Hyperphosphatemia is a common comorbidity in patients on maintenance dialysis. Higher level of plasma phosphorus associated with increased vascular calcification, mineral bone disease cardiovascular events, and all-cause mortality. Food is the major source of phosphorus, and phosphate is the major form absorbed in small intestine. Absorption of phosphate depends on passive diffusion through the epithelial junction and active transport via luminal epithelial sodium phosphate cotransporter (NaPi-IIb).
Previous report suggested significant differences in the abundance of 190 bacterial operational taxonomic units (OTUs) between end-stage renal disease (ESRD) and control groups. Phosphorus is an important element in the survival of microflora. A recent study on pigs indicated that phosphorus might influence the composition and activity of intestinal microflora. The excessive phosphate can be stored as polyphosphates in some types of bacterial cells and used as energy or phosphorus source for metabolism. Thus, it is interesting to know whether gut microbiota participates in phosphorus metabolism in patients with ESRD.
The phosphate-binding agent can bind with phosphate, lower the burden of phosphate in the intestinal lumen, finally reduce phosphate absorption. On the other hand, these medicines may influence the distribution of microbiota in the gut, due to complicated interactions between phosphorus and microbiota. Thus, we took advantage of the cohort who use phosphate-binding agent, to test and analyze stool microbiota of the patients, to investigate the relationship between gut bacteria and phosphate level.
The study was approved by the Ethics Committee of Peking University First Hospital and all individuals signed informed consent.
Study cohort and patient characteristics
From October, 2015 to January, 2016, 21 patients (including 12 men and 9 women, mean age 53.0 ± 9.0 years) with ESRD on maintenance hemodialysis for at least 3 months were recruited from the Hemodialysis Center at Peking University First Hospital. The 21 patients were in a cohort study observing the effect of lanthanum carbonate on lowering serum phosphate level. During the study, seven patients withdrew from the using of lanthanum carbonate because of gastrointestinal side effect. Fourteen patients were treated with the phosphate binder for 12 weeks. Their serum phosphorus levels were over 1.78 mmol/L before taking lanthanum carbonate. The patients have been on regular dialysis three times a week, 4 h each time and single pool urea clearance index (spKt/V) ≥1.2 on regular monitor. All clinical data were collected by standard procedures. Patients with digestive tract diseases, cancer, heart failure, and allergic to lanthanum carbonate were excluded. Moreover, none of them had received lanthanum carbonate within the last 3 months before enrollment. We also got stool samples from volunteers (n = 20) as controls. Mean age was 31.7 ± 9.1 years, including 10 males and 10 females. All these persons are staffs of the unit and had accepted the annual routine medical examination within one year. They had negative urinalysis and normal renal function.
Feces sample collection and DNA extraction
Feces samples were collected from 20 controls, 21 dialysis patients before using lanthanum carbonate, and 14 dialysis patients provided second feces samples after 12 weeks of lanthanum carbonate treatment. The seven patients withdrawn from the drug use did not provide the second feces samples. DNA from the fresh stool samples was extract with QIAamp DNA stool kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. DNA concentration was estimated by absorbance at 260 nm, and the purity of DNA was detected by the A260/A280 ratio. The extracted DNA was used for polymerase chain reaction (PCR) amplification and 454 pyrosequencing.
Polymerase chain reaction amplification
DNA from the stool sample was amplified using primers for the V1-V3 regions of the 16S rRNA gene. The PCR procedure was set by the following cycle conditions: 94°C for 5 min, then 15 cycles at 94°C for 45 s, 55°C for 45 s, 72°C for 45 s followed by a final step of 72°C for 10 min, then stored at 4°C. After the PCR was completed, the products were purified using the QIAquick PCR purification kit (Qiagen Valencia, CA, USA).
Sequencing result estimation
The 454-pyrosequencing data were analyzed in a well-designed pipeline. The sequencing reads were filtered using the Ribosomal Database Project (RDP). OTUs were clustered at 97% sequence identity with CD-HIT, resulting in 44,613 OTUs. After constructing a sample-OTU count matrix, Shannon index was calculated to estimate the species diversity.
