Micro RNA 4301 (miR4301) is an intronic miRNA that resides in the second intron of the primary human DRD2 transcript. Examination of the miR4301 target binding prediction showed a target site in the 3'UTR region of the human DRD2. We hypothesized that miR4301 may regulate the expression of D2R and mediate, in part, the deleterious effects of decreased D2R function, as some common single nucleotide polymorphisms (SNPs), rs6276 and rs6277, of the human DRD2 gene are associated with decreased D2R expression and function, increased renal inflammation/fibrosis, and blood pressure or hypertension. We found that in human renal proximal tubule cells (hRPTCs) transfected with miR4301 mimic showed a 35% (P< 0.03, n=4) reduction in D2R protein compared with the hRPTCs transfected with control miR, showing that the DRD2 transcript is effectively a target of miR4301. Conversely, treatment of the hRPTCs with TGFβ that increases DRD2 transcription also increases miR4301 (1.70±0.07 vs. 1.0±0.03; P< 0.05, n=5). We studied further four hRPTC lines carrying no SNPs (hRPTC-wild-type[WT]) and 4 hRPTC lines with SNPs (hRPTC-SNPs) and found that miR4301 expression was lower in hRPTC-SNPs than hRPTC-WT (0.52±0.07- vs 1.03±0.14-fold, P< 0.05). Silencing D2R via siRNA in hRPTC-WT also decreased miR4301 expression (0.59±0.12-0.12- vs 1.01±0.11-fold, P< 0.05). Moreover, the mRNA expression of the mir4301 target LEF1 in hRPTC-WT transfected with miR4301 mimic was lower (0.070±0.02- vs 1.0±0.05-fold, P< 0.05) than in hRPTCs transfected with control miR. By contrast, transfection of the hRPTCs with miR4301 inhibitor increased the expression of LEF1 mRNA (1.30±0.03- vs 1.0±0.05-fold, P< 0.05), compared with the control miR, indicating that miR4301 represses LEF1 and subsequently regulates the Wnt/β-catenin pathway. These results suggest that D2R function may be dependent, in part, on the regulation of miR4301. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Sodium homeostasis plays a critical role in managing arterial blood pressure, where an increase in renal Na + transport would lead to the development of hypertension. Neuropeptide FF (NPFF) and its two receptors, NPFFR1 and NPFFR2, are found to be expressed in various segments of the nephron such as the renal proximal tubule (RPT) and increase blood pressure (BP). However, the mechanisms by which renal NPFF is involved in raising BP are not fully understood. In human RPT cells (hRPTCs), NPFF (100 nM, 10 min) decreased the lifetime of Na + -binding (τ2) Asante NaTRIUM Green-2 from 3.52 to 3.44 nanoseconds, determined by fluorescence lifetime imaging microscopy, suggesting that NPFF increased Na + transport out of the RPT cells. Notably, in hRPTCs, NPFF increased the protein expressions of Na + /K + -ATPase and sodium-glucose cotransporter 2 (SGLT2) in a time- and concentration-dependent manner. Moreover, the renal-selective infusion of NPFF or NPFFR2 agonist, AC263093 (20 mg/kg/day, 7 days), causes an elevation in systolic BP. By contrast, the renal-selective infusion of RF9, an NPFFR antagonist, or silencing NPFFR1 or NPFFR2 with their siRNAs, decreased the systolic BP of C57Bl/6 mice fed a high-salt diet (4% NaCl). NPFF reduced sodium excretion and Npffr2 but not Npffr1 siRNA slightly decreased renal Na + /K + -ATPase and SGLT2 expression, while NHE3 expression was unchanged. We conclude that renal NPFFR increases BP and decreases sodium excretion that may be related to an NPFFR2-mediated upregulation of Na + /K + -ATPase and SGLT2 protein expressions in the RPT. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Vascular smooth muscle cells (VSMCs), the contractile cells in the tunica media of blood vessels, maintain vascular tone. The proliferation of VSMCs is an important feature of vascular remodeling that contributes to the regulation of blood pressure. Autophagy, an intracellular self-degrading process that delivers cytoplasmic constituents to lysosomes, plays a vital role in VSMC proliferation. This is regulated by the dopaminergic and renin-angiotensin systems but their interplay in their regulation of autophagy in VSMCs is not well-understood. In rat VSMCs, fenoldopam (Fen), a dopamine D1-like receptor agonist, increased autophagy, as determined by the increase in the protein expressions of microtubule-associated protein 1 light chain (LC)3-II and beclin-1 (BECN1), in a time- and concentration-dependent manner. Conversely, angiotensin II (Ang II), the endogenous Ang II type 1 