Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

Coupling Osmotic Efficacy with Biocompatibility in Peritoneal Dialysis: A Stiff Challenge.

Peritoneal dialysis (PD) is a home-based efficacious modality for the replacement of renal function in end-stage kidney failure patients, but it is still under-prescribed. A major limitation is the durability of the dialytic technique. Continuous exposure of the peritoneum to bioincompatible conventional glucose-based solutions is thought to be the main cause of the long-term morpho-functional peritoneal changes that eventually result in ultrafiltration failure. Poor PD solution biocompatibility is primarily related to the high glucose content, which is not only detrimental to the peritoneal membrane but has many potential metabolic side effects. To improve the clinical outcome and prolong the survival of the treatment, PD-related bioincompatibility urgently needs to be overcome. However, combining dialytic and osmotic efficacy with a satisfactory biocompatible profile is proving to be quite difficult. New approaches targeting the composition of the PD solution include the replacement of glucose with other osmotic agents, and the addition of cytoprotective or osmo-metabolic compounds. Other strategies include the infusion of mesenchymal cells or the administration of orally active agents. In the present article, we review the current evidence on efforts to improve the biocompatible and functional performance of PD, focusing on studies performed in vivo (animal models of PD, human subjects on PD).

Renal Oxygen Demand and Nephron Function: Is Glucose a Friend or Foe?

The kidneys and heart work together to balance the body's circulation, and although their physiology is based on strict inter dependence, their performance fulfills different aims. While the heart can rapidly increase its own oxygen consumption to comply with the wide changes in metabolic demand linked to body function, the kidneys physiology are primarily designed to maintain a stable metabolic rate and have a limited capacity to cope with any steep increase in renal metabolism. In the kidneys, glomerular population filters a large amount of blood and the tubular system has been programmed to reabsorb 99% of filtrate by reabsorbing sodium together with other filtered substances, including all glucose molecules. Glucose reabsorption involves the sodium-glucose cotransporters SGLT2 and SGLT1 on the apical membrane in the proximal tubular section; it also enhances bicarbonate formation so as to preserve the acid-base balance. The complex work of reabsorption in the kidney is the main factor in renal oxygen consumption; analysis of the renal glucose transport in disease states provides a better understanding of the renal physiology changes that occur when clinical conditions alter the neurohormonal response leading to an increase in glomerular filtration pressure. In this circumstance, glomerular hyperfiltration occurs, imposing a higher metabolic demand on kidney physiology and causing progressive renal impairment. Albumin urination is the warning signal of renal engagement over exertion and most frequently heralds heart failure development, regardless of disease etiology. The review analyzes the mechanisms linked to renal oxygen consumption, focusing on sodium-glucose management.

Mechanisms of action of SGLT2 inhibitors and their beneficial effects on the cardiorenal axis.

Large clinical studies conducted with sodium-glucose co-transporter 2 inhibitors (SGLT2i) in patients with type 2 diabetes and heart failure with reduced ejection fraction have demonstrated their ability to achieve both cardiac and kidney benefits. Although there is huge evidence on SGLT2i-mediated clinical benefits both in diabetic and non-diabetic patients, the pathophysiological mechanisms underlying their efficacy are still poorly understood. Some favorable mechanisms are likely due to the prompt glycosuric action which is associated with natriuretic effects leading to hemodynamic benefits as well as a reduction in glomerular hyperfiltration and renin-angiotensin-aldosterone system activation. In addition to the renal mechanisms, SGLT2i may play a relevant role in cardiorenal axis protection by improving the cardiomyocyte metabolism, by exerting anti-fibrotic and anti-inflammatory actions, and by increasing cardioprotective adipokine expression. New studies will be needed to better understand the specific molecular mechanisms that mediate the SGLT2i favorable effects in patients suffering diabetes. Our aim is to first discuss about the molecular mechanisms underlying the cardiovascular benefits of SGLT2i in each of the main organs involved in the cardiorenal axis. Furthermore, we update on the most recent clinical trials evaluating the beneficial effects of SGLT2i in treatment of both diabetic and non-diabetic patients suffering heart failure.

