transforming-growth-factor-beta and Reperfusion-Injury

Liver failure after hepatectomy is caused by many factors such as excessive hepatectomy, ischemic-reperfusion injury, postoperative infection, etc. However, apoptosis of hepatocytes is the most important event in the molecular mechanism of liver failure. Liver failure after excessive hepatectomy is characterized by increased apoptosis of hepatocytes and diminished liver regeneration. The former is induced by hypercytokinemia and hyperendotoxemia, and the latter is caused by cell cycle arrest. In ischemic-reperfusion injury of the liver, the apoptosis of hepatocytes is caused by activation of the MMPT and calpain system that are induced by the intracellular accumulation of Ca. The possible mechanism of liver failure in cases with persistent infection is the inhibition of liver regeneration and the induction of apoptosis of hepatocytes due to transforming growth factor-beta 1. The knowledge of these mechanisms will lead to prompt and appropriate treatments for individual patients.

Topics: Animals; Apoptosis; Calcium; Calpain; Cell Membrane Permeability; Cytokines; Endotoxemia; Hepatectomy; Hepatocytes; Humans; Liver Failure; Mitochondria; Postoperative Complications; Reperfusion Injury; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta (TGF-beta) proteins and their antagonists have entered clinical trials. These multi-functional regulators of cell growth and differentiation induce extracellular matrix proteins and suppress the immune system making TGF-betas useful in treatment of wounds with impaired healing, mucositis, fractures, ischemia-reperfusion injuries, and autoimmune disease. In diseases such as keloids, glomerulonephritis and pulmonary fibrosis, excessive expression of TGF-beta has been implicated as being responsible for accumulation of detrimental scar tissue. In these conditions, agents that block TGF-beta have prevented or reversed disease. Similarly, in carcinogenesis, blocking TGF-beta activity may be valuable in stimulating an immune response towards metastasis. As these blocking agents receive approval, we will likely have new therapies for previously recalcitrant diseases.

Topics: Animals; Autoimmune Diseases; Humans; Reperfusion Injury; Transforming Growth Factor beta; Wounds and Injuries

TGF-beta1 has been demonstrated to be up-regulated in response to ischemic events both in animal models and in man. Demonstration of this up-regulation in the kidney following experimentally induced acute renal failure and in renal transplants complements similar findings in coronary and cerebral ischemia. Activation of TGF-beta1 occurs as a direct consequence of hypoxia, angiotensin II signaling and loss of extra cellular matrix (ECM) integrity, all of which occur in renal ischemia-reperfusion injury. TGF-beta1 thus up-regulates the synthesis of extracellular matrix components such as fibronectin and collagen IV providing a basis for the restoration of epithelial coverage in the regenerating tubule. TGF-beta1 also regulates epithelial tubular cell proliferation and differentiation. This response is quickly closed down in response to recovery of the kidney. This review examines the evidence linking TGF-beta1 activity to recovery from renal ischemia thereby constructing a hypothesis for the beneficial role of TGF-beta1 in the post ischemic kidney.

Topics: Acute Kidney Injury; Angiotensin II; Animals; Humans; Ischemia; Kidney; Reperfusion Injury; Transforming Growth Factor beta; Up-Regulation

Cell death by apoptosis is thought to be involved in various pathophysiological situations involving the liver. Indeed, an understanding of apoptosis is becoming increasingly helpful for understanding disease and for patients' care. In this article, we review current scientific and clinical concepts of apoptosis, including death factors such as Fas ligand and tumor necrosis factor, apoptotic signal transduction mechanisms, and the role of intracellular proteinases called caspases. We also discuss apoptosis in the liver, as related to ischemia/reperfusion injury, cholestasis, and cancer, circumstances which physicians often face in the field of the liver surgery.

Topics: Apoptosis; Humans; Liver Diseases; Liver Neoplasms; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

TGF-beta appears to be an important regulatory peptide in cellular physiology. Although all of its actions are not presently known, TGF-beta functions as a cell-switching molecule. In the case of ischemia-reperfusion states, TGF-beta has been shown to exert remarkably effective protective effects. These effects appear to pertain to preservation of endothelial function, particularly to maintenance of EDRF formation by the endothelium. The endothelial protection may be related to actions of TGF-beta opposing the endothelial-destabilizing actions of both TNF and superoxide radicals. However, other important mechanisms will undoubtedly be brought to light with further study of TGF-beta in these situations.

Topics: Animals; Cells, Cultured; Endothelium, Vascular; Free Radicals; Humans; Myocardial Reperfusion Injury; Reperfusion Injury; Splanchnic Circulation; Transforming Growth Factor beta; Vasodilation

Topics: Acute Kidney Injury; Animals; Clodronic Acid; Cytokines; Fibrosis; Interleukin-10; Liposomes; Macrophages; Mice; Mice, Inbred C57BL; Renal Insufficiency, Chronic; Reperfusion Injury; Transforming Growth Factor beta

As a part of natural disease progression, acute kidney injury (AKI) can develop into chronic kidney disease via renal fibrosis and inflammation. LTBP4 (latent transforming growth factor beta binding protein 4) regulates transforming growth factor beta, which plays a role in renal fibrosis pathogenesis. We previously investigated the role of LTBP4 in chronic kidney disease. Here, we examined the role of LTBP4 in AKI.. LTBP4 expression was upregulated in the renal tissues of patients with AKI.. Our study is the first to demonstrate that LTBP4 deficiency increases AKI severity, consequently leading to chronic kidney disease. Potential therapies focusing on LTBP4-associated angiogenesis and LTBP4-regulated DRP1-dependent mitochondrial division are relevant to renal injury.

Topics: Acute Kidney Injury; Animals; Endothelial Cells; Fibrosis; Humans; Inflammation; Kidney; Latent TGF-beta Binding Proteins; Mice; Mice, Inbred C57BL; Mitochondria; Renal Insufficiency, Chronic; Reperfusion Injury; Transforming Growth Factor beta

To elucidate the protective mechanism of lobetyolin on oxygen-glucose deprivation/reperfusion (OGD/R)-induced damage in BV2 microglial cells. The OGD/R model was established using a chemical modeling method to simulate in vivo brain ischemia in lobetyolin-pretreated BV2 cells. The optimum lobetyolin dosage, chemical concentration, and OGD/R modeling duration were screened. The changes in cell morphology were observed, and the levels of immune response-related factors, including tumor necrosis factor-α (TNF-α), interleukin-6, inducible nitric oxide synthase (iNOS), and cluster of differentiation (CD)206, were detected using the enzyme-linked immunosorbent assay. The expression of chemokine-like-factor-1 (CKLF1), hypoxia-inducible factor (HIF)-1α, TNF-α, and CD206, was detected using western blotting. The gene expression of M1 and M2 BV2 phenotype markers was assessed using quantitative polymerase chain reaction (qPCR). The localization of M1 and M2 BV2 markers was detected using immunofluorescence analysis. The results showed that lobetyolin could protect BV2 cells from OGD/R-induced damage. After OGD/R, CKLF1/C-C chemokine receptor type 4 (CCR4) levels increased in BV2 cells, whereas the CKLF1/CCR4 level was decreased due to lobetyolin pretreatment. Additionally, BV2 cells injured with OGD/R tended to be M1 type, but lobetyolin treatment shifted the phenotype of BV2 cells from M1 type to M2 type. Lobetyolin decreased the expression of TNF-α and HIF-1α but increased the expression of transforming growth factor-β (TGF-β) in BV2 cells, indicating a dose-effect relationship. The qPCR results showed that lobetyolin decreased the expression of CD16, CD32, and iNOS at the gene level and increased the expression of C-C-chemokine ligand-22 and TGF-β. The immunofluorescence analysis showed that lobetyolin decreased CD16/CD32 levels and increased CD206 levels. Lobetyolin can protect BV2 cells from OGD/R-induced damage by regulating the phenotypic polarization of BV2 and decreasing inflammatory responses. Additionally, CKLF1/CCR4 may participate in regulating lobetyolin-induced polarization of BV2 cells via the HIF-1α pathway.

Topics: Chemokines; Glucose; Humans; Microglia; Oxygen; Phenotype; Reperfusion; Reperfusion Injury; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Loss of Klotho, an anti-aging protein, plays a critical role in the pathogenesis of chronic kidney diseases. As Klotho is a large transmembrane protein, it is challenging to harness it as a therapeutic remedy. Here we report the discovery of a Klotho-derived peptide 1 (KP1) protecting kidneys by targeting TGF-β signaling. By screening a series of peptides derived from human Klotho protein, we identified KP1 that repressed fibroblast activation by binding to TGF-β receptor 2 (TβR2) and disrupting the TGF-β/TβR2 engagement. As such, KP1 blocked TGF-β-induced activation of Smad2/3 and mitogen-activated protein kinases. In mouse models of renal fibrosis, intravenous injection of KP1 resulted in its preferential accumulation in injured kidneys. KP1 preserved kidney function, repressed TGF-β signaling, ameliorated renal fibrosis and restored endogenous Klotho expression. Together, our findings suggest that KP1 recapitulates the anti-fibrotic action of Klotho and offers a potential remedy in the fight against fibrotic kidney diseases.

Topics: Amino Acid Sequence; Animals; Cell Line; Disease Models, Animal; Fibrosis; Humans; Inflammation; Kidney; Kidney Diseases; Klotho Proteins; Male; Mice, Inbred BALB C; Peptides; Phosphorylation; Protective Agents; Protein Binding; Rats; Receptors, Transforming Growth Factor beta; Reperfusion Injury; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Ureteral Obstruction

Renal ischemia/reperfusion injury (RI/RI) is one of the main driving causes of acute kidney injury. However, effective treatment to limit injury and promote recovery and/or survival is still unavailable. Probenecid (PBN), a drug indicated for refractory gout, exhibits protective activities against several preclinical diseases including cerebral and myocardial I/RI via Pannexin 1 (Panx1) and P2X7 receptors' (P2X7R) inhibition. However, its protective role against RI/RI has not been previously addressed. Accordingly, we subjected rats to bilateral RI/RI with/or without PBN treatment. Twenty-four hours post-reperfusion, PBN showed mild tubular injury and reduced serum nephrotoxicity indices, gene and protein expression levels of Panx 1 and P2X7R, and ATP and pro-inflammatory cytokines' levels. The nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome signaling was also downregulated, as demonstrated by reduced gene and protein expression of NLRP3 and caspase-1, along with suppressed IL-1β maturation. Furthermore, PBN enhanced Tregs activity as indicated by elevated FoxP3 gene expression, IL-10, and TGF-β renal levels. On day 5 post-reperfusion, PBN noticeably enhanced renal recovery, as demonstrated by intact tubular epithelium and restored nephrotoxicity indices, Panx 1 and P2X7R gene and protein expression levels, ATP and pro-inflammatory cytokine levels, and NLRP3 inflammasome signaling. Besides, renal Tregs activity was also significantly increased. Our study elaborates for the first time the effectiveness of PBN in recovering post-ischemic renal injury through synergistic inhibition in Panx1/P2X7R axis leading to inactivation of NLRP3 inflammasome signaling and activation of Tregs in ischemic renal tissues. Therefore, PBN can be considered a promising drug for RI/RI treatment.

Topics: Adenosine Triphosphate; Animals; Caspase 1; Connexins; Cytokines; Forkhead Transcription Factors; Inflammasomes; Interleukin-10; Ischemia; Kidney; Nerve Tissue Proteins; NLR Family, Pyrin Domain-Containing 3 Protein; Nucleotides; Probenecid; Rats; Receptors, Purinergic P2X7; Reperfusion Injury; Transforming Growth Factor beta

Acute kidney injury (AKI) carries high morbidity and mortality, and the inducible nitric oxide synthase (iNOS) is a potential molecular target to prevent kidney dysfunction. In previous work, we reported that the pharmacological inhibitions of iNOS before ischemia/reperfusion (I/R) attenuate the I/R-induced AKI in mice. Here, we study the iNOS inhibitor 1400W [N-(3-(Aminomethyl)benzyl] acetamide, which has been described to be much more specific to iNOS inhibition than other compounds.. We used 30 minutes of bilateral renal ischemia, followed by 24 hours of reperfusion in Balb/c mice. 1400w (10 mg/kg i.p) was applied before I/R injury. We measured the expression of elements associated with kidney injury, inflammation, macrophage polarization, mesenchymal transition, and nephrogenic genes by qRT-PCR in the renal cortex and medulla. The Periodic Acid-Schiff (PAS) was used to study the kidney morphology.. Remarkably, we found that 1400W affects the renal cortex and medulla in different ways. Thus, in the renal cortex, 1400W prevented the I/R-upregulation of 1. NGAL, Clusterin, and signs of morphological damage; 2. IL-6 and TNF-α; 3. TGF-β; 4. M2(Arg1, Erg2, cMyc) and M1(CD38, Fpr2) macrophage polarization makers; and 5. Vimentin and FGF2 levels but not in the renal medulla.. 1400W conferred protection in the kidney cortex compared to the kidney medulla. The present investigation provides relevant information to understand the opportunity to use 1400W as a therapeutic approach in AKI treatment.

Topics: Acetamides; Acute Kidney Injury; Animals; Clusterin; Disease Models, Animal; Fibroblast Growth Factor 2; Interleukin-6; Ischemia; Kidney; Kidney Cortex; Lipocalin-2; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase Type II; Reperfusion Injury; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vimentin

Ischemia-reperfusion injury (IRI) is typically associated with a vigorous inflammatory and oxidative stress response to hypoxia and reperfusion that disturbs the function of the organ. The remote effects of renal IRI on the liver, however, require further study. Renal damage associated with liver disease is a common clinical problem. Colchicine, a polymerization inhibitor of microtubules, has been used as an anti-inflammatory and anti-fibrotic drug for liver diseases. The goal of the current study was to investigate the possible protective mechanisms of colchicine on liver injury following renal IRI. Forty rats were divided randomly into four groups: sham group, colchicine-treated group, IRI group, and colchicine-treated + IRI group. Treatment with colchicine significantly reduced hepatic toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB) transcription factor, myeloid differentiation factor 88 (MyD88), and tumor necrosis factor-alpha (TNF-α) contents; downregulated BCL2 associated X apoptosis regulator (

Topics: Animals; Anti-Inflammatory Agents; Antifibrotic Agents; Apoptosis; bcl-2-Associated X Protein; Colchicine; Gene Expression; Gene Expression Regulation; Kidney Diseases; Male; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Rats, Sprague-Dawley; Reperfusion Injury; Toll-Like Receptor 4; Transforming Growth Factor beta

Indolamine 2,3-dioxygenase (IDO), an enzyme that catalyses the metabolism of tryptophan, may play a detrimental role in ischemia-reperfusion injury (IRI). IDO can be inhibited by 1-methyl-tryptophan, which exists in a D (D-MT) or L (L-MT) isomer. These forms show different pharmacological effects besides IDO inhibition. Therefore, we sought to investigate whether these isomers can play a protective role in renal IRI, either IDO-dependent or independent.. We studied the effect of both isomers in a rat renal IRI model with a focus on IDO-dependent and independent effects.. Both MT isomers reduced creatinine and BUN levels, with D-MT having a faster onset of action but shorter duration and L-MT a slower onset but longer duration (24 h and 48 h vs 48 h and 96 h reperfusion time). Interestingly, this effect was not exclusively dependent on IDO inhibition, but rather from decreased TLR4 signalling, mimicking changes in renal function. Additionally, L-MT increased the overall survival of rats. Moreover, both MT isomers interfered with TGF-β signalling and epithelial-mesenchymal transition. In order to study the effect of isomers in all mechanisms involved in IRI, a series of in vitro experiments was performed. The isomers affected signalling pathways in NK cells and tubular epithelial cells, as well as in dendritic cells and T cells.. This study shows that both MT isomers have a renoprotective effect after ischemia-reperfusion injury, mostly independent of IDO inhibition, involving mutually different mechanisms. We bring novel findings in the pharmacological properties and mechanism of action of MT isomers, which could become a novel therapeutic target of renal IRI.

Topics: Animals; Coculture Techniques; Dendritic Cells; Disease Models, Animal; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Fibrosis; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kidney; Kidney Diseases; Lectins, C-Type; Mice; NIH 3T3 Cells; NK Cell Lectin-Like Receptor Subfamily K; Reperfusion Injury; Signal Transduction; T-Lymphocytes; THP-1 Cells; Toll-Like Receptor 4; Transforming Growth Factor beta; Tryptophan

Topics: Animals; Apoptosis; Base Sequence; Cell Line; Disease Progression; Fibrosis; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Hypoxia; Kruppel-Like Transcription Factors; Male; Mice, Inbred C57BL; MicroRNAs; Myocardial Infarction; Reperfusion Injury; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

We aimed to investigate the role of cMet agonistic antibody (cMet Ab) in preventing kidney fibrosis during acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Additionally, we explored the effect of cMet Ab on TGF-β1/Smad pathway during the pathogenesis of kidney fibrosis. A unilateral ischemia-reperfusion injury (UIRI) mouse model was established to induce AKI-to-CKD transition. Furthermore, we incubated human proximal tubular epithelial cells (hPTECs) under hypoxic conditions as in vitro model of kidney fibrosis. We analyzed the soluble plasma cMet level in patients with AKI requiring dialysis. Patients who did not recover kidney function and progressed to CKD presented a higher increase in the cMet level. The kidneys of mice treated with cMet Ab showed fewer contractions and weighed more than the controls. The mice in the cMet Ab-treated group showed reduced fibrosis and significantly decreased expression of fibronectin and α-smooth muscle actin. cMet Ab treatment decreased inflammatory markers (MCP-1, TNF-α, and IL-1β) expression, reduced Smurf1 and Smad2/3 level, and increased Smad7 expressions. cMet Ab treatment increased cMet expression and reduced the hypoxia-induced increase in collagen-1 and ICAM-1 expression, thereby reducing apoptosis in the in vitro cell model. After cMet Ab treatment, hypoxia-induced expression of Smurf1, Smad2/3, and TGF-β1 was reduced, and suppressed Smad7 was activated. Down-regulation of Smurf1 resulted in suppression of hypoxia-induced fibronectin expression, whereas treatment with cMet Ab showed synergistic effects. cMet Ab can successfully prevent fibrosis response in UIRI models of kidney fibrosis by decreasing inflammatory response and inhibiting the TGF-β1/Smad pathway.

Topics: Acute Kidney Injury; Animals; Fibrosis; Humans; Kidney; Mice, Inbred C57BL; Renal Insufficiency, Chronic; Reperfusion Injury; Smad7 Protein; Transforming Growth Factor beta

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor β (TGF-β) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGF-β (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-β treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-β treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-β.

Topics: Animals; Cytokines; Interleukin-10; Liver; Liver Transplantation; Organ Preservation; Perfusion; Rats; Reperfusion Injury; Transforming Growth Factor beta; Transforming Growth Factors

Acute kidney disease due to renal ischemia/reperfusion (I/R) is a major clinical problem without effective therapies. The injured tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT). It will loss epithelial phenotypes and express the mesenchymal characteristics. The formation of scar tissue in the interstitial space during renal remodeling is caused by the excessive accumulation of extracellular matrix components and induced fibrosis. This study investigated the effect of caffeic acid ethanolamide (CAEA), a novel caffeic acid derivative, on renal remodeling after injury. The inhibitory role of CAEA on EMT was determined by western blotting, real-time PCR, and immunohistochemistry staining. Treating renal epithelial cells with CAEA in TGF-β exposed cell culture successfully maintained the content of E-cadherin and inhibited the expression of mesenchymal marker, indicating that CAEA prevented renal epithelial cells undergo EMT after TGF-β exposure. Unilateral renal I/R were performed in mice to induce renal remodeling models. CAEA can protect against I/R-induced renal remodeling by inhibiting inflammatory reactions and consecutively inhibiting TGF-β-induced EMT, characterized by the preserved E-cadherin expression and alleviated α-SMA and collagen expression, as well as the alleviated of renal fibrosis. We also revealed that CAEA may exhibits biological activity by targeting TGFBRI. CAEA may antagonize TGF-β signaling by interacting with TGFBR1, thereby blocking binding between TGF-β and TGFBR1 and reducing downstream signaling, such as Smad3 phosphorylation. Our data support the administration of CAEA after I/R as a viable method for preventing the progression of acute renal injury to renal fibrosis.

