Xiangmei Chen

MiR-34 modulates Caenorhabditis elegans lifespan via repressing the autophagy gene atg9

Evidence for a regulatory role of the miR-34 family in senescence is growing. However, the exact role of miR-34 in aging in vivo remains unclear. Here, we report that a mir-34 loss-of-function mutation in Caenorhabditis elegans markedly delays the age-related physiological decline, extends lifespan, and increases resistance to heat and oxidative stress. We also found that RNAi against autophagy-related genes, atg4, bec-1, or atg9, significantly reversed the lifespan-extending effect of the mir-34 mutants. Furthermore, miR-34a inhibits Atg9A expression at the post-transcriptional level in vitro, and the miR-34a binding sequences in the 3-UTR of Atg9A contributes to the modulation of Atg9A expression by miR-34a. Our results demonstrate that the C. elegans mir-34 mutation extends lifespan by enhancing autophagic flux in C. elegans, and that miR-34 represses autophagy by directly inhibiting the expression of the autophagy-related proteins Atg9 in mammalian cells.

Hyperuricemia induces endothelial dysfunction via mitochondrial Na+/Ca2+ exchanger-mediated mitochondrial calcium overload.

BACKGROUNDnUric acid (UA) has proven to be a causal agent in endothelial dysfunction in which ROS production plays an important role. Calcium overload in mitochondria can promote the mitochondrial production of ROS. We hypothesize that calcium transduction in mitochondria contributes to UA-induced endothelial dysfunction.nnnMETHODS AND RESULTSnWe first demonstrated that high concentrations of UA cause endothelial dysfunction, marked by a reduction in eNOS protein expression and NO release in vitro. We further found that a high concentration of UA increased levels of [Ca2+]mito, total intracellular ROS, H2O2, and mitochondrial O2·-, and Δψmito but not the [Ca2+]cyt level. When the mitochondrial calcium channels NCXmito and MCU were blocked by CGP-37157 and Ru360, respectively, the UA-induced increases in the levels of [Ca2+]mito and total intracellular ROS were significantly reduced. Mitochondrial levels of O2·- and Δψmito were reduced by inhibition of NCXmito but not of MCU. Moreover, inhibition of NCXmito, but not of MCU, blocked the UA-induced reductions in eNOS protein expression and NO release.nnnCONCLUSIONSnThe increased generation of mitochondrial O2·- induced by a high concentration of UA is triggered by mitochondrial calcium overload and ultimately leads to endothelial dysfunction. In this process, the activation of NCXmito is the major cause of the influx of calcium into mitochondria. Our results provide a new pathophysiological mechanism for UA-induced endothelial dysfunction and may offer a new therapeutic target for clinicians.

Age-related changes in the function of autophagy in rat kidneys

Autophagy is a highly regulated intracellular process for the degradation of cytoplasmic components, especially protein aggregates and damaged organelles. It is essential for maintaining healthy cells. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. In this study, we investigated the effects of age on autophagy in the kidneys of 3-, 12-, and 24-month-old Fischer 344 rats. The results revealed that autophagy-related gene (Atg)7 was significantly downregulated in kidneys of increasing age. The protein expression level of the autophagy marker light chain 3/Atg8 exhibited a marked decline in aged kidneys. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the function of autophagy and proteasomal degradation, increased in older kidneys. The level of 8-hydroxydeoxyguanosine, a marker of mitochondrial DNA oxidative damage, was also increased in older kidneys. Analysis by transmission electron microscope demonstrated swelling and disintegration of cristae in the mitochondria of aged kidneys. These results suggest that autophagic function decreases with age in the kidneys of Fischer 344 rats, and autophagy may mediate the process of kidney aging, leading to the accumulation of damaged mitochondria.

