Background The intestinal microbiome is an appealing target for interventions in ESKD because of its likely contribution to uremic toxicity. Before conducting clinical trials of microbiome-altering treatments, it is necessary to understand the within-person and between-person variability in the composition and function of the gut microbiome in patients with ESKD. Methods We conducted a multicenter, nonrandomized, crossover feasibility study of patients on maintenance hemodialysis consisting of three phases: pretreatment (8 weeks); treatment, during which the prebiotic, p-inulin, was administered at a dosage of 8 g twice daily (12 weeks); and post-treatment (8 weeks). Stool samples were collected 1-2 times per week and blood was collected weekly for 28 weeks. The gut microbiome was characterized using 16S ribosomal-RNA sequencing and metabolomic profiling. Results A total of 11 of the 13 participants completed the 28-week study. Interparticipant variability was greater than intraparticipant variability for microbiome composition (P<0.001 by UniFrac distances) and metabolomic composition (P<0.001 by Euclidean distances). p-Inulin was well tolerated by 12 of 13 participants. Adherence to the frequent sample collection and self-aliquoting of stool samples were both 96%. A change in the microbiome composition from pretreatment to post-treatment was evident by the overall shifts in weighted UniFrac distances (P=0.004) and a progressive decrease in prevalence of high intraclass correlations, indicating an increase in intraparticipant microbiome diversity during and after p-inulin treatment. An effect of p-inulin on the metabolomic profile was not evident. Conclusions The intraparticipant stability of the gut microbiome under no-treatment conditions, the tolerability of p-inulin, the signals of increased diversity of the microbiome with p-inulin treatment, and the willingness of participants to provide stool samples all support the feasibility of a larger trial to investigate interventions targeting the gut microbiome in patients with ESKD. Whether or not p-inulin has sufficient efficacy as an intervention requires evaluation in larger studies. Clinical Trial registry name and registration number Gut Microbiome and p-Inulin in Hemodialysis, NCT02572882.

