Inhibition of Bruton's TK regulates macrophage NF-κB and NLRP3 inflammasome activation in metabolic inflammation.

BACKGROUND AND PURPOSE: There are no medications currently available to treat metabolic inflammation. Bruton's tyrosine kinase (BTK) is highly expressed in monocytes and macrophages and regulates NF-κB and NLRP3 inflammasome activity; both propagate metabolic inflammation in diet-induced obesity. EXPERIMENTAL APPROACH: Using an in vivo model of chronic inflammation, high-fat diet (HFD) feeding, in male C57BL/6J mice and in vitro assays in primary murine and human macrophages, we investigated if ibrutinib, an FDA approved BTK inhibitor, may represent a novel anti-inflammatory medication to treat metabolic inflammation. KEY RESULTS: HFD-feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice decreased the activation of NF-κB and the NLRP3 inflammasome. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS-1/Akt/GSK-3ß pathway, protecting mice against the development of hepatosteatosis and proteinuria. We show that BTK regulates NF-κB and the NLRP3 inflammasome specifically in primary murine and human macrophages, the in vivo cellular target of ibrutinib. CONCLUSION AND IMPLICATIONS: We provide "proof of concept" evidence that BTK is a novel therapeutic target for the treatment of diet-induced metabolic inflammation and ibrutinib may be a candidate for drug repurposing as an anti-inflammatory agent for the treatment of metabolic inflammation in T2D and microvascular disease.

Identification of AnnexinA1 as an Endogenous Regulator of RhoA, and Its Role in the Pathophysiology and Experimental Therapy of Type-2 Diabetes.

Annexin A1 (ANXA1) is an endogenously produced anti-inflammatory protein, which plays an important role in the pathophysiology of diseases associated with chronic inflammation. We demonstrate that patients with type-2 diabetes have increased plasma levels of ANXA1 when compared to normoglycemic subjects. Plasma ANXA1 positively correlated with fatty liver index and elevated plasma cholesterol in patients with type-2 diabetes, suggesting a link between aberrant lipid handling, and ANXA1. Using a murine model of high fat diet (HFD)-induced insulin resistance, we then investigated (a) the role of endogenous ANXA1 in the pathophysiology of HFD-induced insulin resistance using ANXA1-/- mice, and (b) the potential use of hrANXA1 as a new therapeutic approach for experimental diabetes and its microvascular complications. We demonstrate that: (1) ANXA1-/- mice fed a HFD have a more severe diabetic phenotype (e.g., more severe dyslipidemia, insulin resistance, hepatosteatosis, and proteinuria) compared to WT mice fed a HFD; (2) treatment of WT-mice fed a HFD with hrANXA1 attenuated the development of insulin resistance, hepatosteatosis and proteinuria. We demonstrate here for the first time that ANXA1-/- mice have constitutively activated RhoA. Interestingly, diabetic mice, which have reduced tissue expression of ANXA1, also have activated RhoA. Treatment of HFD-mice with hrANXA1 restored tissue levels of ANXA1 and inhibited RhoA activity, which, in turn, resulted in restoration of the activities of Akt, GSK-3ß and endothelial nitric oxide synthase (eNOS) secondary to re-sensitization of IRS-1 signaling. We further demonstrate in human hepatocytes that ANXA1 protects against excessive mitochondrial proton leak by activating FPR2 under hyperglycaemic conditions. In summary, our data suggest that (a) ANXA1 is a key regulator of RhoA activity, which restores IRS-1 signal transduction and (b) recombinant human ANXA1 may represent a novel candidate for the treatment of T2D and/or its complications.

Ethnic differences in the progression of chronic kidney disease and risk of death in a UK diabetic population: an observational cohort study.

