快樂小藥師 Im pharmacist nichts glücklich

Chronic kidney disease (CKD) patients have an increased risk of cardiovascular diseases (CVDs). The present study aimed to investigate the gut microbiota and blood trimethylamine-N-oxide concentration (TMAO) in Chinese CKD patients and explore the underlying explanations through the animal experiment. The median plasma TMAO level was 30.33 μmol/L in the CKD patients, which was significantly higher than the 2.08 μmol/L concentration measured in the healthy controls. Next-generation sequence revealed obvious dysbiosis of the gut microbiome in CKD patients, with reduced bacterial diversity and biased community constitutions. CKD patients had higher percentages of opportunistic pathogens from gamma-Proteobacteria and reduced percentages of beneficial microbes, such as Roseburia, Coprococcus, and Ruminococcaceae. The PICRUSt analysis demonstrated that eight genes involved in choline, betaine, L-carnitine and trimethylamine (TMA) metabolism were changed in the CKD patients. Moreover, we transferred faecal samples from CKD patients and healthy controls into antibiotic-treated C57BL/6 mice and found that the mice that received gut microbes from the CKD patients had significantly higher plasma TMAO levels and different composition of gut microbiota than did the comparative mouse group. Our present study demonstrated that CKD patients had increased plasma TMAO levels due to contributions from both impaired renal functions and dysbiosis of the gut microbiota.

Chronic kidney disease (CKD) is characterized by impaired kidney functions or increased proteinuria confirmed on two or more occasions at least 3 months apart¹. CKD has become an important public health problem in China, with an overall prevalence of 10.8% (~119.5 million people)². CKD is considered an independent cardiovascular risk factor, and a CKD diagnosis implies a very high cardiovascular risk. Thus, cardiovascular disease (CVD) is one of the major comorbidities of CKD. Some studies have revealed a direct correlation between the CVD prevalence and CKD severity³. A Chinese epidemiological study revealed that CVD accounted for nearly 44.2–51.0% of the overall mortality among CKD dialysis patients, which represented a 50-fold increase over the prevalence in the general population⁴. The complex association of CKD with CVD is probably due to several cardiovascular risk factors, including traditional cardiovascular risk factors, such as hypertension, diabetes mellitus, and dyslipidemia⁵, and many novel risk factors, such as anaemia, proteinuria, systemic inflammation, and oxidative stress⁶.

Recently, the gut microbiota has been found to play critical roles in a variety of diet-related chronic inflammatory diseases, including obesity, type 2 diabetes, insulin resistance, atherosclerosis and nonalcoholic fatty liver disease^7,8,9. Dysbiosis of the gut microbial community in the development of CKD has attracted attention. Vaziri et al. found that ESRD patients had higher gut bacterial diversity than the control group¹⁰ and reported an increase in microbes from the phyla Firmicutes, Actinobacteria and Proteobacteria in ESRD patients and a decrease in Bifidobacteria and Lactobacilli. Additionally, a recent study demonstrated that intestinal microbiome dysbiosis led to intestinal barrier dysfunction and bacterial translocation, which finally triggered a state of persistent systemic inflammation in CKD patients¹¹. Within the gut microbiota-derived metabolites, trimethylamine-N-oxide (TMAO) is one of the most important novel risk factors for CVD and related diseases¹². Ingested nutrients, such as phosphatidylcholine and L-carnitine, can be transformed by gut microbes into trimethylamine, which is absorbed and oxidized by hepatic flavin monooxygenase (FMO) to TMAO and finally excreted by the kidneys. A 5-year follow up study demonstrated that circulating TMAO was a forceful prognostic marker for adverse cardiac events¹³. Moreover, elevated TMAO concentrations are positively correlated with the long-term mortality risk in patients with atherosclerosis, heart failure, and CKD^14, 15. Kim et al. reported that the TMAO levels were significantly elevated in CKD patients¹⁶, indicating that TMAO also played a role as a risk factor in these patients. Thus, we could assume that the elevated TMAO levels are caused by the dysbiosis of gut microbiota and impaired renal functions in Chinese CKD patients.

