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From the MRC Molecular Haematology Unit,*
Institute of
Molecular Medicine, University of Oxford, John Radcliffe Hospital,
Oxford, United Kingdom; the Department of Paediatric
Pathology,
John Radcliffe Hospital, Oxford,
United Kingdom; the Service of Nephrology,
University of Barcelona, Barcelona, Spain; and the Division of
Nephrology,§
Department of Medicine, Toronto
Hospital and University of Toronto, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations in two genes, PKD1 and PKD2, account for the vast majority of patients with ADPKD. The identification of these genes3,4 has thus provided a new opportunity to study the pathophysiology of ADPKD. The predicted proteins appear quite different in structure (the PKD1 protein, polycystin-1, is ~4 times larger than its counterpart, polycystin-24,5 ). Nonetheless, they share a significant region of homology in their transmembrane regions, an area also similar to a family of voltage-gated calcium/sodium channels.4,6 Recent evidence indicates that the ADPKD proteins may interact in experimental systems.7,8
PKD2 is less prevalent than PKD1, accounting for ~15% of ADPKD cases,9,10 but preliminary evidence suggests they share the same spectrum of extrarenal manifestations. In one study, the frequency of hepatic cysts was similar in PKD1 and PKD2 patients, although no pancreatic cysts were found in PKD2 patients;10 intracranial aneurysms have also been described in PKD2 families.11 The renal phenotypes are also similar, although PKD2 patients have milder disease and a lower incidence of hypertension.10,12 Consistent with the suggestion that ADPKD proteins have related functions and similar systemic disease phenotypes, expression of PKD113 and PKD24 has been found in most human tissues. To date, however, the different cell types expressing these proteins have not been systematically defined.
One area of uncertainty about ADPKD has been understanding the mutational mechanism underlying cyst initiation. Although the germline mutations at PKD214 and PKD115 are probably inactivating, controversy exists over the additional steps necessary for focal cyst development. Evidence of loss of heterozygosity in individual PKD1 renal16,17 and liver cysts18 and recent data from targeted disruption of the mouse Pkd2 gene19 favor a two-hit mechanism involving somatic inactivation of the normal allele. However, studies of PKD1 cystic tissue have shown polycystin-1 expression13,20,21 . No corresponding studies of human PKD2 tissue have yet been described.
To understand the cellular basis for the renal and extrarenal manifestations of ADPKD, we examined the expression of polycystin-2 and polycystin-1 in human fetal tissues at different ages of gestation, using antisera raised to an epitope in the predicted C-terminal region of PKD2 and two previously described polycystin-1 mAbs13,21 . The expression of polycystin-2 was also examined in PKD2 cystic kidney and liver tissue for clues to the mutational basis of cyst formation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 774-bp segment (2130–2904 nt) of PKD2 was cloned into the vector pET32a+ (Invitrogen, Carlsbad, CA), in frame with the thioredoxin protein and a His tag. This plasmid was introduced into the Escherichia coli strain AD494 DE3 pLysS to synthesize a bacterial fusion protein containing the C-terminal 258 amino acids (aa) of polycystin-2. Following induction with IPTG, recombinant protein was isolated from bacterial lysate by Ni2+ affinity chromatography using a 4.6 x 100 mm POROS MC 20 column on a BioCAD workstation (PerSeptive Biosystems, Framingham, MA). Bound protein was eluted on an imidazole gradient and the relevant fractions dialysed against phosphate-buffered saline before immunizing rabbits.
The production and characterization of two polycystin-1 mAbs, PKS-A and 7e12, has been described previously13,21 . The epitope detected by PKS-A is contained within the final 233 aa of the C-terminus of polycystin-1, whereas 7e12 was raised to an epitope in the N-terminal region (flank-LRR-flank domain) of polycystin-1.
Generation of a Full-Length PKD2 cDNA Expression Construct (PKD2Pk)
A full-length PKD2 cDNA was produced using the IMAGE clone 239P18 (153473) and a 5' PCR product. The clone begins at the initiation codon and ends at 2970 nt.4 The Pk TAG epitope tag recognized by the mAb SV5-Pk (Serotec, UK)22 was incorporated at the C-terminal end of the protein by replacing the stop codon with the sequence 5'-GACTCGGGAAAGCCGATCCCAAACCCTTTGCTGGGATTGGACTCCACCTAGTGA-3'. The PKD2Pk construct was cloned in pCDNA-3 and an internal ribosomal entry site (IRES) from the encephalomyocarditis virus inserted between the cytomegalovirus immediate early promoter and the first in frame ATG.23 This generates a recombinant protein with an open reading frame of 984 aa and predicted MW of 111 kd.
