Vučković, Ivan

BACKGROUND: It is unknown whether cardiac resynchronization therapy (CRT) improves systemic metabolomic profile by enhancing left ventricular function. The observational cohort study aimed to investigate the effect of CRT on left ventricular ejection fraction (LVEF) and circulating plasma metabolites in patients who had heart failure with reduced LVEF. METHODS: We prospectively screened patients with ischemic cardiomyopathy and nonischemic cardiomyopathy who received CRT with a defibrillator for LVEF ≤35% according to current guidelines. Clinical assessment included echocardiography and device interrogation. Blood samples for metabolomic analysis were collected before CRT and at 6-month follow-up. Plasma was subjected to gas chromatography-mass spectrometry and 1 H nuclear magnetic resonance-based metabolomic analysis. RESULTS: Totally 92 patients were enrolled, with a mean age of 67.3±11.3 years (37.0% female). LVEF was significantly improved from 28.9±7.6% at baseline to 36.0±11.3% at 6 months ( P <0.001) and to 40.1±12.6% at 12 months ( P <0.001). After CRT, 42 metabolite features were significantly decreased compared with the baseline. The ketone bodies, including 3-hydroxybutyrate ( P <0.001) and acetone ( P =0.01), were reduced. A branched-chain amino acid, isoleucine, was also decreased after CRT ( P =0.01). These metabolomic changes were mainly observed in the nonischemic cardiomyopathy group, while the metabolomic profile in the ischemic cardiomyopathy group was characterized by enhanced amino acid oxidation and elevated levels of lactate and pyruvate. The improvement in LVEF positively correlated with the ratio of changes in ketone bodies and isoleucine. CONCLUSIONS: CRT may modulate the systemic plasma metabolomic profile, which correlated with improvement in left ventricular function in patients with severe heart failure with reduced ejection fraction.

ABSTRACT The search for long‐standing, drought‐tolerant species with consistent annual yields presents both the demands of sustainable agriculture and a significant challenge. Hungarian clover, an underutilized fodder species with a long lifespan, may be a potential solution to these demands. This study represents the first report on the metabolomic profile of this species. We analyzed 52 samples of Hungarian clover and Swedish clover, used as a control species, employing 1 H NMR spectroscopy combined with multivariate data analysis. A total of 24 metabolites were identified, with dominant signals derived from sugars, amino, eucomic acids, and hydroxyeucomic acids. These acids contributed to the higher antioxidant capacity observed in Hungarian clover samples compared to Swedish clover. The application of nuclear magnetic resonance in the pre‐breeding evaluation of fodder species is a well‐justified approach, providing clear insight into the metabolic variations of the examined material and valuable guidance for future breeding activities.

Combined histopathological and metabolomic analysis of the same tissue specimen can reduce biological variability compared to independent analyses of similar samples. However, conventional workflows are incompatible: tissue fixation leads to metabolite loss through dilution and chemical reactions with fixative, while metabolomic extraction typically destroys the specimen. We propose a method that reconciles these conflicting requirements by enabling metabolite recovery from fixative while preserving tissue for histopathology. In this approach, tissue is initially fixed in a small volume of fixative; an aliquot of the fixative is then collected for nuclear magnetic resonance-based metabolomic analysis before additional fixative is added for standard processing. As a proof-of-concept, mouse kidneys from the same subject were analyzed using two protocols: standard perchloric acid extraction (ensuring complete metabolite recovery but destroying tissue) and metabolite extraction from fixative. Several metabolites were fully recoverable from the fixative, others partially, while some were lost due to chemical reactions. Despite some limitations, this strategy may be useful for analyzing precious specimens, such as human biopsies, that must remain intact for further studies. Moreover, using the same specimen for dual analyses reduces the number of animals required and enhances statistical robustness.

