HMGB1+ microparticles present in urine are hallmarks of nephritis in patients with systemic lupus erythematosus.

Non-classical monocytes infiltrate the kidney parenchyma and participate in tissue damage in patients with lupus nephritis (LN). Circulating microparticles (MPs) seem to play critical roles in the activation of monocytes in systemic lupus erythematosus (SLE) patients. This study aims to characterize the phenotypes of MPs and monocyte subsets in LN patients and to determine their potential to discriminate between SLE patients with and without LN. Blood and urine samples from SLE patients were collected. In monocyte subsets from whole blood samples several phenotypic markers were evaluated. MPs were isolated from platelet-poor plasma and urine by centrifugation. This phenotypic marker characterization was performed using multiparametric flow cytometry. We observed that patients with active LN have lower counts of non-classical monocytes than do those without renal involvement. All monocyte subsets exhibited lower expression of CX3CR1 and ICAM-1 in LN than in patients without LN. High frequencies of MP-HMGB1+ and MP-HLA-DR+ were detected in circulation and urine of LN patients. Although MP-HMGB1+ , MP-HLA-DR+ , and MP-CX3CR1+ from urine were able to discriminate between patients with and without LN, only urinary MP-HMGB1+ were different between patients with active and inactive LN. Therefore, these vesicles may be useful as biomarkers of LN.

Urinary soluble HLA-DR is a potential biomarker for acute renal transplant rejection.

BACKGROUND.: Urine is a potentially rich source of biomarkers for monitoring kidney dysfunction. In this study, we have investigated the potential of soluble human leukocyte antigen (sHLA)-DR in the urine for noninvasive monitoring of renal transplant patients. METHODS.: Urinary soluble HLA-DR levels were measured by sandwich enzyme-linked immunosorbent assay in 103 patients with renal diseases or after renal transplantation. sHLA-DR in urine was characterized by Western blotting and mass spectrometry. RESULTS.: Acute graft rejection was associated with a significantly elevated level of urinary sHLA-DR (P<0.0001), compared with recipients with stable graft function or healthy individuals. A receiver operating characteristic curve analysis showed the area under the curve to be 0.88 (P<0.001). At a selected threshold, the sensitivity was 80% and specificity was 98% for detection of acute renal transplant rejection. sHLA-DR was not exosomally associated and was of lower molecular weight compared with the HLA-DR expressed as heterodimer on the plasma membrane of antigen-presenting cells. CONCLUSIONS.: sHLA-DR excreted into urine is a promising indicator of renal transplant rejection.

Urinary HLA-DR and CD54 expression--indicators for inflammatory activity in decoy cell shedding patients.

BACKGROUND: Polyomavirus (PV) nephropathy may coexist with or follow acute renal transplant rejection. The aim of this study was to evaluate whether HLA-DR and CD54 are useful cellular markers for surveillance of acute rejection in PV-infected patients. METHODS: A prospective study was conducted using 205 renal transplant patients. Urine samples were collected at a regular interval post-transplantation for routine cytology and immunocytochemistry. Urinary levels of tumour necrosis factor alpha, soluble interleukin-2 receptor and interleukin-6 were used as adjunctive markers for acute rejection. RESULTS: Of the 699 total samples, decoy cells were identified in 100 samples of 50 patients. Patients with decoy cell-positive (DCP) samples had higher serum creatinine levels than decoy cell-negative (DCN) samples (1.55 vs 1.41 mg/dl, respectively; P = 0.006). DCP samples were also more likely to be HLA-DR positive (50.0 vs 32.4%; P = 0.029), as well as CD54 positive (17.4 vs 6.9%; P = 0.038). However, serum creatinine levels did not correlate with HLA-DR or CD54 positivity among DCP samples. Instead, CD54 positivity correlated with decoy cell grades. Immunosuppression decreased in 11 DCP patients, and HLA-DR was negatively converted in three of them. None of the patients developed acute clinical rejection. Urinary cytokine levels did not correlate with serum creatinine levels, nor did they correlate with HLA-DR or CD54 status among DCP patients. CONCLUSIONS: Urinary tubular HLA-DR and CD54 expression increased in decoy cell shedding patients but did not indicate a concomitant acute rejection. These markers may instead indicate renal inflammatory activity associated with viral reactivation, which has the potential to progress to PV interstitial nephritis.

Immunocytological urinalysis and monocyte chemotactic peptide-1 in renal transplant recipients with polyomavirus replication.

