Podocytes may be direct target for glucocorticoid therapy in glomerular proteinuric disease. Permeability of podocytes largely depends on their capacity to migrate which involves the contractile apparatus in their foot processes. In this study, we examined the effect of synthetic glucocorticoid dexamethasone (DEX) on the ability of podocytes to produce cyclic guanosine monophosphate (cGMP) in the presence of vasoactive factors, atrial natriuretic peptide (ANP), nitric oxide (NO), and angiotensin II (Ang II). We investigated also the effects of cGMP and DEX on podocyte motility. Primary rat podocytes and immortalized mouse podocytes were pretreated with 1 µM DEX for 4 or 24 h. Glomerular hypertension was mimicked by subjecting the cells to mechanical stress. Total and subcellular cGMP levels were determined in podocytes incubated with 0.1 µM ANP, 1 µM S-nitroso-N-acetyl penicillamine (SNAP), and 1 µM Ang II. Cell motility was estimated by a wound-healing assay. The ANP-dependent production of cGMP increased after 4 h exposition to DEX, but was attenuated after 24 h. Adversely, a 24-h pretreatment with DEX augmented the NO-dependent cGMP synthesis. Ang II suppressed the ANP-dependent cGMP production and the effect was enhanced by DEX in mechanical stress conditions. Mechanical stress reduced total cGMP production in the presence of all stimulators, whereas extracellular to total cGMP ratio increased. 8-Br cGMP enhanced podocyte migration which was accompanied by F-actin disassembly. In the presence of DEX these effects were prevented. We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX. This mechanism may account for the antiproteinuric effect of glucocorticoids.

Many components of oncologic treatment increase serum sIL-2Rα level, which may falsely suggest a relapse. We tried to establish whether granulocyte colony stimulating factor (G-CSF) and central vein catheter (CVC)-related sepsis increase serum sIL-2Rα level to values on relapse of childhood soft tissue sarcomas (STS) and how to distinguish real relapse from a "false" one. Serum sIL-2Rα, B2-M, LDH, CRP and ESR levels and rates of markers' elevated values were determined prospectively in 18 STS children: pre-treatmently (ST1), in complete remission (CR; ST2), in CR during G-CSF therapy (ST3), in CR during CVC-related sepsis (ST4), on relapse (ST5) and after treatment (ST6) and once in 50 healthy pediatric controls. It appeared that pre-treatment serum sIL-2Rα, LDH, CRP and ESR but not B2-M declined significantly with remission (ST2) achievement. At ST5 sIL-2Rα, B2-M, LDH and CRP increased from ST2 to ST1 values. SIL-2Rα levels at ST3 and ST4 rose significantly in all patients from ST2 to ST1 and ST5 values. At ST3 also serum LDH and B2-M increased to values at ST1 and ST5 and exceeded significantly those at ST2 and ST4. At ST4 CRP but not B2-M and LDH, rose significantly in most patients to values at ST1 and ST5. Thus, serum sIL-2Rα monitoring in pediatric STS reflects well response to chemotherapy unless samples are collected during G-CSF therapy or CVC-related sepsis. Determination of serum B2-M, LDH and CRP together with sIL-2Rα may help to distinguish between "real" relapse and "false" sIL-2Rα increase due to G-CSF administration or CVC-related sepsis.

Glutamine:fructose‐6‐phosphate amidotransferase (GFAT) and N‐acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O‐glycosylation, one of key post‐translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT‐PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3 ± 0.2 nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1 ± 0.2 nmol/min/mg protein) in a significant part, involved a sodium‐dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50 µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6 mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577–584, 2010. © 2010 Wiley‐Liss, Inc.

Extracellular purines act via P1 and P2 receptors on podocytes and may influence on their function. This action may be modified under various (patho)physiological conditions leading to development of podocytopathy. Aim of study was to investigate effects of diabetic milieu, represented by high glucose concentration (HG, 30 mM glucose) on purinergic-induced changes of 2-deoxy-D-glucose (2-DG) uptake and on extracellular purines metabolism in cultured rat podocytes. Basal 2-DG uptake was 2.7-fold enhanced in HG compared to normal glucose concentration, NC (1271 +/- 86 vs. 477 +/- 37 nmol/h/mg protein, P < 0.001). ATP stimulated 2-DG uptake by 44 +/- 4% and 29 +/- 5% in NC and HG, respectively. ATP analogues, beta, gamma-methylene ATP and 2-methylthio ATP stimulated 2-DG uptake in range of 18-34% in NC and 16-17% in HG. Benzoylbenzoyl ATP increased 2-DG uptake about 24 +/- 2% in NC however, its effect in HG reached 50 +/- 1%. The antagonists of P2 receptors (suramin, reactive blue 2, PPADS) decreased basal 2-DG uptake in NC and HG; suramin and reactive blue 2 at average of 15 4% in NC but in HG the effect was in following order: suramin 28 +/- 3%; PPADS 20 +/- 3% and RB-2 9 +/- 0.9%. Extracellular adenosine concentration was higher in HG than in NC (0.48 +/- 0.01 vs. 5.05 +/- 0.39 mu M, P < 0.05), however intracellular ATP content and extracellular ATP concentration were not affected. Neither ecto-ATPase nor ecto-5'-nucleotidase activities were affected in FIG. In conclusion, diabetic milieu affects purinergic modulation of glucose transport into podocytes which may play a role in development of diabetic podocytopathy. (C) 2010 Elsevier Inc. All rights reserved.

