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*
University Children's Hospital, Freiburg University, Freiburg,
Germany.
Max-Planck Institute for Immunobiology, Freiburg, Germany.
Correspondence to Dr. Friedhelm Hildebrandt, University Children's Hospital, Mathildenstrasse 1, D-79106, Freiburg, Germany. Phone: +49 761 270 4301; Fax: +49 761 270 4533; E-mail: hildebra{at}kk1200.ukl.uni-freiburg.de
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Disruption of several unlinked genes leads to this renal histopathologic picture. In recessive NPH, at least three different gene loci are known. The gene (NPHP 1) responsible for juvenile nephronophthisis (NPH1; MIM 256100) is localized on human chromosome 2q12-q13 (5, 6). A gene locus for infantile nephronophthisis (NPH2; MIM 602088) is localized on chromosome 9q22-q31 (7), and a third gene locus for adolescent nephronophthisis (NPH3) has been mapped to chromosome 3q21-q22 (H. Omran, C. Fernandez, M. Jung, K. Häffner, B. Fargier, A. Villaquira, R. Waldherr, N. Gretz, M. Brandis, F. Rüschendorf, A. Reis, F. Hildebrandt. Identification of a new gene locus for adolescent nephronophthisis on chromosome 3q22 in a large Venezuelan pedigree, submitted for publication). In addition, recessive forms of NPH with extrarenal involvement are known. In Senior-Løken syndrome (8), NPH is associated with retinitis pigmentosa (MIM 266900) (9), and in Cogan syndrome NPH occurs in combination with oculomotor apraxia (10). Autosomal dominant forms of the NPH/MCD complex lead to end-stage renal failure only in the third decade of life or later and are not associated with extra-renal manifestations (11). For these disease variants, the term "autosomal dominant medullary cystic kidney disease (ADM-CKD)" is used. A gene locus for ADMCKD1 (MIM 174000) has been localized to chromosome 1q21 (12). A second locus was mapped to chromosome 16p12 (13), and the existence of a third locus has been suggested (A. Fuchshuber, personal communication).
Very little is known about the pathogenesis of NPH. As a first step in its analysis, we have recently identified by a positional cloning approach the gene for juvenile nephronophthisis type 1 (5). This gene (NPHP1) encodes a novel gene product termed nephrocystin, which contains a src-homology 3 (SH3) domain. SH3 domains are modular protein-binding domains and are known to interact strongly with proteins, which contain a proline-rich recognition sequence of the consensus "PXXP" and are found in focal adhesion signaling complexes (14, 15). To date, few data are available on the expression pattern of the NPHP1 transcript, as well as on structural motifs and evolutionary conservation of its gene product nephrocystin.
We have previously identified several alternative splice variants of NPHP1 (5). NPHP1 exon 9 possesses a unique strong splice acceptor for different alternative weak splice donors of exon 8. The weak splice donors of exon 8 may be read through to produce a short 5' transcript of NPHP1.
To study evolutionary sequence conservation of NPHP1, in this study we obtained full-length cDNA sequence of the mouse homologue and identified a Caenorhabditis elegans (C. elegans) homologue in a database search. Nephrocystin exhibits an evolutionary conserved and hitherto unknown association of an SH3 domain with other putative domains of protein-protein interaction, such as coiled-coil and E-rich domains.
Expression analysis of human mRNA revealed widespread but weak tissue distribution. In the adult mouse there is very strong expression in testis. In situ hybridization analysis reveals that this expression occurs specifically in cell stages of the first meiotic division and thereafter. In situ hybridization to whole mouse embryos demonstrates widespread and uniform expression at all developmental stages. These data provide the first evidence that nephrocystin is highly conserved between a wide range of species, and might be an important constituent of a majority of cells in the body. Prominent expression in adult testis of the mouse suggests a role in sperm cell differentiation.
