Uremic Toxicity

Asymmetric Dimethylarginine

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are two guanidines with specific biological characteristics.

Since 1992, ADMA has been identified as a uremic retention compound with potential hemodynamic impact because it inhibits nitric oxide synthase (NOS), hence decreasing the endothelial protective effect of nitric oxide (NO). Of note, ADMA concentration is increased in a host of settings, many of which are linked to CKD (such as diabetes mellitus, obesity, and cardiovascular [CV] disease). ADMA concentration has been linked to CV events and mortality in the general population, as well as in CKD. In healthy volunteers, it was shown that infusing ADMA up to concentrations relevant for uremia had definite hemodynamic effects.

The rise in concentration of ADMA in CKD can, to a large extent, be attributed to a decreased activity of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which is responsible for the breakdown of ADMA, rather than to a direct decrease of urinary excretion.

Next to its endothelial impact mentioned earlier, the biologic effects of ADMA have mainly been linked to inflammation, cognitive dysfunction, erythropoietin resistance, and procoagulant effects.

In a more recent experimental study, increasing expression of DDAH in the kidney had a positive effect on diabetic nephropathy.

Symmetric Dimethylarginine

SDMA is an isomer of ADMA that, for a long time, has been considered biologically inert. However, a number of studies subsequently showed that SDMA was also biologically active, essentially via decreasing NO production and proinflammatory effects. Via posttranslational modification of high-density lipoprotein (HDL), SDMA has in addition been shown to generate an abnormal lipoprotein inducing endothelial damage.

On the clinical level, SDMA was more significantly correlated to serum inflammatory markers than ADMA, and it also correlated to mortality and CV events in a post hoc analysis of the Hemodialysis (HEMO) study.

In spite of the structural analogies with ADMA, SDMA is not dependent on DDAH for its clearance from the body as it is largely removed by kidney excretion, with a highly significant correlation to estimated glomerular filtration rate (eGFR).

In a more recent study, Hesse et al. demonstrated that the presence of SDMA in dysfunctional HDL was a mediator to cause glycocalyx breakdown. SDMA was a stronger predictor than ADMA of kidney and CV outcomes both in nondialysis CKD and in elderly White individuals.

Trimethylamine N -Oxide

Trimethylamine N -oxide (TMAO) was also known as a component of uremic biological fluids before a boost in studies on this solute occurred from 2011 onward when Wang et al. suggested a substantial link to CV disease. Intriguingly, in spite of the substantial amount of data supporting the toxicity of TMAO, the compound is also known as a stabilizer of protein structures. Equally paradoxical, fish, as a well-known source of high quantities of TMAO, is protective against CV disease.

The role of TMAO in the spectrum of uremic toxicity has extensively been described in a seminal review published in 2016 by Velasquez et al. TMAO, a small amine oxide, is generated from choline, betaine, and carnitine via intestinal bacterial metabolism after ingestion of certain food products like red meat, shellfish, or cheese. In humans, a positive correlation has been observed between plasma levels of TMAO and CV events and mortality. A number of changes linked to atherogenicity, such as alteration of cholesterol and proinflammatory pathways and foam cell formation, have been described. TMAO plasma levels increase progressively with a decline in kidney function and are associated with mortality in White patients with end-stage kidney disease (ESKD) and the need for CV surgery in CKD.

In more recent studies, in vitro, ex vivo, and in vivo studies demonstrated a role of TMAO in inducing vascular calcification and vascular damage by activation of pro-inflammatory signals and the inflammasome. The relation of TMAO levels and negative CV and kidney outcomes was confirmed in patients with type 1 diabetes mellitus, but no association with outcomes was found in hemodialysis (HD) patients with moderate to severe secondary hyperparathyroidism. In children with early-stage CKD, TMAO levels were associated with abnormal ambulatory blood pressure measurements and vascular stiffness. Finally, inhibition of TMAO production decreased the formation of atherosclerotic lesions in mice fed a high-choline diet and reduced kidney tubule-interstitial fibrosis and functional impairment in a murine model of CKD.

