Identification of a glutathione S-transferase without affinity for glutathione sepharose in human kidney.

To identify kidney glutathione S-transferase (GST) isoenzyme, which does not bind to glutathione affinity column, biochemical characterization was performed by using an array of substrates and by measuring sensitivity to inhibitors. Immunological characterization was done by immunoblotting. Affinity flow-through GST exhibited activity towards 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and cumene hydroperoxide, typical class alpha substrates and high sensitivity towards hematin, an alpha class inhibitor. It cross-reacted with antibodies against alpha class GST. Affinity flow-through GST in human kidney is an alpha class member.

Byproducts of oxidative protein damage and antioxidant enzyme activities in plasma of patients with different degrees of essential hypertension.

Despite evidence that essential hypertension (EH) is a state of increased oxidative stress, the data on oxidative protein modifications is lacking. Besides, the role of extracellular antioxidant enzymes in EH has not been systematically studied. Study was performed in 45 subjects with EH and 25 normotensive controls. Patients were divided into three groups according to the 2003 ESH/ESC guidelines (grade 1-3). Plasma protein reactive carbonyl derivatives (RCD) and SH-groups (as byproducts of oxidative protein damage) as well as antioxidant enzyme activities superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase were studied spectrophotometrically and correlated with blood pressure (BP). RCD levels were increased in EH patients compared to controls and correlated significantly with both systolic blood pressure (SBP) (r = 0.495, P<0.01) and diastolic blood pressure (DBP) (r = 0.534, P<0.01). Plasma SH-groups content was significantly lower in all patients with EH, with no correlation with BP. SOD and catalase activity in patients with grade 1 EH were similar to that of controls. Patients with grade 2 and 3 of EH had lower SOD and catalase activity. However, significant correlation with SBP and DBP was observed for catalase only (r = -0.331; P<0.05 and r = -0.365; P<0.05, respectively). EH patients exhibited higher plasma GPX activity compared to those in controls, and it correlated with SBP (r = 0.328; P<0.05). The results presented show that increased oxidative protein damage is present in all grades of EH. In mild hypertension extracellular antioxidant enzyme activities are not decreased, suggesting they are probably not critical in early EH, but could be important in moderate to severe EH.

Glutathione S-transferase T1-1 activity upregulated in transitional cell carcinoma of urinary bladder.

OBJECTIVES: To perform a systematic functional investigation of different glutathione S-transferase (GST) classes, including GST class Theta (GSTT) member GSTT1-1, in transitional cell carcinoma (TCC) and the surrounding normal uroepithelium of the same individuals. Recently, it was suggested that GSTT1-1 might be an important risk modulator for TCC. METHODS: Tumor samples and surrounding normal uroepithelium were obtained from 24 patients with TCC of urinary bladder. The following substrates with differential specificities were used: 1-chloro-2,4-dinitrobenzene for overall GST activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for GST Alpha; 1,2-dichloro-4-nitro-benzene for GST Mu; 4-vinylpyridine for GST Pi 1-1(GSTP1-1); and 1,2-epoxy-3-(p-nitrophenoxy)propane for GSTT1-1. RESULTS: GSTP1-1 and GSTT1-1 activities were demonstrated in all uroepithelial and TCC samples, and GST Mu activity was detectable in 11 of 24 patients. In the tumor specimens, significant upregulation of all expressed GST subtypes was observed. The mean GSTP1-1 and GSTT1-1 level in TCC was increased 2-fold and 3.6-fold, respectively, compared with the mean level in the normal uroepithelium (P <0.001). Tumor GSTT1-1 activities correlated statistically significantly with the tumor stage (P <0.05). CONCLUSIONS: In tumors and adjacent normal uroepithelium of patients with TCC, three major cytosolic GST classes, Mu, Pi, and Theta, were expressed. Although the GST isoenzyme pattern in TCC was similar to that of the corresponding normal uroepithelium, during cancer progression a clear tendency toward an increase in all the GST subtypes expressed was noted. For the first time, distinct GSTT1-1 activity levels were demonstrated in human uroepithelium, as well as its pronounced upregulation in TCC.

Isoenzyme profile of glutathione S-transferases in human kidney.

Novel glutathione S-transferase (GST) isoenzymes, which do not bind to the glutathione (GSH) affinity column, were recently identified in dog kidney and dog renal cell lines. In humans, similar affinity flow-through GST has been previously found only in the urinary bladder. To ascertain whether these affinity flow-through GST isoenzymes also exist in the human kidney, we separated GST isoenzymes from five kidney samples on the basis of their affinity to GSH affinity resin. GSTs were further purified by anion exchange chromatography and chromatofocusing and characterized with specific substrates. Our results show that the human kidney has both affinity flow-through GST isoenzymes and those which bind tightly to the GSH affinity column. Purification of affinity-bound GST resulted in a rich profile of different isoenzymes with balanced expression of both anionic and cationic forms. Affinity flow-through GST was represented by one isoenzyme (pI-7.9) in all kidney samples tested, but one kidney specimen also contained another GST isoenzyme (pI-7.0). Our results for the first time show the presence of GST isoenzymes that do not bind to GSH-affinity resin in the human kidney. Although the assessment of similarity between the human kidney and urinary bladder affinity flow-through GST requires further elucidation, it can be speculated that these particular GSTs may play an important role in providing protection against the common carcinogens.

