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C-erb B-2 amplification in cystic renal disease
Kidney International, Vol. 40 (1991), PP. 509—5 13
C-erb B-2 amplification in cystic renal disease GUILLERMO A. HERRERA Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, USA
C-erb B-2 amplification in cystic renal disease. Gene amplification 4.8-kD messenger RNA that translates into a 185 kD protein. (overexpression) of c-erb B-2 was tested in a variety of cystic renal The protein product of c-erb B-2 has extracellular, transmemdiseases, renal cell neoplasms (adenomas and carcinomas) and end brane, and intracellular domains; the intracellular domain has stage kidneys without cysts. C-erb B-2 encodes a receptor-like protein that shares homology with, but is distinct from the epidermal growth factor (EGF) receptor. A monoclonal antibody that immunoprecipitates
tyrosine kinase activity. C-erb B-2 encodes a membrane-bound
receptor for an as yet unknown extracellular ligand that is
a protein of approximately 185 kD from a lysate of NLH/3T3 cells
widely assumed to be a cellular growth factor. C-erb B-2 lies on human chromosome 17. Amplification of the c-erb B-2 has been cysts, cystic renal dysplasia, autosomal recessive polycystic kidney demonstrated in human breast, ovarian and pancreatic carcinodisease (ARPKD), and non-cystic, essentially normal kidneys failed to show c-erb B-2 overexpression. In contrast, autosomal-dominant poly- mas and some studies have suggested that c-erb B-2 amplificacystic kidney disease (ADPKD), acquired (dialysis-associated) cystic tion may have significant prognostic implications [8—161. A disease (ACD), non-cystic end stage kidneys and renal cell neoplasms similar factor may play a pivotal role in the genesis, developrevealed overexpression of c-erb B-2 with some frequency (40% or more of cases tested). Three cystic disorders revealing c-erb B-2 ment and neoplastic potential of some cystic renal conditions. overexpression also showed platelet-derived growth factors (PDGFs) Analysis for the c-erb B-2 can be performed by immunohistochemistry. Monoclonal antibodies can recognize the c-erb B-2 expression in similar locations (cyst lining and adjacent tubules). Other growth factors [insulin-like growth factor (IGF-1), fibroblast growth protein and can detect onco-protein overexpression [71. transfected with the c-erb B-2 gene was utilized for testing. Simple renal
factor (FGF) and beta transforming growth factor (TGF/3)J were not noted to be overexpressed in either c-erb B-2 positive or negative cystic diseases. C-erb B-2 may be a marker related to the proliferative/growth capabilities of selected cystic diseases, including potential for associated genesis of benign and malignant renal cell tumors.
Methods
Wedge sections (including cortex and medulla) from both renal poles (2 each) from forty formalin fixed, paraffin-embedded blocks from a variety of cystic renal diseases, end stage
Renal cysts are a central pathologic feature in several renal diseases with varied etiologies. Understanding the formation of renal cysts has received significant attention for centuries; however, the mechanisms involved in their development and neoplastic transformation in a number of them are not well understood [1, 2]. The formation of cysts must be accompanied by hyperplasia of the lining epithelium to keep pace with the enlarging surface area of the cystic cavities. In some cases neoplastic transformation may occur in the lining epithelium and some cystic renal diseases are associated with renal cell adenomas and carcinomas. Understanding of the underlying molecular pathology of renal cystic diseases is just beginning. It appears that elucidation of the role of associated genes and proto-oncogenes involved may be an important issue. The role of epidermal growth factor has been a recent line of investigation [3—5]. C-erb B-2, a closely related proto-oncogene [61, may also play an important role. The erb B proto-oncogene encodes a receptor-like protein that shares homology with but is distinct from the epidermal growth factor [6, 7]. The c-erb B-2 oncogene transcribes a
kidneys, renal cell adenomas and carcinomas were routinely sectioned and stained with hematoxylin and eosin, as well as immunostained using an avidin-biotin peroxidase complex technique according to Hsu, Raine and Fanger [17] with a c-erb B-2
monoclonal antibody from Triton Biosciences Inc., Alameda, California, USA. This antibody stains frozen and paraffinembedded tissues and cell lines overexpressing the c-erb B-2 gene. In all cases the sections included cortical and medullary areas. Four immunostaining sections were cut at 4 microns, deparaffinized and hydrated through xylenes and graded alcohols to distilled water prior to immunostaining. Sections on
slides were then incubated at 37°C for five minutes in 3% hydrogen peroxide to block endogenous peroxidase activity in tissues. The slides were subsequently incubated with the primary antibody (1/9 dilution) for one hour, the linking avidin-
biotin complex (ABC), and later incubated again with the substrate (diaminobenzidine-DAB) for color development. The sections were then counterstained with aqueous hematoxylin and immersed in ammonia water for bluing. Below and in Table 1 are listed details pertaining to the antibody used for testing. Hematoxylin and eosin stained sections were also prepared for
routine morphologic evaluation. At least six sections were Received for publication February 4, 1991 and in revised form April 30, 1991 Accepted for publication May 1, 1991
© 1991 by the International Society of Nephrology
available for evaluation in each case. Three essentially normal, non-cystic kidneys similarly processed, treated and immunostained were also tested for evidence of c-erb B-2 amplification. The various cystic kidneys were classified using well-described combined histopathologic and clinical parameters following
509
Herrera: C-erb B-2 in cystic renal disease
510
Table 2. C-erb B-2 expression
Table 1. Characteristics of antibodies
Antibody to C-erb B-2
Platelet-derived growth factor A Platelet-derived growth factor B Insulin-like growth factor I Fibroblast growth
factor
Transforming
growth factor l
Obtained from
Dilution
Type
Triton Biosciences, Inc., Alameda, California Genzyme, Boston, Massachusetts Genzyme, Boston, Massachusetts Accurate Chemical Corporation, Westbury, New York Genzyme, Boston, Massachusetts Accurate Chemical Corporation, Westbury, New York
1:9
Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal
1:50
1:50 1:50
1:50 1:50
Mouse monoclonal Mouse monoclonal
Specimens Number Percent tested positive positive Non-cystic, essentially normal kidneys Simple cysts Cystic renal dysplasia Infantile polycystic renal disease Adult polycystic renal disease Acquired cystic renal disease End-stage renal disease without cysts Renal cell adenomas Renal cell carcinomas Total a One
3 7 4 3 3 8° 8 3° 5
44
0 0 0 0
0% 0% 0% 0%
3 5
100%
4 2 2 16
63% 50% 66% 40% 36%
case of acquired cystic renal disease also had a renal cell
adenoma (counted as one case but entered twice above).
