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*
Department of Anatomy & Cell Biology, The University of Kansas Medical
Center, Kansas City, Kansas.
Department of Biochemistry & Molecular Biology, The University of
Kansas Medical Center, Kansas City, Kansas.
Correspondence to Dr. Vincent H. Gattone II, Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202. Phone: 317-274-2505; Fax: 317-278-2040; E-mail: gattone{at}anatomy.iupui.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of murine models of ARPKD exist (2). Probably the most used animal model is the C57BL/6J mouse homozygous for the cpk gene, which exhibits renal pathology similar to that seen in human ARPKD. Although the cpk gene on the C57BL/6J background does not show evidence of extrarenal pathology, it does on other genetic backgrounds (2, 3). In the present study, we sought to determine whether the cpk mutation on a BALB/c background would exhibit renal and extra-renal pathology similar to the human disease.
Renal expression of epidermal growth factor (EGF) seems to be significantly diminished in all forms of PKD, including C57BL/6J-cpk/cpk mice (4, 5), DBA-2FG-pcy mice (6), and Han:SPRD cy/+ rats (7). Developmentally, EGF promotes epithelial cell maturation in multiple organs (8, 9). EGF is initially localized in murine renal distal tubule cells at approximately 6 d of age (10, 11). ProEGF remains on the extracellular surface of the apical membrane until it is enzymatically cleaved to EGF, displaced into the distal tubular fluid, and excreted into the urine (8, 12, 13). Therefore, the collecting duct is normally exposed to this growth factor, which may promote maturation of this tubule segment. The immature collecting duct cells have EGF receptors on the apical surface that could bind this protein and result in paracrine stimulation (14). Therefore, in cystic kidneys, the absence of this growth factor could delay epithelial maturation. Previously, exogenous EGF treatment of C57BL/6J-cpk/cpk mice led to an amelioration of collecting duct cysts and decreased renal expression of clusterin (SGP-2) (15), a marker of collecting duct immaturity (16). Exogenous EGF treatment also led to a reduction in tubular dilation and interstitial fibrosis as well as renal SGP-2 expression in neonatal rats with chronic unilateral ureteral obstruction (17). In this study, we evaluated the efficacy of exogenous EGF treatment on the amelioration of renal and extrarenal pathology in ARPKD using the BALB/c-cpk/cpk mouse model.
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Materials and Methods |
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EGF Treatment
BALB/c-cpk litters received subcutaneous injections of EGF (1
µg/g body wt, Catalog #4001, mouse natural culture grade EGF, Collaborative
Biomedical Products, Bedford, MA) on postnatal days 3 through 9. The
EGF-treated litters were killed and evaluated at 15 d of age (see below).
Eyelid opening was assessed to evaluate bioactivity of the EGF with this
treatment (15).
Light and Electron Microscopy
Normal (+/+ or cpk/+) and cystic (cpk/cpk) BALB/c mice
were anesthetized (65 mg/kg sodium pentobarbital, intraperitoneally) and
killed at 0, 5, 10, and 15 d of age. EGF-treated litters were similarly
anesthetized and killed at 15 d of age. Total body weight, total kidney
weight, and transverse bile duct diameter (using a micrometer) were
determined. Blood was collected from the heart, and sera samples were used to
assess kidney function. Serum urea nitrogen (SUN) was determined using a
colorimetric assay (Sigma #640, Sigma Chemical Co., St. Louis, MO). The left
kidney was removed and weighed, and the animals were then perfusion fixed with
4% paraformaldehyde in 0.1 M phosphate buffer for light microscopy or 2.5%
glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer for electron
microscopy (EM). The fixed kidneys were weighed, and the kidney, liver, and
pancreas were processed for microscopy. Segments of the paraformaldehyde-fixed
tissue were embedded in paraffin, and sections were stained with hematoxylin
and eosin or periodic acid-Schiff reagent for histologic examination. Segments
of glutaraldehyde-fixed kidney and liver were processed for transmission EM
(TEM) and scanning EM (SEM). For TEM, samples were rinsed in
phosphate-buffered saline and incubated in 1% osmium tetroxide for 1 h. The
samples were then rinsed with distilled water and dehydrated in graded ethanol
solutions and propylene oxide before being embedded in LX112 (Ladd Research,
Burlington, VT). Thin sections were cut and viewed by JEOL 100S (JEOL USA,
Inc., Peabody, MA) TEM. SEM samples were rinsed in phosphate-buffered saline
and incubated in 1% osmium tetroxide for 30 min. The samples were then rinsed
with distilled water, dehydrated in graded ethanol solutions, and dried in a
critical point drier. The dried samples were mounted onto specimen stubs,
sputter coated with gold-pallidium alloy, and viewed with a Hitachi S2700
(Hitachi Corp., Tokyo, Japan) SEM.
