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ORIGINAL ARTICLE
Year : 2021  |  Volume : 24  |  Issue : 8  |  Page : 1133-1137

Alteration of Intestinal Microflora in Uremia Patients With or Without Blood Purification


1 Department of Nephrology, Minhang Hospital, Fudan University, Shanghai; Department of Geriatrics, First Affiliated Hospital of Soochow University, Suzhou, China
2 Department of Geriatrics, First Affiliated Hospital of Soochow University, Suzhou, China

Date of Submission09-Sep-2019
Date of Acceptance30-Aug-2020
Date of Web Publication14-Aug-2021

Correspondence Address:
Dr. Y Xie
Department of Geriatrics, First Affiliated Hospital of Soochow University, No. 899, Pinghai Road, Soochow - 215 006
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_484_19

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   Abstract 


Aims: To investigate alteration of intestinal microflora in uremia patients with or without blood purification treatments. Methods: The present study included a total of 109 adult patients who were administered in our hospital during 2014 August to 2015 December, 85 cases had already received hemodialysis treatment and 24 cases had not received any renal transplantation treatments. Serum levels of hemoglobin, albumin, creatinine, hypersensitive C reactive protein, and cystatin C, as well as blood urea nitrogen and estimated glomerular filtration rate were determined. 16S rRNA sequencing was conducted to determine the levels of Bifidobacterium, Lactobacillus acidophilus, Escherichia coli, and Enterococcus faecalis. Results: The hemoglobin level in the hemodialysis group was significantly higher than that of the non-hemodialysis patients. The levels of Bifidobacterium and Lactobacillus acidophilus were significantly lower while the levels of Escherichia coli and Enterococcus faecalis were significantly higher in both of the patient groups compared with the healthy control. In all treatment groups, levels of Bifidobacterium and Lactobacillus acidophilus were significantly higher and levels of Escherichia coli and Enterococcus faecalis were significantly lower compared with the non-blood purification treatment group. Conclusions: The intestinal microflora might be influenced by uremia and might also be affected by blood purification treatments.

Keywords: Blood purification, hemodiafiltration, hemodialysis, hemoperfusion, intestinal microflora, uremia


How to cite this article:
He H, Xie Y. Alteration of Intestinal Microflora in Uremia Patients With or Without Blood Purification. Niger J Clin Pract 2021;24:1133-7

How to cite this URL:
He H, Xie Y. Alteration of Intestinal Microflora in Uremia Patients With or Without Blood Purification. Niger J Clin Pract [serial online] 2021 [cited 2022 May 22];24:1133-7. Available from: https://www.njcponline.com/text.asp?2021/24/8/1133/323860




   Introduction Top


Chronic kidney disease (CKD), a globally common chronic disease, influences millions of people worldwide.[1] In China, it is reported that about 119.5 million adults have CKD, and thus has become a huge health burden to society.[2],[3] CKD may progress to end-stage renal disease (ESRD) and finally develops to uremia. At this stage patients may rely on kidney replacement therapies such as hemodialysis (HD), hemodiafiltration (HDF), and hemoperfusion (HP), peritoneal dialysis, and renal transplantation, and have to bear significant economic cost.

CKD may cause toxic effects to many organs as a result of altered kidney function and the inflammatory state. The gastrointestinal tract was once considered as “a forgotten organ” in kidney disease and uremia.[4] The intestinal tract has the largest community of microbiota in the human body.[5] Under normal conditions, the intestinal mucosal barrier will restrict the microorganisms within the intestinal tract.[6] However, CKD may damage the integrity of the intestinal mucosal barrier, leading to the translocation of intestinal bacteria and endotoxin.[7],[8] It has been shown that CKD patients have enteric bacterial overgrowth. The altered bacterial load and its products may contribute to both the progression of CKD and activation of the inflammatory response.[9]

Several studies have recently reported the alteration of intestinal microflora in CKD and uremia;[10] however, certain studies are still lacking and more clinical evidence is always necessary. Besides, few studies focused on the effects of blood purification on intestinal microflora of uremia patients. In the present study, we aimed to investigate alteration of intestinal microflora in uremia patients with or without blood purification. This study may give more clinical basis for the role of intestinal microflora in uremia and may provide some new insights for treatment of CKD patients.