Mesenchymal stem cells protect podocytes from apoptosis induced by high glucose via secretion of epithelial growth factor

IntroductionThe apoptosis and subsequent injury of podocytes plays a pathogenic role in diabetic nephropathy (DN). Mesenchymal stem cells (MSCs) are promising therapeutic cells for preventing apoptosis and reducing cellular injury. Our previous study found that MSCs could protect kidneys from diabetes-induced injury without obvious engraftment. So we evaluated the effects of human adipose-derived MSCs (hAd-MSCs) on podocytic apoptosis and injury induced by high glucose (HG) and the underlying mechanisms.MethodsWe used flow cytometry, Western blot and confocal fluorescence microscopy to study podocytic apoptosis and injury induced by HG at 24 hours, 48 hours, and 72 hours in the presence or absence of MSC-conditioned medium (CM). An antibody-based cytokine array was used to identify the mediating factor, which was verified by adding the neutralizing antibody (NtAb) to block its function or adding the recombinant cytokine to the medium to induce its function.ResultshAd-MSC-CM reduced podocytic apoptosis in a dose-dependent manner, decreased the expression of podocytic cleaved caspase-3, and prevented the reduced expression and maintained the normal arrangement of podocytic synaptopodin and nephrin. However, human embryonic lung cell (Wi38)-CM failed to ameliorate podocytic apoptosis or injury. Twelve cytokines with concentration ratios (MSC-CM/Wi38-CM) >10-fold were identified. Epithelial growth factor (EGF) was singled out for its known ability to prevent apoptosis. Recombinant human EGF (rhEGF) prevented podocytic apoptosis and injury similarly to hAd-MSC-CM but, upon blockade of EGF, the beneficial effect of hAd-MSC-CM decreased dramatically.ConclusionshAd-MSCs prevent podocytic apoptosis and injury induced by HG, mainly through secreting soluble EG.

NAD Blocks High Glucose Induced Mesangial Hypertrophy via Activation of the Sirtuins-AMPK- mTOR Pathway

Background/aims-Since the discovery of NAD-dependent deacetylases, Sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress-response of cells. Here we show that high glucose(HG)-induced mesangial hypertrophy is associated with loss of intracellular levels of NAD. This study was designed to investigate the effect of NAD on HG-induced mesangial hypertrophy. Methods-The rat glomerular mesangial cells (MCs) were incubated in HG medium with or without NAD. Afterwards, NAD+/NADH ratio and enzyme activity of Sirtuins was determined. In addition, the expression analyses of AMPK-mTOR signaling were evaluated by Western blot analysis. Results-We showed that HG induced the NAD+/NADH ratio and the levels of SIRT1 and SIRT3 activity decreased as well as mesangial hypertrophy, but NAD was capable of maintaining intracellular NAD+/NADH ratio and levels of SIRT1 and SIRT3 activity as well as of blocking the HG-induced mesangial hypertrophy in vitro. Activating Sirtuins by NAD blocked the activation of pro-hypertrophic Akt signaling, and augmented the activity of the antihypertrophic AMPK signaling in MCs, which prevented the subsequent induction of mTOR-mediated protein synthesis. By AMPK knockdown, we showed it upregulated phosphorylation of mTOR. In such, the NAD inhibited HG-induced mesangial hypertrophy whereas NAD lost its inhibitory effect in the presence of AMPK siRNA. Conclusion-These results reveal a novel role of NAD as an inhibitor of mesangial hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of mesangial hypertrophy.

Mitochondrial Autophagy Involving Renal Injury and Aging Is Modulated by Caloric Intake in Aged Rat Kidneys

