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.

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.

The renin-angiotensin and endothelin systems regulate blood pressure, in part, by affecting renal tubular sodium transport. In rodents, ETB receptors decrease proximal tubular reabsorption, whereas AT1 receptors produce the opposite effect. We hypothesize that ETB and AT1 receptors interact at the receptor level, and that the interaction is altered in spontaneously hypertensive rats (SHRs). In immortalized renal proximal tubule (RPT) cells from Wistar-Kyoto (WKY) rats, angiotensin II, via AT1 receptors, increased ETB receptor protein in a timeand concentration-dependent manner. In contrast, in SHR RPT cells, angiotensin II (10 8 M/24 hours) had no effect on ETB receptor protein. AT1/ETB receptors colocalized and co-immunoprecipitated in both rat strains but long-term angiotensin II (10 8 M/24 hours) treatment increased AT1/ETB co-immunoprecipitation in WKY but not in SHR cells. Short-term angiotensin II (10 8 M/15 minutes) treatment decreased ETB receptor phosphorylation in both WKY and SHR cells, and increased ETB receptors in RPT cell surface membranes of RPT cells in WKY but not SHRs. Basal cell surface membrane ETB receptor expression was also higher in WKY than in SHRs. We conclude that AT1 receptors regulate ETB receptors by receptor interaction and modulation of receptor expression. The altered AT1 receptor regulation of ETB receptors in SHRs may play a role in the pathogenesis of hypertension. (Hypertension. 2005;46[part 2]:926-931.)

Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.

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.

The renal inflammatory response can be modulated by the dopamine D2 receptor (D2R) subtype. The DRD2 gene is highly polymorphic in humans, and single nucleotide polymorphisms (SNPs), such as rs6276 and rs6277 ( DRD2 SNPs), decrease its expression and function. We hypothesized that the response of human renal proximal tubular cells (hRPTCs) to hormones in the culture medium differs by sex and DRD2 SNPs, leading to differences in the inflammatory response. We determined the effects of dihydrotestosterone (DHT) and estradiol (ES) on hRPTCs genotyped for the presence or absence of DRD2 SNPs. We studied four cell lines from males (M) and females (F) with wild-type (WT) DRD2 (M- DRD2 WT and F- DRD2 WT) and DRD2 SNPs (M- DRD2 SNPs and F- DRD2 SNPs). The cells were cultured in a medium containing charcoal-stripped fetal bovine serum (FBS) for 24 hours, without (control group, CG) and with dihydrotestosterone (DHT, 5 nM) or estradiol (ES, 20 nM). We quantified the renal protein expression (protein of interest/GAPDH) of proinflammatory and profibrotic factors and markers of injury and proliferation. DHT decreased the expression of transforming growth factor (TGF-β) by ≍50% in both M- DRD2 WT (0.4±0.1 vs. 1.0±0.2) and M- DRD2 SNPs (1.0±0.1 vs. 1.6±0.1) (n=5-6, p<0.05). However, there was no significant effect of DHT or ES on the expression of TGF-β in F- DRD2 WT and F- DRD2 SNPs. DHT did not affect the fibronectin 1 (FN1) expression in M- DRD2 WT and M- DRD2 SNPs. In F- DRD2 WT, DHT did not affect FN1 expression but decreased in F- DRD2 SNPs (0.9±0.1 vs. 1.7±0.1, n=6, p<0.05). Similar to DHT, ES did not affect FN1 expression in M- DRD2 WT. However, ES decreased FN1 by 50% in M- DRD2 SNPs (0.5±0.1 vs. 1.1±0.1, n=6, p<0.05). Similar to the studies in males, ES did not affect FN1 expression in F- DRD2 WT but also decreased FN1 (1.0±0.1 vs. 1.7±0.1, n=6, p<0.05) in F-DRD2 SNPs. Neither DHT nor ES affected the expression of Kidney Injury Molecule-1 (KIM-1) in M- DRD2 WT. However, in M- DRD2 SNPs, both DHT and ES decreased KIM-1 (1.4±0.2 vs. 1.8±0.2 and 1.1±0.1 vs. 1.8±0.2, respectively; n=5, p<0.05). Unlike in M- DRD2-WT, in F- DRD2 WT, ES increased KIM-1 expression (1.0±0.2 vs. 0.52±0.05, n=5, p<0.05), while DHT had no significant effect. Unlike in M- DRD2 SNPs, where both DHT and ES decreased KIM-1, in F- DRD2 SNPs, neither DHT nor ES affected KIM-1. DHT did not affect the expression of Ki67 in M- DRD2 WT. However, in M- DRD2 SNPs, DHT decreased Ki67 expression (0.60±0.08 vs. 1.6±0.2, n=6, p<0.05). Similar to M- DRD2-WT, DHT did not affect Ki67 expression in F- DRD2-WT. ES also did not affect Ki67 expression in M- DRD2 WT but decreased Ki67 expression in M-DRD2 SNPs (0.91± 0.18 vs. 1.6±0.2, n=6, p<0.05). Similar to the male data, the treatment with ES did not affect the Ki67 expression in F- DRD2 WT but decreased Ki67 expression in F- DRD2 SNPs (1.5±0.2 vs. 0.9±0.1, n=6, p<0.05). Our results demonstrate similarities (FN1 and Ki67) and differences (TGF-β and KIM-1) in profibrotic and proliferation markers expressed in hRPTCs between males and females. These differences are more pronounced in cell lines expressing DRD2 SNPs than those expressing the DRD2 WT. While both DHT and ES decreased KIM-1 expression in males with DRD2 polymorphisms, they had no effect in females with DRD2 polymorphisms. These results indicate a complex interaction of the effect of hormones and DRD2 polymorphisms in regulating the inflammatory response in hRPTCs. Their roles in regulating renal physiology and blood pressure remain to be defined. R01 DK039308 P01 HL068686 R01 HL023081 R37 HL023081 R01 DK119652/ NIH HH/ US. 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.

