Uremic Toxins

By Toshimitsu Niwa

Reviews all the latest basic and clinical research findings

With contributions from leading international experts in the field, this book is dedicated to all facets of uremic toxins research, including low molecular weight solutes, protein-bound solutes, and middle molecules. Moreover, it covers everything from basic mass spectrometry research to the latest clinical findings and practices.

Uremic Toxins is divided into three sections:

• Section One, Uremic Toxins, explores the definition, classification, listing, and mass spectrometric analysis of uremic toxins
• Section Two, Selected Uremic Toxins, describes key uremic toxins, explaining chemical structures, metabolism, analytical methods, plasma levels, toxicity, clinical implications, and removal methods. Among the uremic toxins covered are indoxyl sulfate, asymmetric dimethylarginine, PTH, ß2-microglobulin, and AGEs
• Section Three, Therapeutic Removal of Uremic Toxins, describes how uremic toxins can be removed by hemodialysis, peritoneal dialysis, and oral sorbent

All chapters are based on the authors' thorough review of the literature as well as their own personal laboratory and clinical experience. References at the end of each chapter provide a gateway to the literature in the field.

Reviewing all the latest basic and clinical research findings, Uremic Toxins will help bench scientists in nephrology advance their own investigations. It will also help clinicians take advantage of the latest tested and proven treatments for the management of chronic kidney disease.

• Language: English
• Publisher: Wiley
• Release date: Sep 4, 2012
• ISBN: 9781118424100

Book preview

Section 1

Uremic Toxins

Chapter 1

Uremic Toxins: AN Integrated Overview of Definition and Classification

Richard J. Glassock and Shaul G. Massry

1.1 Introduction

As the overall function of the kidney declines in the course of chronic kidney disease (CKD) a wide variety of solutes, normally dependent on glomerular filtration, tubular secretion or renal metabolism for elimination, gradually accumulate in the body fluid compartments. Some of these solutes have biological effects that result in the malfunction of various cell types and organ systems. When these biological effects are sufficient to evoke clinically recognizable disturbances, the uremic syndrome is said to be present and the offending molecules are designated uremic toxins.¹,² These uremic toxins exhibit a broad array of physicochemical characteristics and have very diverse pathobiological effects at the cellular level.¹,² A complete characterization of the catalogue of uremic toxins would be very useful in the design of approaches for their removal by dialysis; for ways to enhance their removal by nondialytic methods; for creation of interventions to prevent/mitigate their formation; for synthesis of inhibitors of their adverse effects on cells and organ systems—all directed at subjects with advancing CKD or end-stage renal disease (ESRD). The analysis of the issues surrounding uremic toxicity requires a useful definition and synthesis of a classification of uremic toxins. This brief essay attempts to provide a succinct approach to classification of uremic toxins, derived form a review of the current literature on the subject.¹

1.2 Definition of a Uremic Toxin

In order to define a uremic toxin, one must first define the syndrome of uremia itself. Almost 35 years ago, the late Jonas Bergstrom gave a definition of the uremic syndrome that is just a valid today as it was then.³ He stated that the uremic syndrome is a "toxic syndrome caused by severe glomerular deficiency associated with disturbances in tubular and endocrine functions of the kidney. It is characterized by the retention of toxic metabolites, associated with changes in the volume and composition of the body fluids and an excess or deficiency of various hormones." This very broad definition allows the uremic syndrome to embrace the retention of solutes due to failure of renal excretion (glomerular and/or tubular insufficiency) and hormonal surfeits or deficiencies arising from the disturbances wrought by kidney disease itself, such as enhanced endogenous production or impaired degradation of potential injurious solutes.

From this description of the uremic syndrome, it is clear that uremic toxins must be defined via a connection between the putative toxic substance and one or more of the pathophysiological attributes of the uremic syndrome. Making this connection requires that a series of criteria be fulfilled. These criteria are called the Massry/Koch postulates—so-called because they are a derivative of Koch's postulates for defining a pathogenetic organism as developed by one of the coauthors of this essay (SM) about a quarter century ago.⁴ The requirements for an authentic uremic toxin are as follows:

(i) The toxin must be identified and characterized as a unique chemical entity.

(ii) Quantitative analysis of the toxin in biological fluids must be possible.

(iii) The level of the putative toxin must be elevated in biological fluids of subjects with the uremic syndrome.

(iv) A relationship between the level of the putative toxin in biological fluids and one or more of the manifestations of the uremic syndrome must be present.

(v) A reduction in the levels (or total body burden) of the putative toxin in biological fluids must result in some measurable amelioration of uremic manifestations.

(vi) Administration of the putative toxin to achieve levels similar to that observed in the uremic syndrome must reproduce the uremic manifestations in otherwise normal animals or man (in vitro demonstration of cellular toxicity alone is insufficient to meet this criterion).

A seventh criterion could be added to this list; namely, that a consistent and plausible pathobiologic mechanism should be able to explain the linkage between the putative toxin and the uremic manifestation (e.g., cellular toxicity, inhibition of signal transduction, metabolic perturbations). These postulates are difficult to apply directly to those disturbances that are part and parcel of the uremic syndrome but that emanate from surfeits or deficiencies of certain hormones or biologically active peptides (e.g., erythropoietin, calcitriol) consequent to the loss of renal mass in CKD. Nevertheless, these postulates are quite useful for the definition of uremic toxins resulting from retention of solutes normally excreted by the kidney and substances that arise in enhanced levels endogenously (from excessive synthesis or impaired degradation) as kidney disease progresses to symptomatic uremia (e.g., parathyroid hormone).