Abstract Background A high-calorie (HC) diet induces renal injury and promotes aging, and calorie restriction (CR) may ameliorate these responses. However, the effects of long-term HC and CR on renal damage and aging have been not fully determined. Autophagy plays a crucial role in removing protein aggregates and damaged organelles to maintain intracellular homeostasis and function. The role of autophagy in HC-induced renal damage is unknown. Methods We evaluated the expression of LC3/Atg8 as a marker of the autophagosome; p62/SQSTM1; polyubiquitin aggregates as markers of autophagy flux; Ambra1, PINK1, Parkin and Bnip3 as markers of mitophagy; 8-hydroxydeoxyguanosine (8-OHdG) as a marker of DNA oxidative damage; and p16 as a marker of organ aging by western blot and immunohistochemical staining in the kidneys of 24-month-old Fischer 344 rats. We also observed mitochondrial structure and autolysosomes by transmission electron microscopy. Results Expression of the autophagosome formation marker LC3/Atg8 and markers of mitochondrial autophagy (mitophagy) were markedly decreased in the kidneys of the HC group, and markedly increased in CR kidneys. p62/SQSTM1 and polyubiquitin aggregates increased in HC kidneys, and decreased in CR kidneys. Transmission electron microscopy demonstrated that HC kidneys showed severe abnormal mitochondrial morphology with fewer autolysosomes, while CR kidneys exhibited normal mitochondrial morphology with numerous autolysosomes. The level of 8-hydroxydeoxyguanosine was increased in HC kidneys and decreased in CR kidneys. Markers of aging, such as p16 and senescence-associated-galactosidase, were increased significantly in the HC group and decreased significantly in the CR group. Conclusion The study firstly suggests that HC diet inhibits renal autophagy and aggravates renal oxidative damage and aging, while CR enhances renal autophagy and ameliorates oxidative damage and aging in the kidneys.

High glucose-induced hypertrophy of mesangial cells is reversed by connexin43 overexpression via PTEN/Akt/mTOR signaling

BACKGROUNDnHypertrophy of glomerular mesangial cells (GMC) is one of the earliest pathological abnormalities in diabetic nephropathy, which correlates with eventual glomerulosclerosis. We have previously proved that this hypertrophy is mediated by downregulation of connexin43 (Cx43) and dysfunction of gap junctional intercellular communication, but the mechanism involved is still unclear. This study aims to investigate whether PTEN/Akt/mammalian target of rapamycin (mTOR) was involved as the downstream molecular signaling of Cx43 in regulating high glucose-induced GMC hypertrophy.nnnMETHODSnGMC were isolated from male Wistar rats at the age of 3 months. Gene transfer technique was used to upregulate Cx43 in GMC, which was validated by western blot and immunofluorescent staining. Forward scatter of flow cytometry and total protein/cell number were examined to testify GMC hypertrophy induced by high glucose (30 mM) and the influence of Cx43 overexpression; western blot was performed to demonstrate the changes of Cx43 and signal protein level and flow cytometry and MTT test were carried out to check cell cycle and proliferation rate, respectively.nnnRESULTSnGMC exposed to a high concentration of glucose presented decreased Cx43, inhibited PTEN, triggered Akt phosphorylation and activated downstream mTOR, leading to stagnancy of cell cycle, decline of proliferation rate and occurrence of hypertrophy. Cx43 overexpression could prevent PTEN inhibition, Akt and mTOR phosphorylation, resulting in restoration of cell cycle and proliferation ability and reversion of GMC hypertrophy. GMC with Cx43 inhibition showed similar PTEN/Akt/mTOR change as stimulated by high glucose.nnnCONCLUSIONnPTEN/Akt/mTOR signaling stimulated by high concentration of glucose is regulated by Cx43 overexpression, which unveils part of the molecular mechanism of Cx43 in regulating hyperglycemia-induced hypertrophy.

Autophagy can repair endoplasmic reticulum stress damage of the passive Heymann nephritis model as revealed by proteomics analysis

Membranous nephropathy is a common cause of nephrotic syndrome in adults. Although many mechanisms have been proposed, whole proteomic research is still lacking. We analyzed the passive Heymann nephritis animal model using label-free quantitative proteome technology. Results showed 160 differential proteins between control and PHN model groups at days 14 and 21. The expression level of endoplasmic reticulum stress (ERS)-associated protein GRP78 and GRP94 protein was up-regulated on day 14 or 21, which was confirmed by Western blotting. The results also showed that the autophagy marker LC3 was up-regulated in the models. Furthermore, we used tunicamycin to induce ERS of podocytes in vitro to investigate the mechanism. Results of Western blotting revealed that the expression of GRP78, GRP94, and LC3 was up-regulated, while that of the cytoskeletal protein tubulin-β was down-regulated, and immunofluorescence displayed disordered distribution of tubulin-β. These suggest that ERS plays an important role in podocyte damage. Autophagy can repair the cytoskeleton damage caused by ERS as a protective mechanism. This provides an important basis for a thorough understanding of the mechanism of podocyte damage and the pathogenesis of membranous nephropathy.

