Uremic Toxins Overview

Uremic Toxins

Christos ArgyropoulosRenal Grand Rounds

15/10/2007

General Outline

• Definitions of terms

• Conceptual Pathogenetic Model of Uremia and the “Residual Syndrome”

• Experimental Evidence for the Model

• What can we do?

The “Chosen” Ones

• Review selected compounds from the uremic hall of fame

• Impossible to review everything, since the literature is extensive (includes both gels and small p – values!)

• Focus on less well known culprits (no PTH, CaxP, beta2 microglobulin, K, Phos today)

• Focus on the ones that should make us rethink RRTs

Uremia

• A construct of the 19th century• Literal meaning “Urine in the blood”• Denotes symptoms and signs in

patients with “failing” kidneys irrespective of the initiating cause of renal failure

• Underlying conceptual pathogenetic model: intoxication by accumulation

Uremia

• Manifestations of the uremic syndrome:

• Nausea, vomiting, loss of appetite, wt loss, abnormalities of coagulation cascade, GI bleeding, pruritus, serositis, volume overload, hypertension, soft tissue calcification, pulmonary edema, confusion, lethargy, death

Of theories, measurements and experiments

• All phenomena have a cause (even if it cannot exceed the p-value of 0.05)

• One cannot measure what does not exist• One cannot know what one cannot

measure• One versus multiple toxins (the rise and

fall of urea)• The higher the concentration (signal), the

easier to measure and study• Counting missing limbs is easier than

MALDI-TOF/MS/2D-protein electrophoresis

The “Residual” Syndrome

• Introduction of dialytic therapies provided experimental evidence for the validity of the intoxication model of uremia:– Visual Evidence: uremic frost disappeared – Comatose patients were waking up– Survival was increased

• What we see today is a different life-threatening condition that is known as the “residual uremic syndrome”

• Academically, it has been a rewarding syndrome that has generated publications with multiple, highly significant p-values about itching, depressed, dying, elderly dialysis patients

Clinical Manifestations of the “Residual” Syndrome

• Subtle signs of malnutrition • Increased susceptibility to infection• Increased susceptibility to cardiovascular complications • Low-grade serositis • Impaired vascular reactivity • Hypothermia • Reduced exercise capacity and O2 utilization• Fatigue• Subtle psychological disturbances such as loss of focus

and ambition (or is it depression?)• Sleep disturbances• Restless Legs

Possible Causes of the “Residual” Syndrome

A. Accumulation of:1. large molecular weight solutes that are difficult to

remove by dialysis2. protein-bound small molecular weight solutes that

are difficult to remove by dialysis3. dialyzable solutes that are incompletely removed

B. Indirect phenomena:1. Accelerated protein “aging”2. Inflammation3. Tissue calcification4. Toxic effect of hormone imbalance

C. A toxic effect of the dialysis itself

Semin. Dial. (2001) 14(4):246-251


A. Intoxication By Accumulation

Uremic Solutes (The NEMJ view)

N Engl J Med 357:1316, September 27, 2007

A Systematic Approach To Uremic Retention Solutes

EUTox: workgroup of the ESAO formed in 1999 to 1. systematically categorize,2. experimentally study uremic retention molecules

(general aim)3. More specifically detect the “renal failure specific”

factors which underlie vascular damage (genome, proteome, secretome)

4. design, develop and improve extracorporeal treatment systems (industrial partners)

5. Apply such knowledge to bioartificial reactors and regenerative medicine applications (industrial partners)

http://www.uremic-toxins.org/


• A database of 857 publications between 1966 and 2002 abstracted from Medline

• Database concerns solutes, and inter-individual variability of concentrations

• Manual classification in three categories:– Small solutes with no protein binding– Middle Molecules (>500Da)– Protein Bound Molecules

• Classification scheme emphasizes characteristics that influence removal by extracorporeal therapy modalities

KI, 63 (2003) 1934-1943


http://www.nephro-leipzig.de/eutoxdb/index.php


• Compounds that accumulate the most are the ones which are a) protein bound b) of size that exceeds the cutoff of our dialysis membranes

