Expression of inducible and endothelial nitric oxide synthases, formation of peroxynitrite and reactive oxygen species in human chronic renal transplant failure

Nitric oxide (NO.) is produced by NO syntheses (NOS) and can interact with reactive oxygen species (ROS) to form peroxynitrite, which induces protein damage by formation of nitrotyrosine. NO. has a promotional effect on acute rejection. To investigate the role of NO. during chronic renal transplant failure (CRTF), we studied the expression of eNOS and iNOS in conjunction with H2O2 production and the formation of nitrotyrosines. Nephrectomy material from 10 patients and 10 control kidneys was used in this study. Expression of iNOS, eNOS, nitrotyrosine and the presence of ROS-producing calls and macrophages were determined using Immunohistochemistry. INOS expression in nonsclerosed glomeruli and interstitium was significantly increased in patients with CRTF (p <0.05). Glomerular eNOS expression was decreased in patients with CRTF compared with glomeruli of control kidneys (p <0.01). Nitrotyrosine and ROS positive cells were significantly increased in CRTF in the Interstitium (p <0.05), but not in glomeruli. In summary, we found a marked interstitial increase in iNOS protein expression together with a decrease in glomerular eNOS expression in CRTF patients, associated with a significant increment in ROS and nitrotyrosine-positive cells in the interstitium. Our results suggest that loss of NO. production by glomerular eNOS in conjunction with an increased NO. production by interstitial iNOS, together with the formation of ROS and nitrotyrosine, Is involved in the pathogenesis of CRTF

Nitric oxide production and nitric oxide synthase expression in acute human renal allograft rejection

Background Nitric oxide (NO) is produced by nitric oxide synthases (NOS), which are either constitutively expressed in the kidney or inducible, in resident and infiltrating cells during inflammation and allograft rejection. NO is rapidly degraded to the stable end products nitrite and nitrate, which can be measured in serum and urine, and may serve as noninvasive markers of kidney allograft rejection. Methods. Total nitrite and nitrate levels (NOx) were measured in serum and urine thrice meekly after an overnight fast in 18 consecutive patients following renal cadaveric transplantation. Inducible NOS (iNOS) and endothelial NOS (eNOS) expression was immunochemically determined in renal biopsy specimens with or without acute rejection (AR). Results. Serum NOx levels increased days before AR and were significantly higher at the moment of AR (27 +/- 12.4 mu mol/L) compared with recipients with an uncomplicated course (13 +/- 7.6 mu mol/L), but not compared with recipients with cyclosporine (CsA) toxicity (20 +/- 13.0 mu mol/L), Urinary NOx levels were significantly lower during AR (20 +/- 13.6 mu mol/mmol creatinine) compared with an uncomplicated course (64 +/- 25.2 mu mol/mmol creatinine) or CsA toxicity (53.8 +/- 28.3 mu mol/mmol creatinine). Interstitial and glomerular iNOS expression was significantly increased in biopsy specimens showing AR. Unexpectedly glomerular eNOS expression was significantly decreased in patients with AR. Conclusions. This study reports differences in NOx levels in serum and urine, which may help discriminate AR episodes from an uncomplicated course or CsA toxicity. As expected, renal iNOS expression is increased in acute allograft rejection, The decrease in glomerular eNOS expression suggests an intriguing link between acute and chronic rejection

Long-term dietary L-arginine supplementation attenuates proteinuria and focal glomerulosclerosis in experimental chronic renal transplant failure

Glomerular endothelial nitric oxide synthase expression is decreased in humans during acute rejection and chronic renal transplant failure (CRTF). This may contribute to vascular damage through changes in the renal hemodynamics and enhanced endothelial adhesion of leukocytes and platelets. Dietary supplementation of L-arginine may increase endothelial NO production, thereby protecting the vascular wall and improving renal hemodynamics. We tested the hypothesis that long-term L-arginine supplementation attenuates the development of CRTF in an experimental model for renal transplantation. In the Fisher 344 to Lewis rat model for renal transplantation, renal function and histology of untreated rats was compared with rats receiving L-arginine in the drinking water (10 g/L), starting 2 days before transplantation. Every 4 weeks systolic blood pressure was measured and serum and urine were collected for measurement of nitrite and nitrate (NOx), creatinine, and proteinuria. At 34 weeks the histological renal damage was assessed by scoring focal glomerulosclerosis and measurement of alpha-smooth muscle actin (alpha-SMA) expression. Urinary NOx was significantly increased in treated animals. Proteinuria was significantly lower in L-arginine-treated animals from week 24 onward (p <0.05). Plasma creatinine and creatinine clearance did not differ between the groups. The focal and segmental glomerulosclerosis (FGS) score (max 400) at week 34 was also significantly lower in treated rats arbitrary U (20 +/- 21 vs 61 +/- 67 arbitrary U; p <0.05). The expression of alpha-SMA was lower in L-arginine-treated rats than in untreated rats (1.93 +/- 0.8% area surface vs 3.64 +/- 2.5% area surface). In conclusion, in this experimental model for CRTF, L-arginine administration significantly reduced FGS and proteinuria, without affecting renal function. Our data suggest that dietary L-arginine supplementation attenuates progression of CRTF and may therefore be an additional therapeutic option in human renal allograft recipients. (C) 2002 Elsevier Science (USA). All rights reserved

