Abstract 303: Decreased Adrenomedullin and Interaction with NHE3 in Human GRK4 γ 142v Transgenic Mice

Abstract

The expression of the human G protein-coupled receptor kinase 4γ (GRK4γ) 142V variant (hGRK4γ 142V ) in mice causes hypertension that is related to dysfunction of renal dopaminergic and renin-angiotensin systems. To determine if there are other dysfunctional systems in hGRK4γ 142V mice, we quantified the expression of adrenomedullin (ADM), a natriuretic hormone, and components of its receptor, RAMP2 (receptor activity-modified protein2), CLCR (calcitonin-like calcitonin receptor), and ADMRL2 (ADM receptor L2) in the kidneys of hGRK4 γ 142V and hGRK4 γ WT transgenic mice and non-transgenic (NT) littermates . Systolic blood pressure (measured under anesthesia) was elevated in hGRK4 γ 142V (129.3±10.4, mm Hg) but not in GRK4 γ WT mice (101.7±6.3), relative to NT littermates (99.8±1.8) (n=4/group). The sodium excretion and blood pressure plot in GRK4 γ 142V mice was shifted to the right of GRK4 γ WT mice. Protein expressions of ADM (37±4% of NT by immunoblotting and immunofluorescence) and RAMP2 (36±16%) were decreased in hGRK4 γ 142V mice but unchanged in GRK4 γ WT mice, relative to NT littermates. The protein expressions of CLCR and ADMRL2 were not altered in hGRK4 γ 142V and GRK4 γ WT . In the kidney ADM colocalized with NHE3 but not with NKCC2 and NCC. NHE3 protein expression in renal homogenates and brush border membranes was not altered in GRK4 γ 142V mice. However, the amount of NHE3 that coimmunoprecipitated with ADM was decreased in GRK4 γ 142V (77±3% of NT) mice; a similar decrease was also found in the reverse-coimmunoprecipitation of NHE3 with ADM. In vitro , human ADM inhibited apical sodium transport in polarized human renal proximal tubule cells (1 hr, n=5-6) at concentrations of 10 nM (84±4 % of vehicle) and 100 nM (81±3%), similar to that achieved with the NHE3 inhibitor EIPA (81±6%, 100 nM), suggesting that NHE3 activity was inhibited by ADM. Our data suggest that GRK4γ 142V decreases ADM/RAMP2 expression in the kidney; the natriuretic effect of ADM may depend mainly on the inhibition of the activities of sodium transporters along the nephron (e.g., NHE3). The decreased ADM/NHE3 interaction and dysfunctional natriuretic and antinatriuretic factors may contribute to the impaired sodium excretion and increased blood pressure in GRK4 γ 142V transgenic mice.