Marta Prieto

Metformin prevents experimental gentamicin-induced nephropathy by a mitochondria-dependent pathway

The antidiabetic drug metformin can diminish apoptosis induced by oxidative stress in endothelial cells and prevent vascular dysfunction even in nondiabetic patients. Here we tested whether it has a beneficial effect in a rat model of gentamicin toxicity. Mitochondrial analysis, respiration intensity, levels of reactive oxygen species, permeability transition, and cytochrome c release were assessed 3 and 6 days after gentamicin administration. Metformin treatment fully blocked gentamicin-mediated acute renal failure. This was accompanied by a lower activity of N-acetyl-beta-D-glucosaminidase, together with a decrease of lipid peroxidation and increase of antioxidant systems. Metformin also protected the kidney from histological damage 6 days after gentamicin administration. These in vivo markers of kidney dysfunction and their correction by metformin were complemented by in vitro studies of mitochondrial function. We found that gentamicin treatment depleted respiratory components (cytochrome c, NADH), probably due to the opening of mitochondrial transition pores. These injuries, partly mediated by a rise in reactive oxygen species from the electron transfer chain, were significantly decreased by metformin. Thus, our study suggests that pleiotropic effects of metformin can lessen gentamicin nephrotoxicity and improve mitochondrial homeostasis.

Endoglin regulates nitric oxide-dependent vasodilatation

Endoglin is a membrane glycoprotein that plays an important role in cardiovascular development and angiogenesis. We examined the role of endoglin in the control of vascular tone by measuring nitric oxide (NO)‐dependent vasodilation in haploinsufficient mice (Eng+/−) and their Eng+/+ littermates. The vasodilatory effect of acetylcholine, bradykinin, and sodium nitroprusside was assessed in anesthetized mice; in isolated, perfused hindlimbs; and in aortic rings. The substantial hypotensive and vasodilatory response induced by acetylcholine and bradykinin in Eng+/+ was markedly reduced in Eng+/− mice. Both kinds of animals had similar responses to sodium nitroprusside, suggesting that the deficient vasodilatory effect is not due to a NO response impairment. Urinary and plasma concentrations of nitrites, a NO metabolite, were lower in Eng+/− than in Eng+/+ mice. The levels of endothelial nitric oxide synthase (eNOS) in kidneys and femoral arteries were about half in Eng+/− than in Eng+/+ mice and were also reduced in primary cultures of aortic endothelial cells from Eng+/− compared with those from Eng+/+ mice. Furthermore, overexpression or suppression of endoglin in cultured cells induced a marked increase or decrease in the protein levels of eNOS, respectively. Thus, our results in vivo and in vitro demonstrate a relationship between endoglin and NO‐dependent vasodilation mediated by the regulation of eNOS expression.

Subcellular targets of cisplatin cytotoxicity: an integrated view.

Cisplatin is a chemotherapeutic drug widely used against a variety of cancers. Its clinical utility is severely limited by its toxicity, which mainly affects, but is not limited to, the inner ear and renal tubules. Cisplatin toxicity is determined by target tissue and cell accumulation, subcellular handling and trafficking through diverse subcellular structures, and interaction with macromolecules. Cisplatin accumulates and stresses different organelles from which delay signaling is activated, including mitochondria, lysosomes, the endoplasmic reticulum, the nucleus, the cell membrane and cytoskeleton, and can also be found in the cytosol. This article critically summarizes the available information in order to establish the connection among its known subcellular effects in a hierarchical and integrative framework. Cisplatin causes different types of cell death in a concentration-dependent manner. Knowledge of the events and signaling leading to the different phenotypes is also intertwined within the model, within the scope of the potential utility of this information in the improvement of the pharmacotoxicological profile of this drug. Perspectives for the key aspects that need to be addressed by future investigation are also outlined.

Endoglin Regulates Cyclooxygenase-2 Expression and Activity

The endoglin heterozygous (Eng+/−) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng+/− mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E2 were observed in the Eng+/− mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng+/− but not in Eng+/+ mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N&ohgr;-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng+/+ mice. N&ohgr;-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-β1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng+/− mice.