• Critical limitation: one cannot know what one cannot measure

KI, 63 (2003) 1934-1943


• For people who like p-values, the KI paper has many, small values to explore

• For people who like to think abstractly a few disturbing features emerge:– The non-toxic “stuff” we measure (SCr/Urea)

increases far less than the toxic “stuff” we do not routinely measure

– There is wide inter-individual variability in the retention factors of different solutes

– Conventional renal replacement therapies cannot remove many biologically significant retention solutes

KI, 63 (2003) 1934-1943

Causes and Consequences of Variability

• Differences in laboratory techniques (e.g. p-cresol concentration by GCMS is x6 c.t HPLC)

• Structural modification of molecules• True inter-individual, ethic and geographic

differences in the rate of generation of compounds

• Differences in dialysis modalities• Wide range of biological effects possible (may

account for differences in the epidemiology of complications of CKD/ESRD)

Nephrology Dialysis Transplantation July

15 2007, doi:10.1093/ndt/gfm151

Clinical Implications of Solute Accumulation

• Honestly we do not really know (notable exception: b2 microglobulin)

• However they must do something (or else the patients are “faking” both the uremic residual syndrome and the improvement noted after transplantation)

• Evidence for the accumulation and biological toxicity of individual compounds is indirect and difficult to obtain (but it can be done at least in vitro)

Clinical Implications of Solute Accumulation

• Source of uremic solutes (protein breakdown, environment, dialytic modalities, GIT bacteria)

• Concomitantly prescribed drugs that interfere with the free fraction of uremic solutes

• Residual renal function• Differences in dialysis dose and membrane

flux • Multi-compartmental kinetics and differential

cellular accumulation e.g. differences in organic transporter activity

Urea

• Probably minimal if any toxicity. • Clinical correlates: ICU consults!• In vitro studies: inhibition of volume

sensitive transporters, decreased 2,3 DPG binding to hemoglobin, inhibition of NaK2Cl RBC transporter, macrophage iNOS (at unrealistically high concentrations)

Guanidines

• Large group of metabolites of arginine

• Variable Mechanisms of toxicity.

• Water soluble

• Differential distribution in cellular compartments

Guanidines - Creatinine

• Large group of metabolites of arginine• Variable Mechanisms of toxicity. • The most innocuous one is

Creatinine (has been associated with ICU consults)

• In vitro studies: inhibition of chloride transport activity, reduces contractility of SMCs (at 5 times the concentrations seen in typical cases of uremic coma)

Guanidines – Non Creatinine

• Group of water soluble molecules: Guanidine (G), Guanidinosuccinate (GSA), Methylguanine (MG), Asymmetric Dimethyarginine (ADMA), Guanidino-propionic acid (GPA) and many others

• Neurotoxins - seizures (GPA, MG) through competitive inhibition of GABA/Gly and agonistic effects on NMDA (Glutamate) receptors

• Inferred role in memory disturbances of dialysis patients through interference with Glutamate neurotransmission

Memory, Emotions, Seizures:Kidney v.s. Brain

• Associate memory formation in the mammalian CNS is grounded on the synaptic circuitry of hippocampal area CA1

• Experiments in animals, and fMRI imaging strongly suggest that limbic structures are involved in the pathogenesis and progression of depression

• The same area of the brain is involved in significant proportion of complex partial seizures (target in surgical Tx for Epilepsy)


• Depression in non-uremic individuals is associated with reductions in the volume of the hippocampal/amygdala complex

• Such reductions correlate with disease severity and duration

Neuropsychopharmacology (2004) 29, 952–959


• The effects of GSA administration was recently examined in rats

• The study demonstrated dose dependent effects on:– reductions of hippocampal volume,– impaired performance in cognitive tasks (passive

avoidance, water maze test),– Impaired psychomotor performance (open

exploration, spontaneous activity, social exploration)

Physiology and Behaviour 84(2), 15 February 2005, Pages 251-264

Memory, Emotions, Seizures:Kidney v.s. Hippocampus

http://en.wikipedia.org/wiki/Morris_water_maze

Physiology and Behaviour 84(2), 15 February 2005, Pages 251-264


• Associative, declarative memory formation requires simultaneous activation of pre and post-synaptic neurons in the hippocampus

