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 weekly 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 micromol/L) compared with recipients with an uncomplicated course (13+/-7.6 micromol/L), but not compared with recipients with cyclosporine (CsA) toxicity (20+/-13.0 micromol/L). Urinary NOx levels were significantly lower during AR (20+/-13.6 micromol/mmol creatinine) compared with an uncomplicated course (64+/-25.2 micromol/mmol creatinine) or CsA toxicity (53.8+/-28.3 micromol/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.     

CC-chemokine RANTES is increased in serum and urine in the early post-transplantation period of human renal allograft recipients

BACKGROUND: The chemokine RANTES is a potent chemoattractant for T cells and monocytes that has been shown to enhance inflammation. The aim of our study was to investigate whether RANTES is upregulated within the early post-transplantation period that may influence short-time allograft function rate. METHODS: Serum and urine samples from transplanted renal allograft recipients (n = 17) were obtained from specimens taken for diagnostic reasons. Four patients developed biopsy-proven rejection episodes within the first month. Time course of RANTES was studied within the first 12 days after renal transplantation using ELISA technique. Data were tested for significances between patients with rejection and without rejection, compared to healthy volunteers as controls, and correlated with clinical data. RESULTS: In the control group RANTES concentration was 37.2 +/- 2.7 ng/ml (serum) and 8.1 +/- 1.3 pg/ml (urine), respectively. In transplanted recipients serum RANTES was significantly upregulated up to 132 +/- 28 ng/ml on day 1 after transplantation and remained elevated within the first 12 days (n = 17). Time course of urine RANTES demonstrated elevated concentrations with 754 +/- 115 pg/ml on day 1 followed by an continuous decrease to 22.3 +/- 7 pg/ml on day 12 (n = 17). No significant differences could be detected between patients with rejection and without rejection episodes. CONCLUSIONS: In contrast to data of other urinary marker molecules (like IL-6), there are no significant differences between the rejection and non-rejection group. RANTES is therefore not suitable for early detection of rejection. Nevertheless, serum and urine RANTES concentrations were highly elevated in freshly transplanted renal allograft recipients reflecting an activated immune system.     

Increased nitric oxide production during acute rejection in kidney transplantation: a useful marker to aid in the diagnosis of rejection

BACKGROUND: The diagnosis of acute rejection (AR) relies on biopsy (Bx), with all the noninvasive tests failing to show satisfactory predictive value. Nitric oxide (NO) has been shown to play a role in AR. The aim of this study is to analyze the relationship between NO and (1) biopsy-proven allograft rejection and (2) other reasons of allograft dysfunction. PATIENTS AND METHODS: Fifty consecutive renal allograft recipients ages 23-72 yrs who were transplanted were prospectively recruited. Blood samples were collected for 3 months. Endogenous serum nitrate (SNO(3)) levels were measured with Griess reagent in 1178 samples. Biopsies were performed as clinically indicated. Tacrolimus levels, urinary cultures, and renal function tests were done as per unit protocol. RESULTS: Fifty recipients (mean+/-SD age 45.2+/-2.18 yrs, 24 men and 6 women) underwent 68 biopsies. Forty-five Bx (66.2%) showed AR in 19 recipients (mean age 47+/-8) and 23 (33.8%) Bx in 13 recipients (mean age 43+/-12) showed no AR. SNO(3) in AR was (73+/-8.89 micromol/L) compared with negative Bx (45+/-4.5 micromol/L; P<0.05). There was also a significant difference in SNO(3) during AR and other causes of allograft dysfunction; delayed graft function (54+/-7.8 micromol/L), urinary tract infection (44+/-2.9 micromol/L), tacrolimus toxicity (51+/-2.86 micromol/L), and increase in serum creatinine (44+/-2.36 micromol/L). CONCLUSION: There is a significant increase of serum nitrate with episodes of acute rejection compared with other causes of renal dysfunction. SNO(3) can therefore aid in the diagnosis of acute rejection.     

