Uremic toxins

INTRODUCTION — The uremic syndrome can be defined as the deterioration of multiple biochemical and physiological functions in parallel with progressive kidney dysfunction, thereby resulting in complex but variable symptomatology [1-7]. Normally, healthy kidneys excrete a myriad of compounds. Uremic retention solutes accumulate in patients with chronic kidney disease (CKD), including those with end-stage kidney disease (ESKD) [8]. The retention of these solutes is directly or indirectly attributable to deficient kidney clearance.

These retained solutes are called uremic toxins when they contribute to the uremic syndrome. Apart from inorganic compounds that have an established role, only a few compounds conform to the strictest definition of uremic toxins. However, this does not preclude a potential role for various other retention solutes. (See "Overview of the management of chronic kidney disease in adults".)

Uremic toxins have traditionally been subdivided into three major groups based upon their chemical and physical characteristics, essentially because of the role of those characteristics in removal by conventional dialysis strategies:

●Small, water-soluble compounds with no or minimal protein binding, such as urea

●Small, lipid-soluble compounds with substantial protein binding, such as the phenols

●Larger, so-called middle molecules, such as beta2-microglobulin (beta2-m)

However, more granular classifications have been proposed [9], in part because of the availability of newer dialyzer membranes (eg, medium cut-off [MCO] membranes) that allow for removal of larger compounds than previous membranes. Regardless of classification schemata, uremic retention solutes occupy continuums with regards to protein binding, molecular weight, and presumed toxicity [10,11].

Views on the uremic syndrome and several uremic solutes have changed substantially over time [5,12]. The biochemical, physiologic, and/or clinical impact of the most important and the most extensively studied toxins are presented here [11].

SMALL WATER-SOLUBLE COMPOUNDS

Urea — Many studies demonstrate a toxic (biological) effect of urea at chronic kidney disease (CKD)-relevant concentrations [13,14]:

●Urea inhibits Na-K-2Cl cotransport in human erythrocytes, as well as a number of cell volume-sensitive cellular transport pathways [15]. The Na-K-2Cl cotransport is a ubiquitous process that serves numerous vital functions, including cell volume and extrarenal potassium regulation.

●Urea inhibits macrophage-inducible nitric oxide (NO) synthesis at the posttranscriptional level [16].

●As the most important osmotically active uremic solute, urea may provoke dialysis disequilibrium if the decrease in plasma concentration of urea during dialysis occurs too rapidly. (See "Dialysis disequilibrium syndrome".)

●With increasing kidney dysfunction, increasing amounts of cyanate are spontaneously transformed from urea. The active form of cyanate, isocyanic acid, carbamylates proteins and other molecules, thereby affecting their structure and function [17]. The resulting increase in carbamylated solutes has been linked to endothelial cell death and smooth muscle cell proliferation [18], monocyte adhesion to endothelium [19], and inhibition of endothelial cell repair [20]. All of these factors play a role in vascular damage.

●Treatment of adipocytes with disease-relevant urea concentrations induced radical oxygen species production, caused insulin resistance, and increased modified insulin signaling molecules in one study [21].

●Urea induced in vitro intestinal barrier dysfunction in one study, which could play a role in the increased leakiness of the gut for endotoxin, which has a proinflammatory potential [22].

●In human smooth muscle cells, urea induced apoptosis and cell death, which may contribute atherogenesis and progression of atheromatosis [23].

●An increase in radical oxygen species generation and activation of several other proinflammatory and proatherogenic pathways was demonstrated in arterial endothelial cells [24].

●Insulin resistance was induced in pancreatic beta cells [25].

It has been hypothesized, however, that the possible toxic effects of urea are counterbalanced by the simultaneous retention of methylamines [26].

Findings in in vitro and animal studies that suggest a toxic effect of urea have not been corroborated by clinical studies [27]:

●The lack of correlation with symptoms of the uremic syndrome after addition of urea to the dialysate [28]

●The missing impact in the Hemodialysis (HEMO) and Adequacy of Peritoneal Dialysis in Mexico (ADEMEX) studies of an increase in urea removal on outcome [29,30]

Guanidines — The guanidines are a large group of structural metabolites of arginine. Several of the guanidino compounds modify key biological functions:

●Guanidinosuccinic acid and guanidinopropionic acid inhibit neutrophil superoxide production [31].

●Guanidino compounds exert both pro- and antiinflammatory effects upon leukocytes [32,33].

●Guanidinosuccinic acid, gamma-guanidinobutyric acid, methylguanidine, homoarginine, and creatine induce seizures after systemic and/or cerebroventricular administration in animals [34].

●A mixture of guanidino compounds suppresses the natural killer cell response [35].

●Guanidines cause structural damage to proteins by deamidation/isomerization, which in turn reduces the binding of homocysteine [36].

Although not observed in all studies [37], most investigations have found that NO synthesis is inhibited in patients with end-stage kidney disease (ESKD). This may result in vasoconstriction, hypertension, immune dysfunction, and neurologic abnormalities.

Although arginine (which is also a guanidino compound) markedly enhances the production of NO, some of the other guanidines, as arginine analogs, are strong, competitive inhibitors of NO synthase. Asymmetric dimethylarginine (ADMA), which is significantly increased in ESKD [38], is the most specific endogenous compound with inhibitory effects on NO synthesis. In the brain, ADMA causes vasoconstriction and inhibition of acetylcholine-induced vasorelaxation [39]. It has also been implicated in the development of hypertension, adverse cardiovascular events [40-43], progression of kidney function impairment [44], kidney fibrosis [45], and mortality (see "Risk factors and epidemiology of coronary heart disease in end-stage kidney disease (dialysis)"). Administration of ADMA to healthy volunteers resulted in an increase of vascular resistance and blood pressure in a dose-related manner [43]. It was also demonstrated that ADMA increased vascular stiffness and decreased cerebral perfusion in healthy subjects [46]. ADMA has also been linked to polymorphonuclear activation [47] and to expression of adhesion molecules [48]. It has been suggested, based on solute dialysance data, that ADMA was protein bound [49].

The increase in symmetric dimethylarginine (SDMA), the steric variant of ADMA, is more pronounced than that of ADMA. Although this compound had been considered biologically inactive, SDMA was linked to an increase in reactive oxygen production and an inhibition of NO synthesis [50]. SDMA was also associated with an increase in free radical production by leukocytes; this effect was attributed to an increased calcium influx via store-operated Ca²⁺ channels (SOCs) [51]. In an extensive study that evaluated a panel of guanidino compounds on several cell systems involved in vascular damage, SDMA most consistently induced proinflammatory effects in various types of leukocytes [33]. In addition, SDMA also induced monocytic cytokine production, in contrast to ADMA [52]. In the clinical arm of this study, SDMA in CKD patients was more significantly correlated to interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-alpha) levels compared with ADMA [52]. SDMA also modified high-density lipoprotein (HDL) into an abnormal molecule, inducing endothelial damage [53].

