Types Of Senescence Research Articles

Canagliflozin Inhibits Hedgehog Interacting Protein (Hhip)-Mediated Renal Tubular Cell Senescence in Type 1 Akita Mice

Canagliflozin Inhibits Hedgehog Interacting Protein (Hhip)-Mediated Renal Tubular Cell Senescence in Type 1 Akita Mice

Differential Oxidative Stress Responses in Type II Airway Epithelial Cells Impact Premature Senescence and Lung Fibrosis Susceptibility

Radiation-Induced Pulmonary Fibrosis (RIPF) is a late toxicity characterized by premature senescence in Type II airway epithelial cells (AECII) and accumulation of alternatively activated (M2) macrophages. Differential susceptibility to RIPF is observed across mouse strains. Based on our prior study of the effects of macrophage variation on RIPF, we hypothesized that intrinsic differences in AECII oxidative stress response across mouse strains also impact susceptibility to RIPF. Ten-week-old female mice from C57L, C57BL6 and C3H/HeN strains were exposed to thoracic irradiation (5x6 Gy, n>5 per group). Fifteen weeks after radiation, lung tissue was collected and examined with Masson-Trichrome staining (histologic changes) and β-galactosidase activity assay (senescence). AECII prepared from mice of each strain were exposed to irradiation. To assess differential gene expression, total RNA was extracted and assessed with a multiplex analysis platform and quantitative PCR. Senescence was assessed by β-galactosidase activity assay in primary AECII after irradiation or after co-culture with M2 macrophages polarized with IL13. Susceptibility to radiation-induced lung injury, survival, and premature AECII senescence vary by mouse strain: C57L (fibrosis-prone), C57BL6J (-intermediate) and C3H/HeN (-resistant). Enriched AECII from each strain exhibited differential expression of genes related to inflammatory responses including SASP production after irradiation. Minimal increased expression of Il1r1 was observed in irradiated and unirradiated AECII from C3H/HeN, however Il1rn levels were markedly elevated in response to irradiation. The expression of Thioredoxin (Txn) and Thioredoxin reductase 1 (Txnrd1) in AECII from C3H/HeN was significantly higher than those observed in other strains. In Vivo, C3H/HeN mouse lungs exhibited the least premature senescence in AECII after irradiation. Premature senescence in AECII irradiated In Vitro or co-cultured with superoxide anion-producing M2 macrophages was substantially less in AECII from C3H/HeN compared to other strains. A comparison of primary AECII from three different mouse strains identified intrinsic differences in expression of major inflammatory signaling (IL1R and IL1RN) and redox homeostasis status (TXN and TXNDR1) molecules. This study is the first to demonstrate that intrinsic differences in AECII impacts susceptibility to premature senescence and lung fibrosis after irradiation.

PAI-1 Regulation of p53 Expression and Senescence in Type II Alveolar Epithelial Cells.

Cellular senescence contributes importantly to aging and aging-related diseases, including idiopathic pulmonary fibrosis (IPF). Alveolar epithelial type II (ATII) cells are progenitors of alveolar epithelium, and ATII cell senescence is evident in IPF. Previous studies from this lab have shown that increased expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, promotes ATII cell senescence through inducing p53, a master cell cycle repressor, and activating p53-p21-pRb cell cycle repression pathway. In this study, we further show that PAI-1 binds to proteasome components and inhibits proteasome activity and p53 degradation in human lung epithelial A549 cells and primary mouse ATII cells. This is associated with a senescence phenotype of these cells, manifested as increased p53 and p21 expression, decreased phosphorylated retinoblastoma protein (pRb), and increased senescence-associated beta-galactose (SA-β-gal) activity. Moreover, we find that, although overexpression of wild-type PAI-1 (wtPAI-1) or a secretion-deficient, mature form of PAI-1 (sdPAI-1) alone induces ATII cell senescence (increases SA-β-gal activity), only wtPAI-1 induces p53, suggesting that the premature form of PAI-1 is required for the interaction with the proteasome. In summary, our data indicate that PAI-1 can bind to proteasome components and thus inhibit proteasome activity and p53 degradation in ATII cells. As p53 is a master cell cycle repressor and PAI-1 expression is increased in many senescent cells, the results from this study will have a significant impact not only on ATII cell senescence/lung fibrosis but also on the senescence of other types of cells in different diseases.

Regulation of Cellular Senescence in Type 2 Diabetes Mellitus: From Mechanisms to Clinical Applications.

