Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin
M. Hara
0
2
3
K. Yamagata
0
2
3
Y. Tomino
0
2
3
A. Saito
0
2
3
Y. Hirayama
0
2
3
S. Ogasawara
0
2
3
H. Kurosawa
0
2
3
S. Sekine
0
2
3
K. Yan
0
2
3
0
Y. Tomino Department of Nephrology, Juntendo University
,
Tokyo, Japan
1
) Department of Pediatrics, Yoshida Hospital
, Yoshida 32-14, Tsubame City 959-0242, Niigata,
Japan
2
K. Yan Department of Pediatrics, School of Medicine, Kyorin University
, Mitaka,
Tokyo, Japan
3
A. Saito Department of Medicine, Niigata University
, Niigata,
Japan
Aims/objective Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy.
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Nephropathy is a major complication of diabetes and the
leading cause of end-stage renal disease; it is clinically
characterised by proteinuria and progressive renal insufficiency
[1]. Human podocytes (Pods) have been demonstrated to be
functionally and structurally injured in the natural history of
diabetic nephropathy [2]. Recently, an increase in foot-process
width has been identified in patients with type 1 diabetes and
microalbuminuria, and foot-process width has been shown to
correlate directly with the urinary albumin excretion rate [3].
Furthermore, the number and density of Pods have been
reported to be markedly reduced (podocytopenia) in patients
with diabetes [4]. Pods are located outside the glomerular
basement membrane (GBM). Because of the proximity of
the apical region of Pods to the urinary space, pathological
events occurring in this region are expected to be more easily
detectable in urine than those occurring in the basal or slit
diaphragm regions of Pods [5, 6]. In this study, we report the
establishment of a highly sensitive ELISA for the detection of
urinary (u)-podocalyxin (PCX), one of the primary
glycoconjugates on the Pod surface, for identifying Pod injury in
various glomerular diseases, particularly in the early phase
of diabetic nephropathies.
Patients and samples Urine samples from patients with
glomerular diseases (n 0 142) and type 2 diabetes (n 0 71)
were used for u-PDX quantification by ELISA.
Urine samples were obtained from 69 healthy controls
with laboratory data within normal values. Urine samples
were obtained from urine voided in the morning, and were
stored at 70C within 2 h of collection until quantification
by ELISA. The clinical characteristics of the patients and
healthy controls are shown in Tables 1 and 2. This study was
approved by the ethics committees of all the hospitals
involved. Informed consent was obtained from patients.
Monoclonal antibodies against PCX We used two series of
monoclonal antibodies in this study: antibodies against
native human PCX; and antibodies against recombinant PCX.
Monoclonal antibodies against native human PCX were
produced using, as the immunogen, native PCX prepared
from isolated glomeruli.
The glomeruli were isolated from the normal portion of
the kidneys obtained during nephrectomy. Isolated
glomeruli were extracted in 0.2% (vol./vol.) Triton X-100 in PBS
containing protease inhibitors. The extract was then
incubated with wheat germ agglutinin (WGA)Sepharosel; after
washing, the sialic-acid-rich material that bound to the
WGA column was removed with
N-acetyl--glucopyranoside. BALB/c mice were immunised with 50 g
WGAbound PCX. Spleen cells were fused according to standard
procedures. Clones producing anti-PCX antibody were
screened by indirect immunofluorescence (IF) on cryostat
sections of human kidney and were further characterised by
western blot analysis and immunoprecipitation. A number
of positive clones were identified. Finally, three clones
(22A4, 3H11 and 4D5) were obtained and confirmed as
monoclonal antibodies against native human PCX [7].
The monoclonal antibodies against recombinant PCX were
produced using purified recombinant human
PCXglutathione transferase (GST) fusion protein, according to the method
described by Kershaw et al [8] (electronic supplementary
material [ESM]). BALB/c mice were immunised with purified
recombinant human PCXGST fusion protein prepared by
PCR amplification of the cDNA for base pairs 10041835
of the human PCX (named PC-46, and containing both
intracellular and extracellular domains) by the standard method.
The resulting hybridomas were cultured in 96 well plates and
screened with an ELISA using polystyrene multi-well plates
coated with a WGA-binding fraction in a Triton X-100
glomerular lysate. The PCXGST fusion proteins obtained using
cDNA coding for parts of the extracellular and intracellular
domains of human PCX (named PC-35 and Intra PC,
respectively) were used to characterise the monoclonal antibodies.
The monoclonal antibodies against native P (...truncated)