The demonstration of a linkage between a specific putative uremic toxin and a clinical manifestation of uremia can be a formidable task, as the symptoms and signs of uremia are extraordinarily diverse.⁵ The ability of a specific putative toxin to elaborate a clinical manifestation is governed by a panoply of factors (see Table 1.1). These complicate enormously the task of identifying authentic uremic toxins as they require longitudinal in addition to cross-sectional analysis, body fluid compartmental studies, and the influence of naturally occurring inhibitors and promoters. Some toxins may also exhibit tropism for specific cellular types or organ systems (e.g., neurotropism) (see below).

Table 1.1 Factors Influencing the Toxicity of Substances Accumulating in Uremia.

1.3 Classification of Uremic Toxins by Physicochemical Characteristics

A classification of putative uremic toxins according to their physicochemical characteristics (molecular mass, polarity, protein binding, chemical structure) has been the time-honored and most popular approach.¹,² In this schema, uremic toxins are categorized into four nonoverlapping categories; namely (i) polar, water soluble, nonprotein bound, low molecular mass (<500 Da); (ii) polar, water soluble, protein bound, low molecular mass (<500 Da); (iii) middle molecular mass (>500 and <3000–12,000 Da), nonprotein bound; (iv) high molecular mass (>3000–12,000 Da), nonprotein bound.¹,² The work of the European Uremic Toxin Work Group (EUTox) has been invaluable in creating a uniform approach to classifying uremic toxins, and have pointed out the necessity for standardized schema for analysis of their in vitro effects and the enormous difficulties posed by variability in reported concentrations of putative toxins.² In their landmark review in 2003, the EUTox group created an encyclopedic listing of uremic retention solute (90 total), 68 of which were <500 Da molecular mass, 10 had a molecular mass of >500–12,000 Da, and 12 had molecule mass of >12,000 Da. Twenty-five of the retention solutes were protein bound, all but two had molecular mass of <500 Da. The concentration of these putative toxins in biological fluids ranged broadly from ng/L to g/L. Of all the toxins identified almost 40% were either middle molecules or were protein bound. The development of large-scale, rapid capillary electrophoresis-mass spectrometry analysis of body fluids has greatly enhanced the ability to identify and characterize potential uremic toxins.⁶–⁹

Inorganic substances (H2O, Na+, K+, H+, Mg²+, PO4²−, Ca²+, SO4) and trace metals (Al, Cr, Si, Pb) can also qualify as uremic toxins. For example, retention of sodium chloride and water can evoke disastrous consequences on the cardiovascular system in CKD and ESRD and contribute markedly to organ dysfunction (left ventricular hypertrophy), morbidity (hypertension and congestive heart failure) and mortality (sudden cardiac death).¹⁰ Also, acidosis (retention of H+ ion) can wreak havoc in many cell and organ systems.

Although many potential uremic toxins have elevated plasma concentrations due to impairment of renal excretion, many are also associated with increased synthesis or impaired degradation of normal substances produced endogenously (e.g., parathyroid hormone). It must be emphasized that the plasma concentrations of putative uremic retention solutes are very poorly correlated with the prevailing level of glomerular filtration rate (GFR),¹¹ and the plasma levels of each specific solute may have a unique association with the level of GFR.¹¹ These observations add emphasis to a neglected phenomenon well recognized in the aglomerular kidney of marine teleosts (anglerfish),¹² specifically that the tubules represent an important site for elimination of putative toxic by-products of metabolisms. This phenomenon was pointed out in an elegant essay by Jerome Lowenstein in 2011.¹²

Thus, residual activity of transport systems in tubules of diseased kidneys (specifically the organic anion transporters [OAT] in the proximal tubule) may have important influence on the concentration of toxins at low levels of GFR. This phenomenon gives rise to the notion that enhanced expression of the OAT might be able to limit the accumulation of uremic toxins even with advanced loss of GFR.¹³

The low molecular mass solutes (<500 Da) have attracted a great deal of attention over many years. Urea (a low molecular mass, nonprotein-bound solute) has been used as a surrogate for authentic uremic toxins, although its intrinsic toxicity is greatly limited to very, very high plasma concentrations seldom achieved even in advanced uremia.¹⁴ The evidence that urea per se functions as an authentic uremic toxin is very weak.¹⁴ Nevertheless, its spontaneous degradation to isocyanate can lead to the carbamylation of serum and tissue proteins, such as albumin or hemoglobin.

