Protein gene product 9.5 and ubiquitin are expressed in metabolically active epithelial cells of normal and pathologic human kidney

Francesca Diomedi-Camassei1, Lucilla Ravà2, Evelyne Lerut3, Francesco Callea1 and Boudewijn Van Damme3

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



   Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. In a study initially designed to evaluate the specific protein gene product 9.5 expression in parietal epithelial cells of Bowman's capsule, a marked positivity was also observed in the tubular and collecting duct epithelial cells. Since protein gene product 9.5 is an important enzyme in the ubiquitin system of proteolysis, and plays a regulatory role in cell cycle and proliferation, its presence in specific segments of the nephron was of considerable interest.

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 ubiquitin–proteasome 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



   Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Protein gene product 9.5 (PGP 9.5) is a soluble 27 kDa protein corresponding to an ubiquitin COOH-terminal hydrolase that plays a modulating role in intracellular proteolysis [1].

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 ubiquitin–proteasome one [2–4]. 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 [6–11].

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 [12–15]. 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 Hippel–Lindau disease and neoplastic transformation of renal cells [12–14].

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.



   Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
For the immunohistochemical study, normal kidney obtained from the pole opposite a renal tumour in 5 nephrectomies and 111 consecutive unselected renal biopsies from December 2003 to April 2004 were collected at the Department of Pathology of the Katholieke Universiteit of Leuven (Belgium). The material used was leftover from biopsies taken for clinical reasons. The permission to use the tissue for research purposes is asked in a general way at the moment of admission of the patient to the hospital. No patient data except the final diagnosis were used.

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 (0–5% of positive cells), 1+ (6–20% of positive cells), 2+ (21–50% 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.



   Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The examined series included patients aged from 5 to 82 years (mean 51±18; median 54). Among 111 consecutive renal biopsies, 69 were obtained from a native kidney and 42 from a transplanted graft. The most frequent pathology observed in the native kidney was primary glomerulonephritis (20 crescentic, 8 IgA, 5 minimal changes, 4 membranous and 2 focal and segmental sclerosis), followed by 10 systemic/metabolic diseases (3 diabetes, 2 amyloidosis, 2 systemic lupus, 2 obesity), 7 tubulo-interstitial nephritis, 7 vascular conditions (1 ischaemic, 2 hypertension, 3 acute tubular necrosis, 1 haemolytic-uraemic syndrome), 6 end-stage kidney diseases and 1 recessive polycystic disease. Among transplanted kidneys, 13 were base-line biopsies (all normal), and twenty nine were taken for monitoring the graft (seven without alterations, ten acute rejection, nine chronic rejection/treatment toxicity and two recurrent glomerulonephritis). At immunohistochemical examination, PGP 9.5 and ubiquitin revealed a co-expression and showed a similar pattern of positivity, with the only difference that ubiquitin was more diffusely, but less intensely expressed in comparison to PGP 9.5 (Figure 1A and B). The immunoreaction was diffusely positive throughout the whole series and we could not appreciate important expression differences of the hydrolytic enzyme and its substrate among samples of normal, pathological and transplanted kidneys, except for crescents and tubular atrophy in comparison with parietal epithelial cells and tubules of normal kidney (P = 0.01 and P<0.001, respectively) (Table 1).



View larger version (94K):
[in this window]
[in a new window]
 
Fig. 1. (A) PGP 9.5 (blue/black) and ubiquitin (red) expression in normal kidney surrounding a tumour. There is a strong and diffuse tubular co-expression of both antigens, with a variable intensity. A rim of parietal epithelial cells of Bowman's capsule is positive (star). Podocytes, mesangial cells and vascular structures of the glomerulus are negative, as well as interstitial capillary endothelium and stromal cells. Note two positive small nervous fibres in the interstitum as positive control (arrows) (magnification 20x). (B) PGP 9.5 and ubiquitin coexpression in a tubular structure at higher magnification (40x). Double stain chromogens: 3-amino-9-ethylcarbazole and 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium chloride. No nuclear counterstain.

 

View this table:
[in this window]
[in a new window]
 
Table 1. PGP 9.5 and ubiquitin expression observed in the different renal compartments (0 = –; 0.5 = +/–; 1 = +; 1.5 = +/++; 2 = ++; 2.5 = ++/+++; 3 = +++). Different pathologies with similar microscopic features were grouped together (normal/mild alterations: 18 normal kidney; 8 transplanted kidney with no signs of either rejection or allograft nephropathy; 5 native kidney biopsies with mild alterations or minimal change disease).