Recently, we reported that the TMAO level in Chinese atherosclerosis patients was similar to the level observed in the control group but was deceased in stroke patients, which was different from data reported from studies in the USA^17, 18. This phenomenon suggested that TMAO might be affected by different dietary habitats and gut microbial communities in different regions. In the present study, we evaluated the gut microbial community and the TMAO levels in Chinese CKD patients and explored the underlying explanations through the animal experiment.

CKD patients showed worse clinical manifestations and significantly elevated TMAO concentrations, compared to the healthy participants

To investigate the clinical conditions and plasma TMAO concentrations of the CKD patients, we collected basic clinical indices and measured TMAO concentrations for analysis. The CKD patients and control individuals enrolled in the present study were comparable in age and gender (Table 1). The CKD patients had a relatively higher burden of comorbidities compared with the healthy individuals, including diabetes mellitus, CVD and metabolic syndrome. Additionally, the CKD patients showed obviously impaired renal functions and anaemia symptoms, including significantly elevated blood urea nitrogen and creatinine and reduced red blood cells and haemoglobin. Symptoms related to higher cardiovascular system burdens, including significantly elevated systolic blood pressure and diastolic blood pressure and a reduced plasma high density lipoprotein level, were also observed in the CKD group. Next, we divided the CKD patients into two subgroups according to their glomerular filtration rates (GFRs). The high GFR group had 16 members with GFR values higher than or equal to 7 ml/min/1.73 m², which represented a relatively low level of renal impairment, whereas the low GFR group (GFR < 7 ml/min/1.73 m²) also had 16 individuals. As demonstrated in Table 2, the low GFR group patients had relatively higher levels of L-carnitine and cystatin-C compared to the high GFR group patients.

CKD has become an important public health problem, and our study provides new information about the TMAO levels and gut microbiome in Chinese CKD patients. Our data demonstrated significantly elevated TMAO levels in CKD patients compared to healthy individuals (30.33 ± 27.35 μmol/L and 2.08 ± 1.89 μmol/L, respectively). Missailidis et al. reported that the TMAO levels in CKD 3–4 and CKD 5 patients and controls in Sweden were 73.5 μM/L, 14.6 μM/L and 5.8 μM/L, respectively, and that the renal functions were a major determinant of the TMAO level²². A comparison of the data from two studies showed that the TMAO levels of the healthy group were similar. In contrast, our patients’ TMAO levels were between the CKD 3–4 and CKD 5 patient group data from the previous study. In a clinical study in the USA, people with adverse cardiovascular events expressed significantly higher TMAO levels than the control group (5.0 µM and 3.5 µM, respectively)¹². Compared with our former study, the TMAO level in Chinese atherosclerosis patients was similar to the control group but was deceased in stroke patients (2.92 μmol/L, 2.68 μmol/L, and 1.91 μmol/L, respectively)¹⁷, illustrating that in our study the TMAO level of the healthy controls was relatively normal and comparable, whereas the TMAO level was significantly higher in the CKD patients than in patients with other diseases. According to the findings of Tang et al., elevated TMAO levels in animal models were related to corresponding increases in tubulointerstitial fibrosis and Smad3 phosphorylation, which is an important regulator of fibrosis¹⁵. These data suggested that an extremely high TMAO level might be an important risk factor in CKD patients. Moreover, renal function is a key contributor that affects the TMAO concentration. Our results confirmed that a lower GFR resulted in a higher TMAO level. An inverse association between the TMAO level and the GFR has been observed in several studies^{12, 22,23,24}. The GFR represents the renal filtration function, which is closely related to the amount of toxic metabolite excretion. Interestingly, in the Framingham Heart Study, TMAO was a predicative marker of CKD at 8 years of follow-up that was not related to the baseline GFR²⁵, suggesting that TMAO may be a surrogate marker for the GFR and have nephrotoxic properties.

