Trafficking of ENaC subunits in response to acute insulin in mouse kidney.
Studies done in cell culture have demonstrated that insulin activates the epithelial sodium channel (ENaC) via a variety of mechanisms. However, to date, upregulation of ENaC in native renal tissue by in vivo administration of insulin has not been demonstrated. To address this, we injected 6-mo-old male C57BL/CBA mice (n = 14/group) intraperitoneally with vehicle or 0.5 U/kg body wt insulin and examined short-term (1-2 h) sodium excretion and kidney ENaC subunits (alpha, beta, and gamma) and serum and glucocorticoid-induced kinase (SGK-1) regulation. Insulin resulted in a significant reduction in urine sodium (by approximately 80%) that was restored by intraperitoneal administration of the ENaC antagonist, benzamil (1.4 mg/kg body wt). Differential centrifugation followed by Western blotting of whole kidney revealed significantly increased band densities (by 26-103%) for insulin- relative to vehicle-treated mice for alpha- and gamma-ENaC in the homogenate (H), and plasma membrane-enriched fraction (MF), with no difference in the vesicle-enriched fraction (VF). Similarly, beta-ENaC was significantly increased in MF (by 45%) but no change in the H. It was, however, significantly decreased in the VF (by 28%) with insulin. In agreement, immunoperoxidase labeling demonstrated relatively stronger apical, relative to cytosolic, localization of alpha-, beta-, and gamma-ENaC with insulin, whereas, with vehicle, labeling was fairly evenly dispersed throughout collecting duct principal cells. Furthermore, Western blotting showed insulin increased SGK-1 (by 75%) and phosphorylated-SGK band densities (by 30%) but only in the MF. These studies demonstrate novel in vivo regulation of renal ENaC activity and subunit proteins and SGK-1 by insulin in the acute time frame in the mouse.
Tiwari
Swasti
S
Dept. of Medicine, Georgetown University, Box 571412, Washington, DC 20057-1412, USA.
Nordquist
Lina
L
Halagappa
Veerendra K Madala
VK
Ecelbarger
Carolyn A
CA
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D017874:Immediate-Early Proteins / Q000502:physiology; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009318:Natriuresis / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D012964:Sodium / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D000099184:Serum-Glucocorticoid Regulated Kinases
D000584:Amiloride; D001786:Blood Glucose; D004232:Diuretics; D053503:Epithelial Sodium Channels; D007004:Hypoglycemic Agents; D017874:Immediate-Early Proteins; D007328:Insulin; D017346:Protein Serine-Threonine Kinases; D012964:Sodium; D000099184:Serum-Glucocorticoid Regulated Kinases; C013407:benzamil