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1.
Biochim Biophys Acta Mol Basis Dis ; 1870(5): 167155, 2024 06.
Article in English | MEDLINE | ID: mdl-38579939

ABSTRACT

Tubular proteinuria is a common feature in COVID-19 patients, even in the absence of established acute kidney injury. SARS-CoV-2 spike protein (S protein) was shown to inhibit megalin-mediated albumin endocytosis in proximal tubule epithelial cells (PTECs). Angiotensin-converting enzyme type 2 (ACE2) was not directly involved. Since Toll-like receptor 4 (TLR4) mediates S protein effects in various cell types, we hypothesized that TLR4 could be participating in the inhibition of PTECs albumin endocytosis elicited by S protein. Two different models of PTECs were used: porcine proximal tubule cells (LLC-PK1) and human embryonic kidney cells (HEK-293). S protein reduced Akt activity by specifically inhibiting of threonine 308 (Thr308) phosphorylation, a process mediated by phosphoinositide-dependent kinase 1 (PDK1). GSK2334470, a PDK1 inhibitor, decreased albumin endocytosis and megalin expression mimicking S protein effect. S protein did not change total TLR4 expression but decreased its surface expression. LPS-RS, a TLR4 antagonist, also counteracted the effects of the S protein on Akt phosphorylation at Thr308, albumin endocytosis, and megalin expression. Conversely, these effects of the S protein were replicated by LPS, an agonist of TLR4. Incubation of PTECs with a pseudovirus containing S protein inhibited albumin endocytosis. Null or VSV-G pseudovirus, used as control, had no effect. LPS-RS prevented the inhibitory impact of pseudovirus containing the S protein on albumin endocytosis but had no influence on virus internalization. Our findings demonstrate that the inhibitory effect of the S protein on albumin endocytosis in PTECs is mediated through TLR4, resulting from a reduction in megalin expression.


Subject(s)
Endocytosis , Kidney Tubules, Proximal , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Toll-Like Receptor 4 , Toll-Like Receptor 4/metabolism , Endocytosis/drug effects , Humans , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/virology , Animals , Spike Glycoprotein, Coronavirus/metabolism , SARS-CoV-2/metabolism , HEK293 Cells , Swine , Proto-Oncogene Proteins c-akt/metabolism , Phosphorylation , COVID-19/metabolism , COVID-19/virology , COVID-19/pathology , Albumins/metabolism , LLC-PK1 Cells , Epithelial Cells/metabolism , Epithelial Cells/virology
2.
Peptides ; 146: 170646, 2021 12.
Article in English | MEDLINE | ID: mdl-34500007

ABSTRACT

Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.


Subject(s)
Albumins/metabolism , Bradykinin/pharmacology , Endocytosis/drug effects , Kidney Tubules, Proximal/drug effects , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Animals , Cell Line , Enzyme Activation , Kidney Tubules, Proximal/metabolism , LLC-PK1 Cells , Protein Kinase C/metabolism , Receptor, Bradykinin B2/metabolism , Swine
3.
Toxins (Basel) ; 13(1)2021 01 05.
Article in English | MEDLINE | ID: mdl-33466223

ABSTRACT

Massive, Africanized honeybee attacks have increased in Brazil over the years. Humans and animals present local and systemic effects after envenomation, and there is no specific treatment for this potentially lethal event. This study evaluated the ability of a new Apilic antivenom, which is composed of F(ab')2 fraction of specific immunoglobulins in heterologous and hyperimmune equine serum, to neutralize A. mellifera venom and melittin, in vitro and in vivo, in mice. Animal experiments were performed in according with local ethics committee license (UFRJ protocol no. DFBCICB072-04/16). Venom dose-dependent lethality was diminished with 0.25-0.5 µL of intravenous Apilic antivenom/µg honeybee venom. In vivo injection of 0.1-1 µg/g bee venom induced myotoxicity, hemoconcentration, paw edema, and increase of vascular permeability which were antagonized by Apilic antivenom. Cytotoxicity, assessed in renal LLC-PK1 cells and challenged with 10 µg/mL honeybee venom or melittin, was neutralized by preincubation with Apilic antivenom, as well the hemolytic activity. Apilic antivenom inhibited phospholipase and hyaluronidase enzymatic activities. In flow cytometry experiments, Apilic antivenom neutralized reduction of cell viability due to necrosis by honeybee venom or melittin. These results showed that this antivenom is effective inhibitor of honeybee venom actions. Thus, this next generation of Apilic antivenom emerges as a new promising immunobiological product for the treatment of massive, Africanized honeybee attacks.


