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*
Division of Nephrology, Université
Catholique de Louvain, Medical School, Brussels, Belgium
Center for Human Genetics and Medical Genetics Unit,
Université Catholique de Louvain, Medical
School, Brussels, Belgium
Department of Radiology, Université
Catholique de Louvain, Medical School, Brussels, Belgium
§
Department of Medicine, Division of Renal Diseases, University of Colorado
School of Medicine, Denver, Colorado.
Correspondence to Dr. Olivier Devuyst, Division of Nephrology, Université Catholique de Louvain, 10 Avenue Hippocrate, B-1200 Brussels, Belgium. Phone: 32-2-764-18-55; Fax: 32-2-764-28-36; E-mail: devuyst{at}nefr.ucl.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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F508 in all cases, except for one control subject (1717-1G A). The
frequencies of the 5T, 7T, and 9T intron 8 alleles were also
similar in the ADPKD and control groups. Two additional patients with ADPKD
and the F508 mutation were detected in the families of the two probands
with CF mutations. Kidney volumes and renal function levels were
similar for these four patients with ADPKD and F508 CFTR (heterozygous
for three and homozygous for one) and for control patients with ADPKD
collected in the University of Colorado Health Sciences Center database. The
absence of a renal protective effect of the homozygous F508 mutation
might be related to the lack of a renal phenotype in CF and the variable,
tissue-specific expression of F508 CFTR. Immunohistochemical analysis
of a kidney from the patient with ADPKD who was homozygous for the F508
mutation substantiated that hypothesis, because CFTR expression was detected
in 75% of cysts (compared with <50% in control ADPKD kidneys) and at least
partly in the apical membrane area of cyst-lining cells. These data do not
exclude a potential protective role of some CFTR mutations in ADPKD but
suggest that it might be related to the nature of the mutation and renal
expression of the mutated CFTR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interfamilial phenotypic variability in ADPKD may be accounted for by genetic heterogeneity. Mutations in the PKD1 gene (16p 13.3) cause the disease in approximately 85% of families (4), and mutations in PKD2 (4q21-23) account for the vast majority of remaining cases (5). Although the phenotypes caused by mutations in PKD1 and PKD2 are very similar, they differ in severity, because ESRD occurs an average of 15 yr earlier in type 1 ADPKD (6). Interfamilial phenotypic variability in ADPKD could also result from the nature of the mutation itself (7). The severity of renal disease may differ widely among affected members within given families, even between nonidentical twins (8). This could be explained by at least two mechanisms, i.e., a second hit event or modifying genes. Because cyst formation seems to be triggered by a somatic mutation in the normal PKD1/PKD2 allele (9, 10), the random nature of this phenomenon could provide a valuable explanation. However, the existence of modifying loci has been demonstrated in animal models of polycystic kidney disease (11, 12). These putative modifier genetic backgrounds might affect either cystogenesis itself or clinical conditions (such as hypertension) that are associated with disease progression (13). The first example of the effect of a modifying gene in human ADPKD is the association of the DD genotype of the angiotensin-converting enzyme insertion/deletion polymorphism with worse renal prognoses (14).
Several lines of evidence have demonstrated that abnormal fluid secretion across cystic epithelium is a key factor in ADPKD cyst progression (for review, see reference 15). This process seems to be driven by transepithelial, cAMP-regulated chloride secretion, which is mediated by cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the protein encoded by the CF gene (recently renamed ABCC7 for "ATP-binding cassette, subfamily C, member 7") (16). CFTR is a 1480-amino acid integral membrane protein that contains an ATP-binding cassette domain and functions as both a chloride channel and a conductance regulator (17). CFTR mutations, resulting in functional defects in secretory epithelia (such as that in the respiratory tract, the intestine, or the exocrine pancreas), account for CF, which is the most common lethal autosomal recessive disease among Caucasians (16).
Although significant CFTR expression has been detected in the tubular epithelium lining various nephron segments in fetal and adult kidney (18,19,20), there is no overt renal phenotype in patients with CF (16). In contrast, the frequent occurrence of abnormalities of the Wolffian duct among subjects with various mutations or the T5 polymorphism in intron 8 of CF (21,22,23) suggests that CFTR is involved in the embryonic development of the male genital duct, a structure that includes the ureteric bud (24).
