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.
Highlights
The kidneys play a central role in the metabolism and elimination of a variety of drugs, with the proximal tubule (PT) being exposed to high concentrations of reactive hydrophilic metabolites at both the luminal surface following filtration of plasma at the glomerulus, as well as the basolateral surface following absorption from the peritubular capillaries
The putative endothelial cell networks observed by Hematoxylin and eosin (H&E) and trichrome expressed CD31 and demonstrated that the Human umbilical vein endothelial cells (HUVEC) had organized to form open spaces lined by endothelial cells (Figure 2C)
A variety of systems for 3D culture of renal proximal tubule epithelial cells (RPTEC) in isolation have been developed, including culturing cells in Matrigel, culturing cells as organoids on a variety of scaffolds such as hyaluronic acid or silk, and culture of RPTEC in microfluidic devices (“kidney on a chip”) (Joraku et al, 2009; Subramanian et al, 2010; Astashkina et al, 2012; Jang et al, 2013). These systems lack direct contact between the epithelium and relevant interstitial cell types, including fibroblasts and endothelial cells, that play both a structural role in orienting the epithelium as well as providing a source of growth factors critical for the continued health and organization of the epithelium (Lemley and Kriz, 1991; Kaissling and Le Hir, 2008; Meran and Steadman, 2011). Without these supportive cell types, RPTEC rapidly lose their native phenotype in culture, preventing the ability to perform the chronic, low dose exposure studies necessary to predict how a molecule will perform in the clinic
Summary
The kidneys play a central role in the metabolism and elimination of a variety of drugs, with the proximal tubule (PT) being exposed to high concentrations of reactive hydrophilic metabolites at both the luminal surface following filtration of plasma at the glomerulus, as well as the basolateral surface following absorption from the peritubular capillaries. Due to the action of renal xenobiotic transporters expressed in the PT epithelium, pharmaceutical compounds can accumulate and become concentrated in the PT and may undergo further metabolism by cytochrome P450 enzymes and UDPglucuronyltransferases (Lohr et al, 1998) While this serves a role in detoxifying these compounds to generate more hydrophilic molecules that are secreted into the urine, highly toxic intermediate metabolites can accumulate and cause damage to the tubular epithelium and surrounding cells (Choudhury and Ahmed, 2006). Better predictive tools for identifying nephrotoxic drugs during the drug development process would reduce the costs associated both with bringing a new drug to market and in treating the downstream effects of AKI, as well as improving patients’ lives
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