Inflammation in Children with CKD Linked to Gut Dysbiosis and Metabolite Imbalance.

BACKGROUND: CKD is characterized by a sustained proinflammatory response of the immune system, promoting hypertension and cardiovascular disease. The underlying mechanisms are incompletely understood but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions. METHODS: We analyzed the fecal microbiome, metabolites, and immune phenotypes in 48 children (with normal kidney function, CKD stage G3-G4, G5 treated by hemodialysis [HD], or kidney transplantation) with a mean±SD age of 10.6±3.8 years. RESULTS: Serum TNF-α and sCD14 were stage-dependently elevated, indicating inflammation, gut barrier dysfunction, and endotoxemia. We observed compositional and functional alterations of the microbiome, including diminished production of short-chain fatty acids. Plasma metabolite analysis revealed a stage-dependent increase of tryptophan metabolites of bacterial origin. Serum from patients on HD activated the aryl hydrocarbon receptor and stimulated TNF-α production in monocytes, corresponding to a proinflammatory shift from classic to nonclassic and intermediate monocytes. Unsupervised analysis of T cells revealed a loss of mucosa-associated invariant T (MAIT) cells and regulatory T cell subtypes in patients on HD. CONCLUSIONS: Gut barrier dysfunction and microbial metabolite imbalance apparently mediate the proinflammatory immune phenotype, thereby driving the susceptibility to cardiovascular disease. The data highlight the importance of the microbiota-immune axis in CKD, irrespective of confounding comorbidities.

Bacterial metabolites and cardiovascular risk in children with chronic kidney disease.

Cardiovascular complications are the major cause of the marked morbidity and mortality associated with chronic kidney disease (CKD). The classical cardiovascular risk factors such as diabetes and hypertension undoubtedly play a role in the development of cardiovascular disease (CVD) in adult CKD patients; however, CVD is just as prominent in children with CKD who do not have these risk factors. Hence, the CKD-specific pathophysiology of CVD remains incompletely understood. In light of this, studying children with CKD presents a unique opportunity to analyze CKD-associated mechanisms of CVD more specifically and could help to unveil novel therapeutic targets.Here, we comprehensively review the interaction of the human gut microbiome and the microbial metabolism of nutrients with host immunity and cardiovascular end-organ damage. The human gut microbiome is evolutionary conditioned and modified throughout life by endogenous factors as well as environmental factors. Chronic diseases, such as CKD, cause significant disruption to the composition and function of the gut microbiome and lead to disease-associated dysbiosis. This dysbiosis and the accompanying loss of biochemical homeostasis in the epithelial cells of the colon can be the result of poor diet (e.g., low-fiber intake), medications, and underlying disease. As a result of dysbiosis, bacteria promoting proteolytic fermentation increase and those for saccharolytic fermentation decrease and the integrity of the gut barrier is perturbed (leaky gut). These changes disrupt local metabolite homeostasis in the gut and decrease productions of the beneficial short-chain fatty acids (SCFAs). Moreover, the enhanced proteolytic fermentation generates unhealthy levels of microbially derived toxic metabolites, which further accumulate in the systemic circulation as a consequence of impaired kidney function. We describe possible mechanisms involved in the increased systemic inflammation in CKD that is associated with the combined effect of SCFA deficiency and accumulation of uremic toxins. In the future, a more comprehensive and mechanistic understanding of the gut-kidney-heart interaction, mediated largely by immune dysregulation and inflammation, might allow us to target the gut microbiome more specifically in order to attenuate CKD-associated comorbidities.

Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes.

BACKGROUND: Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes. METHODS: Using Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichiacoli killing and CD4+ T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov. Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov. Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo. RESULTS: Extracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4+ T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of [Formula: see text] and [Formula: see text] respectively, that correlated with decreased monocytic mitochondrial oxygen consumption. CONCLUSIONS: Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease.

[Nutrition and hypertension : What one should pay attention to in addition to the pharmaceutical treatment]. / Ernährung und Hypertonie : Worauf man neben der medikamentösen Therapie achten sollte.