Effect of the Long-Term Treatment with Trandolapril on Endoglin Expression in Rats with Experimental Renal Fibrosis Induced by Renal Mass Reduction

Background: Endoglin is a membrane glycoprotein that regulates TGF-β1 signaling. Previous studies have revealed that endoglin is upregulated in several models of experimental fibrosis, and that endoglin expression can counteract the fibrogenic effects of TGF-β1. As treatment with angiotensin converting enzyme (ACE) inhibitors reduces renal fibrosis by mechanisms that are, in part, not dependent on angiotensin II blockade, we have assessed the hypothesis that this effect could be mediated by endoglin upregulation. Methods: We have used the 5/6-nephrectomy renal mass reduction (RMR) model of renal fibrosis in rats treated (RMR+T) or not treated with the ACE inhibitor trandolapril (0.7 mg/kg/day). One, 3 and 5 months after RMR, mean arterial pressure and renal function were measured. In addition, renal fibrosis was evaluated quantitatively and endoglin, TGF-β1, collagen type I and collagen type IV expression was assessed by Northern blot and immunohistochemistry. Results: RMR induced a progressive increase in mean arterial pressure, urinary protein excretion and glomerular and tubulointerstitial fibrosis, which is accompanied by an increased expression of TGF-β1, endoglin and collagen types I and IV. Trandolapril treatment reduced systemic blood pressure and lessened proteinuria after RMR, as well as expression of TGF-β1, endoglin and collagens. Conclusion: The present study demonstrates an increased TGF-β1, endoglin, collagen type I and collagen type IV expression in rats with severe hypertension and renal damage. The effect of trandolapril to decrease renal fibrosis seems to be based in a reduced TGF-β1 expression but not in an increased expression of endoglin.

Endoglin haploinsufficiency promotes fibroblast accumulation during wound healing through Akt activation.

Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng+/−) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng+/− MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng+/− mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng+/− mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.

Temporal changes in renal endoglin and TGF-β1 expression following ureteral obstruction in rats

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-β1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-β1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-β1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson’s trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen α2(I), TGF-β1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen α2(I), collagen α1(IV), TGF-β1 and endoglin mRNA levels were detected in L kidneysvs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-β1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-β1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.ResumenLa acumulación de proteínas de matriz extracelular en el riñón y la pérdida de la función renal son características de la enfermedad renal crónica. La fibrosis túbulointersticial juega un papel fundamental en la progresión de las enfermedades renales. El factor de crecimiento transformante-beta1 (TGF-β1) es una citoquina profibrótica fundamental para el inicio y desarrollo de la fibrosis renal. La endoglina es una glicoproteína de membrana del sistema de receptores del TGF-β1. El objetivo de este trabajo es determinar la curva de expresión temporal de colágeno I y IV, endoglina y TGF-β1 en riñón de ratas con fibrosis túbulointersticial inducida por 1, 3, 10 y 17 días de obstrucción ureteral unilateral (OUU). En los riñones ligados (L), las tinciones con tricrómico de Masson y rojo Sirio permitieron detectar una marcada fibrosis túbulointersticial a los 17 días de la OUU, acompañada de un incremento de colágeno I detectado por inmunohistoquímica. Los análisis de Northern blot revelaron un incremento progressivo de la expresión de ARNm de colágeno α2(I), TGF-β1 y endoglina en los riñones L comparados con los no ligados (NL). A los 17 días de la OUU, se observó un incremento significativo en los niveles de ARNm de colágeno α2(I), colágeno α1(IV), TGF-β1 y endoglina en los riñones L con respecto a los NL. Se detectó un aumento significativo de endoglina en los riñones L frente a los NL a partir de los 10 días de la obstrucción. Los estudios immunohistoquímica localizaron el aumento de expresión de endoglina y TGF-β1 en el intersticio de los riñones L a los 17 días de la obstrucción. Como conclusión, este estudio demuestra que la expresión renal de endoglina aumenta de forma progresiva y paralela a la de su ligando TGF-β1 en el modelo de fibrosis túbulointersticial inducida por OUU.

