Biomarkers of CVD in Renal Failure. Facts and ?Fiction

A biochemical detective story

ACB SW regional meeting July 4th

John O’Connor

Presentation includes:

• The scale of CVD in CKD patients. The Facts

• Biochemical Markers of Myocycte damage in CKD Patients

• An alternative hypothesis. The ?Fiction

• Diagnostic Dilemmas

• Practical Considerations

Scale of CVD in CKD. The Facts!!

• The 5 level staging system for CKD only came into being in 2002 so outcome vs stage data is still forthcoming

• The overall prognosis for patients with stage 5 CKD is dismal, ACS accounts for 20% of cardiovascular deaths

• Worst still the event is likely to be catastrophic• Only 40% of Dialysis patients survive a year,

27% Two years• 40% of new dialysis patients have left

ventricular abnormalities

n = 1 120 295 between 1996 and 2000 in California (mean age 52 yrs) - Mean follow-up 2.84 yrsAS Go et al., N Engl J Med 2004

Decreases in GFR influence survival rate in patients following MI(VALIANT STUDY)

Rates of Death at 3-years from Cardio-Vascular Causes after MI alongside other adverse outcomes according to GFR at baselineAS Anavekar et al., N Engl J Med 2004

CKD Patients Have additional CVD Risk Factors

•These include;•abnormal calcium and phosphate metabolism,•anaemia, •Inflammation (CRP)•oxidative stress,•thrombogenic factors•uraemic toxins •homocystine

Biochemical Markers of Myocte damage in CKD Patients

•T CK often elevated in Haemodialysis Patients

•Troponin T and I are also raised (why?)

Chemistry Department at the RD&E carried out anChemistry Department at the RD&E carried out an

audit on 174 patients with an impaired GFR (<60). audit on 174 patients with an impaired GFR (<60).

Of these, 75 had measurable cTnT (> 0.02 ng/ml) Of these, 75 had measurable cTnT (> 0.02 ng/ml)

in the absence of any appreciable cardiac damage.in the absence of any appreciable cardiac damage.

Distribution of TrT

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TrT ng/ml

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Biochemical Markers of MI in CKD Patients Hennepin County Medical Center, Minnesota

Cohort: 733 ESRD patients (largest study to date)Cohort: 733 ESRD patients (largest study to date)

Aim : to determined the prevalence of increased Aim : to determined the prevalence of increased cardiac Troponin I (cTnI) and T (cTnT) in ESRDcardiac Troponin I (cTnI) and T (cTnT) in ESRD

Results: A significant increase in TnT (82%) and Results: A significant increase in TnT (82%) and TnI (6%) was observed in ESRD patients compared TnI (6%) was observed in ESRD patients compared to 99to 99thth percentile. percentile.

Biochemical Markers of MI in CKD Patients

Differences between [TnT] and [TnI] not entirely clear Differences between [TnT] and [TnI] not entirely clear from the literature.from the literature.

TnT is more consistently raised than I (about 5:1) TnT is more consistently raised than I (about 5:1) ? Cytosolic ? Cytosolic

[TnT] is greater than [TnI] per gram weight of cardiac [TnT] is greater than [TnI] per gram weight of cardiac musclemuscle

[TnT] increase during dialysis [TnI] decreases [TnT] increase during dialysis [TnI] decreases (absorption onto dialysis membranes)(absorption onto dialysis membranes)

TnI may undergo more significant proteolysis TnI may undergo more significant proteolysis

in the circulation than TnTin the circulation than TnT

Methodological Issues: Troponins

Different forms of troponin which are released from Different forms of troponin which are released from necrosing myocytes. necrosing myocytes. Wu Wu et alet al studied serum troponins separated by studied serum troponins separated by gel filtration (Clin Chem 1998:44;6)gel filtration (Clin Chem 1998:44;6) Troponin T are released as the intact TnT:I:C complex and free cTnT Troponin T are released as the intact TnT:I:C complex and free cTnT One Assay one standard One Assay one standard not a problem not a problem cTnI mainly released in complexed form following cTnI mainly released in complexed form following MI (which most assays measure) but also free cTnI variably measured MI (which most assays measure) but also free cTnI variably measured by different assaysby different assaysAdded issue is TnI hydrophobicityAdded issue is TnI hydrophobicity Free cTnI can bind to other proteins present in blood,Free cTnI can bind to other proteins present in blood,

such as calmodulin, a structural analogue to Troponin C, such as calmodulin, a structural analogue to Troponin C, potentiallypotentially obscuring its antigenic epitopes obscuring its antigenic epitopes It may beIt may be possible that free TnI is lost to the possible that free TnI is lost to the surfaces of blood collectionsurfaces of blood collection tubes, pipette tips, tubes, pipette tips, sample cuvettes, and so forth.sample cuvettes, and so forth.

