Zn2+ deficiency (ZnD) is a common comorbidity of many chronic diseases. In these settings, ZnD exacerbates hypertension. Whether ZnD alone is sufficient to alter blood pressure (BP) is unknown. To explore the role of Zn2+ in BP regulation, adult mice were fed a Zn2+-adequate (ZnA) or a Zn2+-deficient (ZnD) diet. A subset of ZnD mice were either returned to the ZnA diet or treated with hydrochlorothiazide (HCTZ), a Na+-Cl− cotransporter (NCC) inhibitor. To reduce intracellular Zn2+ in vitro, mouse distal convoluted tubule cells were cultured in N,N,N′,N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, a Zn2+ chelator)- or vehicle (DMSO)-containing medium. To replete intracellular Zn2+, TPEN-exposed cells were then cultured in Zn2+-supplemented medium. ZnD promoted a biphasic BP response, characterized by episodes of high BP. BP increases were accompanied by reduced renal Na+ excretion and NCC upregulation. These effects were reversed in Zn2+-replete mice. Likewise, HCTZ stimulated natriuresis and reversed BP increases. In vitro, Zn2+ depletion increased NCC expression. Furthermore, TPEN promoted NCC surface localization and Na+ uptake activity. Zn2+ repletion reversed TPEN effects on NCC. These data indicate that 1) Zn2+ contributes to BP regulation via modulation of renal Na+ transport, 2) renal NCC mediates ZnD-induced hypertension, and 3) NCC is a Zn2+-regulated transporter that is upregulated with ZnD. This study links dysregulated renal Na+ handling to ZnD-induced hypertension. Furthermore, NCC is identified as a novel mechanism by which Zn2+ regulates BP. Understanding the mechanisms of ZnD-induced BP dysregulation may have an important therapeutic impact on hypertension.
BackgroundZn2+ deficiency is a common comorbidity with numerous chronic diseases including type II diabetes and chronic kidney disease. Experimental data show that Zn2+ deficiency exacerbates hypertension in these settings. Moreover, Zn2+ deficiency alone is sufficient to alter blood pressure. However, the mechanisms involved are unknown.ObjectiveThe renal distal convoluted tubule sodium chloride cotransporter (NCC) plays a critical role in blood pressure regulation and hypertension. The objective of this study was to determine if NCC plays a role in Zn2+ deficiency‐induced hypertension.Experimental DesignTo this end, WT mice were pair‐fed a diet with 50 ppm Zn2+ (Zn2+ adequate [ZnA]) or a diet with 1 ppm Zn2+ (Zn2+ deficient [ZnD]) for 6 weeks. A subset of mice was administered 50 mg/kg hydrochlorothiazide, a NCC inhibitor. To reduce intracellular Zn2+ levels in vitro, WT mouse distal convoluted tubular (mDCT‐15) cells were cultured in 1 nM N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN)‐ or vehicle (DMSO)‐containing medium for 24 hours. To replete intracellular Zn2+, TPEN‐exposed cells were then cultured in 1 nM Zn2+‐supplemented medium for 24 hours. Select cell monolayers were treated with 100 nM diphenyliodonium (DPI), a general NADPH oxidase inhibitor, prior to TPEN treatment. In mice, systolic blood pressure was monitored by tail cuff plethysmography. NCC protein expression was examined in vivo and in vitro via western blot and immunohistochemistry. NCC activation was assessed by cellular localization (via biotinylation and immunofluorescence) and Na+ uptake activity (via thiazide‐sensitive assays).ResultsIn ZnD mice, increased blood pressure is accompanied with enhanced NCC protein expression compared to mice receiving a ZnA‐diet. However, NCC inhibition by hydrochlorothiazide treatment reverses blood pressure increases. In the in vitro model of Zn2+ deficiency, Zn2+ chelation with TPEN stimulates NCC protein expression compared to vehicle‐treated cells. Furthermore, TPEN promotes NCC surface localization and Na+ uptake activity. Notably, restoration of intracellular Zn2+ levels by supplementation reverses TPEN effects on NCC. Additionally, NADPH oxidase inhibition with DPI treatment prevents TPEN‐induced NCC upregulation.ConclusionsOur findings indicate that NCC is a Zn2+‐sensitive transporter and is upregulated in settings of Zn2+ deficiency via a NADPH oxidase‐mediated mechanism.SignificanceNCC represents a novel mechanism by which Zn2+ deficiency alters blood pressure and contributes to hypertension in chronic disease settings.Support or Funding InformationCR. Williams (AHA‐SDG), JL. Gooch (VA‐MERIT), RS. Hoover (VA‐MERIT and NIH‐R01)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Our previous work has shown that epithelial sodium channel (ENaC) proteins are present in distal convoluted tubule (DCT) cells of the distal nephron. The purpose of these experiments was to show whether these proteins were functional. We also wish to show whether the channels were stimulated by aldosterone, inhibited by amiloride, and regulated by the ENaC chaperone protein, MLP‐1, as ENaC is regulated in collecting duct principal cells. We used DCT‐15 cells in monolayer culture on permeable polyester supports to measure transepithelial voltage, resistance, and current. The properties of DCT‐15 cells are quite similar to those of in situ cells of the DCT2 segment of the nephron. Our results showed that DCT‐15 cells had relatively low resistances (206 ± 5.71 Ω‐cm2, n=36) and small, but measurable amiloride‐sensitive currents (2.87 ± 0.237 μA, n=36), showing that these cells contain functional ENaC. 100 nM aldosterone significantly increased mean DCT‐15 cell current (from 2.87 ± 0.237, n=36, to 5.33 ± 0.395μA, n=8). Amiloride significantly lowered the current across DCT cells to very low levels (from 2.74 ± 0.219, n=42, to 0.825 ± 0.186μA, n=15). MLP‐1 is a myristoylated protein that when associated with the membrane promotes ENaC activity. When three critical serines are phosphorylated by PKC, MLP‐1 dissociates from the membrane and ENaC activity decreases. We transfected the cells with four MLP FLAG‐tagged mutants: wild type (WT), constitutively active (S3A), constitutively inactive (S3D), and myristoylation negative (GA) and measured DCT‐15 current. For comparison, we examined a principal cell line, mpkCCD cells. Transfection with MLP‐1 modified current in both DCT and CCD cells: median current was highest in S3A mutant and lowest in S3D mutant; the current after transfection with either construct was significantly different from WT. ENaC and MLP construct expression was also studied in lysates of both cell types by Western blotting with FLAG and anti‐ENaC subunit antibodies. In DCT‐15 cells with the highest current, expression of ENaC was greatest. Lastly, we calculated based on an average current of 2 μA/cm2 and ENaC single channel current of 0.4 pA that DCT‐15 cells must have approximately 5 million functional channels per square centimeter.Support or Funding InformationSupported by R01 DK‐110409 to DCE and K01 DK‐115660 and ASN Gottschalk AWARD to BMWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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.