Strong CFTR expression in the apical area of ADPKD cyst-lining cells has been documented (25, 26), and functional studies have demonstrated the role of CFTR in chloride secretion and cyst fluid accumulation (25, 27). Therefore, a loss of CFTR function, such as occurs in CF, would be expected to slow cyst growth and attenuate the ADPKD phenotype. This hypothesis was supported by a recent report of a family with both ADPKD and CF (28); the ADPKD phenotype seemed to be less severe for the two patients affected by both ADPKD and CF, compared with members of the family without CF.
To investigate the potential disease-modifying role of the CF gene
in ADPKD, we performed systematic screening for 12 of the most common
mutations and for the intron 8 polymorphic Tn locus of CF in
a large series of unrelated patients with ADPKD and control subjects. These
analyses allowed the detection of F508 mutations in two families with
ADPKD, which were further investigated for genotype-phenotype correlations. A
molecular mechanism that might account for these correlations is proposed, on
the basis of the expression patterns of wild-type versus F508
CFTR in ADPKD kidneys.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA Extraction and Genotyping
DNA was isolated from peripheral blood lymphocytes using the NaCl
extraction procedure and conventional techniques
(30). Mutation and
polymorphism analyses were performed with different PCR amplifications.
Genomic DNA samples were screened using the Elucigene CF12 kit (based on
Amplification Refractory Mutation System technology; Zeneca Diagnostics,
Abingdon, UK), to detect the following 12 CFTR mutations: 1717-1G A, G542X,
W1282X, N1303K, F508, 3849+10kbC T, 621+1G T, R553X, G551D, R117H,
R1162X, and R334W. The characteristics of these mutations are shown in
Table 1. The studied mutations
accounted for approximately 85% of the alleles causing CF in the Belgian
population (31,
32).
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The length of the intron 8 polythymidine tract (which may contain five, seven, or nine thymidines, corresponding to the 5T, 7T, and 9T alleles, respectively) was investigated using the nested-PCR method, followed by electrophoresis (33). Exon 9 was amplified with primers CFTR.Exon 9.1 and CFTR.Exon 9.2 (34). The PCR conditions were as follows: denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 1 min, for 34 cycles. The reaction mixture contained 2.5 µl of PCR buffer (100 mM Tris-HCL, 15 mM MgCl2, 500 mM KCl [pH 8.3]), 200 µM concentrations of each dNTP, 10 pmol of each primer, and 1 unit of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT), in a final volume of 25 µl containing 100 ng of genomic DNA. To amplify the polypyrimidine sequence, we performed a nested PCR with primers CFTR-19D9 and CFTR-E9R2 (22). The conditions for the nested PCR were as described above, except that 1 µl from the first PCR was amplified with AmpliTaq Gold DNA polymerase and annealing and extension were performed at 54°C for 30 s and 74°C for 40 s, respectively. The stretch was determined using the Gene Scan 672 software of a Perkin-Elmer/Applied Biosystems 373A sequencer.
Linkage analyses for PKD1 and PKD2 were performed for the informative families of the slow progressor subset and for the two families with CF mutations (35, 36). Linkage to the PKD1 locus was investigated with 5'-flanking markers, i.e., 16AC2.5 (D16S291) and CW2 (D16S663) in the telomeric position, VK5.b (D16S94) and 218EP6 (D16S246) in the centromeric position, and the KG8 CA repeat in the 3' untranslated region of the gene (35). The microsatellites 16AC2.5, CW2, and KG8 were amplified using the conditions described previously (37); the former two were analyzed with Gene Scan 672 software. Hybridization with the biallelic probes VK5.b (MspI) and 218EP6 (PvuII) was performed by Southern blotting. Linkage to the PKD2 locus was analyzed with four microsatellite markers that flanked the gene on chromosome 4 (36).
