Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Pharmacological doses of the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduce albuminuria in diabetes. We explored the hypothesis that mTOR mediates podocyte injury in diabetes. High glucose (HG) induces apoptosis of podocytes, inhibits AMP-activated protein kinase (AMPK) activation, inactivates tuberin, and activates mTOR. HG also increases the levels of Nox4 and Nox1 and NADPH oxidase activity. Inhibition of mTOR by low-dose rapamycin decreases HG-induced Nox4 and Nox1, NADPH oxidase activity, and podocyte apoptosis. Inhibition of mTOR had no effect on AMPK or tuberin phosphorylation, indicating that mTOR is downstream of these signaling molecules. In isolated glomeruli of OVE26 mice, there is a similar decrease in the activation of AMPK and tuberin and activation of mTOR with increase in Nox4 and NADPH oxidase activity. Inhibition of mTOR by a small dose of rapamycin reduces podocyte apoptosis and attenuates glomerular injury and albuminuria. Our data provide evidence for a novel function of mTOR in Nox4-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Thus, mTOR and/or NADPH oxidase inhibition may represent a therapeutic modality of diabetic kidney disease.

Abstract. Locally generated angiotensin II (AngII) may be involved in the pathogenic mechanisms of chronic renal diseases. Renal expression of AngII and other components of the renin-angiotensin system (RAS) were analyzed by immunohistochemistry and Western blot in a model of chronic progressive nephropathy induced by inhibition of nitric oxide synthesis. Renal injury was evaluated by histology and albumin excretion. Systemic RAS status was evaluated through plasma renin activity (PRA) and plasma AngII concentration. In addition, the effects of enalapril, losartan, and mycophenolate mofetil (MMF) on AngII expression in animals with chronic renal disease was also analyzed. Plasma renin activity and plasma AngII were not different between rats with nephropathy and controls (2.08 Ϯ 0.7 versus 2.03 Ϯ 0.5 ng/ml/h and 94.3 Ϯ 18 versus 78.9 Ϯ 16 fmol/ml, respectively). However, rats with chronic progressive nephropathy showed augmented renal content of angiotensinogen protein (13.5 Ϯ 3.5 versus 2.2 Ϯ 0.4 pixels in control rats; P Ͻ 0.05), enhanced expression of cathepsin D-a renin-like enzyme-in cortical collecting tubules (103.5 Ϯ 27.0 versus 66.2 Ϯ 3.6 cells/mm 2 in controls; P Ͻ 0.01), and increased expression of AT 1 receptor in interstitium (54.7 Ϯ 7.8 versus 1.3 Ϯ 0

Podocyte injury, a major contributor to the pathogenesis of diabetic nephropathy, is caused at least in part by the excessive generation of reactive oxygen species (ROS). Overproduction of superoxide by the NADPH oxidase isoform Nox4 plays an important role in podocyte injury. The plant extract silymarin is attributed antioxidant and antiproteinuric effects in humans and in animal models of diabetic nephropathy. We investigated the effect of silybin, the active constituent of silymarin, in cultures of mouse podocytes and in the OVE26 mouse, a model of type 1 diabetes mellitus and diabetic nephropathy. Exposure of podocytes to high glucose (HG) increased 60% the intracellular superoxide production, 90% the NADPH oxidase activity, 100% the Nox4 expression, and 150% the number of apoptotic cells, effects that were completely blocked by 10 μM silybin. These in vitro observations were confirmed by similar in vivo findings. The kidney cortex of vehicle-treated control OVE26 mice displayed greater Nox4 expression and twice as much superoxide production than cortex of silybin-treated mice. The glomeruli of control OVE26 mice displayed 35% podocyte drop out that was not present in the silybin-treated mice. Finally, the OVE26 mice experienced 54% more pronounced albuminuria than the silybin-treated animals. In conclusion, this study demonstrates a protective effect of silybin against HG-induced podocyte injury and extends this finding to an animal model of diabetic nephropathy.

Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios.

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