Nitric oxide production and nitric oxide synthase expression in acute human renal allograft rejection

Background Nitric oxide (NO) is produced by nitric oxide synthases (NOS), which are either constitutively expressed in the kidney or inducible, in resident and infiltrating cells during inflammation and allograft rejection. NO is rapidly degraded to the stable end products nitrite and nitrate, which can be measured in serum and urine, and may serve as noninvasive markers of kidney allograft rejection. Methods. Total nitrite and nitrate levels (NOx) were measured in serum and urine thrice meekly after an overnight fast in 18 consecutive patients following renal cadaveric transplantation. Inducible NOS (iNOS) and endothelial NOS (eNOS) expression was immunochemically determined in renal biopsy specimens with or without acute rejection (AR). Results. Serum NOx levels increased days before AR and were significantly higher at the moment of AR (27 +/- 12.4 mu mol/L) compared with recipients with an uncomplicated course (13 +/- 7.6 mu mol/L), but not compared with recipients with cyclosporine (CsA) toxicity (20 +/- 13.0 mu mol/L), Urinary NOx levels were significantly lower during AR (20 +/- 13.6 mu mol/mmol creatinine) compared with an uncomplicated course (64 +/- 25.2 mu mol/mmol creatinine) or CsA toxicity (53.8 +/- 28.3 mu mol/mmol creatinine). Interstitial and glomerular iNOS expression was significantly increased in biopsy specimens showing AR. Unexpectedly glomerular eNOS expression was significantly decreased in patients with AR. Conclusions. This study reports differences in NOx levels in serum and urine, which may help discriminate AR episodes from an uncomplicated course or CsA toxicity. As expected, renal iNOS expression is increased in acute allograft rejection, The decrease in glomerular eNOS expression suggests an intriguing link between acute and chronic rejection

Nitric oxide inhibition enhances platelet aggregation in experimental anti-Thy-1 nephritis

In the present paper we studied the role of nitric oxide radicals (NO) on platelet aggregation, fibrinogen deposition, superoxide formation, peroxynitrite formation, hemodynamics, and leukocyte migration in the Thy-1 model of glomerulonephritis. To first study the baseline kinetics of these parameters, groups of anti-Thy-l-treated rats were sacrificed at 1 h, 4 h, 24 h, 3 days, 7 days, and 14 days and compared to controls. Urinary protein excretion was significantly elevated in Thy-1 nephritis at 3 and 7 days. Glomerular macrophages, PMNs, and superoxide anion-positive cells were significantly increased in Thy-1 nephritis. Nitrotyrosine immunoreactivity was absent during the entire study period. Glomerular platelet aggregation was significantly increased in anti-Thy-1 injected rats at 1 h, 4 h, 24 h, and 3 days. Glomerular fibrinogen deposition was significantly elevated at all time points. To elucidate the role of NO in this process, additional groups of anti-Thy-l-injected rats were treated with the NOS inhibitor L-NAME and studied at 24 h. Urinary protein excretion was significantly higher in L-NAME treated Thy-1 rats compared to nontreated Thy-1 rats. Plasma and urine nitrite/nitrate levels were significantly lower in L-NAME-treated Thy-1 rats compared to nontreated Thy-1 rats. Compared to nontreated Thy-1 rats, there were no differences in intraglomerular leukocyte accumulation after treatment with L-NAME. In contrast, we observed a marked increase in platelet aggregation following L-NAME treatment. From these data we conclude that the inflammatory infiltrate in Thy-1 nephritis develops independent of NO radical production, whereas NO radicals prevent the accumulation of platelet aggregates. (C) 2001 Elsevier Science