynamic studies in repeatedly restrained rats longer habituation is not superior to shorter habituation.

Training differentially regulates elastin level and proteolysis in skeletal and heart muscles and aorta in healthy rats

ABSTRACT Exercise induces changes in muscle fibers and the extracellular matrix that may depend on elastin content and the activity of proteolytic enzymes. We investigated the influence of endurance training on the gene expression and protein content and/or activity of elastin, elastase, cathepsin K, and plasmin in skeletal and heart muscles and in the aorta. Healthy rats were randomly divided into untrained (n=10) and trained (n=10; 6 weeks of endurance training with increasing load) groups. Gene expression was evaluated via qRT-PCR. Elastin content was measured via enzyme-linked immunosorbent assay and enzyme activity was measured fluorometrically. Elastin content was significantly higher in skeletal (P=0.0014) and heart muscle (P=0.000022) from trained rats versus untrained rats, but not in the aorta. Although mRNA levels in skeletal muscle did not differ between groups, the activities of elastase (P=0.0434), cathepsin K (P=0.0343) and plasmin (P=0.000046) were higher in trained rats. The levels of cathepsin K (P=0.0288) and plasminogen (P=0.0005) mRNA were higher in heart muscle from trained rats, but enzyme activity was not. Enzyme activity in the aorta did not differ between groups. Increased elastin content in muscles may result in better adaption to exercise, as may remodeling of the extracellular matrix in skeletal muscle. Summary: Endurance training increases elastin content in muscles but not in the aorta. The activities of enzymes responsible for ECM remodeling increase only in skeletal muscle. These changes seem to be adaptive.

Serum cystatin C and serum and urine NGAL in the kidney function assessment of patients with MGUS

Monoclonal gammopathy of undetermined significance (MGUS) occurs without other symptoms, although monoclonal proteins can cause kidney injuries. Here, we assessed kidney function and identified the best follow‐up parameters in patients with MGUS without kidney damage symptoms.

The Blocking on the Cathepsin B and Fibronectin Accumulation in Kidney Glomeruli of Diabetic Rats

Hyperglycemia results in the activation of tissue angiotensin II. Angiotensin II stimulates the synthesis of ECM proteins and causes a decrease activity of proteolytic enzymes. The aim of this study was to assess the impact of multilevel blocking of the RAAS, cathepsin B activity, and fibronectin accumulation in the glomerular in the rats diabetes model. Sixty male Wistar rats were initially included. Diabetes was induced by intravenous administration of streptozotocin. The animals were randomized to six groups of ten rats in group. Rats in the four groups were treated with inhibitors of the RAAS: enalapril (EN), losartan (LOS), enalapril plus losartan (EN + LOS), and spironolactone (SPIR); another group received dihydralazine (DIH) and the diabetic rats (DM) did not receive any drug. After six weeks, we evaluated blood pressure, 24 h urine collection, and blood for biochemical parameters and kidneys. In this study, fluorometric, ELISA, and immunohistochemical methods were used. Administration of EN + LOS increased activity of cathepsin B in homogenates of glomeruli compared to DM. Losartan treatment resulted in reduction of the ratio kidney weight/body weight compared to untreated diabetic rats. SPIR resulted in the increase activity of cathepsin B in the homogenate of glomeruli. The values of cathepsin B in the plasma of rats in all studied groups were similar and showed no tendency.

The role of the kidney in the systemic elimination of interleukin 6, platelet-derived growth factor and transforming growth factor beta

STUDY GOAL The aim of the study was to assess the role of the kidney in systemic elimination of IL-6 and growth factors (PDGF, TGF-β) by comparison of their concentrations in renal arteries and veins, peripheral veins and urine. MATERIAL AND METHODS 30 brain-dead kidney donors were included in the study. Samples were obtained during the harvesting procedure. 10 healthy volunteers served as controls. A mathematical model of elimination of investigated proteins from systemic circulation was developed. The elimination ratio (ER) formula indicates the predominance of renal synthesis or degradation and also quantifies the renal uptake (UR) and renal pass (PR) of investigated proteins. Serum levels of investigated proteins were estimated using an immunoenzymatic method (ELISA). RESULTS Renal IL-6 uptake ratio (UR) amounted to 6.6%, elimination ratio (ER) amounted to 6.4% and pass ratio (PR) amounted to 0.2%. PDGF ratios amounted to 5.1%, 5.0% and 0.1% and TGF-β ratios amounted to -9%, -9% and 0%, respectively. CONCLUSIONS The kidney takes part in the elimination of IL-6 and PDGF from systemic circulation. The kidney does not take part in the elimination of TGF-β.

Radiotherapy and radiochemotherapy increase serum levels of pro-inflammatory interleukin-6 and C-reactive protein in patients with head and neck cancers

Background: Inflammation influences cancer growth and treatment susceptibility. We investigated the influence of radiochemotherapy and radiotherapy on serum interleukine-6 (IL-6), C-reactive protein (CRP), and transforming growth factor-β (TGF-β) levels in patients with head and neck cancers. Methods: Of 26 patients enrolled, 8 received radiotherapy (SIB-IMRT, 2.25 Gy/fraction, 30 fractions) and 18 received radiochemotherapy [cisplatin 100 mg/m 2 and SIB–IMRT (n=9) or IMRT (2 Gy/fraction, 35 fractions, n=9)]. The control group consisted of healthy individuals. IL-6 and TGF-β concentrations were determined by ELISA and CRP by an immunoturbidimetric method before treatment, 2 and 4 weeks after treatment started, at the end of treatment, and 4 weeks post-treatment. Results: IL-6 levels were higher in the treatment group compared to controls and increased at all-time points compared to pre-treatment levels (P Conclusions: Serum levels of proinflammatory markers, IL-6 and CRP are increased after radiotherapy and radiochemotherapy in patients with head and neck cancers. The adverse effects of IL-6 and CRP could by suppressed by concomitantly decreased TGF-β levels.

Increased TNF-α and TGF-β concentrations in rat liver after intense exercise

Mater ia l and methods : Here we investigated the impact of intense exercise on liver gene expression and concentrations of TNF-α and TGF-β in untrained and trained healthy rats (n = 30 each). The trained rats underwent 6 weeks of endurance training with increasing load. From each group, liver samples were collected before, immediately after, and 3 h after one session of intense exercise. Gene expression was evaluated with quantitative real-time polymerase chain reaction. Protein content was measured by enzyme immunoassay.