The pathophysiological mechanism of Beni-koji Choleste-Help /puberulic acid-induced kidney injury is proximal tubular mitochondrial dysfunction

Abstract Introduction In March 2024, kidney injury, sometimes with Fanconi syndrome, caused by a red yeast rice supplement ( Beni-koji Choleste-Help ), was reported in Japan. By November 24, 2024, 2,628 people had visited medical facilities, making it a social problem. Many patients still show decreased eGFR. However, the previous report only noted that puberulic acid, newly identified for its nephrotoxicity, was present in toxic lots. The pathophysiology of these nephropathies should be clarified. Here, we discovered that mitochondrial dysfunction in renal proximal tubular epithelial cells plays a pivotal role in the nephrotoxicity. Methods To assess the effects of Choleste-Help and puberulic acid, we performed RNA-seq, extracellular flux analysis (Seahorse XF Analyzer), immunofluorescence staining, Western blotting, and other assays across multiple models, including human kidney biopsy specimens, human-derived primary renal proximal tubular epithelial cells (hRPTECs), human renal organoids, and mice. Results A patient renal biopsy sample showed Kidney Injury Molecule-1 (KIM-1) expression in proximal tubules surrounded by activated myofibroblasts, indicating acute tubular damage and interstitial fibrosis. Mice treated with the toxic lot of Choleste-Help and puberulic acid showed kidney injury with Fanconi syndrome-like urinary findings. Pathological sections revealed tubular necrosis and interstitial fibrosis. RNA-seq analysis of whole kidneys showed that Choleste-Help and puberulic acid produced similar RNA patterns, suggesting puberulic acid is a causative agent. Gene Ontology (GO) analysis comparing the normal and toxic lot revealed downregulation of mitochondrial-related pathways. Puberulic acid also showed toxicity to hRPTECs and tubular organoids (tubuloids). In vitro experiments with hRPTECs revealed that it causes mitochondrial damage, especially to the mitochondrial respiratory chain, leading to cell death, mainly by necrosis. Conclusion Puberulic acid and a toxic lot of Choleste-Help cause direct mitochondrial damage to tubular epithelial cells, followed by necrosis. Graphical Abstract Key Points First study shows puberulic acid causes mitochondrial injury preceding nephrotoxicity. First study shows matched renal transcriptomes for puberulic acid and Choleste-Help. Translational study using patient-derived primary cells and organoids for all assays.