Objectives: Deregulated serum IL-10, IL-12 and their reciprocal balance have been stated in malignancies of adults. In children with cancer the issue has not been investigated so far.Design and methods: To determine the diagnostic and prognostic roles of pre-treatment serum levels of IL-10 (Th2 cytokine), IL-12 (Th1) and their ratios (measured by the IL-10 and IL-12p70 ELISA kits; Endogen) in 91 children with soft tissue sarcomas (STS), Hodgkin's lymphomas (HL) and acute lymphoblastic leukemias (ALL).Results: Median IL-10 and IL-12 levels were significantly higher in cancer patients than in healthy controls. Increased IL-10 indicated presence of general symptoms in HL and high risk group in ALL. Elevated IL-10 and IL-10/IL-12 ratios and decreased IL-12 correlated with poor-risk histology in STS, poor response to therapy, relapse and death from cancer. Multivariate analysis identified IL-10AL-12 ratio>0.14 and IL-12<40 pg/mL as significant predictors for shorter EFS and OS, respectively.Conclusion: Pre-treatment serum levels of IL-10, IL-12 and IL-10/IL-12 balance in children with STS, HL and ALL may be of value as additional prognostic tools to predict the response to therapy and probability of EFS and OS. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Hyperglycemia and deriving from glomerular hypertension mechanical stress are the key factors underlying pathogenesis of diabetic nephropathy (DN). Multiple direct and secondary effects of both these factors are mediated by complex signaling pathways with extensive interactions. The common signaling pathways stimulated by high glucose and mechanical insult may act in an additive manner, thereby accelerating the cell damage. Podocytes, the cells covering the outer aspect of glomerular basement membrane (GBM), are subjected not only to the load of filtered glucose but also to diverse mechanical forces. Bulging into the Bowman's space, they have no support from the apical side, which makes them particularly susceptible to the effects of mechanical strain. Both high glucose and mechanical stress may impair the protein systems anchoring the podocyte foot processes in GBM, therefore blunting resistance of these cells to mechanical forces. Modulation by these factors of expression and activity of numerous structural and functional proteins results in the (auto)inflammatory responses, dysfunction, apoptosis or necrosis of the podocytes. Loss of the podocytes is irreversible due to their inability to proliferate and to replenish damaged cells. Podocytes are injured early in the course of DN, which, most likely, underlies further glomerular and renal damage in diabetes. This review summarizes the effects of elevated glucose and mechanical stress that seem to be involved in podocyte impairment in diabetes, with particular focus on the possible interactions between these factors. J. Cell. Physiol. 221: 288–295, 2009. © 2009 Wiley‐Liss, Inc.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.

The podocytes are highly differentiated cells playing a key role in glomerular filtration. Vasoactive factors including angiotensin II (Ang II) and cyclic guanosine 5' monophosphate (cGMP) are synthesized by these cells upon stimulation as well as in the basal state. In this study we have tested whether angiotensin II affects the total synthesis of cGMP in primary culture of rat podocytes. The cells were stimulated with atrial natriuretic peptide (ANP) and/or a nitric oxide (NO) donor, S-nitroso-N-acetyl penicillamine (SNAP), in the absence or presence of Ang II. The cGMP synthesis was determined by radioimmunoassay (RIA). ANP or SNAP alone increased the cGMP synthesis in podocytes although the effects were not additive unless Ang II was present in the medium. Ang II suppressed the ANP-dependent cGMP synthesis whereas SNAP-dependent cGMP production remained unaffected. These effects were prevented by a non-specific antagonist of Ang II receptors (AT), saralasin. Adversely, PD123319, a specific inhibitor of AT2 receptors, augmented inhibition of ANP-dependent and enhanced the NO-dependent cGMP production. Probenecid, an inhibitor of cGMP extrusion from the cells, suppressed the cGMP generation by both ANP and SNAP. We conclude that cGMP synthesis in cultured podocytes is modulated by angiotensin II and that two adversely acting receptors, AT1 and AT2 are involved in this effect. Additionally, production of cGMP might be intrinsically inhibited by cGMP accumulating inside the cells.

