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Animal Model |
From the Max-Delbrueck-Center for Molecular Medicine,*
Berlin, and Childrens Hospital,
Ludwig-Maximilians-University, Munich, Germany; the Departments of
Medical Biochemistry
and Cell
Biology,||
University of Aarhus, Aarhus, Denmark; INSERM
U25, §
Necker Hospital, Paris, France; and the
Institute for Nutritional Research,¶
University of
Oslo, Oslo, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-microglobulin and odorant-binding protein. Megalin
binds these proteins and mediates their cellular uptake. Urinary loss
of carrier proteins in megalin-deficient mice results in concomitant
loss of lipophilic vitamins bound to the carriers. Similar to megalin
knockout mice, patients with low molecular weight proteinuria
as in Fanconi syndrome are also shown to excrete vitamin/carrier
complexes. Thus, these results identify a crucial role of the
proximal tubule in retrieval of filtered vitamin/carrier complexes and
the central role played by megalin in this process.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cell type mainly responsible for the uptake of filtered macromolecules are the epithelial cells of the proximal convoluted tubules. These cells exhibit a number of structural features indicative of a tissue involved in resorption processes.4 Their apical (luminal) plasma membranes form extensive microvilli creating a brush border surface. This enlargement of the apical membranes facilitates endocytosis of macromolecules from the lumen of the tubules via clathrin-coated pits. Furthermore, the cytoplasm of the cells is highly enriched in components of the endocytic apparatus including endosomes, lysosomes, and dense apical tubules (recycling membrane vesicles). Endosomes and lysosomes contain proteins absorbed from the glomerular filtrate.5
The physiological importance of tubular uptake processes is underscored by the pathological changes observed in patients with a resorption deficiency of the proximal tubules. This defect is referred to as the Fanconi syndrome (Lignac-de Toni-Debré-Fanconi syndrome).6-8 In adults, the most frequent cause of Fanconi syndrome is a myeloma-like proliferation of plasma cells secreting immunoglobulin light chains of peculiar toxicity for the proximal tubule epithelium.6 In addition, Fanconi syndrome is acquired by toxification of the proximal tubules by heavy metals and certain therapeutics such as aminoglycosides, anti-tumor drugs (ifosfamide), and anticonvulsants (valproate).6 As a consequence of tubular dysfunction, essential metabolites and proteins cannot be retrieved from the glomerular filtrate but are excreted in the urine instead. One of the earliest symptoms of acquired Fanconi syndrome is the urinary loss of small plasma proteins. Marker proteins of such low molecular weight (LMW) proteinuria are retinol-binding protein, ß2-microglobulin, and lysozyme.9 After prolonged exposure to nephrotoxic compounds, patients also excrete serum potassium and phosphorus, urate, glucose, and amino acids. As a consequence of urinary loss of these solutes, individuals suffer from polyuria, hypokalemia, acidosis, impaired growth, and osteomalacia.6,8
A receptor that might play an important role in uptake processes in the proximal tubules is megalin, also known as gp330 or LRP2.10 Megalin is a 600-kd endocytic receptor and one of the most abundant membrane proteins on the brush border surface of the proximal tubular epithelium.4 This receptor binds several proteins known to be reabsorbed in the proximal tubules (eg, insulin, ß2-glycoprotein, retinol-binding protein).5,11,12 Therefore, it was postulated that megalin might be involved in the clearance of filtered plasma proteins. Alternatively, a role for the receptor in tubular uptake of calcium has been suggested.13,14
To reveal the significance of megalin for tubular uptake processes in vivo, we recently have generated mice carrying a disruption of the receptor genes. Unexpectedly, megalin-deficient mice exhibit severe malformations of the forebrain, and most of them die within minutes after birth.15 These findings emphasize the importance of the receptor for development of the central nervous system. However, the exact role of megalin in embryonic development remains to be elucidated. Fortunately, the severity of forebrain malformations in megalin knockout mice is variable and approximately 2% of the animals survive to adulthood.5 These mice enabled us to study the consequence of receptor deficiency for renal function.
