1 Medical Research Center, Klinikum Mannheim, University of Heidelberg, 68167 Mannheim; 2 Lilly Deutschland, 61350 Bad Homburg, and University Children's Hospital, 35392 Giessen; and 3 Institute for Anatomy and Cell Biology I, University of Heidelberg, 69120 Heidelberg, Germany
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ABSTRACT |
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Because proteinuria has been demonstrated in patients with autosomal-dominant polycystic kidney disease (ADPKD), we have investigated whether proteinuria also occurs in the (cy/+) rat, a widely used model for ADPKD. Increased urinary excretion of proteins, in particular of albumin, can be found in 16-wk-old (cy/+) rats, with a gel electrophoresis pattern compatible with a tubular origin of proteinuria. Using FITC-labeled dextran as an in vivo tracer for renal tubular endosomal function, we could show that portions of cyst-lining epithelia from proximal tubules have lost the ability to endocytose, which is necessary for the reabsorption of low-molecular-weight proteins. By immunohistochemistry, the expression of other proteins implicated in endocytosis, such as the chloride channel ClC-5 and the albumin receptor megalin, correlated well with the presence and absence of FITC-dextran in cysts. As an example of growth factor systems possibly being affected by this endocytosis defect, we could detect increased urinary levels of insulin-like growth factor-I protein in (cy/+) animals. These data indicate that proteinuria and albuminuria in the aforementioned rat model for ADPKD are due to a loss of the endocytic machinery in epithelia of proximal tubular cysts. This may also affect the concentration of different growth factors and hormones in cyst fluids and thus modulate cyst development.
ClC-5; megalin; fluorescein isothiocyanate-dextran; albuminuria; proteinuria; insulin-like growth factor-I
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INTRODUCTION |
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AUTOSOMAL-DOMINANT POLYCYSTIC kidney disease (ADPKD) can be considered as one of the most prevalent tubulointerstitial diseases; it is characterized by the formation of multiple cysts and considerable enlargement of both kidneys. Although it has now been established that mutations in the PKD1 (13a) and PKD2 (27) genes are responsible for most ADPKD cases, there is a high degree of variability in the clinical course of those patients.
Several factors have been considered as being of prognostic value in patients suffering from ADPKD. Importantly, the occurrence of proteinuria and albuminuria has been implicated in a more severe disease progression in ADPKD patients (6, 38). Although there is evidence that human ADPKD may affect all segments of the nephron, including the proximal tubule (4, 10, 14), it has not been addressed whether alterations of this nephron segment contribute to proteinuria and albuminuria. The (cy/+) rat, an animal model for ADPKD, provides a suitable tool for an investigation of the contribution of impaired tubular function to proteinuria and albuminuria, because in this strain cysts originate predominantly from the proximal tubule (8, 31, 37).
Low-molecular-weight plasma proteins such as albumin and
2-microglobulin are reabsorbed via a receptor-mediated
endocytosis pathway by proximal tubules and are then delivered to
lysosomes for degradation (25). The luminal uptake of
albumin requires the presence of binding proteins such as megalin
(gp330) as the first step in the endocytic cascade (9).
The transport of albumin and other low-molecular-weight proteins into
endocytic vesicles also depends on the appropriate endosomal
acidification, which is accomplished by the transport of protons
through a V-type H+-ATPase (25). For the
supply of the required amounts of counterions, channels like the
recently discovered chloride channel ClC-5 are indispensable to
guarantee the appropriate chloride influx into endosomes (17,
25). Defective ClC-5 function caused by mutations in the
respective gene therefore leads to impaired endosomal acidification and
consequently to low-molecular-weight proteinuria, as found in Dent's
disease and related syndromes (22, 23).
In this study we show that, similar to the situation in human patients (6, 38, 44), increased proteinuria and albuminuria can be observed in (cy/+) rats. By the administration of FITC-labeled dextran as an in vivo marker for endocytosis in the proximal tubule, in combination with the immunohistochemical analysis of ClC-5 and the multifunctional receptor megalin, we provide evidence that the partial loss of endocytosis in cysts of (cy/+) rat kidneys contributes to the increased excretion of urinary proteins. We also demonstrate that the impaired tubular endocytic capacity leads to increased urinary excretion of insulin-like growth factor-I (IGF-I). Prolonged exposure of cysts to increased luminal growth factor concentrations may play a secondary role in the enlargement of renal cysts.
