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Rainbow Center for Childhood PKD, Department of Pediatrics, Rainbow Babies and Children's Hospital and Case Western Reserve University, Cleveland, Ohio.
Correspondence to Dr. Ellis D. Avner, Department of Pediatrics, Rainbow Babies and Children's Hospital, 11100 Euclid Avenue, LC 6003, Cleveland, OH 44106-6003. Phone: 216-844-1884; Fax: 216-844-1479; E-mail: eda{at}po.cwru.edu
Abstract
Abstract. c-ErbB2 (also referred to as Neu or HER2), a transmembrane glycoprotein with intrinsic tyrosine kinase activity, is structurally related to epidermal growth factor receptor (EGFR) and forms active heterodimers with EGFR as well as other members of the EGFR family. c-ErbB2 is reported to mediate differentiation and proliferation in epithelial cells and is expressed in a tissue-specific and developmental stage-specific manner. Given the role of EGFR in cystic renal epithelial hyperplasia and the immature phenotype of cystic renal epithelial cells, the segment-specific expression pattern of c-ErbB2 in human autosomal recessive polycystic kidney disease (ARPKD) was examined in nine ARPKD kidney specimens ranging from gestational age 17 wk through postnatal age 4 wk. c-ErbB2 staining of human ARPKD samples showed increased expression with increasing gestational age compared with normal human fetal and postnatal kidneys. This increased c-ErbB2 expression was primarily localized to the apical surfaces of cystic collecting tubule cells, similar to the pattern of EGFR expression, and paralleled collecting tubular cyst formation and growth.
Autosomal recessive polycystic kidney disease (ARPKD) is invariably
characterized by the formation and enlargement of renal collecting tubular
cysts as well as hepatic biliary ectasia and fibrosis
(1,2).
Extensive morphologic analysis and mathematical modeling of cyst formation and
growth indicate that epithelial hyperplasia is a necessary element in the
formation and growth of cystic lesions in polycystic kidney disease (PKD)
(1). Evidence from a number of
laboratories has established a role for the epidermal growth factor
(EGF)-transforming growth factor-
(TGF-)-epidermal growth factor
receptor (EGFR) axis in promoting epithelial hyperplasia and collecting
tubular cyst enlargement in PKD
(3). These studies demonstrate
that EGFR is overexpressed and mislocated to the apical surface of cystic
tubular epithelium in human ARPKD, in human autosomal-dominant PKD (ADPKD),
and in murine models of ARPKD (cpk, bpk, and orpk) and ADPKD
(4,5,6,7,8).
Furthermore, in murine ARPKD, inhibition of EGFR tyrosine kinase through
genetic or pharmacologic manipulation drastically reduces cyst formation and
growth
(5,9).
The c-erbB2 oncogene (also referred to as neu or HER2) encodes a 185-kD transmembrane glycoprotein with intrinsic tyrosine kinase activity. The c-ErbB2 protein is structurally related to EGFR and is able to form active heterodimers with EGFR (10,11). c-ErbB2 is commonly expressed in fetal epithelial cells of the human kidney and to a lesser degree in normal human adult kidneys (12,13). Amplification of c-ErbB2 has been demonstrated in human breast, ovarian, and pancreatic carcinoma, and some studies have suggested that c-ErbB2 amplification may have significant prognostic implications (14,15,16,17,18,19). These findings suggest that c-ErbB2 plays a role in both normal proliferation and neoplastic transformation of epithelial cells. Given these data, as well as the established role of EGFR in cystic renal epithelial hyperplasia, we sought to examine the segment-specific expression pattern of c-ErbB2 in fetal and infantile human ARPKD.
The results demonstrate segment-specific and cyst formation/growth-related c-ErbB2 expression in human ARPKD. These data suggest a potential role for c-ErbB2 in proximal tubular cyst formation and, in concert with EGFR, in progressive collecting tubular cyst formation and enlargement in ARPKD.
Materials and Methods
Specimens: ARPKD Diagnosis
We examined nine human renal ARPKD specimens ranging from gestational age
17 wk (GA17) to postnatal age 4 wk (PA4) from seven families (1 GA17, 2 GA22,
1 GA23, 1 GA26, 1 GA29, 1 GA33, 1 GA34, and 1 PA4). ARPKD was diagnosed in all
cases by consensus clinical, radiographic, and genetic criteria
(20). Five normal kidney
specimens aged GA12, GA13, GA14, GA15, PA4, and PA16 were received as
formalin-fixed, paraffin-embedded blocks and used as controls.