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cDNA Library Screening and cDNA Sequencing
About 1 x 106 plaques of a mouse diaphragm cDNA library
(no. 937393; Stratagene, Heidelberg, Germany), constructed with a Uni-ZAP-XR
phage vector, were screened by hybridization with mouse 3' EST clone
vg40 h08 at normal stringency according to the manufacturer's protocol. This
clone covers the sequence from exon 18 to the end of the coding region (nt
1794-2235). Briefly, the EST fragment was P32 -labeled by random
prime labeling (Amersham, Braunschweign, Germany). Transformants were
replicated onto Hybond N+ filters (Amersham) and hybridized
overnight at 60°C in 20 ml of ExpressHybTM hybridization solution
(Clontech, Palo Alto, CA). Filter membranes were washed for 10 min with 2
x SSC buffer and 0.05% sodium dodecyl sulfate at room temperature and
exposed to x-ray film with two intensifying screens for 16 h. The only
positive cDNA clone MD1 was plaque-purified, subcloned into pBluescript SK +
(Stratagene), and directly sequenced by fluorescently labeled dideoxy
sequencing, using T3 and T7 primers on an ABI 377 Sequencer (Applied
Biosystems, Foster City, CA).
Rapid Amplification of cDNA Ends and PCR
To obtain full length of the mouse Nphp1 transcript, 5'
rapid amplification of cDNA ends (RACE) was carried out using a mouse testis
MarathonTM cDNA amplification kit according to the manufacturer's
protocol (no. 7413-1; Clontech). To obtain clone M-a, primary and nested PCR
were carried out using primers from adaptor AP1 with mouse-specific primer
(derived from clone MD1)
MD1-596-(5'-TCAACTCCTTCAGCATCCTTAGCCAGCC-3'), and in a subsequent
nested PCR with adaptor AP2 and internal primer MD1-505-
(5'-CCAGTCTGCTGAGCAGCGAAGTCCCC-3'). A similar protocol was applied
to generate the murine partial cDNA clone M1357-1774, using primers MD1-1357+
(5'-GGTGACTGTTTTATCAGGTCC-3'); and MD1-1774-
(5'-CATCTCCAAGAATTTGTCGG-3'), which were derived from mouse cDNA
clone MD1.
Northern Blot Analysis
A normalized human poly(A)+RNA dot blot (no. 7770-1; Clontech)
containing 43 different human adult and seven fetal tissues, and a murine
poly(A)+RNA dot blot (no. 7771-1; Clontech) containing 18 murine
adult and four fetal tissues were hybridized with Nphp1 cDNA probes
according to standard (16) and
the manufacturer's protocol. Probes used were a 3'-specific human
NPHP1 cDNA containing exons 9 to 20 (nt 1026-2114), a 5'
-specific human cDNA containing exons 1 to 8 (nt 10-937), and murine
Nphp1 cDNA clone MD1 containing exons 5 to 20 without exons 14 to 16
(nt 456-2217). (All positions are given in relation to the human start codon.)
Optical density was normalized against the background signal on 100 ng of
yeast and Escherichia coli RNA and expressed as relative units.
A multiple tissue Northern blot with human adult poly(A)+RNA (MTN7760-1; Clontech) was hybridized at normal stringency with a 5'-specific human cDNA, which contains exons 1 to 8 only (nt 10-937). Standard (16) and the manufacturer's protocols were applied. A multiple tissue Northern blot containing 20 µg of murine poly(A)+RNA was hybridized at normal stringency with mouse Nphp1 cDNA clone MD1 (nt 465-2217) as described previously (17). Exposure for autoradiography was 3 wk.
In Situ Hybridization
Antisense Nphp1 cDNA from murine clone MD52-2284 was hybridized to
whole mouse embryos and to tissue sections of murine adult testis. Clone
MD52-2284 was constructed by PCR using clones M-a and MD1 as templates with
primers MD52+ (5'-GCAG-GAGCTGAAGCTGCAG-3') and MD2284-
(5'-GCATATGACT-ACGTTCTAACC-3'). This clone encompasses murine
Nphp1 sequence, which is equivalent to human exons 1 to 20 without
exons 14 to 16. Sense sequence was used as negative control. The protocol used
for whole-mount in situ hybridization is based on a procedure
described by Parr et al.
(18) and was modified
according to Knecht et al.
(19). In situ
hybridization on tissue sections was carried out according to Lescher et
al. (20) with the
following modification: Prehybridization and hybridization were carried out at
70°C. After the hybridization, all steps followed the procedure described
for whole-mount in situ hybridization.
