Constipation, Gut Microbiome, and Microbial-derived Uremic Toxins From the Gut Microbiota in HD Patients
Condition: Constipation; Inflammation
MeSH terms: Inflammation; Constipation
Study type: Observational

Summary: 
      Chronic constipation is a prevalent, multifactorial gastrointestinal disorder, and its
      etiology and pathophysiology remain poorly understood. Recently studies using 16S rDNA-based
      microbiota profiling have demonstrated dysbiosis of gut microbiota in chronic constipation.
      In addition, alterations of fecal flora of the a group of severely constipated patients had
      been reported. Constipation, an indicator of gut dysbiosis in dialysis patients, may also
      pose a greater burden in dialysis patients. Some recent findings highlight the plausible link
      between the gut and the kidneys and provide additional insights into the pathogenesis of
      kidney disease progression and development of cardiovascular disease. Yet, the constipation
      in dialysis patients is usually ignored and not even draw the attention of dialysis physician
      as an ominous risk factor of constipated dialysis patients. In view of multiple factors link
      the gut and cardiorenal pathophysiology, and the scarcity of literature on this issue, the
      aim of this study is want to know if constipation can result in any changes to the intestinal
      microbiota and is it associated with inflammation, atherogenic profile and levels of
      microbial derived uremic toxins. Here, the investigators use both self-reported Bristol stool
      form scale (BSFS) scores and Roman IV criteria to diagnose constipation and 16S rDNA Illumina
      amplicon profiles of faecal samples of 90 dialysis patients to assess potential associations
      between microbiota composition and constipation. The relationship between uremic toxins and
      inflammation will also be explored in the dialysis suffering from constipation.
    

Detailed description: 
      Study Design and Population

      Patients and Methods

      This study will include 90 dialysis patients. Patients over age 20 years old and undergoing
      HD for at least 6 months will be enrolled. Patients with inflammatory diseases, cancer, AIDS,
      autoimmune disease, use of a central catheter for hemodialysis access, amputated limbs,
      pregnancy, and patients using catabolic drugs, antioxidant vitamin supplements pre, pro and
      symbiotic and antibiotics in the last 3 months before the start of this study were excluded.
      Dialysis duration was 4 hours per session, three times per week, with a blood flow >250
      mL/min and a dialysate flow of 500 mL/min.

      Analytic Procedures and Sample Processing

      Blood samples will be drawn from each subject in the morning, after overnight fasting (for HD
      patients before a regular HD session). Plasma was separated (15 minutes, 30003 g, 4 C) and
      stored in -80 C until analysis.

      Total concentrations of uremic toxins indoxyl sulfate(IS), p-cresol sulfate(PCS), and
      indoleacetic acid( IAA) are quantified by high-performance liquid chromatography (HPLC) with
      fluorescent detection.. Briefly, for binding competition, 200μl serum to which we added 20μl
      0.50mM 1-naphthalenesulfonic acid (internal standard) was vortex-mixed with 250μl 0.24M
      sodium octanoate (binding competitor).After incubation at room temperature for 5min, we added
      2ml cold acetone to precipitate proteins. Following vortex-mixing and centrifuging at 4 ◦C,
      1860×g for 20 min, the supernatant was transferred to 12mm×100mm, GL 14 glass test tubes and
      2ml dichloromethane was added. After vortex-mixing and centrifuging at 4 ◦C, 1860×g for
      10min, 200μl of the upper layer was transferred to glass autosampler vials, followed by
      addition of 20μl 1M HCl and 15μl was injected onto the HPLC. The HPLC analysis was performed
      on an Agilent 1100 series LC (Santa Clara, CA),and Agilent ChemStations software were used
      for the chromatographic analysis. The separation was carried out on a ZORBAX SB-C18 Solv
      Saver Plus HPLC column (5 μm, 3.0 mm×150 mm).at a flow rate of 0.6 ml/min. Mobile phase A is
      0.2% trifluoroacetic acid in Milli-Q water and mobile phase B is 0.2% trifluoroacetic acid in
      acetonitrile. The analytical method consists of an isocratic run with 92% mobile phase A for
      23 min.. Each analytical run was followed by a 1.3 min washout gradient to 100% B. Column
      temperature was 25 ◦C, and autosampler tray temperature was 6 ◦C. We quantified the analytes
      by using the analyte to standard peak area ratio on a Agilent 1100 High Performance
      Fluorescence detector G1321A and Agilent 1100 Series UV-Visible detectors G1314A. Detector
      settings were λex 260 nm/λem288nm for p-cresyl sulfate and λex 280 nm/λem 390nm for indoxyl
      sulfate and IAA.