Results Results were evaluated in a blind fashion. C-erb B-2 amplifiBernstein criteria 118] into well recognized groups including cation was detected by immunocytochemical demonstration of ARPKD, ADPKD, ACD, simple (retention) cysts and cystic the protein product in the lining epithelium of 30% to more than 90% of the cysts and surrounding tubules in the renal cortex. renal dysplasia. In addition, six fresh, snap-frozen sections from kidney with This percentage was highest for ACD (90% or more), followed cystic and neoplastic disorders (three ACD, one simple cyst, by renal cell carcinoma, (50% or more) and lowest for ADPKD one ADPKD and one renal cell carcinoma) and sections from and end stage kidneys without cysts (approximately 30%). The two essentially normal (non-cystic) kidneys used as controls tubules showing c-erb B-2 overexpression were primarily proxwere sectioned and stained using a similar immunoperoxidase imal tubules with distal tubules comprising a minority of the
procedure as outlined above [detailed in Reference 17] and tested for the expression of insulin-like growth factor (IGF-I), platelet-derived growth factors (PDGFs) A and B, fibroblast growth factor (FGF) and transforming growth factor beta (TGF/3). The monoclonal antibodies to PDGFs A and B and FGF were obtained from Genzyme Co. (Boston, Massachusetts, USA) and those to IGF-I and TGF/3 from Accurate
positive tubules. No medullary staining was identified. The entire cystic nephrons were similarly stained. No staining was
10 minutes and placed in acetone for fixation. The sections were
cytoplasmic matrix of reacting cells. The staining pattern within
detected on glomeruli or in the renal vasculature. The degree of staining was slightly variable from case to case but no repetitive
pattern could be recognized in relationship to specific conditions. No attempt was made at determining the intensity of staining, however, staining was well defined in the positive Chemical Co. (Westbury, New York, USA); all were used at a cases. The presence or absence and the extent of staining were 1/50 dilution. Sections form both poles of the kidneys were the only documented findings. Staining was noted associated examined. Cryostat sections were cut at 4 microns, air dried for with the cell membranes and also identified diffusely in the
incubated for five minutes at 37°C on a stainplate with 3% individual cysts was rather uniform. Two independent observers confirmed the presence and absence of staining in a doubleblind fashion. Staining was noted as detailed in Table 2, and was present in all sections from a given case immunostained. Simple avidin-biotin complex (ABC) and the substrate (DAB) for color (retention cysts), cystic renal dysplasia, ARPKD and some end development. The slides were then counterstained with aque- stage kidneys failed to show c-erb B-2 overexpression (Fig. 1). ous hematoxylin and immersed in ammonia water for bluing. However, the three ADPKD tested (Fig. 2A), 63% of ACD (Fig. Characteristics of the antibodies, including source and dilution 2B, 2C, 3A), 50% of end stage kidneys without cysts, 66% of renal cell adenomas and 40% of renal cell carcinomas (Figs. 2 to used are listed in Table 1. 5) revealed c-erb B-2 overexpression. The one end stage kidney Characteristics of the c-erb B-2 monoclonal antibody with ACD and a renal adenoma showed c-erb B-2 overexpresThe monoclonal antibody c-erb B-2 immunoprecipitates a sion in neoplastic areas, cysts lining and adjacent tubules (Fig. protein of approximately 185 kD from a lysate of NIH/3T3 cells 4). The non-neoplastic renal parenchyma in cases with neotransfected with the c-erb B-2 gene 1191, while an isotype plasms and the non-cystic areas of c-erb B-2 positive kidneys control antibody failed to precipitate this protein. No 185 kD also showed focally c-erb B-2 overexpression in some tubules, protein bands were precipitated from a non-transfected NIH! primarily proximal (Fig. 5). The three non-cystic, functionally 3T3 cell lysate. This monoclonal antibody reacted with cells viable, essentially normal control kidneys failed to show c-erb overexpressing c-erb B-2 and did not immunoprecipitate EGF- B-2 amplification (Fig. 1D). Staining for PDGFs, primarily B, was noted in the cells lining receptor protein (170 kD) from lysates of A 431 cells, nor did it immunocytochemically stain the A 431 cell line [7, 19]. Addi- cysts and in surrounding tubules in three non-neoplastic kidtionally, this monoclonal antibody was shown to recognize the neys (2 ACD and 1 ADPKD) and one renal cell carcinoma also external domain of the c-erb B-2 gene from radiolabeled per- showing staining for c-erb B-2 in similar locations, however, it was much more widespread and intense in the ACD and renal manently transfected CHO cells [71.