Subject(s)
Antivenins/therapeutic use , Bee Venoms/antagonists & inhibitors , Bites and Stings/drug therapy , Melitten/antagonists & inhibitors , Animals , Antibodies/blood , Bees , Brazil , Cell Line , Cell Survival , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Hemolysis/drug effects , Horses , Hyaluronoglucosaminidase/antagonists & inhibitors , Immunoglobulin Fab Fragments/therapeutic use , Injections, Intradermal , LLC-PK1 Cells , Lethal Dose 50 , Male , Mice , Models, Animal , Neutralization Tests , Phospholipases/antagonists & inhibitors , Swine
4.
Molecules ; 25(19)2020 Sep 27.
Article in English | MEDLINE | ID: mdl-32992605

ABSTRACT

Isoliquiritigenin (IsoLQ) is a flavonoid with antioxidant properties and inducer of endoplasmic reticulum (ER) stress. In vitro and in vivo studies show that ER stress-mediated hormesis is cytoprotective; therefore, natural antioxidants and ER stress inducers have been used to prevent renal injury. Oxidative stress and ER stress are some of the mechanisms of damage involved in cisplatin (CP)-induced nephrotoxicity. This study aims to explore whether IsoLQ pretreatment induces ER stress and produces hormesis to protect against CP-induced nephrotoxicity in Lilly Laboratories Cell-Porcine Kidney 1 (LLC-PK1) cells. During the first stage of this study, both IsoLQ protective concentration and pretreatment time against CP-induced toxicity were determined by cell viability. At the second stage, the effect of IsoLQ pretreatment on cell viability, ER stress, and oxidative stress were evaluated. IsoLQ pretreatment in CP-treated cells induces expression of glucose-related proteins 78 and 94 kDa (GRP78 and GRP94, respectively), attenuates CP-induced cell death, decreases reactive oxygen species (ROS) production, and prevents the decrease in glutathione/glutathione disulfide (GSH/GSSG) ratio, free thiols levels, and glutathione reductase (GR) activity. These data suggest that IsoLQ pretreatment has a moderately protective effect on CP-induced toxicity in LLC-PK1 cells, through ER stress-mediated hormesis, as well as by the antioxidant properties of IsoLQ.


Subject(s)
Chalcones/pharmacology , Cisplatin/adverse effects , Endoplasmic Reticulum Stress/drug effects , Hormesis/drug effects , Oxidative Stress/drug effects , Animals , Cisplatin/pharmacology , LLC-PK1 Cells , Swine
5.
Toxicol Appl Pharmacol ; 401: 115074, 2020 08 15.
Article in English | MEDLINE | ID: mdl-32464218

ABSTRACT

The natural naphthoquinones lapachol, α- and ß-lapachone are found in Bignoniaceous Brazilian plant species of the Tabebuia genus (synonym Handroanthus) and are recognized for diverse bioactivities, including as antimalarial. The aim of the present work was to perform in silico, in vitro and in vivo studies to evaluating the antimalarial potential of these three naphthoquinones in comparison with atovaquone, a synthetic antimalarial. The ADMET properties of these compounds were predicted in silico by the preADMET program. The in vitro toxicity assays were experimentally determined in immortalized and tumoral cells from different organs. In vivo acute oral toxicity was also evaluated for lapachol. Several favorable pharmacokinetics data were predicted although, as expected, high cytotoxicity was experimentally determined for ß-lapachone. Lapachol was not cytotoxic or showed low cytotoxicity to all of the cells assayed (HepG2, A549, Neuro 2A, LLC-PK1, MRC-5), it was nontoxic in the acute oral test and disclosed the best parasite selectivity index in the in vitro assays against chloroquine resistant Plasmodium falciparum W2 strain. On the other hand, α- and ß-lapachone were more potent than lapachol in the antiplasmodial assays but with low parasite selectivity due to their cytotoxicity. The diversity of data here reported disclosed lapachol as a promising candidate to antimalarial drug development.