Carbamylated Compounds

Urea, a marker of uremic retention in CKD and adequacy of intradialytic solute removal, has traditionally been considered to be biologically inert. However, a number of recent experimental studies suggest that urea is toxic at concentrations like those observed in CKD patients. At least five studies indicated that urea itself directly induces molecular changes related to insulin resistance, free radical production, apoptosis, and disruption of the protective intestinal barrier. However, in addition, urea also generates cyanate, and via cyanate carbamylated compounds, which all have been linked to biological changes.

The role of carbamylation in CKD has been extensively reviewed elsewhere. Carbamylation is responsible for posttranslational protein modifications resulting in a similar biochemical process as when AGEs are generated (see later). Carbamylated compounds are involved in atherogenesis and other functional changes, such as activation of mesangial cells into a profibrogenic prototype, with the potential to play a role in the progression of kidney failure and modification of leukocyte response to collagens. Carbamylated low-density lipoprotein (LDL) dose dependently induced endothelial cell death and smooth muscle cell proliferation and an increase in monocyte adhesion to endothelium, as well as vascular cell changes of relevance to atherogenesis. In addition, HDL is carbamylated in CKD and inhibits endothelial repair function.

In at least three observational clinical studies focusing on independent HD populations, carbamylated compounds were associated with CV events and overall and CV mortality.

In a more recent study on human vascular smooth muscle cells, protein carbamylation, especially of mitochondrial proteins, exacerbated calcification by suppressing enzymatic inhibition of ectopic calcification. In patients with type 2 diabetes mellitus with eGFR > 60 mL/min/1.73 m ² , the risk of deterioration of kidney function was higher in those with the highest concentration of carbamylated LDL and HDL. Carbamylated LDL was no longer associated with kidney outcome after adjustment for baseline eGFR, but the association with carbamylated HDL remained significant. In a crossover randomized controlled trial (RCT), Di Iorio et al. demonstrated that a urea-lowering diet could decrease protein carbamylation in CKD patients, directly underscoring the deleterious impact of nutrition with urea sources on carbamylation.

Uric Acid

Uric acid has been associated with calcitriol metabolism and inhibition of expression on monocytes of CD14, a lipopolysaccharide receptor, and has also been linked to proinflammatory processes, insulin resistance, and nephropathy. All those mechanisms may contribute to hypertension, uremic vascular disease, progression of CKD, and thus mortality.

In a longitudinal clinical analysis, uric acid was associated with CKD progression and vascular stiffness. In the National Health and Nutrition Survey (NHANES) study, it was related to CV complications and overall CV mortality. In a systematic review and meta-analysis of CKD patients, uric acid levels were significantly associated with mortality, even after adjustment for kidney function. Uric acid has also been associated with an increased incidence of acute ischemic stroke and mild cognitive dysfunction. However, in an observational analysis of HD patients from Taiwan, an inverse relation was found between uric acid concentration and mortality. Similarly, in a large HD patient database, low and not high uric acid was associated with mortality, especially in those patients with low protein intake.

Uric acid is one of the only uremic retention compounds whose concentration can be decreased selectively by specific drugs, such as allopurinol, febuxostat, or probenecid. In an observational study in CKD patients, treatment with allopurinol was associated with improved arterial stiffness and lower overall mortality.

A few RCTs assessing the impact of uric acid–lowering drugs on parameters of endothelial function showed no positive effect, but in at least one other study, allopurinol improved peripheral vasodilator capacity. Allopurinol was shown to decrease blood pressure in young adolescents with newly diagnosed essential hypertension, but in an RCT in overweight adults, uric acid lowering could not lower blood pressure, while a systematic review could also not confirm an impact on blood pressure. In another RCT, in CKD, the group treated with allopurinol had a lower number of CV events and a slower progression of kidney failure, which was confirmed post hoc in a long-term analysis, although, again, a meta-analysis could not support a long-term effect on kidney dysfunction. Finally, an umbrella review including more than 100 studies and 136 outcome parameters, including CKD incidence and progression and mortality in CKD, showed no clear association of uric acid with these parameters.

