In rats undergoing renal mass reduction (RMR) oral supplementation with the nitric oxide (NO) precursor L-arginine increases glomerular filtration rate and ameliorates signs of glomerular injury, suggesting that chronic renal failure in the rats is a condition of low NO formation in the kidney. On the contrary, data are available that in the systemic circulation of uremics, both rats and human beings, NO is formed in excessive amounts and may contribute to platelet dysfunction and bleeding tendency, well-known complications of uremia. The present study was designed to clarify the pathophysiology of renal and systemic NO synthesis in uremia. We showed that renal ex vivo NO generation, measured as the conversion of [3H] L-arginine to [3H] L-citrulline, was lower than normal in RMR rats, seven days after surgery, and progressively worsened with time in close correlation with signs of renal injury. Consistent with these results, urinary excretion of the stable NO metabolites, NO2-/NO3-, significantly decreased in rats with RMR. To go deeper into the cellular origin and biochemical nature of this abnormality we used two histochemical approaches that could locate either NO synthase (NOS) catalytic activity (NADPH-diaphorase) or NOS isoenzyme expression (immunoperoxidase). NADPH-diaphorase documented a progressive loss of renal NOS activity in RMR rats that co-localized with a strong progressive decrease of inducible NOS isoenzyme (iNOS) immunostaining. At variance with iNOS, endothelial cell NOS (ecNOS) staining was rather comparable in RMR and control kidneys. At variance to the kidney, in the systemic circulation of RMR rats the synthesis of NO increased as reflected by higher than normal plasma NO2-/NO3- concentrations. High systemic NO likely derives from vessels as documented by the increased NOS activity and higher expression of both iNOS and ecNOS in the aorta of RMR rats. Up-regulation of systemic NO synthesis might be an early defense mechanism against hypertension of uremia. On the other hand, more NO available to circulating cells may sustain the bleeding tendency, a well-known complication of uremia.

Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact threedimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research.

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Age-related changes of the optic nerve were studied in 3-month-old (young), 12-month-old (adult) and 24-month-old (aged) male Sprague-Dawley rats. Cross sections of the intracranial portion of the optic nerves of animals of different age groups were stained with haematoxylin-eosin and examined under a light microscope at low and high magnification. Other sections were stained with crystal violet for demonstration of glial cells. A third group of sections were stained immunohistochemically to detect glial fibrillary acidic protein (GFAP) which is a marker for localizing and characterizing astrocytes. All morphological results were subjected to the quantitative analysis of images and to statistical analysis to identify significant morphometrical data. Tissue protein concentrations were determined on homogenized fragments of optic nerve. Our results demonstrate the following age-related changes: (1) increase of the optic nerve sheaths (meningeal membranes); (2) increased number of astrocytes; (3) increase of areal density of GFAP immunoreactivity; (4) increased diameter and area of the optic nerve; (5) decreased number of nerve fibres; (6) decreased-size of nerve fibres and (7) decrease of the nerve fibres/meningeal membrane ratio from 3:1 to 1:1. Moreover, the protein amount does not change with age. The rat optic nerve, therefore, appears sensitive to ageing processes and can be considered as a useful model for the studies on neuronal ageing.