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.

Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay.

Therapeutic drug monitoring of immunosuppressive agents is a critical and essential part of patient therapy after organ transplantation. We have developed high-throughput, robust, and rapid liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) methods with common pretreatment procedures for simultaneous quantification of four immunosuppressive agents (everolimus, sirolimus, tacrolimus, and cyclosporin A) in whole blood and one immunosuppressant (mycophenolic acid) in plasma. The new approach used in this work is based on improved sample preparation procedures allowing the analysis of five immunosuppressive drugs. Whole blood was prepared by transferring 100µL of blood into a 1.5-mL silanized conical test tube. Zinc sulfate solution (150µL), containing deuterated internal standards, was added to perform hemolysis. The samples were vortexing for 10s, followed by the addition of 250µL acetonitrile, containing internal standard for cyclosporin A, to precipitate proteins. The mixture was vortexed for 1min and centrifuged for 2min at 14,000rpm. The whole supernatant was transferred to a vial. To prepare blood plasma, the hemolysis step involving the addition of zinc sulfate was omitted and, instead of acetonitrile, methanol was used as the solvent for the internal standard (mycophenolic acid-d3). The volumes of chemicals used in this procedure were the same as those used in the procedure for immunosuppressants in whole blood. The basic validation parameters for the analytical methods were limits of detection (0.5ng/mL for everolimus, sirolimus and tacrolimus, 25ng/mL for cyclosporin A and 100ng/mL for mycophenolic acid), precision (<15%), recovery (>84%), repeatability and reproducibility. Possible mutual ion suppression was eliminated in the presence of internal standards. The method developed for the quantitation of immunosuppressants in whole blood was used to analyze 276 patient samples containing tacrolimus and 55 samples containing cyclosporin A. The results from LC/MS/MS were compared to those obtained from immunoassays of the same samples. Immunoassays significantly overestimated the concentrations of immunosuppressants.

Decreased hypoxia-inducible factor-1α in gastrocnemius muscle in rats with chronic kidney disease.

BACKGROUND/AIMS: Hypoxia-inducible factor (HIF)-1α is responsible for increased expression of genes engaged in angiogenesis. Our previous study indicated capillary rarefaction and atrophy of glycolytic fibers, mainly in locomotor muscles of uremic animals. Perhaps these changes are secondary to disturbances of HIF-1α in skeletal muscles. METHODS: Expression of HIF-1α at mRNA and protein levels, as well as mRNA of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), in gastrocnemius muscle (MG) and longissimus thoracic muscle (ML) were measured by RT-PCR and Western blot. Rats were randomized to subtotal nephrectomy (CKD5/6), uninephrectomy (CKD1/2) or sham operation (controls). RESULTS: For CKD5/6 versus controls, mRNA levels for HIF-1α, VEGF-A, VEGFR-1 and VEGFR-2 were significantly reduced only in MG, while eNOS was significantly decreased and iNOS was significantly increased only in ML. Western blot analysis indicated significantly increased HIF-1α protein levels in MG and ML from CKD1/2 animals versus controls, whereas in the CKD5/6 group, the level of HIF-1α protein decreased significantly in MG and increased significantly in ML versus controls and CKD1/2. CONCLUSION: The reduced expression of HIF-1α mRNA and protein in locomotor muscle from CKD5/6 animals may be involved in the pathogenesis of uremic myopathy. Increased expression of iNOS in the postural muscles may act as a protective factor through HIF-1α stabilization.

The influence of physical exercise on the generation of TGF-ß1, PDGF-AA, and VEGF-A in adipose tissue.

