Anion exchanger 1 interacts with nephrin in podocytes.
The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.
Wu
Fiona
F
Department of Clinical Sciences, South Bristol, UK.
Saleem
Moin A
MA
Kampik
Nicole B
NB
Satchwell
Timothy J
TJ
Williamson
Rosalind C
RC
Blattner
Simone M
SM
Ni
Lan
L
Toth
Tibor
T
White
Graham
G
Young
Mark T
MT
Parker
Mark D
MD
Alper
Seth L
SL
Wagner
Carsten A
CA
Toye
Ashley M
AM
D000328:Adult; D000419:Albuminuria / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis / Q000502:physiology*; D002478:Cells, Cultured; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007678:Kidney Glomerulus / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D050199:Podocytes / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D020798:Two-Hybrid System Techniques; D014981:Xenopus; D000099200:Scaffold Protein ILK
D001457:Anion Exchange Protein 1, Erythrocyte; D008565:Membrane Proteins; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C113267:nephrin