The generation of the guanidines synthesized from arginine in the proximal convoluted tubule, such as guanidinoacetic acid and creatine, is depressed in ESKD [54]. The reported concentration of guanidinoacetic acid is not elevated among uremic individuals [7]. On the other hand, the synthesis of guanidinosuccinic acid, guanidine, and methylguanidine is markedly increased, which appears to be due to urea recycling. The reported concentrations of guanidinosuccinic acid and methylguanidine are increased in uremia compared with normal concentrations (table 1) [7].

Dialytic removal of the guanidines, despite their low molecular weight, is not consistently comparable with that of urea. Many of these compounds display different intradialytic behavior compared with urea, suggesting a pluricompartmental distribution [55,56]. In the absence of protein binding, this behavior points to a retardation in the transfer of these molecules from inside to outside of cells. These findings have been confirmed from direct estimations [57]. Further mathematical analysis revealed that increasing dialysis time was the preferred strategy to improve removal of guanidine compounds that had larger distribution volumes (such as methylguanidine, for example), whereas more frequent dialysis preferably impacted upon compounds with a smaller distribution volume (such as guanidinosuccinic acid) [58]. Optimal results were obtained with a combination of daily and long, slow dialysis [58].

Nondialytic removal of ADMA is less dependent on renal excretion than on metabolic transformation, which is inhibited in the setting of kidney failure. Enhancing metabolism might decrease ADMA concentration. Proof of principle is suggested by studies that showed that genetic overexpression of the ADMA-degrading enzyme, dimethylarginine dimethylaminohydrolase, was cardioprotective in mice following heart transplantation [59]. Furthermore, since the transmethylation reaction catalyzed by this enzyme is stimulated by lowering homocysteine levels, ADMA concentration was decreased by coadministration of intravenous methylcobalamin and oral folate [60]. These data illustrate how uremic solutes may influence each other's concentration and how removal depends not only on kidney function and dialysis but also on metabolism, which can be influenced externally by administration of drugs, vitamins, or nutritional additives.

Oxalate — Massive oxalate retention is uncommon in dialyzed patients, except in primary hyperoxaluria [61]. In this disorder, production is increased due to genetically mediated alterations of oxalate metabolism. (See "Primary hyperoxaluria".)

Among patients with ESKD without primary hyperoxaluria, serum oxalate concentrations are increased approximately 40-fold compared with healthy controls [62]. Secondary oxalosis in such patients is characterized by the deposition of calcium oxalate in multiple tissues, which was mostly observed in early kidney replacement therapy with inefficient dialysis strategies [63]. Oxalate resulted in an increase of intracellular calcium in endothelial cells [64], leading to disturbed endothelial proliferation and repair [64,65]. Oxalate also decreases glucuronidation, a detoxifying conjugation process [66].

Oxalate accumulation may be worsened by excessive intake of oxalate precursors, including ascorbic acid, green leafy vegetables, rhubarb, tea, chocolate, or beets. In addition, those with inflammatory bowel disease are at risk for this complication.

The role of pyridoxine (vitamin B6) in uremic oxalate accumulation remains a matter of debate. In rats with kidney failure, pyridoxine depletion results in increased urine oxalate excretion and depressed kidney function [67]. In hemodialysis patients, pyridoxine at 800 mg/day causes a decrease in oxalate concentration [68]. However, such high doses of pyridoxine may induce gastrointestinal intolerance.

Dialytic removal of oxalate is similar to that of urea and, therefore, is relatively easy with any of the classic dialysis strategies. However, since removal is lower than clearance from the body in healthy controls, serum concentrations of oxalate among dialyzed patients remain higher than normal [69].

Trimethylamine-N-oxide (TMAO) — TMAO has long been recognized as a component of uremic biological fluids [70]. Increased TMAO may be a potentially important risk factor for cardiovascular disease and mortality in the general population [71,72] and in patients with CKD [73,74]. TMAO also may contribute to progression of kidney disease [74,75].

In spite of a substantial number of studies supporting the toxic role of TMAO [75], the compound is also known as a protein stabilizer [76], while fish, one of the major sources of TMAO, is considered cardioprotective [76].

TMAO is generated from digestive breakdown of food products that are further metabolized by the liver [71]. Inhibition of intestinal generation of TMAO precursors not only decreased TMAO concentration, but also experimental indicators of atherogenicity [77]. In animal experiments, the pro-atherogenic effect of TMAO could be inhibited by neutralizing intestinal generation of its precursors [77].

Phosphorus — A high serum concentration of phosphates is related to pruritus and hyperparathyroidism, both manifestations of the uremic syndrome [78]. Phosphorus excess also inhibits 1 alpha-hydroxylase and hence the production of calcitriol, the active vitamin D metabolite [79]. Phosphorus retention also alters polyamine metabolism by causing a decrease in intestinal dysfunction and proliferation of intestinal villi [80]. Hyperphosphatemia is also linked to vascular calcification, proinflammatory mechanisms, and klotho deficiency [81]. This mechanism leads to a rise in fibroblast growth factor-23 (FGF-23), a large molecule that increases in concentration early in the course of CKD [82] and has a strong predictive value for negative outcomes in patients on dialysis [83]. In animal studies, FGF-23 can cause cardiac hypertrophy [84] and impaired recruitment of neutrophils in response to infectious stimuli [85]. (See "Overview of chronic kidney disease-mineral and bone disorder (CKD-MBD)", section on 'Fibroblast growth factor 23'.)

At least in animals, phosphate restriction has an attenuating effect on the progression of kidney disease. The results are less compelling in humans [86]. However, dietary phosphate restriction reduces parathyroid hormone (PTH) levels over a wide range of serum calcium concentrations [87]. In observational studies, high serum phosphorus is linked to mortality [88].

Blood phosphorus concentration is the result of protein catabolism and protein intake, as well as of the ingestion of other dietary sources.

The effect of oral phosphate binders is often insufficient, especially in subjects with high intake. One major effect related to hyperphosphatemia is the increase in Ca deposition in the tissues, especially the vessel wall [88], which is a negative prognostic factor in patients with CKD [89]. This does not exclude the individual roles of both Ca and P in vascular disease of CKD [90]. Application of calcium-containing intestinal phosphate binders may decrease P, but this effect may be counterbalanced by a rise in Ca [91].

The newer generation of phosphate binders, such as sevelamer and lanthanum, may provide a solution to this problem [92]. (See "Management of hyperphosphatemia in adults with chronic kidney disease", section on 'Treatment approach'.)

Hydrogen ions — Metabolic acidosis can contribute to some of the abnormalities in ESKD. Metabolic acidosis can cause muscle wasting and bone mineral loss and, in children, impair growth [93,94]. (See "Pathogenesis, consequences, and treatment of metabolic acidosis in chronic kidney disease".)

PROTEIN-BOUND COMPOUNDS

P-cresol and p-cresyl sulfate — P-cresol, a phenol with a molecular weight of only 108 daltons, has been considered the prototype of lipophilic, uremic, protein-bound toxins. Because of strong protein binding, removal of such compounds by classical dialysis is hampered [95,96]; each hemodialysis session removes only a fraction of p-cresyl sulfate compared to the normal kidneys [97]. It is therefore conceivable that alternative removal methods (eg, adsorption or convective transport) should be developed before adequate elimination of such toxins can be obtained [98,99].