Plasma Membrane Preparation from Tissues and Cells
Plasma membrane fractions from normal human kidney and COS-1 cells were prepared by established methods22,24 . Protease inhibitors (2 mg/ml aprotinin, 1 mmol/L benzamide, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 4 mg/ml pepstatin, 1 mg/ml leupeptin, 1 mg/ml Pefablock; Boehringer Mannheim, Mannheim, Germany) were added to the buffers at all stages of the procedure.
Western Blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 7.5% separating gels using a Bio-Rad Mini Protean II apparatus with molecular weight standards (Rainbow, Amersham Little Chalfont, UK; 14.3–220 kd). Separated proteins were transferred at 30V overnight onto a PVDF membrane (Millipore, Watford, UK). In some experiments, gels were stained with Coomassie, either in parallel to assess migration or after transfer to assess transfer efficiency. Western blotting using enhanced chemiluminescence detection of bound secondary antibody was performed as previously described.13
Immunohistochemistry
Fresh tissue was obtained from fetuses (13–40 weeks gestational
age), fixed in 10% formal saline, and embedded in paraffin for routine
histology. The postmortem findings for each fetus are shown in Table 1
and a summary of the tissues analyzed
for each fetus in Table 2
. In a small
number of cases where single organs were affected, the abnormal tissues
were excluded from analysis. Normal adult kidney was obtained from the
normal pole of three nephrectomy specimens and processed for histology
as for fetal tissues; all three patients (median age, 74 years)
underwent nephrectomy because of renal cell carcinoma.
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Analysis of Cystic Tissue
Cystic tissue was obtained from five PKD2 patients (4 kidneys, 3
livers); the germline mutations in four of these patients have been
defined (see Table 3
). All cysts >2 mm
in diameter were counted and graded as positive (+) or negative (-)
for polycystin-2 and polycystin-1. A small and variable proportion of
cysts, however, showed a heterogeneous pattern of staining with
negative areas clearly present in an otherwise positive cell lining or
vice versa. These were graded as positive/negative (+/-)
cysts. The total number of cysts counted varied between individuals but
reflects the maximum in all available cystic tissue.
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Mouse mAbs to the 
1 subunit of
Na+-K+-ATPase and the
epithelial membrane antigen (EMA) were purchased from Upstate
Biotechnology (Lake Placid, NY) and Sigma (St. Louis,
MO), respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The polyclonal antisera, p30, was raised to the C-terminal 258 aa of polycystin-2. This region was selected because it showed no significant sequence similarity to polycystin-1 or any other known proteins. Recently a polycystin-2-like protein, PKDL, has been described but cross-reactivity with this molecule is unlikely because the antigenic region shows only a low level of identity (26%) and because PKDL appears to have a restricted tissue distribution.25,26
The specificity of p30 was initially tested by Western blotting and
dual immunofluorescent staining of COS-1 cells transiently transfected
with the Pk epitope tagged PKD2 cDNA expression plasmid
(PKD2Pk). Western blot analysis indicated high levels of a 110-kd
protein in PKD2Pk transfected COS-1 cells with the p30 and Pk
antibodies (Figure 1A)
. In addition, p30
detected the endogenous PKD2 protein as a weaker but similarly sized
band in untransfected COS-1 cells (Figure 1A)
. Dual immunofluorescent
labeling of PKD2Pk transfectants also showed that only cells labeling
with the Pk mAb were strongly detected with p30, showing its
specificity for the PKD2 protein (data not shown).
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. A very weak band (<110 kd) was also detected by p30 in both human
kidney membrane fractions and COS-1 transfectants and a faint higher
molecular weight band of >220 kd was sometimes detectable on longer
exposures. The presence of these minor species in the transfected cells
(Figure 1A)
suggests that they are forms of the polycystin-2 protein,
possibly a splice variant or degradation product (<110 kd) or
multimer/aggregate (>220 kd). All three bands disappear when p30 is
preincubated with recombinant polycystin-2, further indicating that the
antisera is polycystin-2-specific (data not shown). Taken together,
these results indicate strong specificity of p30 for both recombinant
and endogenous polycystin-2. Expression of Polycystin-2 and Polycystin-1 in Normal Human Adult Kidney
Initial studies on adult human kidney with p30 showed
polycystin-2 to be expressed mainly in the medullary collecting ducts,
cortical collecting ducts, and distal convoluted tubules (Figure 2A)
with no detectable signal with
preimmune sera from the same rabbit (Figure 2B)
. Similarly, no
detectable signal was seen in human kidney when p30 was preincubated
with the recombinant full-length polycystin-2 protein (data not shown).