Topics: Acute Kidney Injury; Animals; Caffeic Acids; Cell Line; Disease Progression; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibrosis; Kidney; Male; Mice; Mice, Inbred C57BL; Rats; Reperfusion Injury; Transforming Growth Factor beta

Innate lymphoid cells are a recently recognized group of immune cells with critical roles in tissue homeostasis and inflammation. Regulatory innate lymphoid cells are a newly identified subset of innate lymphoid cells, which play a suppressive role in the innate immune response, favoring the resolution of intestinal inflammation. However, the expression and role of regulatory innate lymphoid cells in kidney has not been reported. Here, we show that regulatory innate lymphoid cells are present in both human and mouse kidney, express similar surface markers and form a similar proportion of total kidney innate lymphoid cells. Regulatory innate lymphoid cells from kidney were expanded in vitro with a combination of IL-2, IL-7 and transforming growth factor-β. These cells exhibited immunosuppressive effects on innate immune cells via secretion of IL-10 and transforming growth factor-β. Moreover, treatment with IL-2/IL-2 antibody complexes (IL-2C) promoted expansion of regulatory innate lymphoid cells in vivo, and prevent renal ischemia/reperfusion injury in Rag-/- mice that lack adaptive immune cells including Tregs. Depletion of regulatory innate lymphoid cells with anti-CD25 antibody abolished the beneficial effects of IL-2C in the Rag-/- mice. Adoptive transfer of ex vivo expanded regulatory innate lymphoid cells improved renal function and attenuated histologic damage when given before or after induction of ischemia/reperfusion injury in association with reduction of neutrophil infiltration and induction of reparative M2 macrophages in kidney. Thus, our study shows that regulatory innate lymphoid cells suppress innate renal inflammation and ischemia/reperfusion injury.

Topics: Adoptive Transfer; Animals; Cell Separation; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Flow Cytometry; Homeodomain Proteins; Humans; Immunity, Innate; Interleukin-10; Interleukin-2; Kidney; Lymphocyte Subsets; Macrophages; Male; Mice; Mice, Knockout; Nephritis; Primary Cell Culture; Reperfusion Injury; Transforming Growth Factor beta

Fibrosis is a common pathologic pathway of progressive kidney disease involving complex signaling networks. The deacetylase sirtuin 6 (sirt6) was recently implicated in kidney injury. However, it remains elusive whether and how sirt6 contributes to the regulation of kidney fibrosis. Here, we demonstrate that sirt6 protects against kidney interstitial fibrosis through epigenetic regulation of β-catenin signaling. Sirt6 is markedly upregulated during fibrogenesis following obstructed nephropathy and kidney ischemia-reperfusion injury. Pharmacological inhibition of sirt6 deacetylase activity aggravates kidney fibrosis in obstructed nephropathy. Consistently, knockdown of sirt6 in mouse kidney proximal tubular epithelial cells aggravates transforming growth factor-β-induced fibrosis in vitro. Mechanistically, sirt6 deficiency results in augmented expression of the downstream target proteins of β-catenin signaling. We further show that sirt6 interacts with β-catenin during transforming growth factor-β treatment and binds to the promoters of β-catenin target genes, resulting in the deacetylation of histone H3K56 to prevent the transcription of fibrosis-related genes. Thus, our data reveal the anti-fibrotic function of sirt6 by epigenetically attenuating β-catenin target gene expression.

Topics: Acetylation; Animals; beta Catenin; Disease Models, Animal; Epigenesis, Genetic; Epithelial Cells; Fibrosis; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Histones; Humans; Kidney Tubules; Male; Mice; Primary Cell Culture; Promoter Regions, Genetic; Reperfusion Injury; Sirtuins; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation; Wnt Signaling Pathway

Remote renal injury is a drastic consequence of hepatic ischemia/reperfusion (IR) injury. Vildagliptin (V) is a dipeptidyl peptidase-4 inhibitor that has a hepatorenal protective effect against models of liver and renal IR. This research was done to explore the protective role of vildagliptin against renal injury following hepatic IR injury as well as the possible involvement of transforming growth factor-beta (TGF-β)/Smad/alpha-smooth muscle actin (α-SMA) expressions in the pathophysiological mechanism of the remote renal injury. Three groups of male Wistar rats were organized into: sham group, IR group, and V + IR group in which 10 mg/kg/day of vildagliptin was pretreated for 10 days intraperitoneally. Blood in addition to renal and hepatic tissue samples was used for biochemical and histopathological studies. Hepatic IR induced a marked increase in serum creatinine, blood urea nitrogen, liver enzymes, renal nitric oxide, malondialdehyde, tumor necrosis factor-alpha levels with a marked upregulation of renal mRNA expressions of TGF-β, Smad2, Smad3, and α-SMA in addition to a marked decline in renal catalase content comparing to the sham group. Abnormal histopathological findings of hepatic and renal injury were detected in the IR group. Vildagliptin significantly improved these biochemical markers as well as the histopathological changes. The upregulation of renal TGF-β/Smad/α-SMA mRNA expressions was involved for the first time in the pathogenesis of the renal injury following hepatic IR and vildagliptin ameliorated this renal injury through blocking these expressions.

Topics: Acute Kidney Injury; Animals; Biomarkers; Blood Urea Nitrogen; Creatinine; Ischemic Preconditioning; Liver; Male; Malondialdehyde; Nitric Oxide; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vildagliptin

Despite the fact that histone deacetylase (HDAC) inhibitors have been tested to treat various cardiovascular diseases, the effects of selective HDAC6 inhibitor ACY1215 on infarct size during cardiac ischemia-reperfusion (IR) injury still remain unknown. In the present study we aimed to investigate the effects of ACY1215 on infarct size in rats with cardiac IR injury, as well as to examine the association between HDAC6 inhibitors and the gene expression of hypoxia inducible factor-1α (HIF-1α), a key regulator of cellular responses to hypoxia.. By using computational analysis of high-throughput expression profiling dataset, the association between HDAC inhibitors (pan-HDAC inhibitors panobinostat and vorinostat, and HDAC6 inhibitor ISOX) and their effects on HIF-1α gene-expression were evaluated. The male Wistar rats treated with ligation of left coronary artery followed by reperfusion were used as a cardiac IR model. ACY1215 (50 mg/kg), pan-HDAC inhibitor MPT0E028 (25 mg/kg), and vehicle were intraperitoneally injected within 5 min before reperfusion. The infarct size in rat myocardium was determined by 2,3,5-triphenyltetrazolium chloride staining. The serum levels of transforming growth factor-β (TGF-β) and C-reactive protein (CRP) were also determined.. The high-throughput gene expression assay showed that treatment of ISOX was associated with a more decreased gene expression of HIF-1α than that of panobinostat and vorinostat. Compared to control rats, ACY1215-treated rats had a smaller infarct size (49.75 ± 9.36% vs. 19.22 ± 1.70%, p < 0.05), while MPT0E028-treated rats had a similar infarct size to control rats. ACY-1215- and MPT0E028-treated rats had a trend in decreased serum TGF-β levels, but not statistically significant. ACY1215-treated rats also had higher serum CRP levels compared to control rats (641.6 μg/mL vs. 961.37 ± 64.94 μg/mL, p < 0.05).. Our research indicated that HDAC6 inhibition by ACY1215 might reduce infarct size in rats with cardiac IR injury possibly through modulating HIF-1α expression. TGF-β and CRP should be useful biomarkers to monitor the use of ACY1215 in cardiac IR injury.

Topics: Animals; C-Reactive Protein; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Myocardial Infarction; Myocardium; Pyrimidines; Rats, Wistar; Reperfusion Injury; Transforming Growth Factor beta

The study aimed to investigate the potential attenuation effect of chitosan in liver ischemia/reperfusion injury (I/R), and its relevant protective mechanisms. Chitosan (200 mg/kg) has been administered orally for 30 days, later animals underwent liver 45 min ischemia and reperfusion for 60 min. Following treatment with chitosan, the levels of serum aminotransferases and lactate dehydrogenase were significantly reduced. Similarly, hepatic (GSH, SOD, CAT, GST and GPx) were enhanced, and the level of tissue malondialdehyde (MDA) was decreased. In addition, inflammatory cytokinesis (TNF-α and TGF-β) have recorded a significant decrease in their mRNA expression and protein levels using qPCR and ELISA respectively. Marked reduction of apoptosis has been indicated by the elevation in BCL2, and decreasing in BAX, Caspace-3 and Cytochrome-c expression levels, which furthermore confirmed by DNA fragmentation assay. The enhancement of the previous parameters resulted in a marked improvement in the liver architectures after chitosan administration. In conclusion, chitosan has proved its efficiency as an anti-inflammatory and antioxidant agent through its inhibitory effect of cytokines and reducing ROS respectively. In addition, chitosan could modulate the changes in histological structure and alleviate apoptosis induced by liver I/R, which recommend it as an efficient agent for protection against liver I/R injury.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Aspartate Aminotransferases; bcl-2-Associated X Protein; Caspase 3; Chitosan; Cytokines; Inflammation; Liver; Liver Diseases; Male; Malondialdehyde; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-β administration.. The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-β1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal-regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic-reperfusion injury.. NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dextromethorphan; Dizocilpine Maleate; Epithelial-Mesenchymal Transition; Excitatory Amino Acid Antagonists; Fibrosis; Gene Knockdown Techniques; Humans; In Vitro Techniques; Kidney; Kidney Tubules, Proximal; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Protein Kinase Inhibitors; Receptors, N-Methyl-D-Aspartate; Renal Insufficiency, Chronic; Reperfusion Injury; Sulfonamides; Transforming Growth Factor beta; Ureteral Obstruction

Protease-activated receptor (PAR)-1 has emerged as a key profibrotic player in various organs including kidney. PAR-1 activation leads to deposition of extracellular matrix (ECM) proteins in the tubulointerstitium and induction of epithelial-mesenchymal transition (EMT) during renal fibrosis. We tested the anti-fibrotic potential of vorapaxar, a clinically approved PAR-1 antagonist for cardiovascular protection, in an experimental kidney fibrosis model of unilateral ureteral obstruction (UUO) and an AKI-to-chronic kidney disease (CKD) transition model of unilateral ischemia-reperfusion injury (UIRI), and dissected the underlying renoprotective mechanisms using rat tubular epithelial cells. PAR-1 is activated mostly in the renal tubules in both the UUO and UIRI models of renal fibrosis. Vorapaxar significantly reduced kidney injury and ameliorated morphologic changes in both models. Amelioration of kidney fibrosis was evident from down-regulation of fibronectin (Fn), collagen and α-smooth muscle actin (αSMA) in the injured kidney. Mechanistically, inhibition of PAR-1 inhibited MAPK ERK1/2 and transforming growth factor-β (TGF-β)-mediated Smad signaling, and suppressed oxidative stress, overexpression of pro-inflammatory cytokines and macrophage infiltration into the kidney. These beneficial effects were recapitulated in cultured tubular epithelial cells in which vorapaxar ameliorated thrombin- and hypoxia-induced TGF-β expression and ECM accumulation. In addition, vorapaxar mitigated capillary loss and the expression of adhesion molecules on the vascular endothelium during AKI-to-CKD transition. The PAR-1 antagonist vorapaxar protects against kidney fibrosis during UUO and UIRI. Its efficacy in human CKD in addition to CV protection warrants further investigation.

Topics: Animals; Biomarkers; Cell Hypoxia; Endothelial Cells; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Inflammation; Kidney; Kidney Tubules; Lactones; Macrophages; Mice, Inbred BALB C; Mice, Inbred C57BL; Oxidative Stress; Pyridines; Rats; Reactive Oxygen Species; Receptor, PAR-1; Reperfusion Injury; Smad3 Protein; Thrombin; Transforming Growth Factor beta; Up-Regulation; Ureteral Obstruction

Transforming growth factor β (TGF-β) serves critical functions in brain injury, especially in cerebral ischemia; however, apart from its neuroprotective effects, its role in regulating neurogenesis is unclear. TGF-β acts in different ways; the most important, canonical TGF-β activity involves TGF-β receptor I (TβRI) or the activin receptor-like kinase 5 (ALK5) signaling pathway. ALK5 signaling is a major determinant of adult neurogenesis. In our previous studies, growth arrest and DNA damage protein 45b (Gadd45b) mediated axonal plasticity after stroke. Here, we hypothesized that ALK5 signaling regulates neural plasticity and neurological function recovery after cerebral ischemia/reperfusion (I/R) via Gadd45b. First, ALK5 expression was significantly increased in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Then, we knocked down or overexpressed ALK5 with lentivirus (LV) in vivo. ALK5 knockdown reduced axonal and dendritic plasticity, with a concomitant decrease in neurological function recovery. Conversely, ALK5 overexpression significantly increased neurogenesis as well as functional recovery. Furthermore, ALK5 mediated Gadd45b protein levels by regulating Smad2/3 phosphorylation. Finally, ALK5 coimmunoprecipitated with Gadd45b. Our results suggested that the ALK5 signaling pathway plays a critical role in mediating neural plasticity and neurological function recovery via Gadd45b after cerebral ischemia, representing a new potential target for cerebral I/R injury.

Topics: Animals; Antigens, Differentiation; Brain; Cerebrovascular Disorders; Disease Models, Animal; Gene Expression Regulation; Male; Middle Cerebral Artery; Neurogenesis; Neuronal Plasticity; Neurons; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Recovery of Function; Reperfusion Injury; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Leucine-rich α2-glycoprotein1 (LRG1), a pleiotropic protein, plays a pathogenic role in multiple human diseases. However, its pathophysiological function in ischemia/reperfusion injury remains unclear. In this study, we discussed the function and mechanism of LRG1 in acute ischemic stroke from both basic and clinical research points of view. Mice underwent transient middle cerebral artery occlusion (tMCAO) surgery 2 weeks after LRG1 was overexpressed by the delivery of adeno-associated virus (AAV). For wild-type mice, both the protein and the transcript of LRG1 in the brain tissue were elevated after tMCAO. Meanwhile, the serum levels of LRG1 were decreased after tMCAO. The neuronal injury was shown aggravated in the AAV-LRG1 group (AAV-LRG1 mice with tMCAO) through infarction volume, neurological score, HE, and Nissl staining. Meanwhile, LRG1 significantly enhanced apoptosis and autophagy during tMCAO, as detected by caspase3, Bax, Bcl-2, LC3II/LC3I, Beclin1, p62, and a TUNEL assay. Furthermore, by overexpression of LRG1, the protein of ALK1 was upregulated and the TGFβ-smad1/5 signaling pathway was activated upon tMCAO. We also showed that patients with acute cerebral infarction had lower serum levels of LRG1 compared to healthy controls. In addition, LRG1 levels were associated with infarction volume, stroke severity, and prognosis in patients with supratentorial infarction. Taken together, the data from this study revealed that LRG1 promoted apoptosis and autophagy through the TGFβ-smad1/5 signaling pathway by up-regulating ALK1, which exacerbates ischemia/reperfusion injury.

Topics: Animals; Apoptosis; Autophagy; Biomarkers; Brain; Brain Ischemia; Disease Models, Animal; Female; Glycoproteins; Humans; Male; Mice, Inbred C57BL; Middle Aged; Neurons; Reperfusion Injury; Signal Transduction; Smad1 Protein; Smad5 Protein; Transforming Growth Factor beta

Cellular senescence is associated with renal disease progression, and accelerated tubular cell senescence promotes the pathogenesis of renal fibrosis. However, the underlying mechanism is unknown. We assessed the potential role of Wnt9a in tubular cell senescence and renal fibrosis. Compared with tubular cells of normal subjects, tubular cells of humans with a variety of nephropathies and those of several mouse models of CKD expressed high levels of Wnt9a that colocalized with the senescence-related protein p16

Topics: Animals; Cell Line; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Epithelial Cells; Fibroblasts; Fibrosis; Gene Expression Regulation; Genes, p16; Humans; Kidney; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Rats; Recombinant Proteins; Renal Insufficiency, Chronic; Reperfusion Injury; RNA Interference; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Proteins; Wnt Signaling Pathway

Renal ischemia-reperfusion injury (I/RI) remains a critical clinical situation. Several evidence revealed the potential reno-protective effects of Vitamin D and/or pioglitazone, on renal I/RI. This study addresses the possible involvement of the Wnt4/β-catenin signaling, p-S536NF-κBp65, PPARγ, Ang II/TGF-β, and ACE2 as potential effectors to vitamin D and pioglitazone-mediated renoprotective effects. Two sets of Sprague-Dawley rats (n = 30 rat each), were randomized into sham, I/R, Vit D "alfacalcidol" (5 ng/kg/day), pioglitazone (5 mg/kg/day), and Vit D + pioglitazone groups. In all groups renal biochemical parameters, as well as inflammatory and structural profiles were assessed, besides the expression/contents of Wnt4/β-catenin and pS536-NF-κBp65. All treatments started 7 days before I/RI and animals were killed 24 h after I/RI in the first set, while those in the 2nd set continued their treatments for 14 days. After 24 h, all pre-treatments impeded theI/R effect on neutrophils recruitment, p-S536NF-κBp65, IL-18, NGAL, caspase-3, AngII, ACE-2, PPARγ and TGF-β, besides the expression of Wnt4 and ACE-2 with notable reflection on histological changes. Two weeks after I/RI, except a marked up regulation in Wnt4 expression and a striking elevation in the β-catenin content, the magnitude of the injurious events was relatively less pronounced, an effect that was mostly augmented by the different treatments. The current study pledges a promising and novel reno-protective role of the administration of Vit D and pioglitazone entailing a potential involvement of ICAM-1, MPO, NF-κB, Ang II, ACE2, TGFβ, and a modulation of Wnt4/β-catenin pathway.

Topics: Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; beta Catenin; Cytoprotection; Disease Models, Animal; Hydroxycholecalciferols; Intercellular Adhesion Molecule-1; Interleukin-18; Kidney; Kidney Diseases; Male; Neutrophil Infiltration; NF-kappa B; Peptidyl-Dipeptidase A; Peroxidase; Pioglitazone; Rats, Sprague-Dawley; Reperfusion Injury; Thiazolidinediones; Time Factors; Transforming Growth Factor beta; Wnt Signaling Pathway; Wnt4 Protein

Ischemic stroke is still one of the leading debilitating diseases with high morbidity and mortality. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) play an important role in cerebral ischemia/reperfusion (I/R) injury. However, the mechanism underlying the regulation of ROS generation is still not fully elucidated. This study aims to explore the role of transforming growth beta (TGF-β) signals in ROS generation.. Sprague-Dawley rats were subjected to I/R injury, and PC-12 cells were challenged by hypoxia/reoxygenation (H/R) and/or treated with activin receptor-like kinase (ALK5) inhibitor Sb505124 or siRNA against ALK5. Brain damage was evaluated using neurological scoring, triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, infarct volume measurement, TUNEL staining, and caspase-3 activity measurement. Expression of TGF-β and oxidative stress-related genes was analyzed by real-time polymerase chain reaction and Western blot; NOX activity and ROS level were measured using spectrophotometry and fluorescence microscopy, respectively.. I/R contributed to severe brain damage (impaired neurological function, brain infarction, tissue edema, apoptosis), TGF-β signaling activation (upregulation of ALK5, phosphorylation of SMAD2/3) and oxidative stress (upregulation of NOX2/4, rapid release of ROS [oxidative burst]). However, Sb505124 significantly reversed these alterations and protected rats against I/R injury. As in the animal results, H/R also contributed to TGF-β signaling activation and oxidative stress. Likewise, the inhibition of ALK5 or ALK5 knockdown significantly reversed these alterations in PC-12 cells. Other than ALK5 knockdown, ALK5 inhibition had no effect on the expression of ALK5 in PC-12 cells.. Our studies demonstrated that TGF-β signaling activation is involved in the regulation of NOX2/NOX4 expression and exacerbates cerebral I/R injury.

Topics: Animals; Benzodioxoles; Brain Ischemia; Imidazoles; Male; NADPH Oxidase 2; NADPH Oxidase 4; Oxidative Stress; PC12 Cells; Pyridines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

We have evaluated the protective mechanism of tanshinone IIA in ischemia/reperfusion injury (IRI) induced by liver grafts, revealing novel supplementary immunotherapy for liver transplantation.. The tanshinone IIA preconditioning group (TP group) was pretreated with tanshinone IIA via intraperitoneal injection for 1 week before receiving orthotopic liver transplantation with hepatic arterial ischemia for 30min. The sham-operation group (SO group), control graft group (CG group) and IRI group were pretreated with an equivalent volume of normal saline. The IRI group and CG group received orthotopic liver transplantation with or without hepatic arterial ischemia. Rats were sacrificed at each time point, serum was collected for ELISA detection, and Kupffer cells (KCs) were isolated to extract total protein and RNA for western blotting and real-time PCR, respectively.. The levels of TNF-α and IL-4 in the TP group were significantly lower than those of in the IRI group; meanwhile the IL-10 and TGF-β levels were significantly higher than in the IRI group. The protein and mRNA expression levels of HMGB1 were significantly lower in TP group than in the IRI group at each time point. The TLR-4, Myd88, NLRP3 and p-NF-κb p65 expression levels in the TP groups were significantly lower than those in the IRI group, while the PTEN, PI3K and AKT phosphorylation levels in the TP groups were significantly higher than those in the IRI group.. Tanshinone IIA attenuates IRI caused by liver grafts via down-regulation of the HMGB1-TLR-4/NF-κb pathway in KCs and activation of PTEN/PI3K/AKT pathway, suggesting a potential role for prevention of liver cell IRI during liver transplantation.

