Localization of the polyol pathway in the human kidney.

Sorbitol plays an important role in the osmotic regulation of the mammalian kidney. Sorbitol synthesis is regulated by the enzyme aldose reductase (AR) and its degradation to fructose is catalyzed by the enzyme sorbitol dehydrogenase (SDH). Various data exist on the polyol pathway on the rat kidney, but little is known about the distribution of the polyol pathway enzymes in the human kidney. Determination of enzyme activities and a semiquantitative determination of mRNA expression, immunohistochemistry and in-situ hybridisation in healthy human kidney tissue was carried out. The enzyme activity of AR showed a fourfold increase from cortex to papilla, while SDH-activity dropped from cortex to papilla by a factor of four. Corresponding data was obtained at the mRNA level from the semiquantitative polymerase chain reaction (PCR). Additional differentiation at the cellular level reveals both enzymes in cells of the proximal and distal tubules, thick ascending loop, thin loop and collecting duct. Studies of enzyme activity and expression by immunohistochemistry, PCR and in-situ hybridization presented corresponding results with respect to the localization of the enzymes, which match the experimental data obtained from rats very well. Thus, the established rat model might well represent the situation in the human kidney, too.

Expression of the brain and muscle isoforms of glycogen phosphorylase in rat heart.

Heart glycogen represents a store of glucosyl residues which are mobilized by the catalysis of glycogen phosphorylase (GP) and are mainly destined to serve as substrates for the generation of ATP. The brain isoform of GP (GP BB) was studied in rat heart in comparison with the muscle isoform (GP MM) to find functional analogies to the brain. Western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed that at the protein level, but not at the mRNA level, the content of GP BB is similar in heart and brain. In contrast, GP MM is more abundant in the heart than in the brain. Immunocytochemically GP BB was colocalized with GP MM in cardiomyocytes. GP MM was also detected in interstitial cells identified as fibroblasts. The physiological role of co-expression of GP BB and GP MM in cardiomyocytes and in brain astrocytes is discussed in a comparative way.

Renal expression of the brain and muscle isoforms of glycogen phosphorylase in different cell types.

Kidney contains glycogen. Glycogen is degraded by glycogen phosphorylase (GP). This enzyme comes in three isoforms, one of which, the brain isozyme (GP BB), is known to occur in kidney. Its pattern of distribution in rat kidney was studied in comparison to that of the muscle isoform (GP MM) with the aim to see if for GP BB and GP MM there were functional similarities in brain and kidney. In immunoblotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) experiments, both isozymes and their respective mRNAs were found in kidney homogenates. GP BB was immunocytochemically detected in collecting ducts which were identified by the marker protein aquaporin-2. GP MM was localized exclusively in interstitial cells of cortex and outer medulla. These cells were identified as fibroblasts by their expression of 5'-ectonucleotidase (cortex) or by their morphology (outer medulla). The physiological role of both isozymes is discussed in respect to local demands of energy and of proteoglycan building blocks.

Expression and localization of S-adenosylhomocysteine-hydrolase in the rat kidney following carbon monoxide induced hypoxia.

BACKGROUND/AIMS: Tissue hypoxia induces a variety of functional changes including enhanced transcriptional activity associated with high transmethylation activity (e.g. mRNA cap methylation) in the nucleus. It is well known that the kidney responds to hypoxia with enhanced transcription of erythropoietin (EPO) in the interstitial cells. Since S-adenosylhomocysteine (AdoHcy)-hydrolase regulates most S-adenosylmethionine (AdoMet) dependent transmethylation reactions by hydrolyzing the potent feedback inhibitor AdoHcy to adenosine and homocysteine we studied the effect of hypoxia by carbon monoxide (CO) inhalation (1200 ppm) on AdoHcy-hydrolase gene expression and its localization in rat kidneys. RESULTS: CO lowered renal AdoHcy-hydrolase mRNA expression by 64% whereas AdoHcy-hydrolase activity was not changed during 4h of CO exposure 0.7+/-0.04 mU/mg (control) vs. 0.75+/-0.06 mU/mg protein. Using two-channel immunofluorescence confocal laser scanning microscope AdoHcy-hydrolase was visualized in different cells of the hypoxic rat kidney. A very bright immunofluorescence of AdoHcy-hydrolase was observed in the nuclei of single interstitial cells of renal cortex and outer medulla which respond to hypoxia with increased EPO secretion indicating translocation of AdoHcy-hydrolase from the cytosol to the nucleus. CONCLUSIONS: These data suggest that AdoHcy-hydrolase accumulation in the nucleus of adult mammalian cells is involved in maintaining efficient transmethylation reactions in transcriptionally active cells by removing the product inhibitor AdoHcy.