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. In both cases
weaker proximal tubular expression was also seen (Figure 2, A and C)
.
Colocalization of the two polycystin proteins could be demonstrated
clearly by dual color immunofluorescence (Figure 2, D–F)
in
medullary collecting ducts, which strongly express both proteins. Expression of Polycystin-2 and Polycystin-1 in Human Fetal Kidney and Other Fetal Tissues
Further studies were conducted on fetal tissues of different ages
(13–40 weeks) to assess the expression pattern of both ADPKD proteins
in the developing kidney and other organs. In each case the pattern of
expression was identical for the polycystin-1 antibodies (PKS-A, 7e12)
and the PKD2 antibody (p30) (see Figure 3, B–D
, and Figure 4, A–C
). To illustrate as many fetal ages
and tissues as possible, results of polycystin-2 expression are mainly
illustrated (Figures 3 and 4)
.
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. Faint but detectable expression was seen
in the earliest epithelial precursors (S and comma-shaped bodies and
proximal and distal branches of the ureteric bud) but expression was
most marked in the maturing proximal and distal tubules, collecting
ducts, and transitional epithelium. Striking apical and basal surface
membrane expression was observed in some early medullary collecting
ducts (Figure 3, B–E)
. Proximal tubular expression was prominent up to
28 weeks but appeared to decline in intensity thereafter. In contrast,
medullary collecting duct (Figure 3G)
and cortical collecting duct
(Figure 3H)
expression was maintained right up to 40 weeks.
The results for the other tissues are summarized in Table 2
and
examples of different nonrenal cell types expressing polycystin-2 are
shown in Figure 4
.
Expression of Polycystin-2 and Polycystin-1 in PKD2 Kidney and Liver
Cystic tissue was analyzed for polycystin-2 expression from five
different PKD2 patients (see Table 3
for details). In two cases
(TOR-PKD39 and JRIII:3) the germline mutations are predicted to remove
the area used to raise the p30 antibody (see Table 3
for details),
which consequently will recognize only the protein encoded by the
normal allele. In the other cases the mutation is not known (OX964), is
predicted to generate an in-frame change preserving the p30 epitope
(TOR-PKD6), or is a frame-shifting change 9 aa into the region used to
generate the antibody (TOR-PKD8) and hence, the mutant protein is
probably not recognized by p30.
Overall, the analysis shows that 64.8% of cysts stained with p30,
although only partial staining was seen in 13.2%, and a significant
level of negative cysts (35.2%) was observed. The number of negative
cysts was lower in cystic liver than in cystic kidney. The reason for
this is unclear, although the total number of liver cysts available for
analysis was much lower as a proportion of the total number of cysts
analyzed (Table 3)
. In the two patients in which only the normal
protein was detectable, similar figures were obtained with 63.3% of
cysts positive (16.3% staining partially); we did not observe a
similar pattern of heterogeneous staining in non-cystic tubular
epithelium. Analysis of serial sections for polycystin-1
immunoreactivity showed a striking coordinance, with each
polycystin-2-negative cyst also lacking polycystin-1. Examples of cysts
either positive or negative for polycystin-2 and polycystin-1 are
illustrated in Figure 5
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In fetal and adult kidney, the expression pattern of polycystin-2 was identical to that observed for polycystin-1 and similar to a consensus that is emerging for polycystin-1. This consists of expression early in nephrogenesis, which is most marked in the maturing proximal and distal tubules and collecting ducts but more pronounced in the distal tubules and collecting ducts by 40 weeks. Apart from the study of Griffin et al27 that observed no polycystin-1 expression in the adult, this later distal expression pattern persisting into adult life has been observed in several other studies.20,28,29 The expression of the ADPKD proteins appears only weakly in the earliest nephrogenic precursors, and is consistent with data from the Pkd1-/- knockout mouse,30 where formation of the nephron appears to occur normally; the role of these proteins is thus probably in tubular elongation and the maintenance of tubular architecture rather than in epithelial induction.
The subcellular localization of the ADPKD proteins remains uncertain with evidence for polycystin-1 expression on the apical and/or basolateral plasma membranes20,31 or a predominant intracytoplasmic location.13,27 We observed clear plasma membrane (basal and apical) expression of polycystin-2 (and polycystin-1) in developing medullary collecting ducts at 14–17 weeks, although in other tubules the expression appeared to be intracytoplasmic. Western blot analysis showed membrane associated protein in this study and elsewhere,20 although it is not clear what proportion is located on the plasma membrane. It is possible that there is continuous recycling of both proteins with appropriate membrane targeting dependent on prior interactions with other members of a polycystin complex or on specific cell-cell or cell-matrix interactions.