Peritoneal dialysis (PD) patients have a distinct uremic toxin profile and metabolic abnormalities, which could be related in part to the differences in gut microbiota.1Choi J.-Y. Yoon Y.J. Choi H.J. et al.Dialysis modality-dependent changes in serum metabolites: accumulation of inosine and hypoxanthine in patients on haemodialysis.Nephrol Dial Transplant. 2011; 26: 1304-1313Crossref PubMed Scopus (57) Google Scholar Prebiotics are microbial feed supplements that beneficially affect the host by improving its intestinal microbial balance and re-establishing symbiosis. Treatment with the prebiotic, oligofructose-enriched p-inulin (p-inulin) is believed to enable proliferation of beneficial gut bacteria such as Bifidobacteria, to attenuate inflammation, and to improve metabolic functions.2Gibson G.R. Beatty E.R. Wang X. Cummings J.H. Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin.Gastroenterology. 1995; 108: 975-982Abstract Full Text PDF PubMed Scopus (1215) Google Scholar,3Cani P.D. Neyrinck A.M. Fava F. et al.Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia.Diabetologia. 2007; 50: 2374-2383Crossref PubMed Scopus (1243) Google Scholar The aims of this pilot study are (i) to compare the microbiome profile in PD patients and individuals without kidney disease, and (ii) to evaluate the effect of p-inulin treatment on inflammatory biomarkers and microbiome community composition and to associate it with host metabolomic profile. In a nonrandomized crossover study, 8 PD patients were followed during 3 sequential 8-week phases of no intervention, p-inulin (16 g/d) administration, and a post-intervention phase with no intervention (Supplementary Table S1). We also studied 7 individuals without kidney disease. Patient characteristics are summarized in Supplementary Table S2. Patients’ dietary intake and gastrointestinal symptoms are summarized in Supplementary Tables S3 and S4. We measured several markers of inflammation, but only plasma-level soluble CD14 (sCD14) was significantly lower at week 16 compared to that in the pretreatment period at week 8 (4267 ± 195.8 vs. 4110 ± 191.6 mg/ml, P < 0.02) (Figure 1a− d). The 5 most abundant phyla identified in PD patients, in decreasing order, were Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia (Supplementary Figure S1A). Alpha diversity was not significantly different between controls and PD patients (Supplementary Figure 2A). We also did not observe a significant change in alpha diversity with p-inulin treatment (Supplementary Figure 2B). Intersubject variability in community composition was significantly higher than intrasubject variability (Wilcoxon rank-sum, P < 2e−16) (Figure 2c). With some exceptions, the overall gut microbiome composition of PD patients did not differ from that of controls (adonis P = 1) (Figure 2d). Linear discriminant analysis effect size (LEfSe) analysis revealed that 14 taxa were more abundant in PD patients at baseline compared to healthy controls, whereas 5 taxa were more abundant in controls (Figure 2e). Eight weeks of p-inulin treatment did not cause a reduction in cluster density, indicating that p-inulin intervention did not overcome intersubject variation (Figure 2d). To further evaluate the effect of p-inulin treatment, we performed random-effects mixed models controlling for within-subject autocorrelation. A total of 86 bacterial strains (P < 0.05) were significantly altered by p-inulin intervention, with 55 increased and 31 reduced during the intervention phase compared with baseline (Figure 3). Among 55 bacterial strains that increased during the intervention phase, 17 were significantly altered during the post-intervention phase compared with intervention phase, with 8 still increased and 9 decreased (Figure 3a). Among 31 bacterial strains that were reduced during p-inulin treatment, 8 increased significantly during the post-intervention phase (Figure 3b). To assess the effect of diet on the gut microbiota, we performed multivariate analysis with linear models (MaAsLin2) using default parameters, and found a significant positive correlation among Bacteroides, dietary fiber, and carbohydrate consumption (Supplementary Figure S1B and Supplementary Table S5).Figure 3Impact of p-inulin intervention on bacterial strains. (a) Increased bacterial strains at intervention phase compared with baseline (overall P < 0.05; intervention vs. baseline P < 0.05). Red indicates increased strains at post-intervention (Post) phase compared with intervention phase (overall P < 0.05; Post vs. baseline P < 0.05). Blue indicates decreased strains at Post phase compared with intervention phase (overall P < 0.05; Post vs. baseline P < 0.05). (b) Decreased bacterial strains at intervention phase compared with baseline (overall P < 0.05; intervention vs. baseline P < 0.05). Red text indicates increased strains at Post phase compared with intervention phase (overall P < 0.05; Post vs. baseline P < 0.05). Blue text indicates decreased strains at Post phase compared with intervention phase (overall P < 0.05; Post vs. baseline P < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Kyoto Encyclopedia of Genes and Genomes (KEGG) modules were used to characterize the gene sets linked to microbial metabolic capabilities. Functional alpha diversity was