Topics: Abietanes; Animals; HMGB1 Protein; Interleukin-10; Interleukin-4; Kupffer Cells; Liver; Liver Diseases; Liver Transplantation; Male; Myeloid Differentiation Factor 88; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Toll-Like Receptor 4; Transcription Factor RelA; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

This study aims to explore the effects of the exogenous hydrogen sulfide (H2S)-mediated scavenger receptor A (SR-A) signaling pathway on renal ischemia/reperfusion injury (IRI) by regulating endoplasmic reticulum (ER) stress-induced autophagy in rats.. A total of 48 normal Sprague-Dawley (SD) rats and SR-A knockout rats were selected and divided into six groups (n = 8): wild-type (WT) + sham, WT + ischemia-reperfusion (I/R), WT + I/R + NaHS, SR-A-/- + sham, SR-A-/- + I/R and SR-A-/- + I/R + NaHS. The concentrations of urinary protein, blood urea nitrogen (BUN), serum creatinine (SCR), malondialdehyde (MDA) and H2S in renal tissue were detected. qRT-PCR and Western blotting were used to detect the mRNA and protein levels of IL-6, TGF-β, SR-A, LC3I, LC3II, P62, PERK, ATF6 and IRE1 pathway-related genes. A TUNEL assay was used to detect cell apoptosis. Electron microscopy was applied to observe the structure of renal autophagosomes.. Compared with the WT + sham group, in the rates of the WT + I/R group, the urine volume, urinary protein, BUN, SCR and MDA concentrations, the mRNA and protein expression of IL-6, TGF-β, LC3II/I, and ER stress pathway-related genes, the cell apoptosis index, and the number of autophagosomes were significantly increased 24 h after I/R, while P62 and SR-A protein expression and SOD and H2S concentrations were significantly decreased (all P < 0.05). The levels of renal injury, autophagy and ER stress pathway-related genes were decreased in the WT + I/R + NaHS group but were increased in the SR-A-/- + I/R group relative to the WT + I/R group. No significant differences were observed in the urine volume; the concentrations of urinary protein, BUN, SCR and MDA; the SOD activity; the mRNA and protein expression of IL-6, TGF-β, SR-A, GRP78, SR-A, GPR94, ATF4, IRE1, XBP1, ATF6, and eIF2α; the cell apoptosis index; or the number of autophagosomes in rats of the SR-A-/- + I/R and SR-A-/- + I/R + NaHS groups (all P > 0.05).. These results demonstrate that the exogenous H2S-mediated SR-A signaling pathway reduces renal IRI injury by up-regulating ER stress-induced autophagy in rats.

Topics: Animals; Autophagy; Creatine; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Hydrogen Sulfide; Interleukin-6; Kidney; Male; Malondialdehyde; Microscopy, Electron; Microtubule-Associated Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Scavenger Receptors, Class A; Signal Transduction; Superoxide Dismutase; Transforming Growth Factor beta

Tisp40, a transcription factor of the CREB/CREM family, is involved in cell proliferation, differentiation and other biological functions, but its role in renal tubulointerstitial fibrosis is unknown.. In our study, we investigated the effects of Tisp40 on extracellular matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT) and the underlying molecular mechanisms in transforming growth factor-β (TGF-β)-stimulated TCMK-1 cells by quantitative real-time polymerase chain reaction (qPCR), Western blot analysis and immunofluorescence in vitro, and further explored the role of Tisp40 on renal fibrosis induced by ischemia-reperfusion (I/R) by qPCR, Western blot analysis, hydroxyproline analysis, Masson trichrome staining and immunohistochemistry staining in vivo.. The data showed that Tisp40 was upregulated in a model of renal fibrosis induced by I/R injury (IRI). Upon IRI, Tisp40-deficient mice showed attenuated renal fibrosis compared with wild-type mice. Furthermore, the expression of α-smooth muscle actin, E-cadherin, fibronectin, and collagen I was suppressed. Tisp40 overexpression aggravated ECM accumulation and EMT in the TGF-β-stimulated TCMK-1 cell line, whereas the opposite occurred in cells treated with small interfering RNA (siRNA) targeting Tisp40. Importantly, it is changes in the Smad pathway that attenuate renal fibrosis.. These findings suggest that Tisp40 plays a critical role in the TGF-β/ Smads pathway involved in this process. Hence, Tisp40 could be a useful therapeutic target in the fight against renal tubulointerstitial fibrosis.

Topics: Animals; Cyclic AMP Response Element-Binding Protein; Epithelial-Mesenchymal Transition; Fibrosis; Gene Expression Regulation; Humans; Kidney; Mice; Nephritis, Interstitial; Reperfusion Injury; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

BACKGROUND Tissues fibrosis is caused by ischemia-reperfusion injury (IRI) and results in organ dysfunction. In this study, we aimed to investigate whether fibrosis occurs after uterine ischemia-reperfusion injury, and to investigate the effects of puerarin (Pur) on the fibrosis process in rats. MATERIAL AND METHODS Twenty-four female Wistar rats were randomly divided into three groups (8 in each group): the control group rats only received operation without uterine ischemic, the IRI group and the IRI + Pur group rats received 30-minutes ischemia and 2-weeks of reperfusion. Pur was orally administered at the onset of reperfusion. Picrosirius red staining was used to assess uterine fibrosis. Immunohistochemistry was used to detect the expression levels of transforming growth factor (TGF)-β and a-smooth muscle actin (α-SMA). Western blotting was used to evaluate the expression of chymase, TGF-β, α-SMA, and the activity of the Wnt/β-catenin pathway. RESULTS Uterine fibrosis in the IRI+Pur group was significantly decreased compared with the IRI group. In addition, immunohistochemistry reveals that TGF-β and a-SMA were decreased in the IRI+Pur group compared with the IRI group. Western blotting results showed that Pur significantly suppresses the increase in chymase, α-SMA, TGF-β, and b-catenin expression levels induced by IRI. CONCLUSIONS The results indicated that IRI could induce uterine fibrosis and that Pur had an improvement effect on IRI-induced uterine fibrosis by downregulating the activity of mast cell chymase, TGF-β,  α-SMA, and the Wnt/β-catenin pathway.

Topics: Actins; Animals; beta Catenin; Chymases; Female; Fibrosis; Isoflavones; Kidney; Leiomyoma; Rats; Rats, Wistar; Reperfusion Injury; Transforming Growth Factor beta; Wnt Signaling Pathway

Current treatment options for chronic kidney disease (CKD) are limited and their focus is on slowing its progression by addressing comorbidities. Fibrosis, the common histopathological process in CKD, is a major therapeutic research target. In CKD, fibroblasts are terminally activated due to alterations in their DNA-methylation pattern, particularly hypermethylation. Preventing the copying of pathological DNA-methylation patterns in proliferating fibroblasts could be a new effective therapeutic strategy for treating CKD.. To evaluate the therapeutic effect of short-term treatment with the DNA-methyltransferase (DNMT)-inhibitor decitabine on fibrosis (either developing or already established), male C57Bl/6 mice underwent warm unilateral ischemia-reperfusion injury. Respectively 3 days, 3 and 6 weeks after surgery, decitabine treatment (0.25 mg/kg) was initiated for 10 days after which animals were followed up to 12 weeks after ischemia. The efficacy of therapy on fibrosis was evaluated by collagen I and tgfβ gene expression and histological quantification of collagen I staining. In addition, the effect of decitabine treatment on tubular injury (Kim-1, Ngal), inflammation (TNFa, IL6), DNA-methyltransferases (Dnmt1, 3a, and 3b), and global methylation status was determined.. Following ischemia there was a significant increase in fibrotic, injury, and inflammatory markers as well as an increase of the various dnmts. Although decitabine treatment transiently increased renal injury and had a moderately decreasing effect on dnmt expression and on global DNA-methylation upon immediate treatment, none of the treatment regimens succeeded in preventing, attenuating, or diminishing fibrosis in the long run.. Administration of untargeted nucleoside analogues seems unsuitable as a first-line treatment option in developing or established CKD.

Topics: Animals; Azacitidine; Collagen Type I; Cytokines; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Enzyme Inhibitors; Fibrosis; Kidney; Kidney Diseases; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Reperfusion Injury; Transforming Growth Factor beta

Hypothermic machine perfusion (HMP) has been known as an efficient way to improve kidney graft function, but the underlying mechanisms remain unclear. Here, we adopt a rabbit reperfusion mode to investigate the upstream mechanisms of end-ischemic HMP of kidneys from donors after cardiac death (DCD), with static cold storage (CS) as a control. Eighteen New Zealand healthy male rabbits (12 weeks old, with a weight of 3.0 ± 0.2 kg) were randomly divided into three groups: HMP group, CS group, and Normal group (n = 6). The left kidney of rabbits underwent warm ischemia for 25 min through clamping the left renal pedicle and then reperfusion for 1 h. Then the left kidneys were preserved by CS or HMP (4°C for 4 h) ex vivo respectively, after they were autotransplanted and rabbits were submitted to a right nephrectomy. Twenty-four hours after reperfusion, all left renal specimens were collected. Finally, the expression of Krüppel-like factor 2 (KLF2), transforming growth factor-β (TGF-β) and SMAD4 protein in renal cortical tissue were detected by immunoblotting, and the TGF-β and SMAD4 expressions were further confirmed by immunohistochemistry analysis. We found that expression of KLF2 in HMP group was significantly higher than CS group (P = 0.011), while expression of TGF-β and SMAD4 in HMP group were significantly lower than CS group (P = 0.002, P = 0.01, respectively); Compared with normal group, the expression of TGF-β and SMAD4 in HMP and CS group significantly increased (P<0.05). Compared with CS group, TGF-β and SMAD4 protein were equally down-regulated in glomerular and the tubular epithelial cells in HMP group confirmed by immunohistochemistry. In conclusion, HMP may decrease DCD kidneys inflammation through the pathway of upregulating expression of KLF2 and inhibiting TGF-β signaling after transplantation.

Topics: Animals; Hypothermia, Induced; Inflammation; Kidney; Kidney Transplantation; Kruppel-Like Transcription Factors; Male; Organ Preservation; Rabbits; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta; Transplants

Activins are members of the transforming growth factor-beta (TGF-β) superfamily of cytokines. They play critical roles in the onset of acute and chronic inflammatory responses. The aim of this study was to investigate how activin inhibition affects acute kidney injury and inflammation after transplantation. The study was carried out in kidney transplantation and renal ischemia-reperfusion models in the rat. Soluble activin type 2 receptor (sActRIIB-Fc) was used to inhibit activin signaling. Transplantation groups were as follows: (i) cyclosporine A (CsA) (ii) CsA + sActRIIB-Fc, (iii) CsA+ inactive protein control Fc-G1. IRI groups were as follows: (i) no treatment, (ii) sActRIIB-Fc. Serum activin B concentration was significantly elevated after transplantation and IRI, whereas activin A was produced locally in renal allografts. Activin inhibition efficiently limited neutrophil, macrophage, and dendritic cell infiltration to the allografts measured 72 h after transplantation. In addition, sActRIIB-Fc treatment modulated serum cytokine response after transplantation and reduced the early accumulation of fibroblasts in the graft interstitium. In conclusion activin inhibition reduces the innate immune response early after renal transplantation in the rat. It also limits the accumulation of fibroblasts in the graft suggesting that activins may be involved in the fibrogenic signaling already early after kidney transplantation.

Topics: Activins; Allografts; Animals; Cytokines; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Immunity, Innate; Inflammation; Kidney; Kidney Transplantation; Male; Pilot Projects; Rats; Rats, Wistar; Renal Insufficiency; Reperfusion Injury; Signal Transduction; Time Factors; Transforming Growth Factor beta; Transplantation, Homologous

Acute kidney injury (AKI) is a disease where kidney function is lost almost instantaneously; it can develop very rapidly over few hours to maximum of few days. Despite the advent of technology, the clinical management against this disease is very poor, and most of the time it is life-threatening. AKI has been actively regulated by extracellular matrix proteins (ECM), however, its underlying mechanism of regulation during AKI progression is very poorly understood. In this study, we explored the integrated network of mRNA and microRNAs (miRNAs) that maintains the progression of ECM after induction of AKI by lethal ischemia. To identify key regulators of ECM, we screened large number of transcriptomes using laser capture microdissection (LCM) technique in addition to microarray and RT-qPCR. Our result clearly showed that 9 miRNAs including miR-21, miR-483, miR-5115, miR-204e, miR-128, miR-181c, miR-203, miR-204 and miR-204c were highly regulated, out of which miR-204 expression change (decrease) was most drastic during ischemia/reperfusion. Detail mechanistic study utilizing combined experimental and computational approach revealed that TGF-β signaling pathway was potentially modulated by deregulated miRNA-204 through SP1, where the TGF-β signaling pathway plays a vital role in ECM regulation. Apart from targeting SP1 and antagonizing epithelial-mesenchymal transition (EMT) signaling our result also showed that miR-204 protects interstitial tissue of renal tubules from chronic fibrotic change. Altogether our study provides sufficient details of how miRNA mediated ECM regulation occur during AKI, which can be effectively utilized in future for better AKI management and diagnosis.

Topics: Acute Kidney Injury; Animals; Down-Regulation; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibrosis; Gene Expression Regulation; Kidney Tubules; Laser Capture Microdissection; Male; Mice, Inbred C57BL; MicroRNAs; Reperfusion Injury; Sp1 Transcription Factor; Transforming Growth Factor beta

Testicular torsion is a common problem and, to date, there is no agent to preserve testicular function following detorsion. Platelet-rich plasma (PRP), with its rich growth factor composition, has proven beneficial in regenerative therapy. It is believed that PRP has not been studied in testis for ischemia/reperfusion (I/R) injury.. This study investigated the effect of PRP in an I/R rat model 1 month after detorsion.. Ischemia/reperfusion caused a significant increase in MDA, MPO and caspase-3 activity, and significant decrease in GSH levels and SOD activity. The PRP treatment helped correct the alterations in SOD, caspase-3, and MPO activities and MDA levels. However, the mean MDA level and MPO activity were not totally restored compared with the controls. Serum testosterone levels of the I/R group were significantly lower compared with the control and I/R + PRP groups. TGF-β and caspase-3 protein expressions were significantly higher in the I/R group compared with the control group and were low with PRP administration compared with I/R groups (summary Table).. The findings of the present study suggest that PRP, by inhibiting neutrophil infiltration and oxidative stress and increasing antioxidant defense, exerts protective effects on testicular tissues against I/R. This study had some limitations: a scoring system was not used in the assessment of spermatogenesis in the histopathological findings and specific testis cell types were not histologically assessed.. In light of the biochemical, histological and, especially, hormonal findings, intraparenchymal PRP injection may have a protective effect in testicular tissue against I/R injury.

Topics: Animals; Caspase 3; Hormones; Male; Oxidative Stress; Platelet-Rich Plasma; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spermatic Cord Torsion; Transforming Growth Factor beta

Mangiferin, a xanthone glycoside isolated from leaves of Mangifera indica (Anacardiaceae) is known to modulate many biological targets in inflammation and oxidative stress. The present study was designed to investigate whether mangiferin exerts protection against myocardial ischemia-reperfusion (IR) injury and possible role of Mitogen Activated Protein Kinase (MAPKs) and Transforming Growth Factor-β (TGF-β) pathways in its cardioprotection. Male albino Wistar rats were treated with mangiferin (40 mg/kg, i.p.) for 15 days. At the end of the treatment protocol, rats were subjected to IR injury consisting of 45 min ischemia followed by 1h reperfusion. IR-control rats caused significant cardiac dysfunction, increased serum cardiac injury markers, lipid peroxidation and a significant decrease in tissue antioxidants as compared to sham group. Histopathological examination of IR rats revealed myocardial necrosis, edema and infiltration of inflammatory cells. However, pretreatment with mangiferin significantly restored myocardial oxidant-antioxidant status, maintained membrane integrity, and attenuated the levels of proinflammatory cytokines, pro-apoptotic proteins and TGF-β. Furthermore, mangiferin significantly reduced the phosphorylation of p38, and JNK and enhanced phosphorylation of ERK1/2. These results suggest that mangiferin protects against myocardial IR injury by modulating MAPK mediated inflammation and apoptosis.

Topics: Animals; Apoptosis; Cardiotonic Agents; Gene Expression Regulation; Heart; Hemodynamics; Interleukin-6; Male; MAP Kinase Signaling System; Myocardium; Rats; Rats, Wistar; Reperfusion Injury; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Ventricular Function; Xanthones

Fibroblast growth factor 23 (FGF23) has been reported to induce left ventricular hypertrophy, but it remains unclear whether FGF23 plays a role in cardiac fibrosis. This study is attempted to investigate the role of FGF23 in post-infarct myocardial fibrosis in mice. We noted that myocardial and plasma FGF23 and FGF receptor 4 were increased in mice with heart failure as well as in cultured adult mouse cardiac fibroblasts (AMCFs) exposed to angiotensin II, phenylephrine, soluble fractalkine. Recombinant FGF23 protein increased active β-catenin , procollagen I and procollagen III expression in cultured AMCFs. Furthermore, intra-myocardial injection of adeno-associated virus-FGF23 in mice significantly increased left ventricular end-diastolic pressure and myocardial fibrosis, and markedly upregulated active β-catenin, transforming growth factor β (TGF-β), procollagen I and procollagen III in both myocardial infarction (MI) and ischemia/reperfusion (IR) mice, while β-catenin inhibitor or silencing of β-catenin antagonized the FGF23-promoted myocardial fibrosis in vitro and in vivo. These findings indicate that FGF23 promotes myocardial fibrosis and exacerbates diastolic dysfunction induced by MI or IR, which is associated with the upregulation of active β-catenin and TGF-β.

Topics: Angiotensin II; Animals; beta Catenin; Cells, Cultured; Collagen; Dependovirus; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibrosis; Humans; Hypertrophy, Left Ventricular; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Phenylephrine; Rats; Rats, Sprague-Dawley; Receptor, Fibroblast Growth Factor, Type 4; Reperfusion Injury; RNA, Small Interfering; Transforming Growth Factor beta; Up-Regulation

In most cases of pediatric liver transplantation, the clinical scenario of large-for-size transplants can lead to hepatic dysfunction and a decreased blood supply to the liver graft. The objective of the present experimental investigation was to evaluate the effects of ischemic preconditioning on this clinical entity.. Eighteen pigs were divided into three groups and underwent liver transplantation: a control group, in which the weights of the donors were similar to those of the recipients, a large-for-size group, and a large-for-size + ischemic preconditioning group. Blood samples were collected from the recipients to evaluate the pH and the sodium, potassium, aspartate aminotransferase and alanine aminotransferase levels. In addition, hepatic tissue was sampled from the recipients for histological evaluation, immunohistochemical analyses to detect hepatocyte apoptosis and proliferation and molecular analyses to evaluate the gene expression of Bax (pro-apoptotic), Bcl-XL (anti-apoptotic), c-Fos and c-Jun (immediate-early genes), ischemia-reperfusion-related inflammatory cytokines (IL-1, TNF-alpha and IL-6, which is also a stimulator of hepatocyte regeneration), intracellular adhesion molecule, endothelial nitric oxide synthase (a mediator of the protective effect of ischemic preconditioning) and TGF-beta (a pro-fibrogenic cytokine).. All animals developed acidosis. At 1 hour and 3 hours after reperfusion, the animals in the large-for-size and large-for-size + ischemic preconditioning groups had decreased serum levels of Na and increased serum levels of K and aspartate aminotransferase compared with the control group. The molecular analysis revealed higher expression of the Bax, TNF-alpha, I-CAM and TGF-beta genes in the large-for-size group compared with the control and large-for-size + ischemic preconditioning groups. Ischemic preconditioning was responsible for an increase in c-Fos, IL-1, IL-6 and e-NOS gene expression.. Ischemia-reperfusion injury in this model of large-for-size liver transplantation could be partially attenuated by ischemic preconditioning.

