Water Conservation Overrides Osmotic Diuresis During SGLT2 Inhibition in Patients With Heart Failure.

BACKGROUND: Sodium-glucose cotransporter 2 inhibitors are believed to improve cardiac outcomes due to their osmotic diuretic potential. OBJECTIVES: The goal of this study was to test the hypothesis that vasopressin-driven urine concentration overrides the osmotic diuretic effect of glucosuria induced by dapagliflozin treatment. METHODS: DAPA-Shuttle1 (Hepato-renal Regulation of Water Conservation in Heart Failure Patients With SGLT-2 Inhibitor Treatment) was a single-center, double-blind, randomized, placebo-controlled trial, in which patients with chronic heart failure NYHA functional classes I/II and reduced ejection fraction were randomly assigned to receive dapagliflozin 10 mg daily or placebo (1:1) for 4 weeks. The primary endpoint was change from baseline in urine osmolyte concentration. Secondary endpoints included changes in copeptin levels and solute free water clearance. RESULTS: Thirty-three randomized, sodium-glucose cotransporter 2 inhibitor-naïve participants completed the study, 29 of whom (placebo: n = 14; dapagliflozin: n = 15) provided accurate 24-hour urine collections (mean age 59 ± 14 years; left ventricular ejection fraction 31% ± 9%). Dapagliflozin treatment led to an isolated increase in urine glucose excretion by 3.3 mmol/kg/d (95% CI: 2.51-4.04; P < 0.0001) within 48 hours (early) which persisted after 4 weeks (late; 2.7 mmol/kg/d [95% CI: 1.98-3.51]; P < 0.0001). Dapagliflozin treatment increased serum copeptin early (5.5 pmol/L [95% CI: 0.45-10.5]; P < 0.05) and late (7.8 pmol/L [95% CI: 2.77-12.81]; P < 0.01), leading to proportional reductions in free water clearance (early: -9.1 mL/kg/d [95% CI: -14 to -4.12; P < 0.001]; late: -11.0 mL/kg/d [95% CI: -15.94 to -6.07; P < 0.0001]) and elevated urine concentrations (late: 134 mmol/L [95% CI: 39.28-229.12]; P < 0.01). Therefore, urine volume did not significantly increase with dapagliflozin (mean difference early: 2.8 mL/kg/d [95% CI: -1.97 to 7.48; P = 0.25]; mean difference late: 0.9 mL/kg/d [95% CI: -3.83 to 5.62]; P = 0.70). CONCLUSIONS: Physiological-adaptive water conservation eliminated the expected osmotic diuretic potential of dapagliflozin and thereby prevented a glucose-driven increase in urine volume of approximately 10 mL/kg/d · 75 kg = 750 mL/kg/d. (Hepato-renal Regulation of Water Conservation in Heart Failure Patients With SGLT-2 Inhibitor Treatment [DAPA-Shuttle1]; NCT04080518).

KCNQ5 Controls Perivascular Adipose Tissue-Mediated Vasodilation.

BACKGROUND: Small arteries exhibit resting tone, a partially contracted state that maintains arterial blood pressure. In arterial smooth muscle cells, potassium channels control contraction and relaxation. Perivascular adipose tissue (PVAT) has been shown to exert anticontractile effects on the blood vessels. However, the mechanisms by which PVAT signals small arteries, and their relevance remain largely unknown. We aimed to uncover key molecular components in adipose-vascular coupling. METHODS: A wide spectrum of genetic mouse models targeting Kcnq3, Kcnq4, and Kcnq5 genes (Kcnq3-/-, Kcnq4-/-, Kcnq5-/-, Kcnq5dn/dn, Kcnq4-/-/Kcnq5dn/dn, and Kcnq4-/-/Kcnq5-/-), telemetry blood pressure measurements, targeted lipidomics, RNA-Seq profiling, wire-myography, patch-clamp, and sharp-electrode membrane potential measurements was used. RESULTS: We show that PVAT causes smooth muscle cell KV7.5 family of voltage-gated potassium (K+) channels to hyperpolarize the membrane potential. This effect relaxes small arteries and regulates blood pressure. Oxygenation of polyunsaturated fats generates oxylipins, a superclass of lipid mediators. We identified numerous oxylipins released by PVAT, which potentiate vasodilatory action in small arteries by opening smooth muscle cell KV7.5 family of voltage-gated potassium (K+) channels. CONCLUSIONS: Our results reveal a key molecular function of the KV7.5 family of voltage-gated potassium (K+) channels in the adipose-vascular coupling, translating PVAT signals, particularly oxylipins, to the central physiological function of vasoregulation. This novel pathway opens new therapeutic perspectives.