OBJECTIVE: To determine ethnic differences in the progression of chronic kidney disease (CKD) and risk of end-stage renal failure (ESRF) and death in adults with type 2 diabetes mellitus (T2DM), and to identify predictors of rapid renal decline. DESIGN: Observational community-based cohort study undertaken from 2006 to 2016 with nested case-control study. SETTING: 135 inner London primary care practices contributing to the east London Database. PARTICIPANTS: General practice-registered adults aged 25-85 years with established T2DM and CKD at baseline. OUTCOMES: The annual rate of renal decline was compared between white, south Asian and black groups, and stratified by proteinuria and raised blood pressure (BP) at baseline. Predictors of rapid renal decline were identified in a nested case-control study. Cox proportional hazards regression was used to determine ethnic differences in the risk of ESRF and death. RESULTS: Age-sex adjusted annual decline was greatest in the Bangladeshi population. There was stepwise increase in the rate of decline when stratifying the cohort by baseline proteinuria and BP control, with south Asian groups being most sensitive to the combined effect of proteinuria and raised BP after accounting for key confounders.The odds of rapid renal decline were increased for individuals of Bangladeshi, African and Caribbean ethnicity, those with hypertension, proteinuria, cardiovascular disease and with increasing duration of diabetes. Rapid progression was more frequent in younger age groups. Risk of developing ESRF was highest in the black group compared with the white group (HR 1.88, 95% CI 1.11 to 3.19). Risk of death from any cause was 29% lower in the south Asian group compared with the white group (HR 0.71, 95% CI 0.56 to 0.91). CONCLUSIONS: Proteinuria and hypertension trigger accelerated estimated glomerular filtration rate decline differentially by ethnicity. Active monitoring of younger adults, who have greater odds of rapid progression and the most to gain from interventions, is essential.

Kidney Transplantation in a Patient Lacking Cytosolic Phospholipase A2 Proves Renal Origins of Urinary PGI-M and TX-M.

RATIONALE: The balance between vascular prostacyclin, which is antithrombotic, and platelet thromboxane A2, which is prothrombotic, is fundamental to cardiovascular health. Prostacyclin and thromboxane A2 are formed after the concerted actions of cPLA2α (cytosolic phospholipase A2) and COX (cyclooxygenase). Urinary 2,3-dinor-6-keto-PGF1α (PGI-M) and 11-dehydro-TXB2 (TX-M) have been taken as biomarkers of prostacyclin and thromboxane A2 formation within the circulation and used to explain COX biology and patient phenotypes, despite concerns that urinary PGI-M and TX-M originate in the kidney. OBJECTIVE: We report data from a remarkable patient carrying an extremely rare genetic mutation in cPLA2α, causing almost complete loss of prostacyclin and thromboxane A2, who was transplanted with a normal kidney resulting in an experimental scenario of whole-body cPLA2α knockout, kidney-specific knockin. By studying this patient, we can determine definitively the contribution of the kidney to the productions of PGI-M and TX-M and test their validity as markers of prostacyclin and thromboxane A2 in the circulation. METHODS AND RESULTS: Metabolites were measured using liquid chromatography-tandem mass spectrometry. Endothelial cells were grown from blood progenitors. Before kidney transplantation, the patient's endothelial cells and platelets released negligible levels of prostacyclin (measured as 6-keto-prostaglandin F1α) and thromboxane A2 (measured as TXB2), respectively. Likewise, the urinary levels of PGI-M and TX-M were very low. After transplantation and the establishment of normal renal function, the levels of PGI-M and TX-M in the patient's urine rose to within normal ranges, whereas endothelial production of prostacyclin and platelet production of thromboxane A2 remained negligible. CONCLUSIONS: These data show that PGI-M and TX-M can be derived exclusively from the kidney without contribution from prostacyclin made by endothelial cells or thromboxane A2 by platelets in the general circulation. Previous work relying on urinary metabolites of prostacyclin and thromboxane A2 as markers of whole-body endothelial and platelet function now requires reevaluation.

Membranous nephropathy: a retrospective observational study of membranous nephropathy in north east and central London.