      Total antioxidant activity (TAA) is measured in plasma using the Antioxidant Status Assay Kit
      (Calbiochem, Darmstadt, Germany) according to the manufacturer's protocol. The assay is
      defined as the ability of antioxidants in the plasma samples to prevent oxidation of
      2,2'-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) by metmyoglobin. The amount of
      ABTS+ produced is monitored by reading the absorbance at 600 nm. The inter- and intra-assay
      coefficients of variation are 5.0% and 4.3%, respectively.

      High-sensitivity protein C reactive (CRP), interleukin- 6 (IL-6), MCP-1, and Calprotectin
      were analyzed by immunoenzymatic assay (ELISA; R&D systems ). Routine laboratory parameters
      were measured by standard techniques.

      Laboratory measurements

      Total Antioxidant Status (TAS) kits purchased from Randox Laboratories Ltd. (Crumlin, UK) are
      applied for the assessment of the overall serum antioxidant capacity. It is based on the
      suppression of the formation of 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate; ABTS*+),
      mediated mainly by the subsequent antioxidants: uric acid, protein thiol groups, ascorbic
      acid, and tocopherol. The plasma levels of MCP-1, IL-6, CRP, IL-17A and calprotectin are
      tested by commercially available human ELISA kit respectively, according to the
      manufacturer's instruction.

      Stool DNA Isolation and 16S rDNA Gene Amplicon Sequencing

      QI Aamp DNA Stool Mini Kit (51504; Qiagen, Germantown, MD) is used to extract gDNA from
      freshly collected feces samples from both mouse strains. The 16S rDNA gene variable regions
      V3-V6 are amplified by PCR using fecal gDNA. PCR comprised two consecutive steps. Primers
      targeting the 16S rDNA gene (italic) and specific primers carrying the 59M13/rM13 adapters
      (bold) 338FM13 (GTAAACGACGGCCAGTGCTCCTACGGGWGGCAGCAGT) and 1044R-rM13
      (GGAAACAGCTATGACCATGACTACGCGCTGACGACARCCATG) are used to amplify the V3-V6 region of the
      bacterial 16S rDNA gene. After purification of PCR products using the NucleoSpin Gel and PCR
      Clean-Up Kit per the manufacturer's instructions, concentration and quality of the purified
      PCR products are assessed. To barcode each PCR product with a specific MID sequence and add
      the 454-specific Lib-L tag, a second PCR was performed using M13/rM13-specific primers
      containing the 454-specific Lib-L primers (underlined) A-M13 (CCATCTCATCCCTGCGTGTCTCCGACTCAG
      / MIDsequence/GTAAACGACGGCCAGG) and B-rM13 (CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGGAAACAGCTATGA
      CCATGA).

      Amplicons of the second PCR were pooled and purified by ethanol precipitation. Purified PCR
      products are run on a 0.8% agarose gel, bands corresponding to the barcoded 16S rDNA gene
      sequences are excised, and DNA was extracted using the NucleoSpin Gel and PCR Clean-Up Kit.
      DNA is eluted in ddH2O, further purified using AMPure Beads (Beckman Coulter, Inc., Brea,
      CA), and finally, resuspended in ddH2O. Concentration and quality of the purified barcoded
      sequences are assessed using a Nanodrop (Peqlab Biotechnology). Samples are stored at 220°C.
      Amplicon sequencing is performed at Eurofins on a 454 GS FLX Titanium Platform from one side
      (Lib-L-A) according to the recommended procedures for 454 Roche (Roche, Basel, Switzerland).
    

Observational model: Case-Crossover
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        patients have to be at least 20-years-old and and undergoing hemodialysis (HD) for at least
        6 months.
      
Keywords: constipation, , ,; gut microbiome; uremic toxins; inflammation