Dopamine d4 receptor down-regulates renal sodium chloride cotransporter via ubiquitination-associated lysosome degradation

Background The thiazide-sensitive sodium chloride cotransporter (NCC) is the major apical sodium transporter located in the mammalian renal distal convoluted tubule (DCT). The amount of sodium reabsorbed in the DCT through NCC plays an important role in the regulation of extracellular fluid volume and blood pressure. Dopamine and its receptors constitute a renal antihypertensive system in mammals. The disruption of Drd4 in mice causes kidney-related hypertension. However, the pathogenesis of D4R-deficiency associated hypertension is not well documented. Method We assessed the effects of D4R on NCC protein abundances and activities of DCT in mice with renal or global Drd4 -deficiencies and expressing human D4.7 variant and in cultured mouse DCT cells, and explored the molecular mechanism. Results NCC inhibitor hydrochlorothiazide enhanced the natriuresis in Drd4 -/- mice. Renal NCC protein was greater while ubiquitination of NCC was less in Drd4 -/- than Drd4 +/+ mice. Silencing of D4R in cultured mouse DCT cells increased NCC protein but decreased NCC ubiquitination. D4R agonist had opposite effects that were blocked by the antagonist. In mouse kidneys and DCT cells D4R and NCC colocalized and co-immunoprecipitated. Moreover, D4R-agonist promoted the binding between the two proteins demonstrated by fluorescence resonance energy transfer. D4R agonism internalized NCC, decreased NCC in the plasma membrane, increased NCC in lysosomes and reduced NCC-dependent-intracellular-sodium transport. The lysosomal inhibitor chloroquine prevented the D4R-induced NCC-reduction. A shortened NCC half-life was suggested by its decay under cycloheximide-chase. Ubiquitin-specific-protease 48 (USP48, a deubiquitinating enzyme) was increased in the kidneys and cells with Drd4 -deficiency while D4R stimulation decreased it in vitro and reduction of USP48 with siRNA decreased NCC expression. The mice carrying human D4.7 variant or with renal reduction of D4R developed hypertension with increased NCC. Conclusion Our data demonstrates that D4R downregulates NCC by promoting USP48-associated deubiquitination and subsequent internalization, lysosome relocation and degradation. ### Competing Interest Statement The authors have declared no competing interest.