Thus, in spite of certain arguments for the toxicity of uric acid, essentially based on experimental and observational clinical studies, not all data are consistent, especially in RCTs and meta-analyses considering uric acid-decreasing drugs. This picture may, however, be blurred by the fact that the main pharmaceutical interventions that are effective in decreasing uric acid, allopurinol, and febuxostat, have by themselves a sizeable complication profile.

More recent data confirm these considerations. Publications on pathophysiology point to the role of uric acid in causing inflammation, in injuring the kidneys by reprogramming kidney, immune cell metabolism, and activating the kidney inflammasome. The large majority of publications are, however, observational studies pointing to an association between uric acid concentration and the development or progression of CKD in the general population or in specific groups such as CKD patients with dyslipidemia, early-stage CKD patients, obese adolescents with type 2 diabetes, type 2 diabetics at large, type 1 diabetics, or individuals suffering from dyslipidemia. In addition, the study by Kanbay et al. in the general population also found an association with hypertension, and the one by Pilemann-Lyberg et al. in type 1 diabetics with CKD linked uric acid also to CV events and overall mortality. One meta-analysis found a significant relation between uric acid and a composite endpoint of worsening of kidney function, ESKD, or death, but the study contained only 12 small RCTs representing 832 patients. A recent review pointed to a protective effect on kidney function of uric acid lowering in trials with a decline of GFR in the control group, but not in studies where such a decline was absent.

Summary

Small water-soluble uremic retention solutes can no longer be considered irrelevant to the uremic syndrome, although the role of only a few representatives of this group of compounds in the uremic syndrome is supported by enough evidence to be reported in this chapter ( Table 2.3 ). This is essentially the case for the carbamylated compounds, ADMA, SDMA, and TMAO. In spite of an extensive number of experimental and clinical studies on uric acid, there is continuing debate on its role, mainly related to contradictory results of RCTs and meta-analyses. All four compounds and the group of carbamylated solutes were linked to CV damage, inflammation, and kidney damage. ADMA and uric acid affected the largest number of systems ( Table 2.3 ).

Advanced Glycation End Products

The role of AGEs in CKD has been reviewed extensively elsewhere. AGEs are generated by the reaction of specific amino acids within the protein structure with the carbamyl group of reducing sugars, which is then stabilized by oxidation. AGE compounds and their precursors are numerous and contain N -carboxymethyllysine, pentosidine, hydroxyimidazolone, 3-deoxyglucosone, malondialdehyde, pyrraline, glyoxal, and methylglyoxal. AGE accumulation has first been identified in diabetes mellitus. Because CKD as a condition is also characterized by inflammation, oxidation, and retention, AGE accumulation is a typical feature of CKD, irrespective of diabetic status.

AGEs bind to a number of receptors, among which the AGE-specific receptor (RAGE) plays a central role in several deleterious biological effects. RAGE expression is increased in many inflammatory conditions and also in CKD.

AGEs have a negative impact on body functions and overall outcomes via several mechanisms, among which are induction of oxidative stress, inflammation, and endothelial dysfunction, including quenching of NO. AGEs have also been linked to thrombogenicity, kidney fibrosis, structural bone damage, and neurotoxicity.

With all these functional alterations, it is no surprise that on a more global organic level, AGEs are linked to vascular stiffness, damage, and calcification.

Studies on the links between AGEs and mortality have shown conflicting results, with some studies even showing better outcomes with higher AGE levels. Such results may reflect confounding factors, such as better nutritional status resulting in both higher AGEs and higher survival, or the index AGE may have been a compound with low biological impact or lower vital tissue concentration. In contrast, low serum soluble RAGEs, which are actors protecting against AGE activity, have consistently been associated with CV risk factors and events.

