Subclinical Injury Research Articles

A direct comparison of non-invasive blood markers of liver fibrosis as associates of cardiovascular risk. The Athens cardiometabolic registry

Abstract Background Non-alcoholic fatty liver disease (NAFLD) is associated with increased risk of cardiovascular disease (CVD). This risk increases further with advanced liver fibrosis. To date, non-invasive tests (NIT) for liver fibrosis risk have not been established as diagnostic or prognostic biomarkers of CVD. Purpose The purpose of the study was to compare NIT of liver fibrosis with respect to their associations with vascular injury and cardiovascular (CV) events. Methods Individuals without clinically overt CVD and patients with CVD (total n=1,692), consecutively recruited in the Athens Cardiometabolic Registry, were analysed in this study. We retrospectively evaluated the association of NIT (i.e. Fibrosis-4 index (FIB-4), NAFLD Fibrosis score (NFS), BARD score) with indices of subclinical arterial injury (i.e. carotid wall thickness and atherosclerotic plaque for subclinical atherosclerosis, pulse wave velocity (PWV), for arterial stiffness) and the incidence of CV events. Patients were followed-up for CV events for a median time of 39 months. Results Elevated FIB-4 (>2.67) was associated with vascular damage [adjusted odds ratio (aOR)=2.97, 95% confidence intervals (CI)= 1.71-5.78 for maximal wall thickness (maxWT) and aOR=4.58, 95% CI=2.44-8.60 for PWV] across the entire study population, including those with or without clinically overt CVD. NFS> 0.675 showed similar associations but lacked significant correlation with arterial stiffness in coronary artery disease patients. FIB-4> 2.67 and BARD> 2 were associated with increased risk of the composite endpoint of CV death, acute myocardial infarction, or coronary revascularization [adjusted hazard ratio, (aHR)=2.00, 95% CI= 1.12-3.55 and aHR=3.36, 95% CI=1.03-11.0 for FIB-4 (n=842) and BARD (n=887) respectively]. FIB-4 and BARD as continuous variables confered incremental prognostic value compared to European Systematic Coronary Risk Evaluation 2 (SCORE2). Conclusion In a population with a wide range of cardiometabolic risk, FIB-4 was associated with both markers of atherosclerotic disease and arterial stiffness as well as with adverse CV events. These data support further investigation of FIB-4 as a biomarker of CV risk.

The impact of intravenous agitated saline contrast echocardiography on subclinical neuronal injury determined by neuron-specific enolase

Abstract Introduction Patent foramen ovale (PFO) is a congenital defect in interatrial septum with 25-30% prevelance. Diagnostic methods include transesophageal echocardiography (TEE), magnetic resonance imaging (MRI), and computed tomography (CT), but transthoracic echocardiography (TTE) is the primary diagnostic method. TTE involves the injection of agitated saline intravenously, and the passage of microbubbles from the right atrium to the left atrium is monitored. The reliability of this method has been questioned in various case series, with a low incidence (0.062%) of generally transient, mild clinical cerebrovascular events reported. Silent cerebral ischemia (SSI) is asymptomatic, radiographically identified acute neuronal damage. SSI is associated with an increased risk of stroke, dementia, and cognitive impairment. Neuron-specific enolase (NSE) with high specificity for neural cells can be used to detect SSI. While symptomatic patients have been evaluated in reported case series, there is no study determining the relationship and reliability of intravenous agitated saline echocardiography (SCE) with SSI. This study aims to determine whether TTE, performed on patients with PFO without neurological history and a healthy control group, can cause SSI by evaluating NSE with SCE. Materials and methods 52 patients undergoing SCE for color flow through interatrial septum or prominent interatrial septal aneurysm and are diagnosed with PFO were included. Additionally, 51 control patients without detected PFO or ASA were also included. Serum samples were taken from the patients before and 12 hours after the SCE test, and NSE levels were measured by the ELISA method. Results The mean baseline NSE level was 12.7 ng/mL with a median of 5.3 ng/mL in the PFO group, and in the control group, the mean was 9.1 ng/mL with a median of 6.4 ng/mL. At 12 hours, the mean NSE levels in the PFO group were 9.8 ng/mL with a median of 6.6 ng/mL, and in the control group, the mean was 12.7 ng/mL with a median of 5.8 ng/mL. The increase in NSE levels after SCE, was not statistically significant when comparing the PFO and control groups (p=0.77). No relationship was found between age, presence of interatrial septal aneurysm, presence of mitral annulus calcification, the number of bubbles passed in the SCE test, and the basal and 12-hour change in NSE levels (p=0.926). NSE level changes were found to be correlated between groups (p<0.001). Conclusion This study is the first to evaluate whether SCE causes subclinical neuronal damage. Our study suggests that when evaluated with NSE, the SCE method is safe and does not cause subclinical neuronal damage in patients without underlying neuronal susceptibility.results

Clinical and subclinical acute brain injury caused by invasive cardiovascular procedures.