Cellular senescence is accelerated by hyperglycemia through multiple pathways. Therefore, senescence is an important cellular mechanism to consider in the pathophysiology of type 2 diabetes mellitus (T2DM) and an additional therapeutic target. The use of drugs that remove senescent cells has led to improvements in blood glucose levels and diabetic complications in animal studies. Although the removal of senescent cells is a promising approach for the treatment of T2DM, two main challenges limit its clinical application: the molecular basis of cellular senescence in each organ is yet to be understood, and the specific effect of removing senescent cells in each organ has to be determined. This review aims to discuss future applications of targeting senescence as a therapeutic option in T2DM and elucidate the characteristics of cellular senescence and senescence-associated secretory phenotype in the tissues important for regulating glucose levels: pancreas, liver, adipocytes, and skeletal muscle.

Senescence of alveolar epithelial cells impacts initiation and chronic phases of murine fibrosing interstitial lung disease.

Fibrosing interstitial lung disease (ILD) develops due to the impaired reparative processes following lung tissue damage. Cellular senescence has been reported to contribute to the progression of fibrosis. However, the mechanisms by which these senescent cells initiate and/or drive the progression of lung tissue fibrosis are not yet fully understood. We demonstrated that p21WAF1/CIP1- and p16INK4A-pathway-dependent senescence in type 2 alveolar epithelial cells (AEC2) were both involved in the initiation and progression of lung fibrosis in murine bleomycin (BLM)-induced ILD. p21WAF1/CIP1-senescent AEC2 emerged rapidly, as early as 1 day after the intratracheal instillation of BLM. Their number subsequently increased and persisted until the later fibrosis phase. Very few p16INK4A-senescent AEC2 emerged upon the instillation of BLM, and their increase was slower and milder than that of p21WAF1/CIP1+ AEC2. AEC2 enriched with senescent cells sorted from BLM-ILD lungs expressed senescence-associated secretory phenotype (SASP)-related genes, including Il6, Serpin1, Tnfa, Ccl2, Tgfb, and Pdgfa, at the initiation and chronic phases of fibrosis, exhibiting distinct expression patterns of magnitude that were dependent on the disease phase. Ly6C+ inflammatory monocytes increased in the lungs immediately after the instillation of BLM and interstitial macrophages increased from day 3. The expression of Acta2 and Col1a1 was upregulated as early as day 1, indicating the activation of fibroblasts. We speculated that IL-6, plasminogen activator inhibitor-1 (PAI-1), and TGF-β contributed to the accumulation of senescent cells during the progression of fibrosis in an autocrine and paracrine manner. In addition, CCL2, produced in large amounts by senescent AEC2, may have induced the infiltration of Ly6C+ inflammatory monocytes in the early phase, and TGF-β and PDGFa from senescent AEC2 may contribute to the activation of fibroblasts in the very early phases. Our study indicated that senescent AEC2 plays a role in the pathogenesis of fibrosing ILD throughout the course of the disease and provides insights into its pathogenesis, which may lead to the development of new therapeutic methods targeting senescent cells or SASP molecules.

Endoplasmic Reticulum (ER) Stress and Its Role in Pancreatic β-Cell Dysfunction and Senescence in Type 2 Diabetes.

An increased life span and accompanying nutritional affluency have led to a rapid increase in diseases associated with aging, such as obesity and type 2 diabetes, imposing a tremendous economic and health burden on society. Pancreatic β-cells are crucial for controlling glucose homeostasis by properly producing and secreting the glucose-lowering hormone insulin, and the dysfunction of β-cells determines the outcomes for both type 1 and type 2 diabetes. As the native structure of insulin is formed within the endoplasmic reticulum (ER), ER homeostasis should be appropriately maintained to allow for the proper metabolic homeostasis and functioning of β-cells. Recent studies have found that cellular senescence is critically linked with cellular stresses, including ER stress, oxidative stress, and mitochondrial stress. These studies implied that β-cell senescence is caused by ER stress and other cellular stresses and contributes to β-cells’ dysfunction and the impairment of glucose homeostasis. This review documents and discusses the current understanding of cellular senescence, β-cell function, ER stress, its associated signaling mechanism (unfolded protein response), and the effect of ER stress on β-cell senescence and dysfunction.

