Lactiplantibacillus plantarum from Unexplored Tunisian Ecological Niches: Antimicrobial Potential, Probiotic and Food Applications.

The continued exploration of the diversity of lactic acid bacteria in little-studied ecological niches represents a fundamental activity to understand the diffusion and biotechnological significance of this heterogeneous class of prokaryotes. In this study, Lactiplantibacillus plantarum (Lpb. plantarum) strains were isolated from Tunisian vegetable sources, including fermented olive and fermented pepper, and from dead locust intestines, which were subsequently evaluated for their antimicrobial activity against foodborne pathogenic bacteria, including Escherichia coli O157:H7 CECT 4267 and Listeria monocytogenes CECT 4031, as well as against some fungi, including Penicillium expansum, Aspergilus niger, and Botrytis cinerea. In addition, their resistance to oro-gastro-intestinal transit, aggregation capabilities, biofilm production capacity, adhesion to human enterocyte-like cells, and cytotoxicity to colorectal adenocarcinoma cell line were determined. Further, adhesion to tomatoes and the biocontrol potential of this model food matrix were analyzed. It was found that all the strains were able to inhibit the indicator growth, mostly through organic acid production. Furthermore, these strains showed promising probiotic traits, including in vitro tolerance to oro-gastrointestinal conditions, and adhesion to abiotic surfaces and Caco-2 cells. Moreover, all tested Lpb. plantarum strains were able to adhere to tomatoes with similar rates (4.0-6.0 LogCFU/g tomato). The co-culture of LAB strains with pathogens on tomatoes showed that Lpb. plantarum could be a good candidate to control pathogen growth. Nonetheless, further studies are needed to guarantee their use as probiotic strains for biocontrol on food matrices.

In vivo evaluation of an innovative synbiotics on stage IIIb-IV chronic kidney disease patients.

Background: Microbiota unbalance has been proven to affect chronic kidney disease (CKD) patients and, noteworthy, microbiota composition and activity are implicated in CKD worsening. The progression of kidney failure implies an exceeding accumulation of waste compounds deriving from the nitrogenous metabolism in the intestinal milieu. Therefore, in the presence of an altered intestinal permeability, gut-derived uremic toxins, i.e., indoxyl sulfate (IS) and p-cresyl sulfate (PCS), can accumulate in the blood. Methods: In a scenario facing the nutritional management as adjuvant therapy, the present study assessed the effectiveness of an innovative synbiotics for its ability to modulate the patient gut microbiota and metabolome by setting a randomized, single-blind, placebo-controlled, pilot trial accounting for IIIb-IV stage CKD patients and healthy controls. Metataxonomic fecal microbiota and fecal volatilome were analyzed at the run-in, after 2 months of treatment, and after 1 month of wash out. Results: Significant changes in microbiota profile, as well as an increase of the saccharolytic metabolism, in feces were found for those CKD patients that were allocated in the synbiotics arm. Conclusions: Noteworthy, the here analyzed data emphasized a selective efficacy of the present synbiotics on a stage IIIb-IV CKD patients. Nonetheless, a further validation of this trial accounting for an increased patient number should be considered. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT03815786.

The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.

The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

A Gel-Based Proteomic Analysis Reveals Synovial α-Enolase and Fibrinogen ß-Chain Dysregulation in Knee Osteoarthritis: A Controlled Trial.

BACKGROUND: The identification of synovial fluid (SF) biomarkers that could anticipate the diagnosis of osteoarthritis (OA) is gaining increasing importance in orthopaedic clinical practice. This controlled trial aims to assess the differences between the SF proteome of patients affected by severe OA undergoing Total Knee Replacement (TKR) compared to control subjects (i.e., subjects younger than 35, undergoing knee arthroscopy for acute meniscus injury). METHODS: The synovial samples were collected from patients with Kellgren Lawrence grade 3 and 4 knee osteoarthritis undergoing THR (study group) and young patients with meniscal tears and no OA signs undergoing arthroscopic surgery (control group). The samples were processed and analyzed following the protocol defined in our previous study. All of the patients underwent clinical evaluation using the International Knee Documentation Committee (IKDC) subjective knee evaluation (main outcome), Knee Society Clinical Rating System (KSS), Knee injury and Osteoarthritis Outcome Score (KOOS), and Visual Analogue Scale (VAS) for pain. The drugs' assumptions and comorbidities were recorded. All patients underwent preoperative serial blood tests, including complete blood count and C-Reactive Protein (CRP). RESULTS: The synovial samples' analysis showed a significantly different fibrinogen beta chain (FBG) and alpha-enolase 1 (ENO1) concentration in OA compared to the control samples. A significant correlation between clinical scores, FBG, and ENO1 concentration was observed in osteoarthritic patients. CONCLUSIONS: Synovial fluid FBG and ENO1 concentrations are significantly different in patients affected by knee OA compared with non-OA subjects.