Immunohistochemistry
Sections were incubated for 1 h at room temperature with anti—c-ErbB2
antibody (A485, affinity-purified rabbit polyclonal, 1:100; DAKO, Carpinteria,
CA), and serial sections were incubated for 1 h at room temperature with
anti-EGFR (mouse monoclonal clone 29.1, 1:200; Sigma Chemical Co., St. Louis,
MO) and lectins. Cyst localization was examined using our previously described
segment-specific lectin binding method with biotin-labeled Lotus
Tetragonolobus (Sigma) as a marker for proximal tubules (PT; visualized
with Fast Red) and biotin-labeled Arachis Hypogaea (Sigma) as a
marker for collecting tubules (CT; visualized with DAB)
(4,5,7,9,21,22,23,24).
Staining was scored semiquantitatively with the use of computer video image
analysis as follows: 0, absent; 1 to 3, weak; 4 to 7, moderate; and 8 to 10,
strong.
Results
c-ErbB2 and EGFR Expression in Normal Human Fetal and Postnatal
Kidneys
In normal kidneys GA12, GA13, GA14, GA15, PA4, and PA16, PT demonstrated
weak cytoplasmic expression of c-ErbB2 and CT demonstrated moderate
basolateral as well as apical expression of c-ErbB2
(Table 1, Figure 1, A and C). In normal
kidneys, c-ErbB2 expression was greater in CT than in PT, and expression in
all segments decreased with increasing gestational age
(Figure 1, A and C). EGFR
expression in normal fetal kidney specimens demonstrated apical as well as
basolateral EGFR expression in CT (Figure
1B). However, by PA4, EGFR expression was restricted to the
basolateral membrane domain in all tubular segments
(Figure 1D). The postnatal
expression pattern of c-ErbB2 remained apical as well as basolateral in CT and
primarily cytoplasmic in PT (Figure
1C). The intensity, however, was markedly decreased when compared
with fetal specimens, suggesting a temporal decrease in c-ErbB2
expression.
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Proximal Tubule: c-ErbB2 and EGFR Expression in Human ARPKD
At GA17, both noncystic and cystic PT demonstrated cytoplasmic expression
of c-ErbB2 (Figure 2B,
Table 1). However, expression
of c-ErbB2 was greater in cystic compared with noncystic PT
(Figure 2B). c-ErbB2 expression
in cystic PT decreased with advancing gestational age, paralleling decreasing
proximal tubular cyst numbers and decreasing PT cyst size
(Table 1,
Figure 3)
(22). The difference in
c-ErbB2 expression level between cystic PT and noncystic PT decreased with
advancing gestational age. All ARPKD and normal specimens demonstrated
cystoplastic and basolateral EGFR staining in PT
(Figure 2, C and F).
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Collecting Tubule: c-ErbB2 and EGFR Expression in Human ARPKD
At GA17, both noncystic and cystic CT demonstrated basolateral and apical
expression of c-ErbB2 in equal intensity
(Figure 2B, Table 1). In contrast to
decreasing CT expression with increasing age in normal specimens, c-ErbB2
expression in cystic CT showed little decline with advancing gestational age,
particularly on the apical cell surface of cystic CT
(Figure 2, B insert and E
insert). This increased c-ErbB2 expression in cystic CT paralleled collecting
tubular cyst formation and growth, as well as increased apical EGFR expression
in cystic CT (Table 1, Figures
2, E and F, and
3). c-ErbB2 expression in
noncystic CT was less intense than in cystic CT and decreased with advancing
gestational age similar to that seen in normal fetal specimens
(Figure 2, B and E, Table 1). At GA33, expression
of c-ErbB2 in noncystic CT was weak and the difference of c-ErbB2 expression
between cystic and noncystic CT was striking. At PA4, the expression level of
c-ErbB2 in cystic CT began to decline slightly, whereas expression level of
apical EGFR remained high in cystic CT
(Table 1, Figure 2, E insert and F
insert).
All ARPKD specimens demonstrated apical as well as basolateral expression of EGFR in cystic CT (Figure 2F and insert). This apical expression of EGFR in CT was limited to cystic tubules except in kidneys at GA13, GA17, and GA22 that had apical EGFR expression in noncystic CT (Figure 1B, Table 1).
Discussion
Human ARPKD has been classically described as a cystic kidney disease in which lesions are localized to CT (1). However, studies of the ontogeny of cyst formation in murine models of ARPKD have demonstrated two distinct phases in cystic nephron involvement (4,7,23,24,25). Recently, we established that human ARPKD, like murine ARPKD, has a transient phase of proximal tubular cyst formation during fetal development (22). Given the role of c-ErbB2 and EGFR in mediating proliferation and differentiation in renal epithelium (12,13,21,26,27), we examined segment-specific c-ErbB2 and EGFR expression in normal and ARPKD human fetal kidneys.