Fibrosis of Peritoneal Membrane as Target of New Therapies in Peritoneal Dialysis.

Peritoneal dialysis (PD) is an efficient renal replacement therapy for patients with end-stage renal disease. Even if it ensures an outcome equivalent to hemodialysis and a better quality of life, in the long-term, PD is associated with the development of peritoneal fibrosis and the consequents patient morbidity and PD technique failure. This unfavorable effect is mostly due to the bio-incompatibility of PD solution (mainly based on high glucose concentration). In the present review, we described the mechanisms and the signaling pathway that governs peritoneal fibrosis, epithelial to mesenchymal transition of mesothelial cells, and angiogenesis. Lastly, we summarize the present and future strategies for developing more biocompatible PD solutions.

Mild sodium reduction in peritoneal dialysis solution improves hypertension in end stage kidney disease: a case-report study.

INTRODUCTION: Blood Pressure (BP) control is largely unsatisfied in End Stage Kidney Disease (ESKD) principally due to sodium retention. Peritoneal Dialysis (PD) is the most common type of home dialysis, using a peritoneal membrane to remove sodium, though sodium removal remains challenging. METHODS: This is a case-study reporting two consecutive ESKD patients treated by a novel peritoneal PD solution with a mildly reduced sodium content (130 mmol/L) to treat hypertension. RESULTS: In the first case, a 78-year-old woman treated by Continuous Ambulatory PD (CAPD) with standard solution (three 4 h-dwells per day 1.36% glucose 132 mmol/L) showed resistant hypertension confirmed by ambulatory blood pressure monitoring (ABPM), reporting 24 h-BP: 152/81 mmHg, day-BP:151/83 mmHg and night-ABP: 153/75 mmHg, with inversion of the circadian systolic BP rhythm (1.01), despite use of three anti-hypertensives and a diuretic at adequate doses. No sign of hypervolemia was evident. We then switched from standard PD to low-sodium solution in all daily dwells. A six-months low-sodium CAPD enabled us to reduce diurnal (134/75 mmHg) and nocturnal BP (122/67 mmHg), restoring the circadian BP rhythm, with no change in ultrafiltration or residual diuresis. Diet and drug prescription were unmodified too. The second case was a 61-year-old woman in standard CAPD (three 5 h-dwells per day) suffering from hypertension confirmed by ABPM (mean 24 h-ABP: 139/84 mmHg; mean day-ABP:144/88 mmHg and mean night-ABP:124/70 mmHg). She was switched from 132-Na CAPD to 130-Na CAPD, not changing dialysis schedule. No fluid expansion was evident. During low-sodium CAPD, antihypertensive therapy (amlodipine 10 mg and Olmesartan 20 mg) has been reduced until complete suspension. After 6 months, we repeated ABPM showing a substantial reduction in mean 24 h-ABP (117/69 mmHg), mean diurnal ABP (119/75 mmHg) and mean nocturnal ABP (111/70 mmHg). Ultrafiltration and residual diuresis remained unmodified. No side effects were reported in either cases. CONCLUSIONS: This case-report study suggests that mild low-sodium CAPD might reduce BP in hypertensive ESKD patients.

Endothelial Glycocalyx as a Regulator of Fibrotic Processes.

The endothelial glycocalyx, the gel layer covering the endothelium, is composed of glycosaminoglycans, proteoglycans, and adsorbed plasma proteins. This structure modulates vessels' mechanotransduction, vascular permeability, and leukocyte adhesion. Thus, it regulates several physiological and pathological events. In the present review, we described the mechanisms that disturb glycocalyx stability such as reactive oxygen species, matrix metalloproteinases, and heparanase. We then focused our attention on the role of glycocalyx degradation in the induction of profibrotic events and on the possible pharmacological strategies to preserve this delicate structure.

How to Improve the Biocompatibility of Peritoneal Dialysis Solutions (without Jeopardizing the Patient's Health).