Evidence underscores that conjugates of p-cresol, p-cresyl sulfate and p-cresyl glucuronate, and not p-cresol per se, are present in the blood of patients with chronic kidney disease (CKD) [96,100]. Previous repeated measurements of p-cresol in body fluids were related to the fact that most determination methods applied strong acidification for deproteinization, a measure resulting in deconjugation of p-cresyl sulfate and p-cresyl glucuronate by hydrolysis [101,102]. Since this hydrolysis is conceivably proportional to the quantity of conjugates available, former p-cresol measurements as well as estimations of protein binding are likely proportional to what would have been found by direct measurement of the conjugates.

High-flux dialysis, compared with low-flux dialysis, has no beneficial effect on the removal of these toxins [95,98]. However, compared with peritoneal dialysis, p-cresol is cleared better with high-flux hemodialysis [103]. Nevertheless, the plasma concentrations of protein-bound toxins are lower in patients on peritoneal dialysis compared with those on hemodialysis [103,104]; these findings cannot be explained by differences in dietary protein intake or residual kidney function, suggesting a role for metabolism and/or intestinal handling [105].

P-cresol is an end product of protein catabolism, produced by intestinal bacteria that metabolize tyrosine and phenylalanine [106]. Environmental sources of p-cresol are toluene, menthofuran, and cigarette smoke. Specific concern is warranted regarding menthofuran, which is present in several herbal medicines, flavoring agents, and psychedelic drugs [107]. In view of the known role of the intestine in the generation of p-cresol and its conjugates, several approaches that interfere with intestinal generation of these toxins have been studied and shown to decrease their concentration [108]. (See 'General removal strategies' below.)

In several studies, p-cresol concentration was related to parameters of clinical outcome, including hospitalization rate (particularly due to infection) [102], clinical symptoms of the uremic syndrome [101], mortality [109], and cardiovascular disease [110,111]. The same associations were also demonstrated with p-cresyl sulfate [112,113] and existed among patients with earlier stages of CKD (CKD 2 to 4) [114]. The latter observation suggests that protein-bound solutes may also exert a toxic effect in those not affected by marked kidney disease. In a prospective, observational analysis, p-cresyl sulfate and indoxyl sulfate were independently associated with progression of CKD [115]. A meta-analysis confirmed the negative association of p-cresyl sulfate with hard outcomes [116]. In a study comparing the prognostic weight of several protein-bound uremic toxins, p-cresyl sulfate was the best independent overall predictor of overall mortality and cardiovascular mortality, even after adjustment for estimated glomerular filtration rate (eGFR) [117].

The number of data underscoring the biochemical (toxic) effect of the conjugates is steadily growing [118]. Of note, in most studies, correct concentrations were applied while also taking into account the effect of protein binding. In one study, it was demonstrated that p-cresyl sulfate stimulated baseline leukocyte activity, thereby pointing to a proinflammatory effect, whereas the parent compound p-cresol essentially inhibited activated leukocyte function [119]. Another study found that retained p-cresol and p-cresyl sulfate altered endothelial function [120]. Several studies showed effects related to tubular damage and progression of kidney dysfunction, such as an enhanced expression of cytokine and inflammatory genes in proximal tubular cells [121], epithelial mesothelial transition, fibrosis, and glomerulosclerosis through the activation of the renin-angiotensin-aldosterone system [122] and suppression of klotho gene expression leading to fibrosis [123]. Oxidative stress damage has been demonstrated in rat tubular cells [124], and the induction of insulin resistance and reallocation of adipose cells has been demonstrated in different tissues [125]. The leukocyte-activating impact of p-cresyl sulfate has been demonstrated in whole human blood [126]. In one study, the second conjugate, p-cresyl glucuronide, combined with p-cresyl sulfate, had an additive effect on leucocyte oxidative burst activity [126]. P-cresyl sulfate, administered peritoneally or intravenously, increased rolling of leukocytes along the endothelium of peritoneal vessels, as directly visualized by in vivo microscopy, and p-cresyl sulfate and p-cresyl glucuronide induced vascular leakage for albumin [127]. These data suggest a direct toxic effect of these molecules on vascular endothelium and an induction of the cross-talk between vascular endothelium and leukocytes, which is a primary step of a chain of events leading to vascular damage.

The concern has been expressed that, even if correct uremic concentrations were applied in studies on protein-bound toxins, the (near) absence of albumin may have resulted in an increased percentage of non-protein-bound fraction, which is the fraction that exerts biological activity [128]. However, in a systematic review that was limited to experimental studies that used correct free concentrations of p-cresyl sulfate, several studies confirmed the biological effects of p-cresyl sulfate at uremia-relevant concentrations [129]. The analysis, which also included studies on indoxyl sulfate, in total included 27 publications, of which several complied with high-quality standards.

Although the conjugates are present in the blood stream, it remains unclear whether, under some conditions, they may not be reconverted to p-cresol intracellularly, especially since certain cell types such as the leukocytes contain sulfatases and glucuronidases.

Previously, only a few studies that examined how to improve removal of the phenols by extracorporeal strategies were performed. One study showed superior dialytic clearance of p-cresol by high-flux hemodialysis compared with peritoneal dialysis [103], although, as noted previously, plasma concentrations were lower in peritoneal dialysis patients [105]. Among dialysis strategies, convective approaches were more efficient compared with diffusive ones [130-132]. In another study, increasing dialysate flow and dialyzer surface area while decreasing blood flow in an extended dialysis setting increased removal of protein-bound solutes without altering Kt/V [133]. However, as compared with standard, four-hour dialysis three times per week, a longer session (eight-hour dialysis) three times per week had no significant impact on protein-bound solute concentration; this was in contrast to observations with small, water-soluble compounds and middle molecules [134]. However, a nonsignificant trend for increased removal of protein-bound molecules was observed in this study. In contrast, if hemodialysis was extended without decreasing blood or dialysate flow as compared with standard, a significant increase in removal with extended dialysis could be observed [135].

Removal could be markedly enhanced by applying combined fractionated plasma separation and adsorption, a strategy used by an artificial liver (Prometheus) [136]. Unfortunately, the approach applied in this study resulted in major coagulation disturbances [137]. However, the study offers proof of concept that adsorption may become an additional asset in the removal of this type of compound. In an in vitro setting, embedding adsorptive material on a high-flux membrane markedly enhanced the removal capacity for protein-bound toxins [99,138].

As far as convective strategies are concerned, it was shown that postdilution and predilution hemodiafiltration were not different regarding removal of p-cresyl sulfate. Both hemodiafiltration strategies were superior to predilution hemodiafiltration for p-cresyl sulfate and other protein-bound compounds, whereas postdilution hemodiafiltration was superior to predilution hemodiafiltration for all other types of uremic retention solutes (ie, the small, water-soluble compounds and the larger "middle molecules") [139].

The cresols, as several other protein-bound toxins, are the end product of metabolic transformation of amino acids by the intestinal microbiome and by conjugation processes in the intestinal wall or liver [108]. In advanced chronic kidney disease, the microbiome appears to be modified in favor of species that generate these uremic toxins [140], though studies examining the association between kidney dysfunction and urinary excretion of p-cresol report conflicting results [141,142].