Subject(s)
Antimalarials/administration & dosage , Atovaquone/administration & dosage , Computer Simulation , Drug Delivery Systems/methods , Naphthoquinones/administration & dosage , Plasmodium falciparum/drug effects , A549 Cells , Animals , Caco-2 Cells , Dogs , Drug Evaluation, Preclinical/methods , Female , Hep G2 Cells , Humans , LLC-PK1 Cells , Madin Darby Canine Kidney Cells , Mice , Naphthoquinones/isolation & purification , Plasmodium falciparum/physiology , Swine
6.
Int J Mol Sci ; 19(9)2018 Sep 14.
Article in English | MEDLINE | ID: mdl-30223494

ABSTRACT

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na⁺/K⁺-ATPase inhibition, they activate signal transduction via protein⁻protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 µM (marinobufagin) for pig kidney Na⁺/K⁺-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3ß inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3ß at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/ß-catenin pathway by acting upstream to ß-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.


Subject(s)
Bufanolides/pharmacology , LLC-PK1 Cells/drug effects , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Glycogen Synthase Kinase 3 beta/metabolism , MAP Kinase Signaling System/drug effects , Sodium-Potassium-Exchanging ATPase/metabolism , Swine , Wnt Signaling Pathway/drug effects
7.
Biochem Pharmacol ; 154: 482-491, 2018 08.
Article in English | MEDLINE | ID: mdl-29890144

ABSTRACT

LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, were upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner in the LLC-PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down-regulated following IR in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.


Subject(s)
Endocannabinoids/metabolism , Kidney Tubules, Proximal/metabolism , Reperfusion Injury/metabolism , Signal Transduction/physiology , Sodium-Potassium-Exchanging ATPase/biosynthesis , Animals , Benzoxazines/pharmacology , Benzoxazines/therapeutic use , Endocannabinoids/agonists , Kidney Tubules, Proximal/drug effects , LLC-PK1 Cells , Male , Morpholines/pharmacology , Morpholines/therapeutic use , Naphthalenes/pharmacology , Naphthalenes/therapeutic use , Rats , Rats, Wistar , Reperfusion Injury/drug therapy , Signal Transduction/drug effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Swine
8.
Food Chem Toxicol ; 109(Pt 1): 143-154, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28870684

ABSTRACT

Cisplatin is an effective antineoplastic agent widely used in the treatment of solid tumors, however, it induces nephrotoxicity. Cisplatin-induced nephrotoxicity is associated with increased reactive oxygen species (ROS) production and decreased antioxidant system defense in kidney. Isoliquiritigenin (IsoLQ) is a chalcone, which is characterized by its antioxidant and antiinflammatory properties. Herein, we investigated the protective effect of IsoLQ on LLC-PK1 proximal tubular cells against cisplatin-induced death and its effect on the antineoplastic activity of cisplatin on bladder cancer T24 cell line. It was found that pretreatment with IsoLQ attenuates cisplatin-induced cell death, ROS production, and activation of caspase-3. IsoLQ also induced heme oxygenase-1 (HO-1) expression that may be associated with nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation. The protective effect of IsoLQ was abrogated by tin mesoporphyrin (SnMP), an HO inhibitor. Further, bilirubin and carbon monoxide releasing molecule-2 also showed a protective effect against cisplatin-induced cell death. In addition, IsoLQ induced in a dose-dependent way, death of T24 cells and exacerbated cisplatin-induced cell death. These results suggest that IsoLQ has a protective effect on cisplatin-induced toxicity in LLC-PK1 cells, in part through induction of HO-1, without interfering with the antineoplastic activity of this agent in T24 cells.


Subject(s)
Antineoplastic Agents/adverse effects , Chalcones/pharmacology , Cisplatin/adverse effects , Urinary Bladder Neoplasms/physiopathology , Animals , Apoptosis/drug effects , Caspase 3/genetics , Caspase 3/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Kidney/drug effects , Kidney/metabolism , LLC-PK1 Cells , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Swine , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism
9.
Am J Physiol Renal Physiol ; 313(2): F440-F449, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28468964

ABSTRACT

ANG II has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of ß-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of ß-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.


