Development of human and rat PTC monolayers as in vitro models of nephrotoxicity

Abstract

Nephrotoxicity is a serious side effect of many drugs due to the kidney being one of the major sites for their excretion. In vitro models of nephrotoxicity are therefore paramount in drug development process, which would be used to detect early stages of drug-induced toxicity. Most current proximal tubule models have their limitation, not only due to the lack of expression of some of the major drugs transporters, but also the lack of species-specific properties. This study investigates the use of human and rat primary proximal tubule cells as in vitro models of nephrotoxicity. Human and rat proximal tubule cells (PTCs) were isolated and cultured from cortical tissues of the kidney. The cells form highly polarised monolayers that were exposed to polymyxin B, gentamicin and cisplatin – all well-known nephrotoxins. Cell viability and LDH-based cell death were measured, alongside the production of renal toxicity biomarkers, KIM-1, NGAL and clusterin. As expected, cell viability of the monolayers after polymyxin B, gentamicin and cisplatin challenge deceased. The proportion of cells alive was dependent on the concentration and period of exposure of the nephrotoxin. For example, the cell viability of rat PTCs treated with gentamicin for 48h decreased by 41% than control cells (P < 0.01). Similarly, human PTCs treated with cisplatin showed a significant decrease (39%, P < 0.01) in cell viability when compared with non-treated cells. This relationship was also observed in LDH-based cell viability. Higher levels of KIM-1, NGAL and clusterin secretion were detected when human and rat PTCs were treated with nephrotoxins. All three biomarkers were predominately secreted across the apical membrane of the PTCs monolayers. For instance, KIM-1 level across the apical membrane was 5.25±1.01 ng/ml after polymyxin B treatment for just 24h, which was significantly higher than across the basolateral membrane at 0.81±0.31 ng/ml. The levels of biomarkers secretion were also found to be dependent on concentrations of nephrotoxin and period of exposure. For example, rat PTCs treated with 250 μg/ml polymyxin B for 24h produced 9.8±1.2 ng/ml of KIM-1, which increased to 25.8±2.5 ng/ml with 48h challenge, an increase of more than 2-fold (P <0.01). The mechanisms of toxicity were also investigated by pre-treating the PTCs with supposedly nephron-protectant, rosuvastatin, cilastatin and cimetidine, prior to nephrotoxin exposure. While 50 μM rosuvastatin did not change the cell viability nor KIM-1 expression levels in human and rat PTCs, the pre-treatment of rosuvastatin with polymyxin B increased cell viability by 15 % when compared to polymyxin-B only treatment. Similarly rat PTCs treated with 250 μg/ml gentamicin and 40 μM cilastatin for 48h showed KIM-1 at levels of 28.8±0.27 ng/ml, compared with 28.7±0.5 ng/ml in cells treated with only gentamicin, giving a decrease of 29 % (P < 0.001). Another instance showed human PTCs treated with 250 μg/ml polymyxin B and 50 μM rosuvastatin for 24h produced clusterin at significantly lower level than cells treated with polymyxin B alone (42.3±12.2 ng/ml and 95.6±18.1 ng/ml, P < 0.001, respectively). These data suggest mechanisms of the aminoglycoside uptake could be via megalin/cubilin receptors, which were inhibited by rosuvastatin and cilastatin. Another goal of this project was to investigate the influence of cisplatin treatment on p53, caspase 3, caspase 8 and caspase 9 in human and rat PTCs. These factors are important in the induction of apoptosis and would provide mechanistic data of nephrotoxicity. The activity and mRNA levels of the caspases in PTCs were increased after cisplatin treatment. For example, human PTCs treated with cisplatin, mRNA level of caspase 3 appeared to be increased significantly (P< 0.01) with the treatment of cisplatin (3.51±0.34%) compared to control. The co-treatment of cimetidine with cisplatin did decrease caspase 3 mRNA levels significantly to (2.45±0.32, P< 0.05) when compared only cisplatin treatment. These data revealed the mechanism of how cisplatin may cause nephtoxocitiy in these PTCs. Taken together, these data show the utility of human and rat PTCs as in vitro models for the study of nephrotoxicity, and their potential in elucidating the mechanisms of action of polymyxin-B, gentamicin and cisplatin induced toxicity.