In accordance with the alteration in TMAO, severe dysbiosis of the gut microbial community was observed in the CKD patients. Shifts in the microbial community structure may play a role in the aggravation and development of CKD. In addition to deducing the change in gene functions through bioinformatics analysis, our study confirmed that the mouse group that received the gut microbiota from the CKD patients had a significantly higher TMAO level and different composition of gut microbiota than did the comparative mouse group. This result provided direct evidence for the first time that the increased TMAO levels in CKD patients were partially promoted by gut microbiome dysbiosis; additionally, the results indicated an essential role for the gut microbiota in TMAO generation and metabolism in CKD patients as previously reported¹⁵. However, we transferred the faecal samples from CKD patients into the antibiotic-treated mice for only 7 days and the time interval between the FMT and TMAO measurement was only 3 days. In this short term experiment, we deduced that the alteration of the microbial community mainly affected TMAO metabolism rather than renal functions. Additionally, from the PICRUSt analysis, we concluded that changes occurred in metabolites from the microbial populations and that the dysbiosis of the microbial community increased the TMAO levels, including choline, betaine, L-carnitine and TMA metabolism. Among the 8 genes, we found K07271, K01004, and K00499, which were associated with choline metabolism. The K00130 and K00540 genes were associated with betaine and L-carnitine metabolism, respectively. These genes were all relatively decreased in the CKD patients, suggesting that the altered gut microbiota led to abnormal choline, betaine, and L-carnitine metabolism, resulting in the production of redundant TMA in the intestinal tract. The genes relatively enriched in the CKD patients, including K07811, K07821, and K03532, were related to the increased reduction reaction of TMAO and the decreased decomposition of TMA, resulting in an elevated TMA concentration in the intestinal tract. These results initially suggested that the gut microbiome played a role as a potential source of uremic toxins, including TMAO. Studies employing poorly orally absorbed antibiotics coupled with the dietary intake of isotope-labelled phosphatidylcholine directly demonstrated an obligatory role for gut microbes in TMAO generation in humans¹².

In addition to the causative role in TMAO production, the perturbed gut microbiota in CKD patients provided more information on the probable effect on host physiology. The healthy individuals in our study harboured more diverse and shared more similar microbial communities than did the CKD patients. A stable and diverse microbiota is commonly believed to be essential for host physiological processes and mucosal immune functions²⁶. According to our results, many commensal or beneficial microbes, such as Prevotella, Roseburia, Ruminococcaceae and Coprococcus, were depleted in the patient group. These microbes generally exist in healthy populations belonging to the phylofunctional core of the intestinal microbiota²⁷. These beneficial bacteria produce short chain fatty acids (SCFAs), especially butyrate acid, which has multiple critical roles in the maintenance of human health, including producing intestinal epithelial nutrition and energy components²⁸, maintaining intestinal barrier functions²⁹, reducing the severity of inflammation³⁰, and enhancing colon motility functions³¹. Similarly, ESRD patients in the USA exhibited a reduction of bacterial families capable of producing SCFAs, which might contribute to uremic toxicity, systemic inflammation and adverse consequences in patients with CKD³². In contrast, CKD patients had a greater abundance of Proteobacteria, Enterobacteriaceae, Corynebacteriaceae, and Enterococcus, which are mainly opportunistic pathogens. The enrichment of Proteobacteria indicated an imbalanced and unstable microbial community structure of the host³³. Enterobacteriaceae are among the most commonly overgrown symbionts in many conditions involving inflammation and present as the major harmful members in the human symbiotic microbial community. Inflammation-driven blooms of Enterobacteriaceae are associated with various diseases and potentially contribute to the pathogenesis of disease development³⁴. Corynebacteriaceae belongs to non-pathogenic bacteria that can cause exceptional cases of opportunistic infections in immunocompromised humans³⁵. Under this condition, these bacteria become colitogenic microbes that can trigger inflammatory responses. Similarly, Vaziri et al. found an increased abundance of Enterobacteriaceae, particularly OTUs containing certain clusters of E. coli sequences, in end-stage renal disease (ESRD) patients and a decreased abundance of the Prevotellaceae family in uremic animals¹⁰. Interestingly, no significant differences in the microbial community structure were observed between the GFR groups. By analyzing the raw microbiome data of CKD patients, the low GFR group harbored reduced Streptococcus, Streptococcaceae, and Lactobacillales, compared to the high GFR group. All of them are lactic acid bacteria (LAB) and traditionally considered to be beneficial microorganisms for human beings³⁶. Besides, the low GFR group showed increased Adlercreutzia, which was positively correlated to the TMAO level (Spearman analysis, P = 0.002, R = 0.528), but the abundance of Adlercreutzia was relatively low (<0.00005). These results indicated that the difference of microbial composition between the low GFR group and the high GFR group may be involved in the TMAO separation between the two groups. However, the major contribution of this difference should be from the renal functions.