   Materials and Methods Top


Subjects

The present study included a total of 109 adult patients who were administered in our hospital during 2014 August to 2015 December. All patients were diagnosed as uremia according to the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative (K/DOQI clinical) practice guidelines for CKD.[11] End-stage renal disease (ESRD) was defined as the estimated glomerular filtration rate (eGFR) <15 mL/min/1.73 m2 for 3 months. Among all the patients, 85 cases had already received blood purification treatment and 24 cases had not received any renal transplantation treatments. The patients who received blood purification treatment were further divided into several groups: 1) the hemodialysis (HD) group (n = 30); the HD and hemodiafiltration (HDF) group (n = 29); and HD combined with HDF and hemoperfusion (HP) group (n = 26). All patients who had received other renal transplantation treatments such as peritoneal dialysis and renal transplantation before the study were excluded. Other exclusion criteria included patients with clinical infection (respiratory tract, digestive tract, urogenital system, etc.), liver disease, active autoimmune disease, malignant tumor, right heart failure, three apical insufficiency, diarrhea, or constipation. Patients who were treated with antibiotics or probiotics/prebiotics within 1 month before the study were also excluded.

The HD and HDF was conducted using a 4008B hemodialysis machine, 4008S dialysate filter, and FX60 dialyzer produced by Fresenius, Germany, and a neutral macroporous resin (HA330, Zhuhai Lizhu Medical Bio-Material Co., Ltd., China) was used for HP. Generally, time for HD was set to 4 h, the hemodialysis volume was 4 times the kg body weight, and the dialysate flow rate was 500 mL/min. For patients of the HD group, patients received HD 3 times a week, for 4 h every time; for the HD + HDF group, patients received HD 2 times and HDF 1 time a week, 4 h every time; for the HD + HDF + HP group, patients received HDF 2 times (4 h every time), and HD combined with HP 1 time (HD and HP at the same time for 2.5 h and HD for 1.5 h). Additionally, 30 healthy individuals were recruited as healthy control. All participants signed the written informed consent forms. The present study was approved by the ethics committee of First Affiliated Hospital of Soochow University.

Data collection

Fasting venous blood samples were collected for all participants. Serum levels of hemoglobin, albumin, and creatinine were measured using an automatic biochemical analyzer. Levels of hypersensitive C reactive protein (hs-CRP) and cystatin C were measured using commercial enzyme-linked immunosorbent assay (ELISA) kit. The blood urea nitrogen (BUN) was measured using a modified kinetic Jaffe method. eGFR was determined by the CKD Epidemiology Collaboration (CKD-EPI) equation.

16S rRNA sequencing

The first fresh stool in the morning was collected and stored at -80°C. Stool DNA was extracted using a QIAamp Fast DNA Stool Mini kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instruction. A total of 4 well-known bacteria were chosen in this study, Bifidobacterium, Lactobacillus acidophilus,  Escherichia More Details coli, and Enterococcus faecalis. RNA was converted into cDNA using a Prime-ScriptTM one step qRT-PCR kit (TAKARA, Dalian, China). PCR reactions were performed with using SYBR GREEN mastermix (Solarbio, Beijing, China) on an ABI7500System (Applied Biosystems, Foster City, CA, USA) with the following composition and cycling profile: Pre-denaturation at 95°C for 2 min, then denaturation at 95°C for 15 s; annealing for 20 s 58°C for Bifidobacterium, 58°C for Lactobacillus acidophilus, 60°C for Escherichia coli, and 61°C for Enterococcus faecalis; 68°C for 30 s, and 15 s of 85°C for Bifidobacterium, 83.5°C for Lactobacillus acidophilus, 85.5°C for Escherichia coli, and 82.5°C for Enterococcus faecalis. A total 40 cycles were conducted. The 16S rDNA primers were designed and synthesized by the Beijing Genomics Institute Inc. Sequences were as follows: Bifidobacterium F 5′-TCGCGTC(C/T)GGTGTGAAAG-3′, R 5′-CCACATCCAGC (A/G) TCCAC-3′; Lactobacillus acidophilus F 5′-AGCAGTAGGGAATCTTCCA-3′, R 5′-CACCGCTACACATGGAG-3′; Escherichia coli F 5′-CCCTTATTGTTAGTTGCCATCATT-3′, R 5′-ACTCGTI”GTACTTCCCATTGT-3′; Enterococcus faecalis F 5′-GTTAATACCTTTGCTCATTGA-3′, R 5′-ACCAGGGTATCTTAATCCTGTT-3′. Bacterial quantity was expressed as log10 bacteria per gram of stool.

