Sonia Camaño

Abnormal mitochondrial function in locomotor and respiratory muscles of COPD patients

Several cellular and molecular alterations have been described in skeletal and respiratory muscles of patients with chronic obstructive pulmonary disease (COPD), but information on potential abnormalities of mitochondrial function is scarce. The aim of the present study was to investigate mitochondrial function in the vastus lateralis (VL) and external intercostalis (EI) of COPD patients. Biopsies from VL and EI were obtained during surgery for lung cancer in 13 patients with mild to moderate COPD (age 68±6 yrs, forced expiratory volume in one second (FEV1) 66±15% predicted) and 19 control subjects (age 67±9 yrs, FEV1 95±18% pred). State 3 and 4 mitochondrial oxygen consumption (V′O2,m), ATP synthesis, citrate synthase, cytochrome oxidase (COX) and complex I–III activities, as well as reactive oxygen species (ROS) production, were determined. In COPD patients, in both muscles, COX activity (VL: COPD 3.0±0.8 versus control 2.0±0.8; EI: 3.7±1.6 versus 2.4±0.9 μmol·min−1·mg−1) and ROS production (VL: 1,643±290 versus 1,285±468; EI: 1,033±210 versus 848±288 arbitrary units) were increased, whereas state 3 V′O2,m was reduced (VL: 2.9±0.3 versus 3.6±0.4; EI: 3.6±0.3 versus 4.1±0.4 mmol·min−1·kg−1). Skeletal muscle mitochondria of patients with chronic obstructive pulmonary disease show electron transport chain blockade and excessive production of reactive oxygen species. The concurrent involvement of both vastus lateralis and external intercostalis suggests a systemic (rather than a local) mechanism(s) already occurring in relatively early stages (Global Initiative for Chronic Obstructive Lung Disease stage II) of the disease.

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 μM) in the presence or absence of cilastatin (200 μg/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 α, 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.

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 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.

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.

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.

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.

Novel Strategies in Drug-Induced Acute Kidney Injury

Proximal tubules recover more than 60% of total filtered load, i.e., a single molecule of toxin that is filtered and reabsorbed will pass through the proximal tubule cell more than 50 times per day. Such a high degree of exposure implies a risk of cell damage causing a variety of clinical syndromes, from proximal acidosis and acquired Fanconi syndrome to tubular cell necrosis (Oh, 2010). This spectrum of diseases is known as acute kidney injury (AKI), which also includes cell death by apoptosis, anoikis, necrosis, or cell dysfunction (Lorz et al., 2006).

Abstract 2659: Cilastatin attenuates cisplatin-induced proximal tubular cell damage without compromising antitumoral activity

Introduction: Cisplatin (CDDP) is a very effective and common treatment in solid malignancies. One of the most important side effects of CDDP is the nephrotoxicity. Nephrotoxicity is a limitating side effect on treatment with CDDP, preventing patients with limit kidney function of receiving the drug and stopping treatment once kidney function has worsened. Cilastatin (Cls) is a specific inhibitor of renal dedydrodipeptidase I (DHP-I) which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. In this work we hypothesized that Cls acts as a nephroprotector against CDDP-induced damage without compromising antitumor activity. Methods: Primary cultures of proximal tubular cells (PTCs) and cell lines of different malignancies (colon, breast, ovarian, bladder) were cultured with different concentrations of CDDP (1, 10 and 30 μM) in the presence or absence of Cls. Cell viability was assessed with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. Raft staining was measured with toxine B choleric and FasL by confocal microscopy. Results: Cls interfered with CDDP-induced FasL signalling at raft level on PTCs brush border. Concomitant treatment with Cls reduced CDDP-induced changes. Several tumoral cell lines were tested with CDDP and Cls together. Cls did not increase or decreased tumor growth alone or in combination with CDDP. CDDP sensitivity was not affected by Cls. Conclusion: By binding a DHP-I, Cls blocks CDDP-induced PTCs apoptosis but it does not affect the CDDP antitumoral activity. Our findings suggest that the affinity of Cls for renal DHP-I make this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Cls administration might represent a novel strategy in the prevention of CDDP-induced acute renal injury. Cls treatment could potentially increase the number of patients undergoing CDDP treatment or maintaining treatment. Further clinical trials for renal function preservation in cancer patients are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2659. doi:1538-7445.AM2012-2659