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Tubulointerstitial fibrosis is characterized by an accumulation of extracellular matrix in the renal interstitium, myofibroblast activation, cell infiltration, and tubular cell apoptosis, leading to chronic renal failure. Activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β1 type I receptor with a pivotal role in endothelial proliferation and migration, but its role in the development of renal fibrosis is unknown. To assess this we used the unilateral ureteral obstruction model of tubulointerstitial fibrosis in ALK1 haploinsufficient (ALK1+/−) and wild-type mice. After 15 days, there was an increase in extracellular matrix protein expression in the obstructed kidneys from both ALK1+/+ and ALK1+/− mice, but obstructed kidneys from ALK1+/− mice showed significantly higher expression of type I collagen than those from wild-type mice. Ureteral obstruction increased kidney myofibroblasts markers (α-smooth muscle actin and S100A4), without differences between mouse genotypes. ALK1 expression was increased after ureteral obstruction, and this increased expression was located in myofibroblasts. Moreover, cultured renal fibroblasts from ALK1+/− mice expressed more collagen type I and fibronectin than fibroblasts derived from wild-type mice. Thus, ALK1 modulates obstruction-induced renal fibrosis by increased extracellular matrix synthesis in myofibroblasts, but without differences in myofibroblast number. [ABSTRACT FROM AUTHOR]

Abstract: It is estimated that over 10% of the adult population in developed countries have some degree of chronic kidney disease (CKD). CKD is a progressive and irreversible deterioration of the renal excretory function that results in implementation of renal replacement therapy in the form of dialysis or renal transplant, which may also lead to death. CKD poses a growing problem to society as the incidence of the disease increases at an annual rate of 8%, and consumes up to 2% of the global health expenditure. CKD is caused by a variety of factors including diabetes, hypertension, infection, reduced blood supply to the kidneys, obstruction of the urinary tract and genetic alterations. The nephropathies associated with some of these conditions have been modeled in animals, this being crucial to understanding their pathophysiological mechanism and assessing prospective treatments at the preclinical level. This article reviews and updates the pathophysiological knowledge acquired primarily from experimental models and human studies of CKD. It also highlights the common mechanism(s) underlying the most relevant chronic nephropathies which lead to the appearance of a progressive, common renal phenotype regardless of aetiology. Based on this knowledge, a therapeutic horizon for the treatment of CKD is described. Present therapy primarily based upon renin–angiotensin inhibition, future diagnostics and therapeutic perspectives based upon anti-inflammatory, anti-fibrotic and hemodynamic approaches, new drugs targeting specific signaling pathways, and advances in gene and cell therapies, are all elaborated. [Copyright &y& Elsevier]

Abstract: Strong evidence supports the idea that total peripheral resistance (TPR) is increased in all forms of human and experimental hypertension. Although the etiological participation of TPR in the origin and long-term maintenance of hypertension has been extensively debated, it now seems clear that the renal, nonadaptive, infinite gain-working, pressure-sensitive natriuresis and diuresis is the main mechanism of blood pressure control in the long term. The tissue, cellular, biochemical, and genetic sensors and executors of this process have not been fully identified yet, but the role of the renal medulla has gained growing attention as the physiopathological scenario in which the key regulatory elements reside. Specifically, the functionality of the renomedullary vasculature seems to be highly responsible for blood pressure control. The vasculature of the renal medulla becomes a new and more specific target for the therapeutic intervention of hypertension. Recent data on the effect of baroreceptor-controlled renal sympathetic activity on the long-term regulation of blood pressure are integrated. The renomedullary effects of the main antihypertensive drugs are discussed, and new perspectives for the therapeutic intervention of hypertension are outlined. Comparison of the genetic program of the renal medulla before and after the development of hypertension in spontaneously hypertensive and experimentally induced animal models might provide a mechanism for identifying the key genes that become activated or suppressed in the development of high blood pressure. These genes, their encoded proteins, or other elements related to their signalling and genetic pathways might serve as new and more specific targets for the pharmacological treatment of abnormally elevated blood pressure. Besides, proteins specifically located to the luminal side of the renomedullary vascular endothelium may serve as potential targets for site-directed drug and gene therapy. [Copyright &y& Elsevier]

