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

INTRODUCTION: The 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. METHODS: We 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. RESULTS: hAd-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. CONCLUSIONS: hAd-MSCs prevent podocytic apoptosis and injury induced by HG, mainly through secreting soluble EG.

Mesenchymal stem cells attenuate acute kidney injury via regulation of natural immune system / 中华危重病急救医学

Objective To explore the potential mechanisms of mesenchymal stem cell (MSC) therapy in ischemia/reperfusion injury (IRI)-induced acute kidney injury (AKI). Methods Forty-five C57/BL6 male mice were randomly divided into three groups: sham group, IRI group, and IRI+MSCs group, with 15 mice in each group. The IRI-induced AKI model in mice was reproduced by clamping both renal pedicles for 35 minutes. In the sham group, both kidneys were exposed, but their pedicles were not clamped. Six hours after reperfusion, mice in IRI+MSCs group received 100 μL of MSCs (1×104 /μL) isolated from the bone marrow from C57/BL6 mice via tail vein, while the mice in the IRI group received same amount of normal saline. Blood samples were harvested at 48 hours after reperfusion, and levels of serum creatinine (SCr) and blood urea nitrogen (BUN) were determined. The changes in renal pathology were observed by microscopy with PAS staining, and the tubular injury and acute tubular necrosis (ATN) scores were calculated. The number of leukocytes (CD45+) infiltrated in kidney at 24 hours and 72 hours after reperfusion was measured with flow cytometry. The number of neutrophils (Ly-6G+) and macrophages (F4/80+) infiltrated in kidneys at 24 hours and 72 hours after reperfusion was determined by immunofluorescence. Results There was significant increase in the related parameters in IRI group compared with those of sham group. The levels of SCr (μmol/L) and BUN (mmol/L) were 180.3±8.8 vs. 9.7±3.5, and 1 121.1±8.3 vs. 9.4±2.3, both P < 0.01. The score of tubular injury was 4.80±0.55 vs. 0 at 48 hours after reperfusion. The quantity of leukocyte (CD45+) infiltration in kidney at 24 hours and 72 hours after reperfusion was increased (×105 cells/g: 60.50±2.56 vs. 19.46±4.83, 42.00±1.87 vs. 14.70±3.74, both P < 0.01), and the number of neutrophils (Ly-6G+) and macrophages (F4/80+) infiltrated in kidney at 24 hours and 72 hours after reperfusion was also increase although the number of leukocytes infiltrated in kidney was significantly lower at 72 hours after reperfusion than that at 24 hours. There was significant lowering of the levels of SCr and BUN [SCr (μmol/L): 99.0±8.0 vs. 180.3±8.8, BUN (mmol/L): 84.5±7.6 vs. 112.1±8.3, both P < 0.01] in IRI+MSCs group, compared to IRI group. For the degree of tubular necrosis in two groups, the tubular injury scores were 2.60±0.55 vs. 4.80±0.55 (P < 0.05). The number of leukocytes infiltrated in kidney at 24 hours and 72 hours after reperfusion (×105 cells/g) were 24.20±4.53 vs. 60.50±2.56, 31.70±3.15 vs. 42.00±1.87 (both P < 0.01). The number of neutrophils was lowered despite (the number of macrophages was increased). However, the number of infiltrated leukocytes was significantly more in IRI+MSCs group at 72 hours than that at 24 hours (×105 cells/g: 31.70±3.15 vs. 24.20±4.53, P < 0.05). Conclusion MSCs could protect against IRI induced AKI by reducing the total number of leuckocytes, especially that of the neutrophils infiltrating into ischemic kidney and by recruiting macrophages into ischemic kidney.

The mitochondrial Na+/Ca2+exchanger may reduce high glucose-induced oxidative stress and nucleotide-binding oligomerization domain receptor 3 inflammasome activation in endothelial cells / 老年心脏病学杂志（英文版）

Background The mitochondrial Na+/Ca2+exchanger, NCLX, plays an important role in the balance between Ca2+influx and efflux across the mitochondrial inner membrane in endothelial cells. Mitochondrial metabolism is likely to be affected by the activity of NCLX because Ca2+activates several enzymes of the Krebs cycle. It is currently believed that mitochondria are not only centers of energy produc-tion but are also important sites of reactive oxygen species (ROS) generation and nucleotide-binding oligomerization domain receptor 3 (NLRP3) inflammasome activation. Methods&Results This study focused on NCLX function, in rat aortic endothelial cells (RAECs), induced by glucose. First, we detected an increase in NCLX expression in the endothelia of rats with diabetes mellitus, which was induced by an injection of streptozotocin. Next, colocalization of NCLX expression and mitochondria was detected using confocal analysis. Suppression of NCLX expression, using an siRNA construct (siNCLX), enhanced mitochondrial Ca2+influx and blocked efflux induced by glucose. Un-expectedly, silencing of NCLX expression induced increased ROS generation and NLRP3 inflammasome activation. Conclusions These findings suggest that NCLX affects glucose-dependent mitochondrial Ca2+signaling, thereby regulating ROS generation and NLRP3 in-flammasome activation in high glucose conditions. In the early stages of high glucose stimulation, NCLX expression increases to compensate in order to self-protect mitochondrial maintenance, stability, and function in endothelial cells.

Effect of rapamycin on high glucose-induced autophagy impairment, oxidative stress and premature senescence in rat mesangial cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of rapamycin and 3-methyladenine on autophagy impairment, oxidative stress and premature senescence induced by high-glucose in primarily cultured rat mesangial cells.</p><p><b>METHODS</b>Rat glomerular mesangial cells (GMCs) were isolated and cultured in normal glucose, high glucose, high glucose with 3-methyladenine (3-MA), or high glucose with rapamycin. At 24 h, 72 h and 10 days of culture, the cells were examined for expression levels of autophagy markers LC3 and p62/SQSTM1, malondialdehyde (MDA) and protein carbonyl, β-galactosidase (SA-β-gal) activity and heterochromatin foci (SAHF).</p><p><b>RESULTS</b>Compared with those of normal cell culture, the cells exposed to high glucose for 72 h and 10 days showed down-regulated LC3 expression, up-regulated p62/SQSTM1 expression, elevated MDA and protein carbonyl levels, and increased SAHF formation and percentage of SA-β-gal-positive cells. These changes were reversed in GMCs exposed to high glucose and rapamycin for 72 h and 10 days, but exacerbated in cells incubated with 3-MA.</p><p><b>CONCLUSION</b>High glucose can suppress autophagic function of rat GMCs to result in oxidative damage and cell senescence. Rapamycin can attenuate autophagy impairment, oxidative damage and senescence induced by high glucose, whereas 3-MA can further aggravate high glucose-induced cell injuries in rat GMCs.</p>