Multi-Faceted Role of Luteolin in Cancer Metastasis: EMT, Angiogenesis, ECM Degradation and Apoptosis.

Luteolin (3',4',5,7-tetrahydroxyflavone), a member of the flavonoid family derived from plants and fruits, shows a wide range of biomedical applications. In fact, due to its anti-inflammatory, antioxidant and immunomodulatory activities, Asian medicine has been using luteolin for centuries to treat several human diseases, including arthritis, rheumatism, hypertension, neurodegenerative disorders and various infections. Of note, luteolin displays many anti-cancer/anti-metastatic properties. Thus, the purpose of this review consists in highlighting the relevant mechanisms by which luteolin inhibits tumor progression in metastasis, i.e., affecting epithelial-mesenchymal transition (EMT), repressing angiogenesis and lysis of extracellular matrix (ECM), as well as inducing apoptosis.

Immunomodulatory Activity on Human Macrophages by Cell-Free Supernatants to Explore the Probiotic and Postbiotic Potential of Lactiplantibacillus plantarum Strains of Plant Origin.

Upon dietary administration, probiotic microorganisms can reach as live cells the human gut, where they interact with the microbiota and host cells, thereby exerting a beneficial impact on host functions, mainly through immune-modulatory activities. Recently, attention has been drawn by postbiotics, i.e. non-viable probiotic microbes, including their metabolic products, which possess biological activities that benefit the host. Lactiplantibacillus plantarum is a bacterial species that comprises recognised probiotic strains. In this study, we investigated in vitro the probiotic (and postbiotic) potential of seven L. plantarum strains, including five newly isolated from plant-related niches. The strains were shown to possess some basic probiotic attributes, including tolerance to the gastrointestinal environment, adhesion to the intestinal epithelium and safety. Besides, their cell-free culture supernatants modulated cytokine patterns in human macrophages in vitro, promoting TNF-α gene transcription and secretion, while attenuating the transcriptional activation and secretion of both TNF-α and IL-8 in response to a pro-inflammatory signal, and enhancing the production of IL-10. Some strains induced a high IL-10/IL-12 ratio that may correlate to an anti-inflammatory capacity in vivo. Overall, the investigated strains are good probiotic candidates, whose postbiotic fraction exhibits immunomodulatory properties that need further in vivo studies. The main novelty of this work consists in the polyphasic characterisation of candidate beneficial L. plantarum strains obtained from relatively atypical plant-associated niches, by an approach that explores both probiotic and postbiotic potentials, in particular studying the effect of microbial culture-conditioned media on cytokine pattern, analysed at both transcriptional and secretion level in human macrophages.

Molecular chaperone function of three small heat-shock proteins from a model probiotic species.

Small heat-shock proteins (sHSP) are ubiquitous ATP-independent chaperones that prevent irreversible aggregation of heat-damaged denaturing proteins. Lactiplantibacillus plantarum is a widespread Gram-positive bacterium with probiotic claims and vast potential for agro-food, biotechnological and biomedical applications. L. plantarum possesses a family of three sHSP, which were previously demonstrated to be involved in its stress tolerance mechanisms. Here, the three L. plantarum sHSP were heterologously expressed, purified and shown to have a chaperone activity in vitro, measuring their capacity to suppress protein aggregation, as assayed spectrophotometrically by light scattering. Their anti-aggregative capacity was found to be differently influenced by pH. Differences were also found relative to their holdase function and their capacity to modulate liposome membrane fluidity, suggesting interplays between them and indicating diversified activities. This is the first study assessing the chaperone action of sHSP from a probiotic model. The different roles of the three sHSP can increase L. plantarum's capabilities to survive the various types of stress characterising the diverse habitats of this highly adaptable species. Reported evidence supports the interest in L. plantarum as one of the model species for bacteria that have three different sHSP-encoding genes in their genomes.