In some patients polyomavirus replication induces chronic tubulointerstitial inflammation in the transplanted kidney. The aim of this study was to investigate whether immunocytological urinalysis and monocyte chemoattractant protein-1 (MCP-1) assays could be used for an early diagnosis of nephropathy for patients with polyomavirus replication. We analyzed 1189 urine sediments from 174 renal allograft recipients who were transplanted between 2000 and 2005. Decoy cells were identified by an immunofluorescence method using specific antibodies (JC/BK monoclonal antibody). A similar method was used to detect CD3(+), CD14(+), and HLA-DR(+) cells with appropriate antibodies. The urinary excretion of MCP-1 was assayed by enzyme-linked immunosorbent assay. The results of urine sediment analysis and MCP-1 concentrations were compared with those of patients with stable graft function (control group n = 65). In 17 patients (10%) decoy cells were identified in urine. In 12 patients polyomavirus DNA was detected in plasma or urine by a polymerase chain reaction method. Polyomavirus nephropathy was diagnosed in eight patients by the presence of intranuclear viral inclusions or immunohistochemical staining with SV40 large T-antigen specimens from a renal biopsy, as well as by clinical and histopathological evidence (group I). Polyomavirus replication was diagnosed in four patients by urinary excretion of decoy cells and polyomavirus DNA detection (group III). In five patients only decoy cells were found. The patients of groups I and II showed an increased number of CD3, CD14, HLA-DR surface antigen-positive cells and greater excretion of MCP-1 compared with the control group (P < .02). The number of excreted cells was higher among patients with more severe infiltration. The results of patients from group III were similar to the control group. In conclusion, increased excretion of cells with CD3, CD14, and HLA-DR surface antigens and of MCP-1 were associated with intragraft tubulointerstitial inflammation in patients with polyomavirus nephropathy. Asymptomatic polyomavirus replication was associated with hidden tubulointerstitial inflammation. Monitoring cell excretion and chemokine content may be utilized for early detection of polyomavirus-induced nephropathy.

Urine flow cytometry as a tool to differentiate acute allograft rejection from other causes of acute renal graft dysfunction.

BACKGROUND: Recently, urine flow cytometry (UFC) was introduced as a useful noninvasive tool for the immunological monitoring of renal allograft recipients. The presence of an active urine sediment as determined by UFC was found to be associated with acute rejection (AR) episodes. METHODS: In the present study we assess the value of UFC in the setting of acute graft dysfunction. UFC was performed in 30 patients (32 events) at the time of admission to the hospital for the evaluation of rising creatinine (serum creatinine increment > or =0.6 mg/dl above baseline). UFC analysis was done blinded to the clinical diagnosis, and results were compared with the discharge diagnosis: AR, n=15; chronic rejection (CR), n=8; drug toxicity, n=4; urinary leak, n=2; recurrence of primary disease, n=1; lymphocele, n=1; and unknown, n=1. RESULTS: The presence of at least 5% of HLA-DR-positive cells and intercellular adhesion molecule-1-positive cells was detected in 100% and 53%, respectively, of the samples associated with AR (P<0.01 vs. others). The specificity value for the diagnosis of AR was: 100% for the presence of intercellular adhesion molecule-1 or CD3-positive cells and 88% for the presence of interleukin-2 receptor-positive or HLA-DR-positive cells. Half of the samples associated with CR had CD14-positive cells (P=0.03 vs. others) with a specificity value of 87.3%. The samples associated with drug toxicity, urological problems, or recurrence of primary disease were remarkable for the lack of expression of the antigens studied. CONCLUSION: UFC can clearly differentiate AR from other causes of acute renal allograft dysfunction. HLA-DR is revealed to be the most sensitive and intercellular adhesion molecule-1 the most specific marker for AR. The presence of CD14-positive cells was highly suggestive of CR. Due to its objective nature, UFC should be considered in the evaluation of graft dysfunction.

Urine cytology and urine flow cytometry in renal transplantation--a prospective double blind study.