receptor (AT1R) agonist, decreased the protein expression of LC3-II and BECN1, also in a time- and concentration-dependent manner. The production of cyclic adenosine monophosphate (cAMP) and autophagic LC3-II puncta in VSMCs were increased by Fen and decreased by Ang II. Pre-treatment of VSMCs with Rp-cAMPS, a protein kinase A inhibitor, prevented the Fen-mediated increase and the Ang II-mediated decrease in LC3-II protein expression. Fen decreased, whereas Ang II increased the phosphorylation of P70S6K, a direct downstream mammalian target of rapamycin (mTOR). The inhibitory effect of Fen and stimulatory effect of Ang II on P70S6K phosphorylation were prevented by Rp-cAMPS. Ang II also decreased the Fen-mediated increase in cAMP production, while Fen attenuated the Ang II-mediated increase in cell proliferation, a response that occurs downstream of autophagy. Moreover, Ang II prevented the Fen-mediated inhibition of cell proliferation, an effect that was blocked by losartan, an AT1R antagonist. These results demonstrate that Fen and Ang II counter-regulate autophagy and proliferation of VSMCs via the mTOR pathway, which is cAMP-dependent.

Autophagy, an intracellular degradation process crucial for maintaining cellular homeostasis, is regulated by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs). However, the role of Double FYVE Domain Containing Protein 1 (DFCP1), an early autophagosome protein that inhibits autophagy, particularly in VSMCs, is still largely unknown. We investigated the role of DFCP1 on Ang II-mediated autophagy in VSMCs and found that Ang II (100 nM) decreased beclin-1 and LC3II (a late autophagosome maker) protein expressions. The Ang II-mediated decrease in beclin-1 and LC3-II was attenuated by an AT 1 R antagonist, losartan (10 µM), indicating that Ang II decreased autophagy in the autophagosome formation stage through AT 1 R. Ang II had no effect on the protein expression of Unc-51-like autophagy activating kinase 1 (ULK1) and phospho-ULK1, which are important in the early stages of autophagosome formation. By contrast, Ang II increased DFCP1 and decreased LC3 protein expressions in a concentration (10 nM-1 mM) and time-dependent manner. Ang II (1 nM, 15 min) increased the DFCP1 puncta fluorescence intensity and the colocalization of DFCP1 with Hsp60 (Veh: 5.7±3.1% vs. Ang II: 11.6±6.7%, N=5, p<0.05, Student’s t -test), a heat shock chaperonin in mitochondria that plays a critical role in mitochondria dynamics and homeostasis. We conclude that in VSMCs, Ang II increases DFCP1 protein expression and decreases non-selective autophagy, which is associated with the initiation of mitophagy, a subtype of selective autophagy. National Institutes of Health DK119652 and DK134574. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Sorting nexin 19 (SNX19) is a lipid raft-associated protein that contains binding domains for caveolin-1 and flotillin-1 and plays a regulatory role in dopamine D1 receptor (D 1 R) trafficking in renal proximal tubule cells (RPTCs). However, its role on D 1 R protein expression remains incompletely understood. In human RPTCs, SNX19 colocalized and co-immunoprecipitated with caveolin-1 and flotillin-1, and their interactions were further enhanced by fenoldopam (25 nM, 30 min), a D 1 R/D 5 R agonist. In mouse RPTCs transfected with Snx19 , deletion of the caveolin-1 or flotillin-1 binding sites did not alter D 1 R expression. By contrast, targeted knockdown of SNX19 via siRNA in human RPTCs significantly reduced D 1 R protein abundance (non-silencing siRNA: 100±5.0%; SNX19 siRNA: 45±6.1%; P <0.05) and blunted the fenoldopam-stimulated cAMP generation (mock siRNA: +147.8±4.5 pmol/mg protein; SNX19 siRNA: +56.3±5.4 pmol/mg protein; P <0.05; n =4). Silencing Snx19 also led to a decrease in D 1 R protein levels in mouse kidney homogenates (mock siRNA: 1.0±0.09; Snx19 siRNA: 0.57±0.17; P <0.05; n =5). Consistent with these findings, RPTCs from hypertensive individuals had decreased SNX19 protein expression, relative to normotensive controls (100.0±12.4% vs . 38.7±8.7%; P <0.05; n =4). Furthermore, renal-restricted silencing of Snx19 via renal subcapsular infusion of specific Snx19 siRNA in C57Bl/6J mice decreased D 1 R but increased Na + /K + -ATPase protein and systolic blood pressure (mock siRNA: 101±6 mmHg; Snx19 siRNA: 118±5 mmHg; P <0.05; n =5). Collectively, deficiency of SNX19 decreases D 1 R protein in renal proximal tubules and increases systolic blood pressure in mice.