hydrogen peroxide to block endogenous peroxidase activity in tissues. Test samples, known positive and negative controls were incubated for an hour with the primary antibody, the
511
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Herrera: C-erb B-2 in cystic renal disease
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Fig. I. (A) Cystic dysplastic kidney, (B) simple cyst, (C) end stage kidney without cysts, (13) normal kidney. Avidin-biotin peroxidase complex (ABC) stain for c-erb B-2. x 300. No evidence of c-erb B-2
4
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Fig. 2. (A) Adult Onset polycystic kidney x 400 (B & C). Acquired cystic disease. Non-cystic areas X 200 (B) and cyst lining x 400 (C). Avidin-biotin peroxidase (ABC) stain for c-erb B-2. Staining for c-erb B-2 is noted in the lining of cysts of an adult polycystic kidney (A) and in the non-cystic and cystic areas (B & C) an end stage kidney with acquired cystic disease. Fig. 3. Acquired cystic disease. Avidin-biotin peroxidase complex (ABC) stain for c-erb B-2 (A), and for PDGF (B). x 300. Intense c-erb B-2 staining in cyst lining and adjacent tubules is shown in A. Same kidney stained for PDGF shows similar staining pattern, although less intense (B). Minimal staining is also noted in the interstitial cells. Fig. 4. (A) Renal cell adenoma arising in acquired cystic kidney disease. (B) Adjacent cystic kidney. Avidin-biotin peroxidase complex (ABC) stain for c-erb B-2. x 300. Staining is identified in the majority of the neoplastic cells (A). Scattered tubules in the non-neoplastic kidney also reveal c-erb B-2 overexpression (B). Fig. 5. Renal cell carcinoma. Avidin-biotin peroxidase (ABC) stain for c-erb B-2 (A) and for PDGF (B). Adjacent non-neoplastic kidney c-erb B-2 (C) x 300. Staining is identified in neoplastic cells (A). PDGF staining is also noted in tumor area. (B) Adjacent non-neoplastic kidney shows focal staining for C-erb B-2.
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Herrera: C-erb B-2 in cystic renal disease
cell carcinoma (Figs. 3B, 5B). Conversely, no staining was
Cystic renal disorders and normal kidneys which are not
noted in two kidneys which failed to express c-erb B-2 (one with associated with a neoplastic propensity did not express c-erb ACD and one with a simple retention cyst) or in the two normal B-2, while some cystic disorders with associated neoplastic control kidneys. No staining for IGF-I, FGF or TGFI3 was potential expressed the oncogene, emphasizing the heterogene-
ity of expression in cystic diseases and the selectivity of eases, or in the two normal control kidneys. There was, expression in certain restricted circumstances. The meaning of identified in either c-erb B-2 positive or negative cystic dis-
however, the expected localization of IGF-I in cortical and this finding is not entirely clear at this time; however, it suggests medullary collecting ducts and TGF/3 in proximal tubules in a possible significant role for c-erb B-2 in the pathogenesis and normal areas of renal parenchyma in cystic and non-cystic growth of certain selected cystic disorders. Analysis of the c-erb B-2 protein structure suggests that it serves as a growth control kidneys. factor receptor in normal cells. The binding of an extracellular growth factor induces the c-erb B-2 protein to send a proliferC-erb B-2 encodes a protein that shares homology with but is ation signal to the cells. different from EGF. Previous studies have shown that EGF is a Furthermore, testing for platelet-derived growth factors (A mitogen for adult polycystic kidneys [3—5] and the EGF recep- and B) in a few kidneys overexpressing c-erb B-2 revealed tor is located in the apical portions of the cell membranes of the expression in similar locations, while PDGFs were not exlining epithelium of the cysts. High- and low-affinity EGF pressed in the c-erb B-2 negative kidneys. This finding further Discussion
receptors have been identified in several tissues, including supports the hypothesis that c-erb B-2 may play a role in kidney. In the early stages in ADPKD there appears to be an serving as a growth factor receptor potentiating or facilitating increase in EGF receptor number and a translocation of the the expression and execution of effector signals of other recoghigh-affinity EGF receptors to the apical cell membranes [4]. nized renal growth factors, Two types of PDGFs, A and B, Renal epithelial EGF receptors are overexpressed in renal cell related to different receptors have been characterized. The type carcinomas and also in ACD associated with end-stage renal B receptor, which has been cloned and sequenced, mediates the disease (ESRD) patients on dialysis [3]. Interestingly, plasma major mitogenic effect [24]. PDGF-B expression was much EGF concentration, although elevated in dialysis