Impaired wound repair in adult endoglin heterozygous mice associated with lower NO bioavailability

Endoglin (Eng) is a transmembrane glycoprotein that is mainly expressed in endothelial cells, but it is also present in the epidermis and skin appendages. To address the role of Eng in cutaneous wound healing, we compared the kinetics of reepithelialization in Eng heterozygous null (Eng(+/-)) mice and their normal littermates (Eng(+/+)) following skin wounds. The wound area was significantly larger in Eng(+/-) than in Eng(+/+) mice from 2 to 8 days after injury; overall wound closure was delayed by 1 to 2 days. In Eng(+/-) mice, keratinocytes at the wound edges exhibited impaired proliferation but were more migratory, as shown by their elongated morphology and increased keratin 17 expression. Inhibition of nitric oxide (NO) synthesis delayed healing in Eng(+/+) but not in Eng(+/-) mice. Administration of the NO donor LA-803 accelerated wound closure in Eng(+/-) mice, with no effect on normal littermates. The acute stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced Eng expression in mouse epidermal keratinocytes in vivo and in vitro associated with hyperproliferation. Similarly, the skin of Eng(+/-) mice failed to mount a hyperplastic response to acute stimulation with TPA. These results demonstrate an important involvement of Eng in wound healing that is associated with NO bioavailability.

Key role of oxidative stress in animal models of aminoglycoside nephrotoxicity revealed by a systematic analysis of the antioxidant-to-nephroprotective correlation

The clinical utility of aminoglycoside antibiotics is partly limited by their nephrotoxicity. Co-administration of a variety of candidate nephroprotectants has been tested at the preclinical level. According to a recent meta-analytic study, antioxidants are the only family of compounds with enough preclinical documentation to draw solid conclusions on their class nephroprotective capacity in animal models. In this study a systematic analysis of the relation between the level of antioxidation and the level of nephroprotection was performed. A regression model is presented which crosses the y-axis (i.e. the axis representing the level of nephroprotection) very nearly the zero value, meaning that maximal prevention of the oxidative stress induced by aminoglycosides results in almost maximal nephroprotection. This indicates that oxidative stress plays a central role in the hierarchy of pathophysiological mechanisms underlying aminoglycoside nephrotoxicity. In addition, this model may potentially serve: i) as a standard to evaluate the role of the antioxidant effect of candidate nephroprotectants; ii) to reveal additional, antioxidant-independent effects among those compounds providing more nephroprotection than that expected from its antioxidant activity; and thus iii) to discriminate and focus most effective nephroprotectants on clinical usage.

N-acetylcysteine transforms necrosis into apoptosis and affords tailored protection from cisplatin cytotoxicity

ABSTRACT Nephrotoxicity is the main limitation to the dosage and anticancer efficacy of cisplatin. Cisplatin produces tubular epithelial cell apoptosis and necrosis depending on the concentration of the drug. Protection from cisplatin nephrotoxicity must therefore tackle both cell death modes. For its ability to reduce cisplatin reactivity, in addition to its antioxidant effect, we tested and found that N‐acetylcysteine (NAC) was most effective at inhibiting cisplatin cytotoxicity. NAC has no significant effect on cell death induced by either cycloheximide or Fas activation, indicating a rather selective action. Pt‐DNA‐binding experiments suggest that the differential effectiveness of NAC is due to its capacity to quench cisplatin reactivity inside the cell. NAC abolishes cisplatin‐induced apoptosis, and transforms the necrosis induced by high concentrations of cisplatin into apoptosis. In fact, NAC allows the anti‐apoptotic molecule Bcl‐2 to reduce the cell death caused by pro‐necrotic concentrations of cisplatin, to a significantly greater extent than in the absence of NAC. In rats, a dosage of NAC that significantly ameliorates cisplatin nephrotoxicity, has little effect on gentamicin nephrotoxicity. These characteristics provide NAC with a rationale as a potential nephroprotectant specifically tailored to and especially effective for therapeutic courses with platinated antineoplastics, which prompts to deepening into further preclinical knowledge, and to initiate clinical studies with NAC and mixed therapies composed of NAC and antiapoptotic drugs. HIGHLIGHTSAntioxidant‐independent effects N‐acetylcysteine afford tubular cytoprotection.N‐acetylcysteine binds intracellular cisplatin and reduces its reactivity.N‐acetylcysteine transforms cisplatin‐induced necrosis into apoptosis.N‐acetylcysteine is a potential nephroprotectant of cisplatin nephrotoxicity.