Inverse salt sensitivity, an increase in blood pressure (BP) when sodium intake is reduced, affects about 10-15% of the population, yet the mechanisms underlying this alteration in BP are not well understood. The renal dopamine D2 receptor (D2R) plays a critical role in maintaining normal BP and preventing inflammation and tissue injury. The DRD2 is highly polymorphic, and single nucleotide polymorphisms (SNPs) in this gene impair DRD2 synthesis and stability. Specifically, rs6277 SNP in exon 7 of DRD2 is associated with decreased D2R expression and is present in some individuals with hypertension. We have reported that human renal proximal tubular cells with this SNP have decreased D2R mRNA and protein expressions and increased renal Na+ pump/transporter expression. To study the effects of rs6277 on sodium balance and BP, using CRISPR-Cas9, we generated C57Bl/6 mice lacking their own Drd2 but instead express either the human DRD2 wild-type ( DRD2 WT) or rs6277 ( DRD2 Mut). Male and female mice were placed for one week on three distinct salt diets: normal salt (NS; 0.4% NaCl), high salt (HS; 4% NaCl), and low salt (LS; less than 0.08% NaCl) diets. On NS diet, BPs (measured by tail-cuff plethysmography under pentobarbital anesthesia) were slightly higher in male DRD2 Mut than DRD2 WT mice (79±3 vs 73±±0.5 mm Hg; P<0.04; n=5-7/group) while BPs were similar in female DRD2 Mut and DRD2 WT mice (77±3 vs 75±3 mm Hg: n=8/group). On HS diet, BPs were similar in DRD2 WT and DRD2 Mut mice (males 89±2 vs 92±3; females 79±3 vs 87±5 mm Hg). However, on LS diet DRD2 Mut had higher BPs than DRD2 WT mice (males: 72±2 vs 90±2 P<0.001; females: 68±1 vs 88±3 mm Hg, P<0.001). Thus, in DRD2 WT mice, BP increased on HS diet and decreased on LS, while in DRD2 Mut mice, BP increased on both LS and HS diets. There were no significant differences in urinary sodium excretion between DRD2 WT and DRD2 Mut male and female mice on the different diets. These findings suggest that alterations in DRD2 expression/function may be the underlying cause of inverse salt sensitivity of BP because the presence of DRD2 rs6277 is associated with inverse salt sensitivity in mice and humans. Moreover, the increased BP in DRD2 rs6277 mice on LS is independent of urinary sodium excretion.