Why are Troponins raised?

Renal clearance of Troponin may be impaired?Renal clearance of Troponin may be impaired?

But !! Troponins are large molecules, not easily filteredBut !! Troponins are large molecules, not easily filtered

Kinetic studies (of Troponin fall) in patients with ESRD Kinetic studies (of Troponin fall) in patients with ESRD compared to patients with normal GFR who have MI’s, show no compared to patients with normal GFR who have MI’s, show no differencesdifferences   Current theory is that these patients are having silent microinfarctsCurrent theory is that these patients are having silent microinfarcts   Certainly having a elevated ventricular mass is an independent Certainly having a elevated ventricular mass is an independent variable in having raised levelsvariable in having raised levels

TnT > 0.01 and < 0.25 ng/ml R = 0.282No significant correlationOmitted any patients admitted via A+E

TnT vs GFR

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Uraemic-induced skeletal myopathy ? may be responsible

• Uraemic states may promote re-expression of cardiac troponin isoforms from damaged or regenerating skeletal muscle fibres.

• The uraemic-induced skeletal myopathy hypothesis is also supported by the observation that skeletal muscle from patients treated with maintenance haemodialysis, have significant morphological changes observed by electron and light microscopy 22.

• However, studies using highly specific antibodies have demonstrated absence of cTnT in skeletal muscle immunohistochemically and absence of cTnT mRNA in adult skeletal muscles 23.

• Therefore, there is currently insufficient data to support a skeletal muscle source for serum cardiac cTnT elevations

in patients with ESRD.

Pearson Correlation TnT vs Urea (TnT > 0.01 and < 0.25ng/ml)R = 0.349P = 0.0073 (very significant)Omitted any patients admitted via A+E

TnT vs Urea

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Pointers from Transplant Patients

• TrT was positive in 14 of 26 (54%) pre transplant samples but only 3 out of 26 (12%) post transplant samples.

• But can we assume its all down to clearance??

Another Theory (Currently under investigation at the RDE) ? The Fiction

The presenter gazed at the audience

Will they spot it he mused

The EPO mystery

The EPO Hypothesis

• EPO A hypoxia induced hormone produced by the kidney stimulating hematopoiesis

The EPO Hypothesis

• Role is to inhibit Apoptosis of erythroid precursors

• Clue to its pleiotrpoic cytokine status• There are EPO receptors in the Cardiovascular

system, both in endothelial cells and cardiomyocytes

• Anoxia causes a two fold increase in expression of these receptors

• Apoptosis / mitosis is occurring all the time in healthy individuals but is increased in the absence of EPO as would be the case with CKD

• EPO also improves cardiac vascularity

Apoptosis in Myocytes is via the Mitochondrial pathwayActivated by Oxidative stress (toxic ureamic compounds in CKD)

and Hypoxia

Contain proapoptotic factorsCYT C

If released into cytoplasmactivate Caspase Cascade

Release of CYT is controlled by Bci-2Family members, can open pores

Death

Antipoptotic factors EPO

Keep pores closed

Life

Activation of the Janus Kinase 2 pathwayP I 3 Kinase(AKT) Protein Kinase BUpregulates BcL-x (Anti Apoptotic)

Stabilises mitochondrialMembrane preventing the Release of Cyt CBlocks Caspase 9 protectsGenomic DNA

To induce a local inflammatory response

Not to induce a local inflammatory response

Cell Death

Some more clues

REVIEW Elevation of cardiac troponin I indicates more than myocardial ischemia John P. Higgins, MD, MPhil*Johanna A. Higgins, MD†Clin Invest Med 2003;26(3):133-47.

Apoptosis in myocytes in endstage heart failure Narula J, Haider N, Virmani R, DiSalvo TG, Kolodgie FD, Hajjar RJ, et al.. N Engl J Med 1996;335:1182-9. Comments in: N Engl J Med 1996;335:1224-6; N Engl J Med 1997;336:1025-6. 158.