Urinary epidermal growth factor/monocyte chemotactic peptide 1 ratio as non-invasive predictor of Mayo clinic imaging classes in autosomal dominant polycystic kidney disease.

BACKGROUND: Age- and height-adjusted total kidney volume is currently considered the best prognosticator in patients with autosomal dominant polycystic kidney disease. We tested the ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 for the prediction of the Mayo Clinic Imaging Classes. METHODS: Urinary epidermal growth factor and monocyte chemotactic peptide 1 levels were measured in two independent cohorts (discovery, n = 74 and validation set, n = 177) and healthy controls (n = 59) by immunological assay. Magnetic resonance imaging parameters were used for total kidney volume calculation and the Mayo Clinic Imaging Classification defined slow (1A-1B) and fast progressors (1C-1E). Microarray and quantitative gene expression analysis were used to test epidermal growth factor and monocyte chemotactic peptide 1 gene expression. RESULTS: Baseline ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 correlated with total kidney volume adjusted for height (r = - 0.6, p < 0.001), estimated glomerular filtration rate (r = 0.69 p < 0.001), discriminated between Mayo Clinic Imaging Classes (p < 0.001), and predicted the variation of estimated glomerular filtration rate at 10 years (r = - 0.51, p < 0.001). Conditional Inference Trees identified cut-off levels of the ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 for slow and fast progressors at > 132 (100% slow) and < 25.76 (89% and 86% fast, according to age), with 94% sensitivity and 66% specificity (p = 6.51E-16). Further, the ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 at baseline showed a positive correlation (p = 0.006, r = 0.36) with renal outcome (delta-estimated glomerular filtration rate per year, over a mean follow-up of 4.2 ± 1.2 years). Changes in the urinary epidermal growth factor and monocyte chemotactic peptide 1 were mirrored by gene expression levels in both human kidney cysts (epidermal growth factor: - 5.6-fold, fdr = 0.001; monocyte chemotactic peptide 1: 3.1-fold, fdr = 0.03) and Pkd1 knock-out mouse kidney (Egf: - 14.8-fold, fdr = 2.37E-20, Mcp1: 2.8-fold, fdr = 6.82E-15). CONCLUSION: The ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 is a non-invasive pathophysiological biomarker that can be used for clinical risk stratification in autosomal dominant polycystic kidney disease.

Antimicrobial Properties, Functional Characterisation and Application of Fructobacillus fructosus and Lactiplantibacillus plantarum Isolated from Artisanal Honey.

Honey is a valuable reservoir of lactic acid bacteria (LAB) and, particularly, of fructophilic LAB (FLAB), a relatively novel subgroup of LAB whose functional potential for human and food application has yet to be explored. In this study, FLAB and LAB strains have been isolated from honeys of different floral origins and selected for their broad antimicrobial activity against typical foodborne pathogenic bacteria and spoilage filamentous fungi. The best candidates, two strains belonging to the species Lactiplantibacillus plantarum and Fructobacillus fructosus, were submitted to partial characterisation of their cell free supernatants (CFS) in order to identify the secreted metabolites with antimicrobial activity. Besides, these strains were examined to assess some major functional features, including in vitro tolerance to the oro-gastrointestinal conditions, potential cytotoxicity against HT-29 cells, adhesion to human enterocyte-like cells and capability to stimulate macrophages. Moreover, when the tested strains were applied on table grapes artificially contaminated with pathogenic bacteria or filamentous fungi, they showed a good ability to antagonise the growth of undesired microbes, as well as to survive on the fruit surface at a concentration that is recommended to develop a probiotic effect. In conclusion, both LAB and FLAB honey-isolated strains characterised in this work exhibit functional properties that validate their potential use as biocontrol agents and for the design of novel functional foods. We reported antimicrobial activity, cytotoxic evaluation, probiotic properties and direct food application of a F. fructosus strain, improving the knowledge of this species, in particular, and on FLAB, more generally.

Improving the In Vitro Removal of Indoxyl Sulfate and p-Cresyl Sulfate by Coating Diatomaceous Earth (DE) and Poly-vinyl-pyrrolidone-co-styrene (PVP-co-S) with Polydopamine.

