Anion Exchanger 1 Interacts with Nephrin in Podocytes

Abstract

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 Ϫ/Ϫ mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1 Ϫ/Ϫ 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.

Physical and Functional Links between Anion Exchanger-1 and Sodium Pump

“…Previously reported glomerular staining of kAE1 20 was not detected, possibly owing to methodologic or antibody differences. As expected, in contrast with the restricted IC location of kAE1, b1 was also extensively distributed basolaterally throughout the nephron.…”

“…Several proteins are reported to interact with the C terminus of kAE1, and disruption of these interactions results in abnormal cellular location of kAE1. [18][19][20] We report here the identification and characterization of a novel C-terminal binding partner, the b1 subunit of the Na + ,K + -ATPase for AE1. The sodium pump (Na + ,K + -ATPase) is a ubiquitous plasma membrane ATP-requiring enzyme that transports 3 Na + in exchange for 2 K + .…”

Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis

“…Interestingly, interaction of stomatin with Glut1 also promotes Glut1-mediated transport of dehydroascorbate by red cells (27), secondarily altering transport of electrons and protons. The recent report of kidney AE1's interaction with the podocyte slit-diaphragm protein nephrin (48), another podocinbinding protein, suggests an indirect link between AE1 and Trpc6 [reportedly expressed also in red cells (12)]. However, the conductive nature of stomatocyte red cell cation leak remains controversial.…”

The Murine Cl-/HCO3-Exchanger Ae3 (Slc4a3) is Not Required for Acid-Base Balance but is Involved in Magnesium Handling by the Kidney

“…At present, ten members of the Slc4 gene family have been described and clustered into three major clades of which Ae1, Ae2 and Ae3 belong to the electroneutral and sodium independent Cl - /HCO 3 - exchangers [6,7]. While Ae2 is widely expressed in epithelial and non-epithelial cells, Ae1 is mostly restricted to red blood cells, cardiomyocytes, renal podocytes and type A intercalated cells of the renal collecting ducts (CD) [8,9,10]. In contrast, Ae3 is predominantly found in excitable tissues, such as the central nervous system, retina and heart, where it contributes to the control of intracellular pH and intracellular [Cl - ] [11,12].…”