BACKGROUND: Membranous nephropathy (MN) is the leading cause of nephrotic syndrome in adults. MN is a clinically heterogeneous disease and it is difficult to accurately predict outcomes (including end stage renal failure) at presentation and whom to treat with potentially toxic therapies. We aimed to identify factors predicting outcome in MN in our cohort from two large tertiary London units by undertaking a retrospective data analysis of 148 biopsy-proven MN patients from North East and Central London between 1995 and 2015. METHODS: Review of clinical and biochemistry databases. RESULTS: Surprisingly, patients that reached end stage renal failure (ESRF) had a less severe nephrosis compared to those that did not develop ESRF; serum albumin 33 g/L (3.3 g/dL) versus 24 g/L (2.4 g/dL), p = 0.002 and urinary protein creatinine ratio (uPCR) 550 mg/mmol (5500 mg/g) versus 902 mg/mmol (9020 mg/g), p = 0.0124. The correlation with ESRF was strongest with the presenting creatinine; 215 µmol/L (2.43 mg/dL) compared to 81 µmol/L (0.92 mg/dL), p < 0.0001. Patients presenting with creatinine of >120 µmol/L (1.36 mg/dL; corresponding to an eGFR of ≤60 ml/min in non-Black males) had an increased rate of ESRF and a faster decline. Other traditional risk factors for progression were not significantly associated with ESRF. Black patients presented with higher serum creatinine but no statistically significant difference in the estimated glomerular filtration rate, a higher rate of progression to ESRF and had a poorer response to treatment. CONCLUSIONS: This ethnically diverse cohort does not demonstrate the traditional risk profile associated with development of ESRF. Thus, careful consideration of therapeutic options is crucial, as current risk modelling cannot accurately predict the risk of ESRF. Further studies are required to elucidate the role of antibodies and risk genes.

ABO incompatible renal transplants: Good or bad?

ABO incompatible kidney transplantation (ABOi-KT) was previously considered to be an absolute contraindication for patients with end-stage kidney disease (ESKD) due to hyperacute rejection related to blood type barrier. Since the first successful series of ABOi-KT was reported, ABOi-KT is performed increasingly all over the world. ABOi-KT has led to an expanded donor pool and reduced the number of patients with ESKD awaiting deceased kidney transplantation (KT). Intensified immunosuppression and immunological understanding has helped to shape current desensitization protocols. Consequently, in recent years, ABOi-KT outcome is comparable to ABO compatible KT (ABOc-KT). However, many questions still remain unanswered. In ABOi-KT, there is an additional residual immunological risk that may lead to allograft damage, despite using current diverse but usually intensified immunosuppressive protocols at the expense of increasing risk of infection and possibly malignancy. Notably, in ABOi-KT, desensitization and antibody reduction therapies have increased the cost of KT. Reassuringly, there has been an evolution in ABOi-KT leading to a simplification of protocols over the last decade. This review provides an overview of the history, outcome, protocol, advantages and disadvantages in ABOi-KT, and focuses on whether ABOi-KT should be recommended as a therapeutic option of KT in the future.

Do oral aluminium phosphate binders cause accumulation of aluminium to toxic levels?

BACKGROUND: Aluminium (Al) toxicity was frequent in the 1980s in patients ingesting Al containing phosphate binders (Alucaps) whilst having HD using water potentially contaminated with Al. The aim of this study was to determine the risk of Al toxicity in HD patients receiving Alucaps but never exposed to contaminated dialysate water. METHODS: HD patients only treated with Reverse Osmosis(RO) treated dialysis water with either current or past exposure to Alucaps were given standardised DFO tests. Post-DFO serum Al level > 3.0 µmol/L was defined to indicate toxic loads based on previous bone biopsy studies. RESULTS: 39 patients (34 anuric) were studied. Mean dose of Alucap was 3.5 capsules/d over 23.0 months. Pre-DFO Al levels were > 1.0 µmol/L in only 2 patients and none were > 3.0 µmol/L. No patients had a post DFO Al levels > 3.0 µmol/L. There were no correlations between the serum Al concentrations (pre-, post- or the incremental rise after DFO administration) and the total amount of Al ingested.No patients had unexplained EPO resistance or biochemical evidence of adynamic bone. CONCLUSIONS: Although this is a small study, oral aluminium exposure was considerable. Yet no patients undergoing HD with RO treated water had evidence of Al toxicity despite doses equivalent to 3.5 capsules of Alucap for 2 years. The relationship between the DFO-Al results and the total amount of Al ingested was weak (R² = 0.07) and not statistically significant. In an era of financial prudence, and in view of the recognised risk of excess calcium loading in dialysis patients, perhaps we should re-evaluate the risk of using Al-based phosphate binders in HD patients who remain uric.

Accelerated decline of GFR in diabetic nephropathy predicted by interferon release assay to tuberculosis antigens.