Over the past 50 years, the number and invasiveness of percutaneous cardiovascular procedures globally have increased substantially. However, cardiovascular interventions are inherently associated with a risk of acute brain injury, both periprocedurally and postprocedurally, which impairs medical outcomes and increases health-care costs. Current international clinical guidelines generally do not cover the area of acute brain injury related to cardiovascular invasive procedures. In this international Consensus Statement, we compile the available knowledge (including data on prevalence, pathophysiology, risk factors, clinical presentation and management) to formulate consensus recommendations on the prevention, diagnosis and treatment of acute brain injury caused by cardiovascular interventions. We also identify knowledge gaps and possible future directions in clinical research into acute brain injury related to cardiovascular interventions.

Lung-Kidney Axis in Cystic Fibrosis: Early Urinary Markers of Kidney Injury Correlate with Neutrophil Activation and Worse Lung Function.

Adult people with cystic fibrosis (PwCF) have a higher risk of end-stage kidney disease than the general population. The nature and mechanism of kidney disease in CF are unknown. This study quantifies urinary kidney injury markers and examines the hypothesis that neutrophil activation and lung infection are associated with early kidney injury in CF. Urinary total protein, albumin, and markers of kidney injury and neutrophil activation, normalized to creatinine, as well as urinary immune cells, were quantified in CF (n = 48) and healthy (n = 33) cohorts. Infection burden and chronicity were defined by sputum culture and serum titers of anti-bacterial antibodies. PwCF had increased urinary protein levels, consisting of low-molecular-weight tubular injury markers, independent of glomerular filtration rate (eGFR). This finding suggests subclinical renal injury processes. Urinary analysis of the CF cohort identified different associations of urinary injury markers with aminoglycoside exposure, lung function, and neutrophil activation. High urinary KIM-1 levels and increased prevalence of neutrophils among urine immune cells correlated with decreased lung function in PwCF. The relationship between tubular injury and decreased lung function was most prominent in patients harboring chronic Pseudomonas aeruginosa infection. Increased urinary tubular injury markers in PwCF suggest early subclinical renal injury not readily detected by eGFR. The strong association of high urinary KIM-1 and neutrophils with diminished lung function and high Pseudomonas aeruginosa burden suggests that pulmonary disease may contribute to renal injury in CF.

Associations of Hypertension and Orthostatic Hypotension with Subclinical Cardiovascular Disease

Abstract Background Orthostatic hypotension is associated with cardiovascular disease. It remains unclear if low standing blood pressure or high seated blood pressure is responsible for this association. We compared associations of orthostatic hypotension and hypertension with high-sensitivity cardiac troponin I and N-terminal pro-B-type natriuretic peptide. Methods We performed a secondary analysis of the Study to Understand Fall Reduction and Vitamin D in You (STURDY), a randomized controlled trial funded by the National Institute on Aging, between July 2015 and May 2019. Participants were community-dwelling adults, 70 years or older. Blood tests for high-sensitivity cardiac troponin I and N-terminal pro-B-type natriuretic peptide were drawn at visits concurrent with blood pressure measurements. Secondary analysis occurred in 2023. We determined associations between blood pressure phenotypes and cardiac biomarkers. Results Of 674 participants (mean age 76.5 ± 5.4 years, 43% female, 17.2% Black race), 29.1% had prior cardiovascular disease. Participants with seated hypertension had 10.1% greater high-sensitivity cardiac troponin I (95% CI = 3.8, 16.9) and 11.0% greater N-terminal pro-B-type natriuretic peptide (4.0, 18.6) than those without seated hypertension. Participants with standing hypertension had 8.6% (2.7, 14.9) greater high-sensitivity cardiac troponin I and 11.8% greater N-terminal pro-B-type natriuretic peptide (5.1, 18.9) than those without standing hypertension. Hypotensive phenotypes were not associated with either biomarker. Conclusions Both seated and standing hypertension were associated with greater high-sensitivity cardiac troponin I and N-terminal pro-B-type natriuretic peptide, but hypotensive phenotypes were not. Hypoperfusion may not be the principal mechanism behind subclinical cardiac injury among older adults with orthostatic hypotension.

The transcriptional response of cortical neurons to concussion reveals divergent fates after injury.

Traumatic brain injury (TBI) is a risk factor for neurodegeneration, however little is known about how different neuron types respond to this kind of injury. In this study, we follow neuronal populations over several months after a single mild TBI (mTBI) to assess long ranging consequences of injury at the level of single, transcriptionally defined neuronal classes. We find that the stress responsive Activating Transcription Factor 3 (ATF3) defines a population of cortical neurons after mTBI. We show that neurons that activate ATF3 upregulate stress-related genes while repressing many genes, including commonly used markers for these cell types. Using an inducible reporter linked to ATF3, we genetically mark damaged cells to track them over time. Notably, we find that a population in layer V undergoes cell death acutely after injury, while another in layer II/III survives long term and retains the ability to fire action potentials. To investigate the mechanism controlling layer V neuron death, we genetically silenced candidate stress response pathways. We found that the axon injury responsive kinase MAP3K12, also known as dual leucine zipper kinase (DLK), is required for the layer V neuron death. This work provides a rationale for targeting the DLK signaling pathway as a therapeutic intervention for traumatic brain injury. Beyond this, our novel approach to track neurons after a mild, subclinical injury can inform our understanding of neuronal susceptibility to repeated impacts.

Incremental value of blood-based markers of liver fibrosis in cardiovascular risk stratification.

