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During long-term peritoneal dialysis, peritoneal fibrosis (PF) often happens and results in ultrafiltration failure, which directly leads to the termination of dialysis. The accumulation of extracellular matrix produced from an increasing number of myofibroblasts was a hallmark characteristic of PF. To date, glucose degradation products (GDPs, i.e., methylglyoxal (MGO)) that appeared during the heating and storage of the dialysate are considered to be key components to initiating PF, but how GDPs lead to the activation of myofibroblast in fibrotic peritoneum has not yet been fully elucidated. In this study, mesothelial cell line (MeT-5A) and fibroblast cell line (MRC-5) were used to investigate the transcriptomic and proteomic changes to unveil the underlying mechanism of MGO-induced PF. Our transcriptomic data from the MGO-stimulated mesothelial cells showed upregulation of genes involved in pro-inflammatory, apoptotic, and fibrotic pathways. While no phenotypic changes were noted on fibroblasts after direct MGO, supernatant from MGO-stimulated mesothelial cells promoted fibroblasts to change into proto-myofibroblasts, activated fibroblasts in the first stage toward myofibroblasts. In conclusion, this study showed that MGO-stimulated mesothelial cells promoted fibroblast-to-proto-myofibroblast transition; however, additional involvement of other factors or cells (e.g., macrophages) may be needed to complete the transformation into myofibroblasts. [ABSTRACT FROM AUTHOR]

Author(s): Miguel Crespo [1]; Eduardo Vilar [2, 3, 4]; Su-Yi Tsai [1]; Kyle Chang [2]; Sadaf Amin [1, 5]; Tara Srinivasan [6]; Tuo Zhang [7]; Nina H Pipalia [8]; Huanhuan [...]

Aims  Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs. Methods and results  We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia. Conclusion  We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology. [ABSTRACT FROM AUTHOR]

Renal ischaemia releases reactive oxygen species (ROS) in the kidneys. We hypothesized that the kidneys are more resistant to the insult of ROS in chronically hypoxic rats. We thus compared rats kept at sea level (SL) and those that had been adapted to hypoxia (hypoxia adapted, HA) by exposure to an altitude of 5500 m in an altitude chamber for 15 h day−1 for 4 weeks. Xanthine (X, 0.75 mg kg−1) and xanthine oxidase (XO, 24.8 mU kg−1) were injected intrarenally. A lucigenin‐enhanced chemiluminescence method was employed to detect the amount of free radicals in renal venous blood samples and on the kidney surface. In the renal venous blood samples, 26.05 (± 4.36) × 104 and 10.98 (± 1.79) × 104 counts were detected in the SL and HA rats, respectively, after X‐XO treatment; these figures were significantly different. On the kidney surface of the SL rats, the free radical count amounted to 12.77 (± 1.64) × 104, while that in the HA rats was 8.47 (± 0.42) × 104; these figures were also significantly different. There was a significant increase in urine volume and urinary excretion of Na+, K+ and protein after X‐XO administration in both groups of rats. However, the effect was greater for the SL rats than for the HA rats. The lipid peroxidation of the kidneys was not significantly different in the two groups of rats. Finally, we found that the activity of superoxide dismutase (SOD) and SOD mRNA were higher in the renal tissue of HA rats. We conclude that the renal response to free radicals is attenuated after chronic hypoxia in rats, and that SOD might play an important role in protecting HA rats from oxidative stress. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is increasingly recognized as contributing to glomerular diseases, including those secondary to mitochondrial DNA (mtDNA) mutations and deletions. Mitochondria maintain cellular redox and energy homeostasis and are a major source of intracellular reactive oxygen species (ROS) production. Mitochondrial ROS accumulation may contribute to stress-induced mitochondrial dysfunction and apoptosis and thereby to glomerulosclerosis. In mice, deletion of the gene encoding Mpv17 is associated with glomerulosclerosis, but the underlying mechanism remains poorly defined. Here we report that Mpv17 localizes to mitochondria of podocytes and its expression is reduced in several glomerular injury models and in human focal segmental glomerulosclerosis (FSGS) but not in minimal change disease. Using models of mild or severe nephrotoxic serum nephritis (NTSN) in Mpv17(+/+) wild-type (WT) and Mpv17(-/-) knockout mice, we found that Mpv17 deficiency resulted in increased proteinuria (mild NTSN) and renal insufficiency (severe NTSN) compared with WT. These lesions were associated with increased mitochondrial ROS generation and mitochondrial injury such as oxidative DNA damage. In vitro, podocytes with loss of Mpv17 function were characterized by increased susceptibility to apoptosis and ROS injury including decreased mitochondrial function, loss of mtDNA content, and change in mitochondrial configuration. In summary, the inner mitochondrial membrane protein Mpv17 in podocytes is essential for the maintenance of mitochondrial homeostasis and protects podocytes against oxidative stress-induced injury both in vitro and in vivo., (Copyright © 2014 the American Physiological Society.)

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells by only four transcription factors (Oct4, Sox2, Klf4, and c-Myc) has great potential for tissue-specific regenerative therapies, eliminating the ethical issues surrounding the use of embryonic stem cells and the rejection problems of using non-autologous cells. The reprogramming efficiency generally is very low, however, and the problems surrounding the introduction of viral genetic material are only partially investigated. Recent efforts to reduce the number of virally expressed transcription factors succeeded at reprogramming neural stem cells into iPS cells by overexpressing Oct4 alone. However, the relative inaccessibility and difficulty of obtaining neural cells in humans remains to be resolved. Here we report that dermal papilla (DP) cells, which are specialized skin fibroblasts thought to instruct hair follicle stem cells, endogenously express high levels of Sox2 and c-Myc, and that these cells can be reprogrammed into iPS cells with only Oct4 and Klf4. Moreover, we show that DP cells are reprogrammed more efficiently than skin and embryonic fibroblasts. iPS cells derived from DP cells expressed pluripotency genes and differentiated into cells from all germ layers in vitro and widely contributed to chimeric mice in vivo, including the germline. Our work establishes DP cells as an easily accessible source to generate iPS cells with efficiency and with less genetic material. This opens up the possibility of streamlined generation of skin-derived, patient-specific pluripotent stem cells and of ultimately replacing the remaining two factors with small molecules for safe generation of transplantable cells.