Refinement of renal T lymphocytes enrichment strategies: advancing the in-depth study of renal immune responses.

The kidney T lymphocytes emerge as central agents in both physiological surveillance and pathological processes. In this study, we aimed to establish an optimized protocol for isolating and purifying T lymphocytes from mouse and human kidneys to provide support for renal diseases research. To develop an optimized protocol, we evaluated different tissue fragmentation strategies, enzymatic digestion conditions, digestion times and Percoll density gradients to maximize cell yield and viability while preserving surface markers. Subsequently, we applied this method to various kidney disease models and performed single-cell sequencing analysis by using public database. Our analyses revealed mechanical mincing combined with 0.2% collagenase IV and 0.02% DNase I for 45 min, followed by 33-80% Percoll gradient centrifugation, yielded the highest number of viable mononuclear cells with preserved surface markers. This protocol outperformed mechanical grinding and high concentration collagenase digestion, which caused greater mechanical and chemical damage. Flow cytometry confirmed optimal isolation of CD4+ T cells, CD8+ T cells, and double-negative T cells with minimal impact on cell viability and marker expression. This protocol has been applied in ischemia-reperfusion injury, renal fibrosis, cisplatin-induced acute kidney injury models, and normal human kidney tissue, demonstrating its potential for both preclinical and clinical research. In addition, public single-cell RNA-seq data were used as a complementary reference to support the T lymphocyte subset proportions obtained by our method. This optimized method provides a reliable and scalable approach for renal T lymphocytes isolation, advancing kidney diseases research and potential therapeutic applications.