Clinical Data and Assessment of Renal Volumes
The records for patients with ADPKD were reviewed for gender, geographical
background (Belgium or southern Europe), age at the time of DNA sampling, and,
if pertinent, age at the time of ESRD. Creatinine clearance was calculated
using the Cockcroft-Gault formula. The evolution of renal function
(1/creatinine concentration) with time for the two index patients was compared
with that for five patients with ADPKD who were matched for age and gender and
exhibited negative results in the screening for the 12 CF
mutations.
Assessment of renal volumes for patients from the two families with CF mutations was performed with computed tomographic (CT) scanning. Unenhanced CT scans (200 mA, 120 kV) were obtained with a dual-detector helical CT unit (Twin RTS; Elscint, Haifa, Israel). The protocol consisted of volumetric data acquisition through the kidneys, with 5.5-mm collimation and a pitch of 1.5. The images were reconstructed at 5-mm intervals. Volumetric measurements of the kidneys were performed with the summation-of-areas technique (38), using an Omnipro workstation (Elscint). The cross-sectional area of individual images was automatically calculated after manual tracing of the perimeter of each kidney with a computer mouse-driven stylus. The volume was then determined by multiplying the sum of all areas by the image thickness. Both creatinine clearance values and renal volumes were normalized for body surface area, for comparison with the large database on patients with ADPKD at the University of Colorado Health Sciences Center (28).
Immunocytochemical Localization of CFTR in ADPKD Kidneys
Tissue blocks were prepared from three ADPKD kidneys, as described
(19,
25). The three samples were
obtained at the time of nephrectomy for renal transplantation. One sample was
obtained from the 33-yr-old son of index patient 2, who is affected with both
type 1 ADPKD and CF (F508/F508)
(Figure 1B, subject IV.3). Two
control samples were obtained from patients with ADPKD (a 49-yr-old male
patient and a 55-yr-old female patient) who tested negative for CF
mutations. Before paraffin embedding, kidney samples were fixed for 12 h at
4°C in 4% paraformaldehyde (Boehringer Ingelheim, Heidelberg, Germany) in
0.1 M phosphate buffer (pH 7.4). Sections (6 µm thick) were rehydrated and
incubated for 30 min with 0.3% hydrogen peroxide, to block endogenous
peroxidase. After incubation for 20 min with 10% normal serum in
phosphate-buffered saline, sections were incubated for 45 min with the
anti-CFTR antibodies diluted in phosphate-buffered saline containing 2% bovine
serum albumin. After washing, sections were incubated with the appropriate
biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA),
washed again, and incubated for 45 min with avidin-biotinperoxidase complex
(Vectastain Elite; Vector Laboratories). After washing, antibodies were
detected using aminoethylcarbazole (Vector Laboratories). All incubations were
at room temperature in a humidified chamber. Control experiments included
incubation in the absence of primary antibody or with control rabbit or mouse
IgG (Vector Laboratories). Sections were mounted in glycerol/water and viewed
with a Leica DMR photomicroscope.
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Two antibodies, raised against different domains of human CFTR, were used. Polyclonal antibody 169 (a gift from W. B. Guggino, Johns Hopkins Medical School, Baltimore, MD) was raised against a peptide sequence within the R domain (residues 724 to 746), whereas monoclonal antibody MATG1031 (Transgène, Strasbourg, France) recognized a sequence in the first extracellular loop (residues 107 to 117). These two antibodies have been extensively characterized in human tissues, including normal and ADPKD kidneys (18, 19, 25).
Statistical Analyses
Data are presented as mean ± SD. Comparisons of results from
different groups were performed using 
2 and Fisher's exact
tests, as appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 = 3.08,
P = 0.08) and geographic background (2 = 0.22,
P = 0.63) parameters were similar for the ADPKD and control
groups.
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CF Genotyping
All ADPKD probands and control subjects were screened for the 12
CF mutations detailed in Table
1. Two patients with ADPKD (one in the slow progressor subset) and
five control subjects exhibited a heterozygous CF mutation, which was
identified as F508 for the two patients with ADPKD and four control
subjects and 1717-1G A for one control subject
(Table 2). The prevalence of
CF mutations in the control group (3.7%) is similar to that reported
in the literature (31,
32) and does not differ
significantly from that observed for patients with ADPKD (1.7%) (Fisher's
exact test, 0.46).