Urine macrophage migration inhibitory factor concentrations as a diagnostic tool in human renal allograft rejection.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is a potent activator of macrophages and T cells. Previous studies have shown that local MIF production is increased in acute renal allograft rejection, suggesting that it may play an important role in the rejection process. AIMS: To determine if urine and serum MIF concentrations: (1) are increased in acute rejection, and (2) can be used as noninvasive tools to discriminate between acute rejection (AR) and cyclosporine nephrotoxicity (CyA toxicity). METHODS: In a prospective study of nine renal allograft patients (five acute rejection and four stable), serial urine MIF concentrations were measured by ELISA in the first 14 days after transplantation. In a retrospective study, MIF concentrations in urine and serum were measured in 24 patients who were biopsied for acute renal transplant dysfunction (11 AR, 13 CyA toxicity). Urine and serum MIF were also measured in 23 stable renal transplant patients and 10 normals. RESULTS: MIF was readily detected in the urine of normal healthy controls (106+/-61 pg/micromol creatinine). In the prospective study, the urinary MIF concentration was increased substantially on day 1 posttransplantation and subsequently fell in parallel with the serum creatinine. However, urine MIF increased before episodes of biopsy proven acute rejection. The retrospective study showed that urine MIF concentrations in patients with AR were increased 5-fold compared to normal controls (439+/-313 pg/micromol Cr; P<0.01). In contrast, urine MIF concentrations in CyA toxicity were not significantly different to normal controls (145+/-119 pg/micromol Cr; P=NS). A marked increase in MIF immunostaining was seen in biopsies of AR, but not in CyA toxicity. No significant differences were evident in serum MIF levels between normals and any transplant patient group. CONCLUSIONS: These results suggest that measurement of urine MIF concentration may be useful in monitoring renal transplant patients for acute rejection and as a discriminator from cyclosporine nephrotoxicity.     

Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease

Polyomavirus (PV) exceptionally causes a morphologically manifest renal allograft infection. Five such cases were encountered in this study, and were followed between 40 and 330 d during persistent PV renal allograft infection. Transplant (Tx) control groups without PV graft infection were analyzed for comparison. Tissue and urine samples were evaluated by light microscopy, immunohistochemistry, electron microscopy, and PCR. The initial diagnosis of PV infection with the BK strain was made in biopsies 9+/-2 mo (mean +/- SD) post-Tx after prior rejection episodes and rescue therapy with tacrolimus. All subsequent biopsies showed persistent PV infection. Intranuclear viral inclusion bodies in epithelial cells along the entire nephron and the transitional cell layer were histologic hallmarks of infection. Affected tubular cells were enlarged and often necrotic. In two patients, small glomerular crescents were found. In 54% of biopsies, infection was associated with pronounced inflammation, which had features of cellular rejection. All patients were excreting PV-infected cells in the urine. PV infection was associated with 40% graft loss (2 of 5) and a serum creatinine of 484+/-326 micromol/L (mean +/- SD; 11 mo post-Tx). Tx control groups showed PV-infected cells in the urine in 5%. Control subjects had fewer rejection episodes (P<0.05) and stable graft function (P = 0.01). It is concluded that a manifest renal allograft infection with PV (BK strain) can persist in heavily immunosuppressed patients with recurrent rejection episodes. PV mainly affects tubular cells and causes necrosis, a major reason for functional deterioration. A biopsy is required for diagnosis. Urine cytology can serve as an adjunct diagnostic tool.     

Influence of immunosuppressive agents on tryptophan degradation and neopterin production in human peripheral blood mononuclear cells

The anti-proliferative and immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) degrades the essential amino acid tryptophan via the kynurenine pathway. IDO is stimulated during cellular immune responses preferentially by Th1-type cytokine interferon-γ (IFN-γ). IDO activity is estimated by calculating the kynurenine to tryptophan ratio (Kyn/Trp). In human monocyte-derived macrophages and dendritic cells, GTP-cyclohydrolase I is induced in parallel to IDO and produces neopterin. This study investigated the effects of common immunosuppressants on freshly isolated human peripheral blood mononuclear cells (PBMC) in vitro. PBMC were incubated with compounds for 30 min and then either left unstimulated or stimulated with mitogen phytohaemagglutinin (PHA). Concentrations of tryptophan, kynurenine and neopterin were measured in supernatants after 48 h. Kyn/Trp, neopterin and IFN-γ concentrations were significantly higher in PHA-stimulated vs. unstimulated PBMC. Tacrolimus (FK506), cyclosporine A (CsA), sirolimus and methylprednisolone dose-dependently inhibited tryptophan degradation and neopterin production. FK506, CsA and sirolimus showed significant inhibition at concentrations as low as 0.1 μg/ml, whereas prednisolone and methylprednisolone required higher doses to suppress tryptophan degradation. Mycophenolate-mofetil suppressed neopterin formation more efficiently than Kyn/Trp. All tested drugs also strongly decreased mitogen-induced IFN-γ concentrations. Overall the investigated immunosuppressants are effective to inhibit IDO activity and neopterin production in a similar and dose-dependent manner, however with some differences in IC50s when comparing individual compounds. The corresponding changes of IFN-γ concentrations are in line with its role as a trigger of both biochemical changes.     