 
More in detail (Figures 1 and 2), the most striking feature was the strong positivity of proximal and distal tubules. A variability of immunostaining intensity was observed among different biopsies and among different tubules of the same biopsy; however, we could generally notice that hypertrophic or regenerating tubules showed a strong positivity, whereas atrophic or damaged ones were less immunoreactive. Henle's loops and collecting ducts were variably positive. Pelvic transitional epithelium, wherever observed, was negative. In the glomeruli, the characteristic finding was the positivity of parietal epithelial cells, although with less intensity in comparison with tubules, and the negativity of both podocytes and capillary endothelial cells. Mesangial cells showed a focal and weak staining in few samples, prevalently in intracapillary proliferative conditions. Crescents stained strongly in both cellular and fibrotic types, as the antibodies reacted with parietal epithelial cells and fibroblasts [9], respectively. In sclerotic glomeruli, scattered cells with morphologic features of fibroblasts were positive. In the interstitium, stromal fibroblasts were mildly positive, and became more intensely stained in fibrotic and sclerosing pathologies. Mononuclear and polymorphonuclear inflammatory cells as well as interstitial capillaries were predominantly negative. Larger vessels showed a weak immunostaining of media smooth muscle cells and of thickened intima cells, while endothelial cells were non-reactive.



View larger version (85K):
[in this window]
[in a new window]
 
Fig. 2. (A) PGP 9.5 expression in acute tubular necrosis. Regenerating tubules characterized by cellular dedifferentiation and proliferation, mitoses (arrows) and nuclear polymorphism show positivity of the lining epithelial cells. Two damaged tubules on the lower right (star), characterized by desquamating cells, focal nuclear effacement and swollen cytoplasm are weakly positive. Parietal epithelial cells of Bowman's capsule moderately stain. There is some cellular debris in the urinary space (magnification 20x). (B) Ubiquitin expression in a crescentic glomerulonephritis, vasculitis type. The strong immunoreaction of proliferating parietal cells forming the cellular crescent is evident as well as the positivity of two normal appearing tubules in the upper part. Flattened epithelium of a group of dilated tubules stains less intensely (star). Scattered fibroblasts in the inflamed interstitium show a moderate positivity (magnification 20x). (C) PGP 9.5 expression in a transplanted kidney with chronic rejection. Hypertrophic tubules in the upper part show an intense immunoreaction, while scattered atrophic ones (arrows) are weakly positive or even negative immunoreaction. Some fibroblasts in the sclerotic glomerulus (star), in the thickened vessel (arrowhead) and in interstitium are moderately positive (magnification 10x). (chromogen: 3,3'-diaminobenzidine; nuclear counterstain: haematoxylin).

 
Mean and median values of PGP 9.5 and ubiquitin expression in the functionally different tubular compartments are reported in Table 2. Friedman's two-way analysis of variance evidenced a statistical significant difference in PGP 9.5 and ubiquitin expression among hypertrophic, normal and hypotrophic tubules (P<0.001), confirmed by the pairwise comparison performed through Wilcoxon matched pairs signed-rank tests (P<0.001). The multivariate regression analysis gave no evidence of any effect of diagnosis on PGP 9.5 and ubiquitin expression.


View this table:
[in this window]
[in a new window]
 
Table 2. Tubular PGP 9.5 and ubiquitin (Ubq) expression according tomorphological/functional parameters (see the Methods section; more than one possibility may occur simultaneously). Statistical significant difference of PGP 9.5 and ubiquitin expression among the 3 groups, P<0.001 for both antigenes

 


   Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
This study demonstrated the strong and diffuse expression of the deubiquitinating enzyme PGP 9.5 and its substrate ubiquitin in many epithelial compartments of human kidney, regardless of normal or pathologic conditions.

While much attention has been paid to characterizing the enzymes responsible for adding ubiquitin to proteins [3–5,16], 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 ubiquitin–proteasome 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.



   Acknowledgments
 
The authors are grateful to Dr Gianfranco Rizzoni for critical comment on the manuscript and to Dr Giuseppe Floris for his technical support.

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.