patients, does more intense than PDGF-A. Interestingly, PDGFs are usually not appear to correlate significantly with the presence of ACD mitogenic in vitro primarily for mesangial cells and vessels but [3]. EGF has been noted to be increased in ACD and in ADPKD not for tubular epithelial cells. The tubular action may be direct but not in simple (retention) cysts [51. Increased sensitivity to or indirect by initially stimulating primary target cells to procirculating EGF, perhaps due to overexpression of renal epi- duce mitogenic factors which would then trigger other cells. thelial EGF receptors, appears to be at least one of the The demonstration of PDGFs by immunocytochemistry in mechanisms operating in potentiating the growth of cysts. tubular cells and cysts lining suggests a direct effect. Studies of c-erb B-2 have been primarily in patients with breast Most of the mechanisms by which the known polypeptides cancer. C-erb B-2 amplification has been shown in some studies affect kidney function remain to be elucidated. Cystic renal to be associated with tumor behavior and has been proposed to diseases offer a fascinating "milieu" to study the interaction of be a possible predictor of clinical outcome [8—IS]. It has been various growth factors, as they relate to the genesis and suggested that c-erb B-2 may play an important role in the development of the different cystic disorders. While the accupathogenesis and development not only of human breast, but mulation of growth factors that cannot be excreted may play an also ovarian and pancreatic tumors [15, 16, 20]. Measurement important role in end stage kidneys with ACD, in other cystic of epidermal growth factor receptor and estrogen receptor diseases there may be an oversensitivity to them (perhaps as a status have shown no significant correlation with c-erb B-2 result of increased number of receptors), eventually leading to overexpression in breast tumors [20], further emphasizing dif- the development and progression of cysts [30]. This oversensiferences between EGF and c-erb B-2. tivity is likely genetically determined in some cystic renal Not much is understood about the pathogenetic mechanisms disorders. It has been demonstrated, for example, that EGF is that operate and are responsible for progressive renal injury a more potent mitogen for adult polycystic kidney than for leading to the end stage kidney [21—26]. It is also unclear why age-matched normal epithelia [4]. C-erb B-2 may have unique implications in the understanding kidneys in patients undergoing chronic dialysis may become cystic and are sometimes associated with cellular proliferation, of the biologic potential of renal cystic disorders, especially as including renal cell neoplasms [21, 22, 26—28]. Factors occur- its overexpression relates to the action of renotropic growth ring during progression of renal failure and chronic dialysis factors such as PDGFs which stimulate cellular proliferation. therapy result in release of autocrine and/or paracrine growth Epithelial proliferation, fluid accumulation, and matrix remodfactors that induce cell proliferation [24—26]. The specific eling have been identified as the major processes in the enlargegrowth factors operating have not been entirely elucidated. ment of cysts [29]. C-erb B-2 may play a role in stimulating one Cystic transformation in these kidneys may occur as a result of or any combination of these factors. In the case of ADPKD the accumulation of renotropic factors originating in viable neph- abnormal gene product could lead in some instances to the rons, causing stimulation of the tubular epithelium leading to activation of critical proto-oncogene/s within renal tubular cells cyst formation, and in some cases subsequent epithelial prolif- (that is, c-erb B-2) [291, which in turn may act by stimulating the erative lesions [26, 29]. Among patients with ESRD and ACD, above factors through the action of selected growth factors. An almost 6% have a renal neoplasm, an incidence several times abnormal gene is not the only mechanism whereby critical greater than the general population [26]. The actual incidence of proto-oncogenes can be activated, and in the case of non-cystic kidney tumors may be higher than estimated. end-stage kidneys, ACD and renal cell neoplasms expressing
513
Herrera: C-erb B-2 in cystic renal disease
c-erb B-2 other mechanisms are likely responsible (such as
13. BERGER MS, LOCHER GW, SAURER S, GULLICK WJ, WATERFIELD
MD, GRONER B, HYNES NE: Correlation of c-erb B-2 gene ampli-
endogenous or exogenous substances). Acknowledgments This work was presented in part at the American Association of Nephrology Meeting, December 2—5, 1990.
Reprint requests to Guillermo A. Herrera, M.D., Department of Pathology, The University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505, USA.
