Proximal Tubule Epithelial Cell Reabsorption of the Drug Delivery Protein Elastin‐like Polypeptide

Abstract

Elastin like polypeptide (ELP) is a protein composed of a pentapeptide repeat domain found in human tropoelastin. ELP is used for drug delivery by altering the ELP coding sequence to attach drugs, therapeutic peptides, or therapeutic proteins. One of the focuses of our group has been development of ELP – delivered therapeutics for treatment of kidney disease. Thus, understanding how ELP is filtered, reabsorbed, and distributed within the kidney is of key importance. In vivo studies in our lab showed that ELPs accumulated preferentially in the kidney after systemic administration. Renal deposition and intra‐renal distribution was dependent on ELP size, with smaller ELPs accumulating exclusively in the renal cortex and larger ELPs having a more mixed cortical and medullary distribution. All ELPs were present at high levels in tubular epithelial cells. However, whether the tubular distribution is the result of ELP reabsorption after filtration and whether the amount or rate of reabsorption is dependent on ELP size is unknown.To address this issue, uptake and intracellular distribution of fluorescein‐5‐malemide labeled ELPs of varying sizes were determined in primary human proximal tubule epithelial cells (HRPteC). HRPteC were seeded in a 6 well plate (4x104/well) and incubated with fluorescein labeled ELP (10μM) for various times (24hr, 6hr, 4hr, 2hr and 1hr). HRPteC were collected, and mean cellular fluorescence intensity was measured using flow cytometry. Percentage of ELP internalization was determined by quenching the extracellular fluorescein label with trypan blue (0.13% v/v), then repeating the flow cytometric analysis to measure only intracellular fluorescence. To obtain a qualitative representation of ELP interaction with kidney cells, HRPteC were seeded in an 8‐chamber glass slide (2x103 cells/chamber) and incubated with fluorescein labeled ELP (10μM) for various times as above. Following fixation and staining with 4′,6‐diamidino‐2‐phenylindole (DAPI), cells were imaged by laser scanning confocal microscopy. Our data demonstrate that a longer incubation time led to a greater accumulation of ELP in the proximal tubule epithelial cells. Analysis of internalization rate revealed that all ELPs entered the cells slowly over the course of a 24‐hour time frame. This finding was supported by confocal microscopy. Between 1 and 12 hours, localization of ELP occurred at the cellular membrane of the proximal tubule cells, but at time points between 12 and 24 hours, ELP was visible in punctate cytoplasmic structures likely to be endocytic vesicles. A smaller ELP‐63 (~25 kDa) accumulated in cells at higher levels than did a larger ELP‐159 (~62 kDa) after 24 hours of incubation (390.7 ±5.0 versus 651.6 ±42.7 RFU; p<0.05). These data suggest that there may be a common mechanism for tubular epithelial cell uptake of ELPs of various sizes, but that smaller ELPs may be internalized to a greater extent than larger ELPs. The results of these and future studies to determine the exact renal handling of ELPs of various sizes will be crucial to the design of ELPs for delivery of therapeutics to specific renal compartments or cell types.