Polydopamine (PDA) is a synthetic eumelanin polymer mimicking the biopolymer secreted by mussels to attach to surfaces with a high binding strength. It exhibits unique adhesive properties and has recently attracted considerable interest as a multifunctional thin film coating. In this study, we demonstrate that a PDA coating on silica- and polymer-based materials improves the entrapment and retention of uremic toxins produced in specific diseases. The low-cost natural nanotextured fossil diatomaceous earth (DE), an abundant source of mesoporous silica, and polyvinylpyrrolidone-co-Styrene (PVP-co-S), a commercial absorbent comprising polymeric particles, were easily coated with a PDA layer by oxidative polymerization of dopamine at mild basic aqueous conditions. An in-depth chemical-physical investigation of both the resulting PDA-coated materials was performed by SEM, AFM, UV-visible, Raman spectroscopy and spectroscopic ellipsometry. Finally, the obtained hybrid systems were successfully tested for the removal of two uremic toxins (indoxyl sulfate and p-cresyl sulfate) directly from patients' sera.

Metabolic Fingerprinting of Fabry Disease: Diagnostic and Prognostic Aspects.

Fabry disease (FD) is an X-linked lysosomal disease due to a deficiency in the activity of the lysosomal-galactosidase A (GalA), a key enzyme in the glycosphingolipid degradation pathway. FD is a complex disease with a poor genotype-phenotype correlation. In the early stages, FD could involve the peripheral nervous system (acroparesthesias and dysautonomia) and the ski (angiokeratoma), but later kidney, heart or central nervous system impairment may significantly decrease life expectancy. The advent of omics technologies offers the possibility of a global, integrated and systemic approach well-suited for the exploration of this complex disease. In this narrative review, we will focus on the main metabolomic studies, which have underscored the importance of detecting biomarkers for a diagnostic and prognostic purpose in FD. These investigations are potentially useful to explain the wide clinical, biochemical and molecular heterogeneity found in FD patients. Moreover, the quantitative mass spectrometry methods developed to evaluate concentrations of these biomarkers in urine and plasma will be described. Finally, the complex metabolic biomarker profile depicted in FD patients will be reported, which varies according to gender, types of mutations, and therapeutic treatment.

New Strategies for the Reduction of Uremic Toxins: How Much More We Know.

The importance of uremic toxin (UTx) removal in chronic kidney disease (CKD) is an emerging topic in the literature, widely recognized over time as a strategy to slow-down the disease progression towards end-stage renal disease and, consequentely, the occurence of deleterious effects on cardiovascular (CV) system [...].

Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action.

Lactiplantibacillus plantarum (L. plantarum) is a well-studied and versatile species of lactobacilli. It is found in several niches, including human mucosal surfaces, and it is largely employed in the food industry and boasts a millenary tradition of safe use, sharing a long-lasting relationship with humans. L. plantarum is generally recognised as safe and exhibits a strong probiotic character, so that several strains are commercialised as health-promoting supplements and functional food products. For these reasons, L. plantarum represents a valuable model to gain insight into the nature and mechanisms of antimicrobials as key factors underlying the probiotic action of health-promoting microbes. Probiotic antimicrobials can inhibit the growth of pathogens in the gut ensuring the intestinal homeostasis and contributing to the host health. Furthermore, they may be attractive alternatives to conventional antibiotics, holding potential in several biomedical applications. The aim of this review is to investigate the most relevant papers published in the last ten years, bioprospecting the antimicrobial activity of characterised probiotic L. plantarum strains. Specifically, it focuses on the different chemical nature, the action spectra and the mechanisms underlying the bioactivity of their antibacterial and antiviral agents. Emerging trends in postbiotics, some in vivo applications of L. plantarum antimicrobials, including strengths and limitations of their therapeutic potential, are addressed and discussed.

A neural network for glomerulus classification based on histological images of kidney biopsy.

