AIMS:Molecular clocks in the vasculature contribute to the normal circadian blood pressure (BP) rhythm. Glucocorticoids are known to synchronize molecular clocks within peripheral tissues. Disruption of the endogenous glucocorticoid rhythm causes 'non-dipping', a BP pattern associated with elevated cardiovascular risk. The mechanisms for this are unclear but vascular changes likely contribute. We examined the effect of impaired glucocorticoid rhythmicity on the time-of-day dependent vascular function and define the circadian transcriptome under control conditions and under arrhythmic glucocorticoids. METHODS AND RESULTS:Male C57BL6J mice kept on a 12:12 h light/dark cycle were implanted with a subcutaneous slow-release pellet containing vehicle or corticosterone (∼3.7 mg/kg/day), which flattened the endogenous glucocorticoid rhythm. After 7 days, renal arteries were isolated at 7am and 7pm to measure vasoreactivity using wire myography. Other arteries were taken 2-hourly over a 48 h period for RNA sequencing. In control arteries, endothelium-dependent and independent vasodilation was elevated at wake-phase compared to the sleep-phase. This temporal variation was absent in the renal arteries from corticosterone treated mice. Using CircaCompare and LimoRhyde, we found circadian rhythms in 459 of the 14 225 protein-coding transcripts in control arteries. Following corticosterone treatment, circadian rhythmicity was no longer detected in 156 genes, including genes involved in 'peroxide homeostasis' such as Nox4, and 'TNF signaling' like Mmp14. Paradoxically 492 genes gained rhythmicity with most related to mitochondrial activity. CONCLUSION:This study expands the molecular landscape for understanding circadian vascular physiology and emphasizes the impact of glucocorticoid rhythm on temporal changes in gene expression and vascular function. This is clinically relevant to the pathogenesis of vascular dysfunction associated with perturbed glucocorticoid signaling, for example in metabolic syndrome and chronic stress.

Loss of bone mass has a devastating effect on quality of life. Higher potassium (K+) intake is positively correlated with bone health. Here, we investigated whether kidney calcium (Ca2+) and phosphate (Pi) handling mechanisms mediate dietary K+ effects. Kidney Ca2+ and Pi handling proteins were altered in abundance in mice fed a 0% K+ diet for 2 weeks. In mice fed a 0.1% K+ diet for 4 or 8 weeks, urinary Ca2+ excretion increased, plasma Ca2+ levels were lower and plasma parathyroid hormone (PTH) levels were higher relative to control 1% K+ fed mice. The 0.1% K+ fed mice had greater excretion of the bone resorption marker deoxypyridinoline, increased osteoclast number, and decreased total femoral bone mineral density. During chronic low K+ intake, major changes in renal Ca2+ and Pi transport pathways were absent, except higher abundances of the sodium-potassium-chloride cotransporter (NKCC2) and the sodium-chloride cotransporter (NCC), in line with their role in kidney Ca2+ handling. Low dietary K+ induced hypocalcemia and changes in PTH were absent in mice with constitutively active NCC, supporting its role in mediating low K+ effects on Ca2+ homeostasis. Our study provides insights into the management of bone disorders in conditions of chronic electrolyte imbalance.