All subjects were also genotyped for the intron 8 polymorphic Tn
locus of CF (Table 3).
The vast majority of ADPKD probands and control subjects harbored the
7T allele, with a distribution of the three alleles similar to that
reported for the general population
(21). Each patient with ADPKD
or control subject who exhibited the F508 mutation also bore the
9T allele, at least in the heterozygous state. The one control
subject with the 1717-1G A mutation was homozygous for the 7T allele;
this association was previously reported
(21). Thirteen of the 117
ADPKD probands were heterozygous for the 5T allele. As shown in
Table 4, these patients were
similar to the 104 patients with only the 7T and/or 9T
allele, in terms of demographic characteristics and progression to ESRD. The
characteristics of the two ADPKD probands with the F508 mutation, as
well as further analyses of their families, are described below.
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Additional Studies of the Two ADPKD Families with F508 CFTR
Mutations
Clinical Histories. The index patient from pedigree 1
(Figure 1A, subject III.4) was
a 46-yr-old man. The diagnosis of ADPKD was made fortuitously by
ultrasonographic examination at 30 yr of age, during a sterility evaluation
that revealed poor-quality spermatozoa with mobility abnormalities (30%
spermatozoa with progressive mobility). At the age of 35 yr, hypertension was
detected, requiring treatment with atenolol and enalapril. At that time, serum
creatinine levels were normal (1.2 mg/dl). At the age of 43 yr, serum
creatinine levels were observed to be elevated (1.9 mg/dl) for the first time.
The subsequent history included three episodes of cyst bleeding, without
infection, and a coronary artery bypass graft. At the age of 45 yr, the serum
creatinine levels for the patient were 4.3 mg/dl. His father (subject II.5)
was also affected with ADPKD and began to undergo hemodialysis at the age of
54 yr. Two paternal uncles (subjects II.3 and II.13) and three paternal aunts
(subjects II.2, II.10, and II.11) were also affected with ADPKD. His paternal
grandmother (subject I.2) was reported to be dead as a result of uremia.
The index patient from pedigree 2 (Figure 1B, subject III.1) was a 63-yr-old man. The diagnosis of ADPKD was made at the age of 53 yr, during an evaluation for gross hematuria and lumbar pain. At that time, serum creatinine levels were normal. Two episodes of gross hematuria occurred subsequently. Serum creatinine levels were measured to be 2.9 mg/dl at the age of 59 yr and 4.8 mg/dl at the age of 60 yr; hemodialysis was initiated at the age of 61 yr. The patient experienced no known episodes of cyst infection or nephrolithiasis. His mother (subject II.2), one maternal aunt (subject II.3), and his maternal grandmother (subject I.2) died as a result of uremia, at the ages of 65, 50, and 70 yr, respectively. Index patient 2 has two sons with ADPKD (diagnosed for both by ultrasonographic and CT examinations), of age 37 yr (subject IV.1) and 34 yr (subject IV.3). Of note, subject IV.3 has both ADPKD and CF; the latter condition was diagnosed on the basis of a positive sweat test (performed because of repeated respiratory infections during childhood) and mutation analysis (see below). Because of the deterioration of his pulmonary condition, he underwent heart-lung transplantation at the age of 26 yr. At that time, serum creatinine levels were normal (0.6 mg/dl). After transplantation, renal function progressively deteriorated, leading to the initiation of hemodialysis at the age of 31 yr. Bilateral nephrectomy was performed at the time of renal transplantation, 1 yr later.
Genetic Analyses. Segregation analysis for PKD1 and PKD2 was performed for the two families with CF mutations. For both, linkage analysis allowed exclusion of PKD2 and was compatible with linkage to PKD1 (Figure 1).
Transmission of the F508 mutation of the CF gene was
examined in both families (Figure
2). The F508 allele harbored by index patient 1 (subject
III.4) was transmitted by his mother (subject II.6)
(Figure 2A). The F508
mutation harbored by index patient 2 (subject III.1) was observed in his two
sons, in a heterozygous state in the elder (subject IV.1) and in a homozygous
state in the younger (subject IV.3) (Figure
2B). Genotyping confirmed that their mother (subject III.2)
harbored the F508 mutation in the heterozygous state. All six patients
from these families who were heterozygous (n = 5) or homozygous
(n = 1) for the F508 mutation harbored at least one
9T allele (Figure
1).