Non-alcoholic fatty liver disease (NAFLD) with advanced liver fibrosis is associated with cardiovascular disease (CVD). To examine if markers of vascular injury mediate the link between liver fibrosis non-invasive tests (LFNITs) and CVD events, and to compare the incremental predictive value of LFNITs over established CVD risk scores. Consecutively recruited individuals (n=1,692) with or without clinically overt coronary artery disease (CAD) from the Athens Cardiometabolic Cohort, were analysed. Fibrosis-4 index (FIB-4), NAFLD Fibrosis score (NFS), and BARD score were evaluated for direct and indirect associations with indices of subclinical arterial injury including carotid maximal wall thickness (maxWT) and pulse wave velocity (PWV) and with a composite of major adverse cardiovascular events (MACE) that consisted of cardiac death, acute myocardial infarction, or coronary revascularization (39-month median follow-up). FIB-4 was the only LFNIT which consistently associated with multiple markers of vascular injury, irrespective of CAD presence and after controlling for traditional risk factors, surrogates of insulin resistance or obesity (adjusted p<0.05 for all). FIB-4 also independently associated with CAD presence (adjusted OR 6.55 (3.48-12.3), p<0.001). Increased FIB-4>2.67 was incrementally associated with increased risk for MACE (OR (95% CI) 2.00(1.12, 3.55), deltaAUC (95% CI) 0.014(0.002-0.026)). These associations were mediated by maxWT rather than PWV. Only FIB-4 (>3.25) was independently and incrementally associated with all-cause mortality (adjusted p<0.05). In a cardio-metabolically diverse population, the incremental associations of LFNITs with CVD outcomes were mediated by atherosclerotic burden rather than arterial stiffening. FIB-4 consistently demonstrated associations with all study endpoints. These findings provide mechanistic insights and support the clinical applicability of FIB-4 in CVD prevention.

Abstract 67: Cardiac Biomarkers in Pregnancies with and without Hypertension – a Case Control Study

Background: There is ongoing debate about hypertension (HTN) management during pregnancy with a range of recommended targets. Whether HTN is related to subclinical cardiac injury during pregnancy is unclear. Objective: To determine the relationship between HTN with subclinical cardiac injury during and immediately after pregnancy, measured via high sensitivity troponin I (hs-cTnI). Methods: In a prospective, 1:1 case-control design, we enrolled pregnant women with and without HTN between 24-32 weeks gestation (2019-2022). HTN cases were defined by a clinician diagnosis at &lt;20 weeks gestation or a baseline blood pressure (BP) ≥140/90 mm Hg. The control group (no HTN) had a systolic BP &lt;120 mm Hg. Hs-cTnI was collected at baseline, the delivery admission, and postpartum day 1. We used mixed effects tobit models to compare the groups on log-transformed hs-cTnI, adjusted for age and BMI. Results: The mean age of the HTN group (N=38) was 33.6 (SD 5.4) vs. 30.3 (5.8) years for the no HTN group (N=38). Mean (SD) baseline BP was 131.1 (14.7)/88.2 (13.5) for the HTN group and 110.1 (7.2)/70.9 (8.2) mm Hg for no HTN. At baseline, hs-cTnI was 1.3 (0.2) in the HTN group and 0.6 (0.1) ng/L in the no HTN group ( Figure ). Compared to baseline, hs-cTnI increased 89.1% (95% CI: 27.3-180.8%) and 322% (192-511%) for the HTN group, and 101.6% (41.0-188.3%) and 165.2% (88.6-272.9%) for no HTN at delivery and postpartum, respectively. Across all time points, the HTN group had a higher hs-cTnI of 1.05 (0.07-2.03) ng/L compared to the control group. Discussion: HTN was associated with subclinical cardiac injury during and immediately postpartum. These findings support recommendations to control HTN throughout pregnancy to reduce cardiac injury.

Changes in Mitochondrial Function and Cell Death Patterns in Peripheral Blood Mononuclear Cells during Trastuzumab Treatment Following Doxorubicin Chemotherapy.

Trastuzumab, a monoclonal antibody which works against human epidermal growth factor receptor 2 (HER2), possibly causes cardiotoxicity through mitochondrial dysfunction. The usefulness of isolated peripheral blood mononuclear cells (PBMCs) in the assessment of trastuzumab-induced cardiotoxicity remains uncertain. This study aimed to determine the temporal changes in mitochondrial function, oxidative stress, and cell death in the isolated PBMCs of HER2-positive breast cancer patients during breast cancer treatment and to compare the changes with HER2-negative breast cancer patients who did not receive trastuzumab therapy. Eighteen newly diagnosed HER2-positive breast cancer women who received sequential doxorubicin and trastuzumab were consecutively recruited. Age- and gender-matched controls with HER2-negative breast cancer were selected. Echocardiography was carried out, and blood samples for the study of cardiac biomarkers and PBMCs were collected periodically during treatment. Only one patient in our cohort developed asymptomatic left ventricular dysfunction during trastuzumab treatment. However, trastuzumab following doxorubicin aggravated subclinical cardiac injury, determined by cardiac troponin and echocardiography. Cellular and mitochondrial oxidative stress in isolated PBMCs remained unchanged throughout breast cancer treatment. Regarding mitochondrial respiration, the maximal respiration and spare respiration capacity was significantly increased in controls after doxorubicin treatment but not in patients who received trastuzumab therapy. Moreover, the percentage of apoptosis and necroptosis in isolated PBMCs was dramatically decreased in the control, compared to patients with trastuzumab treatment. In conclusion, trastuzumab caused subtle myocardial injury and impaired mitochondrial respiration and cell viability in isolated PBMCs.