not significantly different (Supplementary Figure S1C). While assessing beta diversity, we noted that that the donor effect persisted irrespective of the study phase (Supplementary Figure S1D). LEfSe analysis showed that 9 KEGG modules were significantly enriched in PD patients compared to control subjects, whereas 2 KEGG modules were significantly decreased (Figure 4a). Furthermore, we examined the time effects on KEGG modules during the 3 phases of the study. Among the 11 KEGG modules, which were different between PD patients at baseline and controls, p-inulin treatment effects were significant (overall P < 0.05; intervention vs. baseline P < 0.05) for 2 KEGG modules (Figure 4b and c). In addition, we noted significant treatment effects on 33 KEGG modules, with 17 KEGG modules increased (Figure 4d) and 16 reduced after p-inulin treatment compared to baseline (Figure 4e). During the post-intervention phase, some of these changes were reversed, whereas others persisted (Figure 4d and e). The interpatient variability was greater than the intrapatient variability (Figure 5a). p-Inulin treatment significantly altered 13 metabolites (overall P < 0.05; intervention vs. baseline P < 0.05), with 7 increased and 6 decreased during the treatment phase compared to baseline (Figure 5b). Using weekly data, significant overall time effects were found for TMAO, IS (both P < 0.0001), and PCS (P = 0.004) (Figure 5c). Top bacterial strain correlated with each metabolite is shown in Figure 5d. Next, we examined the bacterial enzymes involved in IS, PCS, and TMAO biosynthesis pathways in each patient. The trends between bacterial enzymes and respective metabolite changes during 3 phases of the study varied among patients. Interestingly, the changing trajectory of tryptophanase and IS were similar in selected patient (Figure 5e). In addition, a similar changing trajectory was found among amino acid transaminase and PCS, betaine reductase, TMAO reductase, and TMAO (Figure 5e). These results suggest that the functional changes in microbial gene abundance affects the levels of IS, PCS, and TMAO to some extent in some patients. The symbiotic relationship between humans and microbiota allows for co-metabolism of diverse substrates. As the final readout, the plasma metabolome revealed the host and microbiota co-metabolism. We integrated the untargeted metabolomics, lipidomics, and targeted metabolomics (IS, PCS, TMAO) with metagenomics to reveal the microbiota-host co-metabolism. Previous studies have shown that oligofructose inulin significantly reduced PCS generation rates and serum concentrations in hemodialysis patients, but had no effect on IS.4Meijers B.K.I. et al.p-Cresyl Sulfate and Indoxyl Sulfate in Hemodialysis Patients.Clin J Am Soc Nephrol. 2009; 4: 1932-1938Crossref PubMed Scopus (120) Google Scholar A randomized controlled trial showed that resistant starch decreased IS level in patients treated with hemodialysis.5Sirich T.L. Plummer N.S. Gardner C.D. et al.Effect of increasing dietary fiber on plasma levels of colon-derived solutes in hemodialysis patients.Clin J Am Soc Nephrol. 2014; 9: 1603-1610Crossref PubMed Scopus (164) Google Scholar In patients with CKD, synbiotics (a combination of prebiotic and probiotic) did not significantly reduce serum IS but did decrease serum PCS.6Rossi M. et al.Synbiotics Easing Renal Failure by Improving Gut Microbiology (SYNERGY): a randomized trial.Clin J Am Soc Nephrol. 2016; 11: 223-231Crossref PubMed Scopus (176) Google Scholar However, in another small study in hemodialysis patients, synbiotic treatment reduced PCS but not IS.7Nakabayashi I. Nakamura M. Kawakami K. et al.Effects of synbiotic treatment on serum level of p-cresol in haemodialysis patients: a preliminary study.Nephrol Dial Transplant. 2011; 26: 1094-1098Crossref PubMed Scopus (148) Google Scholar In this small but well-designed study with stringent patient selection criteria, we demonstrate that the host co-metabolic pathways are modulated by p-inulin treatment in PD patients. To our knowledge this is one of the first studies to investigate microbial and host metabolic responses to p-inulin supplementation in PD patients. Other strengths include rigorous patient selection criteria, study design, shotgun metagenomic sequencing, and use of targeted and untargeted metabolomic approaches. We show that p-inulin treatment was associated with an array of changes in microbiome, their metabolic pathways, and also plasma metabolome in PD patients. However, there are some study limitations as well. First, the sample size is small. Second, a large number of tests were done, and it is likely that some results will prove to be false positive; however, as we were in a hypothesis-generation mode, we did not try to adjust for multiple testing. The findings reported here are preliminary and need to be validated in a study involving larger numbers of patients with longer duration of follow-up. All the authors declared no competing interests. The study is supported in part by Young Investigator Award from the National Kidney Foundation , Kaleido Biosciences, Jackson GI Medical. DSR is supported by National Institutes of Health grants 1U01DK099924- 01 and 1U01DK099914-01 . Download .pdf (.27 MB) Help with pdf files Supplementary File (PDF)