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays an important role in sodium reabsorption, blood pressure and potassium homeostasis. Dietary potassium (K + ) intake modulates NCC abundance and activity, with high K + intake promoting NCC degradation and reducing BP. We hypothesized in our previous study that ubiquitin-dependent NCC degradation involves the ubiquitin E3 ligase CHIP (C-terminus of Hsc70-interacting protein)(1). In this study, our aim was to elucidate CHIP's role in NCC degradation and its impact on BP under varying dietary K + conditions. Methods: To explore CHIP’s role in regulating NCC and BP we used complementary in vivo and ex vivo experimental approaches. For in vivo studies, CHIP knockout (KO) and wildtype (WT) control mice were fed diets with varying K + content; low (0%), normal (1%), or high (5%) for five days. BP was monitored using telemetry (24 h recordings) or via tail-cuff plethysmography (early evening). To evaluate the contribution of NCC to BP regulation, mice were treated with the NCC inhibitor hydrochlorothiazide (HCTZ, 37.5 mg/kg body weight). For ex vivo studies, renal tubule suspensions isolated from WT mice were treated with a CHIP-specific inhibitor (10 µM) to evaluate its impact on NCC protein abundance. Additionally, tubules from WT and CHIP KO mice were incubated for various time points with cycloheximide and actinomycin to assess NCC half-life using immunoblotting. Results: Pharmacological CHIP inhibition in renal tubules significantly increased NCC protein levels, demonstrating a role for CHIP in NCC biogenesis. In CHIP KO mice on a normal K + diet, NCC protein abundance was significantly higher compared to WT, with no differences in NCC mRNA, suggesting the effects of CHIP on NCC are post-translational. CHIP KO mice had elevated BP that normalized with HCTZ, highlighting a role for CHIP in BP control. CHIP KO mice demonstrated an attenuated reduction in NCC abundance in response to a high K + diet. Ex vivo, NCC half-life was prolonged in tubules isolated from CHIP KO compared to WT, further confirming CHIP’s role in NCC degradation. Conclusion: Our findings highlight CHIP as a key regulator of NCC degradation, linking CHIP activity to BP regulation. Preliminary studies suggest a role for CHIP in mediating the effects of higher dietary potassium intake on NCC. Understanding the mechanisms by which CHIP regulates NCC, especially under varying dietary K + conditions, offers valuable insights into hypertension pathophysiology and may inform future therapeutic strategies targeting BP control. (1) Kortenoeven, MLA. et al., J Biol Chem 297, (2):100915 (2021). M.A. is supported by a Danish Diabetes and Endocrine Academy postdoctoral fellowship (NNF22SA0079901) 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:Potassium (K+)-deficient diets, typical of modern processed foods, increase blood pressure (BP) and NaCl sensitivity. A K+-dependent signaling pathway in the kidney distal convoluted tubule, coined the K+ switch, that couples extracellular K+ sensing to activation of the thiazide-sensitive NaCl cotransporter (NCC) and NaCl retention has been implicated, but causality has not been established. METHODS:To test the hypothesis that small, physiological changes in plasma K+ (PK+) are translated to BP through the switch pathway, a genetic approach was used to activate the downstream switch kinase, SPAK (SPS1-related proline/alanine-rich kinase), within the distal convoluted tubule. The CA-SPAK (constitutively active SPS1-related proline/alanine-rich kinase mice) were compared with control mice over a 4-day PK+ titration (3.8-5.1 mmol) induced by changes in dietary K+. Arterial BP was monitored using radiotelemetry, and renal function measurements, NCC abundance, phosphorylation, and activity were made. RESULTS:As PK+ decreased in control mice, BP progressively increased and became sensitive to dietary NaCl and hydrochlorothiazide, coincident with increased NCC phosphorylation and urinary sodium retention. By contrast, BP in CA-SPAK mice was elevated, resistant to the PK+ titration, and sensitive to hydrochlorothiazide and salt at all PK+ levels, concomitant with sustained and elevated urinary sodium retention and NCC phosphorylation and activity. Thus, genetically locking the switch on drives NaCl sensitivity and prevents the response of BP to potassium. CONCLUSIONS:Low K+, common in modern ultraprocessed diets, presses the K+-switch pathway to turn on NCC activity, increasing sodium retention, BP, and salt sensitivity.

Excessive salt intake raises blood pressure, but the implications of this observation for human health have remained contentious. It has also been recognized for many years that potassium intake may mitigate the effects of salt intake on blood pressure and possibly on outcomes such as stroke. Recent large randomized intervention trials have provided strong support for the benefits of replacing salt (NaCl) with salt substitute (75% NaCl, 25% KCl) on hard outcomes, including stroke. During the same period of time, major advances have been made in understanding how the body senses and tastes salt, and how these sensations drive intake. Additionally, new insights into the complex interactions between systems that control sodium and potassium excretion by the kidneys, and the brain have highlighted the existence of a potassium switch in the kidney distal nephron. This switch seems to contribute importantly to the blood pressure-lowering effects of potassium intake. In recognition of these evolving data, the United States Food and Drug Administration is moving to permit potassium-containing salt substitutes in food manufacturing. Given that previous attempts to reduce salt consumption have not been successful, this new approach has a chance of improving health and ending the 'Salt Wars'.