Perinatal asphyxia leads to acute kidney damage and increased renal susceptibility in adulthood.

Perinatal asphyxia (PA) poses a significant threat to multiple organs, particularly the kidneys. Diagnosing PA-associated kidney injury remains challenging, and treatment options are inadequate. Furthermore, there is a lack of long-term follow-up data regarding the renal implications of PA. In this study, 7-day-old male Wistar rats were exposed to PA using a gas mixture (4% O2; 20% CO2 in N2 for 15 min) to investigate molecular pathways linked to renal tubular damage, hypoxia, angiogenesis, heat shock response, inflammation, and fibrosis in the kidney. In a second experiment, adult rats with a history of PA were subjected to moderate renal ischemia-reperfusion (IR) injury to test the hypothesis that PA exacerbates renal susceptibility. Our results revealed an increased gene expression of renal injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), hypoxic and heat shock factors (hypoxia-inducible factor-1α, heat shock factor-1, and heat shock protein-27), proinflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1), and fibrotic markers (transforming growth factor-β, connective tissue growth factor, and fibronectin) promptly after PA. Moreover, a machine learning model was identified through random forest analysis, demonstrating an impressive classification accuracy (95.5%) for PA. Post-PA rats showed exacerbated functional decline and tubular injury and more intense hypoxic, heat shock, proinflammatory, and profibrotic response after renal IR injury compared with controls. In conclusion, PA leads to subclinical kidney injury, which may increase the susceptibility to subsequent renal damage later in life. In addition, the parameters identified through random forest analysis provide a robust foundation for future biomarker research in the context of PA.NEW & NOTEWORTHY This article demonstrates that perinatal asphyxia leads to subclinical kidney injury that permanently increases renal susceptibility to subsequent ischemic injury. We identified major molecular pathways involved in perinatal asphyxia-induced renal complications, highlighting potential targets of therapeutic approaches. In addition, random forest analysis revealed a model that classifies perinatal asphyxia with 95.5% accuracy that may provide a strong foundation for further biomarker research. These findings underscore the importance of multiorgan follow-up for perinatal asphyxia-affected patients.

#2508 Urinary [TIMP-2]*[IGFBP7] as the ultimate potential biomarker of subclinical Acute Kidney Injury after kidney surgery: a multicentric study

Abstract Background and Aims Nephron-sparing surgery (NSS) represents the preferred technique to treat localized renal lesions and better preserve kidney function. Still it is not exempted from the risk of postoperative acute kidney injury (AKI) to happen, with a reported incidence ranging from 5.5% to 34%. Patients experiencing postoperative AKI, either clinical or subclinical, are more susceptible to chronic kidney disease (CKD). The latter represents a difficult-to-identify subpopulation characterized by tubular damage biomarker's rising without changes in serum creatinine (scr), that after surgery is less likely to be included among the ones deserving a closer follow-up, but actually needing one, as this subpopulation's long term outcomes slightly differ from individuals recovered from clinical postoperative AKI. This is the reason why a standardized tool for early identification of patients suffering from subclinical AKI is required, to allow a customized postoperative surveillance, promote strategies of nephroprotection and reduce the incidence of CKD and end-stage kidney disease. Recent evidences show that by exclusively following outdated consensus criteria for AKI (changes in scr, urine output), 15-20% of patients undergoing through acute tubular injury and suffering from its adverse outcomes are likely to be missed. Only recently, FDA approved NephroCheck® biomarker, which combines two urinary markers, tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7). It represents the first effective test for AKI's risk assessment. In a previous monocentric study [1], our group presented NephroCheck® as a biomarker able to efficiently identify subclinical AKI and predict long-term eGFR decline after robotic NSS. In order to confirm our previous results, we recruited a larger cohort and performed a multicentric study. Method We prospectively observed all patients scheduled for robotic NSS in suspected localized renal cell carcinoma in two different Centers from 2016 to 2018. Samples were collected preoperatively and postoperatively (4 h, 10 h, 24 h, 48 h), while kidney function was re-assessed up to 24 months. A multivariate logistic analysis was performed to evaluate the association between different parameters and eGFR decline at 24 months. Results A total number of 131 patients were included; 55/131 (42%) developed clinical AKI. A significant association between NephroCheck® at 4 h and clinical AKI, as stated by KDIGO Guidelines, is confirmed (p = 0.0055). In the overall cohort, the multivariate analysis shows that only clinical AKI appears as an independent factor for predicting eGFR decline at 24 months (p &amp;lt; 0.0003), while postoperative NephroCheck® alone did not (p = 0.92). Based on NephroCheck® measured 4 h after surgery and clinical AKI, patients can be divided in four groups with significantly different eGFR at 24 months (p = 0.0003) (Fig. 1). Not considering patients who experienced clinical AKI, the independent role of different parameters in predicting severe eGFR decline at 24 months was evaluated by multivariate logistic analysis; in this subgroup of patients, NephroCheck® evaluated at 4 h postoperatively appears as an independent factor able to predict a severe eGFR decline at 24 months (OR 3.76, p = 0.02) (Fig. 2). Conclusion NephroCheck®, in combination with scr, represents an accurate and easy-to-use test, likely able to early identify long-term eGFR decline in patients undergoing NSS surgery that don't develop a clinically evident postoperative AKI (subclinical AKI), making it possible to identify those in need of closer-up surveillance, reducing the incidence and progression of CKD. Still, clinical postoperative AKI is confirmed as the most important prognostic parameter and predictive factor for eGFR decline in the long-term. Further studies are needed, particularly focusing on application of NephroCheck® in setting other than NSS.