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=neurosci.TOC&depth=2


KI (2001) 59(S78): 77-83

GSA competes for the Mg2+ binding site of the NMDA channel leading to over-activation of the channel, Ca2+ influx, neurotoxicity and cell death

Guanidines – Other Effects

• Immunosuppressive properties: inhibition of PMN SOD (GPA, GSA), suppression of NK response to IL-2

• Increased activation induced apoptosis of lymphocytes

• Inhibition of iNOS production (ADMA) leading to vasoconstriction and HTN

• Structural modification of other proteins (esp albumin) and subsequent release of Homocysteine)

P-Cresol and Conjugates

• Phenolic acid derivative, produced by intestinal bacteria as a result of the metabolism of Tyr and Phe

• Other sources: psychedelic drugs, smoking• Clinical Effects:

– Hepato-toxin, – inhibits free O2 generation inside PMNs, – Inhibits expression of adhesion molecules for monocytes and

PMNs in uremic animals,– Protein bound (>98%) in conjugated form– Inefficiently removed by HD, but not by PD

• Oral absorbents (AT120) decrease levels in experimental models and attenuate progression of glomerulosclerosis in the 5/6 rat model

3-carboxy-4-methyl-5-propyl-

2-furanpropionic acid • Urofuranic acid derivative• Strongly lipophilic and protein bound• Major competitive inhibitor of drug binding,

hepatic SAM• Interferes with conversion of T4 to T3• Interferes with oxidation of NADH compounds

in mitochondria• Levels minimally affected by either low flux or

high flux dialysis

Homocysteine (Hcy)

• Sulfur containing aa produced by demethylation of Met• Elevations of Hcy lead to cellular accumulation of S-

adenosyl-Hcy which competes with SAM (a co factor for transmethylation reactions)

• Widespread hypo-methylation (a key epigenetic transcriptional control mechanism) ensues

• Anatomically this leads to accelerated atherosclerosis in rat models and in vitro proliferation of VSMCs

• In the general population, an increase in Hcy concentration is associated with CV mortality

• Note that in both meta-analysis and randomized trials analyzed by ITT, exogenous folate administration did not attenuate CV mortality

• Genetic and epidemiologic studies analyzed by AT disagree

JAMA. 2006;296:2720-2726 http://www.medscape.com/viewarticle/548317

Homocysteine (Hcy)

• Levels are increased x2-4 in ESRD• Mechanism of elevation is unclear

(impairment of Hcy transsulphuration and remethylation)

• Exogenous folate administration may improve metabolic flux in the C1 pathways without modifying Hcy levels

• One study in ESRD patients showed no clinical benefit as far as CV outcomes are concerned (J Am Soc Nephrol 2004; 15: 420–426 )

• HOST study will be completed at the end of this year

Nephrology Dialysis Transplantation 2006 21(5):1161-1166

Small molecular toxins and the dose of dialysis

• Theoretical considerations argue that these compounds should cross the membrane at the same efficiency as Urea

• Even though this is true, total body clearance is vastly different: a RR of 77% for urea amounts to a RR of 49% for GSA and 55% for MG

• URR may be used to estimate the dialyzer extraction ratio (will be the same for all “similar” compounds) ≠ RR

• The degree of accumulation requires knowledge of the degree of generation (which is not the nPCR!) and the order of the corresponding compartmental model

• Dose measurement schemes will have to be reconsidered and validated for known toxins (e.g. is (KT/V)GSA linearly related to (KT/V)UREA?

Am J Kidney Dis. 2007 Aug;50(2):279-88.


B. Indirect Effects and the inflammatory loop model of uremia

Beyond small molecules

• Traditional view of pathophysiology of uremic complication : retention of solutes (small molecules)

• Efficacy of PD attributed to clearance of “middle molecules” of MW between 300Da-12kDA

• Prototypical middle molecule: B2-MG (?PTH)• The existence of other “middle” molecules

could not be demonstrated until the introduction of proteomic techniques (“one cannot know what one cannot measure”)

Proteomics and the Quest for Middle Molecules

• Proteome: “the entire complement of proteins expressed by a genome, cell, tissue or organism “

• Proteomics: The study of the proteome with biochemical techniques (2D electrophoresis, CE coupled with ESI-TOF MS

• Generates an “unbiased” fingerprint of what lies across the twilight zone (the dialysate side!)