BACKGROUND: Computer-aided diagnosis (CAD) systems based on medical images could support physicians in the decision-making process. During the last decades, researchers have proposed CAD systems in several medical domains achieving promising results. CAD systems play an important role in digital pathology supporting pathologists in analyzing biopsy slides by means of standardized and objective workflows. In the proposed work, we designed and tested a novel CAD system module based on image processing techniques and machine learning, whose objective was to classify the condition affecting renal corpuscles (glomeruli) between sclerotic and non-sclerotic. Such discrimination is useful for the biopsy slides evaluation performed by pathologists. RESULTS: We collected 26 digital slides taken from the kidneys of 19 donors with Periodic Acid-Schiff staining. Expert pathologists have conducted the slides preparation, digital acquisition and glomeruli annotations. Before setting the classifiers, we evaluated several feature extraction techniques from the annotated regions. Then, a feature reduction procedure followed by a shallow artificial neural network allowed discriminating between the glomeruli classes. We evaluated the workflow considering an independent dataset (i.e., processing images not used in the training procedure). Ten independent runs of the training algorithm, and evaluation, allowed achieving MCC and Accuracy of 0.95 (± 0.01) and 0.99 (standard deviation < 0.00), respectively. We also obtained good precision (0.9844 ± 0.0111) and recall (0.9310 ± 0.0153). CONCLUSIONS: Results on the test set confirm that the proposed workflow is consistent and reliable for the investigated domain, and it can support the clinical practice of discriminating the two classes of glomeruli. Analyses on misclassifications show that the involved images are usually affected by staining artefacts or present partial sections due to slice preparation and staining processes. In clinical practice, however, pathologists discard images showing such artefacts.

The Pathogenic Role of PI3K/AKT Pathway in Cancer Onset and Drug Resistance: An Updated Review.

The PI3K/AKT pathway is one of the most frequently over-activated intracellular pathways in several human cancers. This pathway, acting on different downstream target proteins, contributes to the carcinogenesis, proliferation, invasion, and metastasis of tumour cells. A multi-level impairment, involving mutation and genetic alteration, aberrant regulation of miRNAs sequences, and abnormal phosphorylation of cascade factors, has been found in multiple cancer types. The deregulation of this pathway counteracts common therapeutic strategies and contributes to multidrug resistance. In this review, we underline the involvement of this pathway in patho-physiological cell survival mechanisms, emphasizing its key role in the development of drug resistance. We also provide an overview of the potential inhibition strategies currently available.

An Innovative Synbiotic Formulation Decreases Free Serum Indoxyl Sulfate, Small Intestine Permeability and Ameliorates Gastrointestinal Symptoms in a Randomized Pilot Trial in Stage IIIb-IV CKD Patients.

Proteolytic dysbiosis of the gut microbiota has been recognized as both a typical feature of chronic kidney disease (CKD) and a risk factor for its progression. Blood accumulation of gut-derived uremic toxins (UTs) like indoxyl sulfate (IS) and p-cresyl sulfate (PCS), intestinal permeability and constipation are typical features accompanying CKD progression and triggering chronic inflammation. In order to verify the efficacy of the innovative synbiotic formulation NATUREN G® in modulating the levels of circulating UTs, intestinal permeability and gastrointestinal symptoms, we set up a randomized, single-blind, placebo-controlled, pilot trial in stage IIIb-IV CKD patients and in healthy controls. Two-month administration of the synbiotic resulted in a decrease of free IS, as compared with the placebo-treated arm, only in the CKD group. The other UTs did not significantly change, although different trends in time (increase in the placebo arm and decrease in the synbiotic arm) were observed. Moreover, after supplementation, reduction of small intestinal permeability and amelioration of abdominal pain and constipation syndromes were observed only in the CKD group. The obtained results suggest the specificity of action of NATUREN G® in CKD and justify further validation in a wider study population.

Protein-Bound Uremic Toxins and Immunity.

Protein-bound uremic toxins (PBUTs) are bioactive microbiota metabolites originated exclusively from protein fermentation of the bacterial community resident within the gut microbiota, whose composition and function is profoundly different in the chronic kidney disease (CKD) population. PBUTs accumulate in the later stages of CKD because they cannot be efficiently removed by conventional hemodialysis due to their high binding affinity for albumin, worsening their toxic effects, especially at the cardiovascular level. The accumulation of uremic toxins, along with oxidative stress products and pro-inflammatory cytokines, characterizes the uremic status of CKD patients which is increasingly associated to a state of immune dysfunction including both immune activation and immunodepression. Furthermore, the links between immune activation and cardiovascular disease (CVD), and between immunodepression and infection diseases, which are the two major complications of CKD, are becoming more and more evident. This review summarizes and discusses the current state of knowledge on the role of the main PBUTs, namely indoxyl sulfate and p-cresyl sulfate, as regulators of immune response in CKD, in order to understand whether a microbiota modulation may be useful in the management of its main complications, CVD, and infections. Summarizing the direct effects of PBUT on immune system we may conclude that PCS seemed to be associated to an immune deficiency status of CKD mainly related to the adaptative immune response, while IS seemed to reflect the activation of both innate and adaptative immune systems likely responsible of the CKD-associated inflammation. However, the exact role of IS and PCS on immunity modulation in physiological and pathological state still needs in-depth investigation, particularly in vivo studies.

