Analysis of patients with inherited hypokalaemic alkalosis resulting from salt-wasting has proved fertile ground for identification of essential elements of renal salt homeostasis and blood-pressure regulation. We now demonstrate linkage of this phenotype to a segment of chromosome 1 containing the gene encoding a renal chloride channel, CLCNKB. Examination of this gene reveals loss-of-function mutations that impair renal chloride reabsorption in the thick ascending limb of Henle's loop. Mutations in seventeen kindreds have been identified, and they include large deletions and nonsense and missense mutations. Some of the deletions are shown to have arisen by unequal crossing over between CLCNKB and the nearby related gene, CLCNKA. Patients who harbour CLCNKB mutations are characterized by hypokalaemic alkalosis with salt-wasting, low blood pressure, normal magnesium and hyper- or normocalciuria; they define a distinct subset of patients with Bartter's syndrome in whom nephrocalcinosis is absent. These findings demonstrate the critical role of CLCNKB in renal salt reabsorption and blood-pressure homeostasis, and demonstrate the potential role of specific CLCNKB antagonists as diuretic antihypertensive agents.
The renal proximal tubule (PT) is a major site for a complete tissue renin-angiotensin system (RAS) and produces endogenous angiotensin II (ANG II). The present studies demonstrate autocrine RAS feedback in a line of origin-defective SV40 plasmid transformed immortalized rat PT cells (IRPTC) designated as line 93-p-2–1, which are highly differentiated and express all RAS components. Receptor competition assays and Southern blot following RT-PCR demonstrated that these IRPTC express AT1 and AT2 angiotensin receptor subtypes. Autocrine RAS feedback was examined following exposure to ANG II (10−8 M), and it was noted that angiotensinogen mRNA increases significantly by 1 h and remains elevated through 24 h. The AT1 blocker losartan prevents this increase. Moreover, ANG II upregulates expression of ANG II receptor mRNA (both AT1 and AT2). Thus the present studies demonstrate positive ANG II feedback with angiotensinogen and ANG II receptors in PTC, suggesting that the main site of such intrarenal feedback in vivo is within PT. ANG II secreted by line 93-p-2–1 is increased by isoproterenol, suggesting β-adrenergic regulation in IRPTC.
A&&act--l l/3-hydroxysterold dehydrogenase (11 P-HSD) 1s expressed m vascular smooth muscle cells (VSMC) but has not been reported to be present m vascular endothehal cells This enzyme assists m regulating the cellular concentration of active endogenous glucocortlcolds (GCs) We have observed that endothelmm intact rat aortlc rings express message for both Type 1 and Type 2 IIP-HSD whereas pnmary cultures of VSMC express only mRNA for the Type I lsoform Since GCs dmunlsh prostacyclm synthesis m endothehal cells, we hypothesized that 11/3-HSD 1s present m vascular endothehal cells In pnmary cultures of rat aortlc endothehal (RAE) cells, mRNA from both lsoforms of II&HSD could be detected by RT-PCR with higher levels of the Type 1 lsoform The oxo-reductase reaction "actlvatmg" 1 I-dehydro metabohtes back to the parent steroid 1s the preferred enzyme direction (12 1 after a 120 mmutes steroid mcubatlon) m intact RAE cells When RAE cells are grown m the presence of antisense ohgonucleotldes specific for Type 1 1 l/3-HSD, oxo-reductase activity 1s decreased by approximately 50% but the dehydrogenase reaction, which mactlvates endogenous GCs and 1s charactenstlc of the Type 2 lsofonn, 1s or reachvate (oxo-reductase 1 e , 11 -dehydrocortlcosterone + comcosterone) GCs The physlologcal correlate of 1 l/3-HSD &rechon has been shown m endothehum intact vascular rings, mhlbltlng the dehydrogenase reactlon 1s associated wth an enhanced contra&e response to catecholammes and angotensm II while blockmg the oxo-reductase reaction 1s lmked to an attenuated contractile response to these same agents 'Vascular endothehal cells contam a number of GC-sensitive mechanisms that directly affect the contractile response of adjacent smooth muscle cells Specifically, the generation of prostacyclm' ' and the activity of atria1 natnuretlc peptldeinduced guanylate cyclase are both Impaired m presence of excess GC ' Thus vasorelaxmg processes active m endothehal cells that normally would counterbalance circulating vasoconstrlctors are blunted Given the role GCs may play m altering endothehal function, the present studies have been designed to determine whether 1 l/3-HSD exists m these cells and if 50 what are its blologlcal characterlstlcs Methods RAE Cell Cultures RAE cell? (passage 6) were ongmally purchased from VEC TEC, Inc , which Isolates these purliied cells from rat aorta usmg a proprietary method The RAE cells demonstrate the typlcal "cobblestone" morphology consistent with eplthehal cells In addmon, these primary cells demonstrate the expected mcorporatlon of acetylated low demlty hpoprotem (LDL) and exprenlon of Factor VIII using polyclonal antibody (DAKO) RAE cells were grown m DMEM (Glbco, BRL), contammg 5'%1 horse serum (Glbco, BRL) and 596 fetal bovme serum (Sigma Chemical) m an atmosphere of 596 CO, at 37'C Primary VSMC CulturesThe aortae from 8 previously sacrificed adult Sprague-Dawley rats were dissected under sterile condltlons and asroclated fat and connective tissue removedThe aortae were next Incubated at 37°C fol 15 min...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.