ADAM19 cleaves the PTH receptor and associates with brachydactyly type E.

Brachydactyly type E (BDE), shortened metacarpals, metatarsals, cone-shaped epiphyses, and short stature commonly occurs as a sole phenotype. Parathyroid hormone-like protein (PTHrP) has been shown to be responsible in all forms to date, either directly or indirectly. We used linkage and then whole genome sequencing in a small pedigree, to elucidate BDE and identified a truncated disintegrin-and-metalloproteinase-19 (ADAM19) allele in all affected family members, but not in nonaffected persons. Since we had shown earlier that the extracellular domain of the parathyroid hormone receptor (PTHR1) is subject to an unidentified metalloproteinase cleavage, we tested the hypothesis that ADAM19 is a sheddase for PTHR1. WT ADAM19 cleaved PTHR1, while mutated ADAM-19 did not. We mapped the cleavage site that we verified with mass spectrometry between amino acids 64-65. ADAM-19 cleavage increased Gq and decreased Gs activation. Moreover, perturbed PTHR1 cleavage by ADAM19 increased ß-arrestin2 recruitment, while cAMP accumulation was not altered. We suggest that ADAM19 serves as a regulatory element for PTHR1 and could be responsible for BDE. This sheddase may affect other PTHrP or PTH-related functions.

Protocol for assessing myogenic tone and perfusion pressure in isolated mouse kidneys.

The isolated perfused kidney is a classic ex vivo preparation for studying renal physiology in general and vascular function. Here, we present a protocol for assessing myogenic tone in isolated mouse kidneys as well as vasodilatory and vasoconstrictive responses, expressed as perfusion pressure. We describe steps for pre-operative preparation, kidney and renal artery isolation, and connection of renal artery with glass cannula. We then detail how to measure pressure changes in perfused kidneys and the myogenic tone. For complete details on the use and execution of this protocol, please refer to Cui et al.1.

Calcineurin inhibition, cardiovascular consequences, vascular resistance, and potential responses.

AIM: To place the consequences of calcineurin inhibition in a cardiovascular context. METHODS: Literature review coupled with personal encounters. RESULTS: Calcineurin is a calcium-binding and calmodulin-binding protein that is conserved across evolution from yeast to mammals. The enzyme functions as a calcium-dependent, calmodulin-stimulated protein phosphatase. Its role in regulating physiology has largely been elucidated by observing calcineurin inhibition. Calcineurin inhibition transformed organ transplantation from an experiment into a therapy and made much of general immunotherapy possible. The function of this phosphatase and how its inhibition leads to toxicity concern us to this date. Initial research from patients and animal models implicated a panoply of factors contributing to hypertension and vasculopathy. Subsequently, the role of calcineurin in regulating the effective fluid volume, sodium reabsorption, and potassium and hydrogen ion excretion was elucidated by investigating calcineurin inhibition. Understanding the regulatory effects of calcineurin on endothelial and vascular smooth muscle cell function has also made substantial progress. However, precisely how the increase in systemic vascular resistance arises requires further mechanistic research. CONCLUSION: Calcineurin inhibition continues to save lives; however, options to counteract the negative effects of calcineurin inhibition should be vigorously pursued.

Sodium as an Important Regulator of Immunometabolism.

Salt sensitivity concerns blood pressure alterations after a change in salt intake (sodium chloride). The heart is a pump, and vessels are tubes; sodium can affect both. A high salt intake increases cardiac output, promotes vascular dysfunction and capillary rarefaction, and chronically leads to increased systemic vascular resistance. More recent findings suggest that sodium also acts as an important second messenger regulating energy metabolism and cellular functions. Besides endothelial cells and fibroblasts, sodium also affects innate and adaptive immunometabolism, immune cell function, and influences certain microbes and microbiota-derived metabolites. We propose the idea that the definition of salt sensitivity should be expanded beyond high blood pressure to cellular and molecular salt sensitivity.

Sixty Years of Confronting Diabetes and Kidney Disease.