Volume Density of Cystic Change
Transverse sections through the entire kidney from the 15-d-old control and
EGF-treated cystic mice were analyzed using point-count stereology as
described previously (15).
RNA Isolation and Northern Hybridization
RNA was extracted and purified from kidneys of 5-, 10-, and 15-d-old normal
and cystic mice as well as 15-d-old EGF-treated normal and cystic mice using
the acid guanidinium thiocyanate-phenol-chloroform extraction method as
described previously (6).
Briefly, the kidneys were snap-frozen in liquid nitrogen and stored at
-80°C until processed for RNA extraction. The kidneys were homogenized in
Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH), and RNA was
isolated according to the manufacturer's instructions. Five or 10 µg of
total renal RNA was added to each lane of a 1% agarose/formaldehyde gel. After
electrophoresis, the RNA was transferred to a Zetabind membrane (Cuno, Inc.,
Meriden, CT) and stained with methylene blue to ensure equal loading and
transfer of RNA from each lane. The blots were baked for 2 h at 80°C and
then hybridized overnight at 65°C in Church's buffer (1% bovine serum
albumin and 7% sodium dodecyl sulfate [SDS] in 0.5 M sodium phosphate with 1
mM ethylenediaminetetraacetate [EDTA])
(18) with
32P-labeled probes. DNA probes for c-myc
(19), clusterin
(SGP-2) (16),
preproEGF (4), and EGF receptor
(obtained from E. Adamson, LaJolla, CA), were labeled using a random prime
labeling method (Rediprime II, Amersham Pharmacia Biotech, Arlington Heights,
IL). Hybridized blots were washed for 10 min each at 65°C; twice in Wash
Buffer A (0.5% bovine serum albumin and 5% SDS in 40 mM sodium phosphate with
1 mM EDTA), and four times in Wash Buffer B (1% SDS in 40 mM sodium phosphate
with 1 mM EDTA). The blots were apposed to x-ray film for autoradiography. The
relative expression of specific mRNA was evaluated by densitometric
quantitation using NIH Image software, including the subtraction of background
and normalization to any RNA loading differences. All of the Northern blot
hybridization studies were performed in duplicate with different sets of
RNA.
Statistical Analyses
Statistical analysis of SUN, total body weight, kidney weight as a
percentage of body weight, and transverse bile duct diameter were performed
using ANOVA or a Mann-Whitney U analysis (P 
0.05) with
the aid of MINITAB software (State College, PA).
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Results |
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The extrarenal pathology includes pancreatic dysplasia (Figure 2a), common bile duct dilation (Figure 2b), and intrahepatic biliary duct cysts (Figure 3). The pancreatic pathology consists of dilated pancreatic ducts and periductal fibrosis with few acini and almost no observable islets of Langerhans (Figure 2a). The intrahepatic biliary duct cysts are associated with periductal hyperplasia and fibrosis (Figure 3, a and b) and are lined by epithelial cells with numerous microvilli and a single cilium (Figure 3, c, e, and f). The cystic intrahepatic bile duct (Figure 3e) is much larger than a normal bile duct (Figure 3d, shown at the same magnification). The cystic pancreatic pathology is present in the newborn (Figure 4a) and may contribute to the overall smaller size of the cystic offspring at 5 to 15 d (Table 1) by limiting digestive capability. Common bile duct and intrahepatic biliary pathology are also evident early in the disease process (Figure 4b, Table 1).
