3D Proximal Tubule Tissues Recapitulate Key Aspects of Renal Physiology to Enable Nephrotoxicity Testing

Abstract

Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current pre-clinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo's proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.

Advances in predictive in vitro models of drug-induced nephrotoxicity

“…Tissue arrays are a form of multicellular 3D culture that has been used for in vitro hepatotoxicity screening, and they have now been adapted for the assessment of nephrotoxicity in renal tissue 65,146 (FIG. 2f).…”

“…2f). To generate these renal tissue arrays, a suspension of renal fibroblasts and HUVECs in a proprietary gel were bioprinted into Transwells to create a 3D interstitial layer 65 , after which a second suspension of primary human proximal tubule cells was bioprinted over the interstitial layer. This method produced a 3D tissue array consisting of a monolayer of proximal tubule cells with a thicker interstitial layer on their basolateral side.…”

“…However, rapid readouts of proximal tubule injury will be required to increase throughput; therefore, further characterization of injury markers is needed. Improved tissue array designs that permit laminar flow (possibly requiring microfabrication similar to that used for kidneys-on-a-chip) might further improve the functional maturity of the proximal tubule cells cultured in these tissue arrays 65 .…”

Bioprinted pluripotent stem cell-derived kidney organoids provide opportunities for high content screening

“…Tissue engineering using 3D bioprinting enables the fabrication of tissues through an automated, spatially controlled, and reproducible deposition of living cells in defined geometric patterns [13][14][15][16] . In addition to establishing some key features of native tissue architecture in vitro, bioprinting may also enhance reproducibility and provide finer resolution and smaller scale than is achievable manually.…”

Hepatocyte CYP2B6 Can Be Expressed in Cell Culture Systems by Exerting Physiological Levels of Shear: Implications for ADME Testing

“…Physiological shear can be reintroduced using perfused Transwells [17], hollow fiber culture devices [28], and 3D bioprinting of cells onto perfusable chips [14, 29]. However, these approaches have limited scalability and may require artificial extracellular matrix and growth factors that introduce ill-defined unintended effects.…”