Topics: Acidosis; Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; bcl-2-Associated X Protein; Biomarkers; Gene Expression; Hepatocytes; Hydrogen-Ion Concentration; Ischemic Preconditioning; Liver; Liver Transplantation; Models, Animal; Nitric Oxide Synthase; Organ Size; Potassium; Random Allocation; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Sodium; Swine; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Glycogen synthase kinase-3β (GSK3β) is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA) expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β-SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

Topics: Animals; beta Catenin; Cell Differentiation; Cell Line; Epithelial Cells; Extracellular Matrix; Fibroblasts; Fibrosis; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Inflammation Mediators; Kidney; Macrophages; Mice, Inbred C57BL; Myofibroblasts; Protein Kinase Inhibitors; Reperfusion Injury; Signal Transduction; Thiadiazoles; Transforming Growth Factor beta

Renal fibrosis is a main cause of end-stage renal disease. Clinically, there is no beneficial treatment that can effectively reverse the progressive loss of renal function. We recently synthesized a novel proteolysis-resistant cyclic helix B peptide (CHBP) that exhibits promising renoprotective effects. In this study, we evaluated the effect of CHBP on renal fibrosis in an in vivo ischemia reperfusion injury (IRI) model and in vitro TGF-β-stimulated tubular epithelial cells (TCMK-1 and HK-2) model. In the IRI in vivo model, mice were randomly divided into sham (sham operation), IR and IR + CHBP groups (n = 6). CHBP (8 nmol/kg) was administered intraperitoneally at the onset of reperfusion, and renal fibrosis was evaluated at 12 weeks post-reperfusion. Our results showed that CHBP markedly attenuated the IRI-induced deposition of collagen I and vimentin. In the in vitro model, CHBP reversed the TGF-β-induced down-regulation of E-cadherin and up-regulation of α-SMA and vimentin. Furthermore, CHBP inhibited the phosphorylation of Akt and Forkhead box O 3a (FoxO3a), whose anti-fibrotic effect could be reversed by the 3-phosphoinositide-dependent kinase-1 (PI3K) inhibitor wortmannin as well as FoxO3a siRNA. These findings demonstrate that CHBP attenuates renal fibrosis and the epithelial-mesenchymal transition of tubular cells, possibly through suppression of the PI3K/Akt pathway and thereby the inhibition FoxO3a activity.

Topics: Animals; Epithelial-Mesenchymal Transition; Fibrosis; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred BALB C; Peptides, Cyclic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reperfusion Injury; Transforming Growth Factor beta

Emerging data have suggested that acute kidney injury (AKI) is often incompletely repaired and can lead to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. However, the underlying mechanisms linking AKI to CKD remain obscure. The present study aimed to investigate the role of cysteine-rich protein 61 (Cyr61) after unilateral kidney ischemia-reperfusion injury (IRI) in mice. After IRI, increased expression of Cyr61 was detected, predominately in the proximal tubular epithelium. This was confirmed by in vitro experiments, which showed that hypoxia stimulated Cyr61 expression in cultured proximal tubular epithelial cells. The proinflammatory property of Cyr61 was indicated by its ability to upregulate monocyte chemoattractant protein-1 and IL-6. Additionally, we found elevated urinary Cyr61 excretion in patients with AKI. Notably, treatment of mice with an anti-Cyr61 antibody attenuated the upregulation of kidney monocyte chemoattractant protein-1, IL-6, IL-1β, and macrophage inflammatory protein-2 and reduced the infiltration of F4/80-positive macrophages on days 7 and 14 after IRI. In addition, blockade of Cyr61 reduced the mRNA expression of collagen, transforming growth factor-β, and plasminogen activator inhibitor-I as well as the degree of collagen fibril accumulation, as evaluated by picrosirius red staining, and levels of α-smooth muscle actin proteins by day 14. Concurrently, in the treated group, peritubular microvascular density was more preserved on day 14. We conclude that Cyr61 blockade inhibits the triad of inflammation, interstitial fibrosis, and capillary rarefaction after severe ischemic AKI. The results of this study expand the knowledge of the mechanisms underlying the AKI-to-CKD transition and suggest that Cyr61 is a potential therapeutic target.

Topics: Acute Kidney Injury; Animals; Antibodies, Anti-Idiotypic; Cells, Cultured; Chemokine CCL2; Cysteine-Rich Protein 61; Disease Models, Animal; Fibrosis; Hypoxia; In Vitro Techniques; Interleukin-6; Kidney; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred ICR; Nephritis; Reperfusion Injury; Serpin E2; Transforming Growth Factor beta

Despite a significant improvement in the management of chronic kidney disease (CKD), its incidence and prevalence has been increasing over the years. Progressive renal fibrosis is present in CKD and involves the participation of several cytokines, including Transforming growth factor-β1 (TGF-β1). Besides cardiovascular diseases and infections, several studies show that Vitamin D status has been considered as a non-traditional risk factor for the progression of CKD. Given the importance of vitamin D in the maintenance of essential physiological functions, we studied the events involved in the chronic kidney disease progression in rats submitted to ischemia/reperfusion injury under vitamin D deficiency (VDD).. Rats were randomized into four groups: Control; VDD; ischemia/reperfusion injury (IRI); and VDD+IRI. At the 62 day after sham or IRI surgery, we measured inulin clearance, biochemical variables and hemodynamic parameters. In kidney tissue, we performed immunoblotting to quantify expression of Klotho, TGF-β, and vitamin D receptor (VDR); gene expression to evaluate renin, angiotensinogen, and angiotensin-converting enzyme; and immunohistochemical staining for ED1 (macrophages), type IV collagen, fibronectin, vimentin, and α-smooth mucle actin. Histomorphometric studies were performed to evaluate fractional interstitial area.. IRI animals presented renal hypertrophy, increased levels of mean blood pressure and plasma PTH. Furthermore, expansion of the interstitial area, increased infiltration of ED1 cells, increased expression of collagen IV, fibronectin, vimentin and α-actin, and reduced expression of Klotho protein were observed. VDD deficiency contributed to increased levels of plasma PTH as well as for important chronic tubulointerstitial changes (fibrosis, inflammatory infiltration, tubular dilation and atrophy), increased expression of TGF-β1 and decreased expression of VDR and Klotho protein observed in VDD+IRI animals.. Through inflammatory pathways and involvement of TGF-β1 growth factor, VDD could be considered as an aggravating factor for tubulointerstitial damage and fibrosis progression following acute kidney injury induced by ischemia/reperfusion.

Topics: Actins; Acute Kidney Injury; Angiotensinogen; Animals; Disease Progression; Fibronectins; Kidney; Male; Rats; Rats, Wistar; Receptors, Calcitriol; Renal Insufficiency, Chronic; Renin; Reperfusion Injury; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vitamin D Deficiency

The present study investigated the effects of ovarian hormone depletion and estrogen administration on ischemia/reperfusion (I/R)-induced bladder damage in female rabbits.. Female New Zealand white rabbits were divided into five groups. A sham surgical procedure was performed on rabbits in group 1. In group 2, both vesical arteries were clamped for 2 hours and then released (I/R surgical procedure). In group 3, 17β-estradiol (100 μg/kg/d) was injected intramuscularly before I/R surgical procedure. In group 4, ovariectomies were performed before I/R surgical procedure. Group 5 had ovariectomy, recovered for 2 weeks, and then received 17β-estradiol for 2 weeks. I/R surgical procedure was performed thereafter. Rabbits were killed 7 days after I/R surgical procedure. Masson's trichrome stain was used, and immunohistochemical experiments were performed to evaluate interstitial fibrosis and intramural nerve changes. Western immunoblots were examined to determine the expressions of markers for inflammation, fibrosis, and oxidative stress.. I/R surgical procedure decreased bladder contractile responses by 30% to 50%. Ovarian hormone depletion further reduced bladder contractile function by 45% to 55% compared with the I/R group members that retained their ovaries. Moreover, I/R surgical procedure significantly decreased intramural neurofilament staining by two thirds compared with the control group. Estrogen replacement after ovariectomy significantly increased the density of nerve terminals. In addition, the expression of transforming growth factor-β and fibronectin increased twofold and fivefold after I/R, respectively. Ovarian hormone depletion further increased the expression of these inflammatory and fibrosis markers. Ovariectomy significantly exacerbated oxidative damage, whereas estrogen replacement diminished oxidative stress to a level approaching that of the control group.. I/R surgical procedure increases oxidative damage, enhances interstitial fibrosis, and results in bladder denervation. Ovarian hormone deficiency exacerbates this I/R-induced bladder damage, whereas estrogen therapy after ovariectomy attenuates this injury. These results reveal estrogen's protective effects on bladders subjected to I/R injury and the potential benefits of estrogen therapy on I/R-induced bladder damage.

Topics: Animals; Estradiol; Estrogen Replacement Therapy; Female; Fibronectins; Gene Expression; Injections, Intramuscular; Muscle Contraction; Ovariectomy; Rabbits; Reperfusion Injury; Transforming Growth Factor beta; Urinary Bladder; Urinary Bladder Diseases

Matrix metalloprotease (MMP) 9 has been always considered as a destructor of extracellular matrix, promoting liver injury and metastasis of carcinoma. In this study, we investigated the role of MMP-9 in liver wound healing from ischemia and reperfusion injury (IRI).. MMP9-/- mice were used to establish partial hepatic IRI model. Serum alanine aminotransferase and hepatic cytokines (tumor necrosis factor alpha, interleukin [IL]-1β, IL-10, and transforming growth factor beta [TGF-β]) levels were analyzed after IRI. Hepatic stellate cells were isolated from wild-type mice to determine the effect of MMP-9 on TGF-β activation. In addition, the effect of TGF-β on liver wound healing from IRI was determined.. Liver recovery from IRI was impaired in MMP9-/- mice, which was described as elevated serum alanine aminotransferase, hepatic tumor necrosis factor alpha, and IL-1β levels. Meanwhile, TGF-β-active protein level was decreased in the liver of MMP9-/- mice. In vitro test, the activation of TGF-β was suppressed in the presence of anti-MMP-9 monoclonal antibody. TGF-β treatment promoted liver recovery from IRI in MMP9-/- mice.. MMP-9 promoted liver recovery from IRI by activating TGF-β. Thus, MMP-9 plays dual roles (bad and good) in liver IRI, depending on the timing.

Topics: Animals; Ischemia; Liver; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Reperfusion Injury; Repressor Proteins; Transforming Growth Factor beta; Wound Healing

In the presence of TGF-β, CD4+CD62L+T cells can be induced to CD4+CD25+FoxP3+ regulatory T cells (iTregs). In our previous work, we have shown that adoptive transfer of iTregs promoted liver recovery from ischemia reperfusion injury (IRI). In this study, we examined the molecular mechanism underlying the liver IRI attenuation by iTregs in a mouse partial hepatic IRI model. We found that the population of hepatic Tregs decreased significantly at 24 h after reperfusion. Adoptive transfer of iTregs before IRI markedly increased the numbers of hepatic Tregs and attenuated liver IRI as indicated by reduced serum aminotransferases and proinflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Ex vivo study indicated that iTregs suppressed IL-1β and TNF-α expression, promoted transcription of interleukin-10 (IL-10), and elevated phosphorylation of SMAD3 in Kupffer cells (KCs). Furthermore, inhibition of TGF-β signaling by anti-TGF-β abolished the effects on KCs. Treatment with TGF-β suppressed matrix metalloprotease (MMP9) production in KCs and protected liver from IRI. In conclusion, our results suggest that iTregs play a critical role in hepatic IRI by regulating pro-inflammatory and anti-inflammatory function of KCs through TGF-β.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cytokines; Disease Models, Animal; Kupffer Cells; Liver; Male; Mice; Mice, Inbred C57BL; Reperfusion Injury; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Hydrogen sulfide (H(2)S) has been shown to display anti-inflammatory and antioxidant activities.. This study was designed to investigate the gastroprotective effect of sodium hydrosulfide (NaHS) on gastric mucosal lesions induced by ischemia-reperfusion (I/R) injury in rats and to determine the possible mechanism involved.. Fifty-sex male Wistar rats were randomly assigned into sham, control (I/R injury), propargylglycine (PAG)-, L-cysteine-, and NaHS-treated groups. To induce I/R lesions, the celiac artery was first clamped for 30 min (ischemia phase), followed by removal of the clamp artery to allow reperfusion for 3 h. Treated rats received PAG [50 mg/kg, intravenous (i.v.)] or NaHS (160, 320, or 640 ng/kg, i.v.) 5 min before reperfusion. The effect of L-cysteine pretreatment was also investigated. Plasma levels of cytokines and cortisol were measured by an enzyme-linked immunosorbent assay. The gastric tissue samples were collected to quantify the mRNA expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and transforming growth factor (TGF-β) by quantitative real-time PCR.. The total area of gastric lesions significantly decreased following the administration of NaHS and L-cysteine. The highest area of mucosal lesions was observed in PAG-treated rats. The mRNA expression and plasma levels of IL-1β and TNF-α were significantly decreased in L-cysteine- and NaHS-treated rats in a dose-dependent manner. Slightly increased levels of TGF-β were observed in these test groups, but the difference was not statistically significant compared with the other groups. The plasma level of cortisol was also not affected by NaHS treatment.. Our findings indicate that a possible mechanism for the gastroprotective effect of H(2)S could be through the decreased mRNA expression and plasma release of proinflammatory cytokines.

Topics: Alkynes; Animals; Biopsy, Needle; Cystine; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gastric Mucosa; Glycine; Immunohistochemistry; Infusions, Intravenous; Interleukin-1beta; Male; Random Allocation; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reference Values; Reperfusion Injury; RNA, Messenger; Sensitivity and Specificity; Sulfides; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-β is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-β signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-β antagonism. Tubule-specific induction of TGF-β led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-β depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-β antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.

Topics: Acute Kidney Injury; Animals; Cell Differentiation; Cells, Cultured; Chronic Disease; Fibrosis; Humans; Kidney Diseases; Kidney Tubules, Proximal; Male; MAP Kinase Kinase 4; Mice; Mice, Transgenic; Models, Animal; Phenotype; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta

Endothelial progenitor cells (EPCs) protect the kidney from acute ischemic injury. The aim of this study was to analyze whether pretreatment of murine "early outgrowth" EPCs (eEPCs) with the hormone melatonin increases the cells' renoprotective effects in the setting of murine acute ischemic renal failure. Male (8-12 wk old) C57Bl/6N mice were subjected to unilateral ischemia-reperfusion injury postuninephrectomy (40 min). Postischemic animals were injected with either 0.5×10(6) untreated syngeneic murine eEPCs or with cells, pretreated with melatonin for 1 h. Injections were performed shortly after reperfusion of the kidney. While animals injected with untreated cells developed acute renal failure, eEPC pretreatment with melatonin dramatically improved renoprotective actions of the cells. These effects were completely reversed after cell pretreatment with melatonin and the MT-1/-2 antagonist luzindole. In vitro analysis revealed that melatonin reduced the amount of tumor growth factor-β-induced eEPC apoptosis/necrosis. Secretion of vascular endothelial growth factor by the cells was markedly stimulated by the hormone. In addition, migratory activity of eEPCs was enhanced by melatonin and supernatant from melatonin-treated eEPCs stimulated migration of cultured mature endothelial cells. In summary, melatonin was identified as a new agonist of eEPCs in acute ischemic kidney injury.

Topics: Acute Kidney Injury; Animals; Antioxidants; Apoptosis; Cell Movement; Cells, Cultured; Endothelial Cells; Male; Melatonin; Mice; Mice, Inbred C57BL; Necrosis; Neovascularization, Physiologic; Recovery of Function; Reperfusion Injury; Stem Cells; Transforming Growth Factor beta

Several beneficial effects have been demonstrated for secretogranin II (SgII) in non-cardiac tissue. As cardiac production of chromogranin A and B, two related proteins, is increased in heart failure (HF), we hypothesized that SgII could play a role in cardiovascular pathophysiology.. SgII production was characterized in a post-myocardial infarction heart failure (HF) mouse model, functional properties explored in experimental models, and circulating levels measured in mice and patients with stable HF of moderate severity. SgII mRNA levels were 10.5 fold upregulated in the left ventricle (LV) of animals with myocardial infarction and HF (p<0.001 vs. sham-operated animals). SgII protein levels were also increased in the LV, but not in other organs investigated. SgII was produced in several cell types in the myocardium and cardiomyocyte synthesis of SgII was potently induced by transforming growth factor-β and norepinephrine stimulation in vitro. Processing of SgII to shorter peptides was enhanced in the failing myocardium due to increased levels of the proteases PC1/3 and PC2 and circulating SgII levels were increased in mice with HF. Examining a pathophysiological role of SgII in the initial phase of post-infarction HF, the SgII fragment secretoneurin reduced myocardial ischemia-reperfusion injury and cardiomyocyte apoptosis by 30% and rapidly increased cardiomyocyte Erk1/2 and Stat3 phosphorylation. SgII levels were also higher in patients with stable, chronic HF compared to age- and gender-matched control subjects: median 0.16 (Q1-3 0.14-0.18) vs. 0.12 (0.10-0.14) nmol/L, p<0.001.. We demonstrate increased myocardial SgII production and processing in the LV in animals with myocardial infarction and HF, which could be beneficial as the SgII fragment secretoneurin protects from ischemia-reperfusion injury and cardiomyocyte apoptosis. Circulating SgII levels are also increased in patients with chronic, stable HF and may represent a new cardiac biomarker.

Topics: Animals; Apoptosis; Female; Heart Failure; Heart Ventricles; Humans; Male; Mice; Middle Aged; Myocardium; Myocytes, Cardiac; Neuropeptides; Norepinephrine; Rats; Reperfusion Injury; Secretogranin II; Transforming Growth Factor beta; Up-Regulation

The tissue-protective effects of erythropoietin (EPO) have been extensively investigated, and EPO administration can raise the hemoglobin (Hb) concentration. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia-reperfusion injury as well as EPO.. To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a tubulointerstitial model rat. We randomized remnant kidney model rats to receive saline, EPO, or CEPO for 8 weeks.. CEPO- and EPO-treated rats had improved serum creatinine levels compared with saline-treated remnant kidney model rats, although the Hb level was significantly increased in EPO-treated rats. Two-photon microscopy revealed that EPO/CEPO significantly ameliorated tubular epithelial cell damage assessed by endocytosis. In addition, CEPO or EPO protected endothelial cells with a sustained blood flow rate. EPO or CEPO suppressed the number of TUNEL-positive apoptotic cells with weak αSMA staining. Furthermore, PCR analysis demonstrated that TGF-β and type I collagen expression was attenuated in EPO- or CEPO-treated rats, accompanied by a significant decrease in interstitial fibrosis.. We established a long-term therapeutic approach to protect tubulointerstitial injury with CEPO, and thus, the therapeutic value of this approach warrants further attention and preclinical studies.

Topics: Animals; Collagen Type I; Creatinine; Disease Models, Animal; Erythropoietin; Fibrosis; Hemoglobins; Kidney; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transforming Growth Factor beta

After ischemia-reperfusion injury (IRI), kidney tubules show activated transforming growth factor β (TGF-β) signaling and increased expression of profibrotic peptides, platelet-derived growth factor-B (PDGF-B) and connective tissue growth factor (CTGF). If tubule repair after IRI is incomplete, sustained paracrine activity of these peptides can activate interstitial fibroblast progenitors and cause fibrosis. We show that lysophosphatidic acid (LPA), a ubiquitous phospholipid that is increased at sites of injury and inflammation, signals through LPA2 receptors and Gαq proteins of cultured proximal tubule cells to transactivate latent TGF-β in a Rho/Rho-kinase and αvβ6 integrin-dependent manner. Active TGF-β peptide then initiates signaling to increase the production and secretion of PDGF-B and CTGF. In a rat model of IRI, increased TGF-β signaling that was initiated early during reperfusion did not subside during recovery, but progressively increased, causing tubulointerstitial fibrosis. This was accompanied by correspondingly increased LPA2 and β6 integrin proteins and elevated tubule expression of TGF-β1, together with PDGF-B and CTGF. Treatment with a pharmacological TGF-β type I receptor antagonist suppressed TGF-β signaling, decreased the expression of β6 integrin, PDGF-B, and CTGF, and ameliorated fibrosis. We suggest that LPA-initiated autocrine signaling is a potentially important mechanism that gives rise to paracrine profibrotic signaling in injured kidney tubule cells.

Topics: Animals; Antigens, Neoplasm; Cell Nucleus; Connective Tissue Growth Factor; Cytokines; Fibrosis; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Integrins; Kidney Tubules, Proximal; Lipids; Lysophospholipids; Male; Mice; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Regeneration; Reperfusion Injury; rho GTP-Binding Proteins; RNA, Messenger; Signal Transduction; Smad2 Protein; Time Factors; Transforming Growth Factor beta

Cerium oxide (CeO₂) represents an important nanomaterial with wide ranging applications. However, little is known regarding how CeO₂ exposure may influence pulmonary or systemic inflammation. Furthermore, how mast cells would influence inflammatory responses to a nanoparticle exposure is unknown. We thus compared pulmonary and cardiovascular responses between C57BL/6 and B6.Cg-Kit(W-sh) mast cell deficient mice following CeO₂ nanoparticle instillation. C57BL/6 mice instilled with CeO₂ exhibited mild pulmonary inflammation. However, B6.Cg-Kit(W-sh) mice did not display a similar degree of inflammation following CeO₂ instillation. Moreover, C57BL/6 mice instilled with CeO₂ exhibited altered aortic vascular responses to adenosine and an increase in myocardial ischemia/reperfusion injury which was absent in B6.Cg-Kit(W-sh) mice. In vitro CeO₂ exposure resulted in increased production of PGD₂, TNF-α, IL-6 and osteopontin by cultured mast cells. These findings demonstrate that CeO₂ nanoparticles activate mast cells contributing to pulmonary inflammation, impairment of vascular relaxation and exacerbation of myocardial ischemia/reperfusion injury.

