Background: Sorting nexin 19 (SNX19) is important in the localization and trafficking of the dopamine D1 receptor (D1R) to lipid raft microdomains. However, the interaction between SNX19 and the lipid raft components caveolin-1 or flotillin-1 and, in particular, their roles in the cellular endocytosis and cell membrane trafficking of the D1R have not been determined. Methods: Caveolin-1 and flotillin-1 motifs were analyzed by in silico analysis; colocalization was observed by confocal immunofluorescence microscopy; protein-protein interaction was determined by co-immunoprecipitation. Results: In silico analysis revealed the presence of putative caveolin-1 and flotillin-1 binding motifs within SNX19. In mouse and human renal proximal tubule cells (RPTCs), SNX19 was localized mainly in lipid rafts. In mouse RPTCs transfected with wild-type (WT) Snx19, fenoldopam (FEN), a D1-like receptor agonist, increased the colocalization of SNX19 with caveolin-1 and flotillin-1. FEN also increased the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1, effects that were prevented by SCH39166, a D1-like receptor antagonist. The FEN-mediated increase in the residence of SNX19 in lipid rafts and the colocalization of the D1R with caveolin-1 and flotilin-1 were attenuated by the deletion of a caveolin-1 (YHTVNRRYREF) (ΔCav1) or a flotillin-1 (EEGPGTETETGLPVS) (ΔFlot1) binding motif. The FEN-mediated increase in intracellular cAMP production was also impaired by the deletion of either the flotillin-1 or caveolin-1 binding motif. Nocodazole, a microtubule depolymerization inhibitor, interfered with the FEN-mediated increase in the colocalization between SNX19 and D1R. Conclusion: SNX19 contains caveolin-1 and flotillin-1 binding motifs, which play an important role in D1R endocytosis and signaling.
Abstract The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.
Sorting nexin 19 (SNX19) belongs to the SNX-PXA-RGS-PXC subfamily of sorting nexins. It controls blood pressure by regulating the endocytosis of dopamine D1 receptor (D 1 R) in renal proximal tubule cells (RPTCs), allowing it to reside in the lipid rafts (LRs) of the plasma membrane. The exclusion of the D 1 R from LRs in RPTCs is associated with hypertension. However, it is unknown if SNX19 interacts with caveolin-1 and flotillin-1, two of the most important LR components. Thus, we studied if SNX19 associates with these two proteins and whether this interaction impacts D 1 R endocytosis. In RPTCs, SNX19 co-immunoprecipitated with caveolin-1 and flotillin-1 in basal conditions. Fenoldopam (FEN, 25nM, 30 min, n=3), a selective D 1 R/D 5 R agonist, increased the co-immunoprecipitation of SNX19 with both caveolin-1 and flotillin-1. The treatment with a D 1 R/D 5 R antagonist, SCH 39166 (SCH, 1 μM, 30 min), prevented the FEN-mediated increase in the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1. In RPTCs, the transfection of SNX19 plasmid with deletion of either caveolin-1 (ΔCav1-SNX19) or flotilin-1(ΔFlot1-SNX19) binding domain impaired the FEN-mediated D 1 R endocytosis compared with the cells that were only transfected with wild-type (WT) SNX19 (WT-SNX19, 100.0±5.0%; ΔCav1-SNX19, 39.1±2.8%; ΔFlot1-SNX19, 59.6±3.4%, n=5). Moreover, the relative mRNA expression of SNX19 did not change in RPTCs transfected with WT-SNX19, ΔCav1-SNX19, or ΔFlot1-SNX19. Thus, SNX19 expression does not depend on its caveolin-1 or flotillin-1 binding motif. In C57Bl/6 mice fed with normal salt diet (0.9% NaCl), the renal subcapsular infusion of Snx19 siRNA (3 μg/day,7 days) significantly increased systolic blood pressure (SBP, mmHg) (mock siRNA: 99.3±1.5, n=8; Snx19 siRNA: 114.1±5.4, n=8). The SBP also increased in mice fed with high salt and aggravated by Snx19 siRNA (4% NaCl; mock siRNA: 119.8 ± 5.6, n=4; Snx19 siRNA: 125.6 ±3.4, n=5). Low salt diet decreased SBP that was not affected by Snx19 siRNA (0.04% NaCl, mock siRNA: 92. 0 ± 3.5, n=3; Snx19 siRNA: 82.7 ±6.7, n=3). Germline deletion of Snx19 in C57Bl/6 mice fed with normal salt diet increased SBP. In conclusion, caveolin-1 and flotillin-1 are important for SNX19-mediated D 1 R endocytosis and blood pressure.