Objective: Renal impairment is a very frequent complication of aortic surgery requiring prolonged suprarenal clamping, especially if it is associated with previous hemorrhage. The aim of this study was to assess the beneficial effect of the administration of a nitric oxide (NO) donor on renal function through a modulation of the systemic inflammatory response in a model of abdominal aortic surgery. Methods: Twenty-five minipigs were divided into five groups. Under anesthesia, the animals were subjected to suprarenal aortic-iliac clamping (for 30 minutes) and bypass with a Dacron-collagen prosthetic graft impregnated in rifampicin, with or without associated hemorrhage (40% of total blood volume). Prophylaxis with cefazolin was implemented. The five groups were (1) the sham group (only aortic dissection), (2) the clamping and bypass (C) group, (3) hemorrhage preclamping and bypass (H+C) group, (4) the same as group C but with the administration of the NO donor molsidomine (4 mg/kg intravenously) (C+NO group), (5) the same as the H+C group but with the administration of the NO donor molsidomine (4 mg/kg intravenously) (H+C+NO group). The following were determined: (1) kidney function (serum creatinine), (2) serum cytokines (tumor necrosis factor α [TNF-α] and interleukin-10 [IL-10]); (3) neutrophil infiltration (myeloperoxidase [MPO]) in the kidney, (4) oxygen free radicals (superoxide anion [SOA] and superoxide dismutase [SOD]) in the kidney, (5) serum nitrites, (6) soluble and kidney tissue cell adhesion molecule (soluble intercellular adhesion molecule-1 [sICAM-1], soluble vascular cell adhesion molecule-1 [sVCAM-1], intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]), (7) inducible nitric oxide synthase (iNOS) in the kidney, and (8) nuclear factor-κB (NF-κB) in the kidney. Determinations were made during ischemia at 15 minutes post-reperfusion; at 24, 48, and 72 hours; and on day 7. Results: The different insults used in the experimental model led to deterioration in kidney function and an increase in the systemic (and renal) inflammatory response at all levels investigated. Treatment with an NO donor, both with and without associated hemorrhage, reduced the inflammatory response at the systemic (TNF-α and IL-10) and kidney (MPO, SOA, and SOD) levels, normalizing kidney function. Likewise, exogenous administration of NO improved the excessive production of NO (nitrites) via iNOS. This was also reflected in a reduction in CAMs and of NF-κB expression. The hypotension induced by molsidomine was transitory and did not elicit hemodynamic repercussions. Conclusion: In our experimental model, prophylactic treatment with the NO donor molsidomine regulates the systemic inflammatory response and minimizes damage at the kidney level. Clinical Relevance: The importance of this article resides in the fact that an experimental study that clarifies the effect of the donors of NO under circumstances as similar as possible to those of the human clinic, such as aortic surgery under hypovolemic shock (ruptured aortic aneurysm) have been little studied, most of these studies being performed in rodents without bypass. Using a model with one or two simultaneous insults (aortic clamping with/without previous hemorrhage) that is very similar to the human clinical situation (abdominal aortic rupture), we confirm the findings of previous work related to the beneficial effects of NO donors. [Copyright &y& Elsevier]

Potential role of platelet activating factor in acute renal failure. The clinical condition of acute renal failure (ARF) can be caused by a diverse number of renal injuries, but it is generally characterized by a sharp reduction in the glomerular filtration rate (GFR). A lipid mediator, platelet activating factor (PAF), may be one of the entities responsible for causing the hemodynamic changes in the ARF kidney because it can act as a vasodilator or vasoconstrictor, depending upon its concentration. This review examines the action and mechanisms of PAF in experimental animal models of ischemia and nephrotoxicity, as well as renal failure associated with extrarenal diseases. While further research is necessary before extrapolating our current knowledge of PAF into the prevention of renal failure or therapeutic intervention using PAF antagonists in human ARF, there is reasonable evidence to support its role as a mediator of the decrease in GFR characteristic of ARF. [ABSTRACT FROM AUTHOR]