The good old days were not good, at least in terms of treating patients with type 2 diabetes. In the 1960s, the development of a radioimmunoassay for insulin permitted determination of the distinguishing features of type 1 and type 2 diabetes. The latter was treated with sulfonylureas and then phenformin, although the mechanisms of action at the time were unknown. The University Group Diabetes Program was a randomized controlled trial experienced by my medical generation, and the results were dramatic, both medically and legally. Next came the thiazolidinediones. All compounds were associated with weight gain and any end point benefits were uncertain. Nevertheless, basic science explained how glucose is sensed and even found a home for sulfonylureas in some patients. Next came the boom in renin-angiotensin-aldosterone system blockade, sacred ground for many, albeit the benefits were less than astounding. Other wonder drugs came and went. Over the decades, great strides were made in defining the pathology of diabetic renal disease, which is appropriate because the condition has become a primary cause of end-stage renal failure. Nonetheless, recent advances have turned around a depressing situation and are reasons for optimism. We now have compounds that actually could help patients with type 2 diabetes. One hundred years after insulin's introduction, it is high time.

Effect of cardiopulmonary bypass on plasma and erythrocytes oxylipins.

BACKGROUND: Oxylipins, the oxidative metabolites of polyunsaturated fatty acids (PUFAs), serve as key mediators of oxidative stress, inflammatory responses, and vasoactive reactions in vivo. Our previous work has established that hemodialysis affects both long chain fatty acids (LCFAs) and oxylipins in plasma and erythrocytes to varying degrees, which may be responsible for excess cardiovascular complications in end-stage renal disease. In this study, we aimed to determine changes in blood oxylipins during cardiopulmonary bypass (CPB) in patients undergoing cardiac surgery to identify novel biomarkers and potential metabolites of CPB-related complications. We tested the hypothesis that CPB would differentially affect plasma oxylipins and erythrocytes oxylipins. METHODS: We conducted a prospective observational study of 12 patients undergoing elective cardiac surgery with expected CPB procedure. We collected venous and arterial blood samples before CPB, 15 and 45 min after the start of CPB, and 60 min after the end of CPB, respectively. Oxylipins profiling in plasma and erythrocytes was achieved using targeted HPLC-MS mass spectrometry. RESULTS: Our results revealed that most venous plasma diols and hydroxy- oxylipins decreased after CPB initiation, with a continuous decline until the termination of CPB. Nevertheless, no statistically significant alterations were detected in erythrocytes oxylipins at all time points. CONCLUSIONS: CPB decreases numerous diols and hydroxy oxylipins in blood plasma, whereas no changes in erythrocytes oxylipins are observed during this procedure in patients undergoing cardiac surgery. As lipid mediators primarily responsive to CPB, plasma diols and hydroxy oxylipins may serve as potential key biomarkers for CPB-related complications.

High tissue-sodium associates with systemic inflammation and insulin resistance in obese individuals.

BACKGROUND AND AIMS: High sodium intake is associated with obesity and insulin resistance, and high extracellular sodium content may induce systemic inflammation, leading to cardiovascular disease. In this study, we aim to investigate whether high tissue sodium accumulation relates with obesity-related insulin resistance and whether the pro-inflammatory effects of excess tissue sodium accumulation may contribute to such association. METHODS AND RESULTS: In a cross-sectional study of 30 obese and 53 non-obese subjects, we measured insulin sensitivity determined as glucose disposal rate (GDR) using hyperinsulinemic euglycemic clamp, and tissue sodium content using 23Na magnetic resonance imaging. Median age was 48 years, 68% were female and 41% were African American. Median (interquartile range) BMI was 33 (31.5, 36.3) and 25 (23.5, 27.2) kg/m2 in the obese and non-obese individuals, respectively. In obese individuals, insulin sensitivity negatively correlated with muscle (r = -0.45, p = 0.01) and skin sodium (r = -0.46, p = 0.01). In interaction analysis among obese individuals, tissue sodium had a greater effect on insulin sensitivity at higher levels of high-sensitivity C-reactive protein (p-interaction = 0.03 and 0.01 for muscle and skin Na+, respectively) and interleukin-6 (p-interaction = 0.024 and 0.003 for muscle and skin Na+, respectively). In interaction analysis of the entire cohort, the association between muscle sodium and insulin sensitivity was stronger with increasing levels of serum leptin (p-interaction = 0.01). CONCLUSIONS: Higher muscle and skin sodium are associated with insulin resistance in obese patients. Whether high tissue sodium accumulation has a mechanistic role in the development of obesity-related insulin resistance through systemic inflammation and leptin dysregulation remains to be examined in future studies. CLINICALTRIALS: gov registration: NCT02236520.