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MATERIALS AND METHODS |
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Animals. Our colony of (cy/+) rats is derived from the Han:SPRD rat strain. It has been inbred for over 20 generations in Mannheim, Germany, and has therefore been registered as follows: polycystic kidney disease, Mannheim (PKD/Mhm; Inbred Strains of Rats, http://www.informatics.jax.org/external/festing/rat/docs/PKD.shtml). Animals were maintained under the control of N. Gretz at the Animal Care Facility in Mannheim. Sixteen-week-old male heterozygous and wild-type animals as well as normal Sprague-Dawley rats were chosen for the different experimental procedures. All rats were allowed free access to tap water and to rat chow containing 19% protein.
Infusion of FITC-labeled dextran and tissue preparation.
FITC-labeled anionic dextran (molecular mass = 10 kDa, catalog no.
D-1821) was purchased from Molecular Probes (Leiden, The Netherlands). Rats were deeply anesthetized with ketamine (100 mg/kg)
and xylazine (5 mg/kg) before a midline incision was made to locate the
abdominal aorta below the renal arteries. Several drops of 0.9% NaCl
were administered to the peritoneal cavity to balance the fluid loss
caused by evaporation. Next, the right jugular vein was carefully
dissected free, and FITC-labeled dextran (1,75 mg/100 g body wt,
dissolved in 0.5 ml 0.9% NaCl) was injected as a bolus into the right
jugular vein over a period of 30 s. Typically, a green
discoloration of the bladder could be observed after 2 min. Six minutes
after the injection of the FITC-dextran bolus, the infrarenal abdominal
aorta was cannulated and the rats were subjected to perfusion-fixation
for subsequent visualization of FITC-labeled dextran and
immunohistochemistry. Animals were perfused with 2% freshly
depolymerized paraformaldehyde in PBS, pH 7.4, at a pressure level of
180 mm Hg for 3 min and subsequently with a 18% sucrose solution in
PBS for another 3 min at the same pressure level. Next, kidneys were
removed, cut into slices, and incubated in a 18% sucrose-PBS solution
(containing 0.02% sodium azide) for another 24 h at 4°C.
Tissues were then snap-frozen in liquid nitrogen-cooled isopentane and
stored at 80°C until further use. Rats without administration of
FITC-dextran were perfused in a similar way, and both kidneys were
processed as described above.
Histomorphological analysis of FITC-dextran uptake and immunohistochemistry. Five- to seven-micrometer cryostat sections from FITC-dextran-infused rat kidneys were transferred onto silane-coated glass slides, air-dried, washed for 30 min in PBS, and mounted in bicarbonate-buffered glycerol (pH 8.6) before being analyzed under the fluorescence microscope.
For additional immunohistochemical experiments, cryostat sections from FITC-dextran-infused rat kidneys were washed in PBS and incubated for 30 min at room temperature in a blocking solution consisting of 2% BSA in PBS. The sections were then incubated with the following primary antibodies diluted in blocking solution as indicated: an affinity-purified rabbit polyclonal antibody against the COOH terminus of rat ClC-5 (diluted 1:200; kind gift of T. Jentsch, Hamburg, Germany) (17); a mouse monoclonal anti-megalin antibody (diluted 1:300; kind gift of D. Kerjaschki, Vienna, Austria) (20); and a mouse monoclonal antibody against dipeptidyl peptidase IV (diluted 1:500; kind gift of W. Reutter, Berlin, Germany) (19). The antibodies were applied for 2 h at room temperature and subsequently overnight at 4°C. The next day, slides were rinsed in PBS and incubated with the appropriate Cy3-coupled secondary antibodies (Dianova, Hamburg, Germany). For double-labeling experiments on noninfused rat kidneys, two primary antibodies developed in different species were applied together, followed by the simultaneous detection using FITC- and Cy3-coupled secondary antibodies.Blood pressure measurements and assessment of blood and urine chemistry. Systolic blood pressure values were monitored by tail plethysmography in awake, trained animals. Sixteen-week-old (cy/+) and (+/+) rats (n = 5/group) were placed into metabolic cages to collect 24-h urine samples. Blood samples were obtained from all animals from the retroorbital plexus under short anesthesia. The (cy/+) or (+/+) carrier status was verified by the histological examination of the kidneys.
