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Mechanisms coupling sodium and magnesium reabsorption in the distal convoluted tubule of the kidney.

ABSTRACT: Hypomagnesaemia is a common feature of renal Na⁺ wasting disorders such as Gitelman and EAST/SeSAME syndrome. These genetic defects specifically affect Na⁺ reabsorption in the distal convoluted tubule, where Mg²⁺ reabsorption is tightly regulated. Apical uptake via TRPM6 Mg²⁺ channels and basolateral Mg²⁺ extrusion via a putative Na⁺ -Mg²⁺ exchanger determines Mg²⁺ reabsorption in the distal convoluted tubule. However, the mechanisms that explain the high incidence of hypomagnesaemia in patients with Na⁺ wasting disorders of the distal convoluted tubule are largely unknown. In this review, we describe three potential mechanisms by which Mg²⁺ reabsorption in the distal convoluted tubule is linked to Na⁺ reabsorption. First, decreased activity of the thiazide-sensitive Na⁺ /Cl^- cotransporter (NCC) results in shortening of the segment, reducing the Mg²⁺ reabsorption capacity. Second, the activity of TRPM6 and NCC are determined by common regulatory pathways. Secondary effects of NCC dysregulation such as hormonal imbalance, therefore, might disturb TRPM6 expression. Third, the basolateral membrane potential, maintained by the K⁺ permeability and Na⁺ -K⁺ -ATPase activity, provides the driving force for Na⁺ and Mg²⁺ extrusion. Depolarisation of the basolateral membrane potential in Na⁺ wasting disorders of the distal convoluted tubule may therefore lead to reduced activity of the putative Na⁺ -Mg²⁺ exchanger SLC41A1. Elucidating the interconnections between Mg²⁺ and Na⁺ transport in the distal convoluted tubule is hampered by the currently available models. Our analysis indicates that the coupling of Na⁺ and Mg²⁺ reabsorption may be multifactorial and that advanced experimental models are required to study the molecular mechanisms.

SUBMITTER: Franken GAC

PROVIDER: S-EPMC7816272 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Mechanisms coupling sodium and magnesium reabsorption in the distal convoluted tubule of the kidney.

Franken Gijs A C GAC Adella Anastasia A Bindels René J M RJM de Baaij Jeroen H F JHF

Acta physiologica (Oxford, England) 20200726 2

Hypomagnesaemia is a common feature of renal Na+ wasting disorders such as Gitelman and EAST/SeSAME syndrome. These genetic defects specifically affect Na+ reabsorption in the distal convoluted tubule, where Mg2+ reabsorption is tightly regulated. Apical uptake via TRPM6 Mg2+ channels and basolateral Mg2+ extrusion via a putative Na+ -Mg2+ exchanger determines Mg2+ reabsorption in the distal convoluted tubule ...[more]

PMID: 32603001

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Project description:The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP2, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH2-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.

Project description:BackgroundThe potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown.MethodsWe used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both.ResultsDeletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice.ConclusionsNedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

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Mechanisms coupling sodium and magnesium reabsorption in the distal convoluted tubule of the kidney.

Publications

Mechanisms coupling sodium and magnesium reabsorption in the distal convoluted tubule of the kidney.

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