BACKGROUND: The QuantiFERON® test (QFT) is a diagnostic tool for active and latent tuberculosis (TB) infections. High rates of positivity to QuantiFERON® have been demonstrated in patients with chronic kidney disease (CKD) and diabetic patients. We performed a pilot study to investigate if QFT positivity in diabetic CKD patients predicted the rate of renal function decline. METHODS: QFT was performed in 38 diabetic patients with CKD 4-5 not on dialysis. The rate of decline in estimated glomerular filtration rate (eGFR) was calculated. RESULTS: 18/38 patients had a positive QFT. Patients with a positive QFT had a steeper decline in eGFR, compared with patients with a negative QFT. Ethnicity (a marker of risk of previous TB exposure), urine protein/creatinine ratio, use of ACE inhibitors/angiotensin II receptor blockers and statins, serum C-reactive protein, vitamin D levels, HbA1c concentration and presenting GFR did not differ significantly. CONCLUSIONS: The finding in this small cohort needs to be replicated in a larger study because our study is susceptible to both type I and type II statistical error. We found that QFT positivity was associated with a more rapid rate of decline in GFR, but this association may be coincidental (with the difference in decline attributed to differences in the blood pressure or proteinuria of the two groups). Moreover, an association does not necessarily mean causality, although it would be interesting to speculate if we are identifying patients with latent TB who have an active interstitial nephritis. Another intriguing possibility is that this assay identifies patients with an immunological phenotype that predisposes to eGFR loss.

Long term outcome of patients with autosomal dominant polycystic kidney diseases receiving peritoneal dialysis.

There is a general perception that patients with polycystic kidney disease on peritoneal dialysis have poor long-term technique survival. In order to test this opinion, we performed a retrospective analysis comparing results of 56 consecutive patients with polycystic kidney disease to 56 non-diabetic patients with bilateral small kidneys. The patient groups were all initiated on peritoneal dialysis over a 12 year period and matched for age, gender and years of end stage renal failure. After a mean follow-up period of 37 months the two groups were statistically indistinguishable in terms of mortality, kidney transplantation-censored technique survival, median death-censored technique survival, the number of patients switched permanently to hemodialysis due to technique failure and the rate of peritonitis. On Cox regression (multivariate) analysis, only the baseline serum albumin level was a significant and independent risk factor of death-censored technique failure. Our study found no difference in long term outcome of peritoneal dialysis therapy in patients with polycystic kidney disease compared to a non-diabetic matched control group.

Race and sex: predictors of the severity of hyperparathyroidism in peritoneal dialysis patients.

BACKGROUND: Uraemic hyperparathyroidism remains a common clinical problem. Conversely, oversuppression of parathyroid hormone (PTH), particularly in diabetic patients on peritoneal dialysis, has been implicated in low bone turnover disease. Race may also be an important factor determining susceptibility to hyperparathyroidism and the different forms of renal osteodystrophy. These compounding factors that might influence the severity of hyperparathyroidism have been studied in US dialysis and predialysis populations. Dialysis-dependant Africans and Afro-Caribbeans (AC) are known to have higher circulating PTH concentrations than comparable Caucasians (C) but Indo-Asians (IA) living in temperate climates have not been studied. METHODS: We performed a cross-sectional study of all patients undergoing peritoneal dialysis at St Bartholomew's and The Royal London Hospital on 1 May 2000. The highest historical recorded PTH was recorded with concurrent biochemical and demographic details. Regression models were used for the analysis of covariance and separate manova was performed incorporating the factors that were shown on univariate analysis to be significant. RESULTS: The current study confirmed that in 50 AC patients on peritoneal dialysis, the mean (+/- SEM) peak PTH concentration (93.9 +/- 9.3 pmol/L) was higher than in 148 C (56.7 +/- 4.3 pmol/L) and 67 IA (60.2 +/- 5.7 pmol/L), P < 0.0001 and P < 0.002, respectively. This is despite there being no significant difference in serum calcium concentrations and AC having a lower serum phosphate concentration at the time of peak hyperparathyroidism. There was no significant difference in mean peak PTH concentration between C and IA. Females were also found to have higher peak PTH concentrations, but the presence of diabetes did not influence the peak PTH concentration in this study. CONCLUSION: Although we have demonstrated that patients of African (but not Asian) descent undergoing peritoneal dialysis have more severe hyperparathyroidism than Caucasians, other studies suggest that Afro-Americans develop low bone turnover at higher PTH. This would suggest that PTH values should be interpreted with care and that bone biopsies to determine histology remain important. It may emerge that there are different optimal PTH concentrations according to race.