Peritoneal dialysis (PD) is an important, if underprescribed, modality for the treatment of patients with end-stage kidney disease. Among the barriers to its wider use are the deleterious effects of currently commercially available glucose-based PD solutions on the morphological integrity and function of the peritoneal membrane due to fibrosis. This is primarily driven by hyperglycaemia due to its effects, through multiple cytokine and transcription factor signalling-and their metabolic sequelae-on the synthesis of collagen and other extracellular membrane components. In this review, we outline these interactions and explore how novel PD solution formulations are aimed at utilizing this knowledge to minimise the complications associated with fibrosis, while maintaining adequate rates of ultrafiltration across the peritoneal membrane and preservation of patient urinary volumes. We discuss the development of a new generation of reduced-glucose PD solutions that employ a variety of osmotically active constituents and highlight the biochemical rationale underlying optimization of oxidative metabolism within the peritoneal membrane. They are aimed at achieving optimal clinical outcomes and improving the whole-body metabolic profile of patients, particularly those who are glucose-intolerant, insulin-resistant, or diabetic, and for whom daily exposure to high doses of glucose is contraindicated.

Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis.

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Proteomic Research in Peritoneal Dialysis.

Peritoneal dialysis (PD) is an established home care, cost-effective renal replacement therapy (RRT), which offers several advantages over the most used dialysis modality, hemodialysis. Despite its potential benefits, however, PD is an under-prescribed method of treating uremic patients. Infectious complications (primarily peritonitis) and bio-incompatibility of PD solutions are the main contributors to PD drop-out, due to their potential for altering the functional and anatomical integrity of the peritoneal membrane. To improve the clinical outcome of PD, there is a need for biomarkers to identify patients at risk of PD-related complications and to guide personalized interventions. Several recent studies have shown that proteomic investigation may be a powerful tool in the prediction, early diagnosis, prognostic assessment, and therapeutic monitoring of patients on PD. Indeed, analysis of the proteome present in PD effluent has uncovered several proteins involved in inflammation and pro-fibrotic insult, in encapsulating peritoneal sclerosis, or even in detecting early changes before any measurable modifications occur in the traditional clinical parameters used to evaluate PD efficacy. We here review the proteomic studies conducted thus far, addressing the potential use of such omics methodology in identifying potential new biomarkers of the peritoneal membrane welfare in relation to dialytic prescription and adequacy.

A Novel Formulation of Glucose-Sparing Peritoneal Dialysis Solutions with l-Carnitine Improves Biocompatibility on Human Mesothelial Cells.

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end-stage renal disease (ESRD) lies in the fact that the currently used glucose-based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and l-carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro-inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro-inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility.

Current Opinion on Usage of L-Carnitine in End-Stage Renal Disease Patients on Peritoneal Dialysis.

The advantages of peritoneal dialysis (PD) over hemodialysis (HD) are well-documented. Notwithstanding, only a small proportion of patients with end-stage renal disease (ESRD) are managed with PD. This may be related to the high glucose load that PD solutions in current use have on the patient. The effects of such excess glucose include the relatively early limitation of the ultrafiltration capacity of the peritoneal membrane, and the metabolic effects associated with hyperglycemia, e.g., decreased insulin sensitivity. This article describes the advantages that may be realized by the glucose-sparing effects of substituting part of the glucose load with other osmotically active metabolites, particularly L-carnitine. The latter is anticipated to have metabolic advantages of its own, especially as in PD patients, high plasma concentrations can be achieved in the absence of renal clearance. Besides its better biocompatibility, L-carnitine demonstrates anti-anemia action due to its effects on erythropoiesis, and positive effects on the longevity and deformability of erythrocytes. Observations from our trials on the use of carnitine-enriched PD solutions have demonstrated the effectiveness of L-carnitine as an efficient osmolyte in PD, and its favorable effect on the insulin sensitivity of the patients. The significance of these findings for future developments in the use of PD in the management of patients with ESRD is discussed.

Acetylcholine and acetylcarnitine transport in peritoneum: Role of the SLC22A4 (OCTN1) transporter.

