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

Introduction

In March 2024, kidney injury sometimes accompanied with Fanconi syndrome caused by a supplement made from red yeast rice ( Beni-koji Choleste-Help ) were reported in Japan. By November 24, 2024, 2,628 people had visited medical facilities, resulting in a social problem. Many patients still have 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 lots. The pathophysiology of those nephropathies should be determined. 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 complementary assays across multiple experimental 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 associated with injured tubules. Mice treated with the toxic lot of Choleste-Help and puberulic acid showed kidney injury with Fanconi syndrome-like urinary findings. Pathological sections showed tubular necrosis and interstitial fibrosis. RNA-seq on whole kidneys of those mice presented the renal damage caused by Choleste-Help and puberulic acid produced similar RNA patterns, indicating 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 on hRPTECs and tubular organoids. In vitro experiments with hRPTECs revealed that it causes mitochondrial damage especially on mitochondrial respiratory chain at first and leads to cell death, mainly necrosis.

Conclusion

puberulic and a toxic lot of Choleste-Help cause direct mitochondrial damage on tubular epithelial cells, followed by necrosis.

Graphical Abstract

Translational Statement

Our research revealed the pathophysiology of puberulic acid nephropathy, which suffers many patients. And we used not only animal models, but human kidney-derived primary cultured tubular epithelial cells and its 3D organoids, which are ones of the closest models of the human body.