Interleukin-6 and neopterin in renal transplant recipients: a longitudinal study

Serum and urine interleukin-6 (IL-6) levels and serum neopterin/creatinine ratios were longitudinally studied in 86 renal transplant recipients until 4 months after transplantation. During acute rejection and acute tubular necrosis (ATN), serum and urine IL-6 levels were elevated compared to during stable transplant function (P<0.001). During acute rejection, serum IL-6 levels increased at least 2 days before plasma creatinine started to rise (P<0.05), indicating its early involvement in the rejection process. During cytomegalovirus (CMV) disease, serum, but not urine, IL-6 levels were higher (P<0.01), and serum neopterin/creatinine values were higher than during stable transplant function, ATN, or acute rejection (P<0.01). No significant differences with stable transplant function occurred during cyclosporin A toxicity. Measurement of serum IL-6 provided a sensitivity of 84% and a specificity of 85% for the diagnosis of acute rejection episodes not coinciding with ATN. All cases of CMV disease could be diagnosed by measurement of serum neopterin/creatinine, which provided a specificity of 73%.     

Serum levels of macrophage-colony stimulating factor (M-CSF): a marker of kidney allograft rejection.

BACKGROUND: Macrophage-colony stimulating factor (M-CSF) is the principal factor for survival of monocytes and macrophages that play an important role in allograft rejection. We studied M-CSF serum levels during successful renal transplantation and acute graft rejection. METHODS: A total of 114 kidney allograft recipients were assessed for M-CSF levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: M-CSF serum levels were elevated in pre-transplant haemodialysis patients (611+/-355 IU/ml vs 168+/-61 in normal controls, P<0.01). Following successful renal transplantation, M-CSF decreased in the first month, stabilizing at 257+/-222 IU/ml (not significantly different from normal controls) in 52 post-transplant stable patients. There was no correlation between M-CSF level and creatinine clearance. M-CSF levels increased significantly (2-5 times) during biopsy-proven acute rejection episodes in 20 of 25 patients. All rejection episodes were successfully treated and serum M-CSF decreased rapidly to pre-rejection levels in 17/20 patients. In contrast, in five patients with cyclosporin toxicity and four patients with other causes of allograft dysfunction, M-CSF serum levels did not change. CONCLUSIONS: M-CSF serum level might be a specific marker of acute rejection. The source of increased production during rejection warrants further investigation, with infiltrating T cells and resident kidney cells being likely candidates.     

Expression of urokinase plasminogen activator and its receptor during acute renal allograft rejection

BACKGROUND: In inflammation, urokinase plasminogen activator (uPA) and its receptor (uPAR) play an important role in fibrinolysis and in activation and chemotaxis of neutrophils and lymphocytes. Moreover, the uPA/uPAR system is involved in processes that affect turnover of the extracellular matrix (ECM). The aim of this study was to determine the local and systemic release of uPAR, and the expression of uPA and uPAR in renal tissues during acute renal allograft rejection. METHODS: Blood, urine, and tissue samples were collected from 33 patients diagnosed with acute allograft rejection and from 14 transplant patients without rejection. From 10 healthy volunteers, blood and urine were collected as a control. In urine and blood samples, the levels of uPAR were determined by enzyme-linked immunosorbent assay (ELISA). Immunostaining and in situ hybridization for uPA and uPAR were performed on renal biopsies. RESULTS: uPAR was detectable at low levels in serum and urine of healthy volunteers and was increased in nonrejecting allograft recipients. Serum and urine levels of uPAR were higher in transplant recipients with rejection compared to nonrejectors. The urine and serum levels of uPAR correlated with the renal function. Immunostaining and in situ hybridization showed an up-regulation of both uPA and uPAR in rejection biopsies. Nonrejected grafts displayed no expression of uPA and uPAR by immunostaining, or of uPAR by in situ hybridization. uPA was detected in a limited number of tubular epithelial cells by in situ hybridization. During rejection, lymphocytes as well as tubular epithelial cells showed uPA and uPAR expression. In the vascular types of rejection, strong expression of uPA was also seen in the entire vessel wall, while uPAR was expressed by the endothelium. CONCLUSION: This study shows that (1) uPA and uPAR are up-regulated during acute renal allograft rejection; (2) uPAR levels in urine and serum correlate with serum creatinine levels, and (3) uPA and uPAR are produced by inflammatory cells, tubular epithelium, and vascular endothelium during acute renal allograft rejection.