Sequence Data
The composite human cDNA NPHP1 sequence is from GenBank (accession
nos. AF023674 and AJ001815). Here we use the sequence of AF023674, based on
clone EO3, which codes for two glutamine residues at positions 313 and 314,
whereas AJ001815 codes for only one glutamine residue. The cDNA sequence of
the C. elegans NPHP1-like sequence was derived from genomic sequence
generated by the Sanger Center (GenBank accession no. Z49911), and from EST
clone yk467b4.5. The sequence of the full-length murine Nphp1 cDNA
isolated in this study was deposited with GenBank (accession no.
AF127180).
Statistical Analyses
For cDNA and amino acid sequence comparisons, the following programs from
the Genetics Computer Group (GCG) Wisconsin were run at the Heidelberg Unix
Sequence Analysis Resources (HUSAR) (URL:
http://genius.embnet.dkfz-heidelberg.de:8080/): BLAST, PILEUP, and CLUSTAL.
Standard parameters were applied. Figure
1 was produced using the programs LINEUP and PRETTYBOX. The
secondary structure prediction program COILS was run at the Baylor College of
Medicine site (URL: http://dot.imgen.bcm.tmc.edu:9331/).
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Within human exon 8, murine clone MD1 was colinear with human splice variant 8A (Figure 3d). In the position equivalent to the exon 9-10 junction in human, there is in mouse clone MD1 an in-frame CAG insertion (encoding an alanine) (Figure 3g). In addition, clone MD1 lacks the human exon equivalents 14 to 16 (Figure 3g). This apparent splicing event of MD1 results in a frameshift of the open reading frame. To determine whether continuity between human exon equivalents 14 to 16 was also present in mouse, we performed PCR using primers to exons 13 and 17 of MD1 on murine testis cDNA, and obtained a single band. Cloning and sequencing revealed that this clone (M1357-1774) contained a splicing product that was completely colinear to the human transcript (Figure 3h). Therefore, we conclude that MD1 represents an aberrantly spliced transcript and that splicing of murine Nphp1 in this region can occur in a manner colinear to the human transcript. The composite Nphp1 mouse cDNA comprises 2332 bp. It terminates with a poly(A) stretch, which indicates a full-length 3' end. The composite murine Nphp1 cDNA sequence was deposited with GenBank (accession no. AF127180). Sequence identity between human- and mouse-deduced amino acid sequences was 83%, when the human exon splice variant 8A (Figure 3d) was used for comparison.
Identification of a C. Elegans Homologue of NPHP1
Sequence comparison of human and mouse Nphp1 cDNA yielded high
similarity with a putative gene of C. elegans (M28.7), which has been
recently identified by the nematode C. elegans genome sequencing
project (GenBank accession no. Z49911). Genomic sequence and exon/intron
structure for this C. elegans NPHP1 homologue suggests the presence
of nine exons. Exon-intron structure was partially conserved between
NPHP1 genes from mammals (human and mouse) and from C.
elegans as indicated in Figures
1 and
3. The C. elegans
NPHP1 homologue shows 23% amino acid identity and 42% similarity to the
human sequence, after adjustments for gaps.
Identification of Conserved Protein Domains
Nephrocystin is a novel gene product. However, it contains a short segment
that is highly similar to known SH3 domains
(5,
6). To further characterize the
amino acid sequence in which the SH3 domain of nephrocystin is embedded,
deduced amino acid sequences of human, mouse, and C. elegans
nephrocystin were compared. Sequence comparison between human and mouse
indicated for both E-rich domains, which flank the SH3 domain (see below), two
flanking short gaps each (Figure
1). This finding suggests that the exact number of negatively
charged residues might not be critical for the function of E-rich domains. An
additional gap results from insertion of "CAG" coding for alanine
in mouse cDNA clone MD1 at the splice junction from exon 9 to exon 10
(Figure 1 and
Figure 3g). After adjustment
for these small gaps, there is between human and mouse amino acid sequence 91%
similarity and 83% identity, whereas between human nephrocystin and the C.
elegans nephrocystin-like sequence amino acid sequence similarity is 42%
(23% identity) (Figure 1).