There are studies suggesting that intestinal bacterial production and/or intestinal uptake of p-cresol can be altered. As an example, prevention of the intestinal absorption of p-cresol by administration of oral sorbents (AST-120) decreased its serum concentration [143]. In a metabolomic study in rats, the same sorbent decreased the concentration of a host of mainly protein-bound uremic solutes, including p-cresyl sulfate [144]. In addition, the prebiotic arabino-xylo-oligosaccharide (AXOS) decreased p-cresyl sulfate in mice, while decreasing its biochemical impact mainly related to insulin resistance [125]. However, in a mouse model of CKD, sevelamer hydrochloride administration had no impact on the concentration of any of the protein-bound compounds [145], and the same was observed in a subsequent study in humans [146]. A subsequent randomized controlled trial (RCT) in patients with nondialysis CKD, however, did show a decrease induced by sevelamer [147].

Dietary intake may alter the generation of p-cresyl sulfate. In one study of 26 individuals with normal kidney function, the average p-cresyl sulfate excretion (reflecting generation) was 62 percent lower among vegetarians than among participants on an unrestricted diet [148]. A metabolomic comparison between hemodialysis patients with and without colon showed striking differences in protein-bound and other uremic toxin concentrations, including p-cresyl sulfate [149].

The quality of studies assessing interventions to modify the intestinal microbiome or its metabolism in CKD is poor [150], and hard outcome studies remain scarce [150]. A randomized, controlled trial on dietary fiber supplementation showed a decrease in indoxyl sulfate concentration but not in p-cresyl sulfate [151]. In another randomized, controlled trial, administration of a symbiotic resulted in a decrease of p-cresyl sulfate concentration, especially in patients who received no antibiotics during the course of the study [152].

Homocysteine — Homocysteine (Hcy) is a sulfur-containing amino acid produced by the demethylation of methionine. Its retention with uremia results in the cellular accumulation of S-adenosyl homocysteine (AdoHcy), an extremely toxic compound that competes with S-adenosyl-methionine (AdoMet) and inhibits methyltransferases [153]. Patients with CKD have total serum Hcy levels two- to fourfold above those observed in normal individuals. In addition to the degree of kidney dysfunction, its serum concentration also depends upon nutritional intake (eg, of methionine), vitamin status (eg, of folate), and genetic factors.

The association of hyperhomocysteinemia with vascular disease is discussed elsewhere. (See "Overview of homocysteine".)

Tryptophan metabolites — Indoxyl sulfate is metabolized by the liver from indole, which is produced by the intestinal microbiome as a metabolite of tryptophan. Indoxyl sulfate, which is secreted in the normal kidney by organic anion transporter 3 [154], enhances drug toxicity by competition with acidic drugs at protein-binding sites [155] and inhibits the active tubular secretion of these compounds as well as the deiodination of thyroxine 4 (T4) by cultured hepatocytes [156,157].

Uremic retention solutes induce glomerular sclerosis [158], with their removal by peritoneal dialysis or by oral sorbent administration retarding the progression of intact nephron loss. Indoxyl sulfate may be one of these possible uremic toxins. The oral administration of indole or of indoxyl sulfate to uremic rats causes a faster progression of glomerular sclerosis and kidney dysfunction [159,160]. In renal tubular cells, indoxyl sulfate induces free radical production, nuclear factor kappa B (NF-kappaB) activation, and upregulation of plasminogen activator inhibitor-1 (PAI-1) expression [161]. Studies cited above demonstrated indoxyl sulfate-associated enhanced expression of cytokine and inflammatory genes [121], epithelial mesothelial transition, fibrosis and glomerulosclerosis through the activation of the renin-angiotensin-aldosterone system [122], and suppression of klotho gene expression leading to fibrosis [123].

Among patients on peritoneal dialysis, indoxyl sulfate was correlated with interleukin (IL)-6 [162]. Risk factors of atherosclerosis are associated with indoxyl sulfate concentration in patients on hemodialysis [163]. In a study that included patients with CKD stages 2 to 5, serum indoxyl sulfate was predictive of mortality, even after adjustment for major cardiovascular risk factors [164]. However, in a multifactorial analysis, the significance of its association with outcomes was inferior to that of p-cresyl sulfate [117].

Endothelial cell dysfunction is common in uremia. Indoxyl sulfate may play a role by inhibiting endothelial cell proliferation and repair [165], while it induces a significant production of free radical species in endothelial cells [166,167]. Endothelial dysfunction induced by indoxyl sulfate is also illustrated by an increase in microparticle release by cultured endothelial cells [168]. Endothelial-leukocyte interaction, resulting in leukocyte adhesion to the endothelial wall, is also activated [169]. In a rat model, indoxyl sulfate stimulates vascular smooth muscle cell proliferation [170]. Indoxyl sulfate also has direct profibrotic, prohypertrophic, and proinflammatory effects on cardiac fibroblasts and myocytes [171].

It has been suggested that indoxyl sulfate plays a role in aortic calcification [172] and elements of bone dysfunction, such as resistance to parathyroid hormone (PTH), osteoblast dysfunction, and downregulation of pathways of PTH gene expression and low-turnover bone disease [173-175].

A thrombogenic effect has also been attributed to indoxyl sulfate, essentially by the demonstration of interference with the generation of thrombogenesis tissue factor 3 via the aryl hydrocarbon receptor pathway [176-178].

In one study, indoxyl sulfate was shown to enhance adhesion and extravasation of leukocytes to the same degree as lipopolysaccharide, as well as to cause an almost total stagnation of blood flow within the studied vessels [127]. In an attempt to unravel the mechanism of these profound alterations, it was shown that, in the presence of indoxyl sulfate, the glycocalyx, a molecular layer protecting the endothelium, was affected. Heparan sulfate, one of the main components of the glycocalyx, was found in increased concentration in the serum of indoxyl sulfate-treated rats. Of note, a study had shown in a similar way increased free-floating heparan sulfate in the plasma of hemodialysis patients [179].

A systematic review that included multiple studies revealed an overall negative biochemical impact of indoxyl sulfate [129]. This was also observed with p-cresyl sulfate (see 'P-cresol and p-cresyl sulfate' above). The data contained in the systematic review are of interest in function of the ongoing discussion that many toxicity studies on protein-bound uremic toxins, of which indoxyl sulfate is a prototype, have been biased by the application of too high concentrations, especially free non-protein-bound concentrations. All 27 studies included in that systematic review had been performed at correct concentrations. The review pointed to biologic effects that all could be linked to vascular damage and progression of kidney failure.

Studies published subsequent to this systematic review and also applying the correct concentrations point to suppression of erythropoietin production [180], monocyte transition into profibrotic macrophages [181], suppression of neovascularization [182], suppression of fetuin-A expression [183], vascular inflammation via activation of the aryl hydrocarbon receptor [184], vascular functional disturbances [185], and cognitive impairment [186]. Many of these studies point to a central role of the aryl hydrocarbon receptor [178,180,184,186].