Hypertension is considered as the most common risk factor for cardiovascular disease. Inflammatory processes link hypertension and cardiovascular disease, and participate in their pathophysiology. In recent years, there has been an increase in research focused on unraveling the role of inflammation and immune activation in development and maintenance of hypertension. Although inflammation is known to be associated with hypertension, whether inflammation is a cause or effect of hypertension remains to be elucidated. This review describes the recent studies that link inflammation and hypertension and demonstrate the involvement of oxidative stress and endothelial dysfunction-two of the key processes in the development of hypertension. Etiology of hypertension, including novel immune cell subtypes, cytokines, toll-like receptors, inflammasomes, and gut microbiome, found to be associated with inflammation and hypertension are summarized and discussed. Most recent findings in this field are presented with special emphasis on potential of anti-inflammatory drugs and statins for treatment of hypertension.

A mechanistic link between trimethylamine N-oxide (TMAO) and atherogenesis has been reported. TMAO is generated enzymatically in the liver by the oxidation of trimethylamine (TMA), which is produced from dietary choline, carnitine and betaine by gut bacteria. It is known that certain members of methanogenic archaea (MA) could use methylated amines such as trimethylamine as growth substrates in culture. Therefore, we investigated the efficacy of gut colonization with MA on lowering plasma TMAO concentrations. Initially, we screened for the colonization potential and TMAO lowering efficacy of five MA species in C57BL/6 mice fed with high choline/TMA supplemented diet, and found out that all five species could colonize and lover plasma TMAO levels, although with different efficacies. The top performing MA, Methanobrevibacter smithii, Methanosarcina mazei, and Methanomicrococcus blatticola, were transplanted into Apoe−/− mice fed with high choline/TMA supplemented diet. Similar to C57BL/6 mice, following initial provision of the MA, there was progressive attrition of MA within fecal microbial communities post-transplantation during the initial 3 weeks of the study. In general, plasma TMAO concentrations decreased significantly in proportion to the level of MA colonization. In a subsequent experiment, use of antibiotics and repeated transplantation of Apoe−/− mice with M. smithii, led to high engraftment levels during the 9 weeks of the study, resulting in a sustained and significantly lower average plasma TMAO concentrations (18.2 ± 19.6 μM) compared to that in mock-transplanted control mice (120.8 ± 13.0 μM, p < 0.001). Compared to control Apoe−/− mice, M. smithii-colonized mice also had a 44% decrease in aortic plaque area (8,570 μm [95% CI 19587–151821] vs. 15,369 μm [95% CI [70058–237321], p = 0.34), and 52% reduction in the fat content in the atherosclerotic plaques (14,283 μm [95% CI 4,957–23,608] vs. 29,870 μm [95% CI 18,074–41,666], p = 0.10), although these differences did not reach significance. Gut colonization with M. smithii leads to a significant reduction in plasma TMAO levels, with a tendency for attenuation of atherosclerosis burden in Apoe−/− mice. The anti-atherogenic potential of MA should be further tested in adequately powered experiments.

The safety and efficacy of spironolactone is uncertain in end-stage renal disease. We randomized 129 maintenance hemodialysis patients to placebo (n=51) or spironolactone 12.5 mg (n=27), 25 mg (n=26), or 50 mg (n=25) daily for 36 weeks in a double-blind, placebo-controlled, multiple dosage trial to assess safety, tolerability and feasibility and to explore cardiovascular efficacy. The primary safety endpoints were hyperkalemia (potassium > 6.5 mEq/L) and hypotension requiring emergency department visit or hospitalization. Diastolic function was assessed by Doppler echocardiography. 125 participants (97%) completed dose escalation, with no significant difference in permanent study drug discontinuation between the groups (27.5% in placebo versus 16.7% in the combined spironolactone groups and 28% in the 50 mg group). Hyperkalemia frequency was similar between spironolactone and placebo (0.49 versus 0.50 events per patient-year) but demonstrated a significant linear trend due primarily to an increased event rate at the 50 mg dose (0.89 events per patient-year). The primary hypotension outcome was infrequent and similar with spironolactone and placebo (0.11 versus 0 events per patient-year). Gynecomastia was rare and did not differ significantly between groups. Change in diastolic function was similar with spironolactone and placebo. Spironolactone appears safe in carefully monitored maintenance hemodialysis patients, but did not affect cardiovascular parameters in this small study. Hyperkalemia occurs more frequently as dosage increases to 50 mg daily.

MicroRNAs (miRNAs) are noncoding RNAs that regulate posttranscriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate, using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGFβ mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGFβ1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR ≥30 vs. <30 ml·min-1·1.73 m-2 Pathway analysis mapped multiple miRNAs to TGFβ signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated, and miR-130a was significantly upregulated, in urine of patients with eGFR <30; miR-1825 and miR-1281 were upregulated in both urine and plasma of patients with decreased eGFR; and miR-423 was significantly downregulated in plasma of patients with decreased eGFR. miRNA expression in urine and plasma of Alb/TGFβ mice generally resembled and confirmed most, although not all, of the observations from the human studies. In response to TGFβ1 exposure, rat renal proximal tubular cells overexpressed miR-1825 and downregulated miR-423. Thus, miRNA are associated with kidney fibrosis, and specific urinary and plasma miRNA profile may have diagnostic and prognostic utility in CKD.

Trimethylamine N-oxide (TMAO) is a small colorless amine oxide generated from choline, betaine, and carnitine by gut microbial metabolism. It accumulates in the tissue of marine animals in high concentrations and protects against the protein-destabilizing effects of urea. Plasma level of TMAO is determined by a number of factors including diet, gut microbial flora and liver flavin monooxygenase activity. In humans, a positive correlation between elevated plasma levels of TMAO and an increased risk for major adverse cardiovascular events and death is reported. The atherogenic effect of TMAO is attributed to alterations in cholesterol and bile acid metabolism, activation of inflammatory pathways and promotion foam cell formation. TMAO levels increase with decreasing levels of kidney function and is associated with mortality in patients with chronic kidney disease. A number of therapeutic strategies are being explored to reduce TMAO levels, including use of oral broad spectrum antibiotics, promoting the growth of bacteria that utilize TMAO as substrate and the development of target-specific molecules with varying level of success. Despite the accumulating evidence, it is questioned whether TMAO is the mediator of a bystander in the disease process. Thus, it is important to undertake studies examining the cellular signaling in physiology and pathological states in order to establish the role of TMAO in health and disease in humans.