Our in vivo studies confirm that megalin is the major
endocytic receptor for the tubular retrieval of filtered plasma
proteins. In particular, it mediates the uptake of small plasma carrier
proteins including vitamin D-binding protein, retinol-binding protein,
1-microglobulin, and odorant-binding protein. Absence of
the receptor in megalin knockout mice results in low molecular weight
proteinuria and urinary loss of lipophilic vitamins bound to the
carrier proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Midstream urine samples were obtained from five patients with myeloma-associated Fanconi syndrome. These patients have been described previously.16 Antisera against human urinary proteins were purchased from DAKO (Copenhagen, Denmark) or The Binding Site (Birmingham, AL). Purified lysozyme and ß2-microglobulin were purchased from Sigma (Deisenhofen, Germany) or Calbiochem (Bad Soden, Germany), respectively. Vitamin D-binding protein and retinol-binding protein were purified from human serum as published.17,18
Immunohistochemistry
Wild-type and megalin-deficient mice were perfusion-fixed through the heart with 4% paraformaldehyde in 0.1 mol/L sodium cacodylated buffer. The tissues were trimmed into smaller blocks, further fixed by immersion for 1 hour in 1% glutaraldehyde, and processed for conventional Epon embedding (for electron microscopy). Alternatively, the tissue blocks were immersion-fixed for 1 hour in 1% paraformaldehyde, infiltrated with 2.3 mol/L sucrose containing 2% paraformaldehyde for 30 minutes, and frozen in liquid nitrogen. For immunolight microscopy, 0.8-µm cryosections were incubated with rabbit anti-human lysozyme antibody (DAKO, 1:1000 dilution) for 1 hour at room temperature, followed by peroxidase-conjugated anti-rabbit IgG (DAKO). Bound IgG was visualized with diaminobenzidine. The sections were counterstained with Meier's stain for 2 minutes. For immunoelectron microscopy, ultrathin kidney sections (70–90 nm) were incubated sequentially with sheep anti-rat megalin antibody (kindly provided by P. Verroust, Hôpital Tenon, Paris; 1:106 dilution), with rabbit anti-sheep IgG (1:20,000 dilution) and with 10 nm goat anti-rabbit gold particles (BioCell, Cardiff, UK).
One- and Two-Dimensional Polyacrylamide Gel Electrophoresis
For one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE), mouse urine samples were separated on 4 to 15% nonreducing polyacrylamide gels. Where indicated, the separated proteins were transferred to nitrocellulose filters and incubated with primary followed by peroxidase-coupled secondary antibodies as described.17 Bound IgG was detected by the enhanced chemiluminescence system (ECL, Amersham, Braunschweig, Germany). For two-dimensional PAGE, 600 µg of mouse urinary proteins were separated in the first dimension on a nonlinear pH 3 to pH 10 gradient (Immobiline Dry Strip, Pharmacia, Uppsala, Sweden), and in the second dimension on a 15% SDS polyacrylamide gel as published.19
Edman Sequencing
Coomassie Blue-stained protein bands were cut from the gels and digested in the gel with sequencing-grade modified trypsin (Promega).20 Tryptic peptide maps were obtained by reverse-phase high performance liquid chromatography on a µRPC C2/C18 SC2.1/10 column using the Smart system (Pharmacia Biotech). The flow rate was 100 µl/minute at 25°C employing a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Peptides of interest were loaded onto a Biobrene-coated glass filter fiber of a Procise sequencer (Perkin Elmer/Applied Biosystems), and sequenced according to standard protocols.
Receptor Binding Assay
Megalin was purified by receptor-associated protein (RAP) affinity
chromatography from rabbit renal cortex according to standard
protocols. The preparation was essentially pure as seen by SDS-PAGE. No
contaminations with other receptors of the low density
lipoprotein receptor gene family or with the RAP were detected
by Western blot analysis (not shown). For megalin affinity
chromatography, 4.5 mg of purified rabbit megalin were coupled to 200
mg CNBr-activated Sepharose 4B (Pharmacia). Two milliliters of mouse
urine were dialyzed against 10 mmol/L Tris-HCl, pH 7.5, lyophilized,
and resuspended in 200 µl of H2O. Then, 100 µl of the
concentrated urine sample was radiolabeled with 125I using
the IODOGEN method21
and circulated over the megalin
Sepharose column for 16 hours at 4°C in incubation buffer (10 mmol/L
Tris-HCl, 150 mmol/L NaCl, 2 mmol/L CaCl2, 2 mmol/L
MgCl2, pH 7.6). The resin was washed with 20 ml of
incubation buffer and specifically bound proteins were eluted in 0.1
mol/L glycin, 10 mmol/L EDTA, pH 2.8. Binding of ligands to megalin was
quantified by BIAcore analysis (Biosensor, Sweden) as described
previously.5
A continuous flow of HBS buffer (10 mmol/L
HEPES, 3.4 mmol/L EDTA, 150 mmol/L NaCl, 0.005% surfactant P20, pH
7.4) passing over the sensor surface was maintained at 5 µl/minute.
