Development of an In Vitro Model for Inflammation Mediated Renal Toxicity Using 3D Renal Tubules and Co-Cultured Human Immune Cells

Abstract

Background: The emergence of various infectious diseases and the toxic effects of hyperinflammation by biotherapeutics have highlighted the need for in vitro preclinical models mimicking the human immune system. In vitro models studying the relationship between hyperinflammation and acute renal injury mainly rely on 2D culture systems, which have shown limitations in recapitulating kidney function. Herein, we developed an in vitro kidney toxicity model by co-culturing 3D engineered kidney proximal tubules cells (RPTEC/TERT1) with human peripheral blood mononuclear cells (PBMC).

Methods: RPTEC/TERT1 were sandwich cultured to form 3D renal tubules for 16 days. The tubules were then co-cultured with PBMC using transwell (0.4 μm pores) for 24 h. Hyperinflammation of PBMC was induced during co-culture using polyinosinic-polycytidylic acid (polyI:C) and lipopolysaccharide (LPS) to investigate the effects of the induced hyperinflammation on the renal tubules.

Results: Encapsulated RPTEC/TERT1 cells in Matrigel exhibited elevated renal function markers compared to 2D culture. The coexistence of PBMC and polyI:C induced a strong inflammatory response in the kidney cells. This hyperinflammation significantly reduced primary cilia formation and upregulated kidney injury markers along the 3D tubules. Similarly, treating co-cultured PBMC with LPS to induce hyperinflammation resulted in comparable inflammatory responses and potential kidney injury.

Conclusion: The model demonstrated similar changes in kidney injury markers following polyI:C and LPS treatment, indicating its suitability for detecting immune-associated kidney damage resulting from infections and biopharmaceutical applications.

Keywords: 3D renal proximal tubule; Hyperinflammation; In vitro renal toxicity model; Peripheral blood mononuclear cells.

Fig. 1

A Development of an in vitro renal toxicity model elucidating hyperinflammation-associated renal damage by co-culturing 3D RPTEC/TERT1 tubules and PBMC. B Scheme of 3D culture of RPTEC/TERT1 cells using the Matrigel sandwich culture system to induce spontaneous formation of renal proximal tubule structures and co-culture with PBMC to induce in vitro hyperinflammation

Fig. 2

A Phase-contrast images of the 3D-cultured RPTEC/TERT1 within the matrix during 16 days of culture (scale bars = 200 μm). Morphological and renal functional evaluation of 3D microtubule constructs. B DAPI staining images obtained by confocal microscopy (upper panel) and cross-sectional observation (lower panel) demonstrating tubular structure formation in the RPTEC/TERT1 cells (scale bars = 20 μm). C Fluorescence images of ZO-1 tight junction protein and Na + K + ATPase stained in 2D cultured cells and 3D microtubule constructs (scale bar = 20 μm). D Expression level of kidney functional markers depending on the culture conditions. “*” indicates statistical significance in comparison with the 2D group (p < 0.05)

Fig. 3

Induction of hyperinflammation through stimulation of co-cultured PBMC by treatment with polyI:C. A Illustrations depicting the experimental groups and quantification data of the secreted inflammatory cytokines IL-1β, IL-6, and TNF-α. “*”, “#”, “$”, “†” and “‡” indicate statistical significance in comparison with the RPTEC_ctl, RPTEC_IC, Co_RPTEC, Co_RPTEC_IC, and PBMC only groups, respectively (p < 0.05). B Expression levels of inflammation markers under various conditions. “*”, “#” and “$” indicate statistical significance in comparison with the RPTEC_ctl, RPTEC_IC, Co_RPTEC groups, respectively (p < 0.05)

Fig. 4

A Multidimensional scaling evaluating the similarity of gene expression patterns of cells cultured under varied conditions. B The number of DEGs in experimental conditions in comparison with the control group. C An overview of the DEGs was described by the MA plot. D Top 10 significantly upregulated biological processes and downregulated cellular components sorted by p-value ranking of the Co_RPTEC_IC group in comparison with those of the Co_RPTEC group. The significant p-values were converted to –log (p-value)

Fig. 5

A A subset of the enriched pathways was chosen and depicted as a network. The node color highlighted the 20 clusters of enriched pathways with similarity. B The color of the node indicated the expression information of DEGs in the enriched pathways. C Heatmap of normalized expression values in PBMC, RPTEC, co-cultured PBMC, and co-cultured RPTEC. D The normalized expression values were compared by plotting each set of genes associated with adaptive immune and cell death. E The normalized expression values were compared by plotting each set of genes associated with cilium development and ciliopathies. “*”, “#”, and “$” indicate statistical significance in comparison with the RPTEC_ctl, RPTEC_IC, and Co_RPTEC groups, respectively (p < 0.05)

Fig. 6

A Expression level of Hedgehog and ciliary development markers. B Fluorescence images of stained ac-tubulin (ac-tub) visualizing primary cilia (scale bar = 20 μm). C Percentage of cells presenting primary cilia. “*”, “#”, and “$” indicate statistical significance in comparison with the RPTEC_ctl, RPTEC_IC and Co_RPTEC groups, respectively (p < 0.05). D Expression level of four representative kidney injury markers. “*”, “#”, and “$” indicate statistical significance in comparison with the RPTEC_ctl, RPTEC_IC and Co_RPTEC groups, respectively (p < 0.05)

Fig. 7

A Quantification data for the IL-1β, IL-6, and TNF-α secreted in response to LPS treatment in the developed model. Expression levels of B inflammatory cytokine genes and C kidney injury markers in the varied immune environment created by LPS treatment. “*”, “#”, “$”, “†”, “‡”,”§”, “&” and “!”, respectively, indicate statistical significance in comparison with the RPTEC_ctl, RPTEC_LPS 1 μg, RPTEC_LPS 10 μg Co_RPTEC, Co_RPTEC_LPS 1 μg, Co_RPTEC_LPS 10 μg, PBMC_ctl, PBMC_LPS 1 μg and PBMC_LPS 10 μg groups, respectively (p < 0.05)