Protective Effect of Targeted Inhibition of Cathepsin S Activity on CKD

Background: Chronic kidney disease (CKD) is a public health issue characterized by vascular rarefaction, marked by reduced capillary density. The molecular mechanisms underlying endothelial cell injury and loss remain unclear. Cathepsin S (CTSS), a lysosomal protease involved in degrading damaged proteins, has been linked to various diseases. However, its role in vascular rarefaction in CKD is not understood. Previous research indicated that limonin, a compound known for its protective effects against acute kidney injury, might also protect against CKD. Methods: We used a bilateral ischemia-reperfusion injury (IRI) model to induce CKD in mice and generated CTSS knockout mice. LY3000328, a specific CTSS inhibitor, was administered three days before IRI. We conducted molecular pathology studies on human umbilical vein endothelial cells (HUVECs) in vitro and screened small molecule inhibitors of CTSS from a natural product library. Molecular docking studies assessed the binding affinity between CTSS and limonin, which was validated by microscale thermophoresis (MST) and cellular thermal shift assays (CETSA). Both in vivo and in vitro studies were performed. Results: CTSS expression and enzyme activity were significantly upregulated in CKD animal models. CTSS knockout and inhibition of CTSS in vivo restored capillary density and reduced renal fibrosis after IRI. Pathologically, CTSS in renal vascular endothelial cells translocated into the nucleus and targeted the degradation of the MCM 2-7 complex, inhibiting the G1/S transition in endothelial cells. This impaired repair mechanisms and worsened CKD progression. Limonin was identified as a natural inhibitor of CTSS. Molecular docking showed a strong binding affinity between CTSS and limonin, confirmed by MST and CETSA. In vitro, limonin bound to CTSS's active center, inhibiting MCM 2-7 complex degradation and promoting the G1/S transition in endothelial cells. In vivo, limonin significantly reduced vascular rarefaction and renal fibrosis. Conclusion: CTSS plays a critical role in CKD by inhibiting HUVEC cell cycle progression through enzymatic hydrolysis of the MCM 2-7 complex. Targeting CTSS activity offers a novel therapeutic strategy for CKD. Limonin effectively inhibits CTSS, promotes the G1/S transition in HUVECs, alleviates renal fibrosis, and shows promise for clinical application in CKD treatment. Funding: Government Support – Non-U.S.