Novel applications of recombinant erythropoietin.

Recombinant erythropoietin (EPO) was introduced into clinical practice after the identification of EPO as the major haemopoietic growth factor determining survival and maturation of erythroid precursors. Advances in our understanding of the novel sites of action of EPO in the vasculature, brain, heart and kidney have opened new avenues of therapeutic potential for EPO, and have led to an increased understanding of the biological roles of EPO and its mechanisms of cell protection.

Mechanisms of disease: Cell death in acute renal failure and emerging evidence for a protective role of erythropoietin.

Acute renal failure--characterized by a sudden loss of the ability of the kidneys to excrete nitrogenous waste, and to maintain electrolyte homeostasis and fluid balance--is a frequently encountered clinical problem, particularly in the intensive care unit. Unfortunately, advances in supportive interventions have done little to reduce the high mortality associated with this condition. Might erythropoietin (EPO) have utility as a therapeutic agent in acute renal failure? This hormone mediates anti-apoptotic effects in the bone marrow, facilitating maturation and differentiation of erythroid progenitors. New evidence indicates that EPO also exerts anti-apoptotic effects in the brain, heart and vasculature, which can limit the degree of organ damage. Here, we review the emerging biological role of EPO in the kidney and the pathophysiology of ischemia-reperfusion injury in an attempt to understand the therapeutic potential of EPO in acute renal failure.

Homocysteine induced impairment of nitric oxide-dependent vasorelaxation is reversible by the superoxide dismutase mimetic TEMPOL.

BACKGROUND: Elevated plasma homocysteine concentrations in renal patients are associated with accelerated cardiovascular disease. The mechanism(s) by which homocysteine acts remains unclear however, evidence implicates a role involving endothelial dysfunction. METHODS: Rat femoral arteries after acute or 4-h pre-incubation with racemic D,L-homocysteine (100 microM) were mounted on a myograph, pre-constricted with phenylephrine (10 microM) and responses to acetylcholine-dependent vasorelaxation examined. The incubations were repeated in the presence of indomethacin (10 microM), omega-nitro-L-arginine methyl ester (100 microM), L-arginine (100 microM), tetrahydrobiopterin (1 microM), catalase (1200 U/ml), ebselen, a peroxynitrite chelator (20 microM) and TEMPOL, a superoxide dismutase mimetic (1 mM). Results are shown as means+/-standard error, expressed as per cent relaxation to acetylcholine added (nmol/l). RESULTS: Increasing concentrations of homocysteine had no affect when added directly to basally relaxed or pre-constricted freshly isolated vessels. However, 4-h pre-incubation with or without homocysteine significantly shifted the acetylcholine EC(50) (EC(50) was defined as the concentration of acetylcholine that caused relaxation of the phenylephrine contracted tissue by 50%), control((4 h)) = 74.7 nmol/l+/-10.5 vs 100 microM D,L-homocysteine((4 h)) = 159.9 nmol/l+/-20.6; P<0.05) without affecting maximal relaxation. Response to endothelial independent relaxation was unaffected. Indomethacin, indomethacin and omega-nitro-L-arginine methyl ester, l-arginine and tetrahydrobiopterin, catalase and ebselen had no effect on the EC(50) in homocysteine-exposed arteries. However, TEMPOL normalized vasorelaxation in homocysteine-treated arteries (75.2 nmol/l+/-14.6) but had no effect on the 4-h control group. Moreover, washing TEMPOL from the treated vessels restored endothelial dysfunction in D,L-homocysteine-treated vessels (163.9 nmol/l+/-34.1). CONCLUSIONS: We conclude that homocysteine causes endothelial dysfunction by up-regulating a potential superoxide generating system resulting in reduced nitric oxide bio-availability.

Structural remodeling of resistance arteries in uremic hypertension.