Topics: Adenosine; Analysis of Variance; Animals; Aorta, Thoracic; Bronchoalveolar Lavage Fluid; Cerium; Chemokine CCL3; Gene Expression Regulation; Histocytochemistry; Interleukin-10; Interleukin-13; Interleukin-6; Lung; Male; Mast Cells; Metal Nanoparticles; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Osteopontin; Particle Size; Pneumonia; Prostaglandin D2; Reperfusion Injury; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Heme oxygenase (HO)-1 protects transplanted organs from ischemia reperfusion injury and immune rejection. This study sought to investigate whether persistent overexpression of HO-1 in donor livers could improve the survival by expanding T regulatory cells in a rat model of orthotopic liver transplantation.. Livers of Dark Agouti rats were intraportally perfused with an AAV expression vector encoding rat HO-1 (AAV-HO-1), and then transplanted into Lewis rats. The survival, HO-1 activity, Banff rejection activity index, serum levels of IL-2 and TNF-α, infiltration of CD4(+), CD8(+), and T(reg) (CD4(+)CD25(+)Foxp3(+)) cells into donor livers, and expression of Foxp3, TGF-β, and IL-10 were examined. A mixed lymphocyte reaction (MLR) was performed.. Intraportal delivery of AAV-HO-1 resulted in persistent expression of HO-1 and increased activity of HO-1 in transplanted livers, leading to prolonged survival of recipients. Overexpression of HO-1 reduced the Banff rejection activity index, and production of IL-2 and TNF-α, inhibited infiltration of CD4(+) and CD8(+) cells, and increased infiltration of T(reg) cells, into donor livers. The spleens of recipients expressed higher levels of Foxp3, TGF-β, and IL-10 than those of control rats, and the transplanted livers expressed higher levels of Foxp3 and TGF-β. Splenocytes from the tolerant recipients had higher percentages of T(reg) cells, and responded poorly to the allogeneic donor splenocytes.. Persistent expression of HO-1 in donor livers by intraportal delivery of AAV-HO-1 improves the survival by expanding T(reg) cells. HO-1-based therapies, as described herein, promise new strategies to prevent the rejection of liver transplants.

Topics: Adenoviridae; Animals; CD8-Positive T-Lymphocytes; Forkhead Transcription Factors; Genetic Therapy; Graft Rejection; Graft Survival; Heme Oxygenase (Decyclizing); Interleukin-10; Liver; Liver Transplantation; Male; Rats; Rats, Inbred Lew; Rats, Wistar; Reperfusion Injury; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Transplantation Conditioning; Transplantation, Homologous

Renal ischemia followed by reperfusion results in kidney injury which in turn produces and releases destructive inflammatory cytokines into the circulation causing subsequent distant organ injury. Little data suggest the immune mechanism of curcumin on protection against ischemia/reperfusion induced injury. We investigated the immunomodulatory and anti-apoptotic effects of curcumin on ischemia/reperfusion (I/R) injury in rats. Thirty-six rats were randomly divided into three experimental groups (sham, I/R and curcumin pretreated I/R, n=12 each). Curcumin was administered orally to curcumin pretreated I/R group. Curcumin can significantly decrease both systemic as well as blood levels of cytokines (p<0.05). Treatment with curcumin also resulted in significant reduction in serum and tissue level of TNF-α, IL-1β, IL-12, IL-18 and INF-γ that were increased by renal I/R injury (p<0.05). Curcumin pretreatment reduce pulmonary apoptotic pathway via significant inhibition of TGF-β and caspase-3 in kidney and lung tissues. Given that pulmonary apoptosis is an important complication of acute renal injury, we identified curcumin protective effect against distant organ I/R induced injury. Based on our results, we concluded that curcumin protects the kidneys and other vital organs against I/R injury via immune-mediated and the new identified anti-apoptotic mechanisms.

Topics: Animals; Apoptosis; Caspase 3; Curcumin; Cytokines; Immunologic Factors; Kidney; Kidney Diseases; Lung; Lung Injury; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transforming Growth Factor beta

Non-hematopoietic CD45+ precursor cells are not known to differentiate into cardiomyocytes. We found that CD45+/CD34-/lin- stromal cells isolated from mouse bone marrow (BMSCs) potentially differentiated into cardiomyocyte-like cells in vitro. Therefore, we hypothesized that the CD45+/CD34-/ lin- BMSCs might protect rat hearts against ischemia/reperfusion (IR) injury following xeno-transplantation. In the present study, BMSCs were isolated by immunoselection and their cellular phenotype and biochemical properties were characterized. The immunological inertness of BMSCs was examined by the allogeneic and xenogeneic mixed lymphocyte reaction (MLR). The potential role of BMSCs for cardioprotection was evaluated by intravenous introduction of 1 x 10(6) cells into rat IR hearts, induced by left coronary ligation for 45 min and released for 72 h. Changes in cardiac contractility and the degree of myocardial injury were assessed. Our findings indicated that BMSCs expressed the muscle-cell marker alpha-actinin after 5-azacytidine treatment. CD45+/CD34-/lin- stromal cells were characterized as mesenchymal progenitor cells based on the expression of Sca-1 and Rex-1. The MLR assay revealed an immunosuppression of BMSCs on mouse and rat lymphocytes. After xeno-transplantation, the BMSCs engrafted into the infarct area and attenuated IR injury. However, increases in intracardial TGF-beta and IFN-gamma contents of IR hearts were not affected by BMSC treatment. Interestingly, ex vivo evidence indicated that CXCR4, SDF-1 and TGFbeta-1 receptors were up-regulated after the cells were exposed to tissue extracts prepared from rat post-IR hearts. In addition, IFN-gamma treatment also markedly increased Sca-1 expression in BMSCs. Mechanistically, these results indicated that CXCR4/SDF-1 and TGF-beta signals potentially enhanced the interaction of BMSCs with the damaged myocardium, and increased IFN-gamma in post-ischemic hearts might cause BMSC to behave more like stem cells in cardioprotection. These data show that CD45+/CD34-/lin- BMSCs possess cardioprotective capacity. Evidently, the accurate production of soluble factors TGF-beta and IFN-gamma in parallel with increased expression of both TGF-beta and Sca-1 receptors may favor BMSCs to achieve a more efficient protective capacity.

Topics: Actinin; Animals; Antigens, CD34; Antigens, Ly; Azacitidine; Bone Marrow Cells; Chemokine CXCL12; Female; Immunosuppression Therapy; Interferon-gamma; Leukocyte Common Antigens; Lymphocytes; Male; Membrane Proteins; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Myocardial Contraction; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Receptors, CXCR4; Receptors, Transforming Growth Factor beta; Reperfusion Injury; Stromal Cells; Transcription Factors; Transforming Growth Factor beta; Transplantation, Heterologous

The prevailing notion is that ischemia reperfusion injury of the small bowel induces transient changes that resolve within a few days post-occurrence. However, chronic injury has been described following a single ischemia reperfusion in the kidney. We proceeded to ascertain if a similar outcome is also witnessed in the small bowel.. ACI rats (n=32) underwent 1, 2 or 3 episodes of ischemia reperfusion by clamping the superior mesenteric artery for 45 minutes at 7-day intervals. Control groups included sham-operated (n=6) or non-operated (n=5) rats. Morphology was examined at day ninety post-ischemia reperfusion and immunostaining was used to evaluate macrophage infiltration, microvascular distribution, and apoptosis. RT-PCR was used to evaluate expression of Inter-Cellular Adhesion Molecule-1 (ICAM-1), transforming growth factor-beta (TGF-beta), Insulin Growth Factor-I (IGF-1), and Insulin Growth Factor-I Receptor (IGF-R). Intestinal function was evaluated by D-xylose performed 24 hours and 4, 8, and 12 weeks after reperfusion.. Chronic morphologic changes were observed with degeneration of crypts, endothelial damage, matrix degeneration, and heightened lymphocyte degeneration within the Payer's patches. Major structural changes were characterized by villous atrophy from partial to total. The grade of histological injuries was significantly increased (P<0.001) after multiple ischemia reperfusion episodes. A higher number of apoptotic cells (P<0.001) and a prominent macrophage infiltration (P<0.05) was also witnessed. Altered expression of ICAM-1, TGF-beta, and IGF-1 was observed. At 24 hours after ischemia reperfusion D-xylose absorption was diminished, returning to baseline values within 4 weeks and becoming abnormal again at 8 and 12 weeks (P<0.05).. Unlike the prevailing conviction, these data demonstrate that transient ischemia reperfusion repeated injuries of the small bowel result in chronic intestinal damage.

Topics: Animals; Apoptosis; Atrophy; Immunohistochemistry; Insulin-Like Growth Factor I; Intercellular Adhesion Molecule-1; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Male; Rats; Rats, Inbred ACI; Receptor, IGF Type 1; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transforming Growth Factor beta

Regeneration of skeletal muscle following injury is dependent on numerous factors including age, the inflammatory response, revascularization, gene expression of myogenic and growth factors and the activation and proliferation of endogenous progenitor cells. It is our hypothesis that oxidative stress preceding a contusion injury to muscle modulates the inflammatory response to inhibit muscle regeneration and enhance fibrotic scar formation. Male F344/BN rats were assigned to one of four groups. Group 1: uinjured control; Group 2: ischaemic occlusion of femoral vessels for 2 h followed by reperfusion (I-R); Group 3: contusion injury of the tibialis anterior (TA); Group 4: I-R, then contusion injury. The acute inflammatory response (8 h, 3 days) was determined by expression of the chemokine CINC-1, TGF-beta1, IFN-gamma and markers of neutrophil (myeloperoxidase) and macrophage (CD68) activity and recruitment. Acute oxidative stress caused by I-R and/or contusion, was determined by measuring GP91(phox) and lipid peroxidation. Muscle recovery (21 days) was assessed by examining the fibrosis after I-R and contusion injuries to the TA with Sirius Red staining and quantification of collagen I expression. Consistent with our hypothesis, I-R preceding contusion increased all markers of the acute inflammatory response and oxidative stress after injury and elevated the expression of collagen. We conclude that ischaemia-induced oxidative stress exacerbated the inflammatory response and enhanced fibrotic scar tissue formation after injury. This response may be attributable to increased levels of TGF-beta1 and diminished expression of IFN-gamma in the ischaemic contused muscle.

Topics: Animals; Contusions; Fibrosis; Inflammation; Interferon-gamma; Male; Muscle, Skeletal; Oxidative Stress; Rats; Rats, Inbred F344; Regeneration; Reperfusion Injury; Transforming Growth Factor beta

Peripheral blood CD34(+) cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation and diminished migratory prowess, largely resulting from decreased nitric oxide (NO) bioavailability. The level of TGF-beta, a key factor that modulates stem cell quiescence, is increased in the serum of type 2 diabetic patients. We asked whether transient TGF-beta1 inhibition in CD34(+) cells would improve their reparative ability.. To inhibit TGF-beta1 protein expression, CD34(+) cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-beta1-PMOs) and analyzed for cell surface CXCR4 expression, cell survival in the absence of added growth factors, SDF-1-induced migration, NO release, and in vivo retinal vascular reparative ability.. TGF-beta1-PMO treatment of diabetic CD34(+) cells resulted in increased expression of CXCR4, enhanced survival in the absence of growth factors, and increased migration and NO release as compared with cells treated with control PMO. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after TGF-beta1-PMO treatment compared with control PMO-treated cells.. Transient inhibition of TGF-beta1 may represent a promising therapeutic strategy for restoring the reparative capacity of dysfunctional diabetic CD34(+) cells.

Topics: Animals; Antigens, CD34; Capillaries; Cell Survival; Diabetes Mellitus; Diabetic Angiopathies; Diabetic Retinopathy; Flow Cytometry; Hematopoietic Stem Cells; Humans; Mice; Morpholines; Morpholinos; Nitric Oxide; Receptors, CXCR4; Reperfusion Injury; Transforming Growth Factor beta; Transforming Growth Factor beta1

After a myocardial infarction (MI), an episode of ischemia-reperfusion (I/R) can result in a greater impairment of left ventricular (LV) regional function (LVRF) than that caused by an initial I/R episode in the absence of MI. Membrane type-I matrix metalloproteinase (MT1-MMP) proteolytically processes the myocardial matrix and is upregulated in LV failure. This study tested the central hypothesis that a differential induction of MT1-MMP occurs and is related to LVRF after I/R in the context of a previous MI. Pigs with a previous MI [3 wk postligation of the left circumflex artery (LCx)] or no MI were randomized to undergo I/R [60-min/120-min left anterior descending coronary artery (LAD) occlusion] or no I/R as follows: no MI and no I/R (n = 6), no MI and I/R (n = 8), MI and no I/R (n = 8), and MI and I/R (n = 8). Baseline LVRF (regional stroke work, sonomicrometry) was lower in the LAD region in the MI group compared with no MI (103 ± 12 vs. 188 ± 26 mmHg·mm, P < 0.05) and remained lower with peak ischemia (35 ± 8 vs. 88 ± 17 mmHg·mm, P < 0.05). Using a novel interstitial microdialysis method, MT1-MMP was directly measured and was over threefold higher in the LCx region and over twofold higher in the LAD region in the MI group compared with the no MI group at baseline. MT1-MMP fluorogenic activity was persistently elevated in the LCx region in the MI and I/R group but remained unchanged in the LAD region. In contrast, no changes in MT1-MMP occurred in the LCx region in the no MI and I/R group but increased in the LAD region. MT1-MMP mRNA was increased by over threefold in the MI region in the MI and I/R group. In conclusion, these findings demonstrate that a heterogeneous response in MT1-MMP activity likely contributes to regional dysfunction with I/R and that a subsequent episode of I/R activates a proteolytic cascade within the MI region that may contribute to a continued adverse remodeling process.

Topics: Animals; Disease Models, Animal; Fibrillar Collagens; Fibrosis; Gene Expression Regulation, Enzymologic; Hemodynamics; Male; Matrix Metalloproteinase 14; Microdialysis; Myocardial Infarction; Myocardium; Reperfusion Injury; RNA, Messenger; Swine; Time Factors; Transforming Growth Factor beta; Ventricular Function, Left; Ventricular Remodeling

Reperfusion injury causes liver dysfunction after warm or cold ischemia. Emerging data suggest a role of T cells as mediators in this ischemia/reperfusion (I/R) injury. In the T cells, a part of CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs) were reported to facilitate recovery from I/R injury. These Tregs can be induced by TGF-beta in vitro. Interestingly, rapamycin was reported to selectively expand these Tregs in vitro. In the present study, addition of rapamycin to cultures containing TGF-beta further increased the frequency and absolute number of functional CD4(+) Tregs. Using a partial (70%) hepatic warm ischemia model, we investigated the effects of liver function recovery under the treatment of Tregs induced by rapamycin and TGF-beta. The treatment of Tregs significantly reduced serum alanine aminotransferase and aspartate aminotransferase compared to I/R control animals at 24 h after reperfusion (P<0.05). They also significantly attenuated the up-regulation of IFN-gamma and IL-17 compared to the I/R control animals (P<0.05). In conclusion, Tregs ameliorate the biochemical of hepatic I/R injury by preventing proinflammatory cytokines following a warm I/R insult. These data may pave the way to use Tregs as cell therapy to prevent hepatic I/R injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; CD4 Antigens; Forkhead Transcription Factors; Interferon-gamma; Interleukin-17; Interleukin-2 Receptor alpha Subunit; Liver; Lymphocyte Activation; Male; Rats; Rats, Wistar; Recovery of Function; Reperfusion Injury; Sirolimus; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Microvascular dysfunction, loss of vascular support, ischaemia and sub-acute vascular instability in surviving blood vessels contribute to secondary injury following SCI (spinal cord injury). Neither the precise temporal profile of the cellular dynamics of spinal microvasculature nor the potential molecular effectors regulating this plasticity are well understood. TGFβ (transforming growth factor β) isoforms have been shown to be rapidly increased in response to SCI and CNS (central nervous system) ischaemia, but no data exist regarding their contribution to microvascular dysfunction following SCI. To examine these issues, in the present study we used a model of focal spinal cord ischaemia/reperfusion SCI to examine the cellular response(s) of affected microvessels from 30 min to 14 days post-ischaemia. Spinal endothelial cells were isolated from affected tissue and subjected to focused microarray analysis of TGFβ-responsive/related mRNAs 6 and 24 h post-SCI. Immunohistochemical analyses of histopathology show neuronal disruption/loss and astroglial regression from spinal microvessels by 3 h post-ischaemia, with complete dissolution of functional endfeet (loss of aquaporin-4) by 12 h post-ischaemia. Coincident with this microvascular plasticity, results from microarray analyses show 9 out of 22 TGFβ-responsive mRNAs significantly up-regulated by 6 h post-ischaemia. Of these, serpine 1/PAI-1 (plasminogen-activator inhibitor 1) demonstrated the greatest increase (>40-fold). Furthermore, uPA (urokinase-type plasminogen activator), another member of the PAS (plasminogen activator system), was also significantly increased (>7.5-fold). These results, along with other select up-regulated mRNAs, were confirmed biochemically or immunohistochemically. Taken together, these results implicate TGFβ as a potential molecular effector of the anatomical and functional plasticity of microvessels following SCI.

Topics: Animals; Endothelial Cells; Female; Microvessels; Neuronal Plasticity; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Spinal Cord Ischemia; Transcriptional Activation; Transforming Growth Factor beta

Advances in the treatment of reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. The authors examined the direct and indirect effects of adipose-derived stem cells on ischemia-reperfusion injury on a skin flap model to determine the in vivo differentiation of adipose-derived stem cells to endothelial cells; the levels of vascular endothelial growth factor (VEGF), transforming growth factor-beta, and fibroblast growth factor; and the ultrastructural changes apparent with scanning electron microscopy to clarify the initial events and the following cascades.. Two identical cranial based random flaps with a dimension of 1 x 5 cm were elevated on the dorsums of 20 ICR mice. The left flap was designated as the control and the right flap was injected with adipose-derived stem cells. The flaps were then subjected to 6 hours of ischemia by clamping the pedicle, and then reperfusion.. The mean viable flap length in the control and experimental groups was 15.2 +/- 3.4 mm and 24.4 +/- 2.9 mm, respectively. The mean viable flap area in the control and experimental groups was 12.9 +/- 4.1 mm and 21.8 +/- 3.7 mm, respectively. The in vivo differentiation of adipose-derived stem cells to endothelial cells was observed. The immunohistochemical stainings, VEGF, transforming growth factor-beta, and fibroblast growth factor revealed increased levels in the experimental groups. Scanning electron microscopy indicated mild injury in the experimental group.. The adipose-derived stem cells could prevent ischemia-reperfusion injury, mainly by regulating the growth factors. Although VEGF was the foremost inhibitor of injury, the overall cascade was enhanced by adipose-derived stem cells, with the help of the other growth factors.

Topics: Adipocytes; Angiography; Animals; Cell Differentiation; Fibroblast Growth Factors; Immunohistochemistry; Mice; Mice, Inbred ICR; Microradiography; Neovascularization, Physiologic; Reperfusion Injury; Stem Cell Transplantation; Surgical Flaps; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The objective of this research is to investigate the influence of rhBMP-2 on the renal tissue of rat with renal ischemia reperfusion injury. In this program the ischemia reperfusion rat model was established and Wistar rats were divided into six groups: sham operation group (S group), renal ischemia reperfusion injury group (R group), rhBMP-2 treatment group (B1, B2, B3 and B4 group). In the rhBMP treatment groups, rhBMP-2 was intravenously administered with different doses before reperfusion. The contents of TNF-alpha, IL-6, IL-8, MDA and SOD in kidney tissue were observed. At the same time, renal function (blood creatine (Scr) and urea nitrogen (BUN)) were measured. As a result, compared with renal ischemia reperfusion group, administration of rhBMP-2 significantly reduced the content of IL-6 and IL-8 (P < 0.05) and ameliorated renal dysfunction cellular damages (P < 0.05). Higher dose of rhBMP-2 may reduce the content of TNF-alpha (P < 0.05) in kidney tissue. rhBMP-2 also increased activity of SOD and reduced the level of MDA, BUN and Scr. So, we can draw a conclusion that rhBMP-2 treatment attenuates renal ischemia reperfusion injury through inhibition of pro-inflammatory cytokines production and anti-oxidation activity.