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Altered Renal mRNA Expression
The renal mRNA expression of SGP-2 and c-myc are
increased in association with the development of ARPKD in
BALB/c-cpk/cpk mice (Figure
5). The expression of both peaks at 10 to 15 d in cystic mice but
remains very low in phenotypically normal mice. Renal EGF mRNA is virtually
undetectable in BALB/c cystic kidney but is present at 10 d and to a much
greater extent at 15 d in normal mice. The EGF-receptor (EGF-R) mRNA
concentration is increased in the cystic kidneys at 10 and 15 d of age
(Figure 5).
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Effects of EGF Treatment
EGF caused precocious eyelid opening (day 8 versus day 12 in
untreated controls) and was therefore biologically effective in vivo.
Fifteen-d-old normal BALB/c mice that were treated with EGF exhibited reduced
body weight compared with untreated controls
(Table 1), which is consistent
with the known effects of EGF given neonatally. Kidney size was therefore
expressed as a percentage of total body weight. Kidneys of the
BALB/c-cpk/cpk mice were smaller after treatment with EGF
(Table 1). However, EGF did not
significantly alter the relative kidney size of the phenotypically normal
BALB/c mice (Table 1). EGF
treatment did not improve renal function in BALB/c-cpk/cpk mice, as
indicated by the elevated SUN levels comparable to those seen in untreated
cystic mice (Table 1).
Treatment with EGF reduced the common bile duct dilation in cystic mice
(Table 1) as well as the
histologic severity of the renal and hepatic cystic pathology
(Figure 6). The volume density
of renal cysts was reduced after EGF treatment in cystic mice (50.3 ±
2.55% versus 59.6 ± 2.56% in sham-treated mice, P
< 0.05 by one-tailed analysis).
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EGF treatment of BALB/c-cpk/cpk mice reduced renal SGP-2 gene expression by 34%; however, renal expression of c-myc mRNA was not significantly altered (Figure 7). EGF treatment had no effect on EGF mRNA expression in cystic kidney but was increased 3.8-fold in normal kidney. Finally, renal EGF receptor mRNA expression was decreased 20% in the EGF-treated cystic mice but was increased 43% in normal mice after EGF treatment (Figure 7).
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The BALB/c-cpk/+ Phenotype
The 1-yr-old BALB/c-cpk/+ heterozygous breeders all developed
massive hepatic cysts (Figure
8). These hepatic biliary ductal cysts were associated with
fibrosis (data not shown). In some of the older breeders, the entire hepatic
parenchyma was almost completely replaced by cysts.
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Discussion |
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The BALB/c cystic mice misexpressed a number of renal mRNA. c-myc, a proto-oncogene associated with increased cellular proliferation (25, 26), and SGP-2 were overexpressed in BALB/c-cpk/cpk mice. Increased expression of SGP-2 and c-myc mRNA was previously localized to the epithelial cells of the cystic collecting duct in C57BL/6J-cpk/cpk mice (16, 19). Furthermore, transgenic mice that overexpressed c-myc develop PKD (27, 28), and the c-myc message is present in the cystic tubules (27). This suggests that c-myc may play an important role in the development of PKD, whereas epithelial cells that line cysts may remain in an immature proliferative state.
As in other PKD models, the kidneys of BALB/c-cpk/cpk mice had severely diminished expression of EGF mRNA (5,6,7, 15) but an increased level of expression of the EGF-R (29). Overexpression of EGF-R mRNA may result from the lack of downregulation by EGF (30, 31).