Protein-bound uremic toxins are of great theoretical and practical importance.¹⁵–¹⁸ Such protein binding may greatly limit the ability of diffusive or convective dialysis therapies to remove the compound efficiently, and this explain the limitations of extra-corporeal therapies using membranes of low molecular mass cutoff for the treatment of uremia. Displacement of the uremic toxin from its protein-binding site might be a very attractive way of enhancing uremic solute removal by dialysis.¹⁵–¹⁸ The most well studied of the protein-bound uremic solutes include p-cresyl sulfate and indoxyl sulfate.¹⁹–²⁴ Both of these uremic solutes originate in the colon from the action of resident bacteria—thus, there is an important contribution of the colon to the uremic state,²⁵ leading to the potential for treatment of uremia by oral adsorbents.²⁶ Other protein-bound uremic solutes include asymmetric dimethylarginine (ADMA), homocysteine, pentosidine, deoxyglucosone, derivatives of nucleosides, and advanced glycation end products.²⁷,²⁸ ADMA appears to accumulate in uremia more as a result of disturbed renal metabolism that from impaired renal excretion.²⁷,²⁸ It is noteworthy that the R² values of ADMA levels in relationship to eGFR is only 0.167 (the R² value to creatinine is 0.737).¹¹ Uric acid and other nucleotide derivatives are emerging as important candidates for low molecular mass uremic toxins.²⁹–³¹

Middle molecules (>500–12,000 Da) have been regarded as important in the uremic syndrome and its response to dialysis treatment, ever since the seminal observations leading up to the middle molecule hypothesis were made by Babb and Scribner 40 years ago.³² As noted above, EUTox identified about 10 such middle molecule uremic toxins in their survey.² These compounds are often glucuronide conjugates, polypeptides (such as β2-microglobulin), carbohydrate derivatives, advanced glycation or oxidation end products, or polypeptide hormones (such as parathyroid hormone or its fragments).²,³³–³⁷ These compounds may exert their toxic effects via engagement of other intermediary processes. The high molecular mass (>12,000 Da) nondialyzable toxins have been less well characterized, but include cytokines, chemokines, Ig light chains, complement factors, advanced glycation or oxidation end products, inhibitor proteins, chemotaxis-inhibiting peptides.²

1.4 Classification of Uremic Toxins According to Pathobiological Processes Underlying Accumulation

The uremic toxins classified by their intrinsic physicochemical properties can accumulate in body fluid compartments though a number of distinct mechanisms. A Type I mechanism represents the accumulation in body fluids of toxic substances normally produced endogenously by metabolic processes largely as a result of reduced renal excretory capacity. A Type II mechanism is a surfeit of toxic substances in body fluids as a result of excess endogenous production or impaired degradation (or both) but not necessarily due to reduced renal excretory capacity. A Type III mechanism is the accumulation of toxic substances in biological fluids from exogenous sources by virtue of reduced renal excretory capacity often combined with continued dietary consumption. A Type IV mechanism is a deficiency or reduced activity of substances normally produced endogenously as a result of decreased synthesis, enhanced degradation, or biological inhibition. Combinations of more than one pathobiological process are possible. For example, urea is a uremic toxin that arises because of a combination of Type I and Type III processes—excessive accumulation due to impaired renal excretion and continued production due to exogenous (dietary) consumption of protein as a precursor of urea. It is helpful to keep this classification of the processes underlying accumulation of uremic toxins when approaching a patient with the uremic syndrome.

1.5 The Relationships of Uremic Toxins to the Pathobiology of Uremia

In recent years, a new concept has emerged that the uremic syndrome is strongly associated with a state of chronic inflammation and enhanced oxidative stress manifested by an increase in positive acute phase reactant proteins (such as CRP, IL-6, fibrinogen, ferritin, and serum amyloid A protein) and a reduction in negative acute phase reactant proteins (albumin, transferrin, prealbumin).³⁸,³⁹ The proposed origins of this inflammatory state include (1) an imbalance between pro- and anti-inflammatory factors; (2) underlying organ-based chronic inflammation (occult infection [periodontal disease, infected vascular access, vulnerable atherosclerotic plaques], kidney inflammation associated with basic disease); (3) exposure to inflammatory promoters (endotoxin-contaminated dialysate, bioincompatible membranes). No doubt in individual patients, multiple factors explain the presence of an inflammatory state.

Certain candidate uremic toxins, such as uric acid or ADMA, may be potent promoters of inflammation, and in turn inflammation can lead to the generation of uremic toxins, such as advanced oxidation products via the generation and inadequate scavenging of toxic oxygen radicals.⁴⁰,⁴¹ Indoxyl sulfate, a putative uremic toxin, can also promote further progression of renal disease by activating harmful mediators such as transforming growth factor-β (TGF-β).⁴² Thus, the accumulation of uremic toxins may also exert a positively reenforcing action on the basic process of tissue and organ damage, in addition to their effect on manifestations of the uremic syndrome per se.⁴²

The toxicity of ADMA has also emerged as an important element in new concepts of the pathobiology of uremic toxicity.⁴⁰,⁴¹ This methylated amino acid is highly protein bound, and its concentration in plasma is elevated in uremia. The elevation is predominantly caused by the inhibition of its major kidney-derived metabolizing enzyme (dimethylarginine dimethylaminohydrolase-1; DDAH-1) rather than by markedly decreased renal excretion. ADMA, along with uric acid, is a potent inhibitor of endothelial cell nitric oxide synthase (eNOS).⁴⁰,⁴¹ Impaired eNOS and reduced nitric oxide production by endothelial cells may lead to vasoconstriction, elevated blood pressure, and vascular damage. Oxidative stress associated with uremia may also impair the effectiveness of DDAH-1, proving a link between endothelial cell dysfunction and inflammation in uremia. DNA methylation and repair may also be adversely affected by putative uremic toxins.⁴³,⁴⁴ These some retention uremic solutes (such as homocysteine and its metabolites) could have profound effects on gene expression and epigenetics.⁴³,⁴⁴

Thus, the pathobiology of uremic toxicity needs to be viewed as a complex, dynamic, interacting system of effector, promoter, and inhibitory molecules occurring in a situation of reduced renal excretory capacity, impaired defensive ability, and superimposed deficiency states. The cumulative adverse effects on cellular and organ system function will depend on the balance of these factors.