Shannon-Weiner curve of different sample groups with increasing number of sequences illustrates the species diversity at different sequencing depth. A smooth curve indicates a good sequencing depth to illustrate the species composition of all the sample groups. In addition, OTUs with statistically significantly different abundance between HC and IBS-D were picked to form a phylogenetic tree by unweighted pair-group method with arithmetic means, and the tree was drawn by iTOL.
Taxonomic composition analysis
We further give taxonomic assignment to every sequence by searching RDP resources with BLAST software, where the e-value threshold was set 10−5. The counts of sequences at different phylogenetic levels (i.e. genus, family, order, class, and phylum) were then generated into a matrix to characterize the microbial composition of each sample. In addition, the Euclidean distance was calculated and mapped into a heat map to further reveal the different abundance distribution of different sample groups.
Correlations between genera and the serum phosphorus were detected with Pearson's correlation. The correlation efficient (R) with P < 0.05 was acceptable; otherwise, R was manually set to be zero. Wilcoxon rank sum test was used to compare the abundance distribution of different taxonomic compositions. P values were adjusted with false discovery rate, and Q < 0.05 were statistically significant. For visualization of the internal interactions and further measurement of the microbial community, SparCC was used to calculate the Spearman correlation coefficient with the corresponding P value between each two genera. The co-occurrence network was then visualized by Circos with the nodes denoting the genera, and connections representing the existence of correlation meeting a given criteria.
Clinical, biochemical traits were compared before and after the use of lanthanum carbonate in patients on maintenance hemodialysis. The serum phosphorus decreased after using lanthanum carbonate for 12 weeks (2.56 ± 0.45 vs. 1.86 ± 0.36, P = 0.002). There was no difference in other biochemical traits, such as serum calcium, potassium, sodium, white blood cell, albumin, blood sugar, triglyceride, creatinine, and urea nitrogen [Table 1].
To characterize the bacterial richness, Shannon-Wiener curve was made by random samples to estimate the total gene numbers that could be identified from these samples. The curve in each group was near saturation, suggesting the sequencing data was enough to reflect the microbial information in the samples [Supplementary Figure 1].
Shannon curves for gene number. The curve in each group is near smooth when the sequencing data are good enough. Green line: Before lanthanum carbonate therapy; Red line: After lanthanum carbonate therapy; Purple line: Healthy controls.
In this study, there were significant variations in the composition of gut microbiota before and after using lanthanum carbonate. At the phylum level, more than 70.00% microbiota was Firmicutes in both groups, but an obvious reduction, from 80.59% to 74.89%, was identified in the patients after using lanthanum carbonate. Bacteroides and Proteobacteria were also decreased after treatment (9.90–8.38% and 2.57–1.01%). At the same time, Actinobacteria increased after the use of lanthanum carbonate from 1.09% to 4.66% as well as other bacteria (5.84–11.06%). No obvious difference was identified between control and patients before using lanthanum carbonate [Figure 1].
At the OTU level, 58 OTUs were different before and after the use of phosphate binder. More decreased OTUs were identified after using the phosphate binder [Figure 2].
At the genus level, seven genera were obviously reduced (P < 0.05), including Centipeda, Chryseobacterium, Gemella, unclassified_Rhodocyclaceae, Pelomonas, Curvibacter, and Parvimonas [Figure 3].
Phosphorus-associated genera in gut microbiome
To further examine the relationship between phosphorus and gut microbiome, correlation analysis showed that 13 genera were closely correlated with serum phosphorus and the correlation coefficient was above 0.4 (P < 0.05). Among them, 11 genera were positively related to serum phosphorus and two genera were negatively related to serum phosphorus [Table 2].
Shannon index based on the genera profile was calculated to estimate diversity. The microbial diversity showed decreasing trends in hemodialysis patients compared with healthy controls and declined further after the phosphate binder therapy [Figure 4].
Co-occurrence network of genera
We constructed the co-occurrence networks of genera in the three different sample groups. Only the strong connection between different genera with the absolute value of the Spearman correlation coefficient larger than 0.6 and the corresponding P < 0.05 was considered valuable and visualized on [Figures 5 and 6]. Compared to the healthy controls, the co-occurrence network of microbial genera in hemodialysis patients gut trend to be more complex [Figure 6]. Similarly, after 12 weeks of treatment with lanthanum carbonate, the co-occurrence network of microbial genera in patients was simplified when compared with that without treatment [Figure 5].