Western diets are typically high in sodium, widely regarded to increase blood pressure (BP). They are also characterised by a relatively low potassium (K) intake, also linked to higher BP. Previous studies have shown that the ability of low dietary K to increase BP is almost fully accounted for by an increase in the abundance and activity of the thiazide-sensitive sodium-chloride transporter (NCC) in the kidney distal convoluted tubule (DCT). This study aims to increase our understanding of the specific effects of low K intake on the molecular landscape of the DCT. Hypothesis: During low dietary K intake unique DCT specific mechanisms account both for the higher NCC abundance and underlie the DCT remodelling processes leading to DCT hypertrophy. Methods: Due to the relatively low abundance of DCT cells vs. whole kidney, many DCT-specific changes are undetectable when analysing whole kidney or cortex samples. To uncover DCT-specific mechanisms, Parvalbumin-GFP+ mice (GFP expressed only in DCT) were fed control (1% K) or a low K (0.2%K, n=7) diet for 4 days. Kidneys were collected on day 5, a single cell suspension prepared and GFP- and GFP+ cells separated using FACs. Cells were prepared for both bulk RNAseq and protein mass spectrometry. In parallel, kidney samples were embedded in paraffin, tubules were isolated using a laser microdissection system (LMD) and a protocol optimized to analyse these samples with mass spectrometry. Results: Compared to control diets, mice on 0.2%K intake had increased NCC (+44%) and phosphorylated NCC (+51%), and decreased expression of α-ENaC (-20%) and cleaved γ-ENaC (-10%). Mice on 0.2%K also had higher BP by tail cuff plethysmography (SBP 121.4 ± 2.1 vs. 110.8 ± 1.1 mmHg), reduced urine volume and reduced urinary K excretion (5.5-fold). Plasma K was slightly decreased compared to control diets (4.06 vs. 4.35mmol/l). FACs of living GFP+ cells was optimized to 95±2% purity and enrichment of DCT cells confirmed at protein and RNA level for DCT specific genes of interest. RNAseq and mass spectrometry studies of these samples are ongoing. For LMD samples, mass spectrometry detected ~1400 proteins from ~40 tubules. Samples from specific segments were enriched for proteins known to be expressed in these regions. Ongoing studies are addressing the proteomes of proximal tubule, DCT and collecting duct cells subsequent to 0.2% K+ intake. Perspectives: Understanding the mechanisms of K handling and their effects on BP may offer new targets for prevention or treatment of hypertension, both through drug and dietary intervention. We have developed a multiplatform approach to study DCT cells by proteomics, transcriptomics and coupled it to functional studies. We have also developed a protocol for processing LMD samples that allows for a good recovery of proteins from a low number of tubules, opening up the possibilities to process a large number of already existing samples from a range of different studies. Novo Nordisk Foundation (NNF21OC0067647), Leducq Foundation (17CVD05). This is the full abstract presented at the American Physiology Summit 2023 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.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

Increased dietary NaCl is associated with higher blood pressure (BP), but dietary potassium supplementation can reduce BP and reduce ‘salt sensitivity’ of BP, improving cardiovascular (CV) outcomes. Rodents fed a high K+ diet often exhibit reduced activity of the sodium‐chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. Recently, the effect of extreme K+ diets on rodent BP seemed inconsistent with patterns of BP modulation seen in humans. Therefore, the aim of this study was to establish how BP is altered during chronic alteration of dietary potassium intake alongside alternative anions and/or NaCl supplementation.

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In ex vivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