Topics: Adrenal Cortex; Animals; Blood Urea Nitrogen; Bone Morphogenetic Protein 2; Creatine; Interleukin-6; Interleukin-8; Kidney; Male; Malondialdehyde; Random Allocation; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We investigated the role of secretory leukocyte protease inhibitor (SLPI) in ischemia/reperfusion injury in cardiac transplantation. SLPI-/- mouse hearts and wild-type (WT) controls were transplanted immediately or after 10 h of cold ischemia (CI). Recombinant SLPI (rSLPI) was added to the preservation solution or given systemically. After evaluation of myocardial performance, grafts were investigated for histology, SLPI, TNF-alpha, TGF-beta, NF-kappaB and protease expression at indicated time points. Early myocardial contraction was profoundly impaired in SLPI-/- hearts exposed to CI and associated with high intra-graft protease expression. Systemic administration of rSLPI had no effect, however, when SLPI was added to the preservation solution, myocardial contraction was restored to normal. At 10 days, inflammation, myocyte vacuolization and necrosis were significantly more severe in SLPI-/- hearts. SLPI gene expression was detected in WT mice at 12 and 24 h and was significantly higher after CI. SLPI protein was observed at 24 h and 10 days. High intra-graft concentrations of SLPI after administration of rSLPI were inversely correlated with protease levels early and TGF-beta expression late after reperfusion. SLPI plays a crucial role in early myocardial performance and postischemic inflammation after cardiac transplantation. A dual inhibitory effect on protease and TGF-beta expression might be the underlying mechanism.

Topics: Animals; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Heart Transplantation; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Contraction; Recombinant Proteins; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Secretory Leukocyte Peptidase Inhibitor; Transforming Growth Factor beta; Transplantation, Isogeneic

Bone marrow-derived cells (BMDC) have been proposed to exert beneficial effects after renal ischemia/reperfusion injury (IRI) by engraftment in the tubular epithelium. However, BMDC can give rise to myofibroblasts and may contribute to fibrosis. BMDC contribution to the renal interstitial myofibroblast population in relation to fibrotic changes after IRI in rats was investigated. A model of unilateral renal IRI (45 min of ischemia) was used in F344 rats that were reconstituted with R26-human placental alkaline phosphatase transgenic BM to quantify BMDC contribution to the renal interstitial myofibroblast population over time. After IRI, transient increases in collagen III transcription and interstitial protein deposition were observed, peaking on days 7 and 28, respectively. Interstitial infiltrates of BMDC and myofibroblasts reached a maximum on day 7 and gradually decreased afterward. Over time, an average of 32% of all interstitial alpha-smooth muscle actin-positive myofibroblasts coexpressed R26-human placental alkaline phosphatase and, therefore, were derived from the BM. BMD myofibroblasts produced procollagen I protein and therefore were functional. The postischemic kidney environment was profibrotic, as demonstrated by increased transcription of TGF-beta and decreased transcription of bone morphogenic protein-7. TGF-beta protein was present predominantly in interstitial myofibroblasts but not in BMD myofibroblasts. In conclusion, functional BMD myofibroblasts infiltrate in the postischemic renal interstitium and are involved in extracellular matrix production.

Topics: Animals; Animals, Genetically Modified; Bone Marrow Transplantation; Collagen Type I; Collagen Type III; Creatinine; Fibroblasts; Humans; Kidney; Male; Rats; Rats, Inbred F344; Reperfusion Injury; RNA; Transforming Growth Factor beta; Transplantation Chimera

The most salient feature of carbon monoxide (CO)-mediated cytoprotection is the suppression of inflammation and cell death. One of the important cellular targets of CO is the macrophage (mphi). Many studies have shown that exposure of mphi to CO results in the generation of an antiinflammatory phenotype; however, these reports have ignored the effect of CO alone on the cell before stimulation. Most investigations have focused on the actions of CO in modulating the response to noxious stimuli. We demonstrate here that exposure of mphi to CO results in a significant and transient burst of reactive oxygen species (ROS) arising from the mitochondria (mitochondria-deficient mphi do not respond to CO to produce ROS). The ROS promote rapid activation and stabilization of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), which regulates expression of genes involved in inflammation, metabolism, and cell survival. The increase in HIF-1alpha expression induced by CO results in regulated expression of TGF-beta, a potent antiinflammatory cytokine. CO-induced HIF-1alpha and TGF-beta expression are necessary to prevent anoxia/reoxygenation-induced apoptosis in mphi. Furthermore, blockade of HIF-1alpha using RNA interference and HIF-1alpha-cre-lox mphi resulted in a loss of TGF-beta expression and CO-induced protection. A similar mechanism of CO-induced protection was operational in vivo to protect against lung ischemia-reperfusion injury. Taken together, we conclude that CO conditions the mphi via a HIF-1alpha and TGF-beta-dependent mechanism and we elucidate the earliest events in mphi signaling that lead to and preserve cellular homeostasis at the site of injury.

Topics: Animals; Apoptosis; Carbon Monoxide; Cell Hypoxia; Cell Survival; Cytoprotection; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-10; Ischemic Preconditioning; Lung; Macrophages; Mice; Mitochondria; Oxygen; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger; Thermodynamics; Transforming Growth Factor beta

Interleukin-1 (IL-1) and transforming growth factor-beta (TGFbeta) are key inhibitors of hepatocyte proliferation after hepatectomy. IL-1 inhibition by heat shock proteins (HSPs) has been reported in inflammatory processes. A recent study indicated the benefits of ischaemic preconditioning in reduced-size orthotopic liver transplantation (ROLT). The present study examined: (a) the effect of ischaemic preconditioning on IL-1 and TGFbeta in ROLT; (b) whether preconditioning protects small liver grafts through HSP induction; and (c) whether the potential benefits of preconditioning on HSP is related to IL-1 inhibition. Our results, obtained with an IL-1 receptor antagonist, indicated the injurious effects of IL-1 in ischaemia-reperfusion (I/R) injury and established a relationship between IL-1 and growth factors. Thus, IL-1 reduced hepatocyte growth factor (HGF) and promoted TGFbeta release, thus contributing to the impaired liver regeneration associated with ROLT. Preconditioning inhibited IL-1 through nitric oxide (NO), thereby protecting against the injurious effects of IL-1. In addition, by another pathway independent of NO, preconditioning induced HSP70 and haem-oxygenase-1 (HO-1). HO-1 protected against I/R injury and liver regeneration, whereas the benefits resulting from HSP70 were mainly related to hepatocyte proliferation. These results suggest a mechanism that explains the effectiveness of preconditioning in ROLT. They suggest, too, that other strategies, in addition to preconditioning, that modulate IL-1 and/or HSPs could be considered in clinical situations requiring liver regeneration such as small liver grafts.

Topics: Animals; Heat-Shock Proteins; Heme Oxygenase-1; Hepatocytes; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Immunohistochemistry; Interleukin-1; Ischemic Preconditioning; Liver; Liver Regeneration; Liver Transplantation; Male; Membrane Proteins; Necrosis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transforming Growth Factor beta

Immunosuppressed individuals undergoing organ transplantation are at increased risk of recurrences of initial cancers, although how immunosuppressive therapy increases early cancer metastasis remains unclear.. The metastatic fate of luciferase-expressing rat metastatic colon cancer cells (luc-RCN-H4) injected intravenously into the liver of syngeneic and allogeneic rats was examined in the presence of the immunosuppressant cyclosporin A (CsA) by in vivo luminescent technique. With respect to potential tumor-progressing factors, contribution of chemokine receptors and transforming growth factor (TGF)-beta1 to early metastasis was evaluated using their specific signaling inhibitors.. F344 rats injected in the liver with luc-RCN-H4 cells did not always exhibit the formation of tumors and showed a dormant state as long as 60 days after inoculation without CsA. However, CsA released early luc-RCN-H4 cells from dormancy within 2 weeks at nearly 100% in liver and preferentially promoted metastasis to the lymph nodes (approximately 40%). A similar dissemination occurred even in minor histocompatibility complex-disparate hosts. As a tumor-progressing factor, RCN-H4 cells aberrantly expressed chemokine receptors CXCR4 and CCR7. The chemokine receptor (CXC) R4-specific antagonist AMD3100 decreased early metastasis of luc-RCN-H4 cells in rats with ischemic liver conditions (P<0.05), but CsA treatment did not enhance early adhesion. Use of CsA was able to facilitate TGF-beta1 expression and the subsequent TGF-beta-mediated random migration was blocked by the use of the specific signaling inhibitor SB431542 in vitro.. Whereas the chemokine receptor expression by cancer cells is implicated with early organotropic dissemination even under CsA-mediated immune suppression, rather, CsA enhances the late-phase progression after tumor adhesion through TGF-beta1 expression.

Topics: Adenocarcinoma; Animals; Benzamides; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclosporine; Dioxoles; Disease Progression; Gene Expression Regulation, Neoplastic; Image Processing, Computer-Assisted; Killer Cells, Natural; Liver Neoplasms; Luminescence; Lymphatic Metastasis; Male; Neoplasm Metastasis; Rats; Rats, Inbred F344; Receptors, Chemokine; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transient renal ischemia induces both inflammatory and fibrotic processes and is a major cause of acute and chronic renal insufficiency. Study of ischemia-reperfusion injury in gene-targeted mice has identified multiple factors responsible for inflammation, whereas mechanisms underlying fibrosis remain poorly defined. Here we demonstrate by both gene inactivation and target protein blockade that a single chemokine receptor subtype, the fractalkine receptor CX3CR1, is able to reduce both inflammation and fibrosis after ischemia-reperfusion injury in the mouse, leading to partially preserved renal function after injury. The mechanism involves selective effects in the outer medulla, including reduced accumulation of macrophages and reduced expression of the macrophage and platelet-derived fibrogenic protein platelet-derived growth factor-B. CX3CR1 is the first chemokine receptor shown to contribute to fibrogenesis in renal ischemia-reperfusion injury.

Topics: Actins; Animals; Antibodies; Chemokine CX3CL1; Chemokines, CX3C; CX3C Chemokine Receptor 1; Fibrosis; Kidney; Kidney Function Tests; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephritis, Interstitial; Neutralization Tests; Platelet-Derived Growth Factor; Receptors, Chemokine; Reperfusion Injury; RNA, Messenger; Time Factors; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta1 and a number of TGF-beta-responsive genes are transiently enhanced following induction of ischemic acute renal failure (ARF) in the rat. The mRNA and protein expression of TGF-beta receptors were analyzed in postischemic rat kidneys by ribonuclease protection, in situ hybridization, and immunohistochemistry. TGF-betaRI and -RII were enhanced within 3 days of ischemia-reperfusion (I/R) injury and remained elevated for up 7 days post-I/R; TGF-beta receptor expression was localized primarily in regenerating tubules within the outer medulla. A neutralizing TGF-beta antibody exacerbated cellular proliferation observed on day 3 postischemia but had no effect on day 1 or 2. TGF-beta antibody treatment had no measurable effect on loss of renal function or the restoration of renal function during the recovery response for up to 35 days postsurgery. However, ischemic injury resulted in modest renal hypertrophy that is due, in part, to in an increase in the number of interstitial cells in the postischemic kidney. Immunohistochemistry showed that several of these cells stained positively for the fibroblast-specific marker, S100A4 positive. Anti-TGF-beta treatment substantially attenuated the renal hypertrophy, interstitial cellularity, and S100A4-positive cells present at 35 days post-I/R. Finally, TGF-beta immunoneutralization attenuated the loss of renal vascular density following recovery from I/R injury. These data suggest that the TGF-beta/TbetaR system is enhanced in the postischemic kidney. However, the current study failed to identify a prominent role for this system in the repair of proximal tubules following ARF. In contrast, the activation of this system may play an important role in the long-term structure of the postischemic kidney by influencing microvascular structure and interstitial cellularity.

Topics: Acute Kidney Injury; Animals; Biomarkers; Cell Proliferation; Epithelial Cells; Immunohistochemistry; In Situ Hybridization; In Vitro Techniques; Kidney; Kidney Function Tests; Male; Neovascularization, Physiologic; Neutralization Tests; Nuclease Protection Assays; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Renal Circulation; Reperfusion Injury; RNA, Messenger; S100 Proteins; Transforming Growth Factor beta

An improved understanding of the early cell injury mechanisms is critical for effective therapy of acute renal failure (ARF).. We utilized cDNA microarrays to identify renal genes that are induced very early after renal ischemia in a mouse model, whose protein products might provide novel information regarding the pathogenesis of ARF. The findings were confirmed by downstream mRNA and protein expression studies, as well as knockdown analysis with antisense primers.. The maximally induced gene (21-fold at 3 hours of reflow) was Zf9, a Kruppel-like transcription factor involved in the regulation of transforming growth factor-beta1 (TGF-beta1). The rapid induction of Zf9 mRNA was confirmed by Northern analysis (14.5-fold at 3 hours of reflow) and that of Zf9 protein by Western analysis (10.5-fold at 3 hours of reflow). Zf9 protein was induced in both proximal and distal tubule cells in a cytoplasmic as well as nuclear distribution. TGF-beta1 protein was also up-regulated in a pattern parallel to that of Zf9. In cultured human proximal tubule cells, induction of ischemia by partial adenosine triphosphate (ATP) depletion resulted in a rapid up-regulation of both Zf9 and of TGF-beta1 proteins. Antisense oligonucleotides to Zf9 markedly blunted the induction of Zf9 and TGF-beta1, and significantly inhibited the apoptotic response to ATP depletion.. Induction of Zf9 and its transactivating factor TGF-beta1 may play a critical and hitherto unrecognized role in the early apoptotic response to ischemic renal injury.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Antisense Elements (Genetics); Apoptosis; Disease Models, Animal; Gene Expression Regulation; Kidney; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Male; Mice; Proto-Oncogene Proteins; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cerebral ischemia rapidly initiates structural and functional changes in brain vessels, including blood-brain barrier disruption, inflammation, and angiogenesis. Molecular events that accompany these changes were investigated in brain microvessels extracted using laser-capture microdissection (LCM) from Sprague-Dawley rats subjected to a 20 min transient global cerebral ischemia followed by 1, 6, or 24 h reperfusion. Proteins extracted from approximately 300 LCM captured microvessels (20-100 microm) were ICAT-labeled and analyzed by nanoLC-MS. In-house software was used to identify paired ICAT peaks, which were then sequenced by nanoLC-MS/MS. Pattern analyses using k-means clustering method classified 57 differentially expressed proteins in 7 distinct dynamic patterns. Protein function was assigned using Panther Classification system. Early reperfusion (1 h) was characterized by down-regulation of ion pumps, nutrient transporters, and cell structure/motility proteins, and up-regulation of transcription factors, signal transduction molecules and proteins involved in carbohydrate metabolism. The up-regulation of inflammatory cytokines and proteins involved in the extracellular matrix remodeling and anti-oxidative defense was observed in late reperfusion (6-24 h). The up-regulation of IL-1beta and TGF-1beta in ischemic brain vessels was confirmed by ELISA, quantitative PCR, and/or immunohistochemistry. A biphasic postischemic (1 and 24 h) BBB opening for (3)H-sucrose was evident in the same model. Differentially expressed proteins identified in brain vessels during reperfusion are likely involved in orchestrating functional vascular responses to ischemia, including the observed BBB disruption.

Topics: Animals; Antioxidants; Blood Vessels; Blood-Brain Barrier; Brain; Brain Ischemia; Carbohydrates; Chromatography, Liquid; Cytokines; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Female; Immunohistochemistry; Inflammation; Interleukin-1; Ischemic Attack, Transient; Lasers; Male; Mass Spectrometry; Microcirculation; Microdissection; Microscopy, Fluorescence; Peptides; Permeability; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sucrose; Time Factors; Transforming Growth Factor beta; Up-Regulation

To observe the effect of astrgaloside IV (Astr) on the long-term consequences of renal ischemia-reperfusion injury (IRI) in rat.. Fifty-four male Sprague-Dawley rats were randomized into 3 equal groups: IRI group, Astr group, and sham operation group. All rats underwent right nephrectomy and isolation of the left renal artery. The left renal arteries of the IRI group and Astr group were gripped by vascular clamp for 60 minutes and that of the sham operation group was only isolated without gripping. Two milliliters of Astr solution (0.1 g/L) was perfused into the stomach of the rats in the Astr group three days before and after the operation respectively. The rats in the IRI and sham operation groups were perfused with normal saline of the same volume. Four, twelve, and twenty-four weeks after the operation 24-hour urine specimens of the rats were collected to detect the urine protein. At each time point 6 rats from each group were anesthetized and blood was collected from the abdominal aorta to measure the level of serum creatinine (Cr), their left kidneys were taken out to undergo pathological examination and extraction of mRNA. Histochemistry was used to detect the expression of tumor growth factor (TGF)-beta1 protein in the renal tissues. RT-PCR was used to detect the expression of TGF-beta1 mRNA. Collagen staining and immunohistochemistry were used to measure the proportion of collagen positive material to the total area.. The level of urine protein was increased progressively, those 12 and 24 weeks after the operation in the IRI group were significantly higher than those in the Astr and sham operation groups (all P < 0.05). The serum Cr 4 weeks after the operation was 36 micromol/L +/- 4 micromol/L, significantly higher than those in the Astr and sham operation groups (31 micromol/L +/- 8 micromol/L and 31 micromol/L +/- 5 micromol/L), and the serum Cr levels 4 weeks 12 and 24 weeks after the operation in the IRI group remained significantly higher than those in the Astr and sham operation groups (all P < 0.05). Collagen staining showed that the glomerular basement membrane, tunica adventitia vasorum, and adventitia of renal tubule were remarkably redder in the IRI than in the Astr and sham operation groups. The expression of TGF-beta1 protein was progressively increased since 12 weeks after the operation in the IRI group, significantly stronger in the Astr and sham operation groups. The expression of TGF-beta1 mRNA was progressively increased since 12 weeks after the operation in the IRI and Astr groups, significantly stronger than that in the sham operation group (P < 0.05). However, the expression of TGF-beta1 mRNA 24 weeks after the operation was significantly stronger in the IRI group than in the Astr group (P < 0.05).. After renal IRI the probability of development of renal fibrosis increases. Astrgaloside IV markedly ameliorates renal injury by downregulating the TGF-beta1 expression.

Topics: Animals; Down-Regulation; Drugs, Chinese Herbal; Kidney; Male; Nephrectomy; Phytotherapy; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Saponins; Transforming Growth Factor beta; Triterpenes

We and others have previously reported that bone morphogenetic protein-7 (BMP-7), given before middle cerebral artery occlusion (MCAO), reduces ischemic injury in brain. Recent studies have indicated that receptors for BMP are upregulated after brain ischemia. It is possible that this upregulation may facilitate endogenous neurorepair in the ischemic brain. The purpose of this study was to determine the neuroregenerative effects of BMP-7 given parenterally after ischemia/reperfusion injury.. Adult Sprague-Dawley rats were anesthetized with chloral hydrate. The middle cerebral artery was transiently occluded by a filament inserted through the right internal carotid artery. The filament was removed after 60-minute ischemia to allow reperfusion. Some animals were killed 24 hours after MCAO to examine BMP-7 mRNA expression. Other animals received a single dose of intravenous BMP-7 or vehicle at 24 hours after MCAO and were used for subsequent behavioral studies and BMP-7 immunostaining.. BMP-7 mRNA was upregulated 24 hours after MCAO in untreated animals. BMP-7 immunoreactivity was dose-dependently increased on the ischemic side of the hippocampus/dentate on day 6 after MCAO in animals receiving intravenous injection of BMP-7. Animals receiving BMP-7 also showed a decrease in body asymmetry from day 7 to day 14 and an increase in locomotor activity on day 14 after MCAO.. Our data indicate that BMP-7, given parenterally after stroke, can pass through the blood-brain barrier on the ischemic side and induce behavioral recovery in stroke animals at longer testing times.

Topics: Animals; Behavior, Animal; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Brain; Disease Models, Animal; Dose-Response Relationship, Drug; Immunohistochemistry; Infarction, Middle Cerebral Artery; Injections, Intravenous; Ischemic Attack, Transient; Male; Motor Activity; Rats; Rats, Sprague-Dawley; Recovery of Function; Reperfusion Injury; RNA, Messenger; Stroke; Survival Rate; Transforming Growth Factor beta

Topics: Angiotensin Receptor Antagonists; Animals; Benzimidazoles; Biphenyl Compounds; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Hepatocyte Growth Factor; Liver; Liver Circulation; Male; Rats; Rats, Inbred Strains; Receptor, Angiotensin, Type 1; Reperfusion Injury; Tetrazoles; Transforming Growth Factor beta

This study evaluated the effects of exogenous vascular endothelial growth factor (VEGF) on the regulation of cytokines in a rat dorsal ischemic skin flap model. Exogenous VEGF (1 microg/ml) was injected subdermally into the flaps of 12 rats before the flaps were sutured back in place. Another 12 rats with flaps received saline injections, as a control group. Biopsy specimens were obtained from the flaps treated with VEGF or saline solution, at positions 2.5, 5.5, and 8.5 cm from the distal edge of the flaps, at 12 hours (n = 6 for each group) and 24 hours (n = 6 for each group) after suturing of the flaps. Expression of cytokine, growth factor, and inducible nitric oxide synthase was measured. The results demonstrated that expression of tumor necrosis factor-alpha and nitric oxide synthase in the distal part of the VEGF-treated flaps was significantly decreased, compared with the control values, at 12 and 24 hours postoperatively. It was concluded that administration of exogenous VEGF could protect flaps from ischemia-reperfusion injury through the regulation of proinflammatory cytokines and the inhibition of cytotoxic nitric oxide production.