Adipose tissue is an important organ that produces and secretes hormones and cytokines, including TGF-ß1, PDGF-AA, and VEGF-A. The goal of the present study was to investigate the influence of a single session of acute exercise, as well as the prolonged endurance training on the production of TGF-ß1, PDGF-AA, and VEGF-A in the subcutaneous white adipose tissue in rats. Rats were randomly divided into two groups: untrained (UT, n = 30) and trained rats (T, subjected to 6-week endurance training with increasing load, n = 29). Both groups were subjected to an acute exercise session with the same work load. The rats were killed before (UTpre, Tpre), immediately after (UT0h, T0h), or 3 h (UT3h, T3h) after exercise and adipose tissue samples collected. Growth factor mRNA was evaluated using RT-PCR; the protein levels were measured before and after training (UTpre and Tpre) using the immunoenzymatic method. TGF-ß1 and PDGF-AA mRNA levels were decreased in the UT3h rats compared to the UTpre rats (P = 0.0001 and P = 0.03, respectively), but the VEGF-A mRNA level remained unchanged in the UT0h and UT3h rats compared to UTpre rats. TGF-ß1, PDGF-AA and VEGF-A mRNA levels were decreased in the T3h rats compared to Tpre (P = 0.0002, P = 0.02, and P = 0.03, respectively). TGF-ß1, PDGF-AA and VEGF-A mRNA levels significantly increased in the Tpre rats compared to UTpre (all P = 0.0002). However, the protein levels remained constant. In conclusion, prolonged physical exercise increases growth factor mRNA in adipose tissue but not protein levels.

Skeletal and heart muscle expression of PDGF-AA and VEGF-A after an acute bout of exercise and endurance training in rats.

BACKGROUND: Physical exercise benefits the heart, but the mechanism is not understood. The mechanism of muscle plasticity after physical training is also unclear. The proangiogenic and mitogenic activity of PDGF and VEGF could be involved in both processes. This study investigated the influence of a single bout of acute exercise on the PDGF-AA and VEGF-A expression in skeletal and heart muscle in untrained rats and in those subjected to prolonged endurance training (trained). MATERIAL/METHODS: Fifty-nine rats were randomly assigned to the untrained (UT) or trained (T, subjected to 6-week endurance training) groups. The groups were subjected to an acute bout of exercise with the same work load a day after final training. Rats of both groups were sacrificed and skeletal and heart muscle samples were collected before, immediately after (0 h), or 3 hours (3 h) after acute exercise. RESULTS: Prolonged training caused an increase in PDGF-AA mRNA (p=0.038) and protein (p=0.016) in rat skeletal muscle (T vs. UT). A similar pattern was observed in VEGF-A, but the changes were not significant. Acute exercise caused a significant decrease in PDGF-AA mRNA after 0 and 3 h (p=0.03 and p=0.04, respectively). In the heart there were no significant changes in either mRNA after acute exercise or after endurance training. CONCLUSIONS: Six-week endurance training in rats increases PDGF-AA mRNA and protein in skeletal, but not in heart muscle.

Proteolytic enzyme activity as a result of aging.

BACKGROUND AND AIMS: The extracellular matrix (ECM) undergoes constant dynamic changes; proteolytic enzymes, particularly the serine proteases plasmin, trypsin and elastase, catalyze critical functions in these processes. Notably, ECM degradation disorders have been reported in various morbid conditions, including cardiac infarction, atheromatosis, and neoplastic diseases, indicating a physiological requirement for proper ECM maintenance. Here we define the role of proteolytic enzymes in the development of aging by assessing changes in proteolytic enzyme activity in serum during aging in rats. METHODS: The activities of trypsin, elastase and plasmin in rat serum were determined by the fluorometric method using AMC-labeled substrates in 34Wistar rats divided into four age groups: 3 month-olds (n=8), 9 month-olds (n=8), 15 month-olds (n=8) and 24 month-olds (n=10). RESULTS: Analysis of proteolytic enzyme activity in four age-dependent groups revealed that in comparison to their 3, 9, and 24 month-old counterparts, the 15 month-old rats exhibited a statistically significant increase in average elastase activity. In accordance with previous studies, a statistically significant increase in trypsin levels was found in the 3 month-old rats, suggesting that trypsin activity decreases with age. Average plasma plasmin activity in the 24 month-old rats was, moreover, statistically significantly higher than that in the other three age groups. CONCLUSIONS: Analysis of combined proteolytic activity indicates that age-dependent patterning of blood serine protease enzyme activity may be related to age-related diseases.