Atrial fibrillation (AF) is the most common sustained arrhythmia in patients with chronic kidney disease (CKD). In this study, we examined the association between inflammation and AF in 3,762 adults with CKD, enrolled in the Chronic Renal Insufficiency Cohort (CRIC) study. AF was determined at baseline by self-report and electrocardiogram (ECG). Plasma concentrations of interleukin(IL)-1, IL-1 Receptor antagonist, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor-β, high sensitivity C-Reactive protein, and fibrinogen, measured at baseline. At baseline, 642 subjects had history of AF, but only 44 had AF in ECG recording. During a mean follow-up of 3.7 years, 108 subjects developed new-onset AF. There was no significant association between inflammatory biomarkers and past history of AF. After adjustment for demographic characteristics, comorbid conditions, laboratory values, echocardiographic variables, and medication use, plasma IL-6 level was significantly associated with presence of AF at baseline (Odds ratio [OR], 1.61; 95% confidence interval [CI], 1.21 to 2.14; P = 0.001) and new-onset AF (OR, 1.25; 95% CI, 1.02 to 1.53; P = 0.03). To summarize, plasma IL-6 level is an independent and consistent predictor of AF in patients with CKD.

More than 100 trillion microbial cells that reside in the human gut heavily influence nutrition, metabolism, and immune function of the host. Gut dysbiosis, seen commonly in patients with chronic kidney disease (CKD), results from qualitative and quantitative changes in host microbiome profile and disruption of gut barrier function. Alterations in gut microbiota and a myriad of host responses have been implicated in progression of CKD, increased cardiovascular risk, uremic toxicity, and inflammation. We present a discussion of dysbiosis, various uremic toxins produced from dysbiotic gut microbiome, and their roles in CKD progression and complications. We also review the gut microbiome in renal transplant, highlighting the role of commensal microbes in alteration of immune responses to transplantation, and conclude with therapeutic interventions that aim to restore intestinal dysbiosis.

NEW FINDINGS:What is the topic of this review? This review addresses the contribution of the altered gut microbiome to uraemic syndrome, with specific reference to gut microbiome-derived uraemic toxins. It also discusses the potential treatment options to normalize the disturbed microbiome in chronic kidney disease (CKD). What advances does it highlight? This review highlights the importance of the gut-kidney connection and how the altered microbial landscape in the intestine contributes to dysmetabolism and inflammation in CKD. Recent findings linking gut-derived uraemic toxins to progression of CKD, cardiovascular disease and mortality are also discussed. Finally, we briefly explain targeted therapies that have been studied to restore intestinal symbiosis in CKD. The human intestine is now recognized as an important metabolic organ powered by gut microbiota. This review addresses the alteration in the gut microbiome in patients with chronic kidney disease (CKD) and its consequence. We describe the major uraemic toxins, p-cresol sulfate, indoxyl sulfate and trimethylamine N-oxide, which are produced by the gut microbiome, and how these metabolites contribute to progression of CKD and associated cardiovascular disease. Translocation of endotoxin from the gut into the systemic circulation contributes to inflammation in CKD. Targeting the gut microbiome to restore symbiosis may prove to be a potent strategy in reducing inflammation and production of these uraemic toxins.