Subject(s)
Angiotensin II/metabolism , Cell Membrane/metabolism , Endocytosis , Endoplasmic Reticulum/metabolism , Kidney/metabolism , Receptor, Angiotensin, Type 1/metabolism , Receptor, Angiotensin, Type 2/metabolism , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 2 Receptor Blockers/pharmacology , Animals , Calcium/metabolism , Cell Membrane/drug effects , Endocytosis/drug effects , Endoplasmic Reticulum/drug effects , Kidney/drug effects , LLC-PK1 Cells , Microtubules/metabolism , Multiprotein Complexes , Phosphorylation , Protein Kinase C-alpha/metabolism , Protein Transport , Receptor, Angiotensin, Type 1/drug effects , Receptor, Angiotensin, Type 2/drug effects , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Swine , beta-Arrestins/metabolism
10.
Toxicol Mech Methods ; 27(6): 435-441, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28372472

ABSTRACT

Drug-induced nephrotoxicity is one of the most frequently observed effects in long-term pharmacotherapy. The effects of nephrotoxicity are commonly discovered later due to a lack of sensitivity in in vivo methods. Therefore, researchers have tried to develop in vitro alternative methods for early identification of toxicity. In this study, LLC-PK1 cells were exposed to gentamicin through MTT and trypan blue assay. Concentrations of 4 (low), 8 (medium) and 12 (high) mM, were used to evaluate differential gene expression. A panel of genes was selected based on gene expression changes. The search for sequences of mRNA encoding proteins previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information (USA). RNA was extracted from the cells, and RT-qPCR was performed to evaluate differential expression profiles of the selected genes. Among the 11 analyzed genes, four proved to be differentially up-regulated in cells exposed to gentamicin: HAVcr1, caspase 3, ICAM-1 and EXOC6. According to this study's results, we suggest that these genes play an important role in the mechanism of in vitro nephrotoxicity caused by gentamicin and can be used as early in vitro biomarkers to identify nephrotoxicity when developing safer drugs.


Subject(s)
Gentamicins/toxicity , Kidney/drug effects , Mutagens/toxicity , Transcriptome/drug effects , Animals , Cell Culture Techniques , Cell Survival/drug effects , Dose-Response Relationship, Drug , Gene Expression Profiling , Genetic Markers , Kidney/metabolism , LLC-PK1 Cells , RNA, Messenger/metabolism , Swine , Up-Regulation
11.
PLoS One ; 11(8): e0161057, 2016.
Article in English | MEDLINE | ID: mdl-27532263

ABSTRACT

Polymyxins have a long history of dose-limiting toxicity, but the underlying mechanism of polymyxin B-induced nephrotoxicity is unclear. This study investigated the link between the nephrotoxic effects of polymyxin B on renal metabolic functions and mitochondrial morphology in rats and on the structural integrity of LLC-PK1 cells. Fifteen Wistar rats were divided into two groups: Saline group, rats received 3 mL/kg of 0.9% NaCl intraperitoneally (i.p.) once a day for 5 days; Polymyxin B group, rats received 4 mg/kg/day of polymyxin B i.p. once a day for 5 days. Renal function, renal hemodynamics, oxidative stress, mitochondrial injury and histological characteristics were assessed. Cell membrane damage was evaluated via lactate dehydrogenase and nitric oxide levels, cell viability, and apoptosis in cells exposed to 12.5 µM, 75 µM and 375 µM polymyxin B. Polymyxin B was immunolocated using Lissamine rhodamine-polymyxin B in LLC-PK1 cells. Polymyxin B administration in rats reduced creatinine clearance and increased renal vascular resistance and oxidative damage. Mitochondrial damage was confirmed by electron microscopy and cytosolic localization of cytochrome c. Histological analysis revealed tubular dilatation and necrosis in the renal cortex. The reduction in cell viability and the increase in apoptosis, lactate dehydrogenase levels and nitric oxide levels confirmed the cytotoxicity of polymyxin B. The incubation of LLC-PK1 cells resulted in mitochondrial localization of polymyxin B. This study demonstrates that polymyxin B nephrotoxicity is characterized by mitochondrial dysfunction and free radical generation in both LLC-PK1 cells and rat kidneys. These data also provide support for clinical studies on the side effects of polymyxin B.