Extensive sequence comparison of nephrocystin-deduced amino acid sequence
to databases resulted in the identification of putative structural domains as
well as segments of significant sequence similarity with known proteins. Exons
1, 2, 3, and 4 in human/mouse and exon 1, 2, and 3 in C. elegans
encode multiple putative coiled-coil domains
(Figures 1,
4, and
5). For their analysis we used
the secondary structure prediction program "COILS," which modeled
with high probability (score > 0.9) three amphipathic 
helices,
termed I, II, and III for human nephrocystin sequence
(Figure 5). The two putative
coiled-coils detected in the C. elegans nephrocystin homologue
correspond to human coiled-coils II and III (data not shown). Coiled-coil
domains are amphipathic -helical structures of heptad repeats, in which
hydrophobic amino acids are found in positions 1 and 4
(Figure 5). This structure
resembles a cylinder, which on one side in a row slightly oblique to its
longitudinal axis exposes hydrophobic amino acid residues (Figures
4 and
5). Two such coiled-coil
cylinders may bind to each other by hydrophobic interaction between these
residues. The structure resembles two aligned rods, which are slightly
inter-twined (21).
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Besides sequence conservation between human, mouse, and C. elegans, the sequence of all three coiled-coil domains of human nephrocystin was found to be conserved in continuity in skeletal muscle myosin, in the region of the myosin S2 segment including the "hinge" region, where a coiled-coil structure is suggested (29% identity, 52% similarity; accession no. P13538). It is also conserved in continuity by many other proteins, which are involved in cytoskeletal scaffolding structures, vesicular traffic, or chromatin binding (list available from the authors).
The nephrocystin SH3 domain, which is highly conserved throughout evolution including yeast (5), is encoded in the human transcript by two exons: the 3' half of exon 5 and all of exon 6 (Figure 1 and 4). We found that the 5' half of exon 5 encodes a glutamate-rich (E-rich) sequence (termed first E-rich domain) (Figure 4). This domain contains from the start of exon 5 to the beginning of the SH3 domain 23 negatively charged amino acid residues (E or D) within 43 consecutive amino acids (53%). Even more strikingly, within a core sequence of 26 amino acids, there are 19 glutamate (E) or aspartate (D) residues (83%). In C. elegans, an equivalent was found for the first E-rich region with amino acid sequence identity of 11/22 (50%) to the human sequence (Figure 1). This finding indicates that the SH3 domain has been conserved between nematode and mammals within the context of the coiled-coil and first E-rich domain (Figures 1 and 4). At closer inspection, the E-rich domain in C. elegans is flanked by short sequences not found in human and mouse (see below), whereas in the two mammals this E-rich domain was in direct continuity with the upstream coiled-coil domain and with the downstream SH3 domain (Figures 1 and 4). Immediately following the SH3 domain, there is in human and mouse a second E-rich domain, which has no equivalent in C. elegans (Figures 1 and 4).
In C. elegans, the E-rich region and the SH3 domain are separated by two unconserved regions (Figures 1 and 4). The first one was a glutamine/proline (QP)-rich domain and the second one a sequence with high similarity to cytochrome P450 (data not shown). The QP-rich sequence over a stretch of 21 amino acids was highly similar (>40% identity) to sequences containing faithful or incomplete triplet repeats of the sequence "PQQ." The SH3 domain was described previously (5). It had been found to be highly homologous to human and mouse proto-oncogenes c-crk. This SH3 domain shows very high amino acid sequence conservation between human and C. elegans (60% similarity and 40% identity) (Figures 1 and 4).
From the data on primary and secondary structure predictions for nephrocystin, we conclude that human and mouse nephrocystin encode putative domain structures in the following order (Figure 4): coiled-coil domains I, II, and III, first E-rich domain, SH3 domain, second E-rich domain. In C. elegans, this domain structure is conserved with three exceptions: There is no equivalent for coiled-coil I, there is an interspersed QP-rich and a cytochrome P450-like region between the first E-rich and SH3 domains, and there is no second E-rich domain.