One-third of individuals without underlying health conditions and 60% of individuals with elevated blood pressure (BP) are salt-sensitive. A shift from low to high Na + diet is linked to higher BP. Inverse salt sensitivity is an increase in BP when Na+ intake is reduced. This affects about 10-15% of the population but the mechanisms underlying this alteration in BP are not understood. The renal dopamine D2 receptor (D2R) plays a critical role in maintaining normal BP and preventing inflammation and tissue injury. The DRD2 gene is highly polymorphic and single nucleotide polymorphisms of this gene have been linked to allelic variations that impair receptor synthesis and stability. In particular, the rs6277 SNP in exon 7 of DRD2 is associated with decreased D2R expression and found in some humans with elevated blood pressure or hypertension. We have also reported that human renal proximal tubular cells with this SNP have decreased mRNA and protein D2R expressions and increased expression of Na+ pump/transporters. To study the effects of this SNP on Na + balance and BP we generated, using CRISP-Cas9, mice expressing either the human D2R wild-type ( DRD2 WT) or the mutant expressing the rs6277 ( DRD2 Mut) in mice lacking their own Drd2. Female mice were placed for one week on three distinct salt diets: normal salt (NS; 0.4% NaCl) diet, high salt (HS; 4% NaCl), and low salt (LS; less than 0.08% NaCl) diets. On NS diet, BPs (measured by tail-cuff under pentobarbital anesthesia) were similar in DRD2 WT and DRD2 Mut mice (79±3 vs 81±5 mm Hg; n=4/group). On HS diet, BPs were also similar in both groups (85±3 vs 86±5 mm Hg; n=4/group). However, on LS diet DRD2 Mut mice had significantly higher BPs than DRD2 WT (89±5 vs 71±1 mm Hg, p<0.05; n=4/group) mice. In DRD2 WT mice, BP increased on HS diet and decreased on LS, while in DRD2 Mut mice, BP increased on LS but their BPs were not different between NS and HS diet. On LS diet, UNaV was lower in DRD2 Mut than DRD2 WT mice (0.003±0.001 vs 0.008±0.001 mEq/day; p<0.05; n=4/group) but on HS diet, UnaV was higher in DRD2 Mut than DRD2 WT (0.91±0.3 vs 0.44±0.07; p<0.05; n=3/group) mice. Preliminary data in male mice show the same alterations. These findings suggest that alterations in DRD2 expression/function may be the underlying cause of inverse salt sensitivity because the presence of DRD2 rs6277 is associated with inverse salt sensitivity in humans. NIH-NIDDK R01Actf DK134574Projectf 01 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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.

Neuropeptide FF (NPFF), an amidated neuropeptide originally isolated from bovine brain, is a pain-modulating peptide with diverse physiological and pathophysiological functions. NPFF and its receptors, NPFFR1 and NPFFR2, are also present in peripheral tissues, including the kidney; NPFF, NPFFR1, and NPFFR2 mRNA and proteins are expressed in mouse and human renal proximal tubules determined by in situ RNA hybridization, immunofluorescence imaging, western blotting, and confirmed by liquid chromatography-tandem mass spectrometry (NPFF, mouse only). In mouse renal proximal tubule cells, NPFF decreased the forskolin-stimulated cAMP production in a concentration (10 -12 -10 -5 M) and time (0-180 min)-dependent manner. NPFF participates in the central and peripheral regulation of blood pressure (BP). The intravenous or renal subcapsular infusion (renal-selective) of NPFF in C57BL/6 mice increased BP, which was prevented by RF-9, an antagonist of NPFF receptors. In C67BL/6 mice fed a high (4%) NaCl diet, the renal subcapsular infusion of Npffr1 or Npffr2 siRNA decreased systolic BP, but urinary sodium excretion was only increased by Npffr2 siRNA. On 4% NaCl diet, the systolic BP of mice with germline deletion of Npffr2 was lower than that of wild-type littermates. In mouse and human renal proximal tubule cells, NPFF decreased intracellular sodium concentration; NPFF (100 nM) decreased the fluorescence lifetime, τ2, using Asante NaTRIUM Green-2 and fluorescence lifetime imaging microscopy. We conclude that renal NPFF, via NPFFR2, positively regulates renal sodium transport and BP. National Institutes of Health (DK119652 and DK134574) and intramural CDRF grant of George Washington University (002655) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background and Objectives: High salt intake is a major contributor to the development and exacerbation of hypertension, partly by inducing an inflammatory response through immune