MiR-34a/SIRT1 Axis Plays a Critical Role in Regulating Chondrocyte Senescence in Type 2 Diabetes Mellitus

Cartilage destruction is central in the pathogenesis of osteoarthritis (OA), an age-related chronic degenerative disease. Cartilage degeneration has been postulated to occur due to chondrocyte senescence. On the other hand, type 2 diabetes mellitus (T2DM) is an independent risk factor for OA initiation and progression, bringing about the concept of “diabetic OA (DM-OA).” Aberrant metabolic pathways in T2DM promote a chronic inflammatory environment that favors cell apoptosis and senescence. However, it is still unclear if the cartilage of diabetic OA patients contains a higher amount of senescent chondrocytes compared to nondiabetic patients.

Role of Cellular Senescence in Type II Diabetes.

Cellular senescence is a cell fate that occurs in response to numerous types of stress and can promote tissue repair or drive inflammation and disruption of tissue homeostasis depending on the context. Aging and obesity lead to an increase in the senescent cell burden in multiple organs. Senescent cells release a myriad of senescence-associated secretory phenotype factors that directly mediate pancreatic β-cell dysfunction, adipose tissue dysfunction, and insulin resistance in peripheral tissues, which promote the onset of type II diabetes mellitus. In addition, hyperglycemia and metabolic changes seen in diabetes promote cellular senescence. Diabetes-induced cellular senescence contributes to various diabetic complications. Thus, type II diabetes is both a cause and consequence of cellular senescence. This review summarizes recent studies on the link between aging, obesity, and diabetes, focusing on the role of cellular senescence in disease processes.

Diabetes Impairs Angiogenesis and Induces Endothelial Cell Senescence by Up-Regulating Thrombospondin-CD47-Dependent Signaling.

Endothelial dysfunction, impaired angiogenesis and cellular senescence in type 2 diabetes constitute dominant risk factors for chronic non-healing wounds and other cardiovascular disorders. Studying these phenomena in the context of diabetes and the TSP1-CD-47 signaling dictated the use of the in vitro wound endothelial cultured system and an in vivo PVA sponge model of angiogenesis. Herein we report that diabetes impaired the in vivo sponge angiogenic capacity by decreasing cell proliferation, fibrovascular invasion and capillary density. In contrast, a heightened state of oxidative stress and elevated expression of TSP1 and CD47 both at the mRNA and protein levels were evident in this diabetic sponge model of wound healing. An in vitro culturing system involving wound endothelial cells confirmed the increase in ROS generation and the up-regulation of TSP1-CD47 signaling as a function of diabetes. We also provided evidence that diabetic wound endothelial cells (W-ECs) exhibited a characteristic feature that is consistent with cellular senescence. Indeed, enhanced SA-β-gal activity, cell cycle arrest, increased cell cycle inhibitors (CKIs) p53, p21 and p16 and decreased cell cycle promoters including Cyclin D1 and CDK4/6 were all demonstrated in these cells. The functional consequence of this cascade of events was illustrated by a marked reduction in diabetic endothelial cell proliferation, migration and tube formation. A genetic-based strategy in diabetic W-ECs using CD47 siRNA significantly ameliorated in these cells the excessiveness in oxidative stress, attenuation in angiogenic potential and more importantly the inhibition in cell cycle progression and its companion cellular senescence. To this end, the current data provide evidence linking the overexpression of TSP1-CD47 signaling in diabetes to a number of parameters associated with endothelial dysfunction including impaired angiogenesis, cellular senescence and a heightened state of oxidative stress. Moreover, it may also point to TSP1-CD47 as a potential therapeutic target in the treatment of the aforementioned pathologies.

The association between Alu hypomethylation and severity of type 2 diabetes mellitus

BackgroundCellular senescence due to genomic instability is believed to be one of the mechanisms causing health problems in diabetes mellitus (DM). Low methylation levels of Alu elements or Alu hypomethylation, an epigenomic event causing genomic instability, were commonly found in aging people and patients with aging phenotypes, such as osteoporosis.ResultsWe investigate Alu methylation levels of white blood cells of type 2 DM, pre-DM, and control. The DM group possess the lowest Alu methylation (P < 0.001, P < 0.0001 adjusted age). In the DM group, Alu hypomethylation is directly correlated with high fasting blood sugar, HbA1C, and blood pressure.ConclusionGenome-wide hypomethylation may be one of the underlining mechanisms causing genomic instability in type 2 DM. Moreover, Alu methylation levels may be a useful biomarker for monitoring cellular senescence in type 2 DM patients.

Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. C57BL/6NCr mice received a diet formulated with rapamycin (14mg/kg food) or a control diet 2days before and continuing for 16weeks after exposure to 5 daily fractions of 6Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction.Senescence was assessed by staining for β-galactosidase activity. Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116days to 156days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9±11.8μg per lung; irradiation plus rapamycin, 21.4±6.0μg per lung; P=.001) and reduced visible fibrotic foci.Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16weeks (3-fold reduction at 16weeks, P<.001). Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

Abstract 3049: Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis

Abstract Introduction: Ionizing radiation (IR) is commonly used in the treatment of thoracic malignancies. Exposure of adjacent normal lung may result in lung injury and fibrosis. Recently, we reported that accelerated senescence of type II pneumocytes, the alveolar stem cell, results in parynchymal depletion and precedes pulmonary fibrosis. Arachidonate 12-lipoxygenase (12-LOX) oxidizes arachidonic acid to form 12-Hydroxyeicosatetraeonic acid (12-HETE), a pro-inflammatory mediator. Increased expression of ALOX-12 has previously been associated with aging. We hypothesized that mice deficient in ALOX-12 would be resistant to IR induced fibrosis and senescence. Methods: C57/Bl6J (WT) and Alox12 homozygous knockout (Alox12-KO) mice (n&amp;gt;3 per group) were exposed to thoracic IR (0Gy, 5Gy, 17.5Gy, 5×5Gy, or 6×5Gy). Fibrotic foci were identified with aniline blue staining of fixed lung sections. Levels of 12-LOX mRNA and protein were assessed in WT lungs after IR with microarray, quantitative PCR, and western blotting. In vitro studies with primary murine pneumocyte cultures and A549 cells included staining for senescence associated β-galactosidase activity and western blotting for NOX4, p21, and PML following IR and 12-HETE treatment. Results: A significant increase in 12-LOX mRNA and protein was observed in murine lungs exposed to fibrogenic doses of IR (17.5 Gy, 5×5 Gy and 5×6 Gy) compared to low dose IR (5 Gy) or controls (0 Gy). 12-LOX deficiency significantly reduced fibrotic foci in murine lungs receiving fibrogenic doses of thoracic IR (6×5 Gy) at 18 weeks. Treatment of murine primary pneumocytes with 12-HETE (1 and 150 nM) increased the rate of pneumocyte senescence (2.1 fold). Treatment of murine primary pneumocytes with 12-HETE increased the expression of NOX4 (a mediator of superoxide generation), p21, and PML (senescence markers), paralleling increases observed after IR. Similarly, human type II pneumocyte A549 cells exhibited increased 12-LOX expression in β-galactosidase-stained senescent cells at 3 days after IR. Conclusion: These studies identify 12-LOX as a critical mediator in radiation lung fibrosis and type II pneumocyte senescence. 12-LOX may serve as a novel therapeutic target in mitigating IR-induced lung fibrosis. Citation Format: Eun Joo Chung, Luca F. Valle, Ayla O. White, Deborah E. Citrin. Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3049.

MiR-34 miRNAs Regulate Cellular Senescence in Type II Alveolar Epithelial Cells of Patients with Idiopathic Pulmonary Fibrosis.

Pathologic features of idiopathic pulmonary fibrosis (IPF) include genetic predisposition, activation of the unfolded protein response, telomere attrition, and cellular senescence. The mechanisms leading to alveolar epithelial cell (AEC) senescence are poorly understood. MicroRNAs (miRNAs) have been reported as regulators of cellular senescence. Senescence markers including p16, p21, p53, and senescence-associated β-galactosidase (SA-βgal) activity were measured in type II AECs from IPF lungs and unused donor lungs. miRNAs were quantified in type II AECs using gene expression arrays and quantitative RT-PCR. Molecular markers of senescence (p16, p21, and p53) were elevated in IPF type II AECs. SA-βgal activity was detected in a greater percentage in type II AECs isolated from IPF patients (23.1%) compared to patients with other interstitial lung diseases (1.2%) or normal controls (0.8%). The relative levels of senescence-associated miRNAs miR-34a, miR-34b, and miR-34c, but not miR-20a, miR-29c, or miR-let-7f were significantly higher in type II AECs from IPF patients. Overexpression of miR-34a, miR-34b, or miR-34c in lung epithelial cells was associated with higher SA-βgal activity (27.8%, 35.1%, and 38.2%, respectively) relative to control treated cells (8.8%). Targets of miR-34 miRNAs, including E2F1, c-Myc, and cyclin E2, were lower in IPF type II AECs. These results show that markers of senescence are uniquely elevated in IPF type II AECs and suggest that the miR-34 family of miRNAs regulate senescence in IPF type II AECs.

