Nephrin is expressed on the surface of insulin vesicles and facilitates glucose-stimulated insulin release.
OBJECTIVE
Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic beta-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in beta-cell function.


METHODS
Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin-transfected cells was used to study nephrin endocytosis.


RESULTS
Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin-transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release.


CONCLUSIONS
Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes.
Fornoni
Alessia
A
Diabetes Research Institute, University of Miami L. Miller School of Medicine, Miami, Florida, USA. afornoni@med.miami.edu
Jeon
Jongmin
J
Varona Santos
Javier
J
Cobianchi
Lorenzo
L
Jauregui
Alexandra
A
Inverardi
Luca
L
Mandic
Slavena A
SA
Bark
Christina
C
Johnson
Kevin
K
McNamara
George
G
Pileggi
Antonello
A
Molano
R Damaris
RD
Reiser
Jochen
J
Tryggvason
Karl
K
Kerjaschki
Dontscho
D
Berggren
Per-Olof
PO
Mundel
Peter
P
Ricordi
Camillo
C
D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D017931:DNA Primers; D003920:Diabetes Mellitus / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000502:physiology*; D016381:Islets of Langerhans Transplantation; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics; D012016:Reference Values; D014162:Transfection; D014183:Transplantation, Heterologous
D017931:DNA Primers; D007328:Insulin; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C113267:nephrin; D005947:Glucose