Supplement-Induced Acute Kidney Injury Reproduced in Kidney Organoids

Abstract

Introduction: Acute kidney injury associated with the consumption of Beni-koji CholesteHelp supplements, which contain red yeast rice (Beni-Koji), has become a significant public health concern in Japan. While renal biopsy findings from several case reports have suggested tubular damage, no definitive causal relationship has been established, and the underlying mechanisms of kidney injury remain poorly understood. The complexity of identifying toxic substances in supplements containing various bioactive compounds makes conventional investigative approaches both time-consuming and challenging. This highlights an urgent need to establish a reliable platform for assessing organ-specific toxicity in such supplements. In this study, we utilized a kidney organoid model derived from adult rat kidney stem cells (KS cells) to assess the potential tubular toxicity of these supplements.

Methods: KS cell clusters were cultured in three-dimensional system supplemented with growth factors to promote kidney organoids. The organoids were subsequently exposed to Beni-koji CholesteHelp supplements or cisplatin, followed by histological and molecular analyses to evaluate structural impacts.

Results: Established organoids had the kidney-like structures including tubular-like structures and glomerulus-like structures at the tips of multiple tubules. Treatment with Beni-koji CholesteHelp supplements induced significant tubular damage in the organoids, characterized by epithelial cell thinning, structural disruption, and increase in cleaved-caspase 3-positive apoptotic tubular cells, similar to the organoids treated with cisplatin.

Conclusion: These findings provide the first evidence suggesting that certain toxicants in specific batches of Beni-koji CholesteHelp supplements cause direct renal tubular injury. This KS cell-based organoid system represents a cost-effective, reproducible, and technically simple platform for nephrotoxicity screening.

Keywords: Acute kidney injury; Drug-induced nephrotoxicity; Kidney organoid; Kidney stem cell.

Fig. 1.

Differentiation and structural characteristics of kidney organoids from KS cells. a–d Stereomicroscopy of control organoids at day 2 (a), day 8 (b), day 14 (c), and day 21 (d). Scale bars, 1 mm. Expression of proximal tubule markers in kidney organoids. e, f Immunostaining images of AQP1 (red) and LTL (green) in control organoids confirm the identity of proximal tubules. g Western blot analysis of KS cells and organoids reveals that AQP1 is present in differentiated organoids but absent in undifferentiated KS cells. Scale bars, 20 µm. LTL, lotus tetragonolobus lectin; AQP1, aquaporin-1.

Fig. 2.

Tubular injury in kidney organoids induced by Beni-koji CholesteHelp supplement and cisplatin. a–c Stereomicroscopy images demonstrate significant tubular thinning (red arrows) in kidney organoids under control conditions and following exposure to Beni-koji CholesteHelp (100 μg/mL) or cisplatin (60 μm). Scale bars, 1 mm. d–f H&E stained sections show luminal accumulation in treated organoids. Scale bars, 100 µm. g–i H&E stained sections show tubular cell thinning (black arrows), structural disruption (white arrows), and luminal accumulation (arrowheads) in treated organoids, indicative of tubular injury. Scale bars, 100 µm.

Fig. 3.

TEM of Beni-koji CholesteHelp-induced tubular injury. a–f TEM images reveal vacuolation (white arrows), tubular cell thinning (black arrows), accumulation of intracellular components within the lumen (arrowhead), separation of tubular cells (yellow arrows), detachment of tubular cells (red arrows) suggesting direct tubular toxicity from Beni-koji CholesteHelp. The brush border-like structure on the luminal side was reduced in organoids treated with Beni-koji CholesteHelp (100 μg/mL) or cisplatin (60 μm). Scale bars, 5 µm (a–c), 2 µm (d–f).

Fig. 4.

Immunofluorescence of Beni-koji CholesteHelp-induced tubular injury. a–e Immunofluorescence staining with cleaved-caspase 3 (red) and LTL (green) in kidney organoids under control conditions (a), after exposure to cisplatin (60 μm) (b), or following treatment with Beni-koji CholesteHelp at concentrations of 100 μg/mL (c), 50 μg/mL (d), and 10 μg/mL (e). The images indicate an increase in apoptotic proximal tubular cells in organoids treated with the cisplatin and Beni-koji CholesteHelp at concentrations of 50 μg/mL and 100 μg/mL. Scale bars, 20 µm. f The number of cleaved-caspase 3-positive cells per field is shown in the graphs. One-way ANOVA followed by Tukey’s test was used to determine statistical significance. **p < 0.01. NS, not significant; LTL, lotus tetragonolobus lectin.