Extrarenal Changes in BALB/c-cpk/cpk Mice
The extrarenal defects in the BALB/c-cpk/cpk mice included common
bile duct dilation, pancreatic cystic dysplasia, and intrahepatic biliary duct
cysts with periductal hyperplasia. Cystic dilation of the common bile duct
associated with pancreatic cysts has also been found in cpk-induced
PKD on other backgrounds (2,
3). However, intrahepatic
biliary duct dilation was limited
(2,
3) or absent
(22) on these other
backgrounds. Mouse strain background has previously been shown to play a role
in the phenotype of PKD. Glucocorticoid-induced PKD is strain dependent
(32), and pcy-induced
PKD progresses differently on the DBA as compared with the C57BL/6 background
(33). This is the first
demonstration that the cpk gene induces hepatic pathology similar to
human ARPKD. Therefore, the BALB/c background seems to be more permissive for
the development of the PKD-associated intrahepatic pathology.
Like PKD-1 knockout mice (24), the BALB/c-cpk/cpk mice were significantly smaller than normal littermates and exhibited pancreatic cysts, which were present at birth. The enormous pancreatic cysts seemed to replace much of the normal pancreatic parenchyma and may have reduced the amount of functional pancreas. The digestive capability of the mice may have been limited, leading to the runted phenotype. However, other cystic mice with common bile duct dilation and pancreatic cystic dysplasia did not exhibit decreased body weight (2, 3). The PKD-1 knockout mice are also runted at birth (24), further suggesting that digestive capability may not completely explain this body size difference. Therefore, other undetermined factors must be contributing to this runted phenotype as well.
BALB/c-cpk/+ Mice
The 1-yr-old BALB/c-cpk/+ breeders also developed extra-renal
pathology, including massive intrahepatic biliary duct cysts. Similar hepatic
pathology has been observed in other heterozygous PKD models
(2,
32,
34). It is interesting to note
that there is a spectrum of phenotypes in human ARPKD
(35). Furthermore, the degree
of extrarenal pathology generally is inversely related to the severity of the
renal pathology (1). The
BALB/c-cpk/+ mice require 1 yr to develop this significant hepatic
pathology, whereas the BALB/c-cpk/cpk mice live only 2 to 3 wk and
exhibit greater renal cystic pathology than hepatic pathology. In the absence
of this morbid renal pathology, the BALB/c-cpk/cpk mice likely would
progress to a phenotype expressing significant hepatic pathology, probably in
a period of a few months rather than a year. However, because
BALB/c-cpk/+ heterozygotes exhibit a cystic phenotype at 1 yr of age,
there seems to be an apparent gene-dosage effect of the cpk gene. The
development of the liver cysts may be due to either haploinsufficiency or a
loss of heterozygosity of the normal allele in the biliary ducts as has been
proposed for human ADPKD (36,
37).
Role of EGF in PKD
The role of EGF in the development of PKD is unclear. EGF can act as a
mitogen to enhance cyst formation and progression by activating EGF-R tyrosine
kinase activity. Two in vivo studies support this role for EGF in
PKD. orpk cystic mice homozygous for defective EGF-R develop a less
severe form of PKD (38).
pcy-induced PKD progresses more rapidly in mice that also express a
transgene for transforming growth factor-
, a member of the EGF family
(39). In addition, EGF-R
tyrosine kinase inhibitors can inhibit the cystic phenotype in bpk
mice with ARPKD both in vitro
(40) and in vivo
(41). However, these
treatments are administered after the first postnatal week. All studies on the
effects of EGF administration earlier in murine ARPKD have found that EGF
inhibits the development of renal
(4) and/or extrarenal cystic
effects (42). Studies have
shown that EGF is important in early development
(43,
44) and renal tubulogenesis
(45). Transgenic CD1 mice with
a targeted disruption of the EGF-R developed cystic dilation of the collecting
ducts as well as hepatic abnormalities in the neonatal period
(44). Maturation of collecting
ducts (46,
47) temporally correlates with
renal EGF expression (10),
whereas the lack of the growth factor is associated with renal cyst
development in C57BL/6J-cpk/cpk mice
(4). Cystic renal and biliary
epithelia are also known to be responsive to EGF in vitro
(48,
49). Administration of
exogenous EGF on days 3 through 9 led to an amelioration of the PKD in
C57BL/6J-cpk/cpk mice in vivo
(15). Therefore, EGF may serve
a crucial role in the maturation of renal and possibly biliary epithelia.
