1 Department of Pathology, 2 Biostatistical Unit, Bambino Gesù Children's Hospital-Research Institute, Rome, Italy and 3 Department of Pathology, U.Z.Katholieke Universiteit, Leuven, Belgium
Correspondence and offprint requests to: Prof. B. Van Damme, Dienst Pathologische Ontleedkunde, U.Z.-K.U.Leuven, MInderbroedersstraat, 12, B3000 Leuven Belgium. Email: bo.vandamme{at}uz.kuleuven.ac.be
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Abstract |
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Methods. We investigated protein gene product 9.5 and ubiquitin expression in both normal and pathologic renal samples (more than 100 cases) using an immunohistochemical technique.
Results. We found that protein gene product 9.5 and ubiquitin were constantly present in Bowman's capsule parietal cells and tubular/collecting duct epithelial cells, with the strongest positivity in metabolically active and proliferative conditions, such as tubular hypertrophy, cellular regeneration and crescent formation. Conversely, the expression of these molecules was attenuated in atrophic tubules. Podocytes were negative.
Conclusion. The diffuse presence of the protein gene product 9.5 and ubiquitin in normal and pathologic metabolically active epithelial cells of the nephron suggests that these proteins (and likely the whole ubiquitinproteasome complex) play a fundamental role in the mechanism upregulating protein metabolism of the kidney and that its expression is correlated with activated cellular functions, like proliferation.
Keywords: immunohistochemistry; kidney; non-lysosomal proteolysis; parietal epithelial cells of Bowman's capsule; protein gene product 9.5; ubiquitin
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Introduction |
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Intracellular protein degradation is a tightly regulated process maintaining the normal cellular homeostasis. Multiple systems exist for proteolysis, and the best-described is the highly conserved ubiquitinproteasome one [24]. This complex is a non-lysosomal proteolytic pathway that degrades diverse cellular proteins in the regulation of cell growth, in the modulation of some membrane receptors, in response to heat shock, and in the turnover of cytoskeletal elements. It comprises the enzymes that ubiquitinate/de-ubiquitinate target proteins and the 26S proteasome complex that degrades ubiquitin-conjugated proteins [5].
Ubiquitination represents the basic cellular process and consists of the covalent attachment of multiple ubiquitin molecules to the protein substrate [3]. This is a reversible step controlled at several levels. One level of regulation involves the large family of de-ubiquitinating enzymes (DUBs) [3]: ubiquitin COOH-terminal hydrolases (UCHs) and ubiquitin-specific processing proteases (UBPs). These enzymes cleave monoubiquitin from proteins and disassemble polyubiquitin chains released from substrates during degradation in the proteasome. Such actions allow recycling of ubiquitin for subsequent use in the conjugation reaction. They can also control the turnover of specific proteins by removing ubiquitin or polyubiquitin chains from the substrate, thus avoiding its degradation by the proteasome.
PGP 9.5 is a member of the UCH family. Immunohistochemical studies revealed its localization in all central and peripheral neurons as well as in cells of the diffuse neuroendocrine system [1,6]. Protein expression has also been described for a small number of non-neuronal tissues, including cells of the ovary, testis, synovial membrane and kidney [611].
Studies involving kidney cells or animal models of renal diseases have been used to highlight the role of UCH family enzymes in physiologic/pathologic conditions [1215]. Some recent papers showed PGP 9.5 expression in the early steps of rat nephrogenesis, and in the newborn and adult rat kidney. A role in kidney development, cellular differentiation of growing renal tubules [12,13] and in the cell cycle of parietal epithelial cells of Bowman's capsule has been suggested [10].
Few data are published concerning DUBs expression and function in the human kidney [6,7,11], except for a generic correlation between alterations in proteolytic/cofactor enzymes and the occurrence of certain renal diseases, such as acute ischaemic renal failure, renal hypertrophy, von HippelLindau disease and neoplastic transformation of renal cells [1214].
The paucity of published data necessitated the present study, in which we report the immunohistochemical localization of PGP 9.5 and ubiquitin in normal human kidney and in an unselected series of more than one hundred renal biopsies, and question its possible role in regulating some cellular functions in this organ.
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Material and methods |
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Immunohistochemistry was carried out on 4 µm sections from formalin-fixed paraffin-embedded tissue and performed according to standard techniques. We applied a polyclonal anti-PGP 9.5 antibody at 4°C overnight (1:1000, Chemicon International) and a polyclonal anti-ubiquitin antibody for 30 min (1:200, Dako). As chromogen, we used 3,3'-diaminobenzidine for single stains, and 3-amino-9-ethylcarbazole plus 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium chloride for double stains (Dako). Sections without primary antibodies served as a negative control. The specificity of PGP 9.5 staining was demonstrated by the following experiments: (a) when the primary antibody was skipped, no stain of the parietal epithelial cells, tubular epithelium or nerves was shown; (b) when a fluoresceinated antibody was used in the second layer, the parietal epithelial cells, tubular cells and nerves were stained; (c) when the primary antibody was incubated with 10 µl of antigen (recombinant human PGP 9.5 protein, ProSpec-Tany TechnoGene LTD, P.O. Box 398 Rehovot 76103, Israel) during 30 min at room temperature, centrifuged and applied to sections, all positive staining was abolished. Absorption of 10 µl of antibody with less than 0.05 µg antigen did not abolish the positive staining.