1.6 Clinical Manifestations of Uremia and the Role of Tropisms

The clinical manifestations of uremic toxicity are broad and diverse. As pointed out previously every organ systems in the body can be affected. Each individual uremic toxin may have its own unique profile of tropisms. That is, each toxin may have a preferential action on only one system (monotropic) or act on only a few systems (oligotropic). Most uremic toxins studied so far have effects on multiple systems (pleiotropic), perhaps by interference with very fundamental common pathways of cellular behavior (elevated cytosolic calcium, nitric oxide synthesis, DNA methylation and repair, defense against oxidative stress), such as exemplified by parathyroid hormone, uric acid, and other derivatives of purine nucleotides and ADMA. However, some toxins (such as guanidino compounds) may exhibit relative specificity for certain organ systems (hematopoiesis, neuronal function, bone metabolism, endothelial cell integrity).⁴⁵ Elucidation of the tropic behavior of individual toxins is an important element in their full characterization and classification.

1.7 Conclusion

An exposition of uremic toxicity requires an integrative analysis of the physicochemical properties of putative toxins (molecular size, protein binding), an understanding of the pathobiological processes responsible for their formation and accumulation, and a mechanistic view of how they alter fundamental cellular and organ behavior. A consideration of both glomerular filtration and tubular secretion is essential for the proper understanding of levels of putative uremic toxins in the body fluids in CKD and ESRD. An explanation of how individual or groups of toxins lead to clinical manifestations of uremia requires a consideration of tropism (monotropic, oligotropic, and pleiotropic toxins). This multidimensional integration allows for a better understanding of the complexity and the potential for mapping of the important elements of uremic toxicity. The long-term importance of better understanding of the chemical basis of uremia is to aid the development of better and more rational methods of treatment including ablation of organ sources of putative toxins, or the medical suppression of the activity of such organs, reduction of exogenous sources of toxic precursors, reduction in (colonic) absorption of putative toxins, enhancement of extra-renal removal of toxins (intra- or extra-corporeal), supplementation for replacement of deficiencies, suppression of toxic effects at the cellular level, replacement of renal tissue or its products.⁴⁶,⁴⁷ Dialytic therapy of uremic toxicity is just one small part of the overall picture of uremia.

References

1. Glassock RJ: Uremic toxins: What are they? An integrated overview of pathobiology and classification. J Ren Nut 18:2–6, 2008.

2. Vanholder R, De Smet R, Glorieux G, Argiles A, Baurmeister U, Brunet P, Clark W, Cohen G, De Deyn PP, Deppisch R, Descamps-Latscha B, Henle T, Jorres A, Lemke HD, Massy Z, Passlick-Deetjen J, Rodriguez M, Stegmayr B, Stenvinkel P, Teta C, Wanner C, Zidek W for the European Uremic Toxin Work Group (EUTox): Review on uremic toxins. Classification, concentration and inter-individual variability. Kidney Int 63:1934–1943, 2003.

3. Bergstrom J, Furst P: Uremic toxins. Kidney Int Suppl S9–S12, 1978.

4. Massry SG: Parathyroid hormone: A uremic toxin. Adv Exp Med Biol 223:1–17, 1987.

5. Merrill JP, Hampers CL: Uremia. Progress in Pathophysiology and Treatment. New York, Grune and Stratton, 1971.

6. Niwa T: Recent progress in the analysis of uremic toxins by mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 877:2600–2606, 2009.

7. Rhee EP, Souza A, Farrell L, Pollak MR, Lewis GD, Steele DJ, Thadhani R, Clish CB, Greka A, Gerszten RE: Metabolite profiling identifies markers of uremia. J Am Soc Nephrol 6:1041–1051, 2010.

8. Niwa T: Update of uremic toxin research by mass spectrometry. Mass Spectrom Rev 3:510–521, 2011.

9. Schiffer E, Mischak H, Vanholder RC: Exploring the uremic toxins using proteomic technologies. Contrib Nephrol 160:159–171, 2008.

10. Glassock RJ, Pecoits-Filho R, Barberato SH: Left ventricular mass in chronic kidney disease and ESRD. Clin J Am Soc Nephrol 1:S79–S91, 2009.

11. Eloot S, Schepers E, Barreto DV, Barreto FC, Liabeuf S, Van Biesen W, Verbeke F, Glorieux G, Choukroun G, Massy Z, Vanholder R: Estimated glomerular filtration rate is a poor predictor of concentration for a broad range of uremic toxins. Clin J Am Soc Nephrol 6:1266–1273, 2011.