Active succinate dehydrogenase (SDH) and lack of SDHD mutations in sporadic paragangliomas.

BACKGROUND: Paragangliomas are benign, slow-growing tumours of the head and neck region. The candidate gene for familial and some sporadic paragangliomas, SDHD (succinate dehydrogenase, subunit D), has been mapped to the PGL1 locus in 11q23.3. MATERIALS AND METHODS: Normal and tumour DNA of 17 patients with sporadic paragangliomas were analysed by sequencing (SDHD, SDHB and SDHC genes), fluorescence in situ hybridisation (FISH). In addition, loss of heterozygosity (LOH) and succinate dehydrogenase (SDH) enzyme activity assays were performed. RESULTS AND CONCLUSION: Only two patients from our collective showed SDH gene mutations, one in SDHD and one in SDHB, respectively. Moreover, SDH activity detected in 5/8 patients confirmed the fact that SDH inactivation is not a major event in sporadic paragangliomas. LOH and FISH analysis demonstrated a frequent loss of regions within chromosome 11, indicating that additional genes in 11q may play a role in tumour genesis of sporadic paragangliomas.

Fluorescence-labeled sphingosines as substrates of sphingosine kinases 1 and 2.

Fluorescently labeled d-erythro-sphingosines have been successfully synthesized in 9 and 12 steps starting from commercially available Garner aldehyde. Determining the influence of the nature, position and linkage of the label on the in vitro phosphorylation rate by sphingosine kinases 1 and 2 resulted in the identification of a pyrene- and a NBD-labeled sphingosine which are both phosphorylated with efficiency comparable to the natural substrate.

Modulation of the endosomal and lysosomal distribution of cathepsins B, L and S in human monocytes/macrophages.

Endosomal and lysosomal fractions of human monocytes/macrophages prepared from buffy coats were analyzed for activities of cathepsins B, L and S, and expression of cathepsin proteins along with major histocompatibility complex class I and class II molecules under control and immunomodulatory conditions. While the total activity of cathepsins B, L, and S together remained unchanged in lysates of control cells during culture for 72 h, the subcellular distribution of cathepsin activities underwent a shift from a predominantly endosomal localization in freshly isolated cells to a lysosomal pattern after 72 h of culture. Interferon-gamma treatment for 72 h resulted in an upregulation of both major histocompatibility complex proteins and cathepsins with differential changes in cathepsin B, L and S activities in endosomes versus lysosomes. These changes suggest a remodeling of the endocytic machinery and imply different functions of cathepsins B, L and S during monocyte differentiation.

Cathepsin S activity is detectable in human keratinocytes and is selectively upregulated upon stimulation with interferon-gamma.

Keratinocytes are an integral component of the skin immune system and function as nonprofessional antigen-presenting cells in pathophysiologic conditions when they express major histocompatibility complex class II molecules, e.g., in psoriasis. In order to analyze further this function we investigated the activity of cathepsin S in comparison with cathepsins B and L. These enzymes were suggested to be involved in antigen presentation. Specific catalytic activities of these cathepsins were determined fluorometrically by hydrolysis of a synthetic substrate (Z-Phe-Arg-7-amido-4-methylcoumarin) in subcellular fractions of human keratinocytes. It was found that the human keratinocyte cell line HaCaT exhibits activities of all three cathepsins investigated. Endosomal/lysosomal compartments show highest cathepsin activities. Normal human keratinocytes in primary culture show a comparable pattern of cathepsin activities. In contrast to this, in syngeneic Epstein-Barr virus-transformed B cells the level of cathepsin B activity was found to be 10% of that in the corresponding keratinocytes, whereas the activities for cathepsins L and S were in a similar range. Interferon-gamma stimulation of primary keratinocytes and HaCaT cells resulted in a selective upregulation of the cathepsin S activity, the extent of which was very similar. The mechanism of this upregulation was demonstrated as induction at the mRNA and protein levels. This report documents that cathepsin S in human keratinocytes is selectively upregulated, in parallel to major histocompatibility complex class II molecules, in response to a pro-inflammatory cytokine. Our observations support the concept of keratinocytes functioning as nonprofessional antigen-presenting cells in states of inflammation.