Anna Długosz

Enzymuria and low molecular weight protein excretion as the differentiating marker of complications in the early post kidney transplantation period

AbstractRenal function in the early post-transplantation period depends largely on factors affecting the kidney prior to implantation. Function of the graft may be also disturbed by the most common complications of the early post-operative period such as acute graft rejection (AGR), acute tubular necrosis (ATN) and may be modified by nephrotoxic action of cyclosporine A (CsA). Evaluation of excretion of enzymes and low molecular weight proteins (LMWP) may help in the differentiation of these complications. Aim Comparison of the urinary excretion of markers of tubular injury in patients with AGR, ATN, or patients with stable graft function (SGF) was made and differences between groups and correlations between markers and cold ischemia time (CIT), warm ischemia time (WIT) and blood trough level of cyclosporine A (CsA0) were determined. Material and methods In 60 cadaveric renal allograft recipients in the early post-transplantation period urinary excretion of N-acetyl-β-d-glucosaminidase (NAG) and B isoenzyme (NAG-B), alanylaminopeptidase (AAP), γ-glutamyltransferase (GGT), α and π isoenzymes of glutathione S-transferase (α-GST, π-GST), retinol binding protein (RBP) and β2- microglobulin (β2M), were analyzed. Results NAG and NAB-B activities were higher in ATN (P<0.05, P<0.01) and in AGR (P<0.005, P<0.02) than in SGF. Excretion of π-GST was higher in AGR than in SGF (P<0.0002) or ATN (P<0.007). CIT and WIT in patients with ATN were higher (P<0.05) than in SGF group. In ATN patients, correlations of CIT with RBP (P<0.05) and π-GST (P<0.05), and WIT with RBP (P<0.05), and π-GST (P<0.001) were found. Conclusions High urinary NAG and NAG B excretion characterizes ATN and AGR patients. Evaluating urinary excretion of π-GST may be helpful in differentiating AGR from ATN. However, taking into account ischemia time is necessary in interpreting the π-GST value in early post transplant period.

Enzymuria and β2-Mikroglobulinuria in the assessment of the influence of proteinuria on the progression of glomerulopathies

The important role of the tubulo-interstitial system for the progression of glomerulonephritis (GN), is the cause of a continuous search for the proper markers of kidney tubules damage, which can be applied in clinical diagnosis.In the present work the activity ofN-acetyl-β-D-glucosamidase (NAG),its isoenzyme NAG-B, alanylaminopeptidase (AAP),γ-glutamyltransferase (GGT), concentration of β2-microglobulin (β2M) and daily protein excretion in the urine of 37 patients with morphologically different glomerulopathies were measured. The serum creatinine was also controlled. The obtained results suggest that activity of NAG in the patients with GN has an intermediate connection with proteinuria and could be a cause of the inflammatory process of the kidney, but the activity of AAP is directly dependent on urine protein concentration. Systemic analysis of both partial and multiple correlation coefficients of the examined indicators creates new, additional possibilities in the estimation of activity and progress of GN.

Toluene and P-Xylene Mixture Exerts Antagonistic Effect on Lipid Peroxidation in vitro

OBJECTIVES Previous research on a group of workers occupationally exposed to styrene, ethylene glycol, toluene, p-xylene and their mixture showed elevated levels of the main products of lipid peroxidation: malondialdehyde and 4-hydroxynonenal (MDA+,4-HNE) in plasma [1]. Moreover, an earlier in vitro study indicated a synergistic interaction between styrene and ethylene glycol on lipid peroxidation [2]. Therefore, it seemed interesting to investigate the effect of combined exposure to toluene and p-xylene on lipid peroxidation and define the type of the interaction. MATERIALS AND METHODS An in vitro model of human placenta mitochondria was used in the study. The concentration of TBARS (thiobarbituric active reagent species) was measured by spectrophotometry, and of hydroxyl radical (.OH) by assessment of deoxyribose degradation. It was investigated whether the administration of coenzyme Q10 (CoQ10) could have a protective function (if given before solvent exposure) or a reparatory function (if given after exposure) in solvent-induced oxidative stress. RESULTS Exposure to p-xylene at concentrations ranging from 5.3 to 265 microg/ml produced an increase in TBARS concentration. The results showed that p-xylene had a stronger influence on lipid peroxidation than toluene. The mixture of toluene and p-xylene induced an antagonistic effect on lipid peroxidation, measured as TBARS concentration. The mechanism connected with .OH generation was found to play an important role in the oxidative damage to lipids resulting from p-xylene exposure. Administration of coenzyme Q10 at the doses of 3.0 and 12.0 microg/ml successfully decreased the TBARS level that was elevated after solvent exposure. CONCLUSIONS In contrast to the synergistic effect that the mixture of styrene and ethylene glycol had on lipid peroxidation (previous study), an antagonism between toluene and p-xylene could be observed. The coenzyme Q10 can be considered a protective agent against lipid peroxidation.

