Abstract Body (Do not enter title and authors here): Global deficiency of the dopamine D4 receptor (D4R) has been associated with kidney-related hypertension and salt sensitivity. To specifically investigate the role of D4R in renal tubules, we generated conditional knockout (CKO) mice using CRISPR/Cas9 to create Drd4-floxed homozygous mice, which were then crossed with CDH16-Cre transgenic mice to achieve tubular-specific deletion.
In CKO mice (male, 4–5 months old), D4R expression was absent in renal tubules as confirmed by immunofluorescence staining. Immunoblotting of whole kidney homogenates revealed a significant reduction in D4R abundance (38 ± 4% of non-CKO levels; n = 5; Student’s t-test, p < 0.05). CKO mice maintained normal systolic blood pressure under standard salt(0.4% NaCl) intake but exhibited a >13% increase under high salt(4% NaCl) conditions (n = 15). Among the renal sodium transporters and channels evaluated, sodium-potassium-chloride cotransporter 2 (NKCC2: 218± 30%; n = 5) and sodium-chloride cotransporter (NCC: 241 ± 39%) were significantly elevated in CKO mice. No significant changes were observed in sodium-hydrogen exchanger 3, epithelial sodium channels, or Na⁺/K⁺-ATPase. Immunofluorescence further confirmed increased NCC expression in CKO kidneys, which colocalized with D4R in non-CKO mice.
Administration of the NCC inhibitor hydrochlorothiazide elicited greater natriuretic and antihypertensive effects in CKO mice, indicating enhanced NCC activity. The increased NCC protein levels were accompanied by reduced NCC ubiquitination (47± 8%; n = 5). In vitro, D4R silencing in cultured mouse distal convoluted tubule (DCT) cells similarly increased NCC expression (155 ± 11%, n=4) and decreased its ubiquitination (43± 8%).
Neither WNK4, Nedd4-2, components of the renin-angiotensin-aldosterone system, nor serum aldosterone levels were altered in CKO mice. However, the expression of ubiquitin-specific protease 48 (USP48), a deubiquitinating enzyme, was significantly increased in both the kidneys (169 ± 19%; n = 5) and DCT cells (202± 29%; n = 6) following Drd4 deficiency. Knockdown of USP48 via siRNA reduced NCC expression and increased NCC ubiquitination in vitro.
In conclusion, renal tubular deletion of Drd4 results in salt-sensitive hypertension in mice, primarily through upregulation of NKCC2 and NCC. The deubiquitinating enzyme USP48 appears to mediate this effect by modulating NCC stability, suggesting a novel mechanism and potential therapeutic target for salt-sensitive hypertension.