Background: The scaffolding protein, caveolin-1 (Cav-1), participates in multiple cellular functions including promotion of sodium excretion from the kidney. Loss of expression of Cav-1 is associated with tumorigenesis of various types of cancer. We have shown the potential link between hypertension and breast cancer via abnormal function of the G protein–coupled receptor kinase type 4 (GRK4). Objective: The current studies tested the hypothesis that Cav-1 acts as a tumor-suppressive factor in breast cancer cells and enhances the sensitivity to the inhibitory effect of the type 1 dopaminergic receptor (D 1 R). Methods: Michigan Cancer Foundation (MCF) MCF-7 cells stably expressing a Cav-1/mCherry fusion protein or mCherry alone were used as models to examine the effect of Cav-1 on cell growth, apoptosis, and senescence. Cell proliferation was determined by cell counting, cell cycle analysis (flow cytometry), and BrdU incorporation. Apoptosis was determined using the Cell Death Detection ELISA kit from Roche Diagnosis. Senescence was determined using the senescence associated beta galactosidase (SA-β-gal) assay. Reactive oxygen species (ROS) was measured using 2′,7′-dichlorodihydrofluorescein diacetate. Western blot analysis was used to measure activation of signaling pathway molecules. All statistical analyses were conducted with Microsoft Excel. Results: Overexpression of Cav-1 in MCF-7 cells reduced cellular growth rate. Both inhibition of proliferation and induction of cell death are contributing factors. Multiple signaling pathways were activated in Cav-1-expressing MCF-7 cells. Activation of Akt was prominent. In MCF-7-expressing Cav-1 (MCF-7 Cav-1) cells, the levels of phosphorylated Akt at S 473 and T 308 were increased 28- and 8.7-fold, respectively. Instead of protecting cells from apoptosis, extremely high levels of activated Akt resulted in increased levels of ROS which led to apoptosis and senescence. The tumor-suppressive effect plus downregulation of GRK4 makes Cav-1-expressing MCF-7 cells significantly more sensitive to the inhibitory effect of the D 1 R agonist, SKF38393. Conclusion: Caveolin-1 acts as a tumor-suppressing factor via extreme activation of Akt and down regulation of survival factors such as GRK4, survivin, and cyclin D1.

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.