INTRODUCTION:Discussion on the frequency of coexistent celiac disease and type 1 diabetes mellitus (DM1) as well as an attempt to standardize diagnostic methods of celiac disease detection among DM1 children have been performed.OBJECTIVES:To assess the incidence of celiac disease among DM1 children in the Pomeranian region of Poland followed by analysis of the putative prognostic factors for celiac disease development in this particular group of children.MATERIALS AND METHODS:70 children aged 9.47+/-4.59 (group 1) de novo diagnosed with DM1 and 223 children aged 10.20+/-3.87 with long-standing diabetes mellitus type 1 (4.47+/-3.16 years from the diagnosis) were enrolled in the study. All the patients had C-peptide, HbA1c, CRP, TSH, fT4, fT3, urinary albumin secretion rate, IgA, level of antigliadin antibodies (AGA), anti-tissue transglutaminase (TGA) IgA and IgG antibodies (ELISA), anti-endomysium (EmA) IgA and IgG antibodies (immunofluorescence) and anti-tyreoglobulin antibodies (TG), anti-thyroid peroxidase (TPO) antibodies (ELISA) evaluated. All the patients had jejunal biopsy and thyroid ultrasound examination.RESULTS:5.7% of group 1 patients were diagnosed with celiac disease based on the positive jejunal biopsy in comparison with 9.4% in the group 2. TGA antibodies were present in 9.52% of group 2, AGA in 7.62%, EmA in 6.19%. 10% of group 1 children had autoimmune thyroiditis versus 24.2% of group 2 children. The group of children with coincident long-lasting DM1 and celiac disease (group A) was characterized by significantly earlier age at diagnosis (p=0.003), higher HbA(1)c (p=<0.001), CRP (p<0.001) and elevated urine albumin secretion in relation to children without celiac disease and autoimmune thyroiditis (group B). Serologic test detecting TGA antibodies was found to be the most sensitive (95.2%) for the detection of celiac disease among DM1 children, while the lowest sensitivity was obtained in the case of the EmA antibody test (61.9%).CONCLUSIONS:The celiac disease morbidity confirmed by jejunal biopsy is high among DM1 children (9.4%). The assessment of the serum TGA appears to be the most sensitive screening marker for the celiac disease detection in DM1 children.

OBJECTIVE:Helicobacter DNA has been detected in the hepatobiliary tree of patients with chronic liver diseases (CLD). The presence of H. pylori in the stomach compared with in the liver of the same patients with CLD has not been studied, therefore to the aim of this study was to investigate the presence of Helicobacter DNA and antigens in the liver and stomach of Polish patients with chronic liver diseases using molecular and immunological methods.MATERIAL AND METHODS:Gastric mucosa and liver tissue samples and sera were collected from 97 Polish patients with CLD. Anti-H. pylori antibodies were detected by enzyme immunoassay (EIA), and H. pylori-like antigens detected by immunohistochemistry. Helicobacter DNA was detected in stomach and liver samples using a semi-nested Helicobacter genus-specific polymerase chain reaction (PCR) assay, and Helicobacter species identified by denaturing gradient gel electrophoresis (DGGE) and sequencing analysis of amplified PCR products.RESULTS:H. pylori was identified by DGGE and sequence analysis in 60/62 (97%) and 25/25 (100%) of the gastric and liver Helicobacter genus-positive samples, respectively, whereas DNA of H. heilmannii was detected in 2/62 (3%) of the Helicobacter genus-positive gastric samples. H. pylori cagA gene was detected in 23/62 (36%) and 3/25 (12%) gastric and liver tissue samples, respectively. H. pylori-like antigens were detected in 61/97 (63%) gastric mucosa and in 40/97 (41%) liver tissue samples.CONCLUSIONS:H. pylori-like organisms appeared to dominate the gastric mucosa and liver tissue of Polish patients with CLD. The prevalence of the cagA gene was higher in stomach compared with liver samples, which suggests a possible role of cagA negative H. pylori-like organisms in CLD. On the other hand, no significant correlation was found between the presence of H. pylori-like DNA and antigens in the liver and liver function tests.