SPSB1-mediated inhibition of TGF-ß receptor-II impairs myogenesis in inflammation.

BACKGROUND: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-ß) is involved in both processes. We uncovered an increased expression of the TGF-ß receptor II (TßRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TßRII signalling impairs myogenic differentiation in response to inflammation. METHODS: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-ß/TßRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. RESULTS: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1ß (IL-1ß), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1ß-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TßRII, resulting in TßRII ubiquitination and destabilization. SPSB1 impaired TßRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. CONCLUSIONS: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TßRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.

Corrigendum: Exercise blood-drop metabolic profiling links metabolism with perceived exertion.

[This corrects the article DOI: 10.3389/fmolb.2022.1042231.].

SLC26A1 is a major determinant of sulfate homeostasis in humans.

Sulfate plays a pivotal role in numerous physiological processes in the human body, including bone and cartilage health. A role of the anion transporter SLC26A1 (Sat1) for sulfate reabsorption in the kidney is supported by the observation of hyposulfatemia and hypersulfaturia in Slc26a1-knockout mice. The impact of SLC26A1 on sulfate homeostasis in humans remains to be defined. By combining clinical genetics, functional expression assays, and population exome analysis, we identify SLC26A1 as a sulfate transporter in humans and experimentally validate several loss-of-function alleles. Whole-exome sequencing from a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in SLC26A1, which has not been previously described to the best of our knowledge. Whole-exome data analysis of more than 5,000 individuals confirmed that rare, putatively damaging SCL26A1 variants were significantly associated with lower plasma sulfate at the population level. Functional expression assays confirmed a substantial reduction in sulfate transport for the SLC26A1 mutation of our patient, which we consider to be novel, as well as for the additional variants detected in the population study. In conclusion, combined evidence from 3 complementary approaches supports SLC26A1 activity as a major determinant of sulfate homeostasis in humans. In view of recent evidence linking sulfate homeostasis with back pain and intervertebral disc disorder, our study identifies SLC26A1 as a potential target for modulation of musculoskeletal health.

The Transcription Factor EB (TFEB) Sensitizes the Heart to Chronic Pressure Overload.

The transcription factor EB (TFEB) promotes protein degradation by the autophagy and lysosomal pathway (ALP) and overexpression of TFEB was suggested for the treatment of ALP-related diseases that often affect the heart. However, TFEB-mediated ALP induction may perturb cardiac stress response. We used adeno-associated viral vectors type 9 (AAV9) to overexpress TFEB (AAV9-Tfeb) or Luciferase-control (AAV9-Luc) in cardiomyocytes of 12-week-old male mice. Mice were subjected to transverse aortic constriction (TAC, 27G; AAV9-Luc: n = 9; AAV9-Tfeb: n = 14) or sham (AAV9-Luc: n = 9; AAV9-Tfeb: n = 9) surgery for 28 days. Heart morphology, echocardiography, gene expression, and protein levels were monitored. AAV9-Tfeb had no effect on cardiac structure and function in sham animals. TAC resulted in compensated left ventricular hypertrophy in AAV9-Luc mice. AAV9-Tfeb TAC mice showed a reduced LV ejection fraction and increased left ventricular diameters. Morphological, histological, and real-time PCR analyses showed increased heart weights, exaggerated fibrosis, and higher expression of stress markers and remodeling genes in AAV9-Tfeb TAC compared to AAV9-Luc TAC. RNA-sequencing, real-time PCR and Western Blot revealed a stronger ALP activation in the hearts of AAV9-Tfeb TAC mice. Cardiomyocyte-specific TFEB-overexpression promoted ALP gene expression during TAC, which was associated with heart failure. Treatment of ALP-related diseases by overexpression of TFEB warrants careful consideration.

Hemodialysis and biotransformation of erythrocyte epoxy fatty acids in peripheral tissue.