#2356 Renal function in pediatric general population from a hot-spot of chronic kidney disease of unknown etiology: a cross-sectional study in Guatemala

Abstract Background and Aims Chronic kidney disease of unknown etiology incidence has been exponentially increasing in Central America. Subclinical kidney injury begins early in life and leads to a higher-than-expected prevalence of chronic kidney disease in adolescents and children. The pediatric population has not had a specific approach to the community with this type of kidney disease and very little data is known in Guatemala. We aim to estimate the glomerular filtration rate (eGFR) using two serum biomarkers (creatinine and cystatin C) and applying the Under 25 equation. Inflammatory and urine biomarkers (Urinary Neutrophil Gelatinase-Associated Lipocalin, albumin/creatinine ratio, and urine dipstick) were determined. Method During 2019, we performed a Cross-Sectional, population-based screening study in children and adolescents between 7-17 years of age, from local schools in a highly CKDu incident rural community in the Pacific Coast of Guatemala. Children with a diagnosis of kidney disease, hepatic and cardiac failure were excluded in the study. Subjects were chosen randomly from 10 different schools and renal function markers were analyzed by sex and age. Results Out of 97 participants of La Democracia, the median age was 11 years and 57% were male. Eighty-seven percent (81/97) of participants had an eGFR between 60 and 90 mL/min/1.73 m2 (Table 1). When comparing eGFR by sex, 63% (51/81) of males and 37% of females had an eGFR between 60 and 90 mL/min/1.73 m2. The difference was statistically significant (p 0.05). When comparing eGFR by age group, the scholar group had an eGFR of 82.7 mL/min/1.73 m2 (SD 7.9), pre-scholars eGFR of 82.2 mL/min/1.73 m2 (SD 6.3), and adolescents of 81.5 (SD 8.1) with no statistically significant difference (p 0.83). Among the twenty-four percent of participants who presented urine dipstick abnormalities, leukocyturia represented 23% (22/97). Twenty-two percent (21/97) of participants had albuminuria more than 0.2 mg/mg and the urinary NGAL/creatinine ratio was higher in females. Conclusion

#3031 Cell free DNA methylation analysis reveals cerebral cell death during hemodialysis

Abstract Background and Aims Patients treated with hemodialysis demonstrate a significant cognitive decline, likely explained by cerebrovascular disease and hypoperfusion. Despite the significant morbidity and mortality associated, no method exists to detect subclinical cerebral injury. This prevents early diagnosis and precludes mitigation strategies. Following cell death, cell free DNA (cfDNA) is released into the peripheral blood. Since DNA methylation is cell-type specific, cfDNA methylation analysis can reveal the cellular origin of cfDNA and cell-type specific changes in cell death. Here, we evaluate if subclinical cerebral ischemia during hemodialysis is detectable through cfDNA methylome analyses. Method Chronic hemodialysis patients with a high cardiovascular risk profile were studied during a single hemodialysis session. Brain tissue oxygenation was measured continuously by near-infrared spectroscopy using two sensors on the forehead. Plasma samples were taken immediately before and after hemodialysis. cfDNA was extracted from the blood plasma and converted into whole genome bisulfite sequencing libraries using tailored protocols. Next, in-solution target enrichment was applied to analyze 4,989 genomic loci, selected for showing specific methylation in one of 15 tissues or cell types. After sequencing, reads were aligned to the hg38 reference genome using BisMark, and deconvoluted using EMeth-Laplace to disclose the relative contribution of the 15 selected tissues or cell types. Results 41 hemodialysis patients participated in the study, with a mean age of 75 ± 8 years (46% male). The median ultrafiltration volume was 2.3 L (IQR 1.6-3.0 L). After targeted bisulfite sequencing and QC-based filtering, 35 patients remained for cfDNA methylation analysis. In 30 patients, cfDNA concentrations were increased following dialysis. Deconvolution analysis revealed a relative increase in granulocyte-derived cfDNA from 36.7% to 55% before versus after dialysis. Consequently, for most other tissues and cell types we observed a relative decrease in their cfDNA contribution. Interestingly, a subset of patients (n = 13) showed an increase in neuron-derived cfDNA from 0% to 0.26%, while no neuron-derived cfDNA was detectable in the remaining 22 patients. Near-infrared spectroscopy showed a significantly reduced cerebral oxygenation in these 13 patients (P &amp;lt; 0.0001) at 2 separate time windows during hemodialysis treatment (at 20-60 and 207-251 minutes into the session) (Fig.). Conclusion In patients with a high cardiovascular risk profile, cfDNA in peripheral blood increased after hemodialysis. cfDNA methylome analyses suggested this to be due to an increased contribution of granulocytes, indicating elevated levels of NETosis. In addition, we observed more neuron-derived cfDNA after dialysis in about 1 in 3 patients. This was most apparent in patients with a reduced brain tissue oxygenation during hemodialysis. Together, these findings suggest that hypoperfusion during hemodialysis can evoke neuronal cell death, detectable through methylome analyses of cfDNA isolated from a minimally invasively obtainable bodily fluid. Follow-up studies are ongoing to assess if this translates to progressive cognitive impairment.