Functional consequences of caspase activation in cardiac myocytes Communal C, Sumandea M, de Tombe P, Narula J, Solaro RJ, Hajjar RJ.. Proc Natl Acad Sci U S A 2002;99:6252-6. 159..

Release of cardiac troponin in acutecoronary syndromes: ischemia or necrosis? Wu AH, Ford L Clin Chim Acta 1999;284:161-74.

Conclusions from review

• Apoptosis (preserved membrane integrity) in addition to necrosis (loss of membrane integrity) may lead to the release of cTnI.

• Apoptosis is associated with the activation of caspases that mediate the cleavage of vital structural proteins.

• In cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium.

• Therefore, activation of apoptotic pathways may lead to contractile dysfunction prior to cell death.

• Other animal studies suggest that release of troponin I can occur in the absence of irreversible ischemia

Further clues: A blast from the past

• Prognostic value of mitochondrial aspartate aminotransferase in acute myocardial infarction Giorgio Annoni Clinical Biochemistry

Volume 19, Issue 4 , August 1986, Pages 235-239

• Demonstrated that 12 hours after admission, serum m-AST and m-AST/AST ratio were significantly higher in the group of non-survivors compared with patients with a favourable prognosis

• ? A reflection of the degree of Apoptosis following MI

• Ischaemia, with mild to moderate hypoxia can lead to apoptosis of myocytes during a MI. This may result in a non-functional region of tissue, which if it is sufficiently large may lead to organ failure.

• Apoptosis following MI begins in endothelial cells EPO has a crucial protective role in these cells

• EPO could be a major determinant in myocardial size and preserving function

• Rats pretreated (24h) with 5000 Iu/Kg EPO and then subjected to I/R showed increased myocardial recovery 

• It could be argued that the damage limitation was due to having increased oxygenated blood, but it was a further 48h before haematocrit increased 

• Potential role in treating patients with MI to prevent CHF

• Bahlmann et al studied CRF patients treated with EPO (Darbepoietin)

• Concluded that EPO conferred tissue protection and preserved the capillary network in the Kidney without raising heamatocrit

 • Could the same be happening to the heart • One could postulate that in CKD patient a low level of EPO would

lead to increased Apoptosis • This could explain the release of biomarkers of cardiac damage

and could explain (Contraversially) the disparity between the bigger rise in Troponin T (Cytostolic) compared with Troponin I. In necrosis caused by ischaemia one would have thought they would be equal

HB vs TnT in Patients with GFR < 50y = -12.114x + 11.585

R = -0.-33N= 69

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In theory patients with low EPO levels should have lower HB levelsFrom the RDE data set Hb vs TnTPearson correlation N=69R = -0.33Two tailed P value = 0.0071 (highly significant)

Clinical Implications

• We know that in CKD patients, anaemia is an established risk for Cardiovascular disease. (but is this because the EPO levels are low)

• Naomichi et al studied patients who suffered their first MI and who had high levels of endogenous EPO. He found that following PCI the infarctions were smaller

• However, there are no RCT’s or other conclusive data for treating CKD patients with EPO (probably explains why only 50% are being prescribed it)

• The TREAT study has just begun to explore this, the outcomes may yield more data about the pleiotropic effects of EPO

• There may be a role for EPO following MI alongside reperfusion therapy, to help avoid CHF

Pointers from Transplant Patients

• cTnT produced positive results in 14 of 26 (54%) pretransplant samples and 3 of 26 (12%) post transplant samples.

• This demonstrates the effect of replacing a fully functional endocrine gland producing Erythropoietin

Prognostic implications

• It may be that if the increased Troponin T levels reflect apoptosis then potentially they could be used as markers of cardioprotection in patients treated with EPO?

Therapeutic implications

• EPO promotes microvascular growth in the heart, suggesting functional cardiac recovery with the formation of new blood vessels

• In patients with resistant CHF EPO plus iv Fe showed a big increase in LV function and a reduction in hospitalization days of 80%

Diagnostic Dilemmas

In CKD patients with low GFR and “Chest Pain ? Cause”

There is a danger that patients get diagnosed NSTEMI on the basis of having a raised Troponin

Solution

Measure 2x Troponin on admission and at 12 hours pocp and see if there is a dynamic change in the concentration

• Pre screen all peritoneal and heamodialysis patients to establish baseline TrT

Thanks for your attention

Any Questions