Description

This study is a prospective observational cohort study designed to screen and validate the predictive value of five serum biomarkers (secretory leukocyte protease inhibitor \[SLPI\], Serpin E1, C-X-C motif chemokine ligand 10 \[CXCL10\], C-X-C motif chemokine ligand 13 \[CXCL13\], and Properdin) for cardiorenal syndrome (CRS) in patients with stable chronic heart failure (CHF). It builds on prior preclinical and clinical evidence from Luminex technology-based analyses, which identified differential expression of these biomarkers in patients with post-cardiac surgery acute kidney injury (AKI)-suggesting their potential role in mediating cardiorenal crosstalk, a key pathophysiological feature of CRS.

The five target biomarkers are selected based on their mechanistic relevance to CRS pathogenesis:SLPI \& Serpin E1: Both are serine protease inhibitors involved in regulating inflammation and endothelial barrier function. Dysregulation of these molecules has been linked to increased renal tubular injury and cardiac interstitial fibrosis-two core processes driving CRS development in CHF. CXCL10 \& CXCL13: These chemokines mediate immune cell recruitment (e.g., T cells, B cells) to sites of cardiac and renal injury. Elevated levels in CHF patients may indicate persistent systemic inflammation, a known trigger for progressive cardiorenal dysfunction.Properdin:As a positive regulator of the alternative complement pathway, Properdin contributes to complement system activation. Reduced Properdin levels (observed in post-cardiac surgery AKI) may reflect impaired complement-mediated tissue repair, increasing susceptibility to renal injury in CHF. This panel addresses a critical gap in current CRS research: existing predictive markers (e.g., NT-proBNP, serum creatinine) lack specificity for cardiorenal crosstalk, while these five biomarkers target pathways uniquely involved in the bidirectional damage between the heart and kidneys.

The study is implemented in two sequential phases, with standardized technical protocols to ensure data reliability:1. Screening Phase (Biomarker Differentiation): this phase focuses on identifying biomarkers with significant expression differences between CHF patients with and without CRS. For serum sample processing: Venous blood samples (5 mL) are collected from participants after an 8-hour fasting period, centrifuged at 3,000 rpm for 10 minutes at 4°C to separate serum, and stored at -80°C within 1 hour of collection to avoid biomarker degradation. Biomarker detection uses a multiplex Luminex xMAP assay (custom panel targeting SLPI, Serpin E1, CXCL10, CXCL13, Properdin) with a lower limit of detection (LLOD) ≤ 0.1 pg/mL for each analyte. Assays are run in duplicate, with inter-assay and intra-assay coefficients of variation (CV) controlled at \< 10% to ensure reproducibility.

2\. Validation Phase (Predictive Efficacy Assessment): this phase validates the predictive performance of screened biomarkers in CHF patients with baseline normal renal function. Key technical procedures include: Longitudinal Sample Tracking: Participants provide serum samples at baseline (enrollment) and 1-year follow-up. Samples are labeled with unique identifiers linked to electronic case report forms (eCRFs) to track renal function (creatinine, eGFR) and biomarker levels over time.

Statistical Validation Framework: Predictive efficacy is evaluated using receiver operating characteristic (ROC) curve analysis, with area under the curve (AUC) calculated to quantify discriminative ability. Biomarker cut-off values for CRS prediction are determined via the Youden index (maximizing sensitivity + specificity). Additionally, Pearson or Spearman correlation analyses are used to assess associations between biomarker levels and clinical indicators (NT-proBNP, creatinine, eGFR), with adjustments for potential confounders (e.g., age, CHF subtype, comorbidities) using multivariable linear regression models.

To account for heterogeneity in CHF pathophysiology, the study stratifies participants by CHF subtype (HFrEF: LVEF ≤ 40%; HFmrEF: LVEF 41-49%; HFpEF: LVEF ≥ 50%) in both phases. This stratification is critical for two technical reasons:1. Biomarker expression may vary by cardiac contractile function (e.g., HFpEF is characterized by myocardial stiffness rather than systolic dysfunction, which may alter inflammatory and complement pathway activation).2. Stratified analyses allow for the evaluation of subtype-specific predictive efficacy-ensuring the identified biomarkers are applicable across the full spectrum of CHF, rather than limited to a single subtype.

All LVEF measurements are standardized using transthoracic echocardiography (TTE) per the 2024 Chinese Heart Failure Diagnosis and Treatment Guidelines, with measurements performed by two independent cardiologists (blinded to biomarker results) to reduce inter-observer variability.

This protocol's technical design prioritizes reproducibility, mechanistic relevance, and clinical applicability-ensuring the validated biomarkers can ultimately be translated into clinical tools for early CRS detection in CHF patients.