References
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MF: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707—712. 1989 16. WILLIAMS TM, WEINER DB, GREENE MI, MAGUIRE HC: Expression of C-erb B-2 in human pancreatic adenocarcinomas. Pathol 59:46—52,
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Dodrecht, Boston, London, Kiuwer Academic Publishers, edited by GARDNER KD, BERNSTEIN J, 1990, pp. 3—17 2. EVAN AP, MCATEER JA: Cyst cells and cyst walls, in The Cystic Kidney, Dodrecht, Boston, London, Kluwer Academic Publishers, edited by GARDNER KD, BERNSTEIN J, 1990, pp. 21—41
3. Mostcownz DW, BONAR SL, PATEL B: Acquired renal cystic disease (ARCD) and plasma epidermal growth factor (EGF) in dialysis patients. (abstract) JAm Soc Nephrol 1:636, 1990 4. Du J, WILSON P: Mislocation of high affinity epidermal growth factor (EGF) receptors in human autosomal dominant polycystic kidney disease (APKD). (abstract) J Am Soc Nephrol 1:627, 1990 5. Moscownz DW, BONAR SL, MARCUS MD, CLAYMAN RV, AVNER
ED: Epidermal growth factor content of human and mouse (CPK) renal cysts (abstract). Am J Kidney Dis 14:435, 1989 6. SCHECHTER AL, HUNG MC, VAIDYANATHAN L, WEINBERG RA, YANG-FENG T, FRANCKE V, ULLRICH A, COUSSENS L: The new
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sion of c-erb B-2 oncoprotein: A prognostic indicator in human breast cancer. Cancer Res 49:2087—2090, 1989 S, TUZI NL, GULLICK WJ: Overexpression of c-erb B-2 oncoprotein in human breast carcinomas: Immunohistological assessment correlates with gene amplification. Lancet 2:69— 71, 1987 10. CLINE Mi, BATTIFORA H, YOKOTA J: Proto-oncogene abnormali9. VENTER DJ, KUMAR
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ALTMAN DG: An immunohistochemical evaluation of c-erb B-2 expression in human breast carcinoma. Br J Cancer 58:448—452, 1988
12. SLAMON Di, CLARK GM, WONG SG, LEVIN WJ, ULLRICH A,
MCGUIRE WL: Human breast cancer: Correlation of relapse and
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C-erb B-2 amplification in cystic renal disease GUILLERMO A. HERRERA Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, USA
C-erb B-2 amplification in cystic renal disease. Gene amplification 4.8-kD messenger RNA that translates into a 185 kD protein. (overexpression) of c-erb B-2 was tested in a variety of cystic renal The protein product of c-erb B-2 has extracellular, transmemdiseases, renal cell neoplasms (adenomas and carcinomas) and end brane, and intracellular domains; the intracellular domain has stage kidneys without cysts. C-erb B-2 encodes a receptor-like protein that shares homology with, but is distinct from the epidermal growth factor (EGF) receptor. A monoclonal antibody that immunoprecipitates
tyrosine kinase activity. C-erb B-2 encodes a membrane-bound
receptor for an as yet unknown extracellular ligand that is
a protein of approximately 185 kD from a lysate of NLH/3T3 cells
widely assumed to be a cellular growth factor. C-erb B-2 lies on human chromosome 17. Amplification of the c-erb B-2 has been cysts, cystic renal dysplasia, autosomal recessive polycystic kidney demonstrated in human breast, ovarian and pancreatic carcinodisease (ARPKD), and non-cystic, essentially normal kidneys failed to show c-erb B-2 overexpression. In contrast, autosomal-dominant poly- mas and some studies have suggested that c-erb B-2 amplificacystic kidney disease (ADPKD), acquired (dialysis-associated) cystic tion may have significant prognostic implications [8—161. A disease (ACD), non-cystic end stage kidneys and renal cell neoplasms similar factor may play a pivotal role in the genesis, developrevealed overexpression of c-erb B-2 with some frequency (40% or more of cases tested). Three cystic disorders revealing c-erb B-2 ment and neoplastic potential of some cystic renal conditions. overexpression also showed platelet-derived growth factors (PDGFs) Analysis for the c-erb B-2 can be performed by immunohistochemistry. Monoclonal antibodies can recognize the c-erb B-2 expression in similar locations (cyst lining and adjacent tubules). Other growth factors [insulin-like growth factor (IGF-1), fibroblast growth protein and can detect onco-protein overexpression [71. transfected with the c-erb B-2 gene was utilized for testing. Simple renal
factor (FGF) and beta transforming growth factor (TGF/3)J were not noted to be overexpressed in either c-erb B-2 positive or negative cystic diseases. C-erb B-2 may be a marker related to the proliferative/growth capabilities of selected cystic diseases, including potential for associated genesis of benign and malignant renal cell tumors.
Methods
Wedge sections (including cortex and medulla) from both renal poles (2 each) from forty formalin fixed, paraffin-embedded blocks from a variety of cystic renal diseases, end stage