Lipid peroxidation stimulated by Solvesso, Bawanol and methanol, and its counteraction by antioxidants in human placental mitochondria

The influence of some solvents used in the paint and lacquer industry, Solvesso 100, 150 and Bawanol, on the process of lipid peroxidation was evaluated. The interactions of the solvents with methanol and the antioxidative effect of coenzyme Q(10) (CoQ(10)) and vitamin E were also examined. The mitochondria isolated from human placenta were used as a model for investigation. The level of peroxidation was evaluated by malonodialdehyde (MDA) measurement. It was observed that Solvesso 150, Bawanol and methanol caused a statistically significant increase of MDA concentration and that the examined solvents had an antagonistic interaction with methanol. Solvesso 100 does not influence on MDA concentration but gives the same type of interaction with methanol as Solvesso 150. CoQ(10) and vitamin E proved to be effective antioxidants to counteract oxidative stress caused by the studied solvents, lowering lipid peroxidation expressed by MDA concentration.

Oestradiol Protects Against the Harmful Effects of Fluoride More by Increasing Thiol Group Levels than Scavenging Hydroxyl Radicals

The aim of the study was to investigate the role of oestrogens in free radical detoxication upon exposure to fluoride. Interactions between xenobiotics and oestrogens need to be investigated, especially as many chemicals interact with the oestrogen receptor. It is still unknown whether free radical-generating xenobiotics can influence the antioxidative ability of oestradiol (E(2)). In an in vitro examination of human placental mitochondria, thiobarbituric active reagent species (TBARS), hydroxyl radical ((*)OH) generation and protein thiol (-SH) groups were detected. 17beta-E(2) was examined in physiological (0.15-0.73 nM) and experimental (1-10 microM) concentrations and sodium fluoride (NaF) in concentrations of 6-24 microM. E(2) in all the concentrations significantly decreased lipid peroxidation measured as the TBARS level, in contrast to NaF, which increased lipid peroxidation. Lipid peroxidation induced by NaF was decreased by E(2). The influence of E(2) on (*)OH generation was not very significant and depended on the E(2 )concentration. The main mechanism of E(2) protection in NaF exposure appeared to be connected with the influence of E(2 )on thiol group levels, not (*)OH scavenging ability. The E(2) in concentrations 0.44-0.73 nM and 1-10 microM significantly increased the levels of -SH groups, in contrast to NaF, which significantly decreased them. E(2) at every concentration reversed the harmful effects of NaF on -SH group levels. No unfavourable interactions in the influence of E(2) and NaF on TBARS production, (*)OH generation, or -SH group levels were observed. The results suggest that postmenopausal women could be more sensitive to NaF-initiated oxidative stress.

Enzymatic Markers of Cyclosporine Nephrotoxicity in Patients after Renal Transplantation

Toxicity of cyclosporine (CsA), an immunosuppressive drug widely used in transplantation, to the transplanted kidney creates a serious side effect. Therefore, searching for sensitive indicators of nephrotoxic action is well worth the effort. In this work we decribe the results of estimation of urine concentration of lysosomal enzymes widely present in the kidney: N-acetyl-β-D-glucosaminidase (NAG), its isoenzyme NAG-B and β-glucuronidase (β-Gr). The studies were conducted in various periods after transplantation of kidneys, on patients under various treatments and receiving different doses of CsA. The results indicate a substantial dependence of the activity of NAG and NAG-B on CsA doses and the period after transplantation. The enzyme proved to be also a sensitive indicator of graft rejection. No such dependence was observed in the case of β-Gr.

The role of oxidative stress in bladder cancer

The review of the knowledge concerning the impact of oxidative and nitrosative stress on signaling pathways and transcription factors involved in the formation of bladder cancer was prepared. In the industrialized countries, bladder cancer is the fourth most frequently occurring malignant tumors. Recent studies indicate the involvement of oxidative and nitrosative stress in the formation and development of this disease. Red-ox disorders are characteristic for both, the initiation and progression of bladder cancer. There are observed changes in the activity of transcription factors, such as nuclear factor NF-kB; transcription factors: AP-1, Nrf2 and STAT3 and hypoxia-inducible factor HIF-1α. In addition, studies indicate a role for oxidative stress in the regulation of MAPK cascade and its involvement in carcinogenesis consisting bladder. Examples of kinases belonging to the MAPK family are ERK kinases, which expression is proportional to the severity and malignant of bladder cancer. Nitric oxide also plays an important role in tumor biology. Overproduction of NO can both inhibit and promote tumor growth, depending on its concentration, duration of action and tumor microenvironment. Numerous studies show that the bladder cancer is characterized by an intensified production of NO. Reactive forms of nitrogen, similar to oxygen free radicals, could cause oxidative and nitrosative damage to DNA and have capacity to post-translational modification of proteins. In contrast to the ROS, which overproduction result from exposure to carcinogenic xenobiotic, nitrogen oxide in high level is produced during inflammation. Sustained iNOS activity therefore plays an important role in carcinogenesis associated with the inflammatory response, characteristic also for bladder cancer.