A suitable experimental tool based on proteoliposomes for assaying Organic Cation Transporter Novel member 1 (OCTN1) of peritoneum was pointed out. OCTN1, recently acknowledged as acetylcholine transporter, was immunodetected in rat peritoneum. Transport was assayed following flux of radiolabelled TEA, acetylcholine or acetylcarnitine in proteoliposomes reconstituted with peritoneum extract. OCTN1 mediated, besides TEA, also acetylcholine and a slower acetylcarnitine transport. External sodium inhibited acetylcholine uptake but not its release from proteoliposomes. Differently, sodium did not affect acetylcarnitine uptake. These results suggested that physiologically, acetylcholine should be released while acetylcarnitine was taken up by peritoneum cells. Transport was impaired by OCTN1 inhibitors, butyrobetaine, spermine, and choline. Biotin was also found as acetylcholine transport inhibitor. Anti-OCTN1 antibody specifically inhibited acetylcholine transport confirming the involvement of OCTN1. The transporter was also immunodetected in human mesothelial primary cells. Extract from these cells was reconstituted in proteoliposomes. Transport features very similar to those found with rat peritoneum were observed. Validation of the proteoliposome model for peritoneal transport study was then achieved assaying transport in intact mesothelial cells. TEA, butyrobetaine and Na(+) inhibited acetylcholine transport in intact cells while efflux was Na(+) insensitive. Therefore transport features in intact cells overlapped those found in proteoliposomes.

A Moderate Carnitine Deficiency Exacerbates Isoproterenol-Induced Myocardial Injury in Rats.

PURPOSE: The myocardium is largely dependent upon oxidation of fatty acids for the production of ATP. Cardiac contractile abnormalities and failure have been reported after acute emotional stress and there is evidence that catecholamines are responsible for acute stress-induced heart injury. We hypothesized that carnitine deficiency increases the risk of stress-induced heart injury. METHODS: Carnitine deficiency was induced in Wistar rats by adding 20 mmol/L of sodium pivalate to drinking water (P). Controls (C) received equimolar sodium bicarbonate and a third group (P + Cn) received pivalate along with 40 mmol/L carnitine. After 15 days, 6 rats/group were used to evaluate function of isolated hearts under infusion of 0.1 µM isoproterenol and 20 rats/group were submitted to a single subcutaneous administration of 50 mg/kg isoproterenol. RESULTS: Isoproterenol infusion in C markedly increased the heart rate, left ventricular (LV) systolic pressure and coronary flow rate. In P rats, isoproterenol increased the heart rate and LV systolic pressure but these increases were not paralleled by a rise in the coronary flow rate and LV diastolic pressure progressively increased. Subcutaneous isoproterenol induced 15 % mortality rate in C and 50 % in P (p < 0.05). Hearts of surviving P rats examined 15 days later appeared clearly dilated, presented a marked impairment of LV function and a greater increase in tumor necrosis factor α (TNFα) levels. All these detrimental effects were negligible in P + Cn rats. CONCLUSIONS: Our study suggests that carnitine deficiency exposes the heart to a greater risk of injury when sympathetic nerve activity is greatly stimulated, for example during emotional, mental or physical stress.

Effect of an L-carnitine-containing peritoneal dialysate on insulin sensitivity in patients treated with CAPD: a 4-month, prospective, multicenter randomized trial.