Arterial hypertension along with a number of dietary risk factors top the global mortality statistics for noncommunicable diseases. The so-called Western diet and an increasingly sedentary lifestyle are partly responsible for the high prevalence of hypertension. A healthier diet has a major role in the prevention and treatment of hypertension. Given the wide range of options for dietary modifications, it is necessary to define important evidence-based cornerstones of a blood pressure-lowering diet and to assess its blood pressure-lowering potential. While extensive evidence has been generated in recent decades and guidelines emphasize healthier diets, implementation of dietary modifications remains a challenge in everyday clinical practice. Information and education as well as medical and nutritional support for patients can help to implement measures, such as weight and sodium restriction in the long term to improve the prognosis of patients with hypertension.

[Gut-heart axis : How gut bacteria influence cardiovascular diseases]. / Darm-Herz-Achse : Wie Darmbakterien kardiovaskuläre Erkrankungen beeinflussen.

The view of humans as holobionts consisting of eukaryotic host cells and associated prokaryotic organisms, has opened up a new perspective on cardiovascular pathophysiology. In particular, intestinal bacteria influence the cell and organ functions of the host. Intestinal bacteria represent a metabolically active community whose composition and function can influence cardiovascular health and disease. The interaction between the intestinal microbiota and the heart occurs via metabolites of bacterial origin, which are resorbed in the intestine and distributed via the circulation. Bacterial metabolites are produced from food components, which in turn emphasizes the importance of nutrition. Some of these metabolites, such as trimethylamine N­oxide (TMAO), can exacerbate cardiovascular pathologies. Short-chain fatty acids (SCFA) in turn are considered to be protective metabolites. The host's immune system is an important target for these metabolites and explains much of their effects. In the future, the targeted manipulation of intestinal bacteria could help to prevent the development and progression of cardiovascular diseases.

The metastasis-promoting phosphatase PRL-3 shows activity toward phosphoinositides.

Phosphatase of regenerating liver 3 (PRL-3) is suggested as a biomarker and therapeutic target in several cancers. It has a well-established causative role in cancer metastasis. However, little is known about its natural substrates, pathways, and biological functions, and only a few protein substrates have been suggested so far. To improve our understanding of the substrate specificity and molecular determinants of PRL-3 activity, the wild-type (WT) protein, two supposedly catalytically inactive mutants D72A and C104S, and the reported hyperactive mutant A111S were tested in vitro for substrate specificity and activity toward phosphopeptides and phosphoinositides (PIPs), their structural stability, and their ability to promote cell migration using stable HEK293 cell lines. We discovered that WT PRL-3 does not dephosphorylate the tested phosphopeptides in vitro. However, as shown by two complementary biochemical assays, PRL-3 is active toward the phosphoinositide PI(4,5)P(2). Our experimental results substantiated by molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. The C104S variant was shown to be not only catalytically inactive but also structurally destabilized and unable to promote cell migration, whereas WT PRL-3 promotes cell migration. The D72A mutant is structurally stable and does not dephosphorylate the unnatural substrate 3-O-methylfluorescein phosphate (OMFP). However, we observed residual in vitro activity of D72A against PI(4,5)P(2), and in accordance with this, it exhibits the same cellular phenotype as WT PRL-3. Our analysis of the A111S variant shows that the hyperactivity toward the unnatural OMFP substrate is not apparent in dephosphorylation assays with phosphoinositides: the mutant is completely inactive against PIPs. We observed significant structural destabilization of this variant. The cellular phenotype of this mutant equals that of the catalytically inactive C104S mutant. These results provide a possible explanation for the absence of the conserved Ser of the PTP catalytic motif in the PRL family. The correlation of the phosphatase activity toward PI(4,5)P(2) with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P(2) dephosphorylation by PRL-3 and its role in cell migration.

Dynamics in the unfolded state of beta2-microglobulin studied by NMR.

Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.

Structural properties of an amyloid precursor of beta(2)-microglobulin.

The population of one or more partially folded states has been proposed as a critical initial step in amyloid formation for several proteins. Here we use equilibrium denaturation measured by (1)H-(15)N NMR to determine the conformational properties of an amyloidogenic intermediate of human beta(2)-microglobulin (beta(2)m) formed at low pH. The data show that this amyloid precursor is a noncooperatively stabilized ensemble that retains stable structure in five of the seven beta-strands that comprise the native fold. The amyloid precursors of beta(2)m and transthyretin have similar properties despite having structurally unrelated native folds. The data offer a rationale as to why these proteins are both amyloidogenic at low pH and suggest that amyloidosis of these and other proteins may involve ordered assembly from a precursor with similar conformational features.