That biological effects of indoles are not restricted to indoxyl sulfate was demonstrated in a study showing that indole-3-acetic acid activated the inflammatory nongenomic aryl hydrocarbon receptor (AhR)/p38MAPK/NF-kappaB pathway, hence inducing the proinflammatory enzyme cyclooxygenase-2 [187]. A clinical arm of this study also showed a higher mortality among patients with the highest indole-3-acetic acid levels [187]. Other studies demonstrated a proinflammatory and a procoagulant effect [176,188,189]. Indole acetic acid has also been associated with kidney damage [158,190] and with osteoblastogenesis [191].

Another group of toxic indoles with emerging interest are the kynurenines. Kynurenines increase leukocyte adhesion to vascular endothelium [192], activate AhR [193], and decrease production of tumor necrosis factor-alpha (TNF-alpha) [194]. Injections of kynurenic acid in rats with normal kidney function impaired their cognitive flexibility [195]. Furthermore, they also modify metabolic functions [66,196].

In patients with ESKD, the kynurenines were associated with oxidative stress, inflammation, cardiovascular disease, and hypercoagulability [197,198].

Indoles are found in various plants and herbs, and a substantial number are produced by the intestinal microbiome. Several metabolites are retained in uremia, and kinetic behavior appears to differ among them. Some of these substances do not even conform to the definition of uremic retention solutes, as their concentration is low in patients with ESKD (eg, tryptophan, melatonin) and/or they have been associated with beneficial biologic effects (eg, indole, indole propionic acid).

Indoxyl sulfate concentration has been associated to mortality and vascular damage in CKD patients [164] and also with postangioplasty stenosis of vascular access grafts [199]. A meta-analysis accounting for several clinical studies confirmed the negative association of indoxyl sulfate with hard outcomes [116]. [200]

Removal of protein-bound tryptophan metabolites is hampered during dialysis [95]. It does not correlate with that of urea or creatinine [201]. In conventional hemodialysis, super-flux triacetate membrane was superior to low-flux cellulose triacetate regarding removal of indoxyl sulfate [202]. Convective strategies are superior to diffusion with regards to removal [132,139]. It is, however, questionable whether these rather subtle differences in removal will be sufficient to have a significant impact on solute concentration. Adsorptive removal strategies have the potential to provide added value [99]. In a group of CKD patients not yet on dialysis, the sorbent AST-120 actively decreased indoxyl sulfate in a dose-dependent way, as appreciated from a short-term, prospective, dose-finding study. However, in that study, there were no differences in decline of kidney function [203].

As in a prospective, observational analysis, p-cresyl sulfate and indoxyl sulfate were independently associated with progression of CKD [115]. A next logical step was to study the impact of AST-120 on the progression of CKD. In small, controlled studies with a long follow-up period, the group on AST-120 showed a slower decline of eGFR, was started later on dialysis, and survived longer once dialysis was started [204-206]. However, a large American/European randomized, controlled trial could not confirm the beneficial impact of AST-120 on the progression of kidney failure [207]. The study contained no control of whether the group on AST-120 showed a decrease in plasma concentration of protein-bound uremic toxins. Also, in another study, no impact of AST-120 on progression of CKD could be observed, but, in this study, the administration of the sorbent was not associated with a marked decrease in plasma indoxyl sulfate [208].

Oral administration of bifidobacteria in gastro-resistant capsules to hemodialysis patients, with the intention of modifying the intestinal microbiome, reduces serum levels of indoxyl sulfate and corrects the microbiome balance [209,210]. Dietary intake may also alter serum levels of indoxyl sulfate. In the study cited above of 26 individuals with normal kidney function, the average indoxyl sulfate excretion (reflecting generation) was 58 percent lower among vegetarians than among participants on an unrestricted diet [148]. In a randomized, controlled trial of hemodialysis patients, dietary fiber supplementation induced a decrease in indoxyl sulfate concentration but not in p-cresyl sulfate [151]. In another randomized, controlled trial, administration of a symbiotic resulted in a decrease of p-cresyl sulfate concentration, especially in patients who received no antibiotics during the course of the study [152]. The differences in response between p-cresyl sulfate and indoxyl sulfate might be explained by diverging metabolic pathways in the generation of these two solutes [211].

Kidney transplantation decreased indoxyl sulfate to a level that was lower than for nontransplanted subjects with similar kidney function [212]. Also, in patients with acute kidney injury (AKI), serum indoxyl sulfate concentrations were lower than in patients with CKD and similar kidney function [213]. These findings suggest an impact of other factors in addition to kidney function (eg, intestinal metabolism).

Furanpropionic acid — 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), a urofuranic fatty acid, is a strongly lipophilic uremic solute and a major inhibitor of drug protein binding [156]. This toxin inhibits the kidney uptake of para-amino hippuric acid (PAH) in rat kidney cortical slices [214] and causes a decrease in renal excretion of various drugs, metabolites, and endogenously produced organic acids that are removed via the PAH pathway. In vivo kidney CMPF clearance in the rat is inhibited by PAH and probenecid [215]. CMPF also inhibits hepatic glutathione-S-transferase [216], deiodination of T4 by cultured hepatocytes [157], and adenosine diphosphate (ADP)-stimulated oxidation of nicotinamide adenine dinucleotide (NADH)-linked substrates in isolated mitochondria [217]. CMPF has been related to inhibition of insulin secretion from pancreatic islets [218,219] but in other studies has been shown to inhibit hepatic steatosis [220,221] and via this route insulin resistance [221].

There is a correlation between neurologic abnormalities and the plasma concentration of CMPF [222]. Since CMPF is almost 100 percent protein bound, its removal by hemodialysis strategies is virtually nonexistent. CMPF levels can be lowered substantially only with peritoneal dialysis [223].

Phenyl acetic acid — Phenyl acetic acid has been linked to inhibition of inducible nitric oxide synthase (iNOS) expression [224], osteoblast proliferation and differentiation [225], enhancement of the formation of oxygen-free radicals in vascular smooth muscle cells [226], and increases of inflammatory response by polymorphonuclear leukocytes [227]. It also inhibits renal cellular solute transport pumps [228] and conjugation by glucuronidation [66]. Finally, in a clinical study, phenyl acetic acid was related to cognitive dysfunction in dialysis patients [229].

MIDDLE MOLECULES — Middle molecules, arbitrarily defined as those of a molecular weight in excess of 500 daltons, were previously thought responsible for the uremic syndrome. However, at the time of the formulation of the middle-molecule hypothesis, it was extremely difficult to isolate specific responsible molecules.

Nevertheless, several clinical, metabolic, and/or biochemical disturbances are caused by uremic compounds with molecular weights in this middle-molecule range:

●Chromatographic fractions of uremic ultrafiltrate with a molecular weight between 1 and 5 kD inhibit appetite and suppress food intake in animals [230].

●A 500 to 2000 dalton subfraction of uremic serum inhibits apolipoprotein (apo) A-I secretion in a human hepatoma cell line [231].

●A compound of molecular weight between 750 and 900 daltons inhibits osteoblast mitogenesis [232].