The carboxylated dextran matrix of the sensor chip flow cell was
activated by injection of 60 µl of a solution containing 0.2 mol/L
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide
and 0.05 mol/L N-hydroxysuccimide in H2O. Then,
180 µl of 10 mmol/L sodium acetate, pH 4.5, containing 10 µg/ml
purified rabbit megalin was injected. The remaining binding sites were
blocked by subsequent injection of 35 µl of 1 mol/L ethanolamine, pH
8.5. The surface plasmon resonance signal from immobilized megalin
generated 22,000 BIAcore response units equivalent to 34 fmol of
megalin/mm.2
To test ligand binding, rabbit megalin
immobilized on the CM5 sensor chip was incubated with the ligands
(0.1–5 µmol/L) in 10 mmol/L HEPES, 150 mmol/L NaCl, 1.5 mmol/L
CaCl2, 1 mmol/L EGTA, pH 7.4, and the relative increase in
response between megalin and control flow channels was determined. For
determination of binding affinities, 6 to 10 different concentrations
of each ligand were subjected to BIAcore analysis on the megalin sensor
chips (as shown in Figure 8A
). The kinetic parameters were determined
by using the BIAevaluation 3.0 software.
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A total of 10 megalin-deficient mice and their control litter mates were available for the studies presented here. For urine collection, these mice were placed in metabolic cages for 16 hours and given 10% sucrose in drinking water. Urine samples (approximately 5 ml/16 hours) were collected on ice and were qualitatively indistinguishable from samples collected without sucrose load. Urine volume per hour and creatinine levels were identical in megalin -/- and in control mice (approximately 0.5 mmol creatinine/L). Creatinine, sodium, potassium, chloride, total calcium, phosphate, urea, uric acid, and glucose were determined on a random access automated clinical chemistry analyzer (Synchron CX5, Beckman, Munich, Germany) by standard clinical chemistry procedures. For amino acid analysis, free amino acids in the urine samples were separated and quantified by ion exchange chromatography followed by ninhydrin reaction on a Biotronik LC3000 amino acid analyzer (Eppendorf, Hamburg, Germany). Retinol and tocopherol were determined by high performance liquid chromatography after solvent extraction (Chromsystem, Munich, Germany). For determination of 25-OH vitamin D3, urine samples were concentrated 30-fold by freeze-drying and analyzed by competitive protein binding assay (Immundiagnostik, Bensheim, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. No receptor protein is detectable in
megalin -/- kidneys (Figure 1A)
. Megalin deficiency does not affect
the overall architecture of the epithelial cells or the appearance of
the brush border surface (Figure 2A)
.
However, absence of megalin results in a significant reduction in the
number of coated pits, endosomes, and lysosomes as compared to
wild-type cells (Figure 2B)
. The histological appearance of other renal
tissues such as glomeruli (Figure 3)
,
distal tubules and collecting ducts (not shown) is indistinguishable
from wild-type controls.
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, urine samples from megalin
-/- animals specifically interacted with the receptor. The
interaction was reversible and dependent on calcium, a characteristic
feature of ligand binding to the receptor. In contrast, little if any
interaction was observed in urine samples obtained from wild-type
animals. These findings demonstrated that normal urine was virtually
devoid of any receptor ligands and that absence of megalin resulted in
a state of renal resorption deficiency.
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). No proteins larger than
approximately 70 kd were present in the urine of control and megalin
-/- animals indicating intact glomerular filtration. However,
knockout mice specifically excreted several low molecular weight
(LMW) proteins that were not seen in control samples.
Potentially, these proteins represented previously unknown ligands for
the receptor. To characterize some of the excreted proteins, we
isolated individual protein bands from the gel and obtained peptide
sequences by Edman degradation (Figure 5
; Table 1
). The 68-kd protein band that was
present in both control and knockout urine was identified as mouse
serum albumin. Apparently, albumin represented the largest plasma
protein filtered in large quantities through the glomerular membranes.