Key Points Experimental autosomal dominant polycystic kidney disease is associated with longitudinal changes in the kidney micro-RNA and mRNA profiles. Alterations in cellular transport, a feature of CKD, and a common biological process within polycystic kidney disease models and human autosomal dominant polycystic kidney disease, is present from early stages. Early versus late micro-RNA expression changes in autosomal dominant polycystic kidney disease could be targeted for a more tailored therapeutic intervention in the disease. Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development and enlargement of bilateral kidney cysts, which often leads to kidney failure. This study comprehensively characterized the expression profile of micro-RNAs (miRNAs) and their target genes in Pkd1 RC/RC mice and further compared them with other murine models of polycystic kidney disease (PKD) and a model of CKD and individuals with ADPKD. Methods Pkd1 RC/RC and wild-type (WT) mice ( n =10 each, five males and five females) were studied at 1, 6, and 12 months. At each time point, kidney volume was determined in vivo (magnetic resonance imaging), followed by ex vivo histomorphometric analyses. In randomly selected Pkd1 RC/RC and WT mouse kidneys ( n =5/genotype, at 1 and 12 months), miRNA-sequencing (seq) was performed, followed by mRNA-seq, integrated (miRNA-seq/mRNA-seq analysis), and functional analysis of miRNA target genes. Venn diagrams were constructed to identify overlapping and novel differentially expressed (DE) miRNAs in Pkd1 RC/RC and other commonly used murine models of PKD, diabetic kidney disease, and individuals with ADPKD. Results miRNA-seq analysis identified 41 and 181 miRNAs DE in Pkd1 RC/RC versus WT kidneys at 1 and 12 months, respectively, which were confirmed by quantitative PCR. Target genes of miRNAs DE in Pkd1 RC/RC at early stages encoded for proteins mainly implicated in cell proliferation and α -ketoglutarate ( α -KG) transport, whereas those DE at late stages encoded for transport proteins involved in proinflammatory and metabolic processes. Urine α -KG concentration ( 1 H nuclear magnetic resonance spectroscopy), its fractional excretion, and tissue levels were higher in Pkd1 RC/RC during the entire course of the disease and associated with decreased protein expression of α -KG transporters sodium-coupled dicarboxylate transporter 3 and organic anion transporter 1. We further identified common DE miRNAs among murine models of PKD, diabetic kidney disease, and previous reports in individuals with ADPKD, as well as several novel miRNAs DE in early and late Pkd1 RC/RC kidneys, which have not been previously reported in either other murine models of PKD or patients with ADPKD. Conclusions Our study demonstrates that the renal miRNA expression profile changes longitudinally with the progression of the disease and might suggest that the post-transcriptional regulation of α -KG transport could represent a novel early feature of the disease.

Our study shows that a brief harvesting-freezing delay associated with organ collection and freezing can significantly alter the kidney metabolic profile of both male and female wild-type and a genetic model of chronic kidney disease. Importantly, given that the effect of this delay differs among genotypes, it is not safe to assume that equally delaying harvesting-freezing in wild-type and polycystic kidney disease kidneys adequately controls this effect, ultimately leading to false positive and negative results among different renal diseases.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

Background Autosomal Dominant Polycystic Kidney Disease (PKD) is the most frequent hereditary renal disease and the fourth leading cause of end‐stage renal disease. However, the exact mechanisms of cystogenesis and disease progression remain to be elucidated. Recent evidence suggests that a metabolic reprograming may underlie the dysregulation of several signaling pathways known to be affected in PKD. To identify the metabolic pathways associated with cystogenesis in the PKD kidney, we characterized the urinary, plasma, and tissue metabolome of PCK (a well established model of PKD) compared to Sprague‐Dawley (SD) wild‐type rats. Methods Male and female PCK and SD rats were treated with dDAVP (PCK‐D, n=16 or SD‐D, n=12) to aggravate the cystic phenotype and achieve animals with different degrees of disease severity for a given age, or saline solution (PCK‐S, n=16 or SD‐S, n=12) as controls. Total kidney volume (TKV) gold standard to assess disease severity, were measured by MRI, and urinary, plasma, and tissue metabolites by proton nuclear magnetic resonance, 1H‐NMR. Kidney metabolites identified from renal tissues collected at euthanasia were correlated with TKV. Mitochondrial structure was assessed by electron microscopy in proximal tubules (PT), medullary thick ascending limb (mTAL), distal tubules (DT), and collecting ducts (CD). Results Consistent with previously published data, administration of dDAVP significantly aggravated the disease in PCK rats (TKV) without inducing a cystic phenotype in SD rats (). Analysis of PCK and SD metabolomes by Partial least squares‐discriminant analysis (PLS‐DA) score plots showed a significant separation between the groups (). Integration of tissue, urine and plasma analysis revealed 5 metabolites (α‐ketoglutarate, fumarate, malate, citrate, and hippurate) consistently differentially expressed, among which 4 participate in the mitochondrial tricarboxylic acid (TCA) cycle (). Moreover, tissue TCA cycle metabolites correlated directly with TKV (). Mitochondrial area and matrix density were preserved in PT, mTAL, and DT of PCK rats, yet disruption of cristae membranes, swelling, and decreased matrix density were observed in their CD cystic cells (). Conclusions Comprehensive multilevel metabolomics analysis showed significant dysregulations of several mitochondrial TCA cycle metabolites in PCK rats, which further aggravated with the more severe phenotype. Notably, these changes were associated with mitochondrial structural abnormalities in the CD. Our observations suggest that mitochondrial damage and metabolic dysregulations may be implicated in cystogenesis, and position mitochondria as important therapeutic targets in PKD. Support or Funding Information This study was supported by the Mayo Translational Polycystic Kidney Disease Center (NIDDK P30 DK090728) and an endowment from Robert M. and Billie J. Pirnie for Kidney Research