Abstract: Fibrosis is a common phenomenon associated with several pathologies, characterized by an excessive extracellular matrix deposition that leads to a progressive organ dysfunction. Thus fibrosis has a relevant role in chronic diseases affecting the kidney, the liver, lung, skin (scleroderma) and joints (arthritis), among others. The pathogenesis of fibrosis in different organs share numerous similarities, being one of them the presence of activated fibroblasts, denominated myofibroblast, which act as the main source of extracellular matrix proteins. Transforming growth factor beta-1 (TGF-β1) is a profibrotic cytokine that plays a pivotal role in fibrosis. The TGF-β1/ALK5/Smad3 signaling pathway has been studied in fibrosis extensively. However, an increasing number of studies involving the ALK1/Smad1 pathway in the fibrotic process exist. In this review we offer a perspective of the function of ALK1/Smad1 pathway in renal fibrosis, liver fibrosis, scleroderma and osteoarthritis, suggesting this pathway as a powerful therapeutical target. We also propose several strategies to modulate the activity of this pathway and its consequences in the fibrotic process. [Copyright &y& Elsevier]

Transforming growth factor-β (TGF-β) is a cytokine known to participate in several processes related to the development of chronic kidney disease (CKD), including tubular degeneration. This is thought to occur mainly through apoptosis and epithelial-to-mesenchymal transition (EMT) of tubule epithelial cells, which give rise to a reduction of the tubular compartment and a scarring-like, fibrotic healing process of the interstitial compartment. In vivo blockade of TGF-β action has been shown to reduce CKD-associated tubular damage. However, a direct action of TGF-β on tubule cells is controversial as the underlying mechanism. On the one hand, TGF-β is known to induce EMT of tubular cells, although its incidence in vivo can hardly explain the extent of the damage. On the other hand, a few publications have reported that TGF-β induces a mild degree of apoptosis in cultured tubular cells. This most likely reflects the consequence of the cell-cycle arrest rather than a direct pro-apoptotic effect of TGF-β. The implications of these observations are analyzed in the pathological context, where normal tubular cells do not normally proliferate, but they might divide for repair purposes. Furthermore, renal fibrosis, a TGF-β-mediated event, is integrated as a potential, indirect effect contributing to tubule deletion. [ABSTRACT FROM AUTHOR]

Abstract: The correlation of erythropoietin (EPO) receptor levels with angiogenesis and progression in some cancers has suggested that EPO could acts directly as an angiogenic factor. The purpose of this study was to assess the effect of treatment with human recombinant erythropoietic (rHuEPO) agents in cancer patients with chemotherapy-induced anaemia on endoglin and vascular endothelial growth factor (VEGF) circulating levels as a possible marker of angiogenesis. Endoglin and VEGF were measured in serum samples from 25 cancer patients with chemotherapy-induced anemia before and after 3–4 weeks of treatment with rHuEPO. A group of 28 healthy voluntaries was used as control. VEGF serum levels were significantly higher in cancer patients than in controls. For endoglin, higher levels were observed without reaching statistical significance. No statistically significant differences in endoglin and VEGF serum levels were found between samples obtained before and after treatment with rHuEPO agents. In conclusion, our result do not support that rHuEpo treatment in anaemic cancer patients induce angiogenesis. [Copyright &y& Elsevier]

Strategies to minimize the nephrotoxicity of platinated antineoplastics without affecting its antitumour efficacy are strongly necessary to improve the pharmacotoxicological profile of these drugs. The natural flavonoid quercetin has been shown to afford nephroprotection without affecting cisplatin antitumour effect. The purpose of the present study has been to assess the differential mechanisms of action of cisplatin and quercetin on kidney and tumour tissues that could explain these effects. Wistar rats bearing subcutaneous tumours were treated with cisplatin and quercetin (and the appropriate controls). Tumour size and renal function evolution was monitored during 6 days. Platinum and quercetin content were also determined in both tissues. All the parameters studied, including blood supply, inflammation, apoptosis, critical MAPK signaling and oxidative stress in the cisplatin-treated animals are almost normalized by quercetin in the kidneys, but unaffected in the tumours. Our results suggest that in a cancer model in vivo , the protection exerted by quercetin on cisplatin nephrotoxicity is related to its antioxidant, vascular, anti-inflammatory and antiapoptotic effects, but these properties do not affect the mechanisms responsible for the antitumour effect of cisplatin. [ABSTRACT FROM AUTHOR]

Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. [ABSTRACT FROM AUTHOR]

The understanding of renal fibrosis in chronic kidney disease (CKD) remains as a challenge. More than 10% of the population of developed countries suffer from CKD. Proliferation and activation of myofibroblasts and accumulation of extracellular matrix proteins are the main features of kidney fibrosis, a process in which a large number of cytokines are involved. Targeting cytokines responsible for kidney fibrosis development might be an important strategy to face the problem of CKD. The increasing knowledge of the signaling pathway network of the transforming growth factor beta (TGF-β) superfamily members, such as the profibrotic cytokine TGF-β1 or the bone morphogenetic proteins (BMPs), and their involvement in the regulation of kidney fibrosis, has stimulated numerous research teams to look for potential strategies to inhibit profibrotic cytokines or to enhance the anti-fibrotic actions of other cytokines. The consequence of all these studies is a better understanding of all these canonical (Smad-mediated) and non-canonical signaling pathways. In addition, the different receptors involved for signaling of each cytokine, the different combinations of type I–type II receptors, and the presence and function of co-receptors that can influence the biological response have been also described. However, are these studies leading to suitable strategies to block the appearance and progression of kidney fibrosis? In this review, we offer a critical perspective analyzing the achievements using the most important strategies developed up till now: TGF-β antibodies, chemical inhibitors of TGF-β receptors, miRNAs and signaling pathways and BMP agonists with a potential role as therapeutic molecules against kidney fibrosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

We studied whether nephrotoxic drug administration sensitizes to acute renal failure (ARF) by administering a sub-nephrotoxic dose of gentamicin. This pre-treatment sensitized animals with no sign of renal injury to develop ARF when exposed to a second potential nephrotoxic drug, also given at sub-nephrotoxic doses that would be otherwise harmless to non-sensitized animals. We identified urinary ganglioside M2 activator protein (GM2AP) as a biomarker of an enhanced sensitivity to suffer ARF following sub-nephrotoxic treatment with gentamicin. Sub-nephrotoxic gentamicin did not alter renal GM2AP gene expression or protein levels, determined by reverse transcriptase-PCR, western blot, and immunostaining, nor was its serum level modified. The origin of increased GM2AP in the urine is thought to be a defective tubular handling of this protein as a consequence of gentamicin action. Hence, markers of acquired sensitivity may improve the prevention of ARF by enhancing our capacity to monitor for this condition, in a preemptive manner. [ABSTRACT FROM AUTHOR]

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-β-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. [ABSTRACT FROM AUTHOR]

Tubulointerstitial fibrosis is characterized by the presence of myofibroblasts that contribute to extracellular matrix accumulation. These cells may originate from resident fibroblasts, bone-marrow-derived cells, or renal epithelial cells converting to a mesenchymal phenotype. Ras GTPases are activated during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-β-induced epithelial–mesenchymal transition. Here we set out to assess the contribution of Ras to experimental renal fibrosis using the well-established model of unilateral ureteral obstruction. Fifteen days after obstruction, both fibroblast proliferation and inducers of epithelial–mesenchymal transition were lower in obstructed kidneys of H-ras knockout mice and in fibroblast cell lines derived from these mice. Interestingly, fibronectin, collagen I accumulation, overall interstitial fibrosis, and the myofibroblast population were also lower in the knockout than in the wild-type mice. As expected, we found lower levels of activated Akt in the kidneys and cultured fibroblasts of the knockout. Whether Ras inhibition will turn out to prevent progression of renal fibrosis will require more direct studies. [ABSTRACT FROM AUTHOR]