The kidney is critical in the overall regulation of fluid and electrolyte balance and blood pressure. Neuropeptide FF (NPFF), a morphine-modulating peptide, regulates cardiovascular function through its interaction with two receptors, NPFFR1 and NPFFR2. We now report that NPFF and its receptors, mainly NPFFR2, are expressed in the kidney. NPFF (9.25 mmol, 0.5 mL/hr, 0.05 nmol/day, 7 days) chronically infused underneath the renal capsule, decreased renal sodium excretion (UNaV) from 0.68±0.07 mEq/day, n=5 to 0.43±0.06 mEq/day (n=6/group) in conscious C57BL/6 mice. This was accompanied by an increase in systolic blood pressure (SBP, 114.5±5.0 mm Hg, n=4); the infusion of vehicle (saline) did not affect the blood pressure (96.4±3.0 mm Hg, n=4). The renal subcapsular injection of a single dose (10 µg in 100 µL) of NPFF also increased SBP within 15-35 min (NPFF: 105.5±3.44 mm Hg, n=6); saline injection did not affect SBP (82.6±5.65 mm Hg, n=4). RF-9 (10 µg in 100 µL), an antagonist of NPFF receptors prevented the NPFF-mediated increase in SBP (NPFF alone: 105.5±3.44 mm Hg, n=6; RF+NPFF: 88.5±5.90 mm Hg, n=5), whereas RF-9, alone, had no effect (Baseline: 97.6±2.70 mmHg; RF-9: 90.9±4.70 mm Hg, n=5). The renal subcapsular injection of scrambled peptide (10 µg in 100 µL) had no effect on SBP (86.1±1.83 mm Hg, n=4). However, SBP was decreased by the renal subcapsular infusion of Npffr2 siRNA (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) in C57Bl/6 mice fed with a high salt diet (4% NaCl). Furthermore, the SBP of conventional germline Npffr2 knockout (KO) mice fed a high salt (4% NaCl) diet was lower than the SBP of wild-type (WT) littermates (WT: 105.6±2.9 mm Hg, n=5; KO: 90.5±5.5 mm Hg, n=4, P < 0.05). By contrast, in mice fed normal salt (0.8% NaCl) diet, SBP was not different between Npffr2 KO mice and WT littermates (WT: 96.5±4.5 mm Hg, n=5; KO: 98.7±11.7 mm Hg, n=4, P > 0.05). Taken together, NPFF and its receptor, NPFFR2, in the kidney cause salt-sensitive hypertension in mice. Uncovering the functional relevance of renal NPFF in the dynamic regulation of renal sodium transport will lead to a better understanding of blood pressure homeostasis.

Globally, hypertension is the number one risk factor for death, affecting more than 1 billion people. Hypertension is the result of the interactions among genetics, epigenetics, environment, and lifestyle. The long-term regulation of blood pressure rests on renal and non-renal mechanisms. The impaired renal sodium handling in hypertension is caused by aberrant counter-regulatory natriuretic/anti-natriuretic pathways. The sympathetic nervous and renin-angiotensin systems are anti-natriuretic pathways. A counter-regulatory natriuretic pathway is the renal dopaminergic system. Aberrant dopaminergic regulation of renal sodium transport in hypertension is caused by a decrease in renal dopamine synthesis and/or dysfunction of any of the 5 dopamine receptors (D1R, D2R, D3R, D4R, & D5R). Normally, an increase in sodium intake increases while a decrease in sodium intake decreases blood pressure, albeit transiently until sodium balance is achieved. However, ~50 % of hypertensive and ~26% of normotensive subjects have increased blood pressure on high sodium intake, a case of salt sensitivity, while ~20 % have increased blood pressure on a low sodium intake, a case of inverse salt sensitivity. Low and high sodium intakes are associated with increased incidence of cardiovascular events/mortality. In humans with inverse salt sensitivity, there is a linear relationship between the number of single nucleotide polymorphisms in DRD2 (rs6276 and 6277) and decreased renal D2R expression. The increase in blood pressure on a low sodium diet may be due to increased activities of the renin-angiotensin and sympathetic nervous systems that cannot be counteracted by D2R. Hypertension may be a cause or consequence of inflammation or oxidative stress. Deficient D2R function causes renal inflammation independently of the increase in blood pressure. Subjects carrying DRD2 single nucleotide polymorphisms have increased inflammation, mediated by decreased regulation of the miR-217-Wnt5a-Ror2 pathway. The D2R, via paraoxonase2 and sestrin2, maintains normal redox balance and blood pressure. In summary, the D2R is important in the maintenance of normal blood pressure by regulating renal sodium transport, vascular reactivity, inflammation, and redox balance.