BACKGROUND:MicroRNAs (miRNAs) are noncoding RNA molecules that play important roles in the pathogenesis of various kidney diseases. We investigated whether patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) have distinct circulating and urinary miRNA expression profiles that could lead to potential development of noninvasive biomarkers of the disease.MATERIALS AND METHODS:Exosome miRNAs were extracted from plasma and urine samples of patients with primary FSGS (n = 16) or MCD (n = 5) and healthy controls (n = 5). Differences in miRNA abundance were examined using Affymetrix GeneChip miRNA 3.0 arrays. QRT-PCR was used to validate the findings from the array.RESULTS:Comparison analysis of FSGS versus MCD revealed 126 and 155 differentially expressed miRNAs in plasma and in urine, respectively. Only 38 of these miRNAs were previously cited, whereas the remaining miRNAs have not been described. Comparison analysis showed that a significant number of miRNAs were downregulated in both plasma and urine samples of patients with FSGS compared to those with MCD. Plasma levels of miR-30b, miR-30c, miR-34b, miR-34c and miR-342 and urine levels of mir-1225-5p were upregulated in patients with MCD compared to patients with FSGS and controls (P < 0.001). Urinary levels of mir-1915 and miR-663 were downregulated in patients with FSGS compared to MCD and controls (P < 0.001), whereas the urinary levels of miR-155 were upregulated in patients with FSGS when compared to patients with MCD and controls (P < 0.005).CONCLUSIONS:Patients with FSGS and MCD have a unique circulating and urinary miRNA profile. The diagnostic and prognostic potential of miRNAs in FSGS and MCD warrants further studies.

