Vol. 283, Issue 2, R339-R346, August 2002
Loss of albumin and megalin binding to renal cubilin in rats
results in albuminuria after total body irradiation
Raghunatha R.
Yammani1,2,
Mukut
Sharma3,
Shakuntla
Seetharam1,2,
John E.
Moulder4,
Nancy M.
Dahms5, and
Bellur
Seetharam1,2,5
1 Department of Medicine, Divisions of
2 Gastroenterology and Hepatology and 3 Nephrology;
and Departments of 4 Radiation Oncology and
5 Biochemistry, Medical College of Wisconsin and
Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226
The role of the
renal apical brush-border membrane (BBM) endocytic receptors cubilin
and megalin in the onset of albuminuria in rats exposed to a
single dose of total body irradiation (TBI) has been investigated.
Albuminuria was evident as immunoblot (IB) analysis of the urine
samples from TBI rats revealed excretion of large amounts of albumin.
IB analysis of the BBM proteins did not reveal any significant changes
in cubilin or megalin levels, but 125I-albumin binding to
BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 µM) in the affinity for albumin. IB analysis of cubilin from
the BBM demonstrated a 75% loss when purified using albumin, but not
intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography.
Immunoprecipitation (IP) of Triton X-100 extract of the BBM with
antiserum to cubilin followed by IB of the immune complex with an
antiserum to megalin revealed a 75% loss of association between
megalin and cubilin. IP studies with antiserum to cubilin or megalin
and IB with antiserum to the cation-independent mannose
6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed
that CIMPR interacted with both cubilin and megalin. In addition, TBI
did not disrupt the association of CIMPR with either cubilin or megalin
in BBM. These results suggest that albuminuria noted in TBI rats is due
to selective loss of albumin and megalin, but not CIMPR or IF-Cbl
binding by cubilin. Furthermore, these results also suggest
that albumin and IF-Cbl binding to cubilin occur at distinct sites and
that in the rat renal BBM, CIMPR interacts with both cubilin and megalin.
endocytic receptors; endocytosis