Alteration in plasma antioxidant capacity in various degrees of chronic renal failure.

AIM, PATIENTS AND METHODS: To obtain a more comprehensive profile of extracellular antioxidant capacity in chronic renal failure (CRF), markers of oxidative stress (malondialdehyde, MDA and hydrogen peroxide), protein SH groups (as an important chain-breaking antioxidant) and activity of antioxidant enzymes (glutathione peroxidase, [GPX], catalase and superoxide dismutase, [SOD]) were studied in plasma of 36 non-dialyzed patients with various degrees of CRF and 10 hemodialyzed (HD) patients. RESULTS: The results show that plasma MDA concentrations significantly increase with the severity of kidney dysfunction (r = -0.543, p < 0.01). A marked and profound fall in plasma thiol group levels was observed in all groups tested, independent of the degree of renal failure (r = 0.082, p > 0.05). Plasma SOD activity increased in CRF patients with the progression of renal insufficiency (r = -0.370, p < 0.05). On the other hand, plasma GPX activity decreased progressively in strong correlation with endogenous CCr (r = 0.712, p < 0.001). However, despite this imbalance between extracellular SOD and GPX activities, plasma concentration of hydrogen peroxide remained unchanged in non-dialyzed CRF patients. Catalase activity in non-dialyzed CRF patients was increased, suggesting the significant involvement of catalase in the regulation of plasma hydrogen peroxide level. CONCLUSION: In hemodialyzed patients significantly lower plasma catalase activity, associated with higher hydrogen peroxide levels, was found. It seems reasonable to assume that the imbalance in the activity of extracellular antioxidant enzymes in chronic renal failure may result in accumulation of free radical species, and in unscheduled oxidation of susceptible molecules.

Markers of oxidative stress after renal transplantation.

An increased degree of oxidative stress (OS) in chronic renal failure (CRF) and a possible role of free radicals in CRF have already been described. However, data on OS after renal transplantation are scarce. The aim of the present study was to estimate the degree of OS in renal transplant patients. The study included four groups: 1) 15 haemodialysis patients (HD group), 2) 11 renal transplant patients with stable function (SF group), 3) 12 renal transplant patients with chronic biopsy-proven rejection (CR group), and 4) 10 healthy controls (C group). Markers of OS (malondialdehyde and thiol group levels) and antioxidant activity (glutathione peroxidase and Cu,Zn-superoxide dismutase) were determined in plasma and in red blood cells of all examined individuals. After successful renal transplantation a significant improvement, but not normalization, of antioxidant enzyme activities accompanied by significantly reduced lipid peroxidation were found. In the CR group the degree of OS was increased, and our results suggest that OS may be a relevant pathophysiological factor for CR development.

Time course of renal glutathione levels in experimental Fanconi syndrome: an enzyme-based approach.

Time-dependent alterations in glutathione (GSH) concentration and the activities of several key enzymes of GSH metabolism were studied in a rat model of experimental Fanconi syndrome induced by i.p. injection of sodium maleate (400 mg/kg BW). The changes in the parameters tested were monitored 0, 2, 4, and 12 h after sodium maleate administration. A significant decrease in renal GSH level was observed 2 and 4 h after sodium maleate treatment (27% and 38% of control values, respectively). The renal GSH depletion did not appear to be due to the decreased production rate or to an increased degradation of the tripeptide. This suggestion is based on the findings that the activities of the GSH synthesis (gamma-glutamyl cysteine synthetase and glutathione reductase) and those of the catabolic pathways (gamma-glutamyl transpeptidase) were unaltered at the same time points. The unchanged activity of gamma-glutamyl transpeptidase also suggests preserved luminal membrane integrity in experimental Fanconi syndrome. The decreased activity of glutathione peroxidase, which utilizes GSH as a cosubstrate in the course of inactivation of free radicals, in the first hours after treatment could facilitate lipid peroxidation reactions in this model of acute renal failure. The observed changes in all parameters tested were transient, with recovery to baseline levels in a period of 12 h after sodium maleate administration. At the same time a pronounced functional impairment still existed. The beneficial effect of fast recovery of renal GSH level on the functional and morphological restitution in experimental Fanconi syndrome is suggested.