Determination of urinary protein and albumin levels. Before protein measurements were taken, all samples were centrifuged for 2 min at 14,000 g, and the supernatants were collected. Protein concentrations were determined according to an improved Bradford method (48) using the Bio-Rad protein assay kit (Bio-Rad, Munich, Germany) with BSA as a standard.
The concentration of albumin in rat urine was determined by using a competitive two-step ELISA, which was developed at the Medical Research Center in Mannheim. During the first step, a chicken anti-rat albumin antibody (catalog no. 55727, Cappel, Eppelheim, Germany) was incubated with the sample. After complexes between albumin and the anti-rat albumin antibody had formed, this mixture was transferred to a 96-well plate coated with rat albumin (catalog no. A-4538, Sigma, Deisenhofen, Germany). The amount of antibody bound to the albumin in the 96-well plates was determined with a peroxidase-coupled antibody directed against chicken IgG (catalog no. A-9792, Sigma) and subsequent enzymatic reaction. The concentration of the reaction product was measured photometrically at 450 nm. Each measurement included rat albumin samples with known concentrations, so that the albumin concentration in the urine could be determined from a standard curve.Measurement of urinary IGF-I protein by radioimmunoassay. Urinary IGF-I levels were determined by radioimmunoassay as described previously (5, 43).
Nonreducing SDS-PAGE of urinary proteins. Equal urine volumes (35 µl, corresponding to 5.5-38.0 µg of protein) were combined with 17.5 µl of 3× SDS-sample buffer (1× SDS-sample buffer is 125 mM Tris · HCl, pH 6.7, 2.5% SDS, 10% glycerol, and 0.01% bromophenol blue), boiled, and loaded onto a denaturing polyacrylamide gel. A broad-range protein marker (New England Biolabs, Schwalbach, Germany) was used as a molecular weight standard. Electrophoresis was followed by staining of the gel with Coomassie brilliant blue R250 for 1 h. The gels were destained, washed in double distilled water, and dried in a cellophane membrane for storage.
Preparation of total RNA.
The kidneys from 16-wk-old male (cy/+) and
(+/+) rats were rapidly removed, and total RNA
was extracted according to the acid guanidinium-phenol-chloroform
protocol of Chomczynski and Sacchi (7). The resulting RNA
pellets were dissolved in diethylpyrocarbonate-treated water, and the
yield was measured by spectrometry at 260 nm. Samples were stored
at 80°C until further use. The integrity of the extracted RNA was
checked by agarose gel electrophoresis.
Ribonuclease protection assay for renal IGF-I mRNA.
RNAse protection analysis was performed according to standard protocols
(3). A 205-bp rat IGF-I cDNA fragment (kind gift of Derek
LeRoith, National Institutes of Health, Bethesda, MD) (1)
and an 80-bp 18S cDNA fragment (Ambion, Austin, TX) were used for in
vitro transcription. Plasmids were digested appropriately, and
radiolabeled antisense cRNA probes were synthesized in vitro with T7
RNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany) in the
presence of [-32P]UTP.
Processing of images and gels. Black and white photographs from interference-phase contrast pictures and immunofluorescence-labeled kidney sections were scanned with a Nikon Coolscan LS-2000 by using Silverfast 4.1 software (LaserSoft, Kiel, Germany). Gels were scanned with a Linotype Saphir ultrascanner using Linocolor 5.1 software. All files were then processed with Photoshop 5.0 (Adobe Systems, San Jose, CA).
Statistics. Statistical evaluation was performed by using the statistical analysis system (SAS Institute, Cary, NC). The procedure PROC TTEST was applied to calculate mean, SD, and t-test statistics.
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RESULTS |
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In the present study, 16-wk-old male (cy/+)
and (+/+) rats were analyzed with respect to
urinary protein and albumin excretion. Table
1 demonstrates that there is a
statistically significant increase in the mean 24-h urinary protein
excretion in the (cy/+) group compared with that
of the (+/+) group. Similarly,
(cy/+) rats excreted higher levels of albumin
than (+/+) rats (Table 1). Serum creatinine and
urea values were already significantly higher in
(cy/+) animals than in their
(+/+) littermates, whereas no differences between
the two groups could be seen with respect to the systolic blood
pressure levels (Table 1).