Phosphorus is an essential element for life and phosphates (containing the phosphate ion, PO43−) are components of DNA, RNA, ATP, and the phospholipids. In certain types of bacteria, phosphates can be stored as polyphosphates in cells and can be used as energy or phosphorus source to meet metabolic demand. The storage process is commonly described as enhanced biological phosphorus removal (EBPR). EBPR was a well-established process and has been used widely to remove phosphorus from wastewater. Several studies suggested that changes in dietary phosphorus could influence intestinal microbial composition and activity in pigs and chickens. On the other side, bacteria who stores phosphate can decrease the phosphate ion burden of the gut epithelium. However, none of the above studies explored whether there was association between gut microbiota and phosphorus level in population.
Phosphate-binding agents can change the intestine phosphate burden by forming phosphate complex. The formation of phosphate complex decreased the phosphate ion level and changes the intestinal microenvironment. Because of the decreased phosphate, the bacteria living on phosphorus may decrease and trigger chain reactions and finally change the community of gut microbiota. Thus, it is possible to find bacteria flora associated with phosphorus metabolism with the model of using phosphate binder. In this study, we took advantage of the model of “phosphate-binder therapy” to perform a longitudinal study, to observe the change of microflora. Elements influencing bacteria flora, such as diet, other drugs and antibiotics, were controlled in the 14 patients before and after the use of phosphate binder.
In this study, we identified seven reduced genera after the use of phosphate binder for 12 weeks, compared with the same cohort of patients before the use of the drug. The result suggested that these genera may have a higher demand for phosphorus, and thus may store more phosphate in the intestinal lumen. We did correlation analysis between genera and the serum phosphorus level, and found 11 genera, positively related to serum phosphorus, indicating that survival of the 11 genera was closely related to phosphorus level in intestinal microenvironment. Among the 11 genera, we identified one genera, Clostridium XIX. Similarly, Metzler-Zebeli et al. found increased numbers of Clostridium cluster XIVa in the distal ileum of pigs fed with high-phosphate diet. Clostridium XIX and Clostridium XIVa belong to the same genus, suggested that Clostridium may correlate with phosphate concentration in the intestine. We found two genera negatively related to serum phosphorus also. Previous studies showed that the intestinal microbiota was a complex ecological network closely related with the microbiology of the host and its homeostasis. The microbes interacted with each other competed for some growth factors and led to increase of some microbes and decrease of others.
With the two main analysis approaches, we found some possible intestinal bacteria associated with serum phosphate level in population. Meta-genomic sequencing is needed to confirm bacterial at the species level and genes participating in phosphorus metabolism.
Other factors, such as phosphorus transformation, absorption, and utilization contribute to the metabolism of phosphorus. Some microbial flora associated with inflammation and epithelial dysfunction, lead to higher permeability of intestinal epithelial cells and destroyed cell junction so that phosphates were easily to get into the blood. Parvimonas among the genera identified in this study has been reported causes of inflammatory diseases, including spinal infections, septic arthritis and periapical periodontitis.
We also found that the co-occurrence network of genera was more complex in patients with ESRD before use of phosphorus-binder compared with normal controls, but the complexity declined after phosphorus binder therapy. Gut microbiota plays a vital role in health and disease, and bacterial network complexity was reported to be associated with various diseases. The gut microenvironment changed a lot due to high uremic toxins in ESRD patients, and the network of bacteria also changed to adapt to the environment. The use of phosphorus binder accompanied by an obviously decreased complexity of microbiota. The clinical value of this change needs to be further investigated.
Advanced renal failure can alter the microbial flora composition due to selection pressures from uremic status. The previous study found marked differences in the abundance of 190 bacterial OTUs between the ESRD and control groups. In our study, we also identified significant differences in the abundance of OTUs. However, in addition to uremia, differences in the underlying diseases therapy and dietary intervention could modify the gut microbiome in the ESRD patients. It is less meaningful to compare the difference between ESRD and normal controls in the gut microbiome.