In more recent studies, methylglyoxal-derived AGE induced matrix metalloprotein expression via RAGE and mitogen-activated protein (MAP) kinase signals contributed to kidney cell dysfunction. In a clinical observational study in kidney transplant patients, AGE concentrations were associated with CV mortality. Administering AGE-breaking drugs to rats with CKD decreased vascular calcification, the CKD-MBD marker fibroblast growth factor-23 (FGF-23) (see later), and AGE content in bone, the latter, however, without changes in bone mechanics. Finally, RAGE was shown to be a key element in the generation of vascular calcification.

p-Cresyl Sulfate

Until 2005, it was thought that the intestinal precursor of p-cresyl sulfate, p-cresol, was the representative cresol during uremic retention. However, later on, this view was shown to be the result of an artifact. Shortly after de Loor et al. and Martinez et al. identified the artifact and had stated that in reality the conjugates p-cresyl sulfate and p-cresyl glucuronide were present, an in vitro study by Schepers et al. for the first time pointed to a role of p-cresyl sulfate in causing oxidative stress by activating leukocytes. Soon after this observation, further exploration started on the biological effect of p-cresyl sulfate. This resulted in extensive data on its toxicity, summarized in a systematic and narrative review article.

The role of p-cresyl sulfate in inflammation and other phenomena linked to CV damage, such as cardiomyocyte apoptosis resulting in diastolic dysfunction, was repeatedly demonstrated. In addition, toxic effects on kidney tubular cells, related to tubular cell damage and kidney fibrosis, have also been reported. Finally, p-cresyl sulfate was also shown to play a role in insulin resistance, changes in adipose tissue metabolism, and fat redistribution through the body.

Hence, p-cresyl sulfate contributes in many ways to the mechanisms responsible for CV damage. In line with these pathophysiologic and experimental findings, p-cresyl sulfate has repeatedly been associated with CV complications and mortality in CKD patients in observational studies as well as in a meta-analysis. Total p-cresyl sulfate has also been linked to uremic pruritus.

More recent studies mainly focused on conditions or interventions with a potential impact on p-cresyl sulfate concentrations. In patients with acute kidney injury (AKI), the rise of plasma p-cresyl sulfate did not match that of creatinine, and for a similar serum creatinine, p-cresyl sulfate was lower in AKI than in CKD. A very-low-protein diet and Mediterranean diet in CKD caused a favorable change in gut microbiota and a reduction of plasma p-cresyl sulfate. A meta-analysis underscored the potential of an increase in fiber intake to cause a decrease in p-cresyl sulfate. Finally, Gryp et al. found no arguments in favor of an increase of intestinal p-cresyl sulfate generation as CKD progressed. The progressive increase in p-cresyl sulfate in CKD seemed largely attributable to the loss of kidney function, while data of fractional clearance suggested that tubular clearance was more affected than glomerular clearance. P-cresyl sulfate also decreased glutathione in porcine tubular cells, rendering them more vulnerable to oxidative stress, especially in combination with other toxic sulfates like indoxyl sulfate and phenyl sulfate.

In a post hoc analysis of the HEMO trial, Shafi et al. failed to confirm the association between p-cresyl sulfate and CV outcomes in dialysis patients. Analytical issues, questioning the accuracy of the free p-cresyl sulfate concentrations and case mix with a majority of Afro-Americans, may explain the discrepant findings with previous outcome studies. Also, in another study by van Gelder et al., levels of p-cresyl sulfate did not associate with mortality and CV events in HD patients enrolled in the Dutch CONvective TRAnsport STudy (CONTRAST). However, total p-cresyl sulfate levels were assessed, in contrast to the previous studies observing an association with free p-cresyl sulfate levels.