Subject(s)
Anti-Bacterial Agents/toxicity , Kidney/drug effects , Polymyxin B/toxicity , Animals , Anti-Bacterial Agents/pharmacokinetics , Apoptosis/drug effects , Cell Survival/drug effects , Cytochromes c/metabolism , Kidney/metabolism , Kidney/pathology , L-Lactate Dehydrogenase/metabolism , LLC-PK1 Cells , Male , Microscopy, Electron, Transmission , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Necrosis , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Polymyxin B/pharmacokinetics , Rats , Rats, Wistar , Swine
12.
Drug Chem Toxicol ; 39(1): 28-34, 2016.
Article in English | MEDLINE | ID: mdl-25690107

ABSTRACT

Amphotericin B is the "gold standard" agent in the management of serious systemic fungal infections. However, this drug can cause nephrotoxicity, which contributes up to 25% of all acute kidney injuries in critically ill patients. Cyclic adenosine monophosphate can protect kidney cells from death due to injury or drug exposure in some cases. Hence, the objective of this work was to evaluate if cAMP could prevent cell death that occurs in renal cell lines subjected to AmB treatment and, if so, to assess the involvement of PKA in the transduction of this signal. Two different renal cell lines (LLC-PK1 and MDCK) were used in this study. MTT and flow cytometry assays showed increased cell survival when cells were exposed to cAMP in a PKA-independent manner, which was confirmed by western blot. This finding suggests that cAMP (db-cAMP) may prevent cell death caused by exposure to AmB. This is the first time this effect has been identified when renal cells are exposed to AmB's nephrotoxic potential.


Subject(s)
Amphotericin B/toxicity , Antifungal Agents/toxicity , Cyclic AMP/administration & dosage , Kidney/drug effects , Animals , Blotting, Western , Cell Survival/drug effects , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Dogs , Flow Cytometry , Kidney/pathology , LLC-PK1 Cells , Madin Darby Canine Kidney Cells , Signal Transduction/drug effects , Swine
13.
Br J Pharmacol ; 172(19): 4615-25, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25537261

ABSTRACT

BACKGROUND AND PURPOSE: The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70-80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na(+) in LLC-PK1 cells. EXPERIMENTAL APPROACH: Changes in Na(+) /K(+) -ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB1 receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na(+) transport. KEY RESULTS: In addition to CB1 and CB2 receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10(-7) M) and HP (10(-6) M) altered Na(+) re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na(+) /K(+) -ATPase activity in a TRPV1 antagonist-sensitive way. WIN's stimulating effect persisted for 30 min, and this effect was partially blocked by a CB1 antagonist or a PKC inhibitor. In contrast, HP inhibited Na(+) /K(+) -ATPase after 30 min incubation, and this effect was attenuated by a CB1 antagonist or a PKA inhibitor. CONCLUSION AND IMPLICATIONS: The ECS is expressed in LLC-PK1 cells. Both CB1 receptors and TRPV1 channels regulate Na(+) /K(+) -ATPase activity in these cells, and are modulated by lipid and peptide CB1 receptor ligands, which act via different signalling pathways.


Subject(s)
Endocannabinoids/metabolism , Kidney/metabolism , Receptor, Cannabinoid, CB1/metabolism , Sodium/metabolism , Animals , Benzoxazines/pharmacology , Biological Transport , Cyclic AMP/metabolism , Hemoglobins/pharmacology , LLC-PK1 Cells , Morpholines/pharmacology , Naphthalenes/pharmacology , Peptide Fragments/pharmacology , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB2/metabolism , Signal Transduction , Sodium-Potassium-Exchanging ATPase/metabolism , Swine , TRPV Cation Channels/metabolism
14.
Rev. panam. salud pública ; 36(5): 342-347, nov. 2014. tab
Article in Spanish | LILACS | ID: lil-733238

ABSTRACT

Este trabajo muestra, desde el punto de vista de la normatividad de la Organización Panamericana de la Salud (OPS), el proceso de gestación, la metodología de implementación y los resultados obtenidos de la iniciativa de formación de recursos humanos en salud vía e-learning a través del Campus Virtual de Salud Pública de la Universidad de Guadalajara, México, a seis años de su inicio. Se trata de un informe especial del trabajo realizado por el comité institucional del campus virtual en la región occidental de México para generar un portal de Internet que se ajustara a los lineamientos del Modelo Estratégico establecido por el Nodo México y la OPS para la Región de las Américas. Este Campus Virtual inició sus actividades en el año 2007. Su filosofía es el uso de software libre y la colaboración entre instituciones. El nodo fue implementado en un año y ha logrado capacitar a más de 500 profesionales de la salud a través de cursos virtuales, su plataforma educativa y un repositorio de recursos virtuales de aprendizaje con interoperabilidad con los repositorios de México y de la Región de las Américas. El comité del Campus Virtual de la Universidad de Guadalajara ha intentado respetar lo más posible al modelo propuesto, lo que ha permitido cumplir la mayoría de los objetivos fijados en el plan de trabajo inicial, aunque ha enfrentado una serie de dificultades administrativas y de motivación de sus integrantes.


