Transformative Research in Diabetic Nephropathy 2.0

TRIDENT 2.0 is a multicenter observational translational study that characterizes kidney molecular and histopathologic features in relation to exposure to kidney-protective therapies, with a focus on sodium-glucose cotransporter-2 (SGLT2) inhibitors. The study leverages archived clinical kidney pathology material and harmonized clinical data to support integrated molecular-histologic analyses across participating sites.

Tissue sources and central repository workflows:

Formalin-fixed, paraffin-embedded (FFPE) kidney tissue sections/slides are generated from archived clinical pathology material (including clinically indicated kidney biopsies and available donor or nephrectomy specimens) and transferred under coded identifiers to a central repository for downstream molecular assays and digital pathology.

Spatial transcriptomics and molecular profiling:

Spatially resolved transcriptomic methods are used to generate high-resolution molecular maps while preserving tissue architecture. FFPE (or fresh-frozen, when available) sections may be processed using commercially available spatial transcriptomics platforms (e.g., 10x Genomics Visium, NanoString CosMx, Xenium) or updated technologies implemented under study governance. Standard quality control procedures evaluate tissue integrity, RNA quality, capture efficiency, and resolution of major kidney compartments and cell types. Sequencing is performed on Illumina platforms with platform-appropriate depth, followed by preprocessing using platform-specific pipelines and downstream analysis in R/Python workflows (e.g., Seurat or equivalent) with normalization and batch correction as needed. Planned analyses include identification of cell-type and sub-cell-type signatures in spatial context, mapping of injury patterns (e.g., fibrosis/inflammation/vascular remodeling), and comparative molecular profiling across disease categories and therapy exposure groups with adjustment for relevant covariates.

Digital pathology and centralized histopathology review:

Digitized clinical stains and available diagnostic images are used for centralized review and standardized lesion scoring by renal pathologists. When applicable, diabetic kidney disease (DKD) is classified using established renal pathology criteria. Specimen adequacy metrics are used to guide analytic inclusion and sensitivity analyses.

Linked clinical data (high level):

Clinical data are harmonized across sites to support clinicopathologic and molecular integration, including medication exposure history and relevant laboratory and diagnostic variables. Longitudinal clinical information is used to contextualize molecular and histologic findings for downstream modeling.

Statistical and integrative analytic approach:

Analytic methods include differential expression and pathway analyses with appropriate multiple-testing control and covariate adjustment. Integrative modeling may combine molecular, histologic, and clinical domains using dimension reduction and regularized approaches to derive molecular signatures associated with disease state and therapy exposure.

• Change in estimated glomerular filtration rate (eGFR) [Time frame: Baseline to 18 months]
• Change in urine protein/creatinine ratio [Time frame: Baseline to 6 months.]
• Descriptive histopathology features and spatial gene expression patterns across cohorts [Time frame: At enrollment/baseline (Single assessment on the archived clinical specimen slide)]
• Associations between histopathological features and clinical outcomes [Time frame: Up to 3 years (longitudinal outcome abstraction, including dialysis/transplant/kidney failure/mortality as captured).]
• Associations between histopathological features and prior medication exposures [Time frame: Baseline (prior/current medication exposure history at enrollment).]
• Feasibility metrics [Time frame: During study accrual and specimen acquisition (up to 3 years).]