Cisplatin is an anticancer agent marred by nephrotoxicity; however, limiting this adverse effect may allow the use of higher doses to improve its efficacy. Cilastatin, a small molecule inhibitor of renal dehydropeptidase I, prevents proximal tubular cells from undergoing cisplatin-induced apoptosis in vitro. Here, we explored the in vivo relevance of these findings and the specificity of protection for kidney cells in cisplatin-treated rats. Cisplatin increased serum blood urea nitrogen and creatinine levels, and the fractional excretion of sodium. Cisplatin decreased the glomerular filtration rate, promoted histological renal injury and the expression of many pro-apoptotic proteins in the renal cortex, increased the Bax/Bcl2 ratio, and oxidative stress in kidney tissue and urine. All these features were decreased by cilastatin, which preserved renal function but did not modify the pharmacokinetics of cisplatin area under the curve. The cisplatin-induced death of cervical, colon, breast, and bladder-derived cancer cell lines was not prevented by cilastatin. Thus, cilastatin has the potential to prevent cisplatin nephrotoxicity without compromising its anticancer efficacy.

A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatininduced injury to renal proximal tubular cells. Cilastatin is a specific inhibitor of renal dehydrodipeptidase I (DHP-I), which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. Primary cultures of proximal cells were treated with cisplatin (1-30 M) in the presence or absence of cilastatin (200 g/ml). Apoptosis and mitochondrial injury were assessed by different techniques. Cisplatin uptake and DNA binding were measured by inductively coupled plasma spectrometry. HeLa cells were used to control the effect of cilastatin on the tumoricidal activity of cisplatin. Cisplatin increased cell death, apoptotic-like morphology, caspase activation, and mitochondrial injury in proximal tubular cells in a dose-and time-dependent way. Concomitant treatment with cilastatin reduced cisplatin-induced changes. Cilastatin also reduced the DNA-bound platinum but did not modify cisplatin-dependent up-regulation of death receptors (Fas) or ligands (tumor necrosis factor ␣, Fas ligand). In contrast, cilastatin did not show any effects on cisplatintreated HeLa cells. Renal DHP-I was virtually absent in HeLa cells. Cilastatin attenuates cisplatin-induced cell death in proximal tubular cells without reducing the cytotoxic activity of cisplatin in tumor cells. Our findings suggest that the affinity of cilastatin for renal dipeptidase makes this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Therefore, cilastatin administration might represent a novel strategy in the prevention of cisplatin-induced acute renal injury.

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 μm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.

Cilastatin reduces cisplatin-induced nephrotoxicity, which is associated with decreased inflammation in vivo. Although the exact role of decreased inflammation in nephroprotection has not been fully elucidated, treatment with cilastatin could be a novel strategy for the prevention of cisplatin-induced acute kidney injury.