This paper discusses the gestation process, implementation methodology, and results obtained from the initiative to use e-learning to train human resources for health, six years after the launch of the Virtual Campus of Public Health of the University of Guadalajara (Mexico); the discussion is framed by Pan American Health Organization (PAHO) standards and practices. This is a special report on the work done by the institutional committee of the Virtual Campus in western Mexico to create an Internet portal that follows the guidelines of the strategic model established by Nodo México and PAHO for the Region of the Americas. This Virtual Campus began its activities in 2007, on the basis of the use of free software and institutional collaboration. Since the initial year of implementation of the node, over 500 health professionals have been trained using virtual courses, the node's educational platform, and a repository of virtual learning resources that are interoperable with other repositories in Mexico and the Region of the Americas. The University of Guadalajara Virtual Campus committee has followed the proposed model as much as possible, thereby achieving most of the goals set in the initial work plan, despite a number of administrative challenges and the difficulty of motivating committee members.


Subject(s)
Animals , Dogs , Iron/toxicity , Kidney Tubules/drug effects , Adenylyl Cyclases/metabolism , /metabolism , Cell Division/drug effects , Cell Line , Epithelium/drug effects , Epithelium/pathology , Epithelium/physiology , Ferric Compounds/toxicity , Iron/metabolism , Kidney Tubules/pathology , Kidney Tubules/physiology , LLC-PK1 Cells , Microscopy, Electron , Swine , Wound Healing/drug effects
15.
Free Radic Res ; 48(11): 1342-54, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25119790

ABSTRACT

The potential protective effect of the dietary antioxidant curcumin (120 mg/Kg/day for 6 days) against the renal injury induced by maleate was evaluated. Tubular proteinuria and oxidative stress were induced by a single injection of maleate (400 mg/kg) in rats. Maleate-induced renal injury included increase in renal vascular resistance and in the urinary excretion of total protein, glucose, sodium, neutrophil gelatinase-associated lipocalin (NGAL) and N-acetyl ß-D-glucosaminidase (NAG), upregulation of kidney injury molecule (KIM)-1, decrease in renal blood flow and claudin-2 expression besides of necrosis and apoptosis of tubular cells on 24 h. Oxidative stress was determined by measuring the oxidation of lipids and proteins and diminution in renal Nrf2 levels. Studies were also conducted in renal epithelial LLC-PK1 cells and in mitochondria isolated from kidneys of all the experimental groups. Maleate induced cell damage and reactive oxygen species (ROS) production in LLC-PK1 cells in culture. In addition, maleate treatment reduced oxygen consumption in ADP-stimulated mitochondria and diminished respiratory control index when using malate/glutamate as substrate. The activities of both complex I and aconitase were also diminished. All the above-described alterations were prevented by curcumin. It is concluded that curcumin is able to attenuate in vivo maleate-induced nephropathy and in vitro cell damage. The in vivo protection was associated to the prevention of oxidative stress and preservation of mitochondrial oxygen consumption and activity of respiratory complex I, and the in vitro protection was associated to the prevention of ROS production.


Subject(s)
Curcumin/pharmacology , Electron Transport Complex I/metabolism , Hemodynamics/drug effects , Kidney Diseases/prevention & control , Mitochondria/drug effects , Oxidative Stress/drug effects , Oxygen Consumption/drug effects , Aldehyde Reductase/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/drug effects , Biomarkers/analysis , Blotting, Western , Electron Transport Complex I/drug effects , Enzyme Inhibitors/toxicity , Kidney Diseases/chemically induced , LLC-PK1 Cells , Lipid Peroxidation/drug effects , Male , Maleates/toxicity , Mitochondria/metabolism , Oxidation-Reduction , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Swine
16.
Rev Esc Enferm USP ; 48(2): 272-7, 2014 Apr.
Article in Portuguese | MEDLINE | ID: mdl-24918886

ABSTRACT

The aim of the study was to characterize the cell damage mechanisms involved in the pathophysiology of cytotoxicity of polymyxin B in proximal tubular cells (LLC - PK1) and discuss about the nurses interventions to identify at risk patients and consider prevention or treatment of nephrotoxicity acute kidney injury. This is a quantitative experimental in vitro study, in which the cells were exposed to 375µM polymyxin B sulfate concentration. Cell viability was determined by exclusion of fluorescent dyes and morphological method with visualization of apoptotic bodies for fluorescence microscopy. Cells exposed to polymyxin B showed reduced viability, increased number of apoptotic cells and a higher concentration of the enzyme lactate dehydrogenase. The administration of polymyxin B in vitro showed the need for actions to minimize adverse effects such as nephrotoxicity.