12. Lowenstein J: The anglerfish and uremic toxins. FASEB J 25:1781–1785, 2011.

13. Toyohara T, Suzuki T, Morimoto R, Akiyama Y, Souma T, Shiwaku HO, Takeuchi Y, Mishima E, Abe M, Tanemoto M, Masuda S, Kawano H, Maemura K, Nakayama M, Sato H, Mikkaichi T, Yamaguchi H, Fukui S, Fukumoto Y, Shimokawa H, Inui K, Terasaki T, Goto J, Ito S, Hishinuma T, Rubera I, Tauc M, Fujii-Kuriyama Y, Yabuuchi H, Moriyama Y, Soga T, Abe T: SLCO4C1 transporter eliminates uremic toxins and attenuates hypertension and renal inflammation. J Am Soc Nephrol 12:2546–2555, 2009.

14. Linthorst GE, Avis HJ, Levi M: Uremic thrombocytopathy is not about urea. J Am Soc Nephrol 5:753–755, 2010.

15. Jourde-Chiche N, Dou L, Cerini C, Dignat-George F, Vanholder R, Brunet P: Protein-bound toxins—Update 2009. Semin Dial 4:334–339, 2009.

16. Vanholder R, Baurmeister U, Brunet P, Cohen G, Glorieux G, Jankowski J; European Uremic Toxin Work Group: A bench to bedside view of uremic toxins. J Am Soc Nephrol 5:863–870, 2008.

17. Raff AC, Meyer TW, Hostetter TH: New insights into uremic toxicity. Curr Opin Nephrol Hypertens 6:560–565, 2008.

18. Lin CJ, Wu CJ, Pan CF, Chen YC, Sun FJ, Chen HH: Serum protein-bound uraemic toxins and clinical outcomes in haemodialysis patients. Nephrol Dial Transplant 11:3693–3700, 2010.

19. Niwa T: Indoxyl Sulfate. In: Textbook of Nephrology, 4th ed., edited by SG Massry, RJ Glassock, Philadelphia, Lippincott, Williams and Wilkins, 2001, pp 1269–1271.

20. Yu M, Kim YJ, Kang DH: Indoxyl sulfate-induced endothelial dysfunction in patients with chronic kidney disease via an induction of oxidative stress. Clin J Am Soc Nephrol 1:30–39, 2011.

21. Glorieux G, Vanholder R: New uremic toxins—Which solutes should be removed? Contrib Nephrol 168:117–128, 2011.

22. Liabeuf S, Barreto DV, Barreto FC, Meert N, Glorieux G, Schepers E, Temmar M, Choukroun G, Vanholder R, Massy ZA;European Uraemic Toxin Work Group (EUTox): Free p-cresyl sulphate is a predictor of mortality in patients at different stages of chronic kidney disease. Nephrol Dial Transplant 4:1183–1191, 2010.

23. Vanholder R, Bammens B, de Loor H, Glorieux G, Meijers B, Schepers E, Massy Z, Evenepoel P: Warning: The unfortunate end of p-cresol as a uraemic toxin. Nephrol Dial Transplant 26:1464–1467, 2011.

24. Schepers E, Meert N, Gloriex G, Goeman J, Van der Eycken J, Vanholder R: p-Cresyl sulphate, the main in vivo metabolite of p-cresol, activates leucocyte free radical production. Nephrol Dial Transplant 22:592–596, 2007.

25. Aronov PA, Luo FJ, Plummer NS, Quan Z, Holmes S, Hostetter TH, Meyer TW: Colonic contribution to uremic solutes. J Am Soc Nephrol 9:1769–1776, 2011.

26. Goto S, Yoshiya K, Kita T, Fujii H, Fukagawa M: Uremic toxins and oral adsorbents. Ther Apher Dial 2:132–134, 2011.

27. Leiper J, Nandi M, Torondel B, Murray-Rust J, Malaki M, O'Hara B, Rossiter S, Anthony S, Madhani M, Selwood D, Smith C, Wojciak-Stothard B, Rudiger A, Stidwill R, McDonald NQ, Vallance P: Disruption of methylarginine metabolism impairs vascular homeostasis. Nat Med 13:198–203, 2007.

28. Kielstein JT, Fliser D, Veldink H: Asymmetric dimethylarginine and symmetric dimethylarginine: Axis of evil or useful alliance? Semin Dial 4:346–350, 2009.

29. Arese M, Strasly M, Ruva C, Costamagna C, Ghigo D, MacAllister R, Verzetti G, Tetta C, Bosia A, Bussolino F: Regulation of nitric oxide synthesis in uraemia. Nephrol Dial Transplant 8:1386–1397, 1995.

30. Heinig M, Johnson RJ: Uric acid in hypertension, renal disease and metabolic syndrome. Cleve Clin J Med 73:1059–1064, 2006.

31. Rutkowski B, Swierczynski J, Slominska E, Szolkiewicz M, Smolenski RT, Marlewski M, Butto B, Rutkowski P: Disturbances of purine nucleotide metabolism in uremia. Semin Nephrol 24:479–483, 2004.

32. Scribner BH, Babb AL: Evidence for toxins of middle molecular weight. Kidney Int Suppl S349–S3511975.

33. Vanholder R, Van Laecke S, Glorieux G: The middle-molecule hypothesis 30 years after: Lost and rediscovered in the universe of uremic toxicity? J Nephrol 2:146–160, 2008.

34. Rodriguez M, Lorenzo V: Parathyroid hormone, a uremic toxin. Semin Dial 4:363–368, 2009.

35. Geara AS, Castellanos MR, Bassil C, Schuller-Levis G, Park E, Smith M, Goldman M, Elsayegh S: Effects of parathyroid hormone on immune function. Clin Dev Immunol pii:418695, 2010.