cell dysfunction. Inflammasomes, key components of the innate immune response, may influence blood pressure regulation. The renal DJ-1 protein is known for its antioxidant and anti-inflammatory properties. To explore novel pharmacological applications of renal DJ-1 pathway, we developed ND-13, a peptide consisting of 13 highly conserved amino acids derived from the DJ-1 sequence. In this study, we investigated the effects of ND-13 and MCC950, a specific NLRP3 inflammasome inhibitor, on blood pressure regulation in C57BL/6J mice on a high-salt diet. Methods: C57BL/6J mice were fed a high-salt diet (HS) for one week and then treated with ND-13 or MCC950, an NLRP3 inflammasome inhibitor. Subsequently, gene expression by qPCR, staining of immune cells, Sirius Red and Periodic Acid-Schiff (PAS) staining were determined in the mice kidneys, as well as the inflammasome activity in peritoneal cells. Results: One week of HS resulted increased in blood pressure, that was prevented by both ND-13 and MCC950 treatments. These treatments also prevented the increase in proteinuria that was accompanied by tubular protein deposits. Renal expression of inflammatory genes, immune cell infiltration, and renal collagen deposition were not observed in the HS group. Peritoneal macrophages isolated from HS treated mice exhibited enhanced IL-1β release upon LPS+ATP stimulation, suggesting activation of the NLRP3 inflammasome. Treatment with ND-13 and MCC950 normalized this activity. Furthermore, ND-13 reduced IL-1β mRNA expression in peritoneal macrophages. Conclusions: Our findings highlight the critical role of the NLRP3 inflammasome in salt-sensitive blood pressure regulation and suggest that ND-13 may serve as a potential therapeutic agent for preventing hypertension and associated inflammatory alterations induced by a high salt intake. ### Competing Interest Statement The authors have declared no competing interest.

Salt-sensitive hypertension is a condition in which blood pressure (BP) increases in response to an increase in a salt intake. This condition is influenced by a complex interplay among environmental, lifestyles, and genetic factors. Salt sensitivity has been shown to be associated with specific genetic variants in the human G protein-coupled receptor kinase 4γ (hGRK4γ) gene. Recent findings from our laboratory demonstrated the expression of neuropeptide FF (NPFF) and its receptors in human and mouse renal proximal tubule cells, variants of which have also been associated with increased BP. Furthermore, the renal-selective, renal subcapsular infusion of physiological concentrations of NPFF led to a decrease in urinary sodium excretion and an increase in BP. These findings suggested the involvement of this system in the regulation of blood pressure. The present study was designed to test the hypothesis that renal NPFF is involved in salt-sensitive hypertension. To this end, a study was conducted in two models of salt sensitivity in mice. The first model involved transgenic mice expressing the hGRK4γ 486 variant (hGRK4γ 486V) and their controls expressing the hGRK4 wild-type (hGRK4γ WT) transgene. The second model involved mice expressing the hGRK4γ 65L variant (hGRK4γ 65L) and their controls, expressing the hGRK4γ wild-type (hGRK4γ WT) transgene. The mice were studied after one week on a normal salt diet (NS; 0.8% NaCl) and after one week on high salt (HS; 4% NaCl) diet. BP was measured by telemetry. On NS diet hGRK4γ 486V had BPs similar to hGRK4γ WT mice while on HS the BPs were higher in hGRK4γ 486V than in hGRK4γ WT mice (124±1.5 vs 105±2.3 mm Hg; n=6-8/ group; P<0.05). On NS diet the renal NPFF mRNA expression was higher (1.5 ± 0.09-fold; P<0.05; n=3-4/group) in hGRK4γ 486V than in hGRK4γ WT mice. A change to HS diet increased NPFF mRNA expression in hGRK4γ WT (1.8±fold; P<0.05; n=6-8/group) but not in hGRK4γ 486V mice. On NS diet hGRK4γ 65L had BPs similar to hGRK4γ WT mice but on HS diet BPs were higher in hGRK4γ 65L than in hGRK4γ WT mice (120±6 vs 104±3.1 mmHg; n=4-6/ group; P<0.05). By contrast, renal NPFF expression was similar in hGRK4γ 65L and hGRK4γ WT on both diets. There were no differences in NPFF mRNA receptors expression in any of the groups. The results of the present study indicate that NPFF may be involved in the pathogenesis of salt sensitivity in hGRK4γ 486V but not in hGRK4γ 65L mice.

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 supcapsular-Drd4-siRNA-delivery 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.