Cardiovascular disease is the leading cause of mortality in patients with renal failure. Red blood cells (RBCs) are potential reservoirs for epoxy fatty acids (oxylipins) that regulate cardiovascular function. Hemoglobin exhibits pseudo-lipoxygenase activity in vitro. We previously assessed the impact of single hemodialysis (HD) treatment on RBC epoxy fatty acids status in circulating arterial blood and found that eicosanoids in oxygenated RBCs could be particularly vulnerable in chronic kidney disease and hemodialysis. The purpose of the present study was to evaluate the differences of RBC epoxy fatty acids profiles in arterial and venous blood in vivo (AV differences) from patients treated by HD treatment. We collected arterial and venous blood samples in upper limbs from 12 end-stage renal disease (ESRD) patients (age 72±12 years) before and after HD treatment. We measured oxylipins derived from cytochrome P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω-1)-hydroxylase pathways in RBCs by LC-MS/MS tandem mass spectrometry. Our data demonstrate arteriovenous differences in LOX pathway metabolites in RBCs after dialysis, including numerous hydroxyeicosatetraenoic acids (HETEs), hydroxydocosahexaenoic acids (HDHAs) and hydroxyeicosapentaenoic acids (HEPEs). We detected more pronounced changes in free metabolites in RBCs after HD, as compared with the total RBC compartment. Hemodialysis treatment did not affect the majority of CYP and CYP ω/(ω-1)-hydroxylase products in RBCs. Our data indicate that erythro-metabolites of the LOX pathway are influenced by renal-replacement therapies, which could have deleterious effects in the circulation.

Bioaccumulation of Blood Long-Chain Fatty Acids during Hemodialysis.

Long-chain fatty acids (LCFAs) serve as energy sources, components of cell membranes, and precursors for signaling molecules. Uremia alters LCFA metabolism so that the risk of cardiovascular events in chronic kidney disease (CKD) is increased. End-stage renal disease (ESRD) patients undergoing dialysis are particularly affected and their hemodialysis (HD) treatment could influence blood LCFA bioaccumulation and transformation. We investigated blood LCFA in HD patients and studied LCFA profiles in vivo by analyzing arterio-venous (A-V) LFCA differences in upper limbs. We collected arterial and venous blood samples from 12 ESRD patients, before and after HD, and analyzed total LCFA levels in red blood cells (RBCs) and plasma by LC-MS/MS tandem mass spectrometry. We observed that differences in arterial and venous LFCA contents within RBCs (RBC LCFA A-V differences) were affected by HD treatment. Numerous saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) n-6 showed negative A-V differences, accumulated during peripheral tissue perfusion of the upper limbs, in RBCs before HD. HD reduced these differences. The omega-3 quotient in the erythrocyte membranes was not affected by HD in either arterial or venous blood. Our data demonstrate that A-V differences in fatty acids status of LCFA are present and active in mature erythrocytes and their bioaccumulation is sensitive to single HD treatment.

Hemodialysis and Plasma Oxylipin Biotransformation in Peripheral Tissue.

Factors causing the increased cardiovascular morbidity and mortality in hemodialysis (HD) patients are largely unknown. Oxylipins are a superclass of lipid mediators with potent bioactivities produced from oxygenation of polyunsaturated fatty acids. We previously assessed the impact of HD on oxylipins in arterial blood plasma and found that HD increases several oxylipins. To study the phenomenon further, we now evaluated the differences in arterial and venous blood oxylipins from patients undergoing HD. We collected arterial and venous blood samples in upper extremities from 12 end-stage renal disease (ESRD) patients before and after HD and measured oxylipins in plasma by LC-MS/MS tandem mass spectrometry. Comparison between cytochrome P450 (CYP), lipoxygenase (LOX), and LOX/CYP ω/(ω-1)-hydroxylase metabolites levels from arterial and venous blood showed no arteriovenous differences before HD but revealed arteriovenous differences in several CYP metabolites immediately after HD. These changes were explained by metabolites in the venous blood stream of the upper limb. Decreased soluble epoxide hydrolase (sEH) activity contributed to the release and accumulation of the CYP metabolites. However, HD did not affect arteriovenous differences of the majority of LOX and LOX/CYP ω/(ω-1)-hydroxylase metabolites. The HD treatment itself causes changes in CYP epoxy metabolites that could have deleterious effects in the circulation.