Nephrocystin cDNA Expression Analysis
To gain insight into tissue specificity of NPHP1 expression,
extensive Northern, Northern dot blot, and in situ hybridization
analyses were performed on multiple human and mouse tissues. A normalized
human poly(A) +RNA dot blot containing 50 different human tissues
was hybridized consecutively with a 3'-specific human NPHP1
cDNA (exons 9 to 20), and with a 5'-specific human cDNA (exons 1 to 8)
(Figure 3). There is widespread
tissue expression, with strongest expression in pituitary gland, spinal cord,
thyroid gland, testis, skeletal muscle, trachea and kidney, in order of
strength (Figure 6a). The
5'-specific probe in addition shows strong expression also in lymph node
(Figure 6b). This
5'-specific expression pattern might be the result of differential
expression of an alternative short 5' transcript, which is
polyadenylated in exon 8' as exemplified by EST yy63e10
(Figure 3e). To demonstrate the
existence of this alternative transcript, a human multiple tissue Northern
blot, which when previously hybridized with the 3'-specific probe
yielded a 4.5-kb full-length transcript
(5), was now probed with the
5'-specific probe and revealed in fact the presence of a short
alternative transcript of 1.2 kb (Figure
7a). To corroborate the NPHP1 tissue expression pattern
in a closely related organism, a murine poly(A) +RNA dot blot
containing 22 different murine tissues, including whole mouse embryo of days
post coitum 7, 11, 15, and 17, was hybridized with mouse Nphp1 cDNA
clone MD1 (exons 5 to 20 without exons 14 to 16). The pattern observed was
similar to results with the 3'-specific probe from human
(Figure 6, a and b), with the
exception that expression was strongest in mouse testis
(Figure 6c). In addition, this
dot blot revealed expression in whole mouse embryo at all developmental stages
tested (Figure 6c).
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In addition, a multiple tissue murine Northern blot was hybridized with a cDNA probe of mouse Nphp1 clone MD1 (Figure 3g). A single transcript of 2.4 kb was detected (Figure 7b), which corresponds well with the murine composite full-length sequence of 2332 kb (Figure 3, g through i). This transcript was expressed very weakly in total embryo at most embryonic stages, as well as in fetal and adult kidney and in adult heart even after 3 wk exposure of the blot. In contrast, there is already very strong expression in adult testis detected after an overnight exposure (Figure 7b). This result parallels the results from dot blot analysis in human and mouse (Figure 6, a and c), and expression in adult kidney and heart is in agreement with previous Northern analysis in adult human tissues (5).
To evaluate tissue-specific expression during mouse embryonic development, in situ hybridization was performed using as a probe murine Nphp1 cDNA clone MD52-2284 (contains exons 1 to 20 without exons 14 to 16) with whole mouse embryos. Expression was found to be widespread but low between days p.c. 7.5 and 11.5 (Figure 8). Because Northern analysis has shown strong Nphp1 expression in testis, in situ hybridization on testis sections of 6-wk-old mice was performed to gain a cellular resolution of Nphp1 expression in this tissue. Strong expression was found in seminiferous tubules (Figure 9, a and c). Differential expression for specific cell types was observed, with strong expression in cell stages at or beyond meiosis, such as, spermatocytes I and II (result of first meiotic division), spermatids (result of second meiotic division), and sperm (Figure 9b). In contrast, there was no expression in spermatogonia A and B, Sertoli cells, and interstitial Leydig cells. Likewise, semithin kidney sections of adult and embryonic stages yielded no signal above sense control for Nphp1 expression, under the conditions used to demonstrate Nphp1 mRNA expression in the testis.
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It is interesting to note that in autosomal dominant polycystic kidney disease, the two gene products (polycystin 1 and 2) of the two disease forms strongly interact with each other through coiled-coil domains (22,23). The finding that several domains of protein-protein interaction (SH3 and coiled-coil) are encoded by NPHP1 suggests that putative binding partners of nephrocystin represent gene products of disease genes of the NPH/MCD complex of diseases.
Regarding potential functions of the putative domains found, one might consider different animal models with an NPH-like phenotype. In this context, three different mouse models of targeted gene disruption of the genes for tensin, angiotensin-converting enzyme (ACE), and Bcl-2 are of interest.
In the tensin knockout mouse model, pathologic changes were only observed in the kidney with a histologic picture highly reminiscent of human NPH: There was marked tubular ectasia and cyst development as well as interstitial inflammatory infiltration. Electron microscopy demonstrated a lack of focal adhesions in cystic tubules (24). Tensin is an F-actinbinding component of focal adhesions, contains a src-homology 2 domain, and plays a central role in focal adhesion signaling. Because most known proteins containing SH3 domains are part of focal adhesion signaling complexes, nephrocystin might also play a role in focal adhesion signaling (25).