Topics: Animals; Cytokines; Endothelial Growth Factors; Gene Expression Regulation; Graft Survival; Growth Substances; Intercellular Signaling Peptides and Proteins; Ischemia; Lymphokines; Male; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Skin; Surgical Flaps; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

We examined changes in mRNA expression patterns for proinflammatory cytokines and growth factors in blood samples after application of a tourniquet to the rat hind limb. Slight upregulations of interferon (IFN)-gamma, macrophage colony-stimulating factor (M-CSF) and transforming growth factor (TGF)-beta1 mRNA began at 2h after tourniquet application and were short-lived. The levels of activating transcription factor (ATF)-3, a stress-inducible gene, had increased at 1h after tourniquet application. No significant expression of interleukin (IL)-6 mRNA was observed in most samples. There were no significant temporal changes in the levels of IL-1beta, cardiotrophin (CT)-1 mRNA compared to the control levels, but, downregulation of gp130, a receptor of the IL-6 family, began at 1h after tourniquet application. Nerve growth factor (NGF) mRNA gradually increased and reached a significantly high level at 4h after application of the tourniquet. Gene expression induction in blood leukocytes occurred soon after application of the tourniquet and was short-lived. The transient mRNA expressions probably trigger secondary events that may be beneficial to wound repair and regeneration.

Topics: Activating Transcription Factor 3; Animals; Gene Expression; Hindlimb; Interferon-gamma; Leukocytes; Macrophage Colony-Stimulating Factor; Male; Muscle, Skeletal; Nerve Growth Factors; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Gene therapy may be an effective strategy for modulating lung graft ischemia-reperfusion injury. We investigated whether recipient intramuscular (IM) naked plasmid gene transfer of transforming growth factor beta1-active (TGF-beta1-active) ameliorates lung graft ischemia-reperfusion injury.. Preliminary studies in F344 rats demonstrated that gastrocnemius muscle transfection of TGF-beta1-active produced muscle and plasma protein expression at 24 and 48 hours after transfection. Recipients (n = 8) received IM injection of naked plasmid-encoding chloramphenicol acetyl transferase (CAT), TGF-beta1-latent or TGF-beta1-active, respectively, at 24 or at 48 hours before left lung transplantation. We did not treat the control group before transplantation (18-hour cold ischemia). Donor lungs were flushed with low-potassium dextran-1% glucose and stored for 18 hours at 4 degrees C. All groups were killed at 24 hours after transplantation. Immediately before killing the animals, we clamped the contralateral right hilum and assessed graft function. We measured wet-to-dry ratio (W/D), myeloperoxidase, pro-inflammatory cytokines (interleukin 1 [IL-1], tumor necrosis factor alpha [TNF-alpha], interferon-gamma [INF-gamma], and IL-2) and performed immunohistochemistry.. Arterial oxygenation was greatest in the recipient group transfected with TGF-beta1-active at 24 hours before transplantation compared with CAT, TGF-beta1-latent, and 18-hour cold ischemia groups (p < 0.01). The W/D ratio and myeloperoxidase decreased in both 24- and 48-hour groups, with TGF-beta1-active compared with CAT, and 18-hour cold ischemia groups (W/D, p < 0.02 and p < 0.004, respectively; myeloperoxidase, p < 0.05 and p < 0.01, respectively). All pro-inflammatory cytokines decreased in the 24-hour TGF-beta1-active group compared with CAT, TGF-beta1-latent, 18-hour and 1-hour cold ischemia, and non-treated lung groups (IL-1beta, p < 0.03; TNF-alpha, p < 0.02; IFN-gamma, p < 0.001; IL-2, p < 0.0001). In 24- and 48-hour groups with TGF-beta1-active, immunohistochemistry showed marked staining of Type I and Type II alveolar cells and of macrophages from the apical to the caudal sections of the lung grafts.. Recipient IM administration of naked plasmid encoding TGF-beta1-active before transplantation ameliorates lung isograft reperfusion injury after prolonged ischemia.

Topics: Animals; Chloramphenicol O-Acetyltransferase; Feasibility Studies; Gene Transfer Techniques; Injections, Intramuscular; Lung Transplantation; Male; Models, Animal; Plasmids; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transgenes

Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in lung graft ischemia-reperfusion injury. In this study we analyzed whether recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 would result in amelioration of lung graft ischemia-reperfusion injury.. Forty-eight hours before transplantation, 6 groups (n = 6) of F344 rats received intramuscular injection of naked plasmid encoding chloramphenicol acetyltransferase, chloramphenicol acetyltransferase plus beta-galactosidase, transforming growth factor beta(1), interleukin 10, or transforming growth factor beta(1) plus interleukin 10 or were not treated. Donor lungs were flushed and stored for 18 hours at 4 degrees C before transplantation. Twenty-four hours later, grafts were assessed immediately before the animals were killed. Arterial oxygenation, wet/dry ratio, myeloperoxidase, and proinflammatory cytokines (interleukin 1, tumor necrosis factor alpha, interferon gamma, and interleukin 2) were measured, and immunohistochemistry was performed.. For lung graft function, the arterial oxygenation was considerably higher in the cotransfected group receiving transforming growth factor beta(1) plus interleukin 10 compared with that in all other groups (P < or =.03). The wet/dry ratio, reflecting lung edema, was reduced in the cotransfected group compared with that in control animals (nontreated, P <.02; chloramphenicol acetyltransferase, P <.03; chloramphenicol acetyltransferase plus beta-galactosidase, P <.01). Myeloperoxidase, which measures neutrophil sequestration, was also reduced with cotransfection compared with that seen in control animals (P < or =.03). All proinflammatory cytokines were decreased in the cotransfected group compared with those in all other groups (interleukin 1beta, P <.04; tumor necrosis factor alpha, P <.002; interferon gamma, P <.0001; interleukin 2, P <.03). These results indicate that cotransfection provides a synergistic benefit in graft function versus either cytokine alone, neutrophil sequestration, or inflammatory cytokine expression. Immunohistochemistry showed positive staining of transforming growth factor beta(1) plus interleukin 10 in type I and II pneumocytes and localized edema fluid.. Recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 provides a synergistic effect in ameliorating lung reperfusion injury after prolonged ischemia.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression; Gene Transfer Techniques; Immunohistochemistry; Interleukin-10; Lung; Lung Transplantation; Male; Peroxidase; Plasmids; Rats; Rats, Inbred F344; Reperfusion Injury; Transfection; Transforming Growth Factor beta

Nitric oxide (NO) has been shown to be neurotoxic while transforming growth factor-beta 1 (TGF-beta1) is neuroprotective in the stroke model. The present study investigates the effects of low energy laser on nitric oxide synthase (NOS) and TGF-beta1 activities after cerebral ischemia and reperfusion injury.. Cerebral ischemia was induced for 1 hour in male adult Sprague-Dawley (S.D.) rats with unilateral occlusion of middle cerebral artery (MCAO). Low energy laser irradiation was then applied to the cerebrum at different durations (1, 5, or 10 minutes). The activity of NOS and the expression of TGF-beta1 were evaluated in groups with different durations of laser irradiation.. After ischemia, the activity of NOS was gradually increased from day 3, became significantly higher from day 4 to 6 (P < 0.001), but returned to the normal level after day 7. The activity and expression of the three isoforms of NOS were significantly suppressed (P < 0.001) to different extents after laser irradiation. In addition, laser irradiation was shown to trigger the expression of TGF-beta1 (P < 0.001).. Low energy laser could suppress the activity of NOS and up-regulate the expression of TGF-beta1 after stroke in rats.

Topics: Animals; Disease Models, Animal; Ischemic Attack, Transient; Low-Level Light Therapy; Male; Middle Cerebral Artery; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Topics: Animals; Animals, Genetically Modified; Hypertension, Renal; Kidney; Proteinuria; Rats; Renal Artery; Renal Veins; Reperfusion Injury; Sirolimus; Time Factors; Transforming Growth Factor beta

Oxidative stress after ischemia-reperfusion of cardiac allografts leads to activation of cardiomyocytes and production of cytokines. Bcl-2, an inhibitor of the apoptotic pathway, also has strong antioxidant properties. Ischemia-reperfusion injury after transplantation leads to decreased bcl-2 and increased tumor necrosis factor (TNF)-alpha levels. Transforming growth factor (TGF)-beta1 is known to attenuate ischemia-reperfusion injury and inhibits apoptosis of myofibroblasts. We hypothesize that TGF-beta1, prevents bcl-2 cleavage and increased TNF-alpha production.. Rat PVG donor hearts were heterotopically transplanted into ACI recipients. Donor hearts were procured and assigned to groups: (1) intracoronary TGF-beta1 (200 ng/ml) perfusion and pressure at 78 psi for 45 minutes (n = 4); (2) intracoronary TGF-beta1 perfusion and incubation for 45 minutes without pressure (n = 4), (3) saline perfusion and incubation for 45 minutes without pressure (n = 4). Hearts were procured 4 hours after transplantation and analyzed by reverse transcriptase-polymerase chain reaction for bcl-2 mRNA expression, ELISA for TNF-alpha, and for myeloperoxidase activity (MPO).. Bcl-2 decreased in untreated animals (bcl-2:G3PDH ratio = 0.85 +/- 0.73 vs 1.16 +/- 0.11, not significant [NS]), whereas TNF-alpha increased to 669.99 +/- 127.09 vs 276.84 +/- 73.65 pg/mg total protein in controls (p < 0.003). In TGF-beta(1) pressure-treated hearts, bcl-2 was up-regulated (2.49 +/- 0.6 vs 1.16 +/- 0.11, controls, p < 0.005), whereas TNF-alpha was unchanged (396.1 +/- 100.38 vs 276.84 +/- 73.65 pg/mg, NS). Hearts treated with TGF-beta1 and pressure showed significant up-regulation of bcl-2 compared with hearts treated with TGF-beta1 without pressure (2.49 +/- 0.6 vs 1.17 +/- 0.6, p < 0.02). MPO showed no differences.. Bcl-2 is down-regulated and TNF-alpha up-regulated in this model of ischemia-reperfusion injury. Furthermore, TGF-beta1 is linked to this process and ameliorates reperfusion injury by up-regulating bcl-2 and inhibiting TNF-alpha. Therapeutic overexpression of myocardial TGF-beta1 may be clinically useful to control ischemia-reperfusion injury associated with cardiac transplantation.

Topics: Animals; California; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Genes, bcl-2; Heart Transplantation; Hyperbaric Oxygenation; Male; NF-kappa B; Oxidative Stress; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Homologous; Treatment Outcome; Tumor Necrosis Factor-alpha; Up-Regulation

Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor-2 (FGF-2) plays an important role in the "self-repair" responses that follow injuries such as trauma and brain ischemia and that FGF-2 contributes to the repair of damaged tissue. Transforming growth factor-beta (TGF-beta) is a potent growth-regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF-2 and TGF-beta1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF-beta1 and FGF-2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron-glia interaction but also to neurochemical conditions involving inhibitory interneurons.

Topics: Animals; Brain Ischemia; Cerebellar Cortex; Cerebral Cortex; Disease Models, Animal; Fibroblast Growth Factor 2; Hippocampus; Male; Nerve Regeneration; Neurons; Rats; Rats, Wistar; Recovery of Function; Reperfusion Injury; Time Factors; Transforming Growth Factor beta

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.

Topics: Animals; Bone Morphogenetic Protein 6; Bone Morphogenetic Proteins; Brain Ischemia; Cerebellum; Cerebral Cortex; Hippocampus; Male; Neurons; Pyramidal Cells; Rats; Rats, Wistar; Reperfusion Injury; Transforming Growth Factor beta; Transforming Growth Factor beta1

Acute episodes of severe renal ischemia result in acute renal failure (ARF). These episodes are followed by a characteristic recovery and repair response, whereby tubular morphology and renal function appear completely restored within approximately 1 mo. However, the chronic effects of such an injury have not been well studied. Male rats were subjected to 60-min bilateral ischemia followed by reperfusion, yielding a characteristic injury. Postischemic animals manifested severe diuresis, peaking at 1 wk postinjury (volume: >45 ml/day, ARF vs. 18 ml/day, sham; P < 0.05). Urine flow subsequently declined but remained significantly elevated vs. sham animals for a 40-wk period. The prolonged alteration in urinary concentrating ability was attributable, in part, to a diminished capacity to generate a hypertonic medullary interstitium. By week 16, proteinuria developed in the post-ARF group and progressed for the duration of the study. Histological examination revealed essentially normal tubular morphology at 4 and 8 wk postinjury but the development of tubulointerstitial fibrosis at 40 wk. Transforming growth factor (TGF)-beta1 expression was elevated at 40 wk, but not at 4 and 8 wk postinjury. Microfil analysis revealed an approximately 30-50% reduction in peritubular capillary density in the inner stripe of the outer medulla at 4, 8, and 40 wk in post-ARF groups vs. sham animals. In addition, post-ARF rats manifested a significant pressor response to a low dose of ANG II (15 ng x kg(-1) x min(-1)). We hypothesize that severe ischemic injury results in a permanent alteration of renal capillary density, contributing to a urinary concentrating defect and the predisposition toward the development of renal fibrosis.

Topics: Acute Kidney Injury; Animals; Blood Pressure; Capillaries; Dehydration; Diuresis; Fibrosis; Ischemia; Kidney; Kidney Concentrating Ability; Kidney Tubules; Male; Natriuresis; Osmolar Concentration; Proteinuria; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urine

The angiopoietin/Tie receptor system may contribute to angiogenesis and vascular remodeling by mediating interactions of endothelial cells with smooth muscle cells and pericytes. The temporal expression of angiopoietin-1 (Angpo-1), angiopoietin-2 (Angpo-2), Tie-1, and Tie-2 mRNA was studied in a focal cerebral ischemia model in rats. The cDNA fragments obtained from reverse transcription polymerase chain reaction amplification were cloned and used as a probe to detect individual genes. Northern blot analysis showed a delayed increase of a 4.4-kb Angpo-1 transcript for up to 2 weeks after ischemia, eightfold higher than the values of the sham-operated controls. A biphasic expression of a 2.4-kb Angpo-2 transcript was noted, peaking at 24 hours (6.4-fold) and 2 weeks (4.6-fold) after ischemia. The expression of Tie-2 mRNA (4.3 kb), a receptor for Angpo-1, and Tie-1 mRNA (4.3 kb) also increased starting 24 hours after reperfusion and remained elevated for up to 2 weeks after ischemia. The temporal profiles of the expression of these genes were different from those of other angiogenic genes such as basic fibrobast growth factor/fibroblast growth factor receptor and vascular endothelial growth factor/vascular endothelial growth factor receptor and proteolytic enzymes (tissue-type plasminogen activator and urokinase plasminogen activator) and their inhibitors (plasminogen activator inhibitor-1). The expression patterns of these genes could be related to progressive tissue liquefaction and neovascularization after ischemia in this stroke model. Differential expression of these angiogenesis genes suggests the involvement of complex regulatory mechanisms that remain to be characterized.

Topics: Angiopoietin-1; Angiopoietin-2; Animals; Blotting, Northern; Brain; DNA Primers; Endothelial Growth Factors; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Ischemic Attack, Transient; Lymphokines; Male; Membrane Glycoproteins; Neovascularization, Physiologic; Proteins; Rats; Rats, Long-Evans; Receptor Protein-Tyrosine Kinases; Receptor, TIE-1; Receptor, TIE-2; Receptors, Cell Surface; Receptors, TIE; Reperfusion Injury; RNA, Messenger; Stroke; Tissue Plasminogen Activator; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.

Topics: Animals; Apoptosis; Astrocytes; DNA Fragmentation; Hippocampus; In Situ Nick-End Labeling; Male; Microglia; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Up-Regulation

Renal ischaemia-reperfusion (IR) injury is an inevitable consequence of transplantation and contributes to later graft fibrosis. This study aimed to elucidate the possible mechanisms by studying the expression of genes associated with extracellular matrix (ECM) turnover.. Male Wistar rats underwent laparotomy, clamping of the right renal pedicle for 45 min, and left nephrectomy. Control animals underwent left nephrectomy only, or had no operation. Animals were killed at 8, 16 and 24 weeks and messenger RNA was extracted from renal tissue. Genes of interest were amplified and then quantified in an enzyme-linked immunosorbent assay system with levels expressed as a ratio to a known housekeeping gene (GAPDH).. Experimental animals developed progressive proteinuria from 16 weeks onwards. At 8 weeks after IR injury, gene levels of matrix metalloproteinase (MMP) 2, an ECM-degrading enzyme, were significantly increased. Levels then fell progressively. This was associated with increasing expression of tissue inhibitor of metalloproteinases (TIMP) 1, an inhibitor of MMP-2, and of transforming growth factor (TGF) beta, a profibrotic cytokine, by 24 weeks following injury.. These results suggest that, after an initial phase of increased ECM turnover following IR injury, the balance turns towards one of reduced degradation. This is likely to be an important mechanism in the subsequent development of fibrosis.

Topics: Animals; Gene Expression; Kidney; Laparotomy; Male; Matrix Metalloproteinase 2; Proteinuria; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Cyclosporin is associated with significant chronic nephrotoxicity, manifest in the long term mainly as renal fibrosis. There have been claims that tacrolimus is a less fibrotic drug than cyclosporin, and this study was designed to determine the effect of the two drugs on the expression of fibrosis-associated genes.. Male Wistar rats underwent clamping of the right renal pedicle for 45 min together with left nephrectomy; this model has previously been shown to be associated with upregulation of fibrosis-associated genes. Experimental groups (six animals per group) received cyclosporin A 10 mg/kg daily, tacrolimus 0.2 mg/kg daily or no treatment. Animals were killed at 16 weeks, and the renal cortical expression of fibrosis-associated genes was studied by means of quantitative reverse transcriptase-polymerase chain reaction.. Tacrolimus-treated animals developed significantly less proteinuria and had lower serum creatinine levels than those receiving cyclosporin. Tacrolimus administration also significantly reduced the expression of transforming growth factor beta and tissue inhibitor of metalloproteinases 1, both the products of genes associated with fibrosis. Although cyclosporin treatment reduced levels of the matrix-degrading enzymes, matrix metalloproteinase (MMP) 2 and MMP-9, this was not statistically significant.. Tacrolimus has less nephrotoxicity than cyclosporin in this model. It also appears to have less fibrogenic potential, and this may have implications for the choice of long-term immunosuppressant in renal transplantation.

Topics: Animals; Creatinine; Cyclosporine; Fibrosis; Gene Expression; Immunosuppressive Agents; Kidney Diseases; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Proteinuria; Rats; Rats, Wistar; Renal Artery; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Tacrolimus; Transforming Growth Factor beta

Proximal pulmonary artery segment transfection may provide beneficial downstream effects on the whole-lung graft. In this study, transforming growth factor-beta1 was transfected to proximal pulmonary artery segments, and the efficacy of transforming growth factor-beta1 transfection was examined in ischemia-reperfusion injury and acute rejection models of rat lung transplantation.. In the ischemia-reperfusion injury model, orthotopic left lung transplantation was performed in F344 rats. In group I, the PPAS was isolated and injected with saline solution. In 2 other groups, lipid67:DOPE:sense (group II) or antisense transforming growth factor-beta1pDNA construct (group III) was injected instead of saline solution. After cold preservation at 4 degrees C for 18 hours, lung grafts were implanted. Graft function was assessed 24 hours later. In the acute rejection model, donor lung grafts were harvested. Proximal pulmonary artery segments were injected with saline solution (group I) or sense (group II) or antisense lipid gene construct (group III) and then implanted. Graft function was assessed on postoperative day 5.. In the ischemia-reperfusion injury study, there were no significant differences in oxygenation, wet-to-dry weight ratios, graft myeloperoxidase activity, or transforming growth factor-beta1 levels in platelet-poor serum or proximal pulmonary artery segment homogenates. In the acute rejection study, oxygenation was significantly improved in group II receiving transforming growth factor-beta1 (group II vs I and III, 136.0 +/- 32.5 vs 54.0 +/- 9.6 mm Hg and 53.8 +/- 14.8 mm Hg; P =.016 and.016). There were no significant pathologic differences. Transforming growth factor-beta1 concentrations from proximal pulmonary artery segment homogenates in group II were significantly higher compared with controls.. Ex vivo transfection of transforming growth factor-beta1 to proximal pulmonary artery segments did not affect reperfusion injury of lung isografts. In acute rejection, however, ex vivo transfection of transforming growth factor-beta 1 to proximal pulmonary artery segments improved allograft function. This suggests that transfection to proximal pulmonary artery segments exerts beneficial downstream effects on the whole-lung allograft.