Renal cysts are a central pathologic feature in several renal diseases with varied etiologies. Understanding the formation of renal cysts has received significant attention for centuries; however, the mechanisms involved in their development and neoplastic transformation in a number of them are not well understood [1, 2]. The formation of cysts must be accompanied by hyperplasia of the lining epithelium to keep pace with the enlarging surface area of the cystic cavities. In some cases neoplastic transformation may occur in the lining epithelium and some cystic renal diseases are associated with renal cell adenomas and carcinomas. Understanding of the underlying molecular pathology of renal cystic diseases is just beginning. It appears that elucidation of the role of associated genes and proto-oncogenes involved may be an important issue. The role of epidermal growth factor has been a recent line of investigation [3—5]. C-erb B-2, a closely related proto-oncogene [61, may also play an important role. The erb B proto-oncogene encodes a receptor-like protein that shares homology with but is distinct from the epidermal growth factor [6, 7]. The c-erb B-2 oncogene transcribes a
kidneys, renal cell adenomas and carcinomas were routinely sectioned and stained with hematoxylin and eosin, as well as immunostained using an avidin-biotin peroxidase complex technique according to Hsu, Raine and Fanger [17] with a c-erb B-2
monoclonal antibody from Triton Biosciences Inc., Alameda, California, USA. This antibody stains frozen and paraffinembedded tissues and cell lines overexpressing the c-erb B-2 gene. In all cases the sections included cortical and medullary areas. Four immunostaining sections were cut at 4 microns, deparaffinized and hydrated through xylenes and graded alcohols to distilled water prior to immunostaining. Sections on
slides were then incubated at 37°C for five minutes in 3% hydrogen peroxide to block endogenous peroxidase activity in tissues. The slides were subsequently incubated with the primary antibody (1/9 dilution) for one hour, the linking avidin-
biotin complex (ABC), and later incubated again with the substrate (diaminobenzidine-DAB) for color development. The sections were then counterstained with aqueous hematoxylin and immersed in ammonia water for bluing. Below and in Table 1 are listed details pertaining to the antibody used for testing. Hematoxylin and eosin stained sections were also prepared for
routine morphologic evaluation. At least six sections were Received for publication February 4, 1991 and in revised form April 30, 1991 Accepted for publication May 1, 1991
© 1991 by the International Society of Nephrology
available for evaluation in each case. Three essentially normal, non-cystic kidneys similarly processed, treated and immunostained were also tested for evidence of c-erb B-2 amplification. The various cystic kidneys were classified using well-described combined histopathologic and clinical parameters following
509
Herrera: C-erb B-2 in cystic renal disease
510
Table 2. C-erb B-2 expression
Table 1. Characteristics of antibodies
Antibody to C-erb B-2
Platelet-derived growth factor A Platelet-derived growth factor B Insulin-like growth factor I Fibroblast growth
factor
Transforming
growth factor l
Obtained from
Dilution
Type
Triton Biosciences, Inc., Alameda, California Genzyme, Boston, Massachusetts Genzyme, Boston, Massachusetts Accurate Chemical Corporation, Westbury, New York Genzyme, Boston, Massachusetts Accurate Chemical Corporation, Westbury, New York
1:9
Mouse monoclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal
1:50
1:50 1:50
1:50 1:50
Mouse monoclonal Mouse monoclonal
Specimens Number Percent tested positive positive Non-cystic, essentially normal kidneys Simple cysts Cystic renal dysplasia Infantile polycystic renal disease Adult polycystic renal disease Acquired cystic renal disease End-stage renal disease without cysts Renal cell adenomas Renal cell carcinomas Total a One
3 7 4 3 3 8° 8 3° 5
44
0 0 0 0
0% 0% 0% 0%
3 5
100%
4 2 2 16
63% 50% 66% 40% 36%
case of acquired cystic renal disease also had a renal cell
adenoma (counted as one case but entered twice above).
Results Results were evaluated in a blind fashion. C-erb B-2 amplifiBernstein criteria 118] into well recognized groups including cation was detected by immunocytochemical demonstration of ARPKD, ADPKD, ACD, simple (retention) cysts and cystic the protein product in the lining epithelium of 30% to more than 90% of the cysts and surrounding tubules in the renal cortex. renal dysplasia. In addition, six fresh, snap-frozen sections from kidney with This percentage was highest for ACD (90% or more), followed cystic and neoplastic disorders (three ACD, one simple cyst, by renal cell carcinoma, (50% or more) and lowest for ADPKD one ADPKD and one renal cell carcinoma) and sections from and end stage kidneys without cysts (approximately 30%). The two essentially normal (non-cystic) kidneys used as controls tubules showing c-erb B-2 overexpression were primarily proxwere sectioned and stained using a similar immunoperoxidase imal tubules with distal tubules comprising a minority of the
procedure as outlined above [detailed in Reference 17] and tested for the expression of insulin-like growth factor (IGF-I), platelet-derived growth factors (PDGFs) A and B, fibroblast growth factor (FGF) and transforming growth factor beta (TGF/3). The monoclonal antibodies to PDGFs A and B and FGF were obtained from Genzyme Co. (Boston, Massachusetts, USA) and those to IGF-I and TGF/3 from Accurate
positive tubules. No medullary staining was identified. The entire cystic nephrons were similarly stained. No staining was
10 minutes and placed in acetone for fixation. The sections were