There are two additional examples of abnormal expression of focal adhesion
components in NPH-like diseases. One comes from studies on integrin expression
in human NPH, in which Rahilly and Fleming described strong 5ß1
integrin expression in proximal tubules, from which this integrin is normally
absent (26). The other comes
from the kd/kd mouse model for NPH
(27), which was characterized
histologically, demonstrating that those proximal tubular cells that were
affected by typical lesions showed no expression of fibronectin receptors
4ß1 (VLA4) and 5ß1 (VLA5), while 6ß1
(VLA6) expression was increased
(28). In both instances,
tubular expression of integrins (as major components of focal adhesion
signaling) is altered. The hypothesis that nephrocystin might be part of focal
adhesion signaling complexes is also supported in analogy by the finding that
coiled-coil-containing proteins are known to be part of cell membrane-attached
protein scaffold complexes and focal adhesion signaling complexes. However,
this hypothesis will have to be tested by studies on nephrocystin subcellular
localization and isolation of protein binding partners.
We found strong similarity between the core E-rich sequences of human and C. elegans, with amino acid sequence identity of 11/22. This finding indicates that the SH3 domain has been conserved between nematode and mammals within the context of the coiled-coil and first E-rich domain (Figures 1 and 4). Interestingly, in plectin (P30427), a protein that is known to interact with vimentin and laminin B and that crosslinks and anchors intermediate filaments to membranes, there was sequence similarity with all three coiled-coil domains of nephrocystin in continuity with an E-rich sequence (29) (data not shown).
The QP-rich sequences found in the C. elegans nephrocystin homologue showed sequence similarity to zyxin, which associates with actin in focal adhesions and may be a component of focal adhesion signal transduction pathways, activating adhesion stimulated changes in gene expression (44% identity; SWISS-PROT accession no. Q15942). In monocytic leukemia zink finger protein, such as in nephrocystin, the QP-rich domain is found in continuity with an N-terminal E-rich domain (43% identity; SWISS-PROT accession no. Q92794).
Knowledge of the presence of these putative structural domains or sequence similarities in nephrocystin can now be used for the generation of functional hypotheses for nephrocystin.
Expression Studies
In nephronophthisis, there is little correlation of the tissue expression
pattern of NPHP1 with the clinical phenotype, which exhibits
pathogenic changes only in the kidney
(5,6).
This is not an unusual situation, however, and has been observed in other
monogenic diseases such as X-linked retinitis pigmentosa, in which the
RPGR gene is widely expressed but detected only in very low levels in
the retina, where the only pathogenic changes occur
(30).
We found tissue-specific Nphp1 expression in adult mouse testis, with restriction to distinct cell stages of meiotic or postmeiotic spermatogenesis. This has been described for a few other genes (31). As an example, cyclin A1 is expressed in mice exclusively in the germ cell lineage, and in testis in cell stages immediately preceding meiosis and thereafter (32,33). This expression pattern is highly reminiscent of Nphp1 expression in mouse testis. It is interesting to note that in cyclin A1 knockout mice (34), spermatocytes undergo apoptotic cell death, resulting in infertility of male cyclin A1 -/- mice. However, current data available to us on 23 male patients with molecular genetically proven NPH1 do not allow assessment of male fertility in human NPH1, due to the relatively young age of NPH1 patients that have received a renal allograft.
In this regard, the mouse model of targeted disruption of the bcl-2 gene might be of relevance. This mouse model exhibits a renal phenotype similar to NPH, although fulminant apoptosis is present and typical basement membrane changes of NPH are lacking. Because Bcl-2 is a modulator of apoptosis, there might be a link of nephrocystin function to processes of apoptosis in renal tubular cells (35). In this light it seems interesting that Lin et al. were able to show in Madin-Darby canine kidney cells, which regularly develop into simple epithelial cell cysts when cultured in type I collagen gel, that Bcl-2 overexpression averts cyst cavitation (36). Their data indicate that apoptosis may be an essential initial event for renal cyst formation.
