Marcin Ufnal

Background: A high-fat diet (HFD) contributes to cardiometabolic disease. Gut microbiota-derived metabolites may participate in this process, but their contribution to lipid regulation is not well defined. Indoxyl sulfate (IS), a microbiota-derived metabolite, has been linked to vascular and metabolic dysfunction. Its role in lipid metabolism remains unclear. Methods: In Part A, plasma and urinary concentrations of IS were measured in plasma and urine from HFD-fed rats in which dyslipidemia had developed, together with controls. In Part B, HepG2 cells were exposed to IS, and cell viability and selected cholesterol metabolism-related transcripts and proteins were assessed. In Part C, 10-week-old, male Sprague–Dawley rats maintained on a standard diet received vehicle or IS at two doses for 8 weeks. Hepatic expression of LDLR, SREBP-2, HMG-CoA reductase, and related cholesterol metabolism markers were measured by quantitative real-time PCR and Western blotting. Results: In Part A, higher plasma IS concentrations and higher daily urinary IS excretion were found in samples collected from HFD-fed rats compared to controls. In HepG2 cells, IS reduced cell viability at higher concentrations and increased LDLR mRNA and protein expression. In IS-treated rats, total cholesterol, LDL-cholesterol, and triglycerides increased in a dose-dependent manner. Hepatic SREBP-2 and HMG-CoA reductase protein levels were increased at both IS doses, whereas LDLR protein abundance was increased at the higher dose. Moreover, serum PCSK9 levels were reduced in IS-treated rats. Conclusion: IS increased in HFD-fed rats. IS altered cholesterol metabolism-related pathways in HepG2 cells and in rats. In vivo IS administration increased circulating lipids and hepatic proteins involved in cholesterol synthesis and uptake. These findings indicate that IS may contribute to disturbed lipid homeostasis, although its role in HFD-induced dyslipidemia requires further mechanistic confirmation.

Hypertension contributes to renal dysfunction, partly through dysregulation of the renin-angiotensin system (RAS). Betaine, an osmolyte that helps maintain medullary osmotic gradients, has demonstrated renoprotective effects in models of nephrotoxicity. Previously, we showed that acute betaine administration increases diuresis—more pronounced in spontaneously hypertensive rats (SHRs) than in normotensive Wistar–Kyoto rats—without affecting blood pressure. In this study, we investigate the effects of chronic betaine supplementation on SHR renal function. Methods: Six-week-old, male SHRs received either 0.5% betaine in drinking water or tap water for 16 weeks. Hemodynamics, serum and urine biochemistry, and renal mRNA expression of RAS components and betaine transporters (SLC6A12, SLC6A20) were assessed. Results: Betaine-treated SHRs demonstrated higher creatinine clearance without significant changes in arterial blood pressure. Renal expression of angiotensinogen and AT1B receptor was reduced, while MAS and AT2 receptor expression increased. Transporter expression showed region-specific regulation: cortical SLC6A20 and medullary SLC6A12 were lower in betaine-treated rats, whereas medullary SLC6A20 was higher. Conclusion: Chronic betaine supplementation was associated with increased creatinine clearance and a renal RAS mRNA profile shifted toward MAS and AT2 receptor predominance. These findings support further investigation of betaine as a potential strategy for early prevention of hypertensive kidney injury. However, direct evidence of improved glomerular filtration and functional activation of protective RAS pathways is still needed.

Unhealthy diet and hypertension constitute major risk factors for the development of cardiometabolic diseases. However, the direct effects of dietary components and high blood pressure on metabolic profiles remain poorly understood. We evaluated concentrations of amino acids and nutrients in the plasma and tissues of animal models subjected to high‐fat and high‐disaccharide diet without excessive caloric intake, in salt‐resistant rats on high salt intake, in spontaneously hypertensive rats, and in angiotensin II–induced hypertensive rats. Using liquid chromatography and mass spectrometry, we identified changes in several analytes across models. We found that a high‐fat diet increased tissue levels of methylglycines (betaine, sarcosine) and glycine, while high salt intake and hypertension were associated with a distinct reduction of methyl/glycine species and the accumulation of taurine in the tissues. We further observed tissue‐specific alterations. For instance, alanine was decreased in the kidneys of rats on high salt and in hypertensive models. Beta‐alanine was higher in the lung and renal cortex of rats on high salt, but lower in the cardiovascular system of hypertensive models. A high‐sugar diet increased circulating levels of betaine and taurine, although its impact on tissues was less pronounced. In conclusion, this study provides a comprehensive evaluation of organic solutes in different animal models and highlights the diverse metabolic alterations associated with diet and hypertension. Further research is required to explore the significance of these findings and their potential implications for understanding disease mechanisms.