In general, a high NaCl intake is associated with increased blood pressure (BP), whereas dietary potassium intake has a negative correlation with BP, cardiovascular (CV) outcomes and reduces the ‘salt sensitivity’ of BP. High K+ intake reduces activity of the sodium‐chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. This study sought to establish an independent role of the anion during chronic potassium supplementation on cardiovascular parameters. Male mice implanted with telemetric BP monitors were initially normalized to a control diet of NaCl (0.3% Na+) and KCl (1.05% K+), and subsequently maintained on this control diet or a high NaCl (1.57% Na+) diet for 7 weeks. Subsequently, mice were stratified to receive either a high KCl (5.25% K+) or a high K‐citrate (KCit, 5.25% K+) diet during control or high NaCl intake for a subsequent 3 weeks. Finally, a subset of mice were switched to a zero KCl diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cllevels were monitored.Fractional excretion (FE) of K+ was significantly increased in both high K+ diets after 3 weeks and was independent of NaCl load. KCl and KCit feeding significantly increased FE of Na+ similarly but did not augment an already increased FE Na+ in mice fed high NaCl. High NaCl intake alone had no significant effect on BP, and chronic high K+ diets did not significantly reduce BP. However, overall plasma K+ levels inversely correlated with BP. Interestingly, plasma Cl− levels also inversely correlated with BP. KCl fed mice exhibited lower heart rate relative to KCit fed animals. Regression analysis points to KCit fed mice having lower BP, of about 5–10mmHg, if at the same heart rate as KCl fed animals. In contrast, 2 weeks of low K+ diet increased BP, more notably in high NaCl fed animals (BP increase of 7mmHg). NaCl‐KCit fed mice exhibited a significant 14% reduction in heart weight tibia length ratio (HW:TL) compared to NaCl‐control fed animals. No significant reduction in HW:TL was evident from NaCl‐KCl fed mice.In summary, in the timeframe of this study, potassium supplementation had limited effects on BP in normotensive mice during normal or high NaCl intake despite increasing the FE Na+. However, chronic KCit feeding may have some benefits to cardiovascular health above those promoted by KCl. The molecular mechanisms underpinning these effects are under investigation.Support or Funding InformationThis work was funded by grants from Novo Nordisk Foundation, Danish Independent Research Council and the Leducq Foundation.

Cardiovascular disease is the world's leading cause of morbidity and mortality, with high blood pressure ( BP ) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATP ase 4 ( PMCA 4) has been previously shown to modulate systemic BP . However, published data are conflicting, with both overexpression and inhibition of PMCA 4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA 4 in the regulation of BP and to further understand how PMCA 4 functionally regulates BP using a novel specific inhibitor to PMCA 4, aurintricarboxylic acid ( ATA ). Our approach assessed conscious BP and contractility of resistance arteries from PMCA 4 global knockout ( PMCA 4 KO ) mice compared to wild‐type animals. Global ablation of PMCA 4 had no significant effect on BP , arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA 4 KO mice. The effect of ATA in vivo and ex vivo was abolished by the neuronal nitric oxide synthase ( nNOS ) inhibitor Vinyl‐ l ‐ NIO . Thus, this highlights differences in the effects of PMCA 4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA 4 and that ATA may be a further experimental tool for elucidating the role of PMCA 4.

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Ischaemic cardiovascular disease is the leading cause of death worldwide. Therapeutic angiogenesis aims to stimulate the growth of new blood vessels from pre-existing ones to reperfuse ischaemic tissues. Our laboratory is characterising the molecular mechanisms that regulate activation of the calcineurin/NFAT pathway during VEGF-induced angiogenesis. We recently showed that the Plasma Membrane Calcium ATPase 4 (PMCA4) negatively regulates angiogenesis by establishing a molecular interaction with calcineurin. The identification of aurintricarboxylic acid (ATA) as an inhibitor of PMCA4 prompted us to hypothesise that ATA will enhance VEGF-induced angiogenesis. Consistent with this hypothesis, we demonstrate in this work that inhibition of PMCA4 by treatment with ATA significantly increases the activity of calcineurin/NFAT signalling, and the subsequent expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in VEGF-stimulated endothelial cells. Targeting PMCA4 with ATA significantly reduces the level of membrane-associated calcineurin, and the amount of calcineurin co-precipitated with PMCA4 in immunoprecipitation assays, indicating that ATA promotes disruption of the PMCA4/calcineurin interaction. ATA robustly enhances endothelial cell motility, and in vitro and in vivo blood vessel formation, with no harmful effects to the cells. Interestingly, incubation of HUVECs with low concentration of ATA had no effect on the viability of the cells or the development of zebra fish embryos. However, higher ATA concentrations were associated with cellular and embryo toxicity. Our study provides evidence for the therapeutic potential of targeting endothelial PMCA4 to improve VEGF-based pro-angiogenic interventions, and highlights possible clinical applications for PMCA4 inhibitors in the treatment of ischaemic cardiovascular disease.

AimsIschaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis.Methods and resultsWe show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs.ConclusionsOur study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors.

Cardiovascular diseases such as ischaemic heart disease, peripheral arterial disease and stroke are leading causes of death worldwide. Therapeutic angiogenesis, which can improve the formation of new blood vessels in the ischaemic organ, provides a valuable tool for treating cardiovascular diseases. Angiogenesis, a complex blood vessel formation process, involves the participation of several pro- and anti-angiogenic factors. Among them, pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) has been identified to play a critical role in pathological angiogenesis. Pre-clinical studies demonstrate that VEGF-based pro-angiogenic therapies result in successful reperfusion of the ischaemic organ in animal models. In our previous study, we reported a novel role for the Plasma Membrane Calcium ATPase 4 (PMCA4) as a negative regulator of angiogenic processes mediated by VEGF. Here, we hypothesised that selective inhibition of PMCA4 with the small molecule aurintricarboxylic acid (ATA) will improve VEGF-driven angiogenesis in vitro and an in vivo model of mouse limb ischaemia. Consistent with this hypothesis, we demonstrate in this work that inhibition of PMCA4 by treatment with ATA significantly increases the activity of calcineurin/NFAT pathway and the subsequent expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in VEGF-stimulated endothelial cells. Additionally, ATA treatment reduces the level of membrane-associated calcineurin, suggesting that enhancement of calcineurin signalling is the result of a disruption of the interaction between PMCA and calcineurin. Moreover, ATA treatment strongly enhances endothelial cell motility and capillary-like formation in matrigel assay. Furthermore, ATA significantly enhances MLEC motility in PMCA4 +/+ (wild type), but not in PMCA4 -/- (knock out) cells, conforming that ATA-mediated inhibition of PMCA4 is implicated in the increase of migration exerted by ATA. Importantly, long-term exposure of endothelial cells to ATA has no changes in cell viability, highlighting the potential clinical application of ATA. In this sense, Post-ischaemic reperfusion of ischaemic limbs in animals treated with ATA is significantly higher than in control-treated animals. The data from this study indicated that modulation of the activity of PMCA4 by treatment with ATA might have important clinical applications to promote blood vessel formation in human diseases associated with insufficient angiogenesis.

The incidence of hypertension, the major modifiable risk factor for cardiovascular disease, is increasing. Thus, there is a pressing need for the development of new and more effective strategies to prevent and treat hypertension. Development of these relies on a continued evolution of our understanding of the mechanisms which control blood pressure (BP). Resistance arteries are important in the regulation of total peripheral resistance and BP; changes in their structure and function are strongly associated with hypertension. Anti-hypertensives which both reduce BP and reverse changes in resistance arterial structure reduce cardiovascular risk more than therapies which reduce BP alone. Hence, identification of novel potential vascular targets which modify BP is important. Hypertension is a multifactorial disorder which may include a genetic component. Genome wide association studies have identified ATP2B1, encoding the calcium pump plasma membrane calcium ATPase 1 (PMCA1), as having a strong association with BP and hypertension. Knockdown or reduced PMCA1 expression in mice has confirmed a physiological role for PMCA1 in BP and resistance arterial regulation. Altered expression or inhibition of PMCA4 has also been shown to modulate these parameters. The mechanisms whereby PMCA1 and 4 can modulate vascular function remain to be fully elucidated but may involve regulation of intracellular calcium homeostasis and/or comprise a structural role. However, clear physiological links between PMCA and BP, coupled with experimental studies directly linking PMCA1 and 4 to changes in BP and arterial function, suggest that they may be important targets for the development of new pharmacological modulators of BP.

