Development of a Cystic Fibrosis Microphysiological Model of the Kidney Proximal Tubule

Abstract

Abstract: Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by a spectrum of mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), whose protein product functions as a chloride channel. The impact of CF on airway and digestive systems is well studied; however, the impact of CFTR deficiency on kidney function is not well characterized. The reasons for a lack of data on kidney function in the CF population is that, historically, patients typically succumbed to other morbidities, particularly opportunistic lung infections. With the development of new therapeutics, including inhaled antibiotics, pancreatic enzyme replacement therapy, lung transplants and, most recently, development of small molecule drug CFTR “correctors and potentiators” the lifespan of a CF patient has increased significantly to a median of 44 years in the United States, as of 2017 with the increased lifespan of CF patients, it is anticipated that they will also now be susceptible to “normal” age-related comorbidities including chronic kidney disease (CKD), which has a prevalence of ~12% in US adults aged 45-64. A unique risk factor that CF patients face regarding CKD is repeated exposure to high dose aminoglycoside and polymyxin antibiotics for treatment of opportunistic lung infections by gram negative bacteria. As these antibiotics are known to induce acute kidney injury, it is reasonable to predict higher than average risk of CKD in CF patient populations. Renal clearance of both antibiotic classes is believed to be mediated by endocytic uptake at the apical membrane of renal proximal tubule epithelial cells via the cubilin/megalin complex. In CF patients, it has been hypothesized that this endocytic complex in the kidney may be impaired, as “shed” cubilin has been observed in the urine of CF patients and mice lacking CFTR have decreased expression of the cubilin receptor in the kidney and low molecular weight proteinuria. Another study suggests that CF patients may be protected from polymyxin-induced acute kidney injury (AKI) relative to normal subjects. Taken together, these observations point to decreased CFTR function as conferring protection against antibiotics renally cleared by the cubilin/megalin pathway. However, with restoration of CFTR function by corrector/potentiator therapeutics, the risk of acute kidney injury and CKD is a possibility that must be considered when managing opportunistic lung infections in CF patients. Thus, we hypothesize that individuals with CF may be protected from antibiotic-induced AKI due to impaired cubilin/megalin endocytic function in the kidney proximal tubule. To address this hypothesis, our aim was to develop a model of CF proximal tubule epithelial cells (PTECs) using a 3D microphysiological system (MPS) cultured with cells from wild type (WT) and CFTR gene-edited ferrets. The rationale for using this preclinical model versus other species is that ferrets lacking CFTR exhibit a similar lung disease phenotype to humans, unlike the CFTR knockout mouse. We isolated, propagated, and characterized ferret PTEC (fPTEC) from ferret kidneys, and optimized the cell culture media to minimize transition from epithelial to fibroblast-like morphology. Furthermore, we established a fPTEC MPS, minimized cell aggregation and improved the epithelial morphology in 3D culture, tested for cubilin shedding in MPS effluents and evaluated endocytic function via uptake of fluorescently labeled albumin. No cubilin shedding was detected by ELISA nor we were able to measure endocytosis of labeled albumin, but optimization of fPTEC MPS development is required. We maintained fPTEC viability of 5 days in MPS and a functional response to SGLT2 inhibition in 2D culture. In conclusion, we have a better understanding of the culture of fPTECs in 2D culture and MPS, supporting their future use in modeling CF kidney function and response(s) to antibiotic-induced AKI