Subject(s)
Anti-Bacterial Agents/adverse effects , Kidney Diseases/chemically induced , Polymyxin B/adverse effects , Animals , Kidney Diseases/nursing , Kidney Diseases/prevention & control , LLC-PK1 Cells , Swine
17.
Rev. Esc. Enferm. USP ; Rev. Esc. Enferm. USP;48(2): 272-277, abr. 2014. tab, graf
Article in English | LILACS, BDENF - Nursing | ID: lil-711793

ABSTRACT

The aim of the study was to characterize the cell damage mechanisms involved in the pathophysiology of cytotoxicity of polymyxin B in proximal tubular cells (LLC - PK1) and discuss about the nurses interventions to identify at risk patients and consider prevention or treatment of nephrotoxicity acute kidney injury. This is a quantitative experimental in vitro study, in which the cells were exposed to 375μM polymyxin B sulfate concentration. Cell viability was determined by exclusion of fluorescent dyes and morphological method with visualization of apoptotic bodies for fluorescence microscopy. Cells exposed to polymyxin B showed reduced viability, increased number of apoptotic cells and a higher concentration of the enzyme lactate dehydrogenase. The administration of polymyxin B in vitro showed the need for actions to minimize adverse effects such as nephrotoxicity.
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El objetivo del estudio fue caracterizar los mecanismos de daño celular implicado en la fisiopatología de la citotoxicidad de la polimixina B en las células tubulares proximales (LLC-PK1) y discutir las propuestas de intervención de enfermería para identificar a los pacientes de riesgo y considerar la prevención o el tratamiento de la lesión renal aguda nefrotóxica. Corresponde a un estudio experimental cuantitativo in vitro, en el cual las células fueron expuestas a sulfato de polimixina B. La viabilidad celular se determinó por exclusión de los colorantes fluorescentes y el método morfológico con la visualización de cuerpos apoptóticos a la microscopía de fluorescencia. Las células expuestas a polimixina B demostraron reducción de la viabilidad, aumento de células apoptóticas y mayor concentración de la enzima lactato deshidrogenasa. La administración de polimixina B in vitro demostró la necesidad de realizar acciones en la práctica clínica para minimizar los efectos adversos como la nefrotoxicidad.


O objetivo do estudo foi caracterizar os mecanismos de lesão celular envolvidos na fisiopatologia da citotoxicidade da polimixina B em células tubulares proximais (LLC-PK1) e discutir as proposições de intervenção do enfermeiro para identificar os pacientes de risco e considerar a prevenção ou o tratamento para lesão renal nefrotóxica. Estudo experimental in vitro , onde as células foram expostas ao sulfato de polimixina B. A viabilidade celular foi determinada pela exclusão dos corantes fluorescentes e o método morfológico com visualização de corpos apoptóticos à microscopia de fluorescência. As células expostas à polimixina B apresentaram redução de viabilidade, aumento do número de células em apoptose e maior concentração da enzima desidrogenase láctea. A administração de polimixina B in vitro demonstrou a necessidade de ações na prática clínica para minimizar os efeitos adversos como a nefrotoxicidade.

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Subject(s)
Animals , Anti-Bacterial Agents/adverse effects , Kidney Diseases/chemically induced , Polymyxin B/adverse effects , Kidney Diseases/nursing , Kidney Diseases/prevention & control , LLC-PK1 Cells , Swine
18.
J Appl Toxicol ; 34(12): 1285-92, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24105867

ABSTRACT

Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling pathway in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B.