Aims: Renal impairment frequently coexists with heart failure (HF) and is associated with increased risk of poor clinical outcomes. This highlights the urgent need for therapies targeting both cardiac and renal dysfunction. AZD3427, a long-acting recombinant fusion protein and relaxin analogue that selectively activates the relaxin family peptide receptor 1 (RXFP1), showed trends of increased stroke volume and estimated glomerular filtration rate (eGFR) in HF patients (NCT04630067). The hypothesis is that AZD3427 may enhance GFR by expanding the functional renal cortex volume and improving renal perfusion. Methods and results: The Re-PERFUSE study (NCT06611423) is a Phase 1b, randomised, double-blind, placebo-controlled trial aimed at evaluating the effects of AZD3427 on renal perfusion in patients with HF and reduced ejection fraction (HFrEF) with reduced eGFR. Patients with HF, EF ≤ 40% and an eGFR of 30 to 90 mL/min/1.73 m2, assessed by the CKD-EPI 2021, will receive a single dose of subcutaneous AZD3427 (n = 6) or placebo (n = 6). Intravenous dopamine will serve as a positive control for increased renal blood flow. Positron emission tomography (PET) imaging with [15O]-labelled water will be used to measure renal cortex blood flow pre- and post-treatment, allowing differentiation between global and focal renal blood flow changes and providing insights into potential nephron recruitment and increased filtration membrane area. Safety and tolerability will be assessed through monitoring of adverse events, clinical laboratory tests and vital signs. Conclusions: This study, alongside other ongoing AZD3427 studies, aims to evaluate the dual effects of AZD3427 in improving both cardiac and renal function. These insights could guide the development of future therapeutic strategies for managing HF and renal impairment.

Trimethylamine N-oxide (TMAO), a bacterial metabolite associated with cardiovascular risk, has a debated role, with evidence suggesting both harmful and protective effects. We hypothesized that chronic TMAO supplementation modulates hypertension-related complications through alterations in the tissue renin-angiotensin system (RAS). Ten-week-old male spontaneously hypertensive rats (SHRs) were divided into two groups, receiving either water or TMAO-supplemented water for 80 weeks. Circulatory parameters, including hemodynamics, echocardiographic measurements, and pathomorphological assessments, tissue RAS expression and biomarker profiles, were evaluated. Rats treated with TMAO exhibited significantly higher plasma TMAO levels accompanied by significantly lower mean and systolic blood pressure and NT-proBNP levels. In the TMAO group, diuresis and natriuresis were numerically increased, and the left atrial size was smaller. No pathological effects of TMAO on the circulatory system were observed in rats. Echocardiographic parameters and age-related histopathological changes, including fibrosis in the heart and kidneys, were comparable between groups. TMAO-treated rats showed upregulated expression of RAS components, including angiotensinogen and AT1b and AT2 receptors, in the heart, kidneys, and colon. TMAO supplementation also increased blood and urinary levels of trimethylamine, a toxic precursor and metabolite of TMAO. In conclusion, lifelong exposure to TMAO improves circulatory function in hypertension, as evidenced by reduced blood pressure, lower NT-proBNP levels, and decreased left atrial size—findings consistent with a reduction in cardiac workload. The upregulation of RAS components may represent a compensatory response to the blood pressure-lowering effect of TMAO. However, the concurrent rise in potentially toxic TMA levels warrants further investigation to fully assess the long-term safety of TMAO supplementation.

Background/Objectives: Trimethylamine (TMA), produced by gut microbiota, and its derivative trimethylamine N-oxide (TMAO) are both associated with cardiometabolic diseases. While the effects of high-fat diets (HFDs) and high-disaccharide diets (HDDs) on gut microbiota in the context of obesity have been well studied, their impact on TMA/TMAO production, particularly alongside physiological caloric intake, remains obscure. This study investigates how standard HFDs and HDDs alongside physiological caloric intake influence gut microbiota composition and TMA/TMAO production in rats. Methods: Sprague Dawley rats were fed one of three diets a standard diet, an HFD, or an HDD for 12 weeks, with chow availability adjusted by age to maintain physiological caloric intake. Gut bacterial diversity was analyzed using 16S rRNA gene sequencing, and metabolites were quantified via High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) in urine and plasma. Results: The HFD group had significantly higher urinary levels of TMA and TMAO compared to the control and HDD groups. Gut bacterial diversity in the HFD group was markedly reduced, displaying the lowest species richness and phylogenetic diversity among all the groups. Notably, Pasteurellaceae (within the order Pasteurellales) and S24-7 (within the order Bacteroidales) were positively correlated with TMAO levels. The demonstrated HDD group increased microbial diversity compared to both the control and HFD groups. Conclusions: A high-fat diet during controlled and physiological caloric intake increases TMA/TMAO production and reduces gut microbial diversity. This underscores the role of diet composition, beyond caloric excess, in shaping gut microbiota and the related cardiometabolic biomarkers.