In summary, our study found that gut flora was related to phosphorus metabolism in hemodialysis patients. The use of phosphate binder lanthanum carbonate leads to decreased microbial diversity and lower network complexity. Our findings indicated a new clue to the therapy of hyperphosphatemia. Since our study was a pilot study with a small sample size. More studies with more samples and different phosphorus binders are needed.
Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website.
Financial support and sponsorship
This study was supported by the grants from the National Natural Science Foundation of China (No. 81570664 and No. 31671366) and the National Key Research and Development Program of China (No. 2017YFC1200205).
Conflicts of interest
There are no conflicts of interest.
1. Cannata-Andía JB, Martin KJ. The challenge of controlling phosphorus
in chronic kidney disease Nephrol Dial Transplant. 2016;31:541–7 doi: 10.1093/ndt/gfv055
2. Kalantar-Zadeh K, Kuwae N, Regidor DL, Kovesdy CP, Kilpatrick RD, Shinaberger CS, et al Survival predictability of time-varying indicators of bone disease in maintenance hemodialysis patients
Kidney Int. 2006;70:771–80 doi: 10.1038/sj.ki.5001514
3. Marks J, Debnam ES, Unwin RJ. The role of the gastrointestinal tract in phosphate homeostasis in health and chronic kidney disease Curr Opin Nephrol Hypertens. 2013;22:481–7 doi: 10.1097/MNH.0b013e3283621310
4. Marks J, Debnam ES, Unwin RJ. Phosphate homeostasis and the renal-gastrointestinal axis Am J Physiol Renal Physiol. 2010;299:F285–96 doi: 10.1152/ajprenal.00508.2009
5. Uribarri J. Phosphorus
homeostasis in normal health and in chronic kidney disease patients with special emphasis on dietary phosphorus
intake Semin Dial. 2007;20:295–301 doi: 10.1111/j.1525-139X.2007.00309.x
6. Vaziri ND, Wong J, Pahl M, Piceno YM, Yuan J, DeSantis TZ, et al Chronic kidney disease alters intestinal microbial flora Kidney Int. 2013;83:308–15 doi: 10.1038/ki.2012.345
7. Heyer CM, Weiss E, Schmucker S, Rodehutscord M, Hoelzle LE, Mosenthin R, et al The impact of phosphorus
on the immune system and the intestinal microbiota with special focus on the pig Nutr Res Rev. 2015;28:67–82 doi: 10.1017/S0954422415000049
8. Wood HG, Clark JE. Biological aspects of inorganic polyphosphates Annu Rev Biochem. 1988;57:235–60 doi: 10.1146/annurev.bi.57.070188.001315
9. Li W, Fu L, Niu B, Wu S, Wooley J. Ultrafast clustering algorithms for metagenomic sequence analysis Brief Bioinform. 2012;13:656–68 doi: 10.1093/bib/bbs035
10. Letunic I, Bork P. Interactive tree of life (iTOL): An online tool for phylogenetic tree display and annotation Bioinformatics. 2007;23:127–8 doi: 10.1093/bioinformatics/btl529
11. Letunic I, Bork P. Interactive tree of life v2: Online annotation and display of phylogenetic trees made easy Nucleic Acids Res. 2011;39:W475–8 doi: 10.1093/nar/gkr201
12. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool J Mol Biol. 1990;215:403–10 doi: 10.1016/S0022-2836(05)80360-2
13. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, et al The ribosomal database project: Improved alignments and new tools for rRNA analysis Nucleic Acids Res. 2009;37:D141–5 doi: 10.1093/nar/gkn879
14. Friedman J, Alm EJ. Inferring correlation networks from genomic survey data PLoS Comput Biol. 2012;8:e1002687 doi: 10.1371/journal.pcbi.1002687
15. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al Circos: An information aesthetic for comparative genomics Genome Res. 2009;19:1639–45 doi: 10.1101/gr.092759.109
16. Abelson PH. A potential phosphate crisis Science. 1999;283:2015 doi: 10.1126/science.283.5410