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

Introduction Angiogenesis, the formation of new blood vessels from pre-existing ones, is a tightly regulated process essential for proper embryonic development, organ growth and tissue repair. Changes in the expression of the pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) have been reported to play a major role in the progression of several human diseases, such as diabetic retinopathy, tumour growth and acute limb ischemia, by altering normal vascularisation. VEGF binding to specific tyrosine kinase receptors located on the surface of endothelial cells activates a variety of signal transduction pathways that switch on the expression of specific target genes. Among them, VEGF-mediated activation of the calcineurin/NFAT signalling pathway has been identified as a crucial regulator of both physiological and pathological angiogenesis. Our laboratory is interested in the characterisation of the molecular mechanisms that regulate the activity of the calcineurin/NFAT pathway during VEGF-induced angiogenesis. In this sense, we have recently identified a novel role for the Plasma Membrane Calcium ATPase 4 protein as a negative regulator of angiogenesis via interaction with calcineurin. We hypothesise that inhibition of PMCA4 will promote angiogenesis, and thus, PMCA4 inhibition might be used to induce therapeutic angiogenesis. Methods To evaluate this hypothesis we have assayed VEGF-dependent proliferation, migration and tube formation in human primary endothelial cells treated with VEGF in the presence or absence of the PMCA4-specific inhibitor aurintricarboxylic acid (ATA). Results We show here that inhibition of PMCA4 by ATA significantly increased the VEGF-induced activation of the calcineurin/NFAT pathway, and the subsequent expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in HUVEC endothelial cells. Furthermore, VEGF-triggered endothelial cell migration and tube formation was also enhanced by treatment of the cells with ATA. Interestingly, ATA had no effect on endothelial cell tubular morphogenesis in response to Fibroblast Growth Factor (a pro-angiogenic protein that induces angiogenesis in a calcineurin-independent manner). Long term incubation of endothelial cells with ATA did not alter the viability of the cells, highlighting its potential use in clinic. Examination of the effect of ATA on the activity of other endothelial molecules regulated by PMCA4 has shown that ATA reduces the phosphorylation of endothelial nitric oxide synthase (eNOS) in the regulatory residue Thr495, suggesting that the ATA-mediated effect on angiogenesis might be consequence of upregulation of several PMCA4-regulated signalling pathways. Conclusion Our results indicate that ATA might be used with therapeutic purposes to treat human diseases that occur with insufficient angiogenesis.

Introduction: Hypertension is a major risk factor for cardiac hypertrophy and heart failure. Genome wide association studies have recently identified single nucleotide polymorphisms in ATP2B1 , the gene encoding the calcium extrusion pump, plasma membrane calcium ATPase (PMCA1), as having a strong association with hypertension risk. Hypothesis: PMCA1 plays an important role in regulation of blood pressure and protection against hypertension and cardiac hypertrophy. Aims: We aim to examine whether there is a functional link between PMCA1 and blood pressure regulation, and the development of hypertension. And to determine the impact this link may have on cardiac structure and function. Methods and Results: To study the role of PMCA1 we generated a global PMCA1 heterozygous knockout mouse (PMCA1 Ht ). PMCA1 Ht mice had 46% to 52% reduction in PMCA1 protein expression compared to the WT, in aorta, heart, kidney and brain. To study the mice under hypertensive stress conditions, 3 month old PMCA1 Ht and wild type (WT) mice were infused via minipump with angiotensin II (1mg/Kg/daily) or water as a control. Upon angiotensin treatment, PMCA1 Ht mice showed a significantly greater increase in systolic (62.24±3.05 mmHg) and diastolic pressure (52.68±4.67 mmHg), in comparison to the WT (33.37±2.91 mmHg and 23.94±4.56 mmHg, respectively), P<0.001, n=12. Moreover, PMCA1 Ht mice showed a significantly greater hypertrophic response as indicated by a greater heart weight to tibia length ratio, cardiomyocyte cell size (410±18.7 μm 2 ), compared to WT mice (340.4±9.8 μm 2 ), and increased expression of B-type natriuretic peptide (BNP), 2.36 ± 0.25 fold change, n =5-6, P< 0.01. Echocardiography showed no significant changes between PMCA1 Ht and WT mice, in heart rate, and in cardiac function, as indicated by fractional shortening and ejection fraction. In addition, PMCA1 Ht mice showed no sign of lung congestion as indicated by lung weight to body weight ratio. Conclusion: ATP2B1 deletion leads to increased blood pressure and cardiac hypertrophy. This provides functional evidence that PMCA1 is involved in blood pressure regulation and protects against the development of hypertension and cardiac hypertrophy.