Urine cytology (UC) has proved to correlate well with core and fine-needle aspiration kidney biopsies of renal allograft recipients undergoing acute rejection (AR). This study was undertaken to compare the relative usefulness of urine flow immunocytometry (UFC) (using fluorescinated antibodies anti-HLA-DR, anti-CD3 and antirenal epithelial cells) with UC in its ability to diagnose AR by analyzing 200 urine specimens during a prospective double-blind study of 40 renal transplant recipients. Clinical diagnosis was retrospectively assigned to one of the following categories: group I--AR, 15; group II--ischemic injury period (first 5 days postop.), 12; group III, 173 (including 168 stable grafts, 1 pyelonephritis and 4 cyclosporine toxicity), by investigators blinded to the urine results. Both tests were highly sensitive for the diagnosis of AR (UC = 86.6% vs. UFC = 100%; P = NS) with a specificity after the ischemic injury period of 78% by UC and 87.9% by UFC. Samples obtained during AR revealed higher levels of expression of HLA-DR as well as higher numbers of CD3-positive cells. These tests had specificity values of 95.3% and 97.6%, respectively, for the diagnosis of AR. The degree of immune activation (established by numbers of lymphocytes/lymphoblasts seen by UC) correlated with the severity of biopsy-proven ARs and with response to antirejection therapy. In conclusion, both test are highly accurate in diagnosing AR. The highest specificity value was obtained when both UC and UFC were utilized together (93%). We suggest that the routine use of these tests can provide an important adjunct to the evaluation of renal transplant recipients.

Quantitation of soluble HLA-DR antigens in human serum and other body fluids.

The existence of soluble forms of MHC class II molecules is well established. To quantify soluble HLA-DR antigens (sHLA-DR) in human serum and other body fluids, we developed an enzyme immunoassay using two non-overlapping HLA-DR-specific monoclonal antibodies (RoDR, BL-la/5) and an immunoaffinity chromatography-purified sHLA-DR standard. In serum of healthy individuals, sHLA-DR levels were found in the range between 0.6 and 3 ng/ml (median 0.85 ng/ml) whereas EDTA plasma samples showed concentrations about 20 times higher (median 21 ng/ml). In tears, saliva, sweat, urine, amniotic fluid, cerebrospinal fluid, and bronchoalveolar lavage, sHLA-DR could also be detected. No association was found between sHLA-DR serum levels and distinct HLA specificities. In the sera of patients with autoimmune diseases, slightly enhanced sHLA-DR values were found (juvenile rheumatoid arthritis: median 2.0 ng/ml, lupus erythematosus: 1.5 ng/ml, diabetes mellitus: 2.1 ng/ml).

Early diagnosis of kidney transplant rejection and cyclosporin nephrotoxicity by urine cytology.

A total of 2000 urine samples from 53 kidney transplant recipients were studied to develop a routine method for the early diagnosis of rejection and cyclosporin (CSA) nephrotoxicity in urine. New-Sternheimer staining and an immunocytochemical technique were used together with classical Papanicolaou staining to differentiate cells in the urine. After cell count and differentiation of second morning urine samples with New-Sternheimer and Papanicolaou stains, immunocytochemistry was performed using antibodies against the following antigens: CD2, CD4, CD8, CD25, CD71 (transferrin receptor), HLA-DR and cytokeratin (Lu-5). Cell counts were obtained for the positively-reacting cells per millilitre of urine. By New-Sternheimer and Papanicolaou staining, CSA nephrotoxicity was characterized by the predominance of proximal tubular cells. During rejection episodes, increased numbers of mononuclear cells and renal epithelial cells were found. Immunocytochemical analysis showed a significant increase in CD2-, CD4-, CD8-, CD25-, CD71-, and HLA-DR-positive epithelial cells and in the ratio HLA-DR/cytokeratin-positive epithelial cells in rejection. CD25-positive cells had the highest sensitivity and specificity for the diagnosis of rejection. Our urine cytology technique proved to be a useful and non-invasive method for the early diagnosis of rejection and CSA nephrotoxicity.

[Differential diagnosis of kidney transplant rejection and ciclosporin nephrotoxicity by urine cytology].

To make the differentiation of kidney transplant acute rejection and ciclosporin (CS) nephrotoxicity urine cytology by classical Papanicolaou with immunocytochemical stain has been performed. Increased numbers of renal tubular cells with lymphocytes and monocytes were found in both rejections and CS toxicities. CS toxicities were associated with increased numbers of proximal tubular cells. In immunocytochemical stain, increased numbers of CD25 and CD8 positive cells as well as increased ratio of HLA-DR/cytokeratin positive cells were typically found in rejections. It is concluded that the proposed analysis of urine cytology is a non-invasive and reliable method for daily graft monitoring of acute rejection and CS toxicity.