Dexamethasone-dependent modulation of cyclic GMP synthesis in podocytes.

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.

Benthic effects on the polarization of light in shallow waters.

Measurements of the upwelling polarized radiance in relatively shallow waters of varying depths and benthic conditions are compared to simulations, revealing the depolarizing nature of the seafloor. The simulations, executed with the software package RayXP, are solutions to the vector radiative transfer equation, which depends on the incident light field and three types of parameters: inherent optical properties, the scattering matrix, and the benthic reflectance. These were measured directly or calculated from measurements with additional assumptions. Specifically, the Lambertian model used to simulate benthic reflectances is something of a simplification of reality, but the bottoms used in this study are found to be crucial for accurate simulations of polarization. Comparisons of simulations with and without bottom contributions show that only the former corroborate measurements of the Stokes components and the degree of linear polarization (DoLP) collected by the polarimeter developed at the City College of New York. Because this polarimeter is multiangular and hyperspectral, errors can be computed point-wise over a large range of scattering angles and wavelengths. Trends also become apparent. DoLP is highly sensitive to the benthic reflectance and to the incident wavelength, peaking in the red band, but the angle of linear polarization is almost spectrally constant and independent of the bottom. These results can thus facilitate the detection of benthic materials as well as future studies of camouflage by benthic biota; to hide underwater successfully, animals must reflect light just as depolarized as that reflected by benthic materials.

Purinergic modulation of glucose uptake into cultured rat podocytes: effect of diabetic milieu.

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, NG (1271 ± 86 vs. 477 ± 37 nmol/h/mg protein, P<0.001). ATP stimulated 2-DG uptake by 44 ± 4% and 29 ± 5% in NG and HG, respectively. ATP analogues, ß, γ-methylene ATP and 2-methylthio ATP stimulated 2-DG uptake in range of 18-34% in NG and 16-17% in HG. Benzoylbenzoyl ATP increased 2-DG uptake about 24 ± 2% in NG however, its effect in HG reached 50 ± 1%. The antagonists of P2 receptors (suramin, reactive blue 2, PPADS) decreased basal 2-DG uptake in NG and HG; suramin and reactive blue 2 at average of 15 ± 4% in NG 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 NG (0.48 ± 0.01 vs. 5.05 ± 0.39 µM, 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 HG. In conclusion, diabetic milieu affects purinergic modulation of glucose transport into podocytes which may play a role in development of diabetic podocytopathy.

G-CSF therapy and catheter-related Gram-positive sepsis increase serum IL-2 receptor α level and may falsely suggest a relapse in children with soft tissue sarcomas unless serum beta2-microglobulin, lactate dehydrogenase and C-reactive protein levels are determined concomitantly.

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.

Expression of GFAT1 and OGT in podocytes: transport of glucosamine and the implications for glucose uptake into these cells.

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 microM), 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.

Metformin induces suppression of NAD(P)H oxidase activity in podocytes.

Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms. The experiments were performed in normal (NG, 5.6mM) and high (HG, 30mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-(3)H]-glucose. ROS production increased by about 27% (187+/-8 vs. 238+/-9 arbitrary units AU, P<0.01) in HG. Metformin (2mM, 2h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58+/-0.07 to. 0.99+/-0.06, and from 0.53+/-0.03 to 0.64+/-0.03; P<0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C. We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.

Hyperglycemia and mechanical stress: targeting the renal podocyte.

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.

[Prognostic factors of celiac disease occurrence in type 1 diabetes mellitus children]. / Czynniki prognostyczne choroby trzewnej u dzieci z cukrzyca typu 1.

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.

Cyclic GMP signaling in podocytes.

Natriuretic peptides (NP), together with nitric oxide (NO) are powerful relaxing factors acting via a common second messenger, cyclic GMP (cGMP). Together with other vasoactive modulators, these vasorelaxing factors play an essential role in regulating the function of kidney glomeruli. The presence of NP receptors in podocytes has been well documented. Recently, also mRNA for soluble guanylate cyclase, the NO receptor, has been shown in these cells. Stimulation of podocytes with atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP), and NO donors results in considerable upregulation of cellular cGMP synthesis. The podocyte foot processes contain a highly organized network of microfilaments adhering to the glomerular basement membrane (GBM). Changes in podocyte cytoskeleton accompanied by detachment of the cells from the GBM are closely associated with many glomerulopathies. The contractile apparatus in the podocyte foot processes seems to be an obvious target for the cyclic GMP signaling cascade. However, little is known about implications of the cGMP synthesis in these cells. We briefly review the current art regarding generation and modulation of cyclic GMP levels in podocytes. We discuss also the possible targets for this secondary messenger as well as its functional role in podocytes.

Pre-treatment serum levels of interleukin-10, interleukin-12 and their ratio predict response to therapy and probability of event-free and overall survival in childhood soft tissue sarcomas, Hodgkin's lymphomas and acute lymphoblastic leukemias.

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-10/IL-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.

Mechanical stress and glucose concentration modulate glucose transport in cultured rat podocytes.