In other organs commonly affected by cysts, such as the liver and pancreas, polycystin-2 expression mirrors that of polycystin-120,27 with protein detected in the ductal epithelial structures of both organs persisting up to 40 weeks of gestational age. Significantly, prominent expression of both polycystin molecules was also found in all other epithelial tissues including the lung, small and large bowel, brain, reproductive organs, placenta, and thymus, indicating a more general role for these proteins in the maintenance of epithelial differentiation and organization. Furthermore, the expression of both proteins by other cell types such as muscle, endothelial, and neuronal cells indicate a more widespread role in the formation and/or maintenance of nonepithelial tissues. This would certainly be consistent with the many systemic non-cystic features described in ADPKD. The function(s) that the ADPKD proteins may have in these diverse cell types can as yet only be guessed at, with structural predictions suggesting a role in signaling, triggered by cell-cell/matrix interactions5 or a possible involvement in ion transport.4,6,32
In general, our results in nonrenal fetal tissues are consistent with those previously described for polycystin-127 but extend them by showing coexpression of the polycystin molecules by a wide variety of different cell types. Nevertheless, some questions remain. We previously described the highest levels of adult PKD1 gene expression in the brain.13 Unlike others,29 we did not observe polycystin-1 (or polycystin-2) expression by astrocytes, but rather by neuronal cell bodies throughout the developing brain. However, no neural phenotype has been reported for ADPKD, indicating that the presence of one mutant allele does not disrupt neural development. It will be interesting to see if there is a neural phenotype in a fuller description of the Pkd1-/- and Pkd2-/- knockout mice.19,30 One clear phenotype affecting the brain and associated with ADPKD, is an increased level of intracranial aneurysms. Interestingly, we found staining for both ADPKD proteins in the endothelial and smooth muscle cells of the arteries in the brain and other organs. Unlike the study of Griffin et al,33 however, staining of muscle cells was evident without the requirement of protease treatment. This pattern of staining suggests a direct role for the polycystin molecules in the pathology of this disease complication.
Our studies of PKD2 cystic tissue showed that the majority of cysts stained for polycystin-2. In the 2 cases with defined germline mutations that eliminate the p30 epitope, this must represent protein encoded by the normal allele. A similar pattern where the majority of cysts stain has been found in analysis of PKD1 cystic tissue with polycystin-1 antibodies,20,27,28,31 including cases with defined mutations that remove the antibody epitope13,21 . The PKD2 results, however, differ from those found in mice heterozygous for a Pkd2 null mutation, where no polycystin-2 expression was detected in cysts, suggesting that somatic loss of the normal product had occurred.19 However, these animals had relatively few cysts and it will be interesting to see if any show polycystin-2 expression in a more detailed analysis. Our results could be consistent with a two-hit hypothesis if the staining is of protein inactivated by a missense change. The variable number of positive cysts between individual tissues suggests that this is a possibility because the nature of the somatic mutation is likely to be a chance event. Alternatively, these results may indicate that cyst initiation can occur without loss of the normal protein by a dosage effect or possibly due to somatic changes at other loci (such as PKD116 ), which may encode proteins that interact with the polycystin complex. Analysis of individual PKD2 cysts for somatic mutations would help resolve whether a second genetic event at this locus is always required for cyst development.
Analysis of PKD2 cystic tissue with a polycystin-1 antibody also showed
staining of most cysts. Interestingly, cysts negative for polycystin-2
were also negative for polycystin-1 (Figure 5)
; a similar concordant
expression pattern was found in PKD1 cysts. Although the loss of
expression of one polycystin molecule may be explained by a germline
and a somatic mutation, it is unlikely that somatic genetic events
could account for the removal of the second ADPKD protein in the same
cyst. More credible, and consistent with the polycystin molecules
forming a multimeric complex, absence of one may lead to rapid
degradation of other binding partner(s). Experimental evidence of
polycystin-2 stabilizing polycystin-1 has been described.8
Note added in proof: Recent evidence of somatic mutations in a proportion of PKD2 renal cysts has been described.35,36
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Acknowledgements |
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Footnotes |
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Supported by grants from the National Kidney Research Fund, the Medical Research Council, the Wellcome Trust, and the Kidney Foundation of Canada. A. C. M. O. is a National Kidney Research Fund Senior Fellow. C. J. W. holds a Wellcome Career Development Fellowship.
Accepted for publication March 2, 1999.
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