17. Hirota R, Kuroda A, Kato J, Ohtake H. Bacterial phosphate metabolism and its application to phosphorus
recovery and industrial bioprocesses J Biosci Bioeng. 2010;109:423–32 doi: 10.1016/j.jbiosc.2009.10.018
18. Harold FM. Inorganic polyphosphates in biology: Structure, metabolism, and function Bacteriol Rev. 1966;30:772–94
19. Lv XM, Shao MF, Li J, Li CL. Metagenomic analysis of the sludge microbial community in a lab-scale denitrifying phosphorus
removal reactor Appl Biochem Biotechnol. 2015;175:3258–70 doi: 10.1007/s12010-015-1491-8
20. Borda-Molina D, Vital M, Sommerfeld V, Rodehutscord M, Camarinha-Silva A. Insights into broilers' gut microbiota
fed with phosphorus
, calcium, and phytase supplemented diets Front Microbiol. 2016;7:2033 doi: 10.3389/fmicb.2016.0
21. Witzig M, Carminha-Silva A, Green-Engert R, Hoelzle K, Zeller E, Seifert J, et al Spatial variation of the gut microbiota
in broiler chickens as affected by dietary available phosphorus
and assessed by T-RFLP analysis and 454 pyrosequencing PLoS One. 2015;10:e0143442 doi: 10.1371/journal.pone.0143442
22. Metzler-Zebeli BU, Vahjen W, Baumgärtel T, Rodehutscord M, Mosenthin R. Ileal microbiota of growing pigs fed different dietary calcium phosphate levels and phytase content and subjected to ileal pectin infusion J Anim Sci. 2010;88:147–58 doi: 10.2527/jas.2008-1560
23. Metzler-Zebeli BU, Zijlstra RT, Mosenthin R, Gänzle MG. Dietary calcium phosphate content and oat β-glucan influence gastrointestinal microbiota, butyrate-producing bacteria and butyrate fermentation in weaned pigs FEMS Microbiol Ecol. 2011;75:402–13 doi: 10.1111/j.1574-6941.2010.01017.x
24. Clemente JC, Ursell LK, Parfrey LW, Knight R. The impact of the gut microbiota
on human health: An integrative view Cell. 2012;148:1258–70 doi: 10.1016/j.cell.2012.01.035
25. Keller L, Surette MG. Communication in bacteria: An ecological and evolutionary perspective Nat Rev Microbiol. 2006;4:249–58 doi: 10.1038/nrmicro1383
26. Lau WL, Kalantar-Zadeh K, Vaziri ND. The gut as a source of inflammation in chronic kidney disease Nephron. 2015;130:92–8 doi: 10.1159/000381990
27. Vaziri ND, Yuan J, Norris K. Role of urea in intestinal barrier dysfunction and disruption of epithelial tight junction in chronic kidney disease Am J Nephrol. 2013;37:1–6 doi: 10.1159/000345969
28. Baghban A, Gupta S. Parvimonas micra
: A rare cause of native joint septic arthritis Anaerobe. 2016;39:26–7 doi: 10.1016/j.anaerobe.2016.02.004
29. Gahier M, Cozic C, Bourdon S, Guimard T, Cormier G. Spinal infections caused by Parvimonas micra
Med Mal Infect. 2015;45:397–8 doi: 10.1016/j.medmal.2015.07.006
30. Ji H, Li H, He Y, Hou B. Study of association between Parvimonas micr
a and pulp dominant pathogens in the infected root canals with chronic periradicular periodontitis (in Chinese) Natl Med J China. 2014;49:495–9
31. Sekirov I, Russell SL, Antunes LC, Finlay BB. Gut microbiota
in health and disease Physiol Rev. 2010;90:859–904 doi: 10.1152/physrev.00045.2009
32. Scher JU, Ubeda C, Artacho A, Attur M, Isaac S, Reddy SM, et al Decreased bacterial diversity characterizes the altered gut microbiota
in patients with psoriatic arthritis, resembling dysbiosis in inflammatory bowel disease Arthritis Rheumatol. 2015;67:128–39 doi: 10.1002/art.38892
33. Giloteaux L, Goodrich JK, Walters WA, Levine SM, Ley RE, Hanson MR. Reduced diversity and altered composition of the gut microbiome in individuals with myalgic encephalomyelitis/chronic fatigue syndrome Microbiome. 2016;4:30 doi: 10.1186/s40168-016-0171-4
Edited by: Ning-Ning Wang
Keywords:© 2018 Chinese Medical Association
Gut Microbiota; Hemodialysis Patients; Lanthanum Carbonate; Phosphorus