Jan Stepinski

Characterization of glucose uptake by cultured rat podocytes.

The nonmetabolizable glucose analogue [3H]-2-deoxy-D-glucose (3H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of 3H-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 3H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a Vmax value of approximately 10 mM and S0.5 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.

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. pyloricagA 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.

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), Hodgkins 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.

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.

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 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.