#2929 Plasma levels of brain injury markers NfL and GFAP are independently predicted by glomerular filtration rate in patients with chronic kidney disease

Abstract Background and Aims Patients with chronic kidney disease (CKD) have a high prevalence of cerebrovascular disease and cognitive impairment. The aim of the present study was to measure brain injury biomarkers in plasma in a cohort of patients with CKD stages 3 and 4, without a diagnosis of cerebrovascular disease, and to determine factors that independently predicted plasma levels of these biomarkers. Method This was a cross-sectional cohort study including 110 patients with CKD stages 3 and 4, and 55 healthy, matched, controls, recruited from the outpatient clinic at Sahlgrenska university hospital, Gothenburg, Sweden. None of the included study subjects had a diagnosis of cerebrovascular disease, cognitive impairment, or any other neurological disease. Plasma concentrations of neurofilament light (NfL), glial fibrillary acidic protein (GFAP) and phosphorylated Tau (p-Tau231) were analyzed with ultrasensitive single molecule array (SIMOA). Measured GFR (mGFR) was determined by plasma clearance of ⁵¹Cr-EDTA. Values are medians [interquartile range]. For linear regression analyses standardised beta coefficients (β) are presented. Results Characteristics of CKD patients and healthy controls are presented in Table 1. Plasma concentrations of NfL (37.5 [22.1-47.5] vs. 13.4 [10.5-16.7] ng/L, p &amp;lt; 0.001), p-Tau231 (25.7 [19.1-38.7] vs. 13.9 [10.5-16.3] ng/L, p &amp;lt; 0.001) and GFAP (190 [140-281] vs. 153 [116-211] ng/L, p = 0.001) were significantly elevated in patients with CKD vs. controls. In CKD patients, mGFR showed statistically significant inverse correlations to plasma concentrations of NfL, p-Tau231, and GFAP (Table 2). In multiple regression analyses, mGFR was associated with plasma levels of NfL (β = −0.457, p &amp;lt; 0.001) and GFAP (β = −0.322, p &amp;lt; 0.001) independently of diabetes, age, troponin-T, b-hemoglobin, and aortic pulse-wave velocity. However, mGFR was not significantly associated with plasma levels of p-Tau231 in a multiple regression analysis. Conclusion Plasma levels of brain injury markers NfL, p-Tau231 and GFAP were elevated in patients with CKD stages 3 and 4, without a history of cerebrovascular disease, compared to healthy controls. Furthermore, mGFR independently predicted plasma concentrations of NfL and GFAP in multiple regression models. These results raise the possibility that reduced GFR per se may be associated with subclinical brain injury.

Acute kidney injury causes subclinical renal and endothelial injury in female rats which predisposes to adverse outcomes of pregnancy

