Nephroprotective Effect of Cilastatin against Gentamicin-Induced Renal Injury In Vitro and In Vivo without Altering Its Bactericidal Efficiency.

Gentamicin is a used antibiotic that causes nephrotoxicity in 10-20% of treatment periods, which limits its use considerably. Our results have shown that cilastatin may be a promising therapeutic alternative in toxin-induced acute kidney injury (AKI). Here, we investigated its potential use as a nephroprotector against gentamicin-induced AKI in vitro and in vivo. Porcine renal cells and rats were treated with gentamicin and/or cilastatin. In vivo nephrotoxicity was analyzed by measuring biochemical markers and renal morphology. Different apoptotic, oxidative and inflammatory parameters were studied at cellular and systemic levels. Megalin, mainly responsible for the entry of gentamicin into the cells, was also analyzed. Results show that cilastatin protects cells from gentamicin-induced AKI. Cilastatin decreased creatinine, BUN, kidney injury molecule-1 (KIM-1) and severe morphological changes previously increased by gentamicin in rats. The interference of cilastatin with lipid rafts cycling leads to decreased expression of megalin, and therefore gentamicin uptake and myeloid bodies, resulting in a decrease of apoptotic, oxidative and inflammatory events. Moreover, cilastatin did not prevent bacterial death by gentamicin. Cilastatin reduced gentamicin-induced AKI by preventing key steps in the amplification of the damage, which is associated to the disruption of megalin-gentamicin endocytosis. Therefore, cilastatin might represent a novel therapeutic tool in the prevention and treatment of gentamicin-induced AKI in the clinical setting.

Cisplatin-induced renal inflammation is ameliorated by cilastatin nephroprotection.

BACKGROUND: Cisplatin is a potent chemotherapeutic drug whose nephrotoxic effect is a major complication and a dose-limiting factor for antitumoral therapy. There is much evidence that inflammation contributes to the pathogenesis of cisplatin-induced nephrotoxicity. We found that cilastatin, a renal dehydropeptidase-I inhibitor, has protective effects in vitro and in vivo against cisplatin-induced renal damage by inhibiting apoptosis and oxidation. Here, we investigated the potential use of cilastatin to protect against cisplatin-induced kidney injury and inflammation in rats. METHODS: Male Wistar rats were divided into four groups: control, cilastatin-control, cisplatin and cilastatin-cisplatin. Nephrotoxicity was assessed 5 days after administration of cisplatin based on blood urea nitrogen, creatinine, glomerular filtration rate (GFR), kidney injury molecule (KIM)-1 and renal morphology. Inflammation was measured using the electrophoretic mobility shift assay, immunohistochemical studies and evaluation of inflammatory mediators. RESULTS: Compared with the control rats, cisplatin-administered rats were affected by significant proximal tubule damage, decreased GFR, increased production of inflammatory mediators and elevations in urea, creatinine and tissue KIM-1 levels. Cilastatin prevented these changes in renal function and ameliorated histological damage in cisplatin-administered animals. Cilastatin also reduced pro-inflammatory cytokine levels, activation of nuclear factor-κB and CD68-positive cell concentrations. CONCLUSIONS: 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.

Protective Effects of Cilastatin against Vancomycin-Induced Nephrotoxicity.

Vancomycin is a very effective antibiotic for treatment of severe infections. However, its use in clinical practice is limited by nephrotoxicity. Cilastatin is a dehydropeptidase I inhibitor that acts on the brush border membrane of the proximal tubule to prevent accumulation of imipenem and toxicity. The aim of this study was to investigate the potential protective effect of cilastatin on vancomycin-induced apoptosis and toxicity in cultured renal proximal tubular epithelial cells (RPTECs). Porcine RPTECs were cultured in the presence of vancomycin with and without cilastatin. Vancomycin induced dose-dependent apoptosis in cultured RPTECs, with DNA fragmentation, cell detachment, and a significant decrease in mitochondrial activity. Cilastatin prevented apoptotic events and diminished the antiproliferative effect and severe morphological changes induced by vancomycin. Cilastatin also improved the long-term recovery and survival of RPTECs exposed to vancomycin and partially attenuated vancomycin uptake by RPTECs. On the other hand, cilastatin had no effects on vancomycin-induced necrosis or the bactericidal effect of the antibiotic. This study indicates that cilastatin protects against vancomycin-induced proximal tubule apoptosis and increases cell viability, without compromising the antimicrobial effect of vancomycin. The beneficial effect could be attributed, at least in part, to decreased accumulation of vancomycin in RPTECs.

Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.

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.

Effects of exercise on mitochondrial DNA content in skeletal muscle of patients with COPD.

BACKGROUND: Exhausting exercise reduces the mitochondrial DNA (mtDNA) content in the skeletal muscle of healthy subjects due to oxidative damage. Since patients with chronic obstructive pulmonary disease (COPD) suffer enhanced oxidative stress during exercise, it was hypothesised that the mtDNA content will be further reduced. OBJECTIVE: To investigate the effects of exercise above and below the lactate threshold (LT) on the mtDNA content of skeletal muscle of patients with COPD. METHODS: Eleven patients with COPD (67 ± 8 years; forced expiratory volume in 1 s (FEV1) 45 ± 8%ref) and 10 healthy controls (66 ± 4 years; FEV1 90 ± 7% ref) cycled 45 min above LT (65% peak oxygen uptake (V'o2peak) and another 7 patients (65 ± 6 years; FEV1 50 ± 4%ref) and 7 controls (56 ± 9 years; FEV1 92 ± 6% ref) cycled 45 min below their LT (50% V'o2peak). Biopsies from the vastus lateralis muscle were obtained before exercise, immediately after and 1 h, 1 day and 1 week later to determine by PCR the mtDNA/nuclear DNA (nDNA) ratio (a marker of mtDNA content) and the expression of the peroxisome proliferator-activated receptor-γcoactivator-1α (PGC-1α) mRNA and the amount of reactive oxygen species produced during exercise was estimated from total V'o2. RESULTS: Skeletal muscle mtDNA/nDNA fell significantly after exercise above the LT both in controls and in patients with COPD, but the changes were greater in those with COPD. These changes correlated with production of reactive oxygen species, increases in manganese superoxide dismutase and PGC-1α mRNA and returned to baseline values 1 week later. This pattern of response was also observed, albeit minimised, in patients exercising below the LT. CONCLUSIONS: In patients with COPD, exercise enhances the decrease in mtDNA content of skeletal muscle and the expression of PGC-1α mRNA seen in healthy subjects, probably due to oxidative stress.

Cilastatin attenuates cisplatin-induced proximal tubular cell damage.

A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatin-induced 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 microM) in the presence or absence of cilastatin (200 microg/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 alpha, Fas ligand). In contrast, cilastatin did not show any effects on cisplatin-treated 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.

Differential proteomic analysis of cyclosporine A-induced toxicity in renal proximal tubule cells.

BACKGROUND: The use of cyclosporine A (CsA) as a potent immunosuppressant has been limited by its severe nephrotoxic effects. The mechanisms involved are haemodynamic but also related to direct toxic effects of CsA on proximal tubule epithelial cells. We focused on defining a proteomic profile in CsA-treated proximal tubule cells to distinguish the direct impact of CsA on these cells from overlapping haemodynamically mediated phenomena that occur in an in vivo system. METHODS: By means of high-throughput differential proteomic analyses and mass spectrometry techniques in CsA and vehicle-treated proximal tubule-derived cell lines of human and mouse origin, we determined proteins that change their expression in the presence of CsA. RESULTS: CsA-induced toxicity analyses revealed that 10 mM CsA for 24 h was the threshold condition to induce significant changes in cell viability and proteomic profile. We identified 38 differentially expressed proteins on CsA-treated mouse PCT3 and human HK-2 cells, related to protein metabolism, response to damage, cell organization and cytoskeleton, energy metabolism, cell cycle and nucleobase/nucleoside/nucleotidic metabolism. 1D and 2D western blot assays in crude extracts from CsA-treated cells or kidneys with impaired function upon CsA treatment revealed a correlation with proteomic changes or differential isoform expression, in randomly selected proteins. CONCLUSIONS: Proteins identified in this work might be useful markers to eventually distinguish CsA toxicity from chronic allograft nephropathy in protocol biopsies of transplanted patients, facilitating the adjustment of CsA doses to non-toxic ranges, as well as to study the impact of potential therapeutic interventions in an animal model.