Elucidation of the Pathophysiology of CholesteHelp and Puberulic Acid Nephropathy

Background: In March 2024, health problems caused by a supplement made from red yeast rice (Cholestehelp) were reported in Japan. By November 24, 2024, 2,628 people had visited medical facilities, resulting in a social problem. Even among patients who have recovered, many still have partial kidney damage and a decline in eGFR. The previous report, however, did not indicate anything other than that puberulic acid which newly identified nephrotoxicity was contained in toxic lot. We need to reveal the pathophysiology of those Nephropathies. Methods: We established the model of Cholestehelp / puberulic acid nephropathy with mice, human-derived tubular organoids and human primary epithelial cells, then analyzed them through RNA-seq, Western Blotting, Immunostaining, and Flow cytometry. Results: We administered PBS, normal lot of Cholestehelp, toxic lot of that, and puberulic acid by oral gavage or intraperitoneal injection to wild-type B6 mice (N= 10) for 6 days and then sacrificed them on day 7. The mice treated with the toxic lot and puberulic acid showed weight loss, renal dysfunction, albuminuria, elevated urinary NAG and urinary glucose, which is consistent with Fanconi syndrome. In addition, pathological sections showed tubular necrosis and interstitial fibrosis. We performed analyses of RNA-seq on whole kidneys of those mice. The renal damage caused by Cholestehelp and puberulic acid produced similar RNA patterns. Gene Ontology (GO) analysis comparing the normal and toxic lot revealed downregulation of mitochondrial-related pathways, including oxidative phosphorylation, aerobic respiration and so on. Puberulic acid also showed toxicity on human primary epithelial cells and tubular organoids. EC50 of puberulic acid is approximately twice that of cisplatin. In vitro experiments with human primary cells, revealed that it causes ATP deficiency and mitochondrial damage at first and leads to cell death. Annexin V assay presented the cell death was mainly necrosis with relatively little apoptosis. Conclusion: Puberulic acid was confirmed as the causative agent for CholesteHelp nephropathy. Both cause direct mitochondrial damage induced by ATP depletion on tubular epithelial cells, followed by necrosis rather than apoptosis.