Statistical analysis

The measurement data was expressed by mean ± SD. Comparisons were conducted using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. It was considered to be statistically significant when the P value was less than 0.05. All calculations were made using SPSS 18.0 (SPSS Inc.; Chicago, IL, USA).


   Results Top


As shown in [Table 1], a total of 109 patients were included in this study with 85 cases having received blood purification with a mean age of 56.8 ± 15.5 and male: female ratio of 44:41, while 24 cases in the non-blood purification group had a mean age of 57.2 ± 15.1 and male: female ratio of 14:11. Among all 109 patients, the cause for renal failure was chronic glomerulonephritis for 58 cases (53%), diabetic nephropathy for 23 cases (21%), hypertensive nephropathy for 9 cases (8%), medicinal nephropathy for 9 cases (8%), polycystic kidney for 6 cases (6%), and other types for 4 cases (4%). No significant difference was found in age, gender, and causes for renal failure in the patient groups.
Table 1: Clinical inflammation for all participants

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Serum levels of hemoglobin, albumin, creatinine, hs-CRP, cystatin C, and BUN, as well as eGFR were also determined. A significant difference was observed for all indexes in both of the patient groups compared with the healthy control, P < 0.05. Only hemoglobin levels in all treatment groups were significantly higher than that of the non-blood purification patients, P < 0.05. However, no significant difference was observed between other serum indexes in the other patient groups.

To investigate the influence of uremia and treatment of hemodialysis on intestinal microflora, 4 well-known bacteria were chosen, Bifidobacterium, Lactobacillus acidophilus, Escherichia coli, and Enterococcus faecalis and 16S rRNA sequencing was determined using RT-qPCR. As shown in [Figure 1], the levels of Bifidobacterium and Lactobacillus acidophilus were significantly lower in both of the patient groups compared with the healthy control, P < 0.05. However, the levels of Escherichia coli and Enterococcus faecalis were significantly higher in both of the patient groups compared with the healthy control, P < 0.05. Meanwhile, in all treatment groups, the levels of Bifidobacterium and Lactobacillus acidophilus were significantly higher and levels of Escherichia coli and Enterococcus faecalis were significantly lower compared with the non-blood purification treatment group, P < 0.05. The level of Lactobacillus acidophilus was significantly higher while Escherichia coli was significantly lower in the HD + HDF + HP group than HD and HD + HDF groups, P < 0.05. No other significant difference was observed between the two groups of HD and HD + HDF, suggesting that uremia might influence the intestinal microflora, and treatment of blood purification might reduce these effects.
Figure 1: Bacterial groups quantified by qPCR expressed as log10 bacteria per gram of stool. qPCR for the hemodialysis patients, non-hemodialysis patients, and healthy control. *P < 0.05, comparison in corresponding groups

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   Discussion Top


Chronic kidney disease (CKD) is a globally common chronic disease which is a significant health and economic burden to public health. It is considered that CKD and uremia impairs intestinal barrier function and promotes inflammation throughout the gastrointestinal tract.[12] The human gastrointestinal tract microbiota is the largest body microbial load, containing a much greater number of microorganisms than that of the host cells.[13] Due to the alteration of intestinal barrier function by CKD, it will result in production of nephrotoxins, contributing to systemic inflammation, and reversely contribute to the progression of CKD. In this respect, metabolic alterations induced by uremia may contribute to gut dysbiosis, pathogen overgrowth, and an increased bacterial translocation, as well as activating innate immunity, and systemic inflammation.[14],[15]

Some studies have proposed several possible mechanisms for the interplay between the gut microbiome and pathogenesis of CKD. Vaziri et al. demonstrated that gut microbiome might help in maintaining an intestinal epithelial barrier, while uremia might damage the barrier and deplete the tight junction protein constituents of intestinal epithelium.[16] Cario et al. showed that gut microbiome could suppress intestinal inflammation mediated by toll-like receptor (TLR) signaling.[17] Mazmanian et al. found gut microbiome might produce molecules to regulate proper T cell population balance.[18] All these studies highlight the importance of and support the potential role for intestinal microflora in CKD progression.