Diuretics are essential for managing fluid overload in heart failure (HF) and controlling blood pressure in hypertension. However, their use is often associated with complications such as electrolyte imbalances and neurohormonal dysregulation, which can limit their effectiveness and contribute to adverse outcomes. These challenges underscore the need for alternative or adjunctive strategies to better manage fluid retention and congestion. Osmolytes are small molecules that help counteract increases in extracellular osmotic and hydrostatic pressure and are naturally present at high concentrations in the renal medulla. Notably, elevated serum levels of osmolytes such as trimethylamine N-oxide (TMAO) and betaine have been observed in patients with HF, although their role in the pathophysiology of the disease remains unclear. Given the known diuretic properties of osmolytes such as urea—historically used in the management of HF—it is plausible that other osmolytes may similarly modulate diuresis and volume status. This review examines the biological actions of several key osmolytes, including urea, TMAO, betaine, and taurine. Emerging evidence supports the need for further preclinical and clinical studies to investigate the potential diuretic and cytoprotective effects of TMAO, betaine, and taurine in the prevention and treatment of HF and hypertension.

Elevated plasma levels of trimethylamine N-oxide (TMAO)—a compound derived from diet and the gut microbiome—have been widely studied for their association with diabetes risk and their potential role in disease pathophysiology and complications. However, clinical studies, both prospective and retrospective, have yielded conflicting results. For example, elevated levels of TMAO are frequently linked to an increased risk of cardiovascular and renal complications in individuals with diabetes. However, the robustness and independence of these associations differ across study populations and are influenced by the degree of adjustment for confounding risk factors. Considering insulin’s regulatory effect on FMO3 activity in liver cells, TMAO may serve as a marker of hepatic insulin resistance, which could partially explain its association with diabetes risk. The role of TMAO in diabetes pathology remains controversial; while some studies emphasize its detrimental impact on insulin sensitivity and the progression of diabetes-related complications, others suggest potential protective effects. Investigating the largely unexplored role of TMAO’s precursor, trimethylamine, may help elucidate these discrepancies. This review consolidates clinical and experimental findings to clarify TMAO’s complex mechanistic contributions to diabetes pathology.

Background: Intestinal microbial metabolites, such as trimethylamine-N-oxide (TMAO) and indoxyl sulfate (IS), have been suggested as markers for the progression of aortic stenosis (AS). However, the impact of transcatheter aortic valve implantation (TAVI) on these intestinal bacterial metabolites has not been evaluated in a multicenter clinical study. The aim of this study was to determine the effect of TAVI on plasma levels of intestinal bacterial metabolites and to assess the predictive value of these metabolites for major adverse cardiovascular events (MACE) following TAVI. Methods: Consecutive patients with AS referred for TAVI were enrolled in this study. Blood samples were collected one day before TAVI and at hospital discharge. The concentrations of intestinal microbial metabolites were measured using ultra performance liquid chromatograph coupled with a mass spectrometer. Results: Plasma levels of TMAO and IS decreased after TAVI, compared to baseline (p ≤ 0.004 for all). Among 128 patients included in the study, 21 patients (16.4%) developed MACE during the median follow-up time of 404 days. Baseline plasma IS level was higher in patients with MACE, compared to those without MACE (p = 0.001). Increased baseline IS level predicted MACE with 75.0% sensitivity and 74.3% specificity independent of other clinical variables (OR 14.264, 95% CI 3.442–59.117, p < 0.001) and decreased the chance of event-free survival (p_{log rank} < 0.001). Conclusions: Plasma concentrations of TMAO and IS decreased after TAVI, compared to baseline. Elevated plasma IS levels were associated with a 14-fold increase in the odds of post-TAVI MACE during a median follow-up period of 404 days. Keywords: aortic stenosis intestinal metabolites indoxyl sulfate transcatheter aortic valve implantation trimethylamine-N-oxide