The Study of Function and Regulation of Renal Drug Transporters in Human Proximal Tubule Epithelial Cells

The study of renal function is crucial to understand and developed novel strategies to cope with renal diseases. This thesis was devoted to study renal proximal tubule function in vitro, focusing on the activity and regulation of renal drug transporters. Chapter 1 offers a rational of the study in hand and an overview on in vitro models in renal research. The role of the ‘breast cancer resistance protein’ (BCRP) in renal function and chemoresistance is reviewed in-depth in chapter 2. Exposing the fact that cancer drugs that are BCRP substrates can themselves influence its expression and function. In chapter 3 assays for studying transport processes over the apical membrane of PTEC are described. Using fluorescence-based transport assays, the function of BCRP, P-glycoprotein (P-gp) and multi-drug resistance protein 4 (MRP4), as well as protein uptake through receptor-mediated endocytosis was determined in conditionally immortalized human proximal tubule cells (ciPTEC). Model substrates and inhibitors were tested, and appeared not specific for the designated transporters, reflecting the complex interactions that can take place in vivo in PTEC. In chapter 4, the effects of uremic solutes p­-cresyl sulfate (pCS) and p­-cresyl glucuronide (pCG) on renal efflux transport and CKD progression are studied. Exposing ciPTEC to pCS and pCG competitively inhibited the activity of both BCRP and MRP4. Further pCS influenced the expression and function of BCRP, and pCG influenced the expression of epithelial markers. These finding support a role for uremic toxins in CKD progression by affecting renal tubule cell phenotype and functionality. The pharmacological interaction between two different anticancer drugs – cetuximab, anti-epidermal growth factor receptor recombinant monoclonal antibody, and methotrexate, a chemotherapeutical and anti-inflammatory agent – was investigated at the renal cellular level, in chapter 5. Overall cetuximab attenuates methotrexate-induced nephrotoxicity by EGFR-mediated transporter regulation, opening possibilities for nephroprotective co-medication therapies. In chapter 6 we investigated the function and differentiation potential of human fetal kidney-specific progenitor cells (KSPC). Functional assays revealed that MRP4 and P-gp activity increased over passage number, while BCRP was functionally inactive despite its gene expression. These results indicated that KSPC can be functionally active in vitro, and although immature, KSPC can differentiate towards a PTEC phenotype over time and passage number. In conclusion, this work pushes forward our understanding of renal xenobiotic transporters activity under physiological and pathological conditions, and provides a novel approach to explore further transporter regulation and renal drug interactions