Trypsin, elastase, plasmin and MMP-9 activity in the serum during the human ageing process.

OBJECTIVE: the aim of this work was to define the influence of the ageing process on the activity of proteolytic enzymes, such as trypsin, elastase, plasmin and active MMP-9 concentration, as well as the inhibitor alpha 1-antitrypsin. Moreover, we assessed associations between enzyme activity and selected clinical and biochemical parameters. METHODS: healthy normotensive volunteers (n = 60, 30 women) aged 20-82 years were split into subgroups: young (aged 20-22), middle-aged (49-52) and elderly (77-82). Serum enzyme activity was assessed using fluorometric methods. RESULTS: overall, active MMP-9 concentration and trypsin activity decreased with age, and alpha1-antitrypsin concentration and plasmin activity increased. Activity of elastase increased with age when compared to the young age group. An inverse correlation was identified between MMP-9 concentration and BMI and a direct correlation found between BMI and elastase, plasmin activity and alpha1-antitrypsin concentration. In the middle-aged group, glucose correlated directly with trypsin activity and inversely with MMP-9 concentration. Trypsin activity and MMP-9 concentration correlated inversely with cholesterol concentration and plasmin and elastase activity, and the alpha1-antitrypsin concentration correlated with cholesterol concentration in the overall group. CONCLUSIONS: the results confirm the influence of the ageing process on the activity of serum proteolytic enzymes. The activity of individual proteolytic enzymes in the serum changes with age.

Urine cytokines profile in renal transplant patients with asymptomatic bacteriuria.

BACKGROUND: The role of asymptomatic bacteriuria in kidney transplant recipients is unknown. There is no clear evidence of its effect on transplanted kidney. METHODS: We studied urine cytokines profile among kidney transplant recipients with bacteriuria found in screening examination. Urine cultures were collected in 269 patients with stable graft function and serum creatinine level <2 mg/dl, during their routine visits. Interleukin (IL)-6 and IL-8 levels were measured in urine samples from patients with asymptomatic bacteriuria, symptomatic urinary tract infection and patients without bacteriuria (control group). Changes in serum creatinine level in patients with asymptomatic bacteriuria and in the control group were observed during 12 months follow up. RESULTS: Urinary tract infection (UTI) was diagnosed in five patients and asymptomatic bacteriuria in 22 patients. Urine IL-6 level was significantly higher in symptomatic UTI group (median 15.71 pg/mg) but there were no differences between group of patients with asymptomatic bacteriuria (3.92 pg/mg) and control group (2.54 pg/mg). Urine IL-8 level was higher in symptomatic UTI group (median 146.8 pg/mg) and was also significantly higher in asymptomatic bacteriuria group (33.49 pg/mg) in comparison to control group (2.97 pg/mg; P=0.0002). During 1-year follow up, incidence of UTI was higher in the asymptomatic bacteriuria group than in the control group but graft function was not different in both groups. CONCLUSIONS: Elevated urine IL-8 level in kidney transplant patients with asymptomatic bacteriuria may reflect impaired immune response to bacterial infection and occult inflammatory process in urinary tract.

[Correlation between the content of transforming growth factor-beta 1, interleukin-1 beta and collagenase activity in proximal femur bone samples obtained from patients with femoral neck fracture]. / Wzajemne relacje pomiedzy zawartoscia transformujacego czynnika wzrostu-beta 1, interleukiny-1 beta i aktywnoscia kolagenazy w bioptatach kostnych blizszego konca kosci udowej u chorych po zlamaniu szyjki kosci udowej.