Preliminary study on application of urine amino acids profiling for monitoring of renal tubular injury using GLC-MS.

BACKGROUND The early diagnosis of the nephrotoxic effect of xenobiotics and drugs is still an unsolved problem. Recent studies suggest a correlation between the nephrotoxic activity of xenobiotics and increased concentration of amino acids in urine. The presented study was focused on the application of GLC-MS method for amino acids profiling in human urine as a noninvasive method for monitoring of kidney condition and tubular injury level. MATERIAL AND METHODS The analytic method is based on the conversion of the amino acids present in the sample to tert-butyldimethylsilyl (TBDMS) derivatives and their analysis by gas-liquid chromatography-mass spectrometry (GLC-MS). The procedure of urine sample preparation for chromatographic analysis was optimized. RESULTS The presence of 12 amino acids in most of the tested healthy human urine samples was detected. The significant differences in the levels of particular amino acids between patients with tubular injury and healthy controls were found, especially for lysine, valine, serine, alanine and leucine (on average 30.0, 7.5, 3.6, 2.9 and 0.5 fold respectively). CONCLUSIONS We found that this approach based on GLC-MS detection can be used in nephrotoxicity studies for urine amino acids monitoring in exposure to xenobiotics and drugs.

The Diagnostic Value of Nuclear Matrix Proteins in Bladder Cancer in the Aspect of Environmental Risk from Carcinogens

Background The interaction of environmental factors with genetic susceptibility and detoxification level seems to be an important causative factor in bladder cancer (BC). The aim of this study was to look for a BC marker panel which reflects the environmental risk. The nuclear matrix protein 22 (NMP22), bladder cancer-4 (BLCA-4), and total level proteins NMP22 and BLCA-4 (NMBL) in BC patients with genetic predisposition NAT2 (classified as slow acetylators, SA), DNA damage (8-OHdG), and detoxification by isoenzyme GSTπ activity were measured. Materials and Methods The urine and blood from 91 BC patients and controls were examined, also according to tumor stage (T) and grade (G). The participants completed a questionnaire in order to evaluate environmental risk. Results Most patients (75.3%) were previous or actual smokers. The levels of 8-OHdG, NMP22, BLCA-4, NMBL, and GSTπ were significantly higher in BC (p ≤ 0.001). The majority of patients (59.3%) were slow acetylators (SA). The highest BLCA-4/8-OHdG correlation was observed in total BC and SA smokers. Conclusions The total pool of nuclear matrix proteins in the urine (NMBL) has a higher diagnostic value in bladder cancer than single proteins. The particular value of BLCA-4 and GSTπ in the aspect of environmental risk was noted.

Evaluation of NMP22 in bladder cancer patients sensitive to environmental toxins

BACKGROUND Bladder cancer (BC) is recognized as environmentally related. The interaction of environmental exposure to chemicals and genetic susceptibility seem to play important roles in BC development. In order to improve diagnosis and the recognition of BC risk, a group of markers which combine genetic susceptibility with detoxification and nuclear matrix protein (NMP22) is proposed. OBJECTIVES The aim of the study was to examine the utility of nuclear matrix protein (NMP22) as a diagnostic marker in BC in genetic susceptibility (NAT2 slow acetylators) combined with detoxification abilities (glutathione S-transferase GST and isoenzyme GST-π). MATERIAL AND METHODS The NMP22 level in urine, N-acetyltransferase 2 (NAT2) genotype and GST activity in hemolysate blood, as well as isoenzyme GST-π level, were determined in the urine and serum of 43 patients with BC and from 25 non-cancer controls. NMP22 and isoenzyme GST-π levels were measured by ELISA. The NAT2 genotype was examined in DNA isolated from whole blood using the PCR (Polymerase Chain Reaction) technique, while the activity of GST was determined with the spectrophotometric method. RESULTS In the BC group, NMP22 (p = 0.005) concentration, GST-π (p = 0.003) in urine and GST (p = 0.009) activity in blood were statistically significantly higher than in the healthy controls. The majority of BC patients were slow acetylators (NAT2 genotype). A correlation between the level of nuclear matrix protein NMP22 and GST was found in all BC group (p = 0.007) and also slow acetylators (p = 0.0147). CONCLUSIONS The results support the utility of a marker combination, which covers the genetic susceptibility to chemicals with the level of detoxification and nuclear matrix protein in BC patients. A relationship between NMP22 level in urine, GST level in blood and NAT2 genotype was observed. Also the isoenzyme GST-π in urine seems useful as a marker of BC.