Truncated Plasminogen Activator Inhibitor-1 Protein Protects From Pulmonary Fibrosis Mediated by Irradiation in a Murine Model

To determine whether the delivery of recombinant truncated plasminogen activator inhibitor-1 (PAI-1) protein (rPAI-1(23)) would protect from the development of radiation-induced lung injury. C57Bl/6 mice received intraperitoneal injections of rPAI-1(23) (5.4 μg/kg/d) or vehicle for 18 weeks, beginning 2 days before irradiation (IR) (5 daily fractions of 6 Gy). Cohorts of mice were followed for survival (n=8 per treatment) and tissue collection (n=3 per treatment and time point). Fibrosis in lung was assessed with Masson-Trichrome staining and measurement of hydroxyproline content. Senescence was assessed with staining for β-galactosidase activity in lung and primary pneumocytes. Hydroxyproline content in irradiated lung was significantly reduced in mice that received rPAI-1(23) compared with mice that received vehicle (IR+vehicle: 84.97 μg/lung; IR+rPAI-1(23): 56.2 μg/lung, P=.001). C57Bl/6 mice exposed to IR+vehicle had dense foci of subpleural fibrosis at 19 weeks, whereas the lungs of mice exposed to IR+rPAI-1(23) were largely devoid of fibrotic foci. Cellular senescence was significantly decreased by rPAI-1(23) treatment in primary pneumocyte cultures and in lung at multiple time points after IR. These studies identify that rPAI-1(23) is capable of preventing radiation-induced fibrosis in murine lungs. These antifibrotic effects are associated with increased fibrin metabolism, enhanced matrix metalloproteinase-3 expression, and reduced senescence in type 2 pneumocytes. Thus, rPAI-1(23) is a novel therapeutic option for radiation-induced fibrosis.

Abstract 852: Enhanced expression of secretory clusterin/apolipoprotein J (sCLU) in pulmonary alveolar stem cells after ionizing radiation exposure

Abstract Ionizing radiation is commonly used in the treatment of broad range of malignancies. However radiation induces DNA damage and oxidative stress in malignant and normal cells and stimulates inflammation in irradiated tissues. These vents may lead to tumor cell death, but also to a range of side effects including self-limited acute toxicities, mild chronic symptoms, or severe organ dysfunction. Recently, we reported that accelerated senescence of type II pneumocytes, the alveolar stem cell, results in depletion of type II pneumocytes and precedes pulmonary fibrosis. Targeted isolated depletion of type II pneumocytes is known to result in fibrosis. To understand the underlying mechanisms between RIPF and senescence, we investigated the role of several molecules which were markedly changed in irradiated lungs with fibrogenic exposures. Secretory Clusterin/Apolipoprotein J (sCLU) is a heterodimeric disulfide-linked glycoprotein encoded by a single gene. sCLU expression is implicated in physiological processes including development, lipid transportation, differentiation, cellular senescence, and in many age-related diseases including neurodegeneration, vascular damage, diabetes and tumorigenesis. It was recently proposed that sCLU is a sensitive cellular biosensor of oxidative stress that functions to protect cells from the deleterious effects of free radicals and their derivatives. We investigated sCLU expression in primary murine pneumocytes after exposure to irradiation. Increased sCLU expression following irradiation was confirmed by immunocytochemical assays and quantitative real-time PCR in primary pneumocyte cultures. At 5 days after irradiation, sCLU expression colocalized with senescence-associated beta-galactosidase (SA-beta-gal) activity. To determine the temporal relationship between sCLU expression and SA-beta-gal activity, sCLU expression and SA-beta-galactosidase activity was measured in murine lung tissues at 2 and 4 weeks after radiation exposures. As observed in pneumocyte cultures, the expression of sCLU mRNA was significantly increased in murine lungs after fibrogenic irradiation, with staining colocalizing to cells staining positive for SA-beta-gal activity. IGF-1, known to induce sCLU expression, was positively correlated with the levels of sCLU in irradiated lungs. Inhibition of IGF-1 signaling reduced sCLU expression in primary pneumocytes exposed to fibrogenic irradiation. Collectively, these data suggest that sCLU may provide a marker or target of type II pneumocyte senescence after irradiation. Citation Format: Eunjoo Chung, Jason Horton, Ayla White, Bradly Scroggins, Kathryn Hudak, Deborah Citrin. Enhanced expression of secretory clusterin/apolipoprotein J (sCLU) in pulmonary alveolar stem cells after ionizing radiation exposure. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 852. doi:10.1158/1538-7445.AM2014-852