[Glutathione and enzymes associated with glutathione metabolism in adriamycin nephropathy]. / Glutation i kljucni enzimi metabolizma glutationa u adriamicinskoj nefropatiji.

Recent data have shown the protective effect of thiol groups on the progression of adriamycin-induced experimental nephropathy, in which reactive oxygen metabolites have been postulated to play an important role. To gain greater insight in the role of glutathione (GSH) in adriamycin-induced nephrosis, we studied changes in reduced GSH level and its associated enzymes in kidney tissue of rats undergoing chronic renal failure produced by i.v. infections of adriamycin (2 x 2 mg/kg b.w.). Kidney damage was characterized by increases in relative kidney weight and BUN levels. The results obtained revealed a 15% drop in renal GSH level in adriamycin-treated animals associated by a similar decrease in the gamma-glutamylcysteine synthetase activity. The activities of kidney glutathione reductase (GR) and glutathione peroxiase (GSH-Px) which are critical constituents of GSH-redox cycle, were significantly decreased (23 an 26%, respectively) in response to adriamycin treatment. Rat kidney glutathione-S transferase and gamma-glutamyl transpeptidase activities were not affected in adriamycin induced nephrosis. We propose that the impairment of renal antioxidant defense, characterized by combined drop in GSH, GR and GSH-Px levels, could permit enhanced free radical induced kidney damage in adriamycin-induced nephropathy.

Erythrocyte glutathione peroxidase and superoxide dismutase activities in different stages of chronic renal failure.

Antioxidant enzyme activities, glutathione peroxidase (GSH-Px) and Cu, Zn superoxide dismutase (SOD-1), were investigated in erythrocytes of non-dialyzed patients with varying degrees of chronic renal failure (CRF), and of patients on regular hemodialysis treatment. The results obtained have shown that GSH-Px and SOD-1 activities were significantly higher in erythrocytes from non-dialyzed CRF patients than in corresponding age-matched healthy controls. Antioxidant enzyme activities increase with the progression of chronic renal insufficiency, and the more pronounced augmentation in GSH-Px and SOD-1 activities were found in erythrocytes from the end-stage renal patients (with Ccr less than 20 ml/min). The increase of GSH-Px and SOD-1 activities seen in non-dialyzed CRF patients were abolished in patients undergoing regular hemodialysis treatment. We propose that the increased GSH-Px and SOD-1 activities could be a protective mechanism for the cells due to the hyperproduction of free radicals in chronic renal failure. The lowering of red blood cell antioxidant activity in uremic patients on chronic dialysis may contribute to the increased oxidative damage in uremia and in the development of uremic complications.

[Significance of laboratory tests for differential diagnosis of acute renal allograft rejection and acute cyclosporine nephrotoxicity]. / Znacaj laboratorijskih testova za diferencijalnu dijagnostiku akutnog odbacivanja transplantisanog bubrega i akutne ciklosporinske nefrotoksicnosti.

The most frequent causes of renal allograft function deterioration in early postransplantation period are aucte rejection (AR) and acute cyclosporine nephotoxicity (CyA NT). In order to contribute to noninvasive diagnostics in differential diagnosis of these two disorders, glomerular and tubular function in 40 patients during 2-3 weeks after renal transplantation, were followed-up. The results showed that ischaemia, during any act of transplantation provoked functional and structural disorders of renal allografts. During acute rejection serum creatinine level was increased diuresis, sodium and beta-2 microglobulin levels were decreased, whyle there was no significant change in the urinary enzymes ativity. In acute CyA NT there was significantly greater fractional excretion of sodium and beta-2 mikroblobulin, as well as activity of N-acetly-beta-d glukosaminidase and alkaline phosphatase in urine in comparison to other examined groups.

Glutathione and its associated enzymes in peripheral blood cells in different stages of chronic renal insufficiency.

Reduced glutathione (GSH) levels and glutathione reductase (GR) and glutathione S-transferase (GST) activities were investigated in the erythrocytes and lymphocytes of non-dialyzed patients with varying degrees of chronic renal insufficiency, and also of patients on regular hemodialysis treatment. GSH, GR and GST levels were higher in erythrocytes and lymphocytes of examined patients as compared to their corresponding age-matched healthy controls. A correlation was found between the degree of renal insufficiency and the above parameters tested. A routine hemodialysis did not significantly affect erythrocyte and lymphocyte GSH content and activities of its associated enzymes. The increased GSH levels as well as GSH-linked enzyme activities of blood cells in uremia may be a protective mechanism for the cells due to the accumulation of toxic, oxidizing, wastes in the blood as a result of the uremic state. This view is supported by the results ofin vitro experiments, which have shown that GR and GST activities of normal human lymphocytes are increased when incubated with plasma from uremic patients.

The effects of hemodialysis with different membranes on middle molecules and uremic neuropathy.

