title: Insulin regulates nitric oxide production in the kidney collecting duct cells.
abstract: The kidney is an important organ for arterial blood pressure (BP) maintenance. Reduced NO generation in the kidney is associated with hypertension in insulin resistance. NO is a critical regulator of vascular tone; however, whether insulin regulates NO production in the renal inner medullary collecting duct (IMCD), the segment with the greatest enzymatic activity for NO production in kidney, is not clear. Using an NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescent dye, we found that insulin increased NO production in mouse IMCD cells (mIMCD) in a time- and dose-dependent manner. A concomitant dose-dependent increase in the NO metabolite (NOx) was also observed in the medium from insulin-stimulated cells. NO production peaked in mIMCD cells at a dose of 100 nm insulin with simultaneously increased NOx levels in the medium. At this dose, insulin significantly increased p-eNOS(Ser1177) levels in mIMCD cells. Pretreatment of cells with a PI 3-kinase inhibitor or insulin receptor silencing with RNA interference abolished these effects of insulin, whereas insulin-like growth factor-1 receptor (IGF-1R) silencing had no effect. We also showed that chronic insulin infusion to normal C57BL/6J mice resulted in increased endothelial NOS (eNOS) protein levels and NO production in the inner medulla. However, insulin-infused IRKO mice, with targeted deletion of insulin receptor from tubule epithelial cells of the kidney, had ∼50% reduced eNOS protein levels in their inner medulla along with a significant rise in BP relative to WT littermates. We have previously reported increased baseline BP and reduced urine NOx in IRKO mice. Thus, reduced insulin receptor signaling in IMCD could contribute to hypertension in the insulin-resistant state.
authors.affiliation: From the Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India and.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Pandey
authors.affiliation: From the Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India and.
authors.forename: Ekta
authors.initials: E
authors.lastname: Makhija
authors.affiliation: From the Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India and.
authors.forename: Nelson
authors.initials: N
authors.lastname: George
authors.affiliation: From the Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India and.
authors.forename: Bandana
authors.initials: B
authors.lastname: Chakravarti
authors.affiliation: From the Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India and.
authors.forename: Madan M
authors.initials: MM
authors.lastname: Godbole
authors.affiliation: the Division of Endocrinology and Metabolism, Department of Medicine, Georgetown University, Washington, D. C. 2007.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Ecelbarger
authors.affiliation: From the Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India and tiwaris@sgpgi.ac.in.
authors.forename: Swasti
authors.initials: S
authors.lastname: Tiwari
mesh_terms: D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000008:administration & dosage / Q000494:pharmacology; D015151:Immunoblotting; D007328:Insulin / Q000008:administration & dosage / Q000494:pharmacology*; D007679:Kidney Medulla / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D034622:RNA Interference; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D005452:Fluoresceins; D007004:Hypoglycemic Agents; D007328:Insulin; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin
keywords: Epithelial Cell; Gene Silencing; Hypertension; Insulin Receptor; Insulin Resistance; Kidney; Kidney Tubule Cells; Nitric Oxide Synthase; Phosphatidylinositide 3-Kinase