Clinical data report a 3-5-fold increase in adverse pregnancy outcomes, including preeclampsia and small for gestational age neonates, among women with a history of acute kidney injury (AKI) despite clinical recovery of renal function. We developed a rat model of pregnancy post-AKI: Sprague-Dawley (SD) rats undergo a bilateral renal-ischemia reperfusion and fully recover renal function as measured by creatinine clearance 30 days post-AKI. Rats are then mated and during pregnancy recovered AKI rats exhibit decreased creatinine clearance, increased uterine artery resistance, reduced plasma volume expansion, and decreased fetal growth compared to pregnant rats that received sham surgery. Healthy pregnancy demands robust renal and vascular adaptations. Whether subclinical renal and vascular injury persists following AKI in females, and whether this is exacerbated in pregnancy to drive the adverse pregnancy outcomes remains unknown. We tested the hypothesis that AKI results in subclinical renal and vascular injury that lingers into pregnancy. Female SD rats were randomized to 45-minute warm bilateral renal ischemia reperfusion or sham (N=6-8/grp) and renal and vascular injury assessments were made 30 days post-AKI/Sham (pre-pregnancy) or during pregnancy on gestational day 20 (GD20). Pre-pregnancy urine volume measured via metabolic cage was greater in post-AKI rats compared to sham (10±1 vs 21±2 ml/daily urine, p&lt;0.05). Urinary kidney injury marker-1 (KIM-1) measured via ELISA 30 days post-AKI/Sham was greater in pre-pregnant AKI rats compared to sham (9685±871 vs. 6399±198 pg/24 hrs urine, p&lt;0.05). Vascular function, measured via wire myography on 3rd order mesenteric arteries 30 days post-AKI/Sham, revealed decreased endothelial-dependent relaxation to acetylcholine (ACh) (concentration-response, 1 nM-10 μM, 2-way ANOVA, RM, *p&lt;0.05). GD20 urine volume (31±5 vs 19±2 ml/daily urine, p&lt;0.05) and KIM-1 levels (9125±673 vs 6380±297 pg/daily urine, p&lt;0.05) were elevated in post-AKI pregnant rats compared to sham pregnant rats. Mason Trichrome renal staining revealed greater glomerular fibrosis (0.93±0.08 vs 0.04±0.04, *p&lt;0.05) and tubular fibrosis (1.7±0.19 vs 0.0±0.0, *p&lt;0.05) in post-AKI pregnant rats vs. sham pregnant. Ultrastructural changes to the kidneys were evaluated via electron microscopy and further revealed extensive brush border loss, loss of mitochondrial structure and mitochondrial swelling in post-AKI pregnant rats compared to sham pregnant. Endothelial-dependent relaxation to ACh was also lower (2-way ANOVA, *p&lt;0.05), suggesting enhanced endothelial dysfunction in post-AKI pregnant rats vs. sham pregnant. Taken together, these data suggest that with a history of AKI, subclinical renal and vascular injury is present despite “recovery” of renal function using clinical definitions and may predispose to adverse pregnancy outcomes in pregnant women with a history of AKI. 1R01HL169576 4R00HL146948 1RO1DK134695 AHA967662. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cholinergic Stimulation Exerts Cardioprotective Effects and Alleviates Renal Inflammatory Responses after Acute Myocardial Infarction in Spontaneous Hypertensive Rats (SHRs).

In this investigation, we explored the effects of pharmacological cholinergic stimulation on cardiac function and renal inflammation following acute myocardial infarction (AMI) in spontaneously hypertensive rats (SHRs). Adult male SHRs were randomized into three experimental groups: sham-operated; AMI + Veh (infarcted, treated with vehicle); and AMI + PY (infarcted, treated with the cholinesterase inhibitor, pyridostigmine bromide (PY)-40 mg/kg, once daily for seven days). Rats were euthanized 7 or 30 days post-surgery. The clinical parameters were assessed on the day before euthanasia. Subsequent to euthanasia, blood samples were collected and renal tissues were harvested for histological and gene expression analyses aimed to evaluate inflammation and injury. Seven days post-surgery, the AMI + PY group demonstrated improvements in left ventricular diastolic function and autonomic regulation, and a reduction in renal macrophage infiltration compared to the AMI + Veh group. Furthermore, there was a notable downregulation in pro-inflammatory gene expression and an upregulation in anti-inflammatory gene expression. Analysis 30 days post-surgery showed that PY treatment had a sustained positive effect on renal gene expression, correlated with a decrease in biomarkers, indicative of subclinical kidney injury. Short-term cholinergic stimulation with PY provides both cardiac and renal protection by mitigating the inflammatory response after AMI.

T1 Mapping in Cardiovascular Magnetic Resonance-A Marker of Diffuse Myocardial Fibrosis in Patients Undergoing Hematopoietic Stem Cell Transplantation.

Introduction: Hematopoietic stem cell transplantation (HSCT) recipients are at increased risk of cardiovascular diseases. In our study, we aimed to find subclinical changes in myocardial tissue after HSCT with the help of cardiovascular magnetic resonance (CMR) tissue imaging techniques. Methods: The data of 44 patients undergoing autologous and allogeneic HSCT in the Hospital of Lithuanian University of Health Sciences Kaunas Clinics from October 2021 to February 2023 were analyzed. Bioethics approval for the prospective study was obtained (No BE-2-96). CMR was performed two times: before enrolling for the HSCT procedure (before starting mobilization chemotherapy for autologous HSCT and before starting the conditioning regimen for allogeneic HSCT) and 12 ± 1 months after HSCT. LV end-diastolic volume, LV end-systolic volume, LV mass and values indexed to body surface area (BSA), and LV ejection fraction were calculated. T1 and T2 mapping values were measured. Results: There was a statistically significant change in T1 mapping values. Before HSCT, mean T1 mapping was 1226.13 ± 39.74 ms, and after HSCT, it was 1248.70 ± 41.07 ms (p = 0.01). The other parameters did not differ significantly. Conclusions: Increases in T1 mapping values following HSCT can show the progress of diffuse myocardial fibrosis and may reflect subclinical injury. T2 mapping values remain the same and do not show edema and active inflammation processes at 12 months after HSCT.