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Coomassie blue-stained protein gels of the individual urine samples run
under nonreducing conditions are shown in Fig.
1. All samples obtained from
(cy/+) animals showed stronger bands for albumin
than the corresponding urine from the (+/+) rats, confirming the results obtained with the ELISA. The urine from (cy/+) rats provided no evidence for a pronounced
increase in the excretion of large proteins such as immunoglobulins,
which should have been present in the 150-kDa range, thus pointing
against a glomerular lesion as the cause of proteinuria. Because these data suggested a tubular defect, the role of impaired endocytosis in
this nephron segment was addressed more closely in subsequent experiments.
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In Fig. 2a the uptake of
filtered FITC-labeled dextran, which is used as a marker for endosomes
(21), can be seen in normal proximal tubular cells, where
it is located in a punctate fashion in the apical cytoplasm. The
resulting fluorescence appears stronger in the epithelial cells of the
proximal convoluted tubules than in cells of the pars recta (S3
segment), indicating a higher rate of endocytosis in the S1 and S2
segments. Immunohistochemical analysis of the same sections with an
antibody against the chloride channel ClC-5, which recently has been
localized to endosomes, yielded a strikingly similar distribution
pattern regarding the staining intensity within different segments of
the proximal tubule (Fig. 2b). Somewhat surprisingly,
however, the distribution of ClC-5 appeared more homogeneous, whereas
that of FITC-dextran was clearly punctate. This finding may be due to
the fact that ClC-5 is present in many endosomal vesicles, which the
light microscope will not be able to resolve into distinct entities.
FITC-dextran, however, has to be reabsorbed from the tubular fluid and
is therefore present in only that subset of endosomes, which has just
been formed, thus resulting in a punctate appearance.
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For a comprehensive analysis, double-labeling immunohistochemical
experiments in normal adult rat kidneys were carried out with the
anti-ClC-5 antibody and an antibody against the enzyme dipeptidyl
peptidase IV, which serves as a marker for the brush border of the
proximal tubule (19). Figure
3 shows that the ClC-5 protein is
expressed in the apical cytoplasm just below the brush border without
any overlap with dipeptidyl peptidase IV (Fig. 3,
a-d). The intracellular distribution of
internalized FITC-dextran and of the ClC-5 chloride channel was
investigated at a higher magnification and revealed a colocalization of
the two signals in proximal convoluted tubules (Fig. 3,
e-h). Only rarely, some
FITC-dextran-containing vesicles did not colocalize with ClC-5. Thus
there is an apparent overlap of both endosomal markers in the proximal
tubule.
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In (cy/+) rat kidneys, infusion of FITC-labeled
dextran resulted in an uptake into the apical cell compartment of
noncystic proximal tubules, comparable to the situation in control
kidneys. However, in cystically modified proximal tubules, many
cyst-lining cells did not show an uptake of FITC-labeled dextran,
whereas in other epithelial cells of the same cyst, endocytosed
FITC-labeled dextran was readily detectable (Figs.
4a and
5a). There was a clear
variability between cysts, with some showing endocytosed FITC-labeled
dextran in only a few or no epithelial cells, whereas other cysts
demonstrated FITC-dextran in almost every epithelial cell. Subsequent
immunohistochemical staining of the sections with the anti-ClC-5
antibody showed complete overlap with the FITC-dextran-containing
cells, whereas the cyst-lining epithelia, which did not internalize
FITC-labeled dextran, also did not express ClC-5 (Fig. 4b).
Next, the presence of gp330/megalin, which has been proposed as a
multiligand receptor for a variety of proteins including albumin
(9, 46), was analyzed by immunohistochemistry on
FITC-dextran-infused (cy/+) rat kidneys. In
cysts, the megalin-expressing tubular cells largely colocalized with
those cells that had endocytosed FITC-labeled dextran. Cells that did
not express megalin were also devoid of FITC-labeled dextran (Fig. 5).
At a higher magnification it could be clearly seen that it was the more
flattened epithelium of the cyst walls that did not express ClC-5 and
megalin any longer (Fig. 6).