• Middle molecules can now be “spotted”

Proteomics and the Quest for Middle Molecules

Nephrology Dialysis Transplantation 2004 19(12):3068-3077; doi:10.1093/ndt/gfh509

http://www.decodon.com/

Effect of Membrane Flux in Maintenance Hemodialysis


Effect of Membrane Flux in Maintenance Hemodialysis

• In UF from uremic plasma 1394 pps were identified in high flux membranes v.s. 1046 (low flux)

• Normal plasma contained 544 and 490 respectively

• Post HF dialysis plasma showed the same distribution of pps as normal plasma

• Mass distribution differed between HF and LF membranes

• “Low flux” membrane used in that study (F10) has a cutoff of 5200 Da


Clinical Application of Proteomics: Defining the Problem• This one had multiple

interesting and statistically significant p-values

• Significant predictors of itchiness were: nationality (Italians topped the list with 55%), duration of ESRD (>3 mo), male sex

• Itchiness was a predictor of mortality (author hypothesized a relation with sleep disturbances) and so was depression

Clinical Application of Proteomics : Proposing an Intervention

• This one also had multiple interesting and statistically significant p-values

• The self-assessed VAS itching strength scores decreased by 15% after 1 month, 30% after 2 months, and 55% after 6 months, and itching duration decreased by, respectively, 10, 22 and 44% at the same time; 2 months after the end of the study, both scores had slightly increased, but ß2-microglobulin levels significantly decreased (P < 0.03); Nephrology Dialysis Transplantation 2007

22(Supplement 5):v8-v12; doi:10.1093 /ndt/gfm293

Clinical Application of Proteomics : Why does the intervention work?

• This one also had multiple interesting trivia

• PMMA membranes are “protein-leaking” and protein adsorbing and have been used succesfully in the Tx of uremic pruritus for the last 10 years

• After eluting the adsorbed protein components, patients with itching showed a band in the 160KDa region that was not an IgG molecule Nephrology Dialysis Transplantation 2007

22(Supplement 5):v13-v19;

doi:10.1093/ndt/gfm295

Clinical Application of Proteomics : Why does the intervention work?

Nephrology Dialysis Transplantation 2007 22(Supplement 5):v13-v19;

doi:10.1093/ndt/gfm295

Clinical Application of Proteomic Research: PD

• A study in 44 PD patients with different transport characteristics

• Screened for differences in protein excretion with 2D-PAGE followed by identification with MALDI-TOF

• Previous episodes (>1mo) of tx peritonitis associated with ↑↑ free light chains in the PD fluid

• High transporters were “leaking” Apo-AI a component of HDL

• Report that the proteome profiles are similar to the ones obtained from normal urine (unfortunately the data were not shown)

J. Proteome Res., ASAP Article 10.1021/

pr0702969 S1535-3893(07)00296-5

Low – Molecular Weight Proteins that accumulate in ESRD

J Am Soc Nephrol 13:S41-S47, 2002

Accumulation of solutes v.s. Cumulative Protein Damage

• So called middle molecules consist of both retention molecules and modified versions of proteins whose total concentration may remain in the normal range

• Such protein damage may provide the missing link between retention and inflammation/malnutrition

Accumulation of solutes v.s. Cumulative Protein Damage

• Initiating Events: oxidative stress of CKD/ESRD/carbonyl stress (from abnormal lipid and sugar metabolism)

• Intermediate molecular changes: hydroxylation, nitration and chlorination of aromatic aa,sulfoxidation of Met in proteins

• End result: deranged protein metabolism and functional alterations

Major Classes of Irreversible Protein Damage Products

1. Advanced Oxidation End Products (AOPP): d/t modification of Tyr residues

2. Advanced Lipoxidation End Products (ALE): d/t Michael reaction of α, β-unsaturated aldehydes to the NH2 group of Lys, imidazolone group of His and the sulphydryl group of Cys

3. Advanced Glyc(oxidation) End Products: predominantly non- fluorescent glyoxal derivatives of Lys rather than the “traditional” fluorescent varieties found in DM.