Moreover, contributory factors that could cause microbial dysbiosis in CKD patients were supported by evidence of clinical manifestations, impaired renal functions and dietary restrictions. According to a study in Belgium, 69 factors correlated significantly with microbiome community variations³⁷, including red blood cell counts, haemoglobin, GFR and HDL cholesterol, which were in line with our discoveries. Impaired renal functions, such as protein assimilation, lead to the proliferation of proteolytic bacteria³⁸, and increased protein fermentation can produce potentially toxic metabolites. Additionally, most of the CKD patients had dietary restrictions in an attempt to prevent hyperkalaemia, and Poesen et al. verified that dietary restrictions contributed to altered gut microbial metabolism in CKD patients to a large extent³⁹. Although a direct mechanistic link between dietary choline or TMAO and the progressive impairment of renal functions has been verified in animal studies¹⁵, nutritional intervention studies investigating potential roles of choline or TMAO are rather limited in humans. Interestingly, Bifidobacterium has been reported to reduce the serum indoxyl sulfate level by correcting the microbial community in haemodialysis patients⁴⁰, thereby modestly improving renal functions⁴¹.

The present study suggests that CKD significantly modifies the composition of the gut microbial community in humans, which also leads to the concentration and decreased excretion of TMAO. Animal experiments and the PICRUSt analysis further confirmed the essential role of the gut microbiota in TMAO generation and metabolism. Above all, we suggest that CKD patients have increased plasma TMAO levels due to contributions from both impaired renal functions and dysbiosis of the gut microbiota. Based on these findings, further research should be performed to identify the roles played by the gut microbiota in different CKD stages in the progression and aggravation of this disease. Significant microbiome differences with altered TMAO levels resulting in predicted perturbations of functional pathways could suggest a metabolite-specific targeted treatment.

Research participants and sample collection

We recruited chronic kidney disease patients in this study. All patients were consecutively recruited from the Department of Nephrology of Nanfang Hospital (a teaching hospital affiliated with Southern Medical University in southern China) during 2014. The following inclusion criteria were used: proven impaired kidney structure or renal function and a measured GFR < 60 mL/min/1.73 m² for a minimum of 3 months. Patients with acute intercurrent illnesses and infections and those who used probiotics or antibiotics within 1 month before admission were excluded. The patient group (n = 32) consisted of thirty-two stable patients (16 men and 16 women, aged 53.34 ± 14.47 years) with abnormal renal functions for a minimum of 3 months. The underlying causes of CKD in this study population included nephrosclerosis in 10 patients, diabetic nephropathy in 5 patients, glomerulonephritis in 4 patients and polycystic kidney disease in 1 patient. The controls (n = 32, 16 men and 16 women, aged 55.03 ± 10.38 years) consisted of age- and gender- matched individuals from the health examination centre of Nanfang Hospital. The only exclusion criterion for the selection of the healthy controls was abnormal renal functions.

In our study, the CKD group were mainly constitutive of CKD4 and CKD5 patients. According to the new KDIGO (Kidney Disease: Improving Global Outcomes) classification, CKD stages are determined at referral and correspond to stages 4 (GFR, 15- < 30 mL/min/1.73 m²), and 5 (GFR < 15 mL/min/1.73 m²)⁴².