36. Glorieux G, Cohen G, Jankowski J, Vanholder R: Platelet/leukocyte activation, inflammation, and uremia. Semin Dial 4:423–427, 2009.

37. Thomas G, Jaber BL: Convective therapies for removal of middle molecular weight uremic toxins in end-stage renal disease: A review of the evidence. Semin Dial 22:610–614, 2009.

38. Stenvinkel P: Inflammation in end-stage renal disease: The hidden enemy. Nephrology 11:36–41, 2006.

39. Palm F, Nangaku M, Fasching A, Tanaka T, Nordquist L, Hansell P, Kawakami T, Nishijima F, Fujita T: Uremia induces abnormal oxygen consumption in tubules and aggravates chronic hypoxia of the kidney via oxidative stress. Am J Physiol Renal Physiol 2:F380–F386, 2010.

40. Kielstein JT, Zocalli C: Asymmetric dimethylarginine: A cardiovascular risk factor and a uremic toxin coming of age. Am J Kidney Dis 46:186–202, 2005.

41. Cardounel AJ, Cui H, Samouilov A, Johnson W, Kearns P, Tsai AL, Berka V, Zweier JL: Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function. J Biol Chem 12:879–887, 2007.

42. Niwa T: Role of indoxyl sulfate in the progression of chronic kidney disease and cardiovascular disease: Experimental and clinical effects of oral sorbent AST-120. Ther Apher Dial 2:120–124, 2011.

43. Perna AF, Ingrosso D, Violetti E, Luciano MG, Sepe I, Lanza D, Capasso R, Ascione E, Raiola I, Lombardi C, Steinvinkel P, Massy Z, De Santo NG: Hyperhomocysteinemia in uremia—A red flag in a disrupted circuit. Semin Dial 4:351–356, 2009.

44. Stenvinkel P, Ekstrom TJ: Does the uremic milieu affect the epigenotype? J Ren Nutr 1:82–85, 2009.

45. De Deyn PP, Vanholder R, Eloot S, Glorieux G: Guanidino compounds as uremic (neuro)toxins. Semin Dial 4:340–345, 2009.

46. Kinugasa E: Markers and possible uremic toxins: Japanese experiences. Contrib Nephrol 168:134–138, 2011.

47. Baurmeister U, Vienken J, Ward RA: Should dialysis modalities be designed to remove specific uremic toxins? Semin Dial 4:454–457, 2009.

Chapter 2

Classification and A List of Uremic Toxins

Nathalie Neirynck, Rita De Smet, Eva Schepers, Raymond Vanholder, and Griet Glorieux

2.1 Introduction

Retention of uremic solutes starts from the moment kidney function declines, evolving in the progressive dysfunction of virtually every organ system. The clinical picture is the uremic syndrome. The kinetics of this retention process are, however, far from clear. Although during the last few years an immense progress has been made in the identification and quantification of uremic solutes,¹ a large number of retention solutes remain unidentified.² The presence of an indefinite number of posttranslational modifications of retention solutes, as a result of oxidation, glycation, cysteination, as well as of several other chemical processes, with each of these structural variants possibly exerting a pathophysiologic impact that differs from the mother compound, hampers the process of mapping the uremic retention solutes even more. Although many compounds and/or their functional role remain unknown, further identification and classification is compulsory before a targeted and possibly also tailored treatment will be possible.

2.2 Classification of Uremic Retention Solutes

For the time being, uremic solutes are preferentially classified according to the physicochemical characteristics affecting their clearance during dialysis that is, as of today, still the main therapeutic option for their removal.

Traditionally, this subdivision focuses on three types of molecules:

1. The small water-soluble compounds, with urea as a prototype.

2. The protein-bound solutes, with the groups of indolic and phenolic compounds as prototypes.

3. The larger peptides (molecular weight: MW >500 Da), also named middle molecules, with β2-microglobulin as a prototype.¹

Many compounds retained during kidney failure exert biological/biochemical activity and contribute to the uremic syndrome, but even if some retention solutes are inert, they may be useful markers of kidney disease or degree of renal dysfunction.

2.3 Listing and Identification of Uremic Retention Solutes

Refined analytical strategies and a better knowledge of biochemistry recently have helped to recognize a growing number of uremic solutes. In a review by the European Uremic Toxin Work Group (EUTox) of 2003, 90 different compounds were tabulated.¹ However, it appears more and more that this review revealed only the tip of the iceberg.²

The identification of uremic retention solutes was accompanied by an increasing number of reports on their concentration, although with unexpectedly large scatter for some compounds.¹

The final aim is to come to a classification of the most important toxins, which could then be combated by specific removal strategies and pharmacologic approaches. Although the moment we will have such a classification is much closer now than a decade ago, it still might be too preliminary to come to straightforward conclusions based on what we have already. Also, some toxins have obviously been investigated more intensively than others, which may be due to many factors, such as whether a certain compound is well known or has been discovered only more recently, whether the knowledge emanated from studies in the general population stimulating more extensive research, or from research in the more restricted uremic population. Nevertheless, the number of citations of toxins may be a barometer of the perception of the relative importance of certain compounds. This may inspire further research that should be focused on therapeutic issues. This research should emphasize pharmacologic neutralization of toxic effects as much as on toxin removal; this will have implications for a much larger population, perhaps as early as chronic kidney disease (CKD) stage 3 (glomerular filtration rate [GFR] < 60 mL/min).

