Functional Genomic Analysis Identifies Indoxyl Sulfate as a Major, Poorly Dialyzable Uremic Toxin in End-Stage Renal Disease

Abstract

BackgroundChronic renal failure is characterized by progressive renal scarring and accelerated arteriosclerotic cardiovascular disease despite what is considered to be adequate hemodialysis or peritoneal dialysis. In rodents with reduced renal mass, renal scarring has been attributed to poorly filtered, small protein-bound molecules. The best studied of these is indoxyl sulfate (IS).MethodsWe have attempted to establish whether there are uremic toxins that are not effectively removed by hemodialysis. We examined plasma from patients undergoing hemodialysis, employing global gene expression in normal human renal cortical cells incubated in pre- and post- dialysis plasma as a reporter system. Responses in cells incubated with pre- and post-dialysis uremic plasma (n = 10) were compared with responses elicited by plasma from control subjects (n = 5). The effects of adding IS to control plasma and of adding probenecid to uremic plasma were examined. Plasma concentrations of IS were measured by HPLC (high pressure liquid chromatography).ResultsGene expression in our reporter system revealed dysregulation of 1912 genes in cells incubated with pre-dialysis uremic plasma. In cells incubated in post-dialysis plasma, the expression of 537 of those genes returned to baseline but the majority of them (1375) remained dysregulated. IS concentration was markedly elevated in pre- and post-dialysis plasma. Addition of IS to control plasma simulated more than 80% of the effects of uremic plasma on gene expression; the addition of probenecid, an organic anion transport (OAT) inhibitor, to uremic plasma reversed the changes in gene expression.ConclusionThese findings provide evidence that hemodialysis fails to effectively clear one or more solutes that effect gene expression, in our reporter system, from the plasma of patients with uremia. The finding that gene dysregulation was simulated by the addition of IS to control plasma and inhibited by addition of an OAT inhibitor to uremic plasma identifies IS as a major, poorly dialyzable, uremic toxin. The signaling pathways initiated by IS and possibly other solutes not effectively removed by dialysis may participate in the pathogenesis of renal scarring and uremic vasculopathy.

The effect of isohydric hemodialysis on the binding and removal of uremic retention solutes

“…In hemodialysis [ 35 ] and in peritoneal dialysis [ 36 ] RRF was associated with better survival independent of the intensity of dialysis. Several studies have compared the concentrations of one or several uremic retention solutes in patients with RRF as compared with patients who are anuric [ 37 , 38 ]. In our small published study we reported that indoxyl sulfate concentration averaged 56 μmol/L in anuric patients and 37 μmol/L in subjects with RRF [ 37 ], a difference of about 33% which may be attributed to the secretion of uremic solutes.…”

“…Several studies have compared the concentrations of one or several uremic retention solutes in patients with RRF as compared with patients who are anuric [ 37 , 38 ]. In our small published study we reported that indoxyl sulfate concentration averaged 56 μmol/L in anuric patients and 37 μmol/L in subjects with RRF [ 37 ], a difference of about 33% which may be attributed to the secretion of uremic solutes. This difference in concentration provides one estimate of a difference that may account for the observed differences in clinical, largely cardiovascular, outcomes.…”

Indoxyl Sulfate Elimination in Renal Replacement Therapy: Influence of Citrate- versus Acetate-Buffering Component during Bicarbonate Dialysis

“…The level of IS in plasma is a significant predictor of major cardiac events [ 6 ], although other authors have found no association at all [ 7 ]. Regardless, a recent study identified IS as a major factor in the dysregulation of almost 2000 genes in vitro [ 8 ]. Moreover, poorly dialyzable uremic toxins are the leading cause of poor survival in dialysis patients.…”

The rebirth of interest in renal tubular function

“…In effect, it may be that the patient with advanced or end-stage renal disease and RRF may excrete toxins in the same manner as the aglomerular anglerfish (33), namely by generating "urine" by tubular secretion rather than filtration. Jhawar et al (26), Klammt et al (28), and Marquez et al (37) have observed that plasma indoxyl sulfate concentration is greater in anuric patients than in patients who retain RRF. Analysis of large data sets suggest that patients with RRF, i.e., continued urine production while undergoing hemodialysis or peritoneal dialysis, have better "quality of life" and less cardiovascular disease than patients who are anuric (2,51).…”

“…These data strongly suggest that factors other than GFR are more powerful determinants of uremic symptoms and outcome. These include renal tubular secretion of protein-bound uremic retention solutes (26,33,37,52), diet content of toxin precursors, toxin production by gut bacteria, and gut transit time (1,13,39,53,55). Furthermore, impaired proximal tubular secretion is now thought to contribute to the increased plasma levels of trimethylamine oxide (23,48,50) and tissue calcification (25), which may influence the outcome in patients with chronic renal disease.…”