Another animal model with an NPH-like phenotype is the knockout mouse for the ACE gene. A testis-specific isoform of this gene transcribed from a promoter within intron 12 is encoded by the 3' region of the gene and is expressed only in testis. Like nephrocystin in adult mouse, it is specifically expressed in postmeiotic spermatogenic cells and sperm (37). ACE knockout mice show reduced fertility in the male and are phenotypically affected by a nephronophthisis-like disease, with renal medullary cysts, corticotubular atrophy, interstitial inflammation, water wasting, and uremia (38). In addition, there is renal vessel wall hypertrophy together with architectural disruption of the renal vascular tree (39). Vessel wall hypertrophy is also seen in human NPH. Furthermore, ACE is known to play a major role in kidney morphogenesis (40). Interestingly, when ACE knockout mice were interbred with transgenic mice specifically expressing ACE from an artificial testis-specific promoter, fertility in males was restored, but mice maintained kidney defects of ACE -/- mice (41). Thus, the testis-specific form of the ACE gene, like the Nphp1 gene, is very specifically expressed in meiotic and postmeiotic cells of spermatogenesis, besides weak expression in kidney and other tissues.
It is interesting to note that a nephronophthisis-like phenotype can be produced by targeted disruption of several different genes, since for human diseases of the NPH/MCD complex at least four different recessive and three different dominant gene loci are known. Isolation of protein-protein binding partners of nephrocystin might therefore lead to the identification of disease genes for other diseases of the NPH/MCD complex.
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M. E. Winkelbauer, J. C. Schafer, C. J. Haycraft, P. Swoboda, and B. K. Yoder The C. elegans homologs of nephrocystin-1 and nephrocystin-4 are cilia transition zone proteins involved in chemosensory perception J. Cell Sci., December 1, 2005; 118(23): 5575 - 5587. [Abstract] [Full Text] [PDF]
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G. Mollet, F. Silbermann, M. Delous, R. Salomon, C. Antignac, and S. Saunier Characterization of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to primary cilia and centrosomes Hum. Mol. Genet., March 1, 2005; 14(5): 645 - 656. [Abstract] [Full Text] [PDF]
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M. T.F. Wolf, J. Lee, F. Panther, E. A. Otto, K.-L. Guan, and F. Hildebrandt Expression and Phenotype Analysis of the Nephrocystin-1 and Nephrocystin-4 Homologs in Caenorhabditis elegans J. Am. Soc. Nephrol., March 1, 2005; 16(3): 676 - 687. [Abstract] [Full Text] [PDF]
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 J. C. Donaldson, R. S. Dise, M. D. Ritchie, and S. K. Hanks Nephrocystin-conserved Domains Involved in Targeting to Epithelial Cell-Cell Junctions, Interaction with Filamins, and Establishing Cell Polarity J. Biol. Chem., August 2, 2002; 277(32): 29028 - 29035. [Abstract] [Full Text] [PDF]
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 V. Chauvet, F. Qian, N. Boute, Y. Cai, B. Phakdeekitacharoen, L. F. Onuchic, T. Attie-Bitach, L. Guicharnaud, O. Devuyst, G. G. Germino, et al. Expression of PKD1 and PKD2 Transcripts and Proteins in Human Embryo and during Normal Kidney Development Am. J. Pathol., March 1, 2002; 160(3): 973 - 983. [Abstract] [Full Text] [PDF]
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H. OMRAN, K. HÄFFNER, S. BURTH, C. FERNANDEZ, B. FARGIER, A. VILLAQUIRAN, H.-G. NOTHWANG, S. SCHNITTGER, H. LEHRACH, D. WOO, et al. Human Adolescent Nephronophthisis: Gene Locus Synteny with Polycystic Kidney Disease in Pcy Mice J. Am. Soc. Nephrol., January 1, 2001; 12(1): 107 - 113. [Abstract] [Full Text]
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F. HILDEBRANDT and E. OTTO Molecular Genetics of Nephronophthisis and Medullary Cystic Kidney Disease J. Am. Soc. Nephrol., September 1, 2000; 11(9): 1753 - 1761. [Abstract] [Full Text]
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 T. Benzing, P. Gerke, K. Hopker, F. Hildebrandt, E. Kim, and G. Walz Nephrocystin interacts with Pyk2, p130Cas, and tensin and triggers phosphorylation of Pyk2 PNAS, August 14, 2001; 98(17): 9784 - 9789. [Abstract] [Full Text] [PDF]
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