A substantial number of compounds have been identified that conform to the strict definition of middle molecules [7,8,11,233]. Several of those have biological effects and especially affect vascular wall integrity by causing inflammation, coagulation, endothelial damage, or smooth muscle cell proliferation [2].

A variety of dialytic strategies increase removal of middle molecules, such as the use of high-flux dialysis membranes, hemodiafiltration, and medium cut-off (MCO) dialyzers. The potential clinical impact of such strategies is discussed elsewhere. (See "Patient survival and maintenance dialysis", section on 'Hemodialysis membranes' and "Alternative kidney replacement therapies in end-stage kidney disease", section on 'Clinical effects of convective therapies' and "Overview of the hemodialysis apparatus", section on 'Flux'.) [234-237]

Beta2-microglobulin — Beta2-m (molecular weight of approximately 12,000 D) is a component of the major histocompatibility antigen (see "Major histocompatibility complex (MHC) structure and function"). Dialysis-related amyloid, which can be observed in patients being maintained on long-term dialysis, is to a large extent composed of beta2-m. Nevertheless, some patients may suffer from amyloidosis after only one to two years of dialysis [238,239]. Dialysis-related amyloidosis is discussed in detail elsewhere. (See "Dialysis-related amyloidosis".)

In a proteomic analysis searching for biomarkers for peripheral vascular disease in a population reportedly without major kidney dysfunction, beta2-m was the only discriminator, suggesting a potential link to the development of vascular disease [240].

Beta2-m has also been shown to impair cognitive function and act as a pro-aging factor [241]. It has also been associated with itching [242] and frailty [243]. However, no activation of circulating leukocytes could be observed in the presence of uremic concentrations of beta2-m in an in vitro study [244]. This does not exclude other biological actions of that molecule. Beta2-m fibrils affect function and/or viability of osteoblasts, osteoclasts, and chondrocytes [245]. An in vitro study showed a link between beta2-m and modifications in the erythrocyte plasma membrane, with the potential to affect cell shape and removal from the circulation [246]. In vivo and in vitro experimental studies showed an impact on osteoclast formation [247] and upregulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 generation [247], epithelial-to-mesenchymal transition [248], and neurotoxicity [249].

Advanced glycosylation end products (AGEs) may affect the pathophysiologic impact of beta2-m. AGE-modified beta2-m has been identified in amyloid of hemodialyzed patients [250]; it also enhances monocytic migration and cytokine secretion [251], suggesting that foci containing AGE beta2-m may initiate inflammatory response, leading to bone/joint destruction [250,251].

Serum beta2-m levels may be lower in peritoneal dialysis patients than in hemodialysis patients [252]. This may be due to a better conservation of endogenous residual kidney function with peritoneal dialysis since peritoneal dialysis alone poorly clears beta2-m [103]. Although the clinical expression of dialysis-related amyloidosis disappears after kidney transplantation, the underlying pathologic processes, such as bone cysts and tissue beta2-m deposits, remain [253].

In prospective studies, a progressive decline of predialysis beta2-m levels has frequently been demonstrated in patients dialyzed with membranes with a larger pore size [254]. The question arises whether this removal is sufficient to prevent the development of beta2-m amyloid. In a retrospective study, AN69 dialyzers with a large pore size were associated with a lower prevalence of amyloid disease than small-pore cuprophane [255]. However, cuprophane also induces a more profound inflammatory reaction, which is known to enhance the development of amyloid disease.

Finally, a post-hoc analysis of the HEMO study found that serum beta2-m levels correlated with mortality, with each 10 mg/L increase in predialysis level being associated with an 11 percent increase for all-cause mortality [256]. Later analysis revealed that this mortality was related to infectious causes [257]. This result may lend support to using beta2-m levels as a marker for middle-molecule clearance. These data may, however, have been confounded by residual kidney function, selection bias, and inflammation.

Parathyroid hormone — Parathyroid hormone (PTH), a middle molecule with a molecular weight of approximately 9000 daltons, is generally recognized as a major uremic toxin. However, its increase in concentration during end-stage kidney disease (ESKD) is attributable to enhanced glandular secretion rather than to decreased removal by the kidneys. Excess PTH gives rise to an increase in intracellular calcium, resulting in disturbances in the function of virtually every organ system [258].

A downregulation of PTH/PTHrP receptor mRNA expression is observed in the liver, kidney, and heart of rats with advanced chronic kidney disease (CKD), thereby blunting the cellular response to excess PTH and creating resistance to PTH [259]. Parathyroidectomy does not entirely prevent PTH/PTHrP receptor downregulation [260], suggesting that this alteration depends on more than elevated PTH alone.

The increased PTH concentration in uremia is the result of a number of compensatory homeostatic mechanisms. Hyperparathyroidism results at least in part from phosphate retention, decreased production of calcitriol (1,25-dihydroxyvitamin D), and hypocalcemia. (See "Overview of chronic kidney disease-mineral and bone disorder (CKD-MBD)".)

Advanced glycosylation end products — The role of AGEs in CKD has extensively been reviewed [261]. Glucose and other reducing sugars react nonenzymatically with free amino groups to be converted after weeks into AGEs through chemical rearrangements and dehydration reactions [262]. Several AGE compounds are peptide-linked degradation products (molecular weight 2000 to 6000 D) [263]. Among the postulated structures for AGE are glyoxal, methylglyoxal, imidazolone, pyrrole aldehyde, pentosidine, and N epsilon-(carboxymethyl)lysine.

The increased accumulation of AGE in CKD is not only the result of enhanced glucose levels or reduced removal of modified proteins by glomerular filtration. With uremia, it is also likely due to increased concentrations of small carbonyl precursors and inflammation. Thus, uremia can be described as a status of increased carbonyl stress, resulting from increased oxidation or decreased detoxification of these carbonyl compounds [264]. Together, these processes result in increased levels of AGEs [264].

AGEs affect multiple processes [261]. These compounds:

●Cause an inflammatory reaction in monocytes by the induction of IL-6, TNF-alpha, and interferon-gamma [265]. Proinflammatory effects had previously been demonstrated with artificially prepared AGEs. A similar proinflammatory impact was present with genuine AGEs as they were accumulated in uremia [266].

●Modify beta2-m, which (as previously mentioned) may play an important role in the formation of dialysis-related amyloid.

●React with and chemically inactivate nitric oxide (NO), a potent endothelium-derived vasodilator, antiaggregant, and antiproliferative factor [267].

●Induce oxidative protein modification [268].

●Cause kidney fibrosis [269].

●Promote thrombogenicity [270], bone disease, [271] and neurotoxicity [272].

In addition to kidney dysfunction, AGEs are also retained in diabetes mellitus and aging, settings in which they have been implicated in tissue damage and functional disturbances. Specific receptors for AGEs have also been identified (RAGE), with their expression already enhanced during moderate uremia [273].

However, it is unclear whether all AGEs retained in uremia have a biological impact. It is also unknown whether one AGE is kinetically representative for the molecules of the group. Pentosidine has been advanced as a marker for these compounds; however, with respect to dialysis, inhomogeneous behavior of pentosidine has been demonstrated, a finding that parallels what is known for uremic toxins in general [274]. In an observational study, high, rather than low, carboxymethyllysine levels were linked to better outcomes [275].