Two pathologists, without knowledge of clinical data, evaluated the immunoreactivity independently and semiquantitatively. The staining was scored using a four-tiered scale: negative (05% of positive cells), 1+ (620% of positive cells), 2+ (2150% of positive cells) and 3+ (>50% of positive cells). In very heterogeneous samples, intermediate values were also considered.
Perirenal/perivascular nervous fibre positivity functioned as an internal positive control and was arbitrarily classified as a strong reaction (3+).
Immunoreactivity was separately assessed for the different nephron compartments: glomeruli (parietal epithelial cells, visceral epithelial cells [podocytes], mesangial cells and capillary tuft endothelial cells); tubular structures (proximal and distal tubules, Henle's loops and collecting ducts); and renal stroma (fibroblasts, inflammatory cells and vessels).
Expression variability was observed in tubular structures, even in the same biopsy, regardless of the diagnosis. We decided to further analyze this aspect dividing the tubular compartment of each sample according to a morphological/functional parameter [15]: normal appearing, hypertrophic/regenerating and atrophic/damaged tubules. Briefly, tubules are considered as hypertrophic when characterized by irregular contours and lined by columnar eosinophilic cells often containing hyaline protein resorption droplets at the apex; nuclei may show margination of chromatin along the nuclear membranes and contain prominent nucleoli. Regenerating tubules show mild dilatation of the lumen, cellular dedifferentiation, nuclear polymorphism and numerous mitoses. Atrophic tubules are characterized by a small lumen, often containing PAS-positive casts, thick and wrinkled basement membrane; cuboidal epithelium with lipofuscin pigment and peritubular fibrosis and inflammation. Damaged tubules show lumen dilatation and cytoplasmic swelling or flattening of epithelium; there may be apoptotic nuclei, individual cell necrosis and focal denudation of the basement membrane; shedding of both necrotic and viable cells into the tubular lumen is a typical feature.
We then gave the same above-mentioned score to each of the three groups for both PGP 9.5 and ubiquitin. The final values were compared by means of Friedman's two-way analysis of variance by ranks and by Wilcoxon matched pairs signed-rank test. A multivariate regression analysis was used in order to take into account the possible effect of diagnosis on PGP 9.5 and ubiquitin expression.
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Results |
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Discussion |
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While much attention has been paid to characterizing the enzymes responsible for adding ubiquitin to proteins [35,16], little is known about deubiquitinating enzymes [1,6]. Indeed, in the biochemical level, the exact chemical reactions catalyzed by each component of the DUB family and the effective sequence of each interaction are still unclear.
Concerning PGP 9.5, we know that it removes ubiquitin from proteins undergoing degradation and allows recycling of free ubiquitin from the conjugation products. Some studies have suggested that PGP 9.5 can induce enhancement of cell metabolism and proliferation by controlling the proteolysis of some cell cycle inhibitors, such as the inhibitor of cyclin-dependent kinases p27 [4,17,18].
The statement of correlation between PGP 9.5 expression and activated cellular functions is in accordance with our results in at least two points.
Firstly, PGP 9.5 expression was present in normal proximal and distal tubules, the part of the nephron characterized by active re-absorption, secretion, endocytosis and molecular transport. PGP 9.5 positivity was even more evident in hypertrophic tubules, a condition of compensatory hyperfiltration and metabolic overcharge. Similar strong immunoreaction was observed in tubules with features of increased proliferative activity and regeneration. Coherently, in conditions of decreased cellular activity, such as in atrophic or damaged tubules, the immunoreaction was weaker.
Concerning the prevalent mesangial cell negativity, our results support the general concept that these elements (and other mesenchymal cells, like fibroblasts, smooth muscle cells and macrophages) use other pathways to control the cellular metabolic state, like activation of phosphatidylinositol 3-kinase and the MAPKs pathway for mesangial cell hypertrophy [19], and of the lysosomal compartment for proteolytic degradation [12]. If ever the proteasome complex is active in these cells, PGP 9.5 expression level is likely below the limit of detectability of the immunohistochemical technique.
The observation of a variable intensity of PGP 9.5 and ubiquitin expression in the same biopsy, in the same tubule and even in the same cell, is likely to be related to a normally variable metabolic state of the epithelial cells in the nephron [15].
Secondly, PGP 9.5 positivity in parietal epithelial cells and in cellular crescents, and its negativity in podocytes paralleled the different proliferative capacity of these cells. In fact, although parietal and visceral epithelial cells develop from the same precursor, the former exhibit an active proliferation and metabolism even in normal conditions, whereas the latter are terminally differentiated and unable to replicate, except for some peculiar conditions [20].
Even though the specific function of PGP 9.5 is not determined in our data, the diffuse expression of this enzyme and its substrate in metabolically active epithelial cells of the nephron, suggests that PGP 9.5, ubiquitin and, likely, the whole ubiquitinproteasome complex play a fundamental role in the mechanism upregulating protein metabolism and proliferation of renal epithelial cells, in both normal and pathologic conditions.
Finally, we would like to stress that, in contrast to the paper of Shirato et al. [10], PGP 9.5 is not a specific cytochemical marker of parietal epithelial cells of Bowman's capsule, at least in human kidney. However, it may be useful in cell cultures from isolated glomeruli for the selective distinction of parietal epithelial cells from podocytes.
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Acknowledgments |
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Conflict of Interest Statement. All authors, Francesca Diomedi-Camassei, Lucilla Ravà, Evelyne Lerut, Francesco Callea Boudewijn Van Damme, declare to not have had any involvements that might raise the question of bias in the work reported or in the conclusions, implications or opinions stated. Leuven, 21 June 2005, Rome, 21 June 2005.
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References |
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