High-throughput sequencing analysis of intestinal flora changes in elderly hemodialysis CKD patients

Background Chronic kidney disease (CKD) disease affects gut flora by causing dysbiosis and lead to systemic inflammatory conditions. Here, we provide intestinal flora changes of CKD patients undertook different hemodialysis therapy.Methods Patients were recruited during 2017-2019 and divided into healthy control group (CT), CKD non-dialysis group (CKD), hemodialysis group (HD) and peritoneal dialysis group (PD). Intestinal flora genome 16S rDNA sequencing and further bio-informatic analysis were performed.Results Decreased diversity and altered communities of intestinal flora in PD patients, in which microbial diversity was positive correlated with the albumin level were observed. A total of 20 intestinal flora phyla were detected in 166 fecal samples, divided into 3 dominant intestinal types including Bacteroides-dominant gut type, Firmicutes-dominant type and Proteobacteria-dominant gut type. Further analyses found 198 genera, the abundance of 86 genera were significantly different. Butyrate-producing taxa as Faecalibacterium in genera level and Bifidobacteriaceae and Prevotellaceae in family level were dominant genus in CT, CKD, and HD groups, while urease containing-, indole- and p-cresol-forming taxa as Escherichia in genera and Enterobacteriaceae , Enterococcaceae in family level was dominated genus in PD group. Number of differential expressed genes in KEGG enrichment pathways were significantly different in PD group in carbohydrate metabolism, amino acid metabolism, energy metabolism, translation, and membrane transport.Conclusion Our results suggest peritoneal dialysis therapy could result in reduced diversity and altered microbial communities, with reduced probiotic butyrate-producing taxa and increased urease containing-, indole- and p-cresol-forming taxa. The disordered intestinal flora can seriously affect the nutrition level in CKD patients with PD therapy.


Background
The human microbiome is comprised of about 100 trillion microbial cells and their encoded genes [1]. The composition of the gut microbiome varies among individuals and remains stable under homeostasis [2] [3]. The most abundant bacterial phyla in a healthy human gut are Bacteroides, Firmicutes, Actinobacteria, and Proteobacteria [4]. Genetic and environmental factors, including disease, diet and antibiotic use, alter the type and abundance of the microbiome [2].This alteration, also known as dysbiosis, causes individuals to become more susceptible to disease [5]. Hormones such as serotonin, dopamine, and norepinephrine, and microbial metabolites including p-cresol sulfate, decyloxysulfate, trimethylamine N-oxide (TMAO), and short-chain fatty acids (SCFAs) secreted by gut microbiota, can influence various bodily functions [6] [7]. Kidney disease, obesity, metabolic syndrome, cancer, and cirrhosis were reported to be associated with changed endogenous flora [8][9] [10]. So far, thorough investigations into gut-microbialmetabolite relationships under disease progression remain unclear.
Uncontrolled metabolic disorders including CKD, affect gut flora, promote intestinal permeability, cause dysbiosis, and can lead to systemic inflammatory conditions [11].
Reduced abundance of Lactobacillaceae and Prevotellaceae in CKD patients has been reported, while Enterobacter and Enterococcus were observed to be 100 times higher [12].
The secretion of uremic toxins is closely related to microbial changes in CKD patients [13].
Intestinal microbiota were associated with inflammatory status and renal function in endstage renal disease (ESRD) patients in southern China, with a decreased proportion of bacteria, and altered intestinal flora from Prevotella to Bacteroides [14]. CKD animal models have shown excessive uremia can result in intestinal dysbiosis, intestinal barrier dysfunction, and bacterial translocation [15]. Intestinal bacterial changes were found in both dialysis and non-dialysis CKD patients. Further investigations indicated that the abundance of Firmicutes and Actinobacteria in peritoneal dialysis patients was reduced, and the abundance of Bacteroides in hemodialysis patients increased [16]. However, the influence of different renal replacement therapies on microbiota remains unclear.
In this study, we examined changes in the intestinal flora of CKD patients by comparing differences in abundance, diversity, and species composition between healthy humans, CKD non-dialysis patients, HD patients, and PD patients, providing evidence for personalized treatment for CKD patients.

16S rDNA sequence intestinal flora analysis
The OTU was compared using RDP classifer (v 2.2). Greengene database was used for 16S bacteria and archaea genome comparison. Sliver database was used for 18S fungus and UNITE database was used for ITS fungus. The Observed Species, Chao 1 index, Ace index, Shannon index, Simpson index, and Good`s coverage was selected to reflect the Alpha diversity of the samples. PICRUSt was used to perform three-level Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and abundance analysis based on different numbers of 16S rRNA copy numbers.

Statistical Analyses
The quantitative data were expressed as mean ± standard deviation. The mean of the two groups was compared by student`s t-test. The mean of multiple groups was compared by ONE-WAY ANOVA analysis. The chi-square test was used to compare the rates between groups. Principal co-ordinates analysis was used to analyze the differences in beta diversity among different groups. The dominant intestinal type was analyzed by Multidimensional cluster analysis and Principal Component Analysis. All statistical analyses were performed using SPSS v22.0 (SPSS Inc., Chicago, IL, USA), with p < 0.05 set as the difference test level.