Abstract: Telomere shortening and redox imbalance have been related to the aging process. We used cultured mouse embryonic fibroblasts (MEF) isolated from mice lacking telomerase activity (Terc−/−) to analyze the redox balance and the functional consequences promoted by telomerase deficiency. Comparison with wild-type (WT) MEF showed that Terc−/− MEF had greater oxidant damage, showing higher superoxide anion and hydrogen peroxide production and lower catalase activity. Restoration of telomerase activity in Terc−/− MEF increased catalase expression and activity. TGF-β1 and collagen type IV levels were higher in Terc−/− than in WT MEF. TGF-β1 promoter activity decreased when Terc−/− MEF were incubated with exogenous catalase, suggesting that catalase deficiency is the cause of the TGF-β1 increase. Similar results were obtained in vivo. Homogenized renal cortex from 6-month-old Terc−/− showed higher oxidant capacity, lower catalase activity, greater oxidative damage, and higher TGF-β1 and fibronectin levels than that from WT mice. In summary, telomerase deficiency reduces catalase activity, determining a redox imbalance that promotes overexpression of TGF-β1 and extracellular matrix proteins. [Copyright &y& Elsevier]

Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.Kidney International (2008) 74, 196–209; doi:10.1038/ki.2008.160; published online 30 April 2008 [ABSTRACT FROM AUTHOR]

Abstract: Gentamicin is an aminoglycoside antibiotic that is very effective in treating different gram negative infections, however, one of its main side effects is nephrotoxicity. Gentamicin-induced decreases in glomerular filtration rate could be mediated by mesangial cell contraction. Resveratrol, a natural hydroxystilbene, has been identified to be a potent antioxidant with many biological activities including protection against kidney diseases. As we have previously demonstrated that gentamicin induced a reduction of planar surface area of cultured rat mesangial cells, and that resveratrol has a protective effect on gentamicin-induced nephrotoxicity in vivo, the aim of this study was to investigate the effect of resveratrol on gentamicin-induced mesangial cell contraction. This study demonstrates that the contractile effect of gentamicin on mesangial cells can be prevented by incubation with resveratrol at an optimal dose of 10 μM, as it blunted the gentamicin-induced reduction in planar cell surface area and the number of contracted cells. Besides, the preincubation with 10−5M diphenylene iodinium (DPI), an inhibitor of the NADP(H) oxidase, also blunted gentamicin-induced cell contraction. This preventive effect was higher when cells were incubated with both substances together. These results strongly suggest that the protective effect resveratrol against gentamicin-induced reduction in renal function in vivo could be mediated by inhibiting gentamicin-induced mesangial cells contraction. [Copyright &y& Elsevier]

Abstract: Reactive oxygen species (ROS) mediate MC contraction, proliferation and apoptosis induced by gentamicin (G) in vitro and in vivo. Sustained increases in cytosolic free calcium, increased iNOS expression and elevated nitric oxide (NO) production are associated with MC apoptosis in vitro. As NO strongly activated c-Jun N-terminal kinase (JNK) and increased AP1 expression, and these two factors are involved in MC proliferation in vitro, we have measured Jun-AP1 expression in rat glomeruli from G-treated rats, and the effect of G on Jun-AP1 expression and JNK activity in cultured MC. Moreover, we studied the expression of inducible (iNOS) and constitutive (cNOS) NO synthases in rat glomeruli. Glomeruli were obtained from rats treated with G (100 mg/kg body weight/day) along 6 days, and MC primary cultures were evaluated after 24, 48 and 72 h incubation with 10−5 M G. G induced an increase in the expression of iNOS, cNOS and Jun-AP1 in rat glomeruli and in MC cultures. Moreover, G activated JNK; JNK activation was reduced by co-incubation with the calcium channel blocker verapamil and with the ROS scavengers superoxide dismutase and catalase. These results strongly suggest a role for reactive oxygen/nitrogen species produced by increased NOS activity in G-induced MC activation. These reactive oxygen molecules and increased intracellular free calcium may mediate the increase in Jun-AP1 expression and JNK activation induced by G treatment in MC. [Copyright &y& Elsevier]