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Correlations Between CF Genotype and ADPKD Phenotype. Renal function
and volumes were assessed for the two patients with ADPKD and a heterozygous
F508 mutation (index patients 1 and 2). The decrease in renal function
(expressed as 1/creatinine concentration with time) for these two patients was
compared with that for five unrelated patients with ADPKD without the
CF mutation, who were matched for age and gender
(Figure 3A). The slope was
steeper for both index patients than for ADPKD control subjects,
i.e., -0.0042 versus -0.0028 for index patient 1
(Figure 3A, upper) and -0.0082
versus -0.0040 for index patient 2
(Figure 3A, lower). With the
exception of the sterility documented for index patient 1, these two patients
experienced classic courses of ADPKD. Representative CT scans obtained at the
age of 45 yr for index patient 1 and 63 yr for index patient 2 are shown in
Figure 3B. Note that hepatic
cysts were absent for both patients.
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When available, creatinine clearance and renal size values for patients with ADPKD and CF mutations were compared with data collected in the large database on patients with ADPKD at the University of Colorado Health Sciences Center (28) (Table 5). The index patient from pedigree 1 (subject III.4) was characterized by relatively poor renal function (only 16% of male patients with ADPKD of that age have lower renal function), together with very large kidneys (99th and 87th percentile for the left and right kidneys, respectively). The index patient from pedigree 2 (subject III.1) was in the average range for both parameters. His two sons exhibited similar impairments of renal function; the elder had smaller ADPKD kidneys than did his younger brother, who was affected by both ADPKD and CF. It must be noted that, for the latter, the renal function and volume parameters presented in Table 5 were recorded before the combined heart-lung transplant, i.e., before the initiation of cyclosporin-based immunosuppressive therapy. Six years later, at the time of nephrectomy (age of 32 yr), the kidneys were even more enlarged (left kidney, 1097 cm3, 93rd percentile; right kidney, 721 cm3, 62nd percentile).
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Pathologic Examination Results and Immunolocalization of CFTR. The pathologic examination of the kidneys removed from subject IV.3 from pedigree 2 (who is affected by both ADPKD and CF) demonstrated polycystic kidneys with multiple dilations of Bowman's spaces and glomerular cysts, as well as areas of atrophic tubules (Figure 4, A to D). Areas of noncystic parenchyma exhibited extensive fibrosis (sclerosis index, 4/6). Multiple small focal adenomas were identified within cysts; some cyst lumina were filled with neutrophils. No signs of acute cyclosporin toxicity were observed. Very mild arteriolar hyalinosis was present.
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Immunohistochemical analysis demonstrated that a majority of cysts were stained for CFTR (Figure 4, E and F); that observation was confirmed by systematic counting within 40 randomly selected fields, which demonstrated a positive immunoreaction in 75% of the cysts (153 of 203 cysts) examined. Typical inter- and intracystic heterogeneity in CFTR staining was observed, regardless of the anti-CFTR antibody used (Figure 4, G and H). No specific reactivity was observed when incubations were performed with control IgG (Figure 4I) or without primary antibody (data not shown). Strong CFTR staining was also observed in some of the glomerular cysts (Figure 4J). The CFTR staining pattern was either diffusely intracellular or concentrated in the apical area (Figure 4, G, H, K, and L). These characteristics are similar to those observed for ADPKD kidneys from patients without detected CF mutations (Figure 4, M and N) and those previously reported (25). The percentage of cysts stained for CFTR was 52% (142 of 273 cysts) in the two ADPKD kidneys without CF mutations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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F508 mutation
in a patient with ADPKD is not associated with a milder renal phenotype in
either the heterozygous or homozygous state; and (3) the presence of
the 5T allele of the intron 8 polymorphic Tn locus, which is
known to be associated with Wolffian duct abnormalities, is not associated
with a milder ADPKD phenotype. In addition, the first detailed examination of
kidneys from a patient with type 1 ADPKD and CF demonstrated not only a marked
cystic phenotype but also the persistence of strong CFTR expression in the
majority of cysts.