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The impaired endocytosis in cysts of the proximal tubule might be of
functional relevance because a variety of proteins are quantitatively
reabsorbed along this nephron segment. For instance, several
circulating hormones and growth factors are filtered and delivered to
the proximal tubule, where binding and uptake play a critical role in
their metabolism (15). By the use of a specific radioimmunoassay for rat IGF-I (5, 43), we have found
increased urinary excretion of IGF-I in (cy/+)
rats compared with the respective controls (Fig.
7). To assess whether the increased
amounts of urinary IGF-I protein were derived from circulating IGF-I or
of renal origin, 16-wk-old (cy/+) as well as
(+/+) rats were analyzed by using a RNAse
protection assay. In three (+/+) kidneys,
abundant IGF-I mRNA could be detected, whereas in five kidney samples
prepared from (cy/+) rats the corresponding
protected bands were weaker (2 animals) or virtually absent (3 animals)
even after a prolonged exposure (Fig. 8).
These results indicate that in (cy/+) rat kidneys IGF-I mRNA is expressed at clearly lower levels than in unaffected (+/+) kidneys, therefore making it unlikely that
cystic kidneys are the origin of increased urinary IGF-I protein
levels.
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DISCUSSION |
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Proteinuria has been identified as a key factor for the prognosis of renal disorders (45), and the occurrence of proteinuria has also been evaluated in ADPKD patients, where it seems to be associated with a more aggressive course of the disease (6, 38). Massive alterations of the tubulointerstitium occur during cyst development, and it is therefore important to address the role of impaired tubular function in the development of proteinuria and albuminuria. Net urinary excretion of filtered proteins critically depends on the structural integrity of the proximal tubule, where most of the proteins are reabsorbed by endocytosis (25). Because in the (cy/+) rat cysts originate predominantly from the proximal tubule (8, 31, 37) and because cyst-lining epithelial cells show distinct changes such as the loss of transporters and channels (31), we focused our attention on this particular animal model of ADPKD. Indeed, we were able to show an increased proteinuria, which was accompanied by albuminuria but not by a pronounced excretion of higher molecular weight proteins such as immunoglobulins. In addition to increased levels of albumin, we also found proteins of ~70 kDa in the urine of the (cy/+) rat, which may not be too surprising considering the fact that normal rats also excrete proteins in that size range (2).
Because those initial observations pointed to a tubular defect, they were followed up by an in vivo approach using FITC-labeled dextran as a fluorescent tracer for endocytosis in proximal tubules (21). Our results clearly show that portions of cyst-lining epithelia of (cy/+) rat kidneys have lost the ability for endocytosis. These findings were extended by demonstrating that the absence of endocytic function is paralleled by the lack of components crucially involved in the endocytic pathway, such as the putative albumin receptor megalin and the endosomal chloride channel ClC-5, in cyst-lining epithelia. A glomerular lesion as the cause of proteinuria is unlikely because of the absence of high-molecular-weight proteins in the urine; furthermore, we could not detect an increase in the systolic blood pressure levels of (cy/+) rats, which may result in hypertensive damage of the glomerular filtration barrier [(37) and this study]. Therefore, the urinary loss of proteins, and especially that of albumin, from cystic kidneys can best be explained as a consequence of disturbed proximal tubular function, i.e., the loss of components of the endocytic machinery, in cyst wall epithelia. In this context it should also be pointed out that in the (cy/+) model the cysts develop predominantly in the cortical portion of the proximal tubule (8, 31, 37), which is also the site with the highest abundance of endocytosed FITC-labeled dextran and the strongest expression of ClC-5 protein. It is conceivable that the loss of at least some proteins could be balanced by the upregulation of endocytosis in the S3 segment, limiting tubular proteinuria to a certain degree. For albumin, however, it has been suggested that an increased load cannot be handled adequately by the proximal tubule, so that a reduced reabsorption capacity may inevitably lead to albuminuria (24).
The importance of the chloride channel ClC-5 and the multifunctional clearance receptor megalin for protein reabsorption in the proximal tubule can also be appreciated by the phenotypes resulting from the inactivation of their respective genes. In Dent's disease and related syndromes, in which the function of the whole proximal tubule is compromised by a germline mutation in the gene encoding ClC-5 (22, 23), excessive low-molecular-weight proteinuria is a prominent feature of the disease. Similarly, proximal tubules from megalin-knockout mice show a reduction of endosomes and lysosomes, which is also accompanied by low-molecular-weight proteinuria (29).
