Role of Gut-derived Uremic Toxins on Oxidative Stress and Inflammation in Patients with Chronic Kidney Disease

Introduction

Several cardiovascular disease (CVD) risk factors have been identified among patients with chronic kidney disease (CKD).[1] The presence of CKD itself has been identified as a CVD risk factor,[2] contributing to endothelial dysfunction. One feature responsible for this is accumulation of uremic toxins. Intestine is an important source of some uremic toxins. Apart from acting as a route for entry of uremic toxins, intestine undergoes changes in the composition of fermenting flora, leading to altered processing of uremic toxin precursors.[3] The gut-derived uremic toxins (GDUT) include endogenously produced molecules without interference by intestinal absorption (uric acid, xanthine), ingested exogenous toxins (advanced glycation end-products), and ingested exogenous products undergoing metabolic modification (derivatives of amino acids, phenol, and indoles). GDUT release inflammatory substances by leukocytes causing endothelial dysfunction.[4] Studying GDUT levels have come to focus with the development of strategies to modify intestinal absorption and metabolism of these toxins with the use of pre/probiotics, dietary modification, and adsorbent therapies. Available literature on GDUT is mostly from the western countries. The GDUT levels can also depend on serum albumin, ethnic variations, and dietary habits,[5] thereby indicating the need for Indian data. However, there are very few Indian studies.[6] Hence, the present study was taken up to estimate GDUT, namely, indoxyl sulfate (IS), para cresyl sulfate (p-CS), indole acetic acid (IAA), and phenol along with the role of GDUT on oxidative stress (OS), inflammation, and mineral metabolism in various stages of CKD in a South Indian cohort.

Material and Methods

The present study was conducted between September 2011 and December 2013 in the Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India. One hundred and twenty patients older than 18 years of age diagnosed to have CKD and willing to participate were enrolled along with forty healthy subjects. The study was approved by the Institutional Ethics Committee. The patients were classified into three groups of forty patients each (G1 = stages 1 and 2 or early CKD; G2 = stages 3 and 4 or advanced CKD; G3 = end-stage renal disease) based on estimated glomerular filtration rate (eGFR) calculated using Cockcroft-Gault formula.[7] Patients undergoing hemodialysis/peritoneal dialysis, those on antioxidant therapy or vitamin supplementation, taking pro/prebiotics, anti-inflammatory/immunosuppressive drugs, and patients with active inflammatory disease or with abnormal liver function were excluded from the study.

Five milliliters of the peripheral venous blood was collected into heparinized bulb. Plasma was separated and was either analyzed immediately or stored at −80°C. GDUT were estimated using high-performance liquid chromatography with a fluorescence detector[8] using Schimadzu HPLC system (LC-20A, Kyoto, Japan). Serum deproteinization and bound uremic solutes displacement were done as part of sample pretreatment. Mobile phase A consisted of 2.76 g/L (20 mM) NaH₂ PO₄, H₂O, and 1.85 g/L (5 mM) tetrabutylammonium iodide in water, and mobile phase B was acetonitrile. For elution, a 1.5 mL/min isocratic flow with 22:78 of A: B mobile phase was used. Twenty microliters of the sample was spiked, and quantification was done using fluorescence detection at specific excitation and emission wavelengths for individual molecules. The concentrations of uremic solutes were calculated using the standard calibration curves using fluorescence detector. The retention times were 4.8 min for phenol, 5.9 min for IAA, 12.5 min for IS, and 16.5 min for p-CS. Three samples spiked to study recovery showed 93% recovery in the normal range and 97% in the uremic range.

To study the effect of OS, malondialdehyde (MDA) and ferric reducing ability of plasma (FRAP) were measured using spectrophotometric methods. The effect of inflammation was assessed by the measurement of high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6). Nitric oxide (NO) levels were estimated to study the status of endothelial function. Calcium (Ca) and phosphorus (P) were estimated for assessing the mineral metabolism status. Urea and creatinine were estimated for assessing renal function. MDA, FRAP, and NO were estimated using spectrophotometric methods.[91011] Urea, creatinine, Ca, P, and hsCRP were quantified using commercially available kits on Beckman Synchron autoanalyzer (Beckman Coulter, USA). IL-6 was estimated by ELISA using commercial kits on ChemWell analyzer (Awareness Technology, USA). A comprehensive score was calculated to have an estimate of the sum effect of GDUT on the pathophysiological factors for CVD risk studied. MDA/FRAP corrected for uric acid (FRAP_U), IL-6, and Ca × P product were included in the generation of the score. Values below or equal to control median level of each of the parameters were taken as no risk (score of 0), up to 25% above control median were given a score of 1, between 25% and 50% above control median were given a score of 2, and more than 50% above control median were given a score of 3.

Results

Demographic data and the biochemical parameters studied are presented in Table 1. The scatterplots showing the distribution of GDUT are shown in Figure 1. There was an increase in all the GDUT across the three groups of CKD. Plasma levels of IS and p-CS were increasing with progression of CKD. The increase found in IS, IAA, and phenol was earlier to p-CS. All patients with CKD had significantly higher levels of MDA, FRAP, hsCRP, IL-6, and calculated comprehensive score (P < 0.001) compared to controls. NO levels showed a significant decrease (P < 0.001) compared to controls.

Table 1 Baseline characteristics and biochemical markers studied in controls and chronic kidney disease patients

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Figure 1

Scatterplots showing the distribution of gut-derived uremic toxins

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The association of GDUT with renal function and CVD risk factors is shown in Table 2. All the four uremic toxins showed significant negative association with eGFR. IS and IAA showed significant positive association with MDA and MDA/FRAP_U. IS and p-CS showed significant positive association with IL-6. IS, IAA, and phenol showed a significant positive association with Ca × P product. Only IS showed a positive association with the comprehensive score, indicating its involvement in all the three pathophysiological factors studied.

Table 2 Association of gut-derived uremic toxins with renal function and cardiovascular disease risk factors

Discussion

Uremic toxins have been identified to be important links in the causation of accelerated atherosclerosis in patients with CKD.[12] GDUT have come to particular focus with the development of strategies to reduce intestinal absorption of these toxins. In the present study, all the GDUT studied, i.e., IS, p-CS, IAA, and phenol, were found to be higher in CKD patients (P = 0.009 for IS, P < 0.001 for p-CS and phenol, P = 0.001 for IAA) compared to controls. The levels of these toxins also increased progressively with increasing stage of CKD (IS: P < 0.001; p-CS: P = 0.003; IAA: P = 0.001; phenol: P = 0.004). However, except for IS which showed a continuous increase, other GDUT studied did not show further increase after Group 2 CKD [Figure 1]. Similar increase was reported for IS,[1314] p-CS,[1415] IAA,[16] and phenol.[17] The mechanisms proposed for increased generation of GDUT include decreased absorption of amino acids resulting in availability of substrate for metabolism by microbes, alteration of gut flora in favor of proteolytic microorganisms producing more toxin precursors, abnormal intestinal motility resulting in increased absorption of bacterial toxins along with decreased renal clearance.[18]