Subject(s)
Amphotericin B/toxicity , Antifungal Agents/toxicity , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytokines/biosynthesis , Kidney/drug effects , Nitric Oxide/biosynthesis , Animals , Cell Culture Techniques , Cell Survival/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , DNA Fragmentation/drug effects , Dogs , Interleukin-6/biosynthesis , Kidney/enzymology , Kidney/immunology , Kidney/pathology , LLC-PK1 Cells , Madin Darby Canine Kidney Cells , Signal Transduction , Swine , Tumor Necrosis Factor-alpha/biosynthesis
19.
Acta paul. enferm ; Acta Paul. Enferm. (Online);26(1): 57-62, 2013. tab
Article in Portuguese | LILACS, BDENF - Nursing | ID: lil-670268

ABSTRACT

OBJETIVO: Caracterizar a toxicidade da polimixina B (PmxB) em células renais em dosagem e tempos diferentes. MÉTODOS: Células LLC-PK1, cultivadas em placas multiwell de 12 poços, foram divididas nos seguintes grupos: Controle (CTL) - células mantidas em meio DMEM suplementado a 5%; G1 - células expostas à concentração de 75mM de PmxB; G2 - células expostas à concentração de 375mM de PmxB. Cada grupo foi avaliado nos tempos de 24, 48 e 72 horas quanto à viabilidade celular (Acridine Orange/Brometo de Etídio) e apoptose (Hoechst 33342). RESULTADOS: Os dados demonstraram a viabilidade celular e a apoptose à exposição de três doses de PmxB em três intervalos de tempo, com um aumento significativo da toxicidade à elevação das doses e ao maior tempo de permanência no antibiótico para apoptose. CONCLUSÃO: A citotoxicidade pela PmxB, no modelo de cultivo celular, se mostrou tempo e dose dependente, aumentando com a maior exposição e maior dose de antibiótico.


OBJECTIVE: To characterize the toxicity of polymyxin B (PmxB) in renal cell in different dosage and times. METHODS: LLC-PK1 cells grown in 12 well multiwell plates were divided into the following groups: Control (CTL) - cells maintained in DMEM supplemented with 5%; G1 - cells exposed to concentration of 75µM PmxB G2 - cells exposed to concentration of 375µM PmxB. Each group was assessed at 24,48 and 72 hours as for cell viability (Acridine orange/ethidium bromide) and apoptosis (Hoechst 33342). RESULTS: The data demonstrate the cell viability and apoptosis exposure of three doses of PmxB in three time intervals, with a significant increase in toxicity to high doses and longer duration of stay in the antibiotic to apoptosis. CONCLUSION: Cytotoxicity by PmxB in cell culture model, showed to be time and dose dependent, increasing with increased exposure and higher dose of antibiotic.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/toxicity , Apoptosis , LLC-PK1 Cells , In Vitro Techniques , Polymyxin B/administration & dosage , Polymyxin B/toxicity , Cell Survival , Evaluation Studies as Topic
20.
Kidney Blood Press Res ; 36(1): 320-34, 2012.
Article in English | MEDLINE | ID: mdl-23235337

ABSTRACT

BACKGROUND/AIMS: Fructose causes a sodium-sensitive hypertension and acutely reduces the urinary Na+ excretion, suggesting that it may regulate the activity of renal tubular sodium transporters. NHE3 is highly expressed in proximal tubule (PT), along with proteins that mediate fructose transport and metabolism. The present work was outlined to investigate whether fructose modulates proximal NHE3 activity and to elucidate the molecular mechanisms underlying this modulation. METHODS/RESULTS: Using in vivo stationary microperfusion, we observed that fructose stimulates NHE3 mediated JHCO3- reabsorption. The MAPK pathway is not involved in this activation, as demonstrated by using of MEK/MAPK inhibitors, whereas experiments using a PKA inhibitor suggest that PKA inhibition plays a role in this response. These results were confirmed in vitro by measuring the cell pH recovery rate after NH4Cl pulse in LLC-PK1, a pig PT cell line, which showed reduced cAMP levels and NHE3 phosphorylation at serine-552 (PKA consensus site) after fructose treatment. CONCLUSIONS: NHE3 activity is stimulated by fructose, which increases proximal tubule Na+ reabsorption. The molecular mechanisms involved in this process are mediated, at least in part, by downregulation of the PKA signaling pathway. Future studies are needed to address whether fructose-stimulated NHE3 activity may contribute to renal injury and hypertension.


Subject(s)
Fructose/pharmacology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Sodium-Hydrogen Exchangers/metabolism , Animals , Cell Line , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Fructokinases/metabolism , Glucose Transporter Type 2/metabolism , Glucose Transporter Type 5/metabolism , Kidney Tubules, Proximal/cytology , LLC-PK1 Cells , Male , Models, Animal , Rats , Rats, Wistar , Signal Transduction/drug effects , Signal Transduction/physiology , Sodium-Hydrogen Exchanger 3 , Swine
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