BACKGROUND: Structural remodeling of the resistance vasculature is present in many forms of human and experimental hypertension. In particular, an increase in the ratio of wall thickness to lumen diameter develops, and might in itself maintain hypertension by increasing vascular resistance. Because uremia is associated with raised peripheral resistance, hypertension, and histologic changes suggestive of vascular remodeling, we sought to formally examine the structural and mechanical (elastic) properties of isolated pressurized resistance arteries in uremic hypertension. METHODS: Cremaster, cerebral and mesenteric arteries from subtotally nephrectomised Wistar-Kyoto rats, normotensive control Wistar-Kyoto rats, and spontaneously hypertensive rats were mounted on a pressure myograph and relaxed in calcium-free buffer. Wall thickness and lumen diameter were measured at increasing lumen pressures from 10 to 200 mm Hg, and from this wall:lumen ratio, wall cross-sectional area, and an index of elasticity were derived. RESULTS: In uremic hypertensive animals increased wall:lumen ratio and decreased lumen diameter was seen in cremaster and mesenteric arteries, although no significant changes were observed in cerebral arteries, compared to normotensive controls. In spontaneously hypertensive animals increased wall thickness and wall:lumen ratio was seen in cerebral and mesenteric arteries, decreased lumen diameter in cremaster and mesenteric arteries, and increased wall cross-sectional area in cerebral arteries, compared to normotensive controls. Elasticity of the arterial wall in uremic and spontaneously hypertensive animals did not differ from normotensive controls. CONCLUSION: Cremaster and mesenteric resistance arteries undergo predominantly eutrophic inward remodeling in uremic hypertension, broadly similar to that seen in spontaneous hypertension.

The myogenic response in uremic hypertension.

BACKGROUND: The constriction of resistance arteries in response to an increase in transmural pressure, the myogenic response, is thought to be an important determinant of peripheral vascular resistance and therefore of arterial blood pressure. Since raised peripheral resistance is known to occur in uremic hypertension, abnormal myogenic constriction might be responsible. We sought to assess the myogenic response of resistance arteries from the subtotal nephrectomy rat model of uremic hypertension. METHODS: Uremic Wistar-Kyoto (WKYU) rats, and sham-operated normotensive (WKYC) and spontaneously hypertensive (SHRC) controls were studied in parallel. Skeletal muscle arteries were mounted on a pressure myograph and allowed to develop myogenic constriction. The active internal diameter was measured at increasing lumen pressures from 20 to 200 mm Hg. Vascular smooth muscle then was relaxed in a calcium free solution containing nitroprusside, and the passive internal diameter measured at the same pressure steps. The ratio of active to passive diameter at any given pressure was used to assess the myogenic response. RESULTS: Myogenic constriction was not increased in either WKYU or SHRC compared to WKYC at pressures up to 180 mm Hg. CONCLUSIONS: Increased myogenic tone is not the cause of uremic hypertension.

Cardiac troponin T and creatine kinase MB content in skeletal muscle of the uremic rat.

BACKGROUND: The assertion that creatine kinase MB (CK-MB) and the developmental isoforms of cardiac troponin T (cTnT) are expressed by skeletal muscle in some clinical settings is an extrapolation from nonuremic rodent studies. We studied the content of CK-MB and cTnT in skeletal muscle of the renal-insufficient rat. METHODS: Skeletal muscles (gastrocnemius) were collected from both five-sixths nephrectomized rats (n = 11) and sham-operated controls (n = 11). cTnT content was analyzed by Elecsys (Roche), immunoblotting, and immunohistochemistry with antibodies M7 and M11-7 (Roche). CK isoenzymes were analyzed electrophoretically. RESULTS: Trace concentrations of cTnT were detected in some of the skeletal muscle samples [controls (3 of 11) and uremic rats (1 of 11)] at concentrations <0.01% of that detected in heart. By contrast, positive staining appeared in both groups with M11-7 by immunoblotting and immunohistochemistry. No immunoreactivity was detected in skeletal muscle using M7 in the immunoblot format, although immunoreactivity was detected by immunohistochemistry in all samples. The median percentages of CK-MB were 6.0% and 4.1% for the skeletal muscle from control and uremic rats, respectively. CONCLUSION: The detection of cTnT and CK-MB in skeletal muscle does not differ for uremic rats compared with sham-operated controls. cTnT isoforms detected by qualitative methods are not detected with the cTnT immunoassay. Observations with rodents should not necessarily be extrapolated to humans.