Modified Hu-lu-ba-wan and its active component trigonelline ameliorate diabetic renal tubular injury via enhancing PACS2-regulated mitochondria-endoplasmic reticulum contacts and mitophagy.

BACKGROUND: Mitochondrial dysfunction and impaired mitophagy in proximal tubule epithelial cells are central to the pathogenesis of Diabetic Kidney Disease (DKD). The mechanisms of Modified Hu-lu-ba-wan (MHLBW), a clinically validated herbal formula for DKD patients, remain to be explored. PURPOSE: This study aimed to investigate the renal protective effects of MHLBW against DKD, identify its bioactive component and elucidate the underlying mechanisms. METHODS: The efficacy of MHLBW was evaluated in DKD mice. Transcriptomics, chemical profiling analysis, and serum metabolomics were employed to uncover the mechanisms of MHLBW and to identify its active components. Molecular docking (MD) and dynamics simulations were conducted to predict the binding affinity between the identified component, trigonelline (TRL), and its putative target, Phosphofurin Acidic Cluster Sorting Protein 2 (PACS2). Gene silence in vitro experiments were further performed for mechanism verification. RESULTS: MHLBW treatment significantly ameliorated hyperglycemia and renal injury in db/db mice. MHLBW restored mitochondrial morphology and function, enhanced mitochondria-endoplasmic reticulum contacts (MERCs), and activated Pink1/Parkin-mediated mitophagy in renal tubules, an effect associated with increased PACS2 expression. Chemical profiling analysis and serum metabolomics identified TRL as a key component of MHLBW. TRL replicated the benefits of MHLBW in HK-2 cells. MD and dynamics simulations demonstrated stable binding between TRL and PACS2. Crucially, knockdown of PACS2 abolished the protective effects of TRL on MERCs and mitophagy. CONCLUSION: MHLBW and TRL can alleviate diabetic renal proximal tubular damage by targeting PACS2 to enhance MERCs and promote Pink1/Parkin-mediated mitophagy, indicating that MHLBW represents a promising therapeutic formula for DKD.