The present results regarding the cellular localization of the ClC-5
protein in (cy/+) and (+/+)
rat kidneys are in general agreement with previous investigations in
normal human, mouse, and rat kidneys. Our own in situ hybridization
study has shown the expression of ClC-5 mRNA in intercalated cells of
connecting tubules and collecting ducts (30), which was
confirmed subsequently both by others (11, 17, 36) and by
this study using anti-ClC-5 antibodies. Using nonradioactive in situ
hybridization, we could not, however, unambiguously detect ClC-5 mRNA
in the proximal tubule (30), which is in contrast to our
present study and previous results obtained with anti-ClC-5 antibodies
(11, 17, 36). This discrepancy can probably be explained
by the lower sensitivity achieved with the nonradioactive in situ
hybridization technique. On a subcellular level, the localization of
the ClC-5 protein in endosomes has been demonstrated biochemically
(11, 36), ultrastructurally (17, 36), and by
double staining with endosomal markers (17) and
endocytosed 2-microglobulin,
2-macroglobulin, and albumin (11, 17). We
are able to confirm the previous studies employing FITC-dextran uptake
by proximal tubules and its colocalization with ClC-5, although one
investigation failed to colocalize ClC-5 with a fluid-phase marker of
endocytosis using a permanent opossum kidney cell line
(11).
The finding of an endocytosis defect in cyst-lining epithelial cells also raised the question of whether growth factors, which eventually could have an impact on cyst development, are affected by the endocytosis defect in proximal tubules. We were able to demonstrate that increased amounts of IGF-I, which probably do not originate in the kidney itself, are excreted by (cy/+) rats. Although no direct evidence has yet been presented that IGF-I contributes to cyst formation, IGF-I shows certain features that make it an interesting candidate. First, a previous report has presented evidence for the upregulation of IGF-I in the pcy mouse model of polycystic kidney disease (28). Second, binding studies (16, 18) and functional data (33) support the view that the receptor for IGF-I is located on both the apical and the basolateral side of proximal tubules. And, third, systemic administration of IGF-I results in a higher mitotic index in proximal tubules of rat kidneys (26) [an increased proliferation rate is also observed in polycystic kidneys (28, 34)]. Even if IGF-I did not play a key role in cyst formation, our study leaves open the possibility that other growth factors also accumulate in cyst fluid, which is one possible explanation for previous results showing that cyst fluid stimulates cell proliferation (47).
The importance of growth factors acting through the luminal side of cyst-lining epithelial cells is exemplified by epidermal growth factor (EGF). A relocation of the EGF receptor from the basolateral to the apical side of cyst wall epithelia has been repeatedly demonstrated (13, 32, 35, 39). The importance of these findings was emphasized by a study demonstrating that the expression of an EGF receptor mutant with decreased tyrosine kinase activity results in a significant decrease in cyst formation (35). Furthermore, the specific blockade of EGF receptor tyrosine kinase activity has recently been shown to reduce collecting duct cysts in vitro (41) and in vivo (40). In addition to the action of growth factors, proteinuria per se is supposed to aggravate tubulointerstitial disease (45), and some investigations also hint at a mitogenic role of albumin on proximal tubular cells in the pathophysiology of proteinuric states (12).
In summary, we have provided functional evidence that in the (cy/+) rat model of ADPKD, albuminuria and proteinuria result from the disappearance of components of the endocytic apparatus such as the chloride channel ClC-5 and the scavenger receptor megalin. This endocytic defect may play a secondary role in cyst development.
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ACKNOWLEDGEMENTS |
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We thank Dennis Brown (Renal Unit, Massachusetts General Hospital, Charlestown, MA) for advice regarding the FITC-dextran studies. We are also thankful for the superb arrangement of the figures by Rolf Nonnenmacher and the expert photographic work of Ingrid Ertel. Jutta Christophel skillfully performed the albumin ELISAs.
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FOOTNOTES |
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Parts of these studies were presented at the International Society of Nephrology Forefronts in Nephrology Symposium on Gene Therapy at Beaver Creek, CO (June 5-6, 2000).
Address for reprint requests and other correspondence: R. Witzgall, Institute for Anatomy and Cell Biology I, Univ. of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany (E-mail: ralph.witzgall{at}urz.uni-heidelberg.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 April 2000; accepted in final form 10 October 2000.
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