The clinical features and outcome of crush patients with acute kidney injury after the Wenchuan earthquake: Differences between elderly and younger adults

BACKGROUNDnOn May 12, 2008, a devastating earthquake hit Wenchuan county of Chinas Sichuan province. Acute kidney injury (AKI) is one of the most lethal but reversible complications of crush syndrome after an earthquake. However, little is known about the epidemiological features of elderly crush patients with AKI. The aim of the present study is to compare clinical features and outcome of crush related AKI between elderly and younger adults in the Wenchuan earthquake.nnnMATERIALS AND METHODSnA questionnaire was sent to 17 reference hospitals that treated the victims after the earthquake. Clinical and laboratory characteristics of crush patients with AKI were retrospectively analysed.nnnRESULTSn228 victims experienced crush related AKI, of which 211 were adults, including 45 elderly (age ≥ 65 years) and 166 younger adults (age, 15-64 years). Compared with the resident population, the percentage of patients was higher amongst elderly (19.7% versus 7.6%, P<0.001). The distribution of gender was similar in elderly and younger adults. Mean systolic blood pressure was higher in elderly groups. Although no statistical differences in number of injury and injury severity score were observed between elderly and younger adults, elderly victims had lower frequency of extremities crush injury; higher incidences of thoracic traumas, limb, rib, and vertebral fractures; lower serum creatinine, potassium and creatinine kinase levels; lower incidence of oliguria or anuria; lower dialysis requirement; underwent less fasciotomies and amputations, received less blood and plasma transfusions. Mortality were 17.8% and 10.2% in elderly and younger adults, respectively (P=0.165). Stratified analysis demonstrated the elderly receiving dialysis had higher mortality rate compared with younger patients (62.5% versus 10.5%, P<0.001). Multivariate logistic regression analysis indicated that need for dialysis and sepsis were independent risk factors for death in the elderly patients.nnnCONCLUSIONSnElderly crush victims more frequently developed AKI in the Wenchuan earthquake, and they differ from younger adults in injury patterns and treatment modalities. The elderly patients with AKI requiring dialysis were at a relatively high risk of mortality.

SIRT1 is required for the effects of rapamycin on high glucose-inducing mesangial cells senescence.

The mTOR deregulation has a role in chronic kidney disease including diabetic nephropathy. SIRT1 is an important participant in renal cytoprotective responses to aging and stress. However, whether both mTOR and SIRT1 are involved in high glucose-inducing mesangial cells (MCs) senescence still remains to be explored. Hence we investigate the potential functional interrelationship between these two proteins in high glucose-inducing MCs senescence. High glucose increased mTOR expression and activity, but decreased SIRT1 expression and activity. The level of mTOR was increased significantly, while the SIRT1 expression and activity was declined significantly with serial cell culture passage. The siRNA-SIRT1 and nicotinamide promoted MCs senescence. NAD or resveratrol arrested high glucose-inducing MCs senescence. Meanwhile, the effects of NAD or resveratrol on high glucose-inducing MCs senescence were also completely blocked by SiRNA-SIRT1. Rapamycin arrested MCs senescence induced by high glucose and prevented MCs senescence with serial cell culture passage, and meanwhile increased the SIRT1 expression and activity. Moreover, the effects of rapamycin on MCs senescence induced by high glucose were also completely blocked by treating cells with niacinamide or siRNA-SIRT1. These findings provide support for the hypothesis that SIRT1 is required for the effects of rapamycin on high glucose-inducing MCs senescence.