BACKGROUND: Recent studies show that mechanical stress modifies both morphology and protein expression in podocytes. Ambient glucose is another factor modulating protein synthesis in these cells. In diabetes, podocytes experience elevated glucose concentrations as well as mechanical strain generated by high intracapillary pressures. Both these factors are responsible for podocyte injury, leading to impairment of kidney glomerular function. In the present study, we examined the effects of glucose concentration and mechanical stress on glucose uptake in podocytes. METHODS: Following a 24 h pre-incubation in low (2.5 mM, LG), normal (5.6 mM, NG) or high (30 mM, HG) glucose media, cultured rat podocytes were exposed to 4 h mechanical stress. We used the labelled glucose analogue, [3H]2-deoxy-D-glucose, to measure glucose uptake. The distribution of facilitative glucose transporters GLUT2 and GLUT4 was assessed by flow cytometry. RESULTS: In the control (static) cells, glucose uptake was similar in the three glucose groups. In mechanically stressed podocytes, glucose uptake increased 2-fold in the LG and NG groups but increased 3-fold in the HG group. In the NG cells, mechanical load increased the membrane expression of GLUT2 and reduced the membrane-bound GLUT4. In stretched HG cells, the membrane expression of both GLUT2 and GLUT4 was decreased. High glucose decreased the plasma membrane GLUT2 content in the stretched cells, whereas both static and stretched podocytes showed an elevation in GLUT4. CONCLUSION: Mechanical stress potentiated glucose uptake in podocytes and this effect was enhanced by high ambient glucose. The decreased expression of GLUT2 and GLUT4 on the surface of stretched cells suggests that the activity of other glucose transporters may be regulated by mechanical stress in podocytes.

Detection of Helicobacter species in liver and stomach tissues of patients with chronic liver diseases using polymerase chain reaction-denaturing gradient gel electrophoresis and immunohistochemistry.

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.

Characterization of glucose uptake by cultured rat podocytes.

The nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose ((3)H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of (3)H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the (3)H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a V(max) value of approximately 10 mM and S(0.5)of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.

C-type natriuretic peptide as a podocyte hormone and modulation of its cGMP production by glucose and mechanical stress.

BACKGROUND: High glucose and mechanical strain resulting from capillary hypertension are relevant risk factors affecting glomerular cells in diabetes. Altered activity of the natriuretic peptide (NP) system acting via vasorelaxing cyclic guanosine 5' monophosphate (cGMP) has been proposed to be one of the reasons for diabetes-dependent impairment of kidney function. Podocytes possess the NP receptors (NPRs) coupled to particular guanylyl cyclase. We investigated whether mechanical stress and high ambient glucose influence cGMP generation in podocytes stimulated with NPs. Additionally, the C-type natriuretic peptide (CNP) system has been characterized in these cells. METHODS: Conditionally immortalized mouse podocytes were stimulated with NP for 15 minutes and cGMP was determined by enzymatic immunoassay. The mRNA expression for CNP and CNP-specific NPR-B was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, cGMP synthesis was measured in cells exposed to mechanical stress and to 30 mmol/L glucose for 3 days. RESULTS: Production of cGMP upon stimulation of the NPR-B receptor with CNP and inhibition by an antagonist HS-142-1 was dose-dependent. RT-PCR showed that podocytes express mRNA not only for NPR-B but also for CNP. Mechanical stress reduced the cGMP response by 50%, both to atrial natriuretic peptide (ANP) and to CNP. Conversely, upon high glucose, the CNP-induced production of cGMP was elevated twofold in stretched and in control cells. Furthermore, as compared to ANP, stimulation with CNP caused a larger increase in cGMP levels in stretched as well as in nonstretched cells. CONCLUSION: Expression of CNP together with potent NPR-B receptors suggests that in podocytes, CNP may act in an autocrine and/or paracrine manner. Furthermore, in a diabetic kidney, high glucose and mechanical stress may modulate the CNP-dependent cGMP production in podocytes in an opposite manner.

Effect of angiotensin II on ANP-dependent guanylyl cyclase activity in cultured mouse and rat podocytes.

The presence of a well-developed contractile apparatus is the feature determining major roles of podocytes in the renal glomeruli. Receptors for a variety of vasoactive hormones are expressed in these cells; however, most of the signaling pathways are still unknown and remain to be elucidated. Angiotensin II (Ang II) and atrial natriuretic peptide (ANP), due to their opposite action, are the major modulators of glomerular filtration. In podocytes, Ang II induces rise in intracellular calcium concentration, whereas ANP stimulates generation of cGMP. The present study was designed to check whether ANP-stimulated cGMP synthesis in podocytes might be affected by Ang II. Cultured rat (RP) and mouse (MP) podocytes were stimulated with ANP, in the absence or presence of Ang II and cyclic GMP was determined by RIA method. Co-incubation of podocytes with ANP and Ang II caused significant (p < 0.01) suppression of ANP-dependent cGMP generation. The effect was prevented by saralasin, an inhibitor of angiotensin receptors. Phorbol-12-myristate-13-acetate (PMA) mimicked, whereas chelerythrine reversed inhibitory effect of Ang II. In conclusion, angiotensin II counteracts ANP-stimulated cGMP synthesis in cultured podocytes. It seems likely that the protein kinase C pathway is involved in this effect.