Further than that, efforts should go to recognize new compounds and new mechanisms, to further broaden the therapeutic perspective. The new tools made available to us by the advent of genomic, proteomic, and metabolomic research techniques are of invaluable help to find new elements and mechanisms.

In this chapter, we offer an update on the concentrations of the compounds listed by the EUTox workgroup in 2003. In addition, lists are extended with uremic retention solutes newly identified by means of mass spectrometry (MS).

2.4 Method

The listing of the uremic retention solutes in the present chapter is based on the list published by the EUTox workgroup in 2003. The present table includes the molecular weight of the solute and its normal and uremic concentration reported at that time. The list was updated based on a PubMed search performed between 2003 and 2011, with as reference words (search items),uremic toxins/uremic toxicity and/or specific names of known uremic retention solutes as previously reviewed,¹,³–⁶ collecting at a maximum 30 new publications. The highest and lowest mean uremic concentration and its corresponding normal concentration (only when available in the same publication) are reported, next to the method of quantification (Tables 2.1–2.3). Finally, the list was extended reporting the mean normal and uremic concentration of known and newly identified uremic retention solutes determined by MS (Table 2.4). Only plasma/serum concentrations were taken into consideration. Data were collected only from studies evaluating subjects treated by dialysis or subjects with advanced renal dysfunction (GFR < 30 mL/min—corresponding to CKD stages 4 and 5). For patients treated by hemodialysis (HD) or by related strategies, concentrations in samples collected before the start of the dialysis session were retrieved.

Table 2.1 Small Water-Soluble Solutes (MW < 500)

Table 2.2 Protein-Bound Solutes.

Table 2.3 Middle Molecules (MW > 500)

Table 2.4 Newly Identified Uremic Solutes (2003–2011) by Mass Spectrometry.

2.5 Results

The 95 listed uremic solutes identified until 2003¹ are summarized in Tables 2.1–2.3 according to their physicochemical properties. In the tables, per uremic solute, the mean uremic concentration as reported in 2003 (Cur-2003), the minimum (Cur-min) and maximum (Cur-max) mean uremic concentrations as reported in publications in the period 2003–2011 are listed next to each other. If available in the same study, a normal value is mentioned. In addition, the ratios Cur-min/Cur-2003 and Cur-max/Cur-2003 were calculated. Major differences between the earlier and more recent published concentrations are reflected by a ratio with arbitrary cutoff of ≥10 (increase) or ≤ 0.1 (decrease). These solutes are indicated with (∗) in the respective tables.

The number of studies evaluated per solute is also listed, as this can be of additive value to appreciate difference between Cur-min and Cur-max. In addition, the ratio Cur-max/Cur-min was calculated and a ratio of >5 was arbitrarily considered as a large concentration difference between Cur-min and Cur-max, suggesting a broad concentration range for the respective solute in uremia. This is indicated with (#) next to the individual solutes in the tables.

For 12 of these compounds there are also concentrations measured by MS in the period 2003–2011, indicated with (§) in the table. Newly identified solutes by MS are summarized in Table 2.4.

2.5.1 Small Water-Soluble Compounds (MW < 500 Da)

The small water-soluble compounds, as found in the classification of 2003¹ are listed in Table 2.1, grouped according their chemical family: ribonucleosides, guanidines, purines, pyrimidines, polyols, and some other single compounds. The two most important groups are the guanidines (14/45) and the ribonucleosides (9/45). Only 19 of the 45 compounds had newly published concentrations between 2003 and 2011, and these were mainly found for guanidino compounds, which accounted for 13/19 updates.

Evaluation of the ratios Cur-min/Cur-2003 and Cur-max/Cur-2003, showed that only the Cur-min of some solutes (asymmetric dimethylarginine (ADMA), creatine, methylguanidine, and malondialdehyde) was remarkable lower with a ratio of ≤0.1 (∗). The sole newly found concentration of β-guanidinopropionic acid was remarkably lower than the earlier reported one, the ratio being 0.06 (∗). ADMA, dimethylguanidine, and malondialdehyde had a Cur-max/Cur-min > 5 (#), indicating a broad concentration range.

The standardization of the creatinine assay to an IDMS (isotope dilution mass spectrometry) reference measurement procedure⁹⁵ in 2006 is implemented in most routine laboratories, which makes it possible to compare serum creatinine values measured by different methods.

2.5.2 Protein-Bound Solutes

In 2003, 25 protein-bound solutes were summarized,¹ for 21/25 compounds recently reported concentrations were found (Table 2.2). 3-Deoxyglucosone, N-(carboxymethyl)lysine, indoxyl sulfate, leptin, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) have a broad mean uremic concentration range with Cur-max/Cur-min > 5 (#).

When comparing the more recent concentrations to the ones reported before 2003, some points need special considerations.

The Cur-min of deoxyglucosone, quinolinic acid, leptin and CMPF are substantially lower than before (Cur-min/Cur-2003 ≤ 0.1). None of the updated concentrations are considerably higher.