Basic clinical characteristics
A total of 166 patients were enrolled in the study, with 17 in CKD group, 47 in CT group, 49 in HD group, and 53 in PD group. No significant differences in age, gender, and body weight were found among the four groups (p > 0.05). Renal function tests, including uric acid, blood urea, nitrogen, and serum creatinine in the healthy control group were significantly lower than those in the other three groups (p < 0.001). Metabolic status, including the expression levels of albumin and TC, were significantly higher in CT group (p < 0.001), while FBG showed no difference among groups (p = 0.29) ( Table 1) (Table 1).

Alpha-beta diversity analysis of intestinal flora
Alpha and beta diversity analysis showed that the intestinal flora structure diversity (including Sob index, Chao index, Ace index, Shannon index, and Simpson index) and species diversity distance in PD group was significantly lower than CKD, CT, and HD groups under the same measurement depth (p < 0.001) (Fig. 1). Further analysis showed that the express of albumin affects the alpha diversity, patients showed with lower albumin level present lower intestinal flora structure diversity and vice visa (Fig. 2).

Relative abundance of indole and p-cresol producing taxa in family level
Bifidobacteriaceae and Prevotellaceae was significantly decreased, while Enterobacteriaceae, Enterococcaceae were significantly increased in patients with PD compared with the other groups. A trend of increased level was found in the relative abundance of Verrucomicroblaceae (Fig 6).

Analysis of intestinal microbial function
High-abundance bacteria KEGG level 1 pathways in the four groups were significantly enriched in metabolism, genetic information processing, and environmental information processing. The following KEGG level 2 pathways were significantly enriched in carbohydrate metabolism, amino acid metabolism, energy metabolism, translation, and membrane transport. Number of differential expressed genes in KEGG enrichment pathways: starch and sucrose metabolism, alanine aspartate and glutamate metabolism, arginine and proline metabolism, oxidative phosphorylation, ribosome, aminoacyl tRNA biosynthesis, and ABC transporters were significantly different in PD group compared with CT, CKD, and HD groups ( Fig. 7 & Table S1).

Discussion
Gut microbiota affect physiological functions in CKD patients by modulating genes involved in host immunity, cell proliferation, and metabolism, the pattern of renal replacement therapy also appears to influence gut microbiota [17] Qualitative and quantitative changes in host microbiome profile and disruption in gut barrier resulting in gut dysbiosis was commonly seen among CKD patients [21]. A Chinese study observed no significant differences in intestinal flora diversity between CKD patients and healthy control groups, suggesting that bacterial diversity was not seriously damaged in this population [14]. Microbiota dysbiosis, which was differed between modes of dialysis, was considered a main risk factor in promoting chronic systemic inflammation in CKD patients [22]. Besides dialysis modes, age and dialysis vintage also contributed to the microbiome diversity [20]. Another Chinese study found that probiotic bacteria was less frequently detected in PD patients, which may impair host intestinal barrier and increase the risk of enteric organism invasion [23]. Diabetes was thought to be the main cause of kidney disease, whereas kidney disease is an important risk factor of cardiovascular [24] [25]. Studies have shown that diabetic cardiomyopathy influenced the bacterial metabolism and presented as a risk factor of cardiovascular events [26]. Considered that diabetic patients were exclusive and no significantly different was found on cardiac dysfunction, suggested that different dialysis modes were critical contributors to microbiota alterations found in our study.
High-throughput sequencing in our study found that the intestinal flora diversity of PD patients was lower than that of HD and non-dialysis CKD patients, suggested that intestinal flora was seriously damaged by PD as a renal replacement therapy. Other investigation further revealed that alpha diversity was closely related to the patient's inflammatory condition [20] [14]. Significant relationship between diversity and inflammatory factors was not found in our study, however, we revealed that patients who have higher albumin level showed with more abundance intestinal flora. This result suggested that people with better alpha diversity of the flora could have better nutrition.
Improve the diversity of bacteria could be an effective way to improve the malnutrition status of dialysis patients.
Colonic bacteria ferment indigestible carbohydrates and proteins and form short chain fatty acids (SCFAs), so that distal guts can absorb more rapidly. SCFAs were also used as cross-feeding nutrients for microorganisms, which were unable to digest macromolecules [27][28] SCFAs can bind to G-protein-coupled receptors GPR41 and GPR43 for regulating

Consent for publication:
We demonstrated that our manuscript have followed the principle of anonymity, no direct or indirect identifiers of our participants were used for publication.

Availability of data and materials:
All data generated or analyzed during this study are included in this published article.

Competing interests:
The authors declare that they have no relevant financial interests.

Authors` contribution
Jianguang Hu designed the study protocol, analyzed data and wrote the main manuscript, Xiaoshi Zhong, Jing Yan and Daoyuan Zhou collecting data, Danping Qin, Xiao Xiao and Yuanyuan Zheng did the statistical analysis, Yan Liu was a guarantor of integrity of the entire study. All authors read and approved the final manuscript.

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