Abstract P107: The DASH Diet Reduces Markers of Subclinical Cardiac Injury Over an 8-week Period: Results From an Individual Level Meta-Analysis

Introduction: The Dietary Approaches to Stop Hypertension (DASH) diet has previously been shown to reduce blood pressure and low-density lipoprotein (LDL) cholesterol as compared to the typical American diet. However, direct evidence linking the DASH diet with clinical markers of cardiovascular disease (CVD) is limited. Objectives: To perform a systematic review of trials examining the DASH diet and sensitive markers implicated in CVD: high-sensitivity cardiac troponin (hs-cTnl), N-terminal pro-brain natriuretic peptide (NT-proBNP), and high-sensitivity c-reactive protein (hs-CRP) to determine the effects of the DASH diet on subclinical cardiac injury. Methods: We systematically reviewed 1122 abstracts from PubMed, EMBASE, Web of Science, and the Cochrane Central Register of Controlled Trials (date range 1997 to 2022). We retrieved data from two trials (DASH and DASH-Sodium, using 8-week biomarker measurements), and combined data from the trials to determine subgroup effects. We used mixed effects tobit models to determine the effects of the DASH diet (versus an isocaloric typical American diet) on hs-cTnl, NT-proBNP, and hs-CRP. Effects were determined overall and in subgroups of age, sex, race, hypertension status, obesity, and smoking status. Results: Of the 632 participants included, mean age (SD) was 47 (10.5); 54% were female and 55% were Black. Compared to an isocaloric control diet, the DASH diet reduced hs-cTnl (β = -17.7%, 95% CI: -26.3, -8.1), NT-proBNP (β = -8.6%, 95% CI: -16.8, 0.4), and hs-CRP (β = -11.1%, 95% CI: -20.7, 0.2). Effects were consistent across demographic groups, although the DASH diet had a larger effect on hs-CRP among men ( P -interaction = 0.016) ( Figure ). Conclusion: The DASH diet reduced subclinical cardiac damage and inflammation over an 8-week period. These data provide evidence of the direct benefits of the DASH diet on cardiac health.

Serum Prestin and Otolin-1 Levels in Pilots of Helicopter-Based Emergency Medical Services: Potential Markers for Ear Injury.

Occupational noise exposure is a major public health concern, impacting a large workforce worldwide. In this study, we sought to evaluate the serum concentrations of prestin, a cochlear protein that diminishes following noise exposure, and otolin-1, a protein secreted into the bloodstream subsequent to inner ear damage, among three diverse professional categories, each exposed to varying degrees of noise. Helicopter emergency medical service (HEMS) pilots and construction workers were considered high-risk groups due to their elevated exposure to occupational noise, whereas office workers were regarded as a low-risk group, reflecting their comparatively minimal noise exposure. The study sample included 60 males, encompassing helicopter pilots, construction laborers, and office workers (n=20, each). Recruitment occurred during standard occupational health visits, with all participants presenting normal clinical audiograms. Serum levels of prestin and otolin-1 were measured in duplicate using commercially available immunoassays and compared across the three professional categories. HEMS pilots had the lowest mean serum prestin level at 211±27 pg/mL, followed by construction workers at 234±29 pg/mL, and office workers at 269±42 pg/mL (p<0.001, one-way analysis of variance), with all inter-group differences statistically significant (p<0.05, Tukey'spost hoctests). For otolin-1, HEMS pilots showed the highest mean at 216±20 pg/mL, with construction workers at 196±22 pg/mL, and office workers at 181±20 pg/mL (p<0.001, one-way analysis of variance). Statistically significant differences were found between HEMS pilots and both other groups for otolin-1 levels (p<0.05, Tukey'spost hoctests), but not between construction workers and office workers. Serum concentrations of prestin and otolin-1 may differ among healthy individuals according to their occupational noise exposure and have the potential to act as indicators of subclinical inner ear injury. To substantiate these preliminary observations, incorporating exposure assessment, especially via direct measurements of noise and vibration exposure, would markedly improve the reliability of our findings.

Cardiac Function in Pediatric Patients with MIS-C Using Speckle Tracking and Conventional Echocardiography: A Longitudinal, Single-Center Study.

Cardiovascular involvement in Multisystem Inflammatory Syndrome in Children (MIS-C), a potential consequence of coronavirus disease-2019 (COVID-19), is common. Conventional transthoracic echocardiography (TTE) provides primary data on the function of the left and right ventricles, while Speckle Tracking Echocardiography (STE) is more sensitive. This study aims to assess longitudinal cardiac function using STE in these patients. This longitudinal study was conducted from late 2021 to early 2022 at Imam Hossein Children's Hospital, Isfahan. Cardiac function was assessed by STE at the time of diagnosis and again two months later. Demographics, clinical characteristics, ECG interpretations, imaging studies, and serum cardiac marker levels were collected. Thirty-five pediatric patients with a mean age of 5.1 years (range: 4 months to 17 years) were included and prospectively followed. Twenty-nine of them, comprising 14 males (48.3%) and 15 females (51.7%), underwent STE and were compared with 29 healthy age- and sex-matched children. Factors related to adverse events included reduced myocardial function, enlarged left atrium or ventricle, and mitral regurgitation (MR). Patients with comorbidities affecting strain measurements were excluded from the strain analyses. A significant difference was observed between the groups in regional strains in the basal and apical septal and middle lateral regions. Global strain rate (GLS) and strain rates were not significantly different but were still lower than the control group. Twenty percent of patients had abnormal GLS but normal left ventricular ejection fraction (LVEF). All patients exhibited reduced segmental myocardial strain in at least one segment. Four out of 26 recovered patients without comorbidities had abnormal GLS at follow-up, despite normal LVEF. STE proves more useful than conventional echocardiography in patients with MIS-C, revealing subclinical cardiac injury in the acute and post-acute phases.