Furthermore, the lack of EGF could contribute to the persistence of epithelial
immaturity in neonatal mice with ARPKD
(50). After this neonatal
period, EGF treatment seems to have distinctly different effects on the kidney
and the cystic phenotype, which may explain the data from the orpk
(38), transforming growth
factor-/pcy
(39), and EGF-R tyrosine
kinase inhibitor (40,
41) studies.
EGF Treatment Effects in BALB/c Mice
In the present study, neonatal EGF treatment ameliorated the renal and
extrarenal pathology in the BALB/c-cpk/cpk mice; however, the
pancreatic pathology and the degree of azotemia were unaffected. It is unclear
why the neonatal cystic kidney responds differently than the adult kidney to
the affects of EGF. In another model, the BALB/c-bpk/bpk mouse, the
SUN levels and common bile duct dilation were reduced, whereas relative kidney
weight was unchanged after treatment with exogenous EGF
(42). In general, the degree
of cystic change has been shown to correlate with deterioration in renal
function in human ADPKD (51).
However, there may be exceptions to this rule as the progression to renal
failure is highly variable in PKD and is affected by multiple factors
(51). In studies on the effect
of testosterone on ADPKD in rats
(52), castration of cystic
male rats led to a reduced kidney size but not to a change in the degree of
azotemia. Conversely, when bpk mice were treated with EGF, renal
function improved without any change in the amount of cystic pathology
(42). Therefore, factors other
than the amount of cystic change may influence the amount of residual renal
function in PKD.
The role of EGF in promoting maturation of the distal tubule is supported by studies that show that EGF causes decreased proliferation of this segment (11). It has also been shown that exogenous EGF increases differentiation of the distal segment of the kidney (53). Therefore, a possible explanation for the upregulation of EGF mRNA after EGF treatment in normal distal tubule cells is that the exogenous EGF is advancing the development of the kidney to a later stage of maturation in which higher EGF synthesis would be expected. Although the expression level of the renal SGP-2 message remains higher in cystic than in normal mice, the expression is consistently lower in cystic kidneys after EGF treatment. This finding is similar to that seen when C57BL/6J-cpk/cpk mice (15) or neonatal rats with unilateral ureteral obstruction were treated with EGF (17). This suggests that neonatal EGF treatment stimulates maturation of cystic epithelia.
BALB/c-cpk/cpk mice, like many other models of PKD, had dramatically reduced expression of EGF (5,6,7, 15), whereas the EGF-R mRNA expression was correspondingly upregulated. After treatment with exogenous EGF, EGF mRNA expression remained diminished, whereas EGF-R mRNA expression was downregulated. Others have shown that EGF-R is decreased after addition of EGF to renal epithelial cells (30) and enterocytes (31) in vitro. Our data are consistent with an EGF-induced downregulation of EGF-R in the EGF-treated mice similar to other systems. In this study, EGF treatment of neonatal BALB/c-cpk/cpk mice also led to a reduction in renal as well as common and hepatic biliary duct pathology. However, EGF treatment did not alter the pancreatic pathology or the expression of c-myc mRNA in the kidney. Because the pancreatic change is prominent at birth, it is not surprising that EGF was ineffective in preventing this pathology. In general, neonatal EGF treatment seems to partially ameliorate aspects of ARPKD, whereas its effect later in the cystic process may be different or even opposite what was seen neonatally. These findings suggest that EGF may function differently in various aspects and/or phases of PKD (54). Glucocorticoids given neonatally cause an infantile form of PKD (32) but when given later in the disease process actually ameliorates PKD (55). Neonatal kidneys do not necessarily respond to agents in the same way as mature kidneys do. Therefore, caution should be exercised with any PKD intervention because the effect may be dependent on the stage of kidney development.
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Acknowledgments |
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References |
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on rabbit proximal tubule solute transport.
Am J Physiol 266:F459
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Matrigel cultures in serum-free medium. Proc Natl Acad Sci
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