Influence of donor age on bovine pancreatic islet isolation.

BACKGROUND: Pancreatic islets from pigs are largely used for experimental studies. However, pancreas harvesting requires modification of conventional slaughtering to reduce ischemia time. It has been shown that bovine pancreatic islets can be more easily obtained and they show satisfactory in vitro and in vivo function. To improve the isolation procedure we compared the effect of bovine donor age on islet isolation. METHODS: Islets were isolated by collagenase digestion and sequential sieving from calves (6 months of age) and from adult bovine (> 16 months of age). After isolation the number of islet equivalents was calculated and histological and immunohistochemical studies performed. The purity and viability of islet for each preparation was also estimated. In vitro function of islets was evaluated by static insulin secretion assay, and alginate encapsulated islets were transplanted in streptozotocin-induced diabetic rats for in vivo functional evaluation. RESULTS: A significantly higher number of islets were obtained from calf pancreas, compared with adult bovine pancreas. Hystological examination showed intact morphologic features of islets. The purity of islet preparations was higher from calf pancreas than from adult pancreas. Cell viability, and insulin production in presence of high glucose concentration, were not affected by donor age. All animals receiving microencapsulated islets from calves showed normoglycemia for prolonged periods (17-40 days). CONCLUSIONS: These results indicate that pancreatic islet isolation is more efficient from juvenile bovine than from adult. Calf pancreas is a good and convenient source of tissue for massive islet isolation for experimental studies.

17beta-estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases.

Conjugated estrogens shorten the prolonged bleeding time in uremic patients and are similarly effective in a rat model of uremia. We have previously demonstrated that the shortening effect of a conjugated estrogen mixture or 17beta-estradiol on bleeding time was abolished by the nitric oxide (NO) precursor L-arginine, suggesting that the effect of these drugs on hemostasis in uremia might be mediated by changes in the NO synthetic pathway. The present study investigated the biochemical mechanism(s) by which conjugated estrogens limit the excessive formation of NO. 17beta-estradiol (0.6 mg/kg), given to rats made uremic by reduction of renal mass, significantly reduced bleeding time within 24 h and completely normalized plasma concentrations of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS) catalytic activity, determined by NADPH-diaphorase staining in the thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS) immunoperoxidase staining in the endothelium of uremic aortas of untreated rats was significantly more intense than in control rats, while in uremic rats receiving 17beta-estradiol staining was comparable to controls. Thus 17beta-estradiol corrected the prolonged bleeding time of uremic rats and fully normalized the formation of NO by reducing the expression of ecNOS and iNOS in vascular endothelium. These results provide a possible biochemical explanation of the well-known effect of estrogens on primary hemostasis in uremia, in experimental animals and humans.

Upregulation of renal and systemic cyclooxygenase-2 in patients with active lupus nephritis.

In lupus nephritis (LN), renal thromboxane A2 (TXA2) production is increased, and inhibition of TXA2 activity improves renal function. In patients with LN, renal function depends very much on vasodilatory prostaglandins, and indeed inhibiting the prostaglandin-forming enzyme cyclooxygenase (COX) with aspirin or related compounds was detrimental on renal hemodynamics in these patients. There are no data so far on whether the excessive TXA2 production in LN derives from upregulation of type I or type II isoforms of COX. It was found that TXB2 synthesis and COX-2 gene expression were higher in peripheral blood mononuclear cells from patients with active LN compared to patients in the inactive form of the disease and to healthy subjects. Unlike COX-2, levels of COX-1 mRNA were comparable in lupus patients and control subjects and were not influenced by the disease activity. Immunoperoxidase studies on kidney biopsies showed COX-1 staining in glomerular arterioles and other renal vessels, with no evident difference between lupus biopsies and control specimens taken from either individuals who were free of renal disease or patients with non-lupus nephropathies. In contrast, COX-2 staining was definitely stronger in specimens from patients with active LN than control specimens. In active LN, COX-2-specific staining was localized mainly in the glomeruli, with a weaker signal on tubuli and in the interstitium. Double-staining studies with an antibody against the macrophage marker CD68 and an anti-COX-2 antibody definitely showed that COX-2 and CD68 often colocalized on the same cell, with only occasional glomerular COX-2-stained mesangial areas. Patients with non-lupus nephropathies had no increase in renal COX-2 expression. These results indicate that COX-2 upregulation is a specific finding of active LN and that monocytes infiltrating the glomeruli contribute to the exaggerated local synthesis of TXA2. If this is correct, COX-2 may soon become a target for therapeutic intervention in this disease.

Renal and systemic nitric oxide synthesis in rats with renal mass reduction.

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.