Gut Microbiota, the Immune System, and Cytotoxic T Lymphocytes.

Gut microbiota, the largest microbial community living in the human body, exerts a variety of metabolic, structural, and functional actions. In particular, it is essential for the full immune system development and maturation, as demonstrated by studies on germ-free animals, showing immune impairment at different levels. Gut microbiota shapes the immune responses by promoting immune tolerance toward food antigens and commensals in the steady state. This process is orchestrated by a complex network of both microbial and human cells and molecular mediators. Microbiota eubiosis is fundamental in establishing a correct balance between tolerance and immunity. Contrarily, microbiota dysbiosis is correlated with alterations in the immune balance, as evidenced in intestinal pathologies characterized by aberrant immune responses, such as inflammatory bowel disease and celiac disease, in which either break of tolerance against commensals or microbial dysbiosis is reported. On the other hand, a role for gut microbiota in stimulating the cytotoxic immune response in contexts of immunosuppression, like the ones featuring tumors and vaccinations, is emerging. The bifaceted role of gut microbiota in the delicate balance between tolerance and immunity could be exploited in order to develop pioneering therapeutic strategies, complementary to the pharmacological ones, thus representing a field worthy of further studies specifically focused on this topic.

The Ambivalent Role of miRNAs in Carcinogenesis: Involvement in Renal Cell Carcinoma and Their Clinical Applications.

The analysis of microRNA (miRNAs), small, non-coding endogenous RNA, plays a crucial role in oncology. These short regulatory sequences, acting on thousands of messenger RNAs (mRNAs), modulate gene expression at the transcriptional and post-transcriptional level leading to translational repression or degradation of target molecules. Although their function is required for several physiological processes, such as proliferation, apoptosis and cell differentiation, miRNAs are also responsible for development and/or progression of several cancers, since they may interact with classical tumor pathways. In this review, we highlight recent advances in deregulated miRNAs in cancer focusing on renal cell carcinoma (RCC) and provide an overview of the potential use of miRNA in their clinical settings, such as diagnostic and prognostic markers.

Ketoanalogs' Effects on Intestinal Microbiota Modulation and Uremic Toxins Serum Levels in Chronic Kidney Disease (Medika2 Study).

Nutritional therapy (NT) is a therapeutic option in the conservative treatment of chronic kidney disease (CKD) patients to delay the start of dialysis. The aim of this study was to evaluate the specific effect of ketoanalogs (KA)-supplemented diets for gut microbiota modulation. In a previous study we observed that the Mediterranean diet (MD) and a KA-supplemented very-low-protein diet (VLPD) modulated beneficially gut microbiota, reducing indoxyl- and p-cresyl-sulfate (IS, PCS) serum levels, and ameliorating the intestinal permeability in CKD patients. In the current study, we added a third diet regimen consisting of KA-supplemented MD. Forty-three patients with CKD grades 3B-4 continuing the crossover clinical trial were assigned to six months of KA-supplemented MD (MD + KA). Compared to MD, KA-supplementation in MD + KA determined (i) a decrease of Clostridiaceae, Methanobacteriaceae, Prevotellaceae, and Lactobacillaceae while Bacteroidaceae and Lachnospiraceae increased; (ii) a reduction of total and free IS and PCS compared to a free diet (FD)-more than the MD, but not as effectively as the VLPD. These results further clarify the driving role of urea levels in regulating gut integrity status and demonstrating that the reduction of azotemia produced by KA-supplemented VLPD was more effective than KA-supplemented MD in gut microbiota modulation mainly due to the effect of the drastic reduction of protein intake rather than the effect of KA.