In this study, the GFR of each healthy participant was beyond 90 ml/min/1.73 m². According to the Initiating Dialysis Early and Late (IDEAL) study⁴³, we further divided the CKD patients into 2 subgroups. The high GFR group was determined as those patients whose GFR was higher than or equal to 7 ml/min/1.73 m² and the low GFR group was determined lower than 7 ml/min/1.73 m². Specifically, the low GFR group contained 16 CKD5 patients, and the high GFR group contained 15 CKD5 patients and 1 CKD4 patient.

For all participants, we collected fasting EDTA-plasma samples, fresh faecal samples and associated clinical data. Plasma aliquots were collected before dialysis treatment, centrifuged, and then immediately frozen at −40 °C. The faecal sample aliquots were frozen at −40 °C immediately after collection. The Ethics Committee of Southern Medical University approved the study protocol, and informed written consent was obtained from all participants. We confirmed that all methods were performed in accordance with the relevant guidelines and regulations.

Faecal DNA extraction

DNA was extracted from the microbial flora contained in the stool samples using the PowerSoil DNA Extraction Kit (Shenzhen Bioeasy Biotechnologies. Co., Ltd) following the manufacturer’s specifications⁴⁴.

Polymerase chain reaction amplification of bacterial 16S rRNA genes

All 64 individually processed human faecal gDNA extractions were PCR amplified. The V4F (GTGTGCCAGCMGCCGCGGTAA) and V4R (CCGGACTACHVGGGTWTCTAAT) primers were used to amplify the 16S rRNA gene V4 variable region from the bacteria. The polymerase chain reaction cycle conditions were described previously⁴⁵. Amplifications were performed using a step cycling protocol consisting of 94 °C for 2 minutes, 30 cycles of 94 °C for 30 seconds, 54 °C for 30 seconds, and 72 °C for 45 seconds. A condition of 72 °C for 5 minutes was used for the final elongation. Each 25-μL PCR reaction contained 0.5 μL of template DNA, 2.0 μL of dNTP Mix (2.5 mmol/L; TaKaRa), 2.5 μL of TaKaRa 10× Ex Taq buffer (Mg2+ free), 1.5 μL of Mg²⁺ (25 mmol/L), 0.25 μL of TaKaRa Ex Taq DNA polymerase (2.5 units), 0.5 μL of 10 μmol/L bar code primer 514 F, 0.5 μL of 10 μmol/L primer 805R, and 17.25 μL of double-distilled water.

Sequencing and microbiome data analysis

According to the manufacturer’s protocol, all polymerase chain reaction amplicons were mixed together and sent to the Beijing Genomic Institute for sequencing using Illumina Miseq (PE 150). The raw sequences were preprocessed based on the BIPES protocol⁴⁵. The sequences were analysed using QIIME version 1.8.0⁴⁶. All samples were normalized for the subsequent analysis. The sequences were stored in the Sequence Read Archive database under accession numbers ERS725509 to ERS725741.

Sequences with a distance-based similarity of 97% or greater were grouped into operational taxonomic units (OTUs) using the Usearch algorithm⁴⁷. According to the sequence frequency, we determined the representative sequences for each OTU, which were aligned using the PyNAST algorithms⁴⁸. The phylogenetic relationships were determined based on a representative sequence alignment using Fast-Tree⁴⁹. The taxonomic assignments for each representative sequence were measured, and the above information was combined to construct a BIOM file⁵⁰ using the command pick_de_novo_otus.py –i clean.fasta –o output_dir. We performed principal coordinate analysis (PCoA) using the command beta_diversity_through_plots.py –i otu.biom –o output_dir.

To determine the significantly differential taxa between the CKD patients and the healthy individuals, we applied linear discriminant analysis effect size (LEfSe) to compare samples between these 2 groups⁵¹. The threshold of the linear discriminant was set to 3. LEfSe is an algorithm for high-dimensional biomarker discovery that also identifies genomic features that characterise differences between two or more biological conditions. LEfSe allows researchers to identify differentially abundant features that are also in accord with biologically meaningful categories by emphasizing statistical significance, biological consistency and effect relevance. For each of the differential features detected by LEfSe, we calculated a linear discriminant analysis value, which represented the differences of the feature between the tested groups.