Neuropeptide FF (NPFF), an octapeptide originally found in the brain, participates in the regulation of blood pressure because it is present with its receptors, NPFFR1 and NPFFR2, in the cardiovascular regulatory center in the hypothalamus. However, the role of NPFF and its receptors in the kidney on blood pressure regulation is not known. In both the human and mouse renal proximal tubule, NPFFR2, rather than NPFFR1, is the predominant NPFF receptor determined by RNA in situ hybridization and immunofluorescence microscopy. In mouse renal proximal tubule cells, AC263093 (10 -6 M, 15 min), a specific NPFFR2 agonist and NPFFR1 antagonist, inhibited the forskolin-stimulated cAMP production (Vehicle: 100±6.14%, n=6; AC263093: 72.4±6.53%, n=6, p<0.05), which was reversed by pretreatment with RF-9, an antagonist of both NPFFR1 and NPFFR2 (98.2±9.10%, n=6); RF-9, by itself, had no significant effect (100.8±11.44%, n=6), indicating linkage of NPFFR2 to the inhibitory G protein Gai in renal proximal tubule cells. In human renal proximal tubule cells, NPFF also increased Na + /K + -ATPase protein expression, determined by immunoblotting, in a time- and concentration-dependent manner. Protein expression of Na + /K + -ATPase was decreased in NPFFR2 -deficient human renal proximal tubule cells caused by specific NPFFR2 siRNA (Mock: 100±1.71%, NPFFR2 siRNA: 58.4±4.93%, n=4; P < 0.05). Furthermore, blood pressure, measured by telemetry, was increased and sodium excretion was decreased in conscious C57Bl/6 mice infused with NPFF (9.25 mmol, 0.5 mL/hr/7 days) underneath the renal capsule. The effect was probably via NPFFR2 because the blood pressure of mice was increased by the intraperitoneal injection of AC263093 (20 mg/kg/day, 7 days) and fed a high salt diet (4% NaCl), which is consistent with the decrease in blood pressure (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) by the renal subcapsular infusion of Npffr2 siRNA in C57Bl/6 mice also fed the high salt diet. Taken together, NPFFR2 activation increases blood pressure and decreases sodium excretion that is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule.
Peroxiredoxin-4 (PRDX4), an endoplasmic reticulum (ER)-localized antioxidant enzyme, plays an essential role in cellular redox homeostasis by reducing hydrogen peroxide from thiol-containing compounds. The dopamine D5 Receptor (D5R) also plays a protective role against oxidative stress. We hypothesize that D5 R interacts with the PRDX4 to reduce oxidative stress in the kidney. In D5 R-HEK 293 cells, fenoldopam (FEN, 25 nM/12 hr, n=4), a D1 R and D5 R receptor agonist, increased PRDX4 protein expression (1.92±0.12-fold over basal level, n=4), mainly in non-lipid raft (LR) fractions (LRs: 24.9±11.4%, non-LRs: 75.1±11.4%, baseline; LRs: 30.9±13.9%, non-LRs: 174.1±16.7%, FEN). By contrast, fenoldopam did not affect PRDX4 protein expression in D1 R-HEK 293 cells, indicating a D5 R-specific effect on PRDX4 protein expression. In human renal proximal tubule cells (hRPTCs) and D5 R HEK293 cells, fenoldopam increased the co-immunoprecipitation between PRDX4 and D5 R and their co-localization in the ER. SiRNA-mediated silencing of PRDX4 increased hydrogen peroxide production in both the vehicle (Veh)- and fenoldopam-treated hRPTCs [(scrambled siRNA: 100 ±15.1% and 55.2± 7.2% with Veh and FEN, respectively; PRDX4 siRNA: 161.8±15.3% and 145.1±14.6 % with Veh and FEN, respectively, n=4)]. The D5 R protects against inflammation and siRNA silencing of PRDX4 increased the production of the pro-inflammatory cytokines, interleukin-1β [26.88±3.8 and 46.40±4.2 pg/mL (n=3, D5 R-HEK 293); 15.87±1.2 and 37.9±1.4 pg/mL (n=3, hRPTCs), and tumor necrosis factors [131.7±6.5 and 271.2±18.1 pg/mL (n=4, D5 R-HEK 293); 108.8±11.8 and 240.1±13.7 pg/mL (n=4, hRPTCs)]. In D5 R-HEK293 and hRPTCs, the fenoldopam-mediated decrease in ER-resident caspase-12 was also impaired by gene silencing of PRDX4. Furthermore, PRDX4 protein expression was reduced in the kidney cortices of Drd5-/- mice, relative to Drd5 WT mice (WT: 1.00±0.12, n=4; Drd5-/- : 0.64±0.13, n=4; P<0.05). We conclude that D5 R positively interacts with PRDX4 to reduce ER stress in the kidney.
Autophagy, a crucial cellular homeostatic process, has protective roles in the pathogenesis of hypertension. Dopamine, a monoamine neurotransmitter, regulates autophagy in the kidney and vascular system. However, the exact roles and mechanisms of dopaminergic system on the regulation of autophagy is not well known. In rat aortic vascular smooth muscle cells, fenoldopam (FEN, 25 nM/15 min), a D1-like receptor agonist, increased the ATG5 and LC3-II (a late autophagosome maker) protein expression. Consistently, FEN decreased p62/SQSTM1 protein expression. The FEN-mediated increase in ATG5 was attenuated by SCH39166 (1 μM/15 min), a D1- like receptor antagonist, indicating that FEN increased autophagy in the autophagosome formation stage through its D1-like receptors. FEN increased the protein expression of WD Repeat Domain, Phosphoinositide Interacting 2 (WIPI2) in a concentration- and time-dependent manner, which is important in the early steps of autophagosome formation. FEN (25 nM, 15 min) increased the WIPI2 puncta fluorescence intensity and the colocalization of WIPI2 with EEA1 (Veh: 7.12±1.1% vs FEN: 9.96±0.9%, N=5, P < 0.001, Student’s t test), a typical phosphatidylinositol-3-phosphate binding protein in early endosomes, indicating that WIPI2 recruitment in early endosomes is important in the early stage of FEN-mediated autophagic process. Taken together, FEN increases autophagy with WIPI2 recruitment in the early endosomes in vascular smooth muscle cells. This work was supported by US National Institutes of Health grants R01DK134574 and R01DK119652. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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