This group of molecules highlights growing interest to dialytically remove more of the larger molecules that are retained in uremia, perhaps including more specific removal via adsorption columns.

Removal is also more efficient with membranes with a larger pore size [276]. This includes protein-leaking dialyzers [277]. This was shown in one study in which two groups of 13 hemodialysis patients were treated for six months with protein-leaking and non-protein-leaking dialyzers [277]. A significant decrease in total pentosidine values was observed with the protein-leaking dialyzer (-43 percent), a decrease not observed with the other dialyzer.

This removal could counterbalance some of the deleterious effects of AGE accumulation (eg, apolipoprotein B production) [278]. Whether this removal will be sufficient to neutralize the clinical complications potentially attributed to the AGEs is unclear.

Several nondialytic therapeutic approaches to reduce AGE load have achieved variable success, such as crosslink breakers and AGE-receptor inhibitors [261]. Studies with a glyoxylase-1 activator showed a decrease of methylglyoxal and an increase in insulin sensitivity [279].

Other middle molecules — Several peptides of molecular weight above 12 kD, which can be recovered from uremic sera, ultrafiltrate, or peritoneal dialysis dwell fluid, dampen various polymorphonuclear cells functions involved in the killing of invading bacteria [280,281]. The exact concentrations in uremic sera or biological fluids were not reported in these studies.

Leptin, a 16 kD plasma protein that suppresses appetite [282] and induces weight reduction in mice [283], is retained in kidney dysfunction [284]. The increase in serum leptin levels is almost entirely due to a rise in free (non-protein-bound) concentration [284]; it has been suggested to play a role in the decreased appetite of uremic patients (see "Pathogenesis and treatment of malnutrition in patients on maintenance hemodialysis"). However, there are conflicting findings concerning the possible biochemical role of leptin in kidney dysfunction:

●Increased leptin is associated with low protein intake and loss of lean tissue [285]. Data suggest an inverse correlation in uremia between leptin and indices of nutritional status such as serum albumin or lean body mass [286] and a direct correlation with C-reactive protein (CRP) [287].

●However, leptin levels are also elevated in obese people and are not necessarily related to reduced appetite. Body fat and serum leptin correlate positively in uremia [287].

The dinucleoside polyphosphates are molecules characterized by the presence of two nucleotides at the extremes, linked by a variable number (mostly two to six) of phosphates. They have a molecular weight of approximately 1000 daltons and are protein bound. The diadenosine polyphosphates induce proliferation of smooth muscle cells [288,289] and enhance free radical production by leukocytes [290]. Uridine adenosine tetraphosphate is a strong vasoconstrictor, which is released by endothelium [291].

Fibroblast growth factor-23 (FGF-23) is mostly considered as a regulator of calcium/phosphate metabolism associated with outcome parameters [292] but also has a direct biological impact by inducing left ventricular hypertrophy [84] and leukocyte dysfunction [85].

Further middle molecules of potential importance are complement factor D, adrenomedullin, atrial natriuretic peptide (ANP), resistin, immunoglobulin light chains, neuropeptide Y, and various cytokines. Plasma IL-6 is independently associated with mortality in patients with CKD [293]. In vitro, however, neither IL-6 nor any of the other major cytokines induced leukocyte activation at concentrations observed in dialysis patients [294]. The only cytokine that produced an effect was TNF-alpha, however, only at high concentrations, which are no longer observed with dialysis strategies employed today. On the other hand, blockade of the interleukin-6 receptor in clinical studies significantly reduced cardiovascular risk [295].

LARGE MOLECULES — Newer classifications of uremic toxins have added larger molecules as a novel category [9]. The term refers to molecules with a molecular weight above 58,000 daltons (ie, above the cut-off value of the pores of the kidney glomerular basement membrane). Thus, kidney insufficiency has not much impact on their concentration, but their biological impact may be affected by post-translational modifications of their structure due to uremic conditions. These large molecules include albumin, lipoproteins, or other proteins that may be linked to a variety of smaller moieties that accumulate in patients with impaired kidney function. Examples of such ligands are malondialdehyde, guanidines (including symmetric dimethylarginine), or carbonyl compounds [296].

GENERAL REMOVAL STRATEGIES — The main strategies that have been used to decrease uremic solute concentration are conventional hemodialysis and peritoneal dialysis. However, dialysis is nonspecific and also removes essential compounds. In addition, lipophilic compounds, which may be responsible at least in part for functional alterations in uremia, are inadequately removed by current dialysis strategies. Finally, the complex multicompartmental behavior of uremic toxins results in the removal of only a small fraction of the solutes that are accumulated in the body.

With maintenance hemodialysis, treatment with high-flux membranes was suggested to provide superior removal of middle molecules, possibly resulting in improved survival. However, the Hemodialysis (HEMO) study found no overall survival difference with high- versus low-flux membranes at primary analysis [29]. On the other hand, differences were found for the entire cohort in cardiovascular mortality and in the subgroup treated for >3.7 years upon enrollment for overall mortality with secondary analysis [297]. In the same database, a direct relation between beta2-microglobulin (beta2-m) concentration and mortality was also demonstrated [256]. In addition, the Membrane Permeability Outcome (MPO) trial subsequently demonstrated better outcomes associated with high-flux compared with low-flux membranes in the population with serum albumin <4 g/dL [298]. Of note, this low serum albumin group composes a large section of the current population on dialysis. (See "Patient survival and maintenance dialysis", section on 'Hemodialysis membranes' and "Overview of the hemodialysis apparatus", section on 'Flux'.)

Adding convection by increasing ultrafiltration and equivoluminous substitution with sterile saline or ultrapure dialysate (hemodiafiltration) adds to middle-molecule removal [139,299-307]. (See "Alternative kidney replacement therapies in end-stage kidney disease", section on 'Clinical effects of convective therapies'.)

Compounds may be cleared more efficiently with continuous or long-lasting strategies because removal is more gradual (see "Technical aspects of nocturnal hemodialysis"). In a strictly comparable setting for all dialysis parameters except treatment length, prolonging high-flux hemodialysis from four to eight hours resulted in a time-dependent increase in beta2-m and phosphorus removal [308]. This implied decreasing blood and dialysis flows in proportion to the increase of dialysis length. However, prolonging hemodialysis did not decrease protein-bound solutes in a similar study [134] unless blood and dialysate flows were kept high [135].

Optimal removal for individual types of molecules may be obtained with different types of extracorporeal treatment (eg, by using large-pore membranes and/or dialyzers or devices with a high adsorptive capacity for some or several of the uremic toxins). This includes, for example, protein-leaking membranes, which are designed to allow passage of albumin, other similarly sized proteins, and uremic toxins in the 35 to 60 kD size range [309].

Medium cut-off (MCO) dialyzers contain larger pores and remove more middle molecules than high-flux dialyzers, with albumin losses comparable with high-flux hemodiafiltration [310]. They may equal or even surpass removal by hemodiafiltration with classical high-flux membranes, even when used in a hemodialysis mode [310].