In 1907, Elie Metchnikoff hypothesized that "autointoxication" by "putrefactive" bacteria accelerated aging and caused disease. Emerging science from the Human Microbiome Project and the Metagenomics of Human Intestinal Tract projects has brought in a paradigm shift in our perception about the gut microbiome.1 The human microbiome has coevolved with the host and established a symbiotic relationship, which has expanded our metabolic and biosynthetic capabilities well beyond what is coded in our genomes. Numbers of signaling molecules, receptors, and effectors from the microbiome that regulate host functions are being constantly unraveled.2 Short–chain fatty acids (SCFAs) are organic fatty acids with one to six carbons, which are products of bacterial fermentation of complex polysaccharides in the colon. The most abundant SCFAs are acetate, propionate, and bytyrate. SCFAs are shown to have physiologic functions and beneficial effects on the human host, but they are essentially waste products to the microbes, which are required to balance redox in the anaerobic environment of the colon.3 These molecules are partly metabolized by colonic epithelial cells, and a proportion enters the portal and peripheral circulation, where they exert their systemic effects through the G protein–coupled receptors, such as GPR41 and GPR43. An observation that has intrigued researchers is that germfree mice have increased susceptibility to ischemia and reperfusion injury (IRI), which is reversed by colonization with commensal bacteria.4 The mechanism by which the gut microbiome confers protection against IRI is the focus of the study by Andrade-Oliveira et al.,5 which appears in this issue of JASN. In this exciting study, Andrade-Oliveira et al.5 have expanded the role of SCFAs beyond their well known role as nutrient for colonic epithelium and regulators of intracellular pH, ion transport, and cell proliferation to explain the gut-kidney connection in IRI. In a series of well designed in vivo and in vitro experiments, Andrade-Oliveira et al.5 show that treatment with SCFAs reduces IRI–induced kidney injury. Among the SCFAs, acetate treatment offered the best protection. Andrade-Oliveira et al.5 believe that the key mechanism that confers protection against AKI is reduction in inflammation mediated by an epigenetic mechanism. Andrade-Oliveira et al.5 also noticed an increase in autophagy, a reduction in apoptosis, and an improvement in mitochondrial biogenesis in response to treatment with SCFA. Furthermore, treatment with acetate-producing bacteria protected the mice kidneys from IRI.5 This study clearly shows that SCFA protects against IRI through convergence of multiple mechanisms, but it also provokes a number of questions.5 Considering the complexity of the communication between microbiome, cells, genes, and the ecosystem, it is often challenging to clearly define the role of individual components, which is the case in this study. Inflammation plays a critical role in induction, maintenance, and resolution of AKI. Innate pattern recognition receptors, including Toll-like receptors (TLRs) and the inflammasome, trigger inflammation in response to tissue injury and pathogens.6 The composition of the microbiome influences the balance between immune regulatory (Treg) and proinflammatory (TH17) T cells. For instance, segmented filamentous bacterium residing in the terminal ileum in mice recruits CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. Another commensal bacteria in the gut, Bacteroides fragilis, induces accumulation of Foxp3+ Treg cells. This effect was dependent on the expression of a capsular polysaccharide known as polysaccharide A by the bacteria.7 Smith et al.8 showed that feeding germfree mice with SCFAs, acetate, propionate, and butyrate increased the abundance of Foxp3+ Treg cells in the large intestine in a GPR43-dependent manner. Immune cells express the SCFA receptors GPR41 and GPR43.9 SCFAs may modulate the magnitude and direction of the immune responses by influencing the differentiation and proliferation of T cells and reducing proinflammatory cytokine expression initiated by TLR signaling.8 In the study by Andrade-Oliveira et al.,5 acetate treatment reduced inflammatory cell infiltration and expression of TLR-4 and its endogenous ligand, Biglycan. However, among the SCFAs, acetate is not the most potent activator of these receptors.9 Immune response is a highly coordinated multistep process that involves sequential epigenetic changes. Transition from euchromatin to transcriptionally silent heterochromatin is mediated by histone deacetylases (HDACs). Butyrate plays a role in modulating immune responses of intestinal macrophages by inhibiting HDAC, leading to a decreased production of proinflammatory mediators, such as NO, IL-6, and IL-12.10 HDAC also plays an important role in cell survival and cell proliferation. Recent studies have shown that a significant proportion of surviving, proliferating renal tubular epithelial cells undergo G2/M arrest after injury, which delays recovery from AKI. Hypermethylation of renin-angiotensin system protein activator like-1, which encodes renin-angiotensin system oncoprotein, perpetuates fibroblast activation and fibrogenesis in the kidney, and thus, it may lead to progressive loss of kidney function. In vitro studies have shown that butyrate regulates expression of genes that arrest growth and induces cellular differentiation.11 Furthermore, in the HT-29 carcinoma cell line, butyrate inhibited proliferation and increased apoptosis but had no effect on the normal epithelial cell line,12 suggesting that the action of SCFAs may depend on the state of activation of the target cells. Future studies should consider examining the effect of different SCFAs at different stages of IRI injury. The hallmark of IRI is profound depletion of intracellular ATP content. In fact, adenine nucleotides infusion enhanced recovery from AKI after an ischemic insult. Mitochondria are the principal generators of cellular ATP. Two mechanistically distinct forms of programmed cell deaths (autophagy and apoptosis) may be induced by cellular stress. Mitochondria regulate the transition between apoptosis and autophagy, with low-intensity stress favoring autophagy and high intensity of cellular stress leading to apoptosis. Autophagy is an evolutionarily conserved cell survival mechanism that recycles cellular constituents to sustain bioenergetics. Jiang et al.13 showed that hypoxia induces autophagy in cultured renal proximal tubular cells. Blocking autophagy by 3-methyladenine or knockdown of autophagic genes (Becline-1 and ATG5) sensitized the cells to apoptosis. Providing butyrate or colonizing with butyrate-producing bacteria (Butyrivibrio fibrisolvens) improved oxidative phosphorylation and ATP synthesis and prevented autophagy.14 It is important to remember that autophagy is the lesser of two evils. The decrease in apoptosis and increase in autophagy observed in the study by Andrade-Oliveira et al.5 may be caused by improved mitochondrial energetics with acetate treatment. Although the increase in mitochondrial DNA content reported in the study provides some clue, future studies should be designed to examine mitochondrial function by assessing mitochondrial membrane potential, ATP content, and mitochondrial dynamics.5 The 2013 World Kidney Day Steering Committee focused on AKI: directing awareness to its effect and calling for campaigns to promote early detection, prevention, and implementation of evidence-based therapies. The study by Andrade-Oliveira et al.5 is very important in that it shows that SCFA confers kidney protection against IRI through multiple potentially biologically interrelated mechanisms. When analyzed critically, it seems that the common factor that underpins the kidney-protective effect of SCFA is through energy conservation in the oxygen-deprived kidney and by improvement of mitochondrial energetics. SCFAs provide about 10% of the daily caloric requirement in humans. They affect lipid, glucose, and cholesterol metabolism in various tissues. It is important to remember that mounting an immune response is an energy-consuming process, which may compete for meager energy resources in the setting of AKI. It is tempting to hypothesize that, other than providing an energy source to the kidney, SCFAs may be reducing the energy consumption by reducing inflammation, promoting apoptosis, and thereby, diverting the much-desired energy toward cellular regeneration. Although the pioneering study by Andrade-Oliveira et al.5 is an important step in our comprehension of the effect of SCFAs on the ischemic kidney, it has left a number of questions to be addressed by future researchers. (1) Is the kidney-protective effect confined to acetate alone or applicable to other SCFAs as well? Convincing evidence indicates that propionate and butyrate are more effective than acetate in reducing inflammation and improving mitochondrial energetics, but then, why is acetate most effective in kidney IRI? (2) Do the signals from the ischemic kidney influence the generation of the amount and type of SCFAs produced by the gut microbiome? (3) Is there a mechanism by which the body traffics the SCFAs from the gut to the kidneys in IRI? (4) Does the kidney express specific receptors in response to IRI, or is it is solely through receptors present in the invading inflammatory cells? (5) Is there a role for the vascular effects of SCFAs mediated through Olfr78 and Gpr41 in protection against the IRI?15 Answering these questions will improve our understanding and propel the SCFAs to prime time in the management of AKI. In this state of knowledge, it is important that we do not overemphasize the importance of a single metabolite without fully understanding how the modulations of these biologic effects interact to confer protection against a complex disease process, such as AKI. Although the results from the study are encouraging, caution must be exercised when in vitro and animal studies are extrapolated to human disease. Disclosures None. D.S.R. was supported by National Institutes of Health Grants 1R01-DK073665-01A1, 1U01-DK099924-01, and 1U01-DK099914-01.