The relatively high prevalence of heterozygous CF mutations in the
general population in European countries
(16,
32) is in keeping with the
3.7% prevalence of mutations observed for our control group. Similarly, the
allelic frequencies for the 5T, 7T, and 9T alleles of the
intron 8 polymorphic Tn locus of CF observed for our control
subjects, as well as the association between these alleles and the two
CF mutations described here, confirm the findings of a previous
report (21). The prevalence of
CF mutations among our series of 117 ADPKD probands (1.7%) was more
than two times lower than that among our control subjects (3.7%). Although
this difference did not reach statistical significance, the question arises of
whether the association of ADPKD with CF might have a lethal effect. The
existence of sterility resulting from spermatozoa of poor quality for one of
the subjects with a F508 mutation (index patient 1) must be noted.
Indeed, the frequency of heterozygous carriers of CF mutations is
increased among men with congenital bilateral absence of the vas deferens
(CBAVD), as well as among subjects with azoospermia resulting from other
causes or with sperm of poor quality
(39). The variable penetrance
of the phenotype of infertility among heterozygous carriers of CF
mutations could depend on the association with other variants of the gene,
such as the intron 8 polymorphic Tn locus. Whereas allele 5T
of this locus seems to be involved in CBAVD
(21,22,23),
in conjunction with CF mutations or other CF variants
(40), the genetic background
involved in the occurrence of fertility problems among male heterozygous
carriers of CF mutations without CBAVD is still unknown.
The existence of a F508 mutation in two ADPKD probands allowed us to
identify two other patients with ADPKD who harbored the same mutation in the
heterozygous state. With the exception of documented sterility for one
patient, the ADPKD phenotypes of these four patients were unremarkable.
Compared with the University of Colorado Health Sciences Center database,
their kidney size and creatinine clearance values displayed wide variability,
ranging from the 16th to the 99th percentile
(Table 5). This suggests that a
heterozygous F508 mutation in these patients does not affect the ADPKD
phenotype, which is in agreement with the report by O'Sullivan et al.
(28).
Examination of our patient with ADPKD and a homozygous F508 mutation
demonstrated that kidney involvement was similar to that observed for patients
with ADPKD without CF. This patient exhibited an average ADPKD phenotype, as
evidenced by kidney volume (particularly in comparison with his affected
brother), creatinine clearance values, and morphologic examination results
(extended cystic involvement). This absence of a protective effect of CF on
ADPKD is in contrast to a previous report
(28). Several explanations
might account for this discrepancy. First, in view of the significant number
of CF variants and the potential modifying role of variants located
inside
(21,40)
or outside
(14,41)
the CF gene, the discrepancy could be attributable to different
genetic backgrounds in the subjects described by O'Sullivan et al.
(28) and us. Second, it could
be related to the specific nature of the CF mutations detected.
Indeed, the two patients with CF described by O'Sullivan et al.
(28) each harbored a mutation
known to reduce CFTR mRNA levels (stop codon within exon 3 and severe
reduction in transcription for E60X; abnormal splicing for 3849+10kbC T)
(16,28);
for our F508/F508 patient, the problem involved abnormal protein
folding, resulting in a presumed lack of CFTR at the apical membrane
(42). Third, it is possible
that the less severe phenotypes of the two subjects described by O'Sullivan
et al. (28) were
attributable to their younger age, compared with their heterozygous or
wild-type relatives. This argument must be considered, keeping in mind that
the aggregate median survival time for CF is still <30 yr
(16).
In view of the well established role of CFTR in intracystic fluid secretion
in ADPKD (15), the existence
of a protective role of the F508 mutation because of decreased
expression of the mutated protein in cyst-lining epithelial cells was an
attractive hypothesis. However, decreased expression of F508 CFTR was
demonstrated only in sweat glands and submucosal glands of the upper airways
(43,44).
The inference that decreased expression also occurs in other tissues was
recently challenged by Kälin et al.