Research centers on alternative measures adding to the removal capacity of classical dialysis. These measures include adsorption [99] or changing the physical conditions within the dialyzer (hemodiafiltration with increased plasma ionic strength) and enhancing the free fraction of protein-bound toxins [311]. Clinical tests with the latter strategy yield discrepant results with regard to protein-bound toxin removal [312].

Removal is also influenced by intestinal intake (especially for the protein-bound solutes, but also for some small water-soluble compounds such as trimethylamine-N-oxide) and preservation of kidney function [313]:

●Intestinal uptake can be reduced by influencing dietary uptake or by oral administration of absorbents. Approaches that have been shown to result in a decrease in concentration include a low-protein diet [148,314], administration of prebiotics such as resistant starch [315], or probiotics such as Bifidobacterium [108,316]. The active intestinal sorbent AST-120 has essentially been associated with absorption of indoxyl sulfate [203] and subsequent preservation of kidney function, but this substance also absorbs p-cresol as well as other compounds [143,144]. However, in large randomized controlled trials (RCTs), the expected positive impact on progression of kidney function impairment has not been confirmed [112,207]. Administration of a symbiotic resulted in a decrease of p-cresyl sulfate and indoxyl sulfate concentration, especially in patients who received no antibiotics during the course of the study [152].

●Preservation of residual kidney function may also be an important manner to pursue additional removal of retention solutes. In a number of studies, a correlation was found between concentration of several uremic toxins and residual kidney function [317,318]. This relationship appeared stronger than that to adequacy of dialysis. Many of the uremic retention solutes are secreted in the normal kidneys by the organic acid transporters (OATs) of the renal tubular cells [319]. Enhancing this removal (eg, by applying bioartificial kidneys seeded with living tubular cells) may increase removal of these compounds [320].

Metabolic pathways generating uremic toxins also generate beneficial compounds. For example, tryptophan metabolites generate notorious uremic toxins like indoxyl sulfate or the kynurenines, but also beneficial compounds like indole of indole propionic acid [200]. It is conceivable that dialysis as a removal strategy clears all compounds from the blood without distinction between harmful and beneficial compounds.

Finally, it should be considered that, in uremia, not only strategies decreasing solute concentration are important, but interventions countering their biological impact also play a role [6]. Typically, this can be obtained with already developed drugs, such as angiotensin-converting enzyme (ACE) inhibitors in neutralizing Ca influx due to symmetric dimethylarginine (SDMA) [321], but also with drugs still to be developed and would reach a much broader population than with removal strategies (ie, not only the 0.1 to 0.2 percent of the global population with stage 5 chronic kidney disease [CKD], but all those with stage 3 CKD or more [5 to 10 percent]). Similarly, interventions increasing metabolic degradation of toxic solutes may be considered [60]. Some experimental studies suggest that the concentration of indoxyl sulfate can be decreased by interventions decreasing the activity of sulfotransferase, the enzyme responsible for the sulfation of the parent compound, indole [322].

SUMMARY — The uremic syndrome is a complex mosaic of clinical alterations that may be attributable to several of the different solutes retained in uremia/chronic kidney disease (CKD). Knowledge about the nature and kinetic behavior of the responsible compounds may help when new therapeutic options are considered in the future.

●Factors affecting uremic retention solutes – The following factors may interfere with uremic solute concentrations and their impact on biological functions:

•In addition to classical sources of uremic solutes such as dietary protein breakdown, alternative sources such as environmental contact, food additives, natural stimulants (coffee, tea), intake of herbal medicines, or addiction to psychedelic drugs may play a role in uremic toxicity.

•Many solutes with toxic capacity enter the body through the intestine. Changes in the composition of the intestinal microbiome, or changes in intestinal production, absorption, transfer, and metabolization may alter serum concentration of these toxins.

•Some uremic solutes interfere with functions that directly affect the biochemical action of other solutes (eg, the expression of parathyroid hormone [PTH] receptors, the response to 1,25(OH)2 vitamin D3, protein binding, breakdown, tubular transport, etc).

•Most patients with kidney failure use multiple medications. Interference of drugs with protein binding and/or tubular secretion of uremic solutes influences their biological effect.

•Lipophilic (protein-bound) compounds may be responsible at least in part for functional alterations in uremia; they are inadequately removed by current dialysis strategies.

•The main strategy that has been used to decrease uremic solute concentration is dialysis. However, dialysis is nonspecific and also removes essential compounds.

•Uremic solutes accumulate not only in the plasma but also in the cells, where most of the biological activity is exerted. Removal of intracellular compounds during dialysis through the cell membrane may be hampered, resulting in multicompartmental kinetics and inadequate detoxification, unless dialysis length and/or frequency are increased.

●Spectrum of uremic retention solutes – Biochemical alterations are provoked by a broad spectrum of compounds:

•Some are small and water soluble (eg, urea, the guanidines, phosphate, oxalate, trimethylamine-N-oxide). (See 'Small water-soluble compounds' above.)

•Some are lipophilic and/or protein bound (eg, p-cresyl sulfate, 3-carboxy-4-metyl-5-propyl-2-furanpropionic acid [CMPF], homocysteine [Hcy], indoxyl sulfate, kynurenine). (See 'Protein-bound compounds' above.)

•Some are larger and in the middle-molecule range (eg, beta2-microglobulin [beta2-m], PTH, advanced glycosylation end products [AGEs], the cytokines, fibroblast growth factor-23). (See 'Middle molecules' above and 'Large molecules' above.)

•Some compounds from one group may impact generation and behavior of compounds from another group (eg, guanidines increasing generation of tumor necrosis factor-alpha [TNF-alpha]).

●Removal with extracorporeal therapies – Optimal removal for each type of molecule may be obtained with a different type of extracorporeal treatment (eg, by using large-pore membranes, convection, and/or dialyzers or devices with a high adsorptive capacity for some or several of the uremic toxins). Adsorption with the dialysis devices currently available is of little importance. More specific strategies using large adsorptive surface areas must be developed before adequate adsorption can be obtained. (See 'General removal strategies' above.)

●Importance of intestinal uptake and residual kidney function – Solute concentration is also influenced by intestinal intake and preservation of kidney function:

•Intestinal uptake can be reduced by influencing dietary intake, by oral administration of sorbents, or by influencing the intestinal microbiome by administration of prebiotics, probiotics, or synbiotics.

•Preservation of residual kidney function is also an important manner to pursue additional removal of retention solutes.

●Need for better markers of uremia – A selection of appropriate marker molecules for uremic retention and dialytic removal should be reconsidered. It has become increasingly evident that the current markers, which are all small, water-soluble compounds (urea, creatinine), are not always representative in their kinetic behavior for middle molecules, lipophilic/protein-bound compounds, and even some other hydrosoluble compounds. On the other hand, it may be derisory to seek a marker that is representative for all solutes or even groups of solutes responsible for the uremic syndrome. Thus, a panel of solutes representative for a broad array of physicochemical characteristics with solid evidence of toxicity is desirable.