cytoplasmic matrix of reacting cells. The staining pattern within
detected on glomeruli or in the renal vasculature. The degree of staining was slightly variable from case to case but no repetitive
pattern could be recognized in relationship to specific conditions. No attempt was made at determining the intensity of staining, however, staining was well defined in the positive Chemical Co. (Westbury, New York, USA); all were used at a cases. The presence or absence and the extent of staining were 1/50 dilution. Sections form both poles of the kidneys were the only documented findings. Staining was noted associated examined. Cryostat sections were cut at 4 microns, air dried for with the cell membranes and also identified diffusely in the
incubated for five minutes at 37°C on a stainplate with 3% individual cysts was rather uniform. Two independent observers confirmed the presence and absence of staining in a doubleblind fashion. Staining was noted as detailed in Table 2, and was present in all sections from a given case immunostained. Simple avidin-biotin complex (ABC) and the substrate (DAB) for color (retention cysts), cystic renal dysplasia, ARPKD and some end development. The slides were then counterstained with aque- stage kidneys failed to show c-erb B-2 overexpression (Fig. 1). ous hematoxylin and immersed in ammonia water for bluing. However, the three ADPKD tested (Fig. 2A), 63% of ACD (Fig. Characteristics of the antibodies, including source and dilution 2B, 2C, 3A), 50% of end stage kidneys without cysts, 66% of renal cell adenomas and 40% of renal cell carcinomas (Figs. 2 to used are listed in Table 1. 5) revealed c-erb B-2 overexpression. The one end stage kidney Characteristics of the c-erb B-2 monoclonal antibody with ACD and a renal adenoma showed c-erb B-2 overexpresThe monoclonal antibody c-erb B-2 immunoprecipitates a sion in neoplastic areas, cysts lining and adjacent tubules (Fig. protein of approximately 185 kD from a lysate of NIH/3T3 cells 4). The non-neoplastic renal parenchyma in cases with neotransfected with the c-erb B-2 gene 1191, while an isotype plasms and the non-cystic areas of c-erb B-2 positive kidneys control antibody failed to precipitate this protein. No 185 kD also showed focally c-erb B-2 overexpression in some tubules, protein bands were precipitated from a non-transfected NIH! primarily proximal (Fig. 5). The three non-cystic, functionally 3T3 cell lysate. This monoclonal antibody reacted with cells viable, essentially normal control kidneys failed to show c-erb overexpressing c-erb B-2 and did not immunoprecipitate EGF- B-2 amplification (Fig. 1D). Staining for PDGFs, primarily B, was noted in the cells lining receptor protein (170 kD) from lysates of A 431 cells, nor did it immunocytochemically stain the A 431 cell line [7, 19]. Addi- cysts and in surrounding tubules in three non-neoplastic kidtionally, this monoclonal antibody was shown to recognize the neys (2 ACD and 1 ADPKD) and one renal cell carcinoma also external domain of the c-erb B-2 gene from radiolabeled per- showing staining for c-erb B-2 in similar locations, however, it was much more widespread and intense in the ACD and renal manently transfected CHO cells [71.
hydrogen peroxide to block endogenous peroxidase activity in tissues. Test samples, known positive and negative controls were incubated for an hour with the primary antibody, the
511
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Herrera: C-erb B-2 in cystic renal disease
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expression is noted on any of the cystic kidneys or normal kidney illustrated.
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Fig. I. (A) Cystic dysplastic kidney, (B) simple cyst, (C) end stage kidney without cysts, (13) normal kidney. Avidin-biotin peroxidase complex (ABC) stain for c-erb B-2. x 300. No evidence of c-erb B-2
4
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Fig. 2. (A) Adult Onset polycystic kidney x 400 (B & C). Acquired cystic disease. Non-cystic areas X 200 (B) and cyst lining x 400 (C). Avidin-biotin peroxidase (ABC) stain for c-erb B-2. Staining for c-erb B-2 is noted in the lining of cysts of an adult polycystic kidney (A) and in the non-cystic and cystic areas (B & C) an end stage kidney with acquired cystic disease. Fig. 3. Acquired cystic disease. Avidin-biotin peroxidase complex (ABC) stain for c-erb B-2 (A), and for PDGF (B). x 300. Intense c-erb B-2 staining in cyst lining and adjacent tubules is shown in A. Same kidney stained for PDGF shows similar staining pattern, although less intense (B). Minimal staining is also noted in the interstitial cells. Fig. 4. (A) Renal cell adenoma arising in acquired cystic kidney disease. (B) Adjacent cystic kidney. Avidin-biotin peroxidase complex (ABC) stain for c-erb B-2. x 300. Staining is identified in the majority of the neoplastic cells (A). Scattered tubules in the non-neoplastic kidney also reveal c-erb B-2 overexpression (B). Fig. 5. Renal cell carcinoma. Avidin-biotin peroxidase (ABC) stain for c-erb B-2 (A) and for PDGF (B). Adjacent non-neoplastic kidney c-erb B-2 (C) x 300. Staining is identified in neoplastic cells (A). PDGF staining is also noted in tumor area. (B) Adjacent non-neoplastic kidney shows focal staining for C-erb B-2.