Bile duct ligation (BDL) in rats induces portal fibrosis. This process has been linked to changes in the oxidative state of the hepatic cells and in the production of nitric oxide. Our objective was to find possible temporal connections between hepatic redox state, NO synthesis and liver injury. In this work we have characterized hepatic lesions 17 and 31 days after BDL and determined changes in hepatic function, oxidative state, and NO production. We have also analyzed the expression and localization of inducible NO synthase (NOS2) and constitutive NO synthase (NOS3). After 17 and 31 days from ligature, lipid peroxidation is increased and both plasma concentration and biliary excretion of nitrite+nitrate are rised. 17 days after BDL both NOS2 and NOS3 are expressed intensely and in the same regions. 31 days after BDL, the expression of NOS2 remains elevated and is localized mostly in preserved hepatocytes in portal areas and in neighborhoods of centrolobulillar vein. NOS3 is localized in vascular regions of portal spaces and centrolobulillar veins and in preserved sinusoids and although its expression is greater than in control animals (34%), it is clearly lower (50%) than 17 days after BDL. The time after BDL is crucial in the study of NO production, intrahepatic localization of NOS isoforms expression, and cell type involved, since all these parameters change with time. BDL-induced, peroxidation and fibrosis are not ligated by a cause-effect relationship, but rather they both seem to be the consequence of common inductors. [Copyright &y& Elsevier]

Gentamicin treatment induces simultaneous mesangial proliferation and apoptosis in rats. Background. Gentamicin (G)-induced acute renal failure is characterized by an impairment of glomerular function without apparent changes in glomerular structure. However, G stimulates reactive oxygen species (ROS)-mediated mesangial cell proliferation in vitro. We studied whether G promotes mesangial cell apoptosis in vitro, and if apoptosis and proliferation in parallel may occur in glomerular cells in vivo after a renal damage induced by G treatment. Methods. For in vivo studies, rats were treated with G (100 mg/kg body weight/day) for 6 days, and functional and histologic studies were performed. For in vitro studies, mesangial cell proliferation and apoptosis were evaluated after 24, 48, and 72 hours of 10−5 mol/L G incubation. Results. After G injections, the number of nuclei per glomerulus did not change, whereas proliferating and apoptotic cell numbers increased. G increases DNA synthesis and cell number in cultured mesangial cells, and increases markedly the apoptotic cell number. ROS scavengers superoxide dismutase and catalase reduce G-induced mesangial cell apoptosis, whereas the incubation with the ROS donor system xanthine plus xanthine oxidase increases apoptosis to levels similar to G. G-induced cellular proliferation and apoptosis either in vitro or in vivo is associated to an early increase in the pro-apoptotic protein Bax and a delayed increase in the survival protein Bcl-2. Conclusion. G simultaneously induces proliferation and apoptosis of mesangial cells in vitro and glomerular mesangial cells in vivo. ROS may mediate G-induced mesangial apoptosis in vitro. The equilibrium proliferation/apoptosis may maintain mesangial cell number within normal limits after a G-induced glomerular insult. [ABSTRACT FROM AUTHOR]