No major difference in the amount of albumin excretion was observed in
knockout compared to control samples by SDS-PAGE. The second protein
band that was present in all samples was the 21-kd major urinary
protein 6 (MUP-6), a pheromone carrier that is abundant in urine from
male rodents (Figure 5A
, lanes 1–3). Besides MUP-6 and serum albumin,
the urine of knockout mice displayed three major protein bands that
were not seen in control urine. These proteins were identified by
peptide sequencing as the vitamin D-binding protein (DBP),
1-microglobulin (1-M), and the
retinol-binding protein (RBP) (Figure 5A
; Table 1
). Because massive
amounts of MUP-6 in the urine of adult male mice obscured analysis of
additional protein bands in the same molecular weight range, we further
examined urine samples from wild-type and knockout females (Figure 5B)
.
In these samples, pancreatitis-associated protein (PAP-1), lysozyme,
and ß2-microglobulin (ß2-M) were identified
as novel proteins in urine from megalin -/- mice (Table 1)
. Finally,
we resolved mouse urine samples by two-dimensional SDS-PAGE and
discovered another major protein spot present in knockout urine
exclusively (Figure 6)
. This protein was
identified as the odorant-binding protein IA (OBPIA, Table 1
).
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, we hypothesized that proteins
exclusively present in knockout samples represented receptor ligands.
This hypothesis was confirmed when iodinated urinary proteins were
incubated with megalin coupled to Sepharose resin (Figure 7)
. Specifically bound proteins were
eluted from the receptor column and analyzed by SDS-PAGE and
autoradiography. Albumin, which was abundantly present in the urine
sample (lane 1), did not bind to the megalin column to any major
extent, while a number of smaller proteins interacted with the receptor
and were enriched in the column eluate (lanes 2 and 3). No specific
binding was seen with wild-type urine samples (not shown). To
investigate whether these megalin-binding proteins represented some of
the LMW proteins exclusively seen in knockout urine, we directly tested
receptor binding by BIAcore analysis. Similar to the findings obtained
with megalin affinity chromatography (Figure 7)
, serum albumin did not
bind to the receptor whereas DBP, RBP, lysozyme, and ß2-M
interacted with the receptor with a Kd of 0.1 to
1.8 µmol/L (Table 2)
. The binding of
ligands was concentration dependent (Figure 8A)
and blocked completely by the
receptor-associated protein, a receptor antagonist (Figure 8B)
.
Furthermore, ligand binding was abolished by adding EDTA or by reducing
the receptor (Figure 8C)
. Taken together, these results demonstrated
specificity of ligand-receptor interaction.
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.
In contrast, no uptake of lysozyme was detected in knockout kidneys
(Figure 9A)
. Similar results were obtained with antibodies directed
against DBP and RBP (not shown). No murine specific antibodies against
other identified proteins were available to extend these
investigations.
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. No
significant differences were observed in the concentrations of glucose,
electrolytes, urea, uric acid, and amino acids. Furthermore, the total
amount of protein excreted was also unchanged. The latter finding
reflects the fact that mice excrete massive amounts of MUP-6 and that
the additional loss of LMW proteins in the knockout mice does not
increase the total urinary protein concentration significantly.
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. In contrast, vitamin E (tocopherol) was not
detectable in any of the urine samples. This vitamin is transported in
lipoprotein particles which are too large to be filtered through the
glomerulus. Therefore, tocopherol was not expected to be present in the
glomerular filtrate.
Finally, we wanted to test whether phenotypic similarities exist
between megalin-deficient mice and patients with Fanconi syndrome. We
focused our analysis on individuals with multiple myeloma, a malignant
proliferation of immunoglobulin producing plasma cells. Multiple
myeloma is characterized in a majority of cases by the appearance of
massive amounts of immunoglobulin light chains in the glomerular
filtrate (Bence-Jones protein).6
By yet unknown
mechanisms, certain light chain molecules impair proximal tubular
function causing Fanconi syndrome.6,16
We obtained urine
samples from five patients with multiple myeloma-associated Fanconi
syndrome. As described previously, these patients presented with a low
tumor mass myeloma (2 to 9% marrow plasma cells) and mild but
progressive renal insufficiency. Renal biopsies showed epithelial
lesions, some with crystal formation in the cytoplasm of proximal
tubular cells. No glomerular lesions were observed.16
No
difference in the urinary excretion of megalin fragments was seen when
comparing these patients with control subjects (unpublished
observations). By analysis of their urinary protein profiles, these
patients were shown to exhibit low molecular weight proteinuria (Figure 10
, lanes 3 and 4) and to excrete the
characteristic pattern of small plasma proteins9
including
DBP, 1-M, RBP, and ß2-M (lanes 7 and 8).