In agreement with previous studies, all five normal kidney specimens in the current study demonstrated weaker c-ErbB2 expression in PT compared with CT (12). However, in human fetal ARPKD kidneys at GA17, c-ErbB2 expression in cystic PT was equivalent to that in cystic CT and was more intense than that in noncystic PT. The expression level in cystic PT after GA17 decreased with advancing gestational age, paralleling decreases in PT cyst formation and enlargement (22). These findings suggest that c-ErbB2 may be involved in the developmentally regulated pathophysiology of PT cyst formation in ARPKD. The contribution of cystic PT to the total population of kidney cysts decreases during disease progression. This is most likely due to restriction of proximal tubular cystogenesis to a specific developmental stage in the context of ongoing nephrogenesis and progressive formation of collecting tubular cysts (25). The shift in nephron involvement during renal organogenesis suggests that there may be a developmentally regulated pattern of cystic gene expression or tubular responsiveness to cyst-promoting processes. Regulation of PT c-ErbB2 expression may be one factor involved in this process.
In cystic CT from human fetal ARPKD kidney specimens, c-ErbB2 expression remained strong with advancing gestational age, particularly on the apical cell surface. This increased c-ErbB2 expression in CT paralleled collecting tubular cyst formation and growth as well as apical EGFR expression in cystic CT lesions. In contrast, c-ErbB2 expression in non-cystic CT decreased with advancing gestational age. However, at PA4, expression level of c-ErbB2 in cystic CT began to decrease, whereas expression level of apical EGFR remained high.
These findings suggest that c-ErbB2 may also be involved, in a developmentally regulated manner, in the pathophysiology of early to mid-CT cyst formation. Herrera (28) reported c-ErbB2 amplification in ADPKD (3 of 3 specimens) but not ARPKD specimens (0 of 3 specimens). Ages of ARPKD specimens were not described in that report, and differences between those and the current findings may relate to stage or severity of the cystic lesions and/or ages of the specimens.
EGFR is overexpressed and mislocated to the apical surface of cystic tubular epithelium in human ARPKD and ADPKD and in the murine models of ARPKD (cpk, bpk, and orpk) and ADPKD (3,4,5,6,7,8). In this study, we report apical EGFR expression in noncystic CT in specimens at GA17 and GA22 and in four normal human fetal kidney specimens aged between GA12 and GA15 as well as in cystic CT in all ARPKD kidney specimens regardless of age. Apical EGFR expression in noncystic CT is not seen in human ARPKD older than GA22. As previously reported, apical EGFR expression may be a normal pattern in CT of developing human kidneys (3,6). These findings confirm that cystic renal CT demonstrates features of an immature epithelial phenotype (29,30).
In summary, developmental expression patterns of c-ErbB2 suggest a potential role for this glycoprotein in proximal tubular cyst formation and enlargement and in concert with EGFR, in progressive collecting tubular cyst formation and enlargement in ARPKD. These findings suggest that c-ErbB2, like EGFR, may be a potential target for cyst reduction therapy in ARPKD.
Acknowledgments
We thank Dr. Lisa M. Guay-Woodford of the University of Alabama Birmingham; Dr. Klaus Zerres of Institute for Human Genetics, Technical University of Aachen, Germany, for providing some of the ARPKD specimens; and Dr. Yasuhiro Iwai of the Department of Pathology, Takatsuki General Hospital, Japan, Dr. Norishige Yoshikawa, Professor of Health Sciences, Kobe University School of Medicine, Kobe, Japan, and Dr. Cynthia G. Goodyer, Montreal Children's Hospital for providing normal human fetal kidney specimens.
This study was supported by funding from the Polycystic Kidney Research Foundation 97002 and the National Institute of Diabetes, Digestive and Kidney Diseases Grant DK 57306-01.
References
This article has been cited by other articles:
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  W. B.W.H. Melenhorst, G. M. Mulder, Q. Xi, J. G.J. Hoenderop, K. Kimura, S. Eguchi, and H. van Goor Epidermal Growth Factor Receptor Signaling in the Kidney: Key Roles in Physiology and Disease Hypertension, December 1, 2008; 52(6): 987 - 993. [Full Text] [PDF]
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 Y. Zhou, P. Bouyer, and W. F. Boron Role of a tyrosine kinase in the CO2-induced stimulation of HCO3- reabsorption by rabbit S2 proximal tubules Am J Physiol Renal Physiol, August 1, 2006; 291(2): F358 - F367. [Abstract] [Full Text] [PDF]
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