br Materials and methods br

Materials and methods
Results The immunoblot analysis demonstrated the presence of similar immunoreactivity by liver FBPase in hepatic and renal extracts. These results indicate that FBPase is present in a similar amount in both tissues. Moreover, the analysis revealed that the antibody detected two single bands with a MW of 36 000 and 40 000, in kidney and liver extracts, respectively (Fig. 1). The immunoreactive bands migrated at the predicted subunit molecular weight, demonstrating the high specificity of the prepared antibody. Minimal degradation of these proteins was observed on both total tissue extracts. The antibody does not react with other proteins and does not recognize the muscle isoform (Fig. 1, lane 4). Immunohistochemical detection of FBPase in kidney, using the peroxidase reaction (DAB) and light microscopy, yielded a strong signal at the proximal straight and convoluted tubules located at the cortex but not in Thirty percent of of the kidney medulla (Fig. 2). The very low immunoreaction detected in the distal convoluted tubules indicated low or non-existent expression of FBPase (Fig. 2), with the distal cells providing an internal control for the primary and secondary antibodies. At the subcellular level, FBPase showed a strong reaction in the nuclei of the proximal convoluted cells. The cytosol was also stained and showed an apical localization of the enzyme. This unusual organization of FBPase correlates with a subcellular compartmentalization of metabolic enzymes. FBPase exhibited a similar immunoreaction level in the liver. This technique is unable to quantify the amount of expressed enzymes; however, qualitative values show that FBPase is similarly expressed in proximal tubule cells and periportal hepatocytes. FBPase immunostaining was observed in the cytoplasm of the hepatocytes; however, the periportal vein and endothelial cells were negative (Fig. 2). Particularly intense staining was seen in the plasma membranes of adjacent cells (Fig. 2, Fig. 4), suggesting a special subcellular distribution of the FBPase, which could be related to the glycogen synthesis. In addition, a prominent perinuclear and nuclear staining was also observed in hepatocytes from periportal regions. Immunostaining controls replacing the primary antibody by non-immune rabbit serum showed no immune reaction (data not shown). The immunostaining pattern seen with conventional light microcopy showed the association of FBPase to the nuclei of rat liver and kidney cells. To identify the presence of FBPase inside the nuclei, we performed immunofluorescence and confocal analysis taking optical sections of 1 μm. The results revealed a clear nuclear localization of FBPase in kidney proximal tubule cells. The white arrow (Fig. 3) shows several stained nuclei through the 1 μm optical series, confirming this unexpected observation. Moreover, this analysis demonstrated that FBPase is concentrated in the apical membrane region. The nuclear localization of FBPase was detected in several kidney samples; however, not all the cells of this epithelium exhibited this staining. These results indicate that not all the proximal tubule cells are in the same metabolic state that allows the translocation of FBPase from the cytosol to the nucleus. Similar results were observed in liver, confirming the relative abundance of this enzyme in the cytoplasm of hepatocytes (Fig. 4A). The nuclear localization of FBPase in these cells was also clearly observed (Fig. 4A). Additionally, the nuclear staining was not uniform and the nucleolus was negative for FBPase staining. Furthermore, the rotation of a three-dimensional reconstruction of optical sections from hepatocytes clearly shows that FBPase is concentrated at the hepatocyte cell periphery forming uneven clumps, close to the plasma membrane (Fig. 4B). No specific staining was detected in any of the negative controls without primary or secondary antibodies against rabbit IgG (data not shown).
Discussion