Topics: Acute Disease; Animals; Graft Rejection; Lung Transplantation; Male; Organ Preservation; Pulmonary Artery; Rats; Rats, Inbred F344; Reperfusion Injury; Transfection; Transforming Growth Factor beta

Platelets protect myocardium against ischemia-reperfusion injury. This study examined the role of platelet-derived TGF-beta1 in cardioprotection during ischemia-reperfusion. Isolated Sprague Dawley rat hearts were perfused with K-H buffer and subjected to 25 min of global ischemia followed by 30 min of reperfusion. Ischemia-reperfusion resulted in myocardial dysfunction indicated by increase in CPP and LVEDP, and decrease in dLVP. Perfusion of hearts with washed platelets or supernatant of aggregated platelets attenuated (P < 0.01) of myocardial dysfunction following ischemia-reperfusion. Ischemia-reperfusion resulted in a decrease in myocardial TGF-beta1 determined by immunohistochemistry. ELISA showed an increase in latent TGF-beta1, but a decrease in active TGF-beta1. Perfusion of hearts with platelets or aggregated platelet supernatant preserved myocardial TGF-beta1 content upon ischemia-reperfusion. Perfusion of hearts with recombinant TGF-beta1 also resulted in cardioprotection following ischemia-reperfusion qualitatively similar to that observed with platelets or aggregated platelet supernatants. RT-PCR analysis showed an increase in myocardial TGF-beta1 mRNA following ischemia-reperfusion. These observations indicate that platelets protect the myocardium against ischemia-reperfusion-mediated dysfunction at least in part by releasing TGF-beta1. Increase in both TGF-beta1 mRNA and latent TGF-beta1 does not indicate a defect in the translation of mRNA. Reduction in myocardial TGF-beta1 following ischemia-reperfusion suggests a defect in the conversion of latent TGF-beta1 to active TGF-beta1.

Topics: Animals; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Heart; Male; Myocardium; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transforming Growth Factor beta

Accelerated hepatic apoptosis was first described in portal vein-ligated livers but has since been reported in a variety of liver injuries. Because porto-prival states can induce apoptosis we sought to determine whether transient ischemic periods followed by reperfusion would trigger such cell death. The cytokines TNF-alpha and TGF-beta are known to facilitate apoptosis and are released in response to a number of stimuli including ischemia. We also investigated alterations in plasma and tissue levels of these cytokines which might lend support to their role in increased apoptotic activity following ischemia/reperfusion. Female pigs were used as the experimental model. Inflow occlusion of portal and hepatic arterial blood was performed to a portion of the swine liver directing the entire splanchnic flow to the remaining hepatic lobes for a period of 2 h. The livers were then reperfused and plasma and tissue samples taken for determination of apoptotic activity utilizing cell death immunoperoxidase staining of 3'-OH DNA ends generated by fragmentation and ELISA assay of histone-associated DNA fragments. Plasma and tissue levels of TNF-alpha and plasma levels of TGF-beta were determined by ELISA assay. An increase in apoptotic activity following reperfusion was seen at Day 2 and Day 4 compared to preischemic values by the cell death stain. The ELISA cell death assay showed an increase in apoptotic activity at 60 min, Day 2, and Day 4. Moreover, the ELISA cell death assay showed enhanced apoptotic activity in "hyperperfused" hepatic lobes compared to preischemic, or resting, liver. This was also observed when compared to sham-operated animals. Surprisingly, there was no detectable increase in plasma TNF-alpha or TGF-beta levels following ischemia/reperfusion, although homogenized liver TNF-alpha levels were increased at 60 min and Day 2 following reperfusion. We conclude that transient hepatic inflow occlusion followed by reperfusion can induce increased apoptotic activity in the swine model. Furthermore, increased apoptotic activity also occurs in the hyperperfused liver raising the possibility of a locally active factor or global hepatic expression of receptor activity in response to ischemia/reperfusion. This period of ischemia/reperfusion did not produce a detectable increase in circulating cytokine levels, and accelerated apoptosis could not be linked to heightened TNF-alpha or TGF-beta plasma activity. Higher tissue levels of TNF-alpha could reflect enh

Topics: Animals; Apoptosis; Female; Ischemia; Liver; Liver Circulation; Reperfusion Injury; Swine; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

A major contributor to the development and progression of ischemia-reperfusion (IR)-induced acute renal failure (ARF) is the loss of functioning tubular epithelial cells by means of various cell deletion or death processes. Although the term "acute tubular necrosis" is still used to describe the pathology of ARF, this is a misnomer because apoptotic cell death, as well as necrosis, occurs [1, 2] along with desquamation and loss of viable epithelial cells [3]. Apoptosis was first described in renal disease in 1987 in an animal model of hydronephrosis [4]. In ARF, with reference to only the death processes, the relative contribution of necrosis or apoptosis possibly depends on the extent of the initiating events. For example, after prolonged total renal ischemia, necrosis or "accidental cell death" occurs from the resultant negation of the cell's energy and protein levels. In apoptosis, the cells use their own energy processes and proteins to die, and often the initiating ischemia is more mild [5]. Finally, despite prolonged ischemia, within the heterogeneous renal cell populations there are those that are more sensitive to ischemia, such as the proximal straight tubule and to some extent the thick ascending limb (TAL) of the loop of Henle. It may be hypothesized that these cells tend to undergo necrosis in comparison with the less sensitive segments that undergo apoptosis. Because apoptosis is gene driven, its identification is important because of the possibility of its modulation via molecular controls. However, despite these new concepts of ARF, patient death remains high, at approximately 30 to 50% of ARF cases. Recovery from ARF depends not only on the replacement or regeneration of cells deleted by death, the theme of many recent studies, but also on protection of cells from death. Both processes are dependent on many of the cellular and molecular controls that have evolved in multicellular organisms to manage normal development, differentiation and growth processes, but that then become involved in the pathogenesis and progression of many renal diseases, including ARF.

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-X Protein; Body Weight; Cell Division; Cell Survival; Epidermal Growth Factor; Epithelial Cells; Insulin-Like Growth Factor I; Loop of Henle; Male; Necrosis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; Transforming Growth Factor beta

The outcome of myocardial ischemia-reperfusion has been partially attributed to the degree of apoptosis in cardiomyocytes. Aggregating platelets by release of transforming growth factor-beta(1) (TGF-beta(1)) protect the isolated heart against ischemia-reperfusion injury and preserve myocardial TGF-beta(1) content. To gain more insight into the modulation of hypoxia-reoxygenation-induced injury (apoptosis and necrosis) to myocytes by TGF-beta(1) and aggregating platelets, cultured adult rat myocytes were exposed for 48 or 72 h to hypoxia alone, or to hypoxia followed by 3 h of reoxygenation. Apoptosis in the cells was determined by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining and DNA fragmentation on gel electrophoresis. Hypoxia alone caused a time-dependent increase in myocyte apoptosis (number of apoptotic cells: 19+/-3% at 48 h and 39+/-5% at 72 h compared with 5+/-1% in control cells, based on a 500-cell count). Three hours of reoxygenation after 48 h of hypoxia further increased the number of apoptotic cells (34+/-8 versus 19+/-3% in hypoxia for 48 h), but reoxygenation after 72 h of hypoxia did not additionally increase the number of apoptotic cells, perhaps because of extensive cell necrosis on prolonged hypoxia. Forty-eight hours of hypoxia followed by 3 h of reoxygenation also resulted in a decrease in Bcl-2 and an increase in Fas protein level. Incubation of myocytes with either recombinant TGF-beta(1) (0.5-5 ng/ml) or aggregated platelet supernatant (from 2-3 x10(7) platelets/ml, containing approximately 0.5 ng/ml of TGF-beta(1)) markedly (P<.01) decreased the number of apoptotic cells after hypoxia-reoxygenation. Incubation with TGF-beta(1) also reduced myocyte necrosis as evident from lactate dehydrogenase release and trypan blue dye exclusion. These data demonstrate that hypoxia-reoxygenation results in apoptosis and necrosis in cultured adult rat myocytes; this can be attenuated by TGF-beta(1). Similarity of data with TGF-beta(1) and aggregated platelet supernatant suggests that platelet-mediated cardioprotection during hypoxia-reoxygenation may relate in part to the release of TGF-beta(1).

Topics: Animals; Apoptosis; Cells, Cultured; Electrophoresis, Agar Gel; fas Receptor; Genes, bcl-2; Heart Ventricles; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Necrosis; Platelet Aggregation; Precipitin Tests; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Transforming Growth Factor beta; Trypan Blue

The phospholipid platelet-activating factor (PAF) participates in the pathogenesis of renal ischemia/reperfusion injury, and in vitro, it induces synthesis of extracellular matrix proteins by mesangial and tubular epithelial cells. This study investigated the long-term effects of the potent orally active PAF antagonist UR-12670 in warm ischemic uninephrectomized rats, which was given according to different therapeutic schedules.. Uninephrectomized male Sprague-Dawley rats were divided into five groups and were followed for 52 weeks: rats without ischemia (SK); ischemic kidney for 60 minutes (SIK); ischemic kidney and UR-12670 from 0 to the 7th day (UR 0-7); ischemic kidney and UR-12670 from day 0 to 52 weeks (UR 0-E); and ischemic kidney and UR-12670 from day 8 to week 52 (UR 8-E). Two more groups (ischemic and UR treated) served to evaluate the UR-12670-protective effect on ischemic acute renal failure at one week.. UR-12670 administration exerted functional and morphological protection against post-ischemic acute renal failure. The ischemic untreated (SIK) group developed progressive proteinuria from week 12. The onset of proteinuria in ischemic UR-12670-treated groups was delayed to the 24th week, and it was significantly lower than in SIK group throughout the study. Only SIK and ischemic-treated UR 0-7 rats presented with chronic renal failure, as shown by creatinine, creatinine clearance, glomerular filtration rate (GFR), and renal plasma flow (GFR 52 weeks: SK, 2525 +/- 267; SIK, 992 +/- 149; UR 0-7, 1551 +/- 385 microliter/min). Kidneys from the short-term treated group (UR 0-7) showed a reduction of glomerulosclerosis (SK, 14.4 +/- 3.7; SIK, 75.7 +/- 7.7; UR 0-7, 41. 5 +/- 8.5%) and vascular myointimal hyperplasia, but the tubulointerstitial damage (tubulointerstitial score: SK, 0.2 +/- 0. 2; SIK, 4.4 +/- 0.5; UR 0-7, 3.7 +/- 0.7) was similar to that in the ischemic untreated group. Long-term ischemic treated rats (UR 0-E, UR 8-E) did not develop chronic renal failure (GFR: UR 0-E, 2059 +/- 314; UR 8-E, 2410 +/- 208 microliter/min). In these groups, glomerulosclerosis (UR 0-E, 32.8 +/- 5.8; UR 8-E, 24.3 +/- 3.0%), tubulointerstitial damage (tubulointerstitial score: UR 0-E, 2.1 +/- 0.5; UR 8-E, 1.9 +/- 0.3) and vascular myointimal hyperplasia were significantly lower than in the ischemic untreated group. By in situ hybridization, an increase of transforming growth factor-beta1 mRNA expression in glomerular and tubular cells was observed in ischemic untreated and ischemic treated UR 0-7 rats. UR-12670 long-term treated rats showed a clear reduction of transforming growth factor-beta1 mRNA-positive glomerular cells.. The chronic administration of the PAF antagonist UR-12670 attenuates the long-term effects of ischemia-reperfusion injury in uninephrectomized rats. The beneficial effect of this agent, even when given beyond the initial ischemia/reperfusion injury, suggests that PAF plays a role in the mechanisms of progression to late renal damage in this model.

Topics: Acute Kidney Injury; Animals; Imidazoles; Ischemia; Kidney; Male; Nephrectomy; Platelet Activating Factor; Pyridines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor beta

Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor, have been correlated with hypertension and neuronal damage in ischemic/reperfusion injury. The presence of polymorphonuclear cells (PMNs) in the brain has been shown to be directly responsible for this observed pathology. To address the question of whether Et-1 plays a role in this process, human brain-derived endothelial cells (CNS-ECs) were cultured with Et-1. The results demonstrate that Et-1 induces production of the neutrophil chemoattractant interleukin-8 (IL-8) twofold to threefold after 72 hours; mRNA was maximal after 1 hour of stimulation. Conditioned culture medium derived from Et-1-stimulated CNS-ECs induced a chemotactic response in the PMN migration assay. The inflammatory cytokines tumor necrosis factor-alpha (TNF) and IL-1beta functioned additively with Et-1 in increasing IL-8 production. In contrast, transforming growth factor-beta (TGF-beta), but not IL-10, completely abolished the effect of Et-1 on IL-8 production. However, Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1) expression. These data demonstrate that Et-1 may be a risk factor in ischemic/reperfusion injury by inducing increased levels of the neutrophil chemoattractant IL-8.

Topics: Brain Ischemia; Cells, Cultured; Cerebral Arteries; Cerebral Veins; Chemotaxis, Leukocyte; Culture Media, Conditioned; Drug Synergism; Endothelin-1; Endothelium, Vascular; Gene Expression Regulation; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-10; Interleukin-8; Protein Isoforms; Reperfusion Injury; Risk Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Ischemic injury to cadaver organs is a major risk factor for development of chronic organ dysfunction. We have recently shown that the B7 costimulatory pathway plays a critical role in early organ dysfunction developing after renal cold ischemia/reperfusion injury. We extended these observations to investigate the role of this pathway in the development and progression of chronic organ dysfunction following such injury. Uninephrectomized rats which underwent cold ischemia/reperfusion injury developed progressive proteinuria as compared to uninephrectomized controls. Animals treated with CTLA4Ig, which blocks B7 costimulation, starting on the day of injury had significantly better long-term survival and developed significantly less proteinuria than control animals treated with control Ig. RT-PCR analysis of kidney tissue showed significant reduction in expression of activation and inflammatory cytokines, chemoattractants, and growth factors, as compared to controls. Delaying administration of CTLA4Ig for one week, but not four weeks, after injury was still effective in ameliorating development of progressive proteinuria. Interestingly, selective blockade of B7-1 by a mutant form of CTLA4Ig had no effect on early or chronic organ dysfunction. These findings indicate the long-term functional and molecular consequences of experimental cold ischemia/reperfusion injury, and suggest that B7-2 is critical in the development of organ dysfunction following ischemic injury, even in the absence of alloantigen.

Topics: Abatacept; Animals; Antigens, CD; Antigens, Differentiation; B7-1 Antigen; CD28 Antigens; Chemokine CCL2; Chemokine CCL5; Cold Temperature; Cryopreservation; CTLA-4 Antigen; Disease Progression; Endothelins; Gene Expression; Immunoconjugates; Immunosuppressive Agents; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukins; Kidney; Male; Nephrectomy; Proteinuria; Rats; Rats, Inbred Lew; Receptors, Interleukin-2; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Increasing clinical evidence suggests that delayed initial function secondary to ischemia/reperfusion injury alone, and particularly in combination with early episodes of acute rejection, reduces kidney allograft survival over time.. We investigated changes developing over the long term following a standardized ischemia/reperfusion insult in a Lewis rat model. The left kidney was isolated in a uninephrectomized host and cooled, and the pedicle was clamped for 45 min. Animals were followed for 48 weeks after initial renal injury. Organs were removed serially (4, 8, 16, 24, 32, 40, and 48 weeks) for immunohistology and reverse transcriptase polymerase chain reaction.. Progressive proteinuria developed after 8 weeks. By immunohistology, CD4+ leukocytes and ED-1+ macrophages infiltrated the ischemic organs in parallel with up-regulation of major histocompatibility complex class II antigen expression. Because macrophages have been shown to be critical in chronic changes in other models, they were examined primarily in these studies. By reverse transcriptase polymerase chain reaction, macrophage-derived, fibrosis-inducing factors (transforming growth factor-beta, interleukin 6, and tumor necrosis factor-alpha) remained highly and constantly expressed throughout the follow-up period. The long-term influence of initial treatment with the soluble form of P-selectin glycoprotein ligand-1, a soluble ligand for P- and E-selectin, was then examined. All functional and structural changes remained at relative baseline, similar to uninephrectomized controls.. These data suggest that blocking the initial selectin-mediated step after ischemia/reperfusion injury, which triggers significant early cellular and molecular events, also reduces later renal dysfunction and tissue damage over time. In part, the findings may be explained by the sparing of functioning nephron units, which if destroyed or compromised by the original insult, may contribute to long-term graft failure. This approach may be important clinically in the transplantation of kidneys from non-heart-beating or marginal donors or organs experiencing prolonged ischemic times.

Topics: Animals; Chemokine CCL2; Endothelin-1; Interleukin-6; Ischemia; Kidney; Ligands; Male; Membrane Glycoproteins; Mucins; P-Selectin; Polymerase Chain Reaction; Proteinuria; Rats; Rats, Inbred Lew; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Peptide growth factors have been reported to contribute to the atherogenic process, and they are known to mediate signals for vascular remodeling. Using syngeneic and allogeneic rat aorta transplant models, we analyzed the impact of cold ischemia time up to 24 hours and reperfusion injury on development of transplant arteriosclerosis during the first 2 months after transplantation. The expression of the transforming growth factor-beta (TGF-beta) family as well as the platelet-derived growth factor (PDGF) and its receptors was studied by use of immunohistochemistry, followed by semiquantitative evaluation and multivariate analysis. In the syngeneically transplanted aortas, the expression of TGF-beta 1, PDGF, and the two PDGF receptors in the neointima increased significantly with the extent of cold ischemia time. Furthermore, there was a significant induction of the latent TGF-beta binding protein in the neointima as well as TGF-beta 2 in the media, both correlating with the observation time after transplantation. In the allogeneic grafts, all examined proteins were already induced strongly 2 weeks after transplantation, even at the shortest ischemic period studied (1 hour). However, no positive correlation between growth factor expression and cold ischemia or observation time could be found. Double immunohistochemistry revealed that macrophages express PDGF and its receptors as well as TGF-beta 1. Smooth muscle cells express both types of PDGF receptors, and a few T cells express TGF-beta 1 as well as PDGF receptors. In summary, TGF-beta and PDGF are induced by allogeneic as well as ischemic stimuli in transplanted aortas, suggesting a role in the pathogenesis of transplant arteriosclerosis and representing a potential target for therapeutic intervention.

Topics: Animals; Aorta; Arteriosclerosis; Immunohistochemistry; Male; Platelet-Derived Growth Factor; Rats; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Reperfusion Injury; Transforming Growth Factor beta; Transplantation, Homologous; Transplantation, Isogeneic

The influence of the somatostatin analogue angiopeptin on transplant arteriosclerosis was investigated using two aortic transplantation rat models. One was characterized by ischemia/reperfusion-induced changes in syngeneic transplants while immunologically induced changes dominated in the other allogeneic model. Angiopeptin, 100 micrograms/kg per day, was administered continuously until the sacrifice of the rats after 8 weeks. No additional immunosuppression was used in either model. An image analysis system was used to quantify the intimal and medial thicknesses of the grafts. In the syngeneic grafts, the intimal thickness was less than 50% of that of control grafts (P < 0.05), but no difference was seen in the allogeneic model. The expression of selected cells, TGF-beta s, and PDGF and PDGF alpha-receptors was detected immunohistochemically and displayed a similar picture in control and angiopeptin-treated grafts in both models. We conclude that angiopeptin has no clear immunosuppressive properties but may counteract ischemia-induced transplant arteriosclerosis.

Topics: Animals; Aorta; Arteriosclerosis; Image Processing, Computer-Assisted; Male; Oligopeptides; Peptides, Cyclic; Platelet-Derived Growth Factor; Rats; Reperfusion Injury; Somatostatin; Transforming Growth Factor beta; Transplantation, Homologous; Transplantation, Isogeneic; Tunica Intima; Tunica Media

Anesthesized male rabbits having a resting mean arterial pressure of 81 +/- 4 mm Hg and superior mesenteric artery blood flow of 91 +/- 7 mL min-1 were subjected to 60 min of splanchnic ischemia followed by 60 min of reperfusion. Upon reperfusion, mean arterial pressure fell. Splanchnic blood flow also decreased but not in parallel with blood pressure; consequently, vascular resistance was increased over the reperfusion period. This increase in splanchnic vascular resistance was not affected by intravenous t-PA (0.5 mg kg-1 + 5 mg kg-1 hr-1) for 30 min prior to and throughout the reperfusion period or by intravenous L-NAME (1 mg kg-1 x 2). However, intravenous infusions of TGF-beta (18 or 54 micrograms kg-1) at the time of reperfusion dose dependently attenuated the increases in vascular resistance (p < 0.05). This effect of TGF-beta was enhanced by coadministration of t-PA and inhibited by the coadministration of L-NAME. We propose that the effects of TGF-beta are ultimately mediated via nitric oxide release, and conclude that this may be useful therapy for the prevention of reperfusion-associated injury following surgery or as an adjunct to thrombolytic therapy.

Topics: Animals; Arginine; Blood Flow Velocity; Blood Pressure; Intestines; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rabbits; Recombinant Proteins; Reperfusion Injury; Shock; Splanchnic Circulation; Tissue Plasminogen Activator; Transforming Growth Factor beta; Vascular Resistance