The aim of this study was to determine correlations between the content of TGF-beta1, IL-1beta and collagenase activity in bone samples of the femoral neck obtained from patients with femoral neck fracture. The material consisted of 42 samples of cancellous bone of femoral neck collected during hemiarthroplasty or total hip replacement procedure. 24 samples of cancellous bone from patients with osteoarthritis of the hip harvested during total hip replacement served as a control group. The content of TGF-beta1, IL-1beta was measured using ELISA and collagenase activity was measured with spectrofluorometry. In patients with fracture there was found a distinct inversely proportional correlation between the content of TGF-beta1 and IL-1beta. This correlation was not observed in the control group. In addition, a directly proportional relation between the content of TGF-beta1 and IL-1beta was discovered. These results confirm mutual correlations between examined cytokines in bone.

Lack of relationship between interleukin-6 and CRP levels in healthy male athletes.

Interleukin-6 (IL-6) is the main cytokine involved in the induction of acute phase response, which includes synthesis of certain proteins in the liver, one of which is C-reactive protein (CRP). The aim of this study was to assess the impact of IL-6 released during physical exercise on CRP generation in healthy male athletes. Fourteen young cyclists were enrolled in the study, which involves the performance of strenuous physical exercise. Serum levels of IL-6 and CRP were measured at rest before exercise, and immediately after and 2h after cessation of exercise. IL-6 level was increased 2.42-fold immediately after, and 21.67-fold 2h after exercise. Serum CRP level did not change significantly over the course of observation: it was 3.25 mg/dl before, 2.36 mg/dl immediately after and 2.71 mg/dl 2h after exercise and unrelated to IL-6 level. No correlation between serum levels of IL-6 and CRP was observed during the period of observation. We conclude that under certain circumstances, acute, pulsatile release of IL-6 does not stimulate synthesis of CRP.

Urinary levels of interleukin-6 and interleukin-8 in children with urinary tract infections to age 2.

BACKGROUND: The aim of this study was to assess relations between the clinical course of UTI, IL-6 and IL-8 levels, and the presence of inflammatory changes detected by renal scintigraphy using 99mTc-DMSA (DMSA). MATERIAL/METHODS: We studied 33 children aged 1-24 months (mean 7.1+/-5.8 months) with first-time UTI. The subjects were divided in two groups: with fever (group I, n=10) and without fever (group II, n=23). Inflammatory markers (ESR, CRP, leukocyte count), urinary IL-6 and IL-8 level, and DMSA scan were evaluated in all children. RESULTS: Urinary IL-6 and IL-8 levels [IL-6, IL-8/creatinine (pg/mg)] were significantly higher in group I than in group II (Il-6 level: 39.4+/-41.1 vs. 6.3+/-13.7, p<0.01; IL-8 level: 791.1+/-1143.6 vs. 36+/-87.9, p<0.001). We found positive correlation between urinary IL-6 and IL-8 levels and ESR, CRP, and leukocyte count (IL-6: r=0.43, p<0.05; r=0.46, p<0.05; and r=0.59, p<0.001, respectively; IL-8: r=0.55, p<0.05; r=0.72, p<0.0001; and r=0.44, p<0.05, respectively). We found no relation between urinary cytokine levels and the presence of inflammatory changes detected by DMSA scanning, despite slightly higher mean urinary cytokine levels in children with inflammatory changes in DMSA scan. CONCLUSIONS: We found significantly higher IL-6 and IL-8 levels in children with febrile UTI and elevated inflammatory markers. IL-6 and IL-8 levels do not differentiate between acute pyelonephritis and UTI in children to age 24 months.

Serum growth factors in hemodialyzed patients.

The contact of chronic hemodialysis patients' blood with components of extracorporeal circulation leads to mobilization of several systemic reactions. The purpose of this study was to determine the activity of transforming growth factor (TGF-beta1) and platelet derived growth factor (PDGF) in serum of patients on long-term hemodialysis program and to compare these results with ones obtained in healthy volunteers. Twenty-five patients on dialysis carried out on polysulfone membrane dialyzer, and 16 patients on dialysis with the used of cuprophan membrane dialyzer on long-term hemodialysis program participated in the study. TGF-beta1 level in serum of healthy volunteers (12.06 +/- 7.56 ng/mL) was lower than in serum of patients dialyzed on polysulfone membrane dialyzers (26.56 +/- 14.83 ng/mL). Differences in PDGF concentrations in serum of control group and hemodialyzed patients were not statistically significant. The strong correlation between TGF-beta1 and PDGF in serum of both studied groups of patients was demonstrated.