Recent findings demonstrated that p-cresol itself is not present in uremia, but was generated due to strong acidification for deproteinization in the preparative analytical phase.⁹⁶ In this classification p-cresol is replaced by its two in vivo present conjugates; p-cresyl sulfate, which is the most abundant and the recently quantified p-cresyl glucuronide.³¹

Regarding the polyamines, the Cur-min of putrescine is almost a 10-fold lower than earlier reported.⁹⁷ Spermine and spermidine on the other hand, are suppressed in uremia, according to more recently published concentrations,⁵⁸ for this reason they are withdrawn from the list as uremic solutes.

Methionine-enkephalin, classified as middle molecules in 2003, was no longer studied as a uremic solute in the period 2003–2011, while the amino acid methionine was repeatedly found to be elevated in uremia. Therefore, in this classification, methionine was withheld as a protein-bound solute and methionine-enkephalin was withdrawn.

Retinol-binding protein is a low molecular weight protein of 21 kDa (middle molecule) that is in the circulation for >90% bound to transthyrethin,⁹⁸ it is listed here as a protein-bound peptide.

Melatonin is a hormone secreted by the pineal gland with a marked circadian rhythm in a healthy population. The morning and daytime concentrations are the lowest in a normal population, so that only morning concentrations were considered. It is of note that there are conflicting results about the circadian concentration variations in uremia.⁵²,⁹⁹,¹⁰⁰

2.5.3 Middle Molecules (MW > 500 Da)

There are 19 solutes listed as middle molecules in Table 2.3, which can be divided in peptides and cytokines. Cur-min is remarkably lower for atrial natriuretic peptide (ANP), cholecystokinine (CCK), endothelin, and the cytokines (Cur-min/Cur-2003 ≤ 0.1, ∗). The single recent concentration found for clara cell protein on the other hand is considerably higher than the earlier reported concentrations, the ratio being Cur-max/Cur-2003 > 10. The Cur-max/Cur-min is > 5 (#) for ANP, CCK, endothelin, and neuropeptide Y.

A lot of progress has been made in the determination of the immunoglobulin light chains, which are polyclonally elevated in CKD. Until recently, reported concentrations covered the free light chains plus the light chains bound to the heavy chains. More recently, only the free immunoglobulin light chains κ and λ were measured in CKD and dialysis patients, which both are elevated and show a change in κ/λ ratio in comparison to a normal population.⁷⁷,⁷⁶ Therefore, both concentrations for total and free immunoglobulin light chains are listed in Table 2.3.

The molecular weights of these middle molecules can vary depending on the amount of amino acids in the peptide, often without loss of in vivo activity. In the case of hyaluronic acid the molecular weight of the disaccharide is reported, as the resulting molecular weight depends on the number of disaccharide repeats, which can vary widely.

2.5.4 Newly Identified Solutes with Mass Spectrometry (MS)

Twenty-two uremic solutes are newly identified with MS (Table 2.4), most of them protein bound. For each component, the molecular weight, number of publications, and the maximum mean uremic concentration are reported. Many additional solutes have already been identified with MS, often without reporting concentrations.¹⁰¹ Therefore, they are not summarized in the present list.

2.6 Conclusion

Uremic solute concentrations reported in the literature are frequently subject to scatter, owing to the influence of various factors, above all to inaccuracies in methodological approaches. Deviations in measured concentrations may lead to incorrect interpretations of the pathophysiological role of uremic solutes and/or to erroneous therapeutic decisions. When choosing solute concentrations for in vitro tests or when comparing newly determined concentrations with those reported previously, potential discrepancies among available values should be carefully analyzed and taken into account. To come to an objective approach to this problem, variability analysis of reported concentrations may be of help. Striking outliers either should be discarded or analyzed together with other values when more consistent with the majority of reported data. Introduction of more sophisticated analytical methods, such as MS, could increase the accuracy of reported concentrations in the future.

References

1. Vanholder R, De Smet R, Glorieux G, Argiles A, Baurmeister U, Brunet P, Clark W, Cohen G, De Deyn PP, Deppisch R, scamps-Latscha B, Henle T, Jorres A, Lemke HD, Massy ZA, Passlick-Deetjen J, Rodriguez M, Stegmayr B, Stenvinkel P, Tetta C, Wanner C, Zidek W: Review on uremic toxins: Classification, concentration, and interindividual variability. Kidney Int 63:1934–1943, 2003.

2. Weissinger EM, Kaiser T, Meert N, De SR, Walden M, Mischak H, Vanholder RC: Proteomics: A novel tool to unravel the patho-physiology of uraemia. Nephrol Dial Transplant 19:3068–3077, 2004.

3. Vanholder R, De Smet R: Pathophysiologic effects of uremic retention solutes. J Am Soc Nephrol 10:1815–1823, 1999.

4. Vanholder R, Argiles A, Baurmeister U, Brunet P, Clark W, Cohen G, De Deyn PP, Deppisch R, Descamps-Latscha B, Henle T, Jorres A, Massy ZA, Rodriguez M, Stegmayr B, Stenvinkel P, Wratten ML: Uremic toxicity: Present state of the art. Int J Artif Organs 24:695–725, 2001.

5. Vanholder R, De Smet R, Lameire NH: Redesigning the map of uremic toxins. Contrib Nephrol 133:42–70, 2001.

6. Vanholder R, Glorieux