Angiotensin II type-2 receptor (AT 2 R) stimulation with Ang III leads to the dopamine D1 Receptor (D 1 R) plasma membrane recruitment in normally cAMP coupled human renal proximal tubule cells (nRPTC). While extending these studies to include D 1 R cAMP uncoupled (uRPTC), we determined that Ang III stimulation did not lead to D 1 R plasma membrane recruitment. We then hypothesized that local production and secretion of dopamine may be stimulated by sodium stress as well as in this AT 2 R to D 1 R recruitment pathway. A stable clone of Dlight 1.3 HEK293 cell line expressing the GFP dopamine biosensor functioned properly through multiple titration experiments using fenoldopam (D 1 R/D 5 R agonist) direct binding, showing dose dependent increase in the Dlight biosensor fluorescence, as well as a dose dependent increase and decrease of intracellular dopamine production in high or low sodium conditions respectively. Dopamine secretion was examined in a co-culture experiment using a stable clone of D 5 R and ICUE3 cAMP reporter HEK293 cell line. Local extracellular dopamine produces a change in the FRET biosensor. Both nRPTCs and uRPTCs make and secrete dopamine and this is increased further with incubation with L-Dopa. Interestingly 8Br-cGMP lead to an increase in dopamine secretion only in nRPTCs by 18.28±3.2 fold FRET nRPTC vs uRPTC (p<0.001, N=9 per group). Further experiments examined autocrine D 1 R signaling second messenger cAMP levels after Ang III stimulation at differing sodium levels. Only nRPTCs produced more cAMP (8.53±0.73 fold increase vs NS VEH control, p<0.01, N=8 per group) when stimulated with Ang III in high sodium conditions, this being from local dopamine production, secretion and D 1 R stimulation. These studies provide new insights into the autocrine dopamine production by RPTCs and D 1 R/AT 2 R trans-regulation that play a role in maintaining sodium and blood pressure homeostasis.

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.

OBJECTIVE:Macrophages are abundantly detected at sites of disc herniation, however, their function in the disease progression is unclear. We aim to investigate the functions of macrophages in acute disc herniation using a macrophage Fas-induced apoptosis (MaFIA) transgenic mouse strain. METHOD:To transiently deplete macrophages, a dimerizer, AP20187, or vehicle solution was administered via intraperitoneal injection to MaFIA mice immediately, day 1 and 2 after annular puncture induced disc herniation. Local infiltrated tissues at disc hernia and DRGs at corresponding levels were harvested to analyze immune cells and neuroinflammation on postoperative day (POD) 6 by flow cytometry and/or immunostaining. Mouse spines were harvested to analyze structures of degenerated discs and adjacent vertebrae and to assess osteoclast activity by histology and tartrate-resistant acid phosphatase (TRAP) staining on POD 6, 13, and 20, respectively. RESULTS:On POD 6, abundant macrophages were confirmed at disc hernia sites. Compared to vehicle control, AP20187 significantly reduced GFP+ cells in blood, spleen, and local inflammatory tissue. At disc hernia sites, AP20187 markedly reduced macrophages (CD11b+, F4/80+, GFP+CD11b+, CD11b+F4/80+) while increasing neutrophils and B cells. Transient macrophage depletion decreased ectopic bone formation and osteoclast activity in herniated discs and adjacent cortical bones for up to 20 days post herniation. Disc herniation elevated expressions of TNF-α, IL-6, substance P, calcitonin gene-related peptide, accompanied by increasing GFP+, CD11b+ and F4/80+ macrophages. Macrophage depletion did not attenuate these markers of neuroinflammation. CONCLUSIONS:Transient depletion of macrophages altered local inflammatory response at the site of disc herniation.