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512
Herrera: C-erb B-2 in cystic renal disease
cell carcinoma (Figs. 3B, 5B). Conversely, no staining was
Cystic renal disorders and normal kidneys which are not
noted in two kidneys which failed to express c-erb B-2 (one with associated with a neoplastic propensity did not express c-erb ACD and one with a simple retention cyst) or in the two normal B-2, while some cystic disorders with associated neoplastic control kidneys. No staining for IGF-I, FGF or TGFI3 was potential expressed the oncogene, emphasizing the heterogene-
ity of expression in cystic diseases and the selectivity of eases, or in the two normal control kidneys. There was, expression in certain restricted circumstances. The meaning of identified in either c-erb B-2 positive or negative cystic dis-
however, the expected localization of IGF-I in cortical and this finding is not entirely clear at this time; however, it suggests medullary collecting ducts and TGF/3 in proximal tubules in a possible significant role for c-erb B-2 in the pathogenesis and normal areas of renal parenchyma in cystic and non-cystic growth of certain selected cystic disorders. Analysis of the c-erb B-2 protein structure suggests that it serves as a growth control kidneys. factor receptor in normal cells. The binding of an extracellular growth factor induces the c-erb B-2 protein to send a proliferC-erb B-2 encodes a protein that shares homology with but is ation signal to the cells. different from EGF. Previous studies have shown that EGF is a Furthermore, testing for platelet-derived growth factors (A mitogen for adult polycystic kidneys [3—5] and the EGF recep- and B) in a few kidneys overexpressing c-erb B-2 revealed tor is located in the apical portions of the cell membranes of the expression in similar locations, while PDGFs were not exlining epithelium of the cysts. High- and low-affinity EGF pressed in the c-erb B-2 negative kidneys. This finding further Discussion
receptors have been identified in several tissues, including supports the hypothesis that c-erb B-2 may play a role in kidney. In the early stages in ADPKD there appears to be an serving as a growth factor receptor potentiating or facilitating increase in EGF receptor number and a translocation of the the expression and execution of effector signals of other recoghigh-affinity EGF receptors to the apical cell membranes [4]. nized renal growth factors, Two types of PDGFs, A and B, Renal epithelial EGF receptors are overexpressed in renal cell related to different receptors have been characterized. The type carcinomas and also in ACD associated with end-stage renal B receptor, which has been cloned and sequenced, mediates the disease (ESRD) patients on dialysis [3]. Interestingly, plasma major mitogenic effect [24]. PDGF-B expression was much EGF concentration, although elevated in dialysis patients, does more intense than PDGF-A. Interestingly, PDGFs are usually not appear to correlate significantly with the presence of ACD mitogenic in vitro primarily for mesangial cells and vessels but [3]. EGF has been noted to be increased in ACD and in ADPKD not for tubular epithelial cells. The tubular action may be direct but not in simple (retention) cysts [51. Increased sensitivity to or indirect by initially stimulating primary target cells to procirculating EGF, perhaps due to overexpression of renal epi- duce mitogenic factors which would then trigger other cells. thelial EGF receptors, appears to be at least one of the The demonstration of PDGFs by immunocytochemistry in mechanisms operating in potentiating the growth of cysts. tubular cells and cysts lining suggests a direct effect. Studies of c-erb B-2 have been primarily in patients with breast Most of the mechanisms by which the known polypeptides cancer. C-erb B-2 amplification has been shown in some studies affect kidney function remain to be elucidated. Cystic renal to be associated with tumor behavior and has been proposed to diseases offer a fascinating "milieu" to study the interaction of be a possible predictor of clinical outcome [8—IS]. It has been various growth factors, as they relate to the genesis and suggested that c-erb B-2 may play an important role in the development of the different cystic disorders. While the accupathogenesis and development not only of human breast, but mulation of growth factors that cannot be excreted may play an also ovarian and pancreatic tumors [15, 16, 20]. Measurement important role in end stage kidneys with ACD, in other cystic of epidermal growth factor receptor and estrogen receptor diseases there may be an oversensitivity to them (perhaps as a status have shown no significant correlation with c-erb B-2 result of increased number of receptors), eventually leading to overexpression in breast tumors [20], further emphasizing dif- the development and progression of cysts [30]. This oversensiferences between EGF and c-erb B-2. tivity is likely genetically determined in some cystic renal Not much is understood about the pathogenetic mechanisms disorders. It has been demonstrated, for example, that EGF is that operate and are responsible for progressive renal injury a more potent mitogen for adult polycystic kidney than for leading to the end stage kidney [21—26]. It is also unclear why age-matched normal epithelia [4]. C-erb B-2 may have unique implications in the understanding kidneys in patients undergoing chronic dialysis may become cystic and are sometimes associated with cellular proliferation, of the biologic potential of renal cystic disorders, especially as including renal cell neoplasms [21, 22, 26—28]. Factors occur- its overexpression relates to the action of renotropic growth ring during progression of renal failure and chronic dialysis factors such as PDGFs which stimulate cellular proliferation. therapy result in release of autocrine and/or paracrine growth Epithelial proliferation, fluid accumulation, and matrix remodfactors that induce cell proliferation [24—26]. The specific eling have been identified as the major processes in the enlargegrowth factors operating have not been entirely elucidated. ment of cysts [29]. C-erb B-2 may play a role in stimulating one Cystic transformation in these kidneys may occur as a result of or any combination of these factors. In the case of ADPKD the accumulation of renotropic factors originating in viable neph- abnormal gene product could lead in some instances to the rons, causing stimulation of the tubular epithelium leading to activation of critical proto-oncogene/s within renal tubular cells cyst formation, and in some cases subsequent epithelial prolif- (that is, c-erb B-2) [291, which in turn may act by stimulating the erative lesions [26, 29]. Among patients with ESRD and ACD, above factors through the action of selected growth factors. An almost 6% have a renal neoplasm, an incidence several times abnormal gene is not the only mechanism whereby critical greater than the general population [26]. The actual incidence of proto-oncogenes can be activated, and in the case of non-cystic kidney tumors may be higher than estimated. end-stage kidneys, ACD and renal cell neoplasms expressing
513
Herrera: C-erb B-2 in cystic renal disease
c-erb B-2 other mechanisms are likely responsible (such as
13. BERGER MS, LOCHER GW, SAURER S, GULLICK WJ, WATERFIELD
MD, GRONER B, HYNES NE: Correlation of c-erb B-2 gene ampli-
endogenous or exogenous substances). Acknowledgments This work was presented in part at the American Association of Nephrology Meeting, December 2—5, 1990.
Reprint requests to Guillermo A. Herrera, M.D., Department of Pathology, The University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505, USA.
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