BACKGROUND: In peritoneal dialysis, the high glucose load absorbed from dialysis fluid contributes to several metabolic abnormalities, including insulin resistance. We evaluate the efficacy of a peritoneal dialysis solution containing l-carnitine as an additive to improve insulin sensitivity. STUDY DESIGN: Multicenter parallel randomized controlled trial. SETTING & PARTICIPANTS: Nondiabetic uremic patients on continuous ambulatory peritoneal dialysis enrolled in 8 peritoneal dialysis centers. INTERVENTION: Patients were randomly assigned to receive peritoneal dialysis diurnal exchanges with either a standard glucose-based solution (1.5% or 2.5% according to the patient's need) or a glucose-based solution (identical glucose amount) enriched with l-carnitine (0.1%, weight/volume; 2 g/bag) for 4 months, the nocturnal exchange with icodextrin being unmodified. OUTCOMES & MEASUREMENTS: The primary outcome was insulin sensitivity, measured by the magnitude of change from baseline in glucose infusion rate (in milligrams per kilogram of body weight per minute) during a euglycemic hyperinsulinemic clamp. Secondary outcomes were safety and tolerability, body fluid management, peritoneal dialysis efficiency parameters, and biochemistry tests. RESULTS: 35 patients were randomly assigned, whereas 27 patients (standard solution, n=12; experimental solution, n = 15) were analyzed. Adverse events were not attributable to treatment. Glucose infusion rates in the l-carnitine-treated group increased from 3.8 ± 2.0 (SD) mg/kg/min at baseline to 5.0 ± 2.2 mg/kg/min at day 120 (P = 0.03) compared with 4.8 ± 2.4 mg/kg/min at baseline and 4.7 ± 2.4 mg/kg/min at day 120 observed in the control group (P = 0.8). The difference in glucose infusion rates between groups was 1.3 (95% CI, 0.0-2.6) mg/kg/min. In patients treated with l-carnitine-containing solution, urine volume did not change significantly (P = 0.1) compared to a significant diuresis reduction found in the other group (P = 0.02). For peritoneal function, no differences were observed during the observation period. LIMITATIONS: Small sample size. CONCLUSIONS: The use of l-carnitine in dialysis solutions may represent a new approach to improving insulin sensitivity in nondiabetic peritoneal dialysis patients.

Overproduction, Purification, and Stability of the Functionally Active Human Carnitine Acetyl Transferase.

Human Carnitine Acetyl Transferase (hCAT) reversibly catalyzes the transfer of the acetyl-moiety from acetyl-CoA to L-carnitine, modulating the acetyl-CoA/CoA ratio in mitochondria. Derangement of acetyl-CoA/CoA ratio leads to metabolic alterations that could result in the onset or worsening of pathological states. Due to the importance of CAT as a pharmacological target and to the European directive for reducing animal experimentation, we have pointed out a procedure to produce a recombinant, pure, and functional hCAT using the E. coli expression system. The cDNA encoding for the hCAT was cloned into the pH6EX3 vector. This construct was used to transform the E. coli Rosetta strain. The optimal conditions for the overexpression of the fully active hCAT include induction with a low concentration of IPTG (0.01 mM) and a low growth temperature (25 °C). The recombinant protein was purified from bacterial homogenate by affinity chromatography. The pure hCAT is very stable in an aqueous solution, retaining full activity for at least two months if stored at - 20 °C. These results could be helpful for a broad set of functional studies on hCAT, including drug-design applications.

L-carnitine is an osmotic agent suitable for peritoneal dialysis.

Excessive intraperitoneal absorption of glucose during peritoneal dialysis has both local cytotoxic and systemic metabolic effects. Here we evaluate peritoneal dialysis solutions containing L-carnitine, an osmotically active compound that induces fluid flow across the peritoneum. In rats, L-carnitine in the peritoneal cavity had a dose-dependent osmotic effect similar to glucose. Analogous ultrafiltration and small solute transport characteristics were found for dialysates containing 3.86% glucose, equimolar L-carnitine, or combinations of both osmotic agents in mice. About half of the ultrafiltration generated by L-carnitine reflected facilitated water transport by aquaporin-1 (AQP1) water channels of endothelial cells. Nocturnal exchanges with 1.5% glucose and 0.25% L-carnitine in four patients receiving continuous ambulatory peritoneal dialysis were well tolerated and associated with higher net ultrafiltration than that achieved with 2.5% glucose solutions, despite the lower osmolarity of the carnitine-containing solution. Addition of L-carnitine to endothelial cells in culture increased the expression of AQP1, significantly improved viability, and prevented glucose-induced apoptosis. In a standard toxicity test, the addition of L-carnitine to peritoneal dialysis solution improved the viability of L929 fibroblasts. Thus, our studies support the use of L-carnitine as an alternative osmotic agent in peritoneal dialysis.