Peroxiredoxin-4 (PRDX4), an endoplasmic reticulum peroxiredoxin protein, plays a protective role against oxidative stress and inflammation by reducing hydrogen peroxide to water. The dopamine D5 receptor (D 5 R) is also important in protecting against oxidative stress, but the interaction between PRDX4 and D 5 R in regulating oxidative stress in the kidney is not known. In D 5 R-HEK 293 cells, fenoldopam (FEN, 25 nM/12 hr, n=4), a D1-likereceptor agonist, increased PRDX4 protein expression (1.92±0.12-fold over basal level, n=4), mainly in non-lipid rafts (LRs: 24.9±11.4%, non-LRs: 75.1±11.4%, baseline; LRs: 30.9±13.9%, non-LRs: 174.1±16.7%, FEN). FEN also increased the co-immunoprecipitation of D 5 R and PRDX4 and their colocalization, particularly in the endoplasmic reticulum. In human renal proximal tubule cells (hRPTCs), FEN (25 nM/12 hr, n=3) increased PRDX4 and D 5 R interaction in non-LRs, also. Si-RNA silencing of PRDX4 increased reactive oxygen species (ROS) production and impaired the inhibitory effect of FEN on ROS production (scrambled siRNA: 100.0±11.6% and 65.4±5.6% for Vehicle (Veh) and FEN, respectively; PRDX4 siRNA: 147.7±11.8% and 134.8±11.2% for Veh and FEN, respectively, n=4/group) detected by Amplex Red. In addition in both D 5 R-HEK 293 and hRPTCs, siRNA silencing of PRDX4 increased the production of interleukin-1β (26.88±3.8 and 46.40±4.2 pg/mL [n=3, D 5 R-HEK 293]; 15.87±1.2 and 37.9±1.4 pg/mL [n=3 in hRPTCs]), tumor necrosis factor (131.7±6.5 and 271.2±18.1 pg/mL [n=4, D 5 R-HEK 293]; 108.8±11.8 and 240.1±13.7 pg/mL [n=4 in hRPTCs]), and caspase-12 (15.21±3.8 and 40.78±4.3 ng/mL [n=4,n D 5 R-HEK 293]; 8.8±1.1 and 27.9±2.0 ng/mL [n=4, hRPTCs]). Furthermore, the protein expression of D 5 R was decreased in PRDX4 siRNA-treated D 5 R-HEK293 (~41.2%, n=3) and hRPTCs (~39.6%, n=3). PRDX4 protein was also reduced in the kidney homogenates from D 5 R -/- mice (WT: 1.00±0.18, n=5; D 5 R -/- : 0.686±0.14, n=4; P<0.05). Taken together, PRDX4 interacts with D 5 R to decrease oxidative stress and inflammation in the kidney.

Clozapine (CLZ) is the most effective antipsychotic for treatment-resistant schizophrenia which affects approximately 24 million people worldwide. Schizophrenics are 3-4 times more likely to die from cardiovascular disease due to increased incidence of hypertension. CLZ is a high affinity antagonist for the dopamine type 4 receptor (D 4 R). Studies have shown that germline deletion of D 4 R in mice caused hypertension, and CLZ administered subcutaneously in mice induced hypertension. Since the proximal tubule (PT) regulates about 75% of excreted Na + we cultured renal proximal tubule cells excreted in the urine (uRPTCs) from individuals treated with and without clozapine and previously found that CLZ decreased D 4 R recruitment to cell surface and increased reactive oxygen species in healthy controls (HC). Recently, western blot analysis showed D 4 R expression was reduced in CLZ treated patients (CLZ 0.85±0.08 n=12 vs HC 1.09±0.09 n=11, p<0.05 t-test). The total D 4 R protein was also decreased under CLZ treatment in RPTCs from HC. This suggests that the reduction in D 4 R occurred in PTs when treated with CLZ in-vivo but this was a durable effect still measurable through the process of culturing the cells, and is replicated in-vitro using healthy control cells. In In-vivo CLZ treated patient cells, D 4 R expression is not further reduced when treated in-vitro. Moreover, the natriuretic dopamine D1 receptor (D 1 R) was significantly increased under ClZ treatment of uRPTCs from HC, but not RPTCs from CLZ treated patients (VEH 0.37±0.07 vs CLZ 0.5±0.08 n=11, p<0.05 paired t-test; CLZ/VEH, HC 1.51±0.18 n=11 vs CLZ 0.99±0.01 n=12, p<0.05 t-test). Interestingly, high salt (HS) treatment on these uRPTCs showed a similar trend with CLZ treatment (VEH 0.33±0.06 vs CLZ 0.47±0.08, n=10, p<0.05 paired t-test; HS/VEH, HC 1.55±0.29 n=11 vs CLZ 0.99±0.01 n=12, p<0.05 t-test), indicating these CLZ treated patients may have a blunted dopamine response to excess sodium and therefore develop salt sensitive hypertension. In Summary, CLZ induced hypertension in these schizophrenia patients may be caused by a disrupted dopaminergic system. Further investigations also need to be done including response of sodium transporters and modulation of the renin-angiotensin system.