Quantification of the plasma TMAO concentrations

Plasma aliquots were stored at −40 °C prior to analysis. The plasma TMAO concentrations were quantified using stable isotope dilution liquid chromatography tandem mass spectrometry (6460 Series Triple Quadrupole LC/MS; Agilent) as previously described⁵².

The molecular weight of TMAO is 75.22 g/mol. The extracted plasma aliquots were spiked with internal standards comprised of d9-TMAO in methanol. The samples were mechanically vortexed for 1 minute and centrifuged at 15,000 × g for 25 minutes at 4 °C. The analytes were separated on a phenomenex Luna Silica column (4.6 mm × 250 mm, 5 μm particle size) at room temperature. The mobile phase consisted of 80% solvent A (0.1% formic acid in water) and 20% solvent B (methanol) at a flow rate of 0.5 mL/min. The MS/MS analyses for TMAO and d9-TMAO were conducted in the positive multiple reaction monitoring mass spectrometry mode at m/z 76 → 58 and m/z 85 → 66, respectively. To calculate the TMAO concentration, various concentrations of a TMAO standard were added to control plasma to prepare calibration curves. The data were collected and analysed using the chemistry station provided by Agilent Technology.

Animal experiments

The animal experiment protocols were reviewed and approved by the Ethical Committee of Southern Medical University (SMU, Guangzhou, China). All procedures were performed in accordance with the relevant guidelines and regulations. Male C57BL/6 mice were maintained at the Experimental Animal Research Centre at Southern Medical University. The mice were given the same autoclaved standard chow and autoclaved water ad libitum. The animal room was controlled at 23 °C and 40% humidity with a 12-h light/12-h dark cycle. The general health status of the mice, including their physical appearance, was checked every day. All mice in this study had comparable normal health statuses before the experiment.

At the start of the experiment, all mice were 4 weeks old. All 25 mice were treated with a mixture of broad spectrum, poorly absorbed antibiotics, including vancomycin (100 mg/Kg), neomycin sulfate (200 mg/Kg), metronidazole (200 mg/Kg), and ampicillin (200 mg/Kg) for 1 week, in accordance with a previous study⁵³. After this procedure, the mice were randomized into two groups with 12 and 13 mice in each group, respectively. Both groups were treated with faecal microbiota transplantation (FMT) and transplanted with the faeces of the CKD patients or the healthy controls. These two groups of mice were separately bred in different isolators to prevent normalization of the gut microbiota.

Faecal samples from randomly chosen CKD patients (n = 5, male) and healthy controls (n = 5, male) were used. Each faecal sample (0.1 g) was suspended in 1.5 mL of reduced sterile phosphate-buffered saline, and pools were made from equal volumes of the donor suspensions. Adult (5 weeks old) male antibiotic-treated C57BL/6 mice were administered pooled samples derived from either the CKD patients or the healthy controls for 1 week. After centrifugation of the suspensions at 2000 rpm for 10 minutes, the supernatants were collected, and 200 μL was administered to each mouse by gastric gavage.

On day 3 post-FMT, plasma samples were obtained from the mice and immediately stored at −40 °C. These samples were analysed by stable isotope dilution liquid chromatography tandem mass spectrometry to determine the TMAO levels in the mice. The cecum contents were collected when the mice were sacrificed and immediately stored at −40 °C.

Bioinformatics analysis and statistical analyses

Here, we used PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to gain insights into the possible functional pathways that differed between groups⁵⁴. PICRUSt uses evolutionary modelling to predict metagenomes from 16S rDNA-derived bacterial data and a reference genome database.

Statistical analysis of the clinical data was implemented using SPSS 20.0. We used the mean (standard deviation) to express measurement data that obeyed a normal distribution, the median (interquartile range) to express measurement data that obeyed a skewed distribution, and a percentage to express enumeration data. The Normality test was used to determine the distribution of the data, and the Mann-Whitney U test was used to determine significance between different groups. The Wilcoxon rank sum test was used to determine significance in α-diversity. The results of Mann-Whitney U test was two-tailed. P < 0.05 was considered significant in the compared groups.