Abstract 505: An Iron Chelator, Deferasirox, Prevents Renal Iron Accumulation and Macrophage Infiltration But Not Cell Senescence in Type 1 Diabetic Mice

We previously reported that streptozotocin (STZ)-induced type 1 diabetes caused renal tubular cell senescence through blood glucose- and p21-dependent pathway at week 4 of STZ administration in mice. We investigated the involvement of the cell senescence on accumulation of iron in the renal tubular cells in the present study. STZ mice (n=5) did not show the significant iron accumulation in the kidney at week 4. STZ mice (n=10) showed increases in renal tubular iron accumulation (Perl’s Prussian blue: 278.1±71.9 fold vs. non-diabetic control, p&lt;0.01), F4/80-positive macrophage infiltration (2.6±0.6 fold vs. non-diabetic control, p&lt;0.01), and cell senescence (senescence-associated β galactosidase: 3.7±0.4 fold, p16 mRNA: 2.3±0.2 fold vs. non-diabetic control, respectively, p&lt;0.01, respectively) at week 28. Administering oral iron chelator, deferasirox (10 mg/kg/day, n=10), that removes only dietary iron, significantly attenuated the iron accumulation (38.1±12.4 fold vs. non-diabetic control, n.s.) in proximal tubules and the F4/80-positive cells number (1.3±0.3 fold, vs. non-diabetic control, n.s.) without the effect on blood glucose, hematocrit and hemoglobin levels. On the other hand, deferasirox did not affect renal cell senescence (senescence-associated β galactosidase: 3.9±0.5 fold, p16 mRNA: 2.4±0.3 fold vs. non-diabetic control, respectively, n.s.). The functional deletion of p21, a cyclin-dependent kinase inhibitor, in mice decreased the renal tubular iron accumulation (14.0±5.5 fold vs. non-diabetic control, p&lt;0.01), and did not change the cell senescence at week 28. The animals showed an increase in p16, another cyclin-dependent kinase inhibitor, suggesting that p16 rather than p21 plays important role on cell senescence in the kidney of STZ mice at this time point. The results indicate that the chelation of dietary iron attenuates renal iron accumulation and macrophage infiltration through p21-dependent pathway in STZ mice. It seems likely that there is little interaction between renal tubular cell senescence and iron accumulation in the type 1 diabetic kidney.

Abstract 53: Telmisartan But Not Tempol Improves the Already Established Renal Cell Senescence in Type 2 Diabetic Rats

We previously reported that hyperglycemia induces renal tubular cell senescence in type 1 diabetic animals. In the present study, we evaluated the tubular cell senescence in type 2 diabetic animals and investigated whether the renal cell senescence could regress in response to treatment with a reno-protective dosage of an angiotensin receptor blocker, telmisartan (10 mg/kg/day). Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneously developed type 2 diabetes, showed an age-dependent increase in renal cell senescence analyzed by p21 expression (6 weeks old: 1.3±0.3 fold, 22 weeks old: 2.6±0.3 fold, vs. age-matched lean control rats, n=8, respectively) and senescence-associated β galactosidase (SABG) staining. OLETF rats were separated into the following 3 groups at 22 weeks of age and were kept under treatment until 34 weeks of age (n=10 per group): 1) un-treated, 2) telmisartan, and 3) tempol (an anti-oxidant, 3 mmol/L in drinking water). Neither treatment affected the blood glucose levels. Telmisartan, but not tempol, markedly reduced SABG staining by 34 weeks of age compared with that at 22 weeks of age or with that of age-matched un-treated rats. Increase in the expression of p21 was significantly smaller in telmisartan-treated rats (1.3±0.2 fold vs. lean rats) than in un-treated rats (2.9±0.2 fold, p&lt;0.01) and tempol-treated rats (3.0±0.2 fold, p&lt;0.01). Un-treated OLETF rats showed a greater intrarenal expression of TNF-α than lean rats, and the increase was suppressed by telmisartan. On the other hand, ED-1-positive macrophages rarely colocalized with p21-positive tubules at any stage of diabetes in any treated groups. These results indicate that type 2 diabetes prompts the renal cell senescence and that accumulation of renal senescent cells is reversible. Macrophage infiltration seems unlikely to contribute to the reduction of senescent cells. In addition, while there are many reports showing the involvement of oxidative stress in cell senescence, anti-oxidant tempol did not affect the established cell senescence.

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L.

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O 2 ) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40°C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.