Induction of DNA synthesis by ligation of the CD53 tetraspanin antigen in primary cultures of mesangial cells. Background. The interaction of mesangial cells with the extracellular matrix plays a major role in kidney biology. Tetraspanin proteins modulate cell interaction with the extracellular matrix. Tetraspanins form supramolecular structures on the cell membrane that send signals after engagement by unknown ligands, modulate different signaling processes, and regulate cell adhesion and motility. Methods. CD53 was determined by immunohistochemistry, and on the cell surface of cultured rat mesangial cells by flow cytometry. Mesangial cell cultures were stimulated with MRC OX-44 antibody. DNA synthesis was measured by thymidine incorporation. Extracellular signal-regulated kinase (ERK) activation was determined by Western blot. Results. CD53 was present in mesangial cells in vivo and in culture. Ligation of CD53 antigen with a monoclonal antibody triggered the induction of DNA synthesis, which was not sensitive to inhibitors of signaling pathways that use phosphatidylinositol 3-kinase (PI3K) and protein kinase C, or to calcium channel inhibitors, such as thapsigargin and verapamil. The DNA synthesis was inhibited by PD98059, a specific inhibitor of MEK that prevents ERK1/ERK2 activation. In addition, ERK1 and ERK2 activation by phosphorylation occurred following CD53 antigen ligation. The DNA synthesis was due to de novo synthesis and not to DNA repair as a consequence of the initiation of apoptosis, determined by flow cytometry, and lack of proteolytic activation of PARP by caspase 3. CD53 antigen ligation also induced an increase in mitochondrial activity. Conclusions. To our knowledge this is the first identification of a tetraspanin protein in mesangial cells. CD53 antigen delivers a signal that initiates DNA synthesis. This signal is mediated by ERK1/ERK2 activation, but it is not sufficient to complete the cell cycle. [ABSTRACT FROM AUTHOR]

Background. Reactive oxygen species (ROS) have been shown to be involved in the reduction of glomerular filtration rate observed after gentamicin (Genta) treatment in vivo, a phenomenon directly related with mesangial cell (MC) contraction. Our previous study reported that Genta induces concentration-dependent MC contraction and proliferation in vitro. Methods. To study the possible mediation of ROS in the effect of Genta, ROS production was measured in primary cultures of rat MC stimulated with Genta (10[sup -5] mol/L). In addition, the MC response to Genta in the presence of the ROS scavengers superoxide dismutase (SOD) and catalase (CAT) was studied. MC activation and O[sub 2, sup -] production were studied in the presence of an inhibitor of the NADP(H) oxidase, diphenylene iodinium (DPI), and in the presence of L-NAME, an inhibitor of nitric oxide synthases (NOS). Finally, the effects of Genta on SOD activity and mRNA expression were examined. Results. Genta (10[sup -5] mol/L) induced an increase in O[sub 2, sup -] production and SOD activity that was neither accompanied by an elevation in cytosolic Cu/Zn-SOD mRNA expression nor by H[sub 2]O[sub 2] accumulation. Genta induced MC contraction and proliferation that were inhibited by SOD plus CAT. Both the extracellular and intracellular ROS donor systems, xantine + xantine oxidase (X + XO) and dimethoxinaphtoquinone (DMNQ), respectively, also stimulated MC contraction and proliferation. Genta-induced MC activation and O[sub 2, sup -] production were inhibited by DPI. Genta-induced O[sub 2, sup -] production was inhibited by L-NAME. Furthermore, Genta did not induce detectable changes in membrane fluidity and lipid peroxidation. Conclusions. These results strongly suggest that an oxidative-mediated pathway exists in Genta-induced MC activation. A portion of the production of O[sub 2, sup -] may be due to NADP(H) oxidase and NOS activation. The amount of ROS produced, rather than having a toxic effect, might... [ABSTRACT FROM AUTHOR]

Glomerular cell proliferation and apoptosis in uninephrectomized spontaneously hypertensive rats. We studied renal function, glomerular cell proliferation and apoptosis for three months after uninephrectomy (UNX) in young, male, spontaneously hypertensive rats (SHR). Apoptosis was assessed by in situ dUTP biotin nick-end labeling method (TUNEL) and by propidium iodide staining. Proliferation rate was determined by immunohistochemistry to proliferating cell nuclear antigen (PCNA). Glomerular bcl-2 expression was assessed by Northern blot analysis. Our results indicate a parallel increase in proliferation and in apoptotic rates in glomerular cells from the first to the second month after UNX. In the third month after UNX, PCNA-labeled cell number continues increasing, whereas TUNEL-labeled cells did not increase. Bcl-2 expression was negative in the first and second months and increased in the third month. Glomerular size and proteinuria increased progressively along the three months of follow-up. Our observations demonstrate a different profile of cell proliferation and apoptosis during the genesis of early glomerular damage in UNX- SHR. [ABSTRACT FROM AUTHOR]