4. Tissue fluorescent AGEs: measure of cumulative exposure to pro-inflammatory and oxidative stress. Skin autofluorescence is a predictor of mortality in ESRD patients

Nephrology Dialysis Transplantation 2007

22(Supplement 5):v20-v36; doi:10.1093/ndt/gfm294

J Am Soc Nephrol 16: 3687-3693, 2005

Tissue AGEs and mortality in ESRD

Putting it all together

• A multi-step “inflammatory loop” model for the pathogenesis of uremia was proposed recently

1. Carbonyl+Oxidation Stress lead to protein damage2. Protein End products are recognized by the RAGE

superfamily of membrane receptors3. Initiation of inflammatory cascade4. Generation of reactive oxygen and nitrogen species5. Further protein damage leading to more inflammation and

production of reactive radicals (positive loop)6. An important independent source of both AGE and

inflammatory mediators may be dialysis itself (PD and HD respectively)

7. The loop will be broken by increases in the GFR

Nephrology Dialysis Transplantation 2007 22(Supplement 5):v20-v36

Putting it all together

Nephrology Dialysis Transplantation 2007 22(Supplement 5):v20-v36

Knowing is not enough, we must apply. Willing is not enough, we must do.

Goethe

What do we know?

• Not much, but certainly we have ended the beginning of the quest for knowledge

• The pathogenesis of the uremic residual syndrome has evolved from the accumulated toxicity model of the 19th century

• Both accumulation of “stuff” and cumulative damage from “stuff” are to blame

• Accumulation is they key word … we’ve got to give more “GFR” back

What can we do?

• The best way of giving GFR back is a transplant

• If the organ shortage could be solved, then the precise nature of the uremic toxins can be left unexplored

• It is unlikely that we can achieve this goal any time soon for all our patients so we’ve got to improve our dialysis Tx

What can we do?

• The (unsatisfactory) answer is that we have got to give more “machine GFR” back

• One cannot know (that one gives more GFR) what one cannot measure (dose of dialysis)

• However kinetic models of the dose of dialysis based on (KT/V)urea have to be abandoned or drastically modified

• Possible solution: analysis of large cohorts of people who do well and people who do not do well with detailed kinetic model and validation of population PK/PD models (we did that with FK/CSA!)

What can we do?

• In light of the inadequacy of our current kinetic constructs, we should re-examine the major dialysis trials (they assume the kinetic constructs are correct) because more dialysis (and higher flux) may in fact be better

• Itching, depressed, fatigued dying elderly dialysis patients may help us “spot” the correct dialysis dosing schedules (ASK for symptoms, study what goes down the drain and what stays behind)

• Follow up with more efficient dialysis along or even instead of SSRIs and moisturizing lotions)

What can we do?

• The molecular analysis of what goes down the drain points out that no matter how much (KT/V)UREA/GSA/etc we give there will be bigger bad guys that stay behind

• Ideas to test in RCTs (should not be analyzed by the ITT):– Hemo-diafiltration (adds convective clearance)– Protein Leaking/Absorbent Membranes (removes

larger “stuff”)– More frequent dialysis (improves small moleculer

clearance, BP and volume control)– Biocompatible membranes and solutions (reduces

inflammation from the machine/PD solution)

If you are interested to find out more …

• EuTOX database: – http://www.uremic-toxins.org/

• Lowrie EG. The kinetic behaviors of urea and other marker molecules during hemodialysis. Am J Kidney Dis. 2007 Aug;50(2):181-3

• Galli F. Protein Damage and inflammation in uraemia and dialysis patients Nephrology Dialysis Transplantation, 2007 22 Supplement 5 20-36 (Special Issue on uremic toxins)

• Weissinger et al Proteomics: a novel tool to unravel the pathophysiology of uremia. Nephrology Dialysis Transplantation (2004) 19: 2068-3077

• Depner T.A. Uremic Toxicity: Urea and Beyond. Seminars in Dialysis (2001),14(4): 246-251

• Locatelli F. et al. A critical assessment of Uremia Research Blood Purif 2006,24:71-78

• Vanholder R et al. What is uremia? Retention versus Oxidation Blood Purif 2006,24:33-38

Uremic Toxins Overview

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