(45), who demonstrated that
the expression of F508 CFTR is strikingly tissue-specific, ranging from
undetectable in sweat glands to indistinguishable from that of the wild-type
protein in the respiratory and intestinal tracts. These data suggest that the
variable severity of CF in different organs might reflect heterogeneity of
expression of the mutated CFTR. It is currently unknown whether the latter
hypothesis applies to the kidney. However, given the lack of major renal
involvement in CF
(16,46),
it is not unlikely that the expression of F508 CFTR is normal or only
moderately decreased in the kidney.
Our study substantiates this hypothesis, because immunocytochemical
examination of CFTR in a kidney sample from a patient with type 1 ADPKD and CF
demonstrated F508 CFTR expression in approximately 75% of cysts. This
fraction of CFTR-positive cysts is greater than that observed for patients
with ADPKD without CF mutations (52%) and for patients with ADPKD in
general (60%) (25). At the
level of resolution achieved here, at least part of the F508 CFTR
immunoreactivity is concentrated in the apical membrane area. The staining
pattern, including the significant inter- and intra- cystic heterogeneity, is
similar to that previously reported
(25) and that observed in
patients with ADPKD without CF mutations
(Figure 4). Keeping in mind the
limitations of a single-sample examination, it is tempting to argue that the
lack of a protective effect of the F508 mutation in ADPKD reflects the
fact that mutated CFTR expression remains stable in ADPKD cysts. By analogy
with the studies of Kälin et al.
(45), our findings might
suggest that the F508 mutation does not significantly impair the
processing of CFTR in the kidney.
The amount of CFTR mRNA without exon 9 depends on the number of thymines in the polymorphic Tn sequence of intron 8. The 5T allele causes an increase in the number of CFTR mRNA transcripts that lack exon 9, which leads to a nonfunctional protein (47). The 5T allele has been associated with various genital, respiratory, and pancreatic phenotypes (21,22,48). Our study is the first to investigate the distribution of these alleles in patients with ADPKD. As shown in Table 4, we did not observe evidence for a protective effect of the 5T allele in ADPKD. However, we did not find patients homozygous for this rare allele or patients with both a 5T allele and a classic CF mutation. We did not look for the presence of alleles belonging to other loci, such as (TG)m and M470V, the combination of which might explain the penetrance of some mutations (40). Definitive exclusion of a protective effect of a particular combination of CF alleles would be a daunting task, given the highly polymorphic nature of that gene (32).
Even if it is confirmed, the lack of effect of the F508 mutation on
ADPKD phenotypes does not exclude a potential modifying role of the
CF gene in ADPKD. It merely indicates that subjects with the
F508 mutation are not good models for assessment of this effect, as
suggested by the possibility of tissue-specific regulation of mutated CFTR
expression (45) and our
demonstration of high F508 CFTR reactivity in ADPKD cysts. The effects
of a CF mutation affecting the single-channel properties of CFTR,
rather than its processing, would be very interesting to examine. However,
because F508 represents 66% of the mutated alleles throughout the world
(32), the chance of
identifying an association between ADPKD and a rare mutation of CF is
extremely small. Considerations of the role of CFTR in cyst growth must also
take into account alternative mechanisms of chloride secretion in ADPKD cysts.
Most of the morphologic and functional studies that support the role of CFTR
in ADPKD have documented a striking heterogeneity in CFTR expression in the
cells or cysts examined
(25,27).
It is possible that the expression and activity of CFTR in some cyst-lining
cells are sufficient only to maintain a filled cyst or, alternatively, that
transepithelial chloride secretion in ADPKD is mediated by different types of
transporters
(15,25).
In conclusion, we performed a wide screening of ADPKD probands, to assess
the effects of CF mutations on kidney involvement. We did not observe
a protective effect of F508 (the most common CF mutation) on
the renal ADPKD phenotype. This was also true for the 5T allele of
the intron 8 polymorphic Tn locus, which has been previously
associated with Wolffian duct abnormalities. Furthermore, we demonstrated, for
the first time, nearly normal levels of expression of the F508 CFTR in
kidney cysts. These data do not exclude a potential protective role of some
CFTR mutations in ADPKD but suggest that it might be related to the nature of
the mutation and renal expression of the mutated CFTR.
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