Similar to megalin-deficient mice, excretion of RBP resulted in
concomitant loss of retinol in the urine. All 5 patients excreted
between 0.08 and 0.18 mg retinol/mmol creatinine (mean, 0.132 mg
retinol/mmol creatinine). No retinol was detectable in control urine
samples (n = 9). In 2 of the patients, we also
determined the levels of 25-OH vitamin D3. Significantly
more 25-OH vitamin D3 (0.2–0.3 nmol/mmol creatinine) was
detected in patients urine as compared to control samples (0.01–0.06
nmol/mmol creatinine). The three other patients received vitamin D
supplementation and could not be used for analysis of the vitamin D
metabolism. Tocopherol was not present in any of the urine samples.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of animal models are available to study the consequence of proximal tubular dysfunction in vivo. For example, intravenous injection of maleic acid into rats results in severe renal toxicity and Fanconi syndrome.22,23 Furthermore, several genetic disease models have been reported including Basenji dogs24 and mice carrying a targeted disruption of the hepatocyte nuclear factor 1 (HNF1) genes.25 Similar to patients with acquired Fanconi syndrome, these animal models are characterized by combined defects in several tubular uptake processes resulting in urinary excretion of proteins, electrolytes, and metabolites. Basenji dogs exhibit an altered brush border membrane fluidity, which apparently affects transport across the plasma membrane.24 HNF1 knockout mice are characterized by glucosuria, polyuria, amino aciduria, and phosphaturia.25 Detailed studies suggest that these animals have an impaired function of the Na+/K+-ATPase and fail to maintain a proper Na+ electrochemical gradient across the apical brush border surface. As a consequence, cellular uptake processes dependent on this electrochemical gradient are defective. In contrast to aforementioned animal models, megalin-deficient mice exhibit a specific defect in one tubular uptake pathway only, the endocytosis of filtered plasma proteins. Uptake of electrolytes, amino acids, and glucose is not affected by the receptor gene defect. Thus, these mice constitute an important animal model with which to dissect the various tubular uptake pathways and to elucidate the significance of tubular uptake of LMW proteins.
In humans, hundreds of milligrams of plasma proteins are filtered daily through the glomerulus into the lumen of the proximal tubules.1,2 Despite this massive influx of protein, human urine is virtually devoid of significant amounts of protein. The reasons for such massive uptake of proteins in the proximal tubules and the receptors involved in this process remain unclear. Because internalized proteins are subjected to lysosomal degradation, it is hypothesized that protein uptake is essential to salvage amino acids. Alternatively, the clearance of proteins from the glomerular filtrate is believed to remove metabolites that might serve as food source for bacteria and hence increase the risk of bacterial infections of the urogenital tract.2,4
Our studies now have identified megalin as a central receptor pathway
for tubular retrieval of small plasma proteins. The pattern of proteins
excreted in the urine of megalin-deficient mice is similar to the
pattern in patients with general tubular resorption
deficiency.9
DBP, RBP, lysozyme, and ß2-M
bind to the receptor with similar affinities as previously identified
ligands (Kd of 0.1–1.8
µmol/L).26
No purified 1-M and OBPIA were
available to test their binding. Taken together, our findings suggest
that megalin constitutes a low affinity but high capacity receptor
pathway for scavenging proteins from the glomerular filtrate. Besides
direct ligand binding, megalin may alternatively mediate the endocytic
uptake of some of the identified proteins via co-receptor systems.
Studies by Moestrup and colleagues have identified a 460-kd receptor in
the epithelium of the proximal tubules called intrinsic factor/vitamin
B12 receptor or cubilin.27
This receptor
mediates the tubular uptake of intrinsic factor/vitamin B12
complexes from the glomerular filtrate. Interestingly, cubilin does not
contain a transmembrane domain like other endocytic receptors. Because
it exhibits high affinity for megalin and co-localizes with this
receptor in the endocytic pathway of proximal tubular cells, cubilin is
believed to mediate ligand uptake by binding to megalin.27
Such a co-receptor system would also be defective in megalin knockout
kidneys. Thus, megalin could act as a central receptor pathway in the
kidney either by direct ligand binding or via co-receptors.