Twenty-four hemodialysis patients, 14 with uremic neuropathy and 10 symptom-free, were studied over 12 months. Cuprophan and AN 69 membrane dialyzers were used in their treatment in order to investigate the influence of different membranes on plasma levels of middle molecular weight substances (MMS) and uremic neuropathy. Hemodialysis with the cuprophan membrane caused no significant changes in plasma MMS levels or in the neurological condition of patients. The effect of dialysis with AN 69 membrane depended on initial plasma MMS levels. Initially high plasma MMS levels decreased significantly and significant improvement of neuropathy was achieved. In neuropathic patients with plasma MMS levels similar to those of symptom-free patients, hemodialysis with AN 69 membrane had no effect. These results suggest that hemodialysis with MMS high-permeability membranes may be recommended for neuropathic patients with high plasma MMS levels.

Erythrocyte and plasma glutathione levels in patients with chronic renal insufficiency.

Reduced glutathione (GSH) levels were investigated in the erythrocytes and plasma of nondialyzed patients with varying degrees of renal insufficiency and also of patients on regular hemodialysis treatment. GSH levels were from 19 to 70% higher in the erythrocytes of examined patients as compared to their corresponding age-matched controls. A correlation was found between the degree of renal insufficiency and the erythrocyte GSH level. No variations in plasma GSH levels which could be related to the degree of renal deterioration were observed. A routine hemodialysis did not significantly affect erythrocyte and plasma GSH levels. No significant differences in GSH levels between anemic and nonanemic uremic patients were observed.

Cellular distribution of protein-disulfide interchange enzyme activity in mouse spleen.

In an attempt to precise the cellular distribution of protein-disulfide interchange enzyme activity within different compartments of the splenic lymphoid tissue, we have analyzed the protein-disulfide interchange (PDI) enzyme activity in adherent and nonadherent cell populations of normal and T-cell depleted CBA mice. In vivo depletion of T-cells, as evaluated by functional and cytotoxic tests, was achieved by two i. v. injections of anti-T monoclonal antibodies (Mab F7D5). Nonadherent cell populations were found to have levels of protein-disulfide interchange enzyme activity significantly higher than that of the adherent cells. Pretreatment with F7D5 monoclonal antibodies enhanced the protein-disulfide interchange activity in nonadherent cell population, thus indicating that the major source of the enzyme activity are nonadherent spleen cells, which do not bear T-cell marker, probably B-cells.

Characterization of low-molecular-weight acid phosphoprotein phosphatase associated with rat splenic cell nucleoli.

Evidence is presented on the existence of an acid phosphoprotein phosphatase (APPase) associated with rat splenic cell nucleoli. The enzyme is purified 1250-fold from 0.3 M NaCl nucleolar extract by means of chromatography on P cellulose and Sephacryl S-200. The nucleolar acid phosphoprotein phosphatase is a very basic protein (pI 8.3) and shows maximal activity at pH 5.8. It dephosphorylates acidic phosphoproteins (casein and phosvitin), ATP, and p-nitrophenyl phosphate, but not basic phosphoproteins (histones and protamine phosphate). The enzyme activity is very dependent on reducing agents, especially on ascorbic acid. Divalent and monovalent cations did not affect phosphatase activity, but heavier divalent metals, Co2+ and Zn2+, strongly inhibit the enzyme activity. The activity was also inhibited by N-ethylmaleimide, indicating a requirement for free sulfhydryl groups. The estimated molecular weight of the purified enzyme is approximately 38,000 by gel filtration and sedimentation in sucrose gradient concentration.

The ontogenetic evolution of acidic phosphoprotein phosphatase activity in the lymphatic tissue and the liver of the rat.

We have shown that an acidic phosphoprotein phosphatase (APP-ase) has a different pattern of postnatal maturation in the spleen, thymus and liver of rats and mice. The APP-ase activity increases during the first eight months of postnatal life in the spleen of rats (when it attains an 8--10 times higher value than at birth) and up to the sixth month of life in the spleen of mice. It increases considerably during the first two weeks of postnatal life in the thymus of rats and mice; in the liver of rats it reaches maximum activity before birth, but continues to increase up to the sixth month of postnatal life in the liver of mice. The results show also that the APP-ase from the spleen, thymus and liver of rats is equally active in dephosphorylating ATP and phenyl phosphate during the whole life span of rats, but not in relation to beta-glycerol phosphate. After analyzing its substrate specificity, its pH dependence in relation to different substrates, its kinetic properties, as well as its behaviour towards ascorbic acid and different inhibitors (sodium tungstate and sodium molybdate, L-tartrate, L-phenylalanine and L-cysteine) we have come to the conclusion that the rat spleen APP-ase is different from "nonspecific" acid and alkaline phosphatases and very similar to the EC 3.1.3.16 acid phosphoprotein phosphatase.