[Predictive value of plasma interleukin 1, interleukin 6, interleukin 8 and C-reactive protein (CRP) in patients with myocardial infarction]. / Rokownicze znaczenie osoczowego stezenia interleukiny 1, interleukiny 6, interleukiny 8 oraz CRP w zawale serca.

Long-term risk of mortality in patients with myocardial infarction is thought to be linked with plasma concentrations of proinflammatory cytokines and CRP (markers of inflammation). The aim of our study was to analyze plasma levels of interleukin (IL) 1, interleukin 6, interleukin 8 and C-reactive protein (CRP) in patients with myocardial infarction. One hundred and seven (107) patients with myocardial infarction hospitalized at the Cardiac Care Unit of St. Elizabeth's Sisters' Hospital in Warsaw and a control group of 10 subjects were enrolled in our study. The samples of peripheral venous blood were withdrawn from the patients on 2nd and 7th of infarction and plasma levels of IL-1, IL-6, IL-8 and CRP were determined. The patients were followed-up for a year. The analysis of survivals and deaths caused by acute coronary syndrome allowed to determine the predictive value of IL-1, IL-6, IL-8 and CRP in myocardial infarction. Twenty-two (22) of the total 107 patients died of acute coronary syndrome during one-year follow-up. Plasma IL-6 and CRP levels were higher in non-survivors as compared to the levels of IL-6 and CRP in living subjects, whereas plasma levels of IL-1 and IL-8 were comparable in both groups. IL-6 and CRP proved to be of predictive value in patients with myocardial infarction during one-year follow-up. It has also been found that plasma IL-6 level correlates with plasma CRP concentration and that there is a positive correlation between the former and CK-MB levels. IL-6 and CRP levels were higher in patients with Q wave infarction in comparison with non-Q wave infarction. Plasma levels of IL-1 and IL-8 have not been found to be good predictors of death during 12-month follow-up.

[Urine TGF-beta1 concentration in patients with type II diabetes mellitus--prognostic values]. / Stezenie TGF-beta 1 w moczu u pacjentów z cukrzyca typu 2--znaczenie prognostyczne.

Type 2 diabetes mellitus is a social and civilization-related disease that leads to various micro- and macroangiopathic complications, including diabetic nephropathy. At present, the most sensitive and non-invasive indicator of the progression of diabetic nephropathy is microalbuminuria. Morphological features such as accumulation of extracellular matrix proteins, thickening of glomerules' basement membranes are prior to microalbuminuria. The aim of our clinical study was to establish whether urine and serum TGF beta 1 levels may be significant in prognosing and evaluating a risk for developing diabetic nephropathy. The trial was carried out in 68 patients with type 2 diabetes mellitus and a group of 10 healthy subjects served as control. Urine and serum TGF beta 1 concentrations were evaluated, as well as basic laboratory parameters. After one-year-observation serum creatinine level and microalbuminuria value were investigated in 60 patients with type 2 diabetes mellitus. In patients with type 2 diabetes mellitus both urine and serum TGF beta 1 concentration were elevated. After one-year-observation of patients with type 2 diabetes mellitus it was established that the increase of serum creatinine concentration and that of microalbuminuria value were higher in those patients, whose initial TGF beta 1 levels exceeded normal values. A positive correlation between urine TGF beta 1 level and the progression of renal failure measured by the increase of serum creatinine level was observed. In conclusion, our findings indicate that urine TGF beta 1 level may be a good prognostic factor of the development of diabetic nephropathy in the course of diabetes mellitus.