BACKGROUND:The nitric oxide synthases (NOS) have been observed in human tumour cell lines as well as in solid tumours. Neuronal isoform of NOS (NOS1) was particularly abundant in low-differentiated gynaecological, breast and central nerve system tumours, suggesting that it may characterize poorly differentiated tumours. So far, little is known about expression of the neuronal NOS isoform in non-small cell lung cancer. Our aim was to compare NOS1 expression in non-small lung carcinomas with respect to tumor staging and p53 protein expression.MATERIAL AND METHODS:Thirty-two cases of non-small lung carcinomas of all grades of malignancy and ten control lung specimens with neoplastic lesions were examined. Paraffin-embedded tissues were stained with hematoxylin and eosin, or with antibodies to NOS1 and p53, and evaluated under light microscope.RESULTS:No statistical correlation between expression of p53, NOS1 and degree od tumour differentiation was observed.CONCLUSION:Expression of NOS1 can not serve as a marker for highly malignant tumours in the non-small cell lung carcinomas.

ANP and NO act via different receptors, although inducing the common intracellular messenger - cyclic GMP. However, interaction between both factors remains unclear. Our observations suggested that in the rat kidney glomeruli, activities of the ANP- and NO-dependent guanylyl cyclase systems may be mutually compensated. To check this, we have tested effects of ANP and sodium nitroprusside (SNP) on cGMP synthesis and relaxation of glomeruli contracted with angiotensin II. The glomeruli were isolated from Wistar rats receiving saline (Control), dexamethasone (DEX), deoxycorticosterone (DOCA) or N-c-nitro-L-arginine methyl ester (NAME) for 1 or 2 days. In the DEX glomeruli exposed to 100 microM SNP, rate of cGMP synthesis was significantly higher then in the Control (26.3 vs 16.0 pmol/mg.prot./2 min., P<0.05), while 1 microM ANP was markedly less effective (2.8 vs 16.7 pmol/mg.prot./2 min in Control, P<0.01). On the contrary, in NAME group 1 microM ANP stimulated cGMP synthesis up to 35.6 pmol/mg.prot./2 min whereas efficacy of SNP was slightly suppressed. High correlation coefficient (r = 0.979, p<0.01) indicates interrelationship between NO- and ANP-dependent cGMP synthesis. Ability of the glomeruli to relax in response to ANP or SNP was in accord to their ability to cGMP generation. This was confirmed by high correlation (r = 0.845, p<0.001) between degree of relaxation and rate of cGMP synthesis. Our results support strongly the hypothesis that both, ANP and NO dependent systems co-operate in regulation of the function of kidney glomeruli.

Objective: The aim of this study was to analyze association between HLA-DRB1 alleles and pulmonary tuberculosis (PTB) in the Polish population.

The distinctive feature of the renal function and metabolism implicate a possibility of excessive ATP degradation during insufficient oxygen supply. Protection of the purine ring against degradation is one among other functions of the purine nucleotide cycle (PNC). The purpose of this study was to estimate the activity of PNC in cytosol of rat renal cortex and medulla under conditions that mimic normal and low oxygen supply in vivo. In normoxic-like condition the rate of AMP deamination was 1.7 and 2.0 nmol/mg protein/min in the cytosol of cortex and medulla, respectively. Under this condition, the rate of IMP reamination was similar to that of AMP deamination. In a hypoxia-like condition the rate of AMP deamination increased by 41% in cytosol from both parts of the kidney, while the rate of IMP reamination remained unchanged in the cytosol of medulla and decreased by 46% in the cortex cytosol. Distribution of the other enzymes of the PNC, that is, adenylosuccinate synthetase and adenylosuccinate lyase, in the cytosol of cortex and medulla correlated with that observed for AMP deamination and IMP reamination potentials. At 150 microM IMP, the activity of adenylosuccinate synthetase in the cortex and medulla was 0.34 and 1.24 nmol/mg protein/min, respectively. Activity of the adenylosuccinate lyase was severalfold greater than the respective activity of the adenylosuccinate synthetase. These results show that the efficiency of PNC is about twice as high in the medulla cytosol as in the cortex cytosol, and that the activity of PNC in kidney is mainly limited by the activity of adenylosuccinate synthetase and supply of AMP.

We measured capability for guanosine 3',5'-cyclic monophosphate (cGMP) synthesis by isolated renal glomeruli from steroid-treated rats. Glomeruli obtained from animals treated with dexamethasone showed 5-fold higher sensitivity to sodium nitroprusside (SNP) than glomeruli from deoxycorticosterone- or saline-treated rats. Similar increase in activity of the glomerular soluble guanylate cyclase in response to SNP was observed. Dexamethasone markedly suppressed stimulatory effect of atrial natriuretic factor (ANF) on glomerular cGMP synthesis suggesting existence of cross-regulatory system(s) generating cGMP in renal glomeruli. We presume that glucocorticoid-induced hyperfiltration can be partially caused by sensitization of soluble guanylate cyclase to the nitric oxide, a potent natural dilatory agent.