More recently, Jiang et al. studied alteration of the gut microbiota in Chinese population with CKD and found that the total bacteria in feces were reduced in patients with ESRD compared to that in healthy individuals.[19] Janice et al. investigated intestinal microbiota in pediatric patients with ESRD and found ESRD children had altered intestinal microbiota and increased bacterially derived serum uremic toxins.[20] Vitetta et al. reviewed the relationship between uremia and CKD and pointed out the crucial role for gut microflora in uremia.[21]

Based on these studies, we investigated the alteration of intestinal microflora in uremia patients with or without hemodialysis. The present study showed that levels of Bifidobacterium and Lactobacillus acidophilus were significantly lower in both of the patient groups compared with the healthy control group, while the levels of Escherichia coli and Enterococcus faecalis were significantly higher. Besides, a significant difference was also found in blood purification treatment and non-treatment groups. These results indicated that the intestinal microflora might be influenced by uremia and also might be affected by hemodialysis treatment. The present study also had some limitations, first we chose only several bacteria to determine their level but did not perform bioinformatics analysis; second, the study size was small and all Chinese; at last deeper insights were still needed for the alteration we observed. Thus, further studies are still needed to confirm our results.


   Conclusion Top


We investigated alterations of intestinal microflora in uremia patients with or without blood purification and found the intestinal microflora might be influenced by uremia and may be affected by blood purification treatments. Further studies are still needed to confirm our results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Hill NR, Fatoba ST, Oke JL, Hirst JA, O'Callaghan CA, Lasserson DS, et al. Global prevalence of chronic kidney disease – A systematic review and meta-analysis. PloS One 2016;11:e0158765.  Back to cited text no. 1
    
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Nugent RA, Fathima SF, Feigl AB, Chyung D. The burden of chronic kidney disease on developing nations: A 21st century challenge in global health. Nephron Clin Pract 2011;118:269-77.  Back to cited text no. 2
    
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Tan S-J, Yu C, Yu Z, Lin Z-L, Wu G-H, Yu W-K, et al. High-fat enteral nutrition controls intestinal inflammation and improves intestinal motility after peritoneal air exposure. J Sur Res 2016;201:408-15.  Back to cited text no. 7
    
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Mafra D, Lobo JC, Barros AF, Koppe L, Vaziri ND, Fouque D. Role of altered intestinal microbiota in systemic inflammation and cardiovascular disease in chronic kidney disease. Future Microbiol 2014;9:399-410.  Back to cited text no. 9
    
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Mafra D, Barros AF, Fouque D. Dietary protein metabolism by gut microbiota and its consequences for chronic kidney disease patients. Future Microbiol 2013;8:1317-23.  Back to cited text no. 10
    
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K/DOQI Workgroup. K/DOQI clinical practice guidelines for cardiovascular disease in dialysis patients. Am J Kidney Dis 2005;45:S1-153.  Back to cited text no. 11
    
12.
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.  Back to cited text no. 12
    
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Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. Diversity of the human intestinal microbial flora. Science 2005;308:1635-8.  Back to cited text no. 13
    
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Winslow MM, Gallo EM, Neilson JR, Crabtree GR. The calcineurin phosphatase complex modulates immunogenic B cell responses. Immunity 2006;24:141-52.  Back to cited text no. 14
    
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Anders HJ, Andersen K, Stecher B. The intestinal microbiota, a leaky gut, and abnormal immunity in kidney disease. Kidney Int 2013;83:1010-6.  Back to cited text no. 15
    
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Vaziri ND, Goshtasbi N, Yuan J, Jellbauer S, Moradi H, Raffatellu M, et al. Uremic plasma impairs barrier function and depletes the tight junction protein constituents of intestinal epithelium. Am J Nephrol 2012;36:438-43.  Back to cited text no. 16
    
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Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function. Gastroenterology 2007;132:1359-74.  Back to cited text no. 17
    
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Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 2005;122:107-18.  Back to cited text no. 18
    
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Jiang S, Xie S, Lv D, Wang P, He H, Zhang T, et al. Alteration of the gut microbiota in Chinese population with chronic kidney disease. Sci Rep 2017;7:2870.  Back to cited text no. 19
    
20.
Crespo-Salgado J, Vehaskari VM, Stewart T, Ferris M, Zhang Q, Wang G, et al. Intestinal microbiota in pediatric patients with end stage renal disease: A midwest pediatric nephrology consortium study. Microbiome 2016;4:50.  Back to cited text no. 20
    
21.
Vitetta L, Gobe G. Uremia and chronic kidney disease: The role of the gut microflora and therapies with pro- and prebiotics. Mol Nutr Food Res 2013;57:824-32.  Back to cited text no. 21
    


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