   References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

  1. Wilkinson KD, Lee KM, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J. The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 1989; 246: 670–673[ISI][Medline]
  2. Pickart CM. Back to the future with ubiquitin. Cell 2004; 116: 181–190[CrossRef][ISI][Medline]
  3. Hershko A, Ciechanover A, Varshavsky A. Basic Medical Research Award. The ubiquitin system. Nat Med 2000; 6: 1073–1081[CrossRef][ISI][Medline]
  4. Duncan JC. Proteolysis and the cell cycle. Cell Cycle 2002; 1: 233–234[Medline]
  5. Itoh H, Matsuoka M, Steber CM. A role for the ubiquitin-26S-proteasome pathway in gibberellin signaling. Trends Plant Sci 2003; 8: 492–497[CrossRef][ISI][Medline]
  6. Bradbury JM, Thompson RJ. Immunoassay of the neuronal and neuroendocrine marker PGP 9.5 in human tissues. J Neurochem 1985; 44: 651–653[ISI][Medline]
  7. Wilson PO, Barber PC, Hamid QA et al. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol 1988; 69: 91–104[ISI][Medline]
  8. Kitamura HP, Yanase H, Kitamura H, Iwanaga T. Unique localization of protein gene product 9.5 in type B synoviocytes in the joints of the horse. J Histochem Cytochem 1999; 47: 343–352[Abstract/Free Full Text]
  9. Olerud JE, Chiu DS, Usui ML, Gibran NS, Ansel JC. Protein gene product 9.5 is expressed by fibroblasts in human cutaneous wounds. J Invest Dermatol 1998; 111: 565–572[CrossRef][ISI][Medline]
  10. Shirato I, Asanuma K, Takeda Y, Hayashi K, Tomino Y. Protein gene product 9.5 is selectively localized in parietal epithelial cells of Bowman's capsule in the rat kidney. J Am Soc Nephrol 2000; 11: 2381–2386[Abstract/Free Full Text]
  11. D’Andrea V, Malinovsky L, Berni A et al. The immunolocalization of PGP 9.5 in normal human kidney and renal cell carcinoma. G Chir 1997; 18: 521–524[Medline]
  12. Franch HA. Pathways of proteolysis affecting renal cell growth. Curr Opin Nephrol Hypertens 2002; 11: 445–450[CrossRef][ISI][Medline]
  13. Debigaré R, Price SR. Proteolysis, the ubiquitin-proteasome system, and renal diseases. Am J Physiol Renal Physiol 2003; 285: F1–F8[Abstract/Free Full Text]
  14. Kanayama H, Tanaka K, Aki M et al. Changes in expressions of proteasome and ubiquitin genes in human renal cancer cells. Cancer Res 1991; 51: 6677–6685[Abstract]
  15. Jenette JC, Olson JL, Schwartz MM, Silva FG. Heptinstall's Pathology of the Kidney. Section I, Chapters 3 and 4. Lippincott-Raven Publishers, Philadelphia, PA: 1998: 85–167
  16. Contino G, Amati F, Pucci S et al. Expression analysis of the gene encoding for the U-box-type ubiquitin ligase UBE4A in human tissues. Gene 2004; 328: 69–74[CrossRef][ISI][Medline]
  17. Pagano M, Tam SW, Theodoras AM et al. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 1995; 269: 682–685[ISI][Medline]
  18. Giambanco I, Bianchi R, Ceccarelli P et al. ‘Neuron-specific’ protein gene product 9.5 (PGP 9.5) is also expressed in glioma cell lines and its expression depends on cellular growth state. FEBS Lett 1991; 290: 131–134[CrossRef][ISI][Medline]
  19. Goruppi S, Bonventre JV, Kyriakis JM. Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy. EMBO 2002; 21: 5427–5436[Abstract/Free Full Text]
  20. Laurinavicius A, Rennke HG. Collapsing glomerulopathy-a new pattern of renal injury. Semin Diagn Pathol 2002; 19: 106–115[ISI][Medline]
Received for publication: 26. 7.05
Accepted in revised form: 12. 8.05





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
20/12/2714    most recent
gfi124v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Diomedi-Camassei, F.
Articles by Van Damme, B.
PubMed
PubMed Citation
Articles by Diomedi-Camassei, F.
Articles by Van Damme, B.