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  • Impaired natriuretic response to high-NaCl diet plus aldosterone infusion in mice overexpressing human CD39, an ectonucleotidase (NTPDase1).

Impaired natriuretic response to high-NaCl diet plus aldosterone infusion in mice overexpressing human CD39, an ectonucleotidase (NTPDase1).

American journal of physiology. Renal physiology (2015-04-17)
Yue Zhang, Simon C Robson, Kaiya L Morris, Kristina M Heiney, Karen M Dwyer, Bellamkonda K Kishore, Carolyn M Ecelbarger
ABSTRACT

Extracellular nucleotides acting through P2 receptors facilitate natriuresis. To define how purinergic mechanisms are involved in sodium homeostasis, we used transgenic (TG) mice that globally overexpress human CD39 (hCD39, NTPDase1), an ectonucleotidase that hydrolyzes extracellular ATP/ADP to AMP, resulting in an altered extracellular purine profile. On a high-sodium diet (HSD, 3.5% Na(+)), urine volume and serum sodium were significantly higher in TG mice but sodium excretion was unaltered. Furthermore, TG mice showed an attenuated fall in urine aldosterone with HSD. Western blot analysis revealed significantly lower densities (∼40%) of the β-subunit of the epithelial sodium channel (ENaC) in medulla, and the major band (85-kDa) of γ-ENaC in TG mice cortex. To evaluate aldosterone-independent differences, in a second experiment, aldosterone was clamped by osmotic minipump at 20 μg/day, and mice were fed either an HSD or a low-sodium diet (LSD, 0.03% Na(+)). Here, no differences in urine volume or osmolality, or serum aldosterone were found, but TG mice showed a modest, yet significant impairment in late natriuresis (days 3 and 4). Several major sodium transporters or channel subunits were differentially expressed between the genotypes. HSD caused a downregulation of Na-Cl cotransporter (NCC) in both genotypes; and had higher cortical levels of NCC, Na-K-ATPase (α-1 subunit), and α- and γ-ENaC. The Na-K-2Cl cotransporter (NKCC2) was downregulated by HSD in wild-type mice, but it increased in TG mice. In summary, our data support the concept that extracellular nucleotides facilitate natriuresis; they also reveal an aldosterone-independent downregulation of major renal sodium transporters and channel subunits by purinergic signaling.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-ENTPD1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Na+/K+ ATPase α-1 Antibody, clone C464.6, clone C464.6, Upstate®, from mouse
Sigma-Aldrich
Hematoxylin
Sigma-Aldrich
Aldosterone, ≥95% (HPLC)
Sigma-Aldrich
Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
Sigma-Aldrich
Sodium phosphate, 96%
Sigma-Aldrich
Hematoxylin, certified by the Biological Stain Commission