In a previous study, uptake of albumin microinfused into rat proximal tubules was blocked by the addition of RAP, a megalin antagonist.28 These results suggested a prominent role of megalin as a receptor for tubular clearance of albumin. In experiments presented here, albumin did not bind to megalin to any significant extend and no major difference in albumin clearance was seen in megalin-deficient as compared to control mice. Taken together both studies suggest that an alternative RAP-sensitive receptor may be mainly responsible for the tubular uptake of albumin.
The identification of ligands internalized via megalin (or
co-receptors) has uncovered the importance of tubular protein uptake
for homeostasis of lipophilic compounds. Many of the ligands taken up
via megalin-dependent pathways are plasma carriers for lipophilic
substances.29
DBP is the transporter for vitamin
D3 metabolites; RBP and 1-M carry retinoids;
and OBPIA binds lipophilic odors. Excretion of these carrier proteins
results in concomitant loss of lipids (eg, 25-OH vitamin
D3, retinol) in the urine of megalin -/- mice. Additional
studies demonstrate that the urinary loss of 25-OH vitamin
D3 in megalin knockout mice results in plasma vitamin D
deficiency and impaired bone formation.17
Similar to
megalin knockout mice, humans with multiple myeloma also excrete
complexes of retinol and RBP in their urine (Figure 10)
. In addition,
enhanced urinary loss of 25-OH vitamin D3 was seen in two
patients not receiving vitamin D supplementation. If confirmed in a
larger number of patients, these findings might have important
implications for our understanding of the pathology of the Fanconi
syndrome. Individuals with this disease are characterized by
osteomalacia and vitamin D-resistant rickets.6
Such
defects might be explained in part by the enhanced urinary loss of
vitamin D3 metabolites bound to DBP. Also, urinary loss of
retinoids and other essential lipids yet to be identified may
contribute to the multiple defects in Fanconi patients.6
One protein found in the urine of megalin knockout mice does not represent a normal plasma protein. This is the pancreatitis-associated protein 1. PAP-1 is a secretory protein that is induced in the acute phase of pancreatitis.30 Because PAP-1 is not present in the circulation of healthy mice, it is not likely to be a physiological ligand for megalin. Instead, the presence of PAP-1 may indicate pancreatitis in receptor-deficient animals and point to additional roles of the receptor in other tissues than the kidney. Due to the poor viability and the complex phenotype of the knockout mice, such investigations are difficult to perform at present. The generation of mice carrying tissue-specific receptor gene defects will be helpful in the future in dissecting the multiple functions of the receptor in various tissues.
In conclusion, our studies have identified megalin as the central receptor for the tubular retrieval of several low molecular weight plasma proteins. Megalin-mediated uptake of plasma carrier proteins is required for reuptake of filtered lipid/carrier complexes. These findings have uncovered a crucial role of tubular protein uptake for systemic vitamin homeostasis.
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Acknowledgements |
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Footnotes |
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Supported by Grant Wi 1158/3–1 and a Heisenberg fellowship (to TEW) from the Deutsche Forschungsgemeinschaft and by the Danish Medical Research Council and the Novo Nordic Foundation.
Accepted for publication June 10, 1999.
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M. Yamagata, K. Ozono, Y. Hashimoto, Y. Miyauchi, H. Kondou, and T. Michigami Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter J. Am. Soc. Nephrol., August 1, 2005; 16(8): 2338 - 2345. [Abstract] [Full Text] [PDF]
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T. J. Jentsch Chloride Transport in the Kidney: Lessons from Human Disease and Knockout Mice J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1549 - 1561. [Abstract] [Full Text] [PDF]
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 J. Prakash, A. M. van Loenen-Weemaes, M. Haas, J. H. Proost, D. K. F. Meijer, F. Moolenaar, K. Poelstra, and R. J. Kok RENAL-SELECTIVE DELIVERY AND ANGIOTENSIN-CONVERTING ENZYME INHIBITION BY SUBCUTANEOUSLY ADMINISTERED CAPTOPRIL-LYSOZYME Drug Metab. Dispos., May 1, 2005; 33(5): 683 - 688. [Abstract] [Full Text] [PDF]
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