Also known as the "second human genome," the gut microbiome plays important roles in both the maintenance of health and the pathogenesis of disease. The symbiotic relationship between host and microbiome is disturbed due to the proliferation of dysbiotic bacteria in patients with chronic kidney disease (CKD). Fermentation of protein and amino acids by gut bacteria generates excess amounts of potentially toxic compounds such as ammonia, amines, thiols, phenols, and indoles, but the generation of short-chain fatty acids is reduced. Impaired intestinal barrier function in patients with CKD permits translocation of gut-derived uremic toxins into the systemic circulation, contributing to the progression of CKD, cardiovascular disease, insulin resistance, and protein-energy wasting. The field of microbiome research is still nascent, but is evolving rapidly. Establishing symbiosis to treat uremic syndrome is a novel concept, but if proved effective, it will have a significant impact on the management of patients with CKD.

In 1907, Elie Metchnikoff hypothesized that “autointoxication” by “putrefactive” bacteria accelerated aging and caused disease. Emerging science from the Human Microbiome Project and the Metagenomics of Human Intestinal Tract projects has brought in a paradigm shift in our perception about

Protein carbamylation may result from chronic exposure to elevated levels of urea in patients with chronic kidney disease. Carbamylation could cause conformational changes in proteins resulting in alterations in binding sites and disturbances in cellular functions. Elevated levels of carbamylated protein have been shown to be associated with increased risk of death from cardiac causes in patients with end-stage renal disease. The precise mechanism by which carbamylated proteins mediate toxicity in uremia needs further investigation.

BACKGROUND AND OBJECTIVES CD14 plays a key role in the innate immunity as pattern-recognition receptor of endotoxin. Higher levels of soluble CD14 (sCD14) are associated with overall mortality in hemodialysis patients. The influence of kidney function on plasma sCD14 levels and its relationship with adverse outcomes in patients with CKD not yet on dialysis is unknown. This study examines the associations between plasma levels of sCD14 and endotoxin with adverse outcomes in patients with CKD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We measured plasma levels of sCD14 and endotoxin in 495 Leuven Mild-to-Moderate CKD Study participants. Mild-to-moderate CKD was defined as presence of kidney damage or eGFR<60 ml/min per 1.73 m(2) for ≥3 months, with exclusion of patients on RRT. Study participants were enrolled between November 2005 and September 2006. RESULTS Plasma sCD14 was negatively associated with eGFR (ρ=-0.34, P<0.001). During a median follow-up of 54 (interquartile range, 23-58) months, 53 patients died. Plasma sCD14 was predictive of mortality, even after adjustment for renal function, Framingham risk factors, markers of mineral bone metabolism, and nutritional and inflammatory parameters (hazard ratio [HR] per SD higher of 1.90; 95% confidence interval [95% CI],1.32 to 2.74; P<0.001). After adjustment for the same risk factors, plasma sCD14 was also a predictor of cardiovascular disease (HR, 1.30; 95% CI, 1.00 to 1.69; P=0.05). Although plasma sCD14 was associated with progression of CKD, defined as reaching ESRD or doubling of serum creatinine in models adjusted for CKD-specific risk factors (HR, 1.24; 95% CI, 1.01 to 1.52; P=0.04), significance was lost when adjusted for proteinuria (HR, 1.19; 95% CI, 0.96 to 1.48; P=0.11). There was neither correlation between plasma endotoxin and sCD14 (ρ=-0.06, P=0.20) nor was endotoxin independently associated with adverse outcome during follow-up. CONCLUSIONS Plasma sCD14 is elevated in patients with decreased kidney function and associated with mortality and cardiovascular disease in patients with CKD not yet on dialysis.