[Enzymatic activity of cathepsin B, cathepsin B and L, plasmin, trypsin and collagenase in hepatocellular carcinoma]. / Aktywnosc enzymów proteolitycznych: katepsyny B, katepsyny B i L, plazminy, trypsyny i kolagenazy w raku watrobowokomórkowym.

UNLABELLED: Hepatocellular carcinoma is one of the most common malignancies worldwide. Invasiveness of this tumour seems to be related to degradation of extracellular matrix. Such proteolytic enzymes as: cathepsin B and L, plasmin, collagenase and trypsin are thought to play a pivotal role in this process. Enzymatic activity depends on balance between enzymes and their inhibitors and--moreover--on interactions among these enzymes. The purpose of our study was to evaluate enzymatic activity of cathepsin B, cathepsin B and L, plasmin, collagenase and trypsin in patients with hepatocellular carcinoma in liver tissue and in peripheral blood. Then correlations between activity of enzymes (mentioned above) and clinical status, pathological findings and laboratory tests were assessed. Our study was conducted on 14 patients who underwent surgery because of hepatocellular carcinoma. Tissue samples were obtained during surgery from neoplastic area and from non-neoplastic area. Peripheral blood was withdrawn before surgery and within early post-operative period. Proteolytic activity of these enzymes was determined with use of fluorometric assay. Enzymatic activity in tissue samples was referred to protein concentration (BCA assay) and to DNA concentration (fluorometric assay). RESULTS: Proteolytic activity of plasmin and trypsin in neoplastic tissue were significantly lower as compared to non-neoplastic area of these patients (p = 0.0356; p = 0.0412, respectively). Activity of the remaining enzymes: cathepsin B, cathepsin B and L and collagenase did not differ significantly. No difference was demonstrated between activity of enzymes in peripheral blood withdrawn before surgery and in postoperative period. There was a statistically significant inverse correlation between serum AFP level and enzymatic activity of cathepsin B, cathepsin B and L and collagenase in tumor tissue. Lower activity of all investigated enzymes was observed in tumor tissue of HBV related hepatocellular carcinoma in comparison with the remaining tissue samples. Correlation between patients age and activity of enzymes was not statistically significant. CONCLUSION: Although the evaluation of presented enzymatic profile did not allow for the assessment of associations between investigated enzymes, our results demonstrated correlations between proteolytic activity of enzymes and serum AFP level, viral status, but it requires further investigations.

[Cytokines in noninvasive diagnostics of diabetic nephropathy progression]. / Cytokiny w nieinwazyjnej diagnostyce nefropatii cukrzycowej.

Diabetic nephropathy is major long-term complication of diabetes mellitus a social and civilization-related disease. At present, the most sensitive and non-invasive indicator of the progression of diabetic nephropathy is microalbuminuria. Morphological features such as accumulation of extracellular matrix proteins, thickening of glomerules' basement membranes are prior to microalbuminuria. The aim of our clinical study was to establish whether urine and serum TGF-beta 1 and IL-6 levels may be significant in prognosing and evaluating a risk for developing diabetic nephropathy. The trial was carried out in 68 patients with type II diabetes mellitus and a group of 10 healthy subjects served as control. Urine and serum TGF-beta 1 concentrations were evaluated, as well as, basic laboratory parameters. After one-year-observation serum creatinine level and microalbuminuria value were investigated in 60 patients with type II diabetes mellitus. In patients with type II diabetes mellitus both urine and serum TGF-beta 1 and IL-6 were elevated. After one-year-observation of patients with type II diabetes mellitus it was established that the increase of serum creatinine concentrations values were higher in those patients, whose initial TGF-beta 1 levels exceeded normal values. A positive correlation between urine TGF-beta 1 level and the progression of renal failure measured by the increase of serum creatinine level was observed. In conclusion, our findings indicate that urine TGF-beta 1 and IL-6 levels may be a good prognostic factor of the development of diabetic nephropathy in the course of diabetes mellitus.