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Background: Acute rejection of allograft is one of the most serious complications of renal transplantation that requires fast and precise diagnostic approach. In this paper our experience in cytologic urinalysis as a diagnostic method of the acute renal allograft rejection was reviewed., Methods: The study group included 20 of 56 patients with transplanted kidneys who were assumed for the acute allograft rejection according to allograft dysfunction and/or urine cytology findings. Histological findings confirmed allograft rejection in 4 patients. Urine sediment obtained in cytocentrifuge was air-dried and stained with May-Grunwald-Giemsa. Acute allograft rejection was suspected if in 10 fields under high magnification 15 or more lymphocytes with renal tubular cells were found., Results: Acute transplant rejection occurred in 32.1% patients. In 15 patients clinical findings of the acute renal allograft rejection corresponded with cytological and histological findings (in the cases in which it was performed). Three patients with clinical signs of the acute allograft rejection were without cytological confirmation, and in 2 patients cytological findings pointed to the acute rejection, but allograft dysfunction was of different etiology (acute tubular necrosis, cyclosporine nephrotoxicity). In patients with clinical, cytological and histological findings of the acute allograft rejection urine finding consisted of 58% lymphocytes, 34% neutrophilic leucocytes and 8% monocytes/macrophages on the average. The accuracy of cytologic urinalysis related to clinical and histological finding was 75%., Conclusion: Urine cytology as the reliable, noninvasive, fast and simple method is appropriate as the a first diagnostic line of renal allograft dysfunction, as well as for monitoring of the graft function.

Background: Proteinuria is associated with an increased risk of renal failure. In chronic kidney transplant failure it is associated with poorer graft outcome., Materials and Methods: In our Unit 405 renal transplants were performed between April 1992 and December 2001. We analysed 1) the main causes of post-transplant proteinuria and 2) the prognostic significance for graft outcome in patients with a minimum follow-up of 6 months., Results: Early proteinuria was associated with a higher incidence of chronic allograft nephropathy (CAN) and de novo/recurrent nephropathies. Graft outcome was poorer in patients with early persistent proteinuria., Conclusions: Proteinuria after renal transplantation increases the risk of graft failure. We can, therefore, hypothesize that a graft biopsy is the best way to reveal the causes of proteinuria so that therapeutic interventions, which have been shown to reduce proteinuria, can be applied immediately.

A total of 9000 urine samples from 69 kidney transplant recipients were studied for differential diagnoses of transplant rejection and cyclosporin/tacrolimus toxicity. New-Sternheimer and Papanicolaou staining were used to differentiate cells in urine. We also employed an immunocytochemical technique for further identification of exfoliated cells. With New-Sternheimer and Papanicolaou staining, the predominance of proximal tubular cells was useful to differentiate cyclosporin/tacrolimus toxicity from acute rejection in cases of increased serum creatinine level. During rejection episodes, an increased number of mononuclear cells and renal epithelial cells were found. Immunocytochemical analysis showed a significant increase of CD2-, CD4- CD8-, CD25- and HLA-DR-positive cells with rejection. However, there was no relationship between Banff criteria rejection grade and the increase of mononuclear cells.

Intrigued by the rapid clearance of free fetal DNA from the maternal circulation, we have investigated whether this fetal genetic material could be cleared via the kidney. For this purpose, we examined for the presence of Y chromosome-specific DNA sequences in urine samples obtained from 8 women pregnant with male fetuses. No male-specific sequences could be detected, despite the use of a very sensitive nested PCR assay nor a highly reproducible real-time PCR assay. We did, however, detect maternal DNA sequences. To determine if this cell-free DNA was derived from the kidney or another source, we next examined urine from female kidney transplant patients who had received male kidneys. Y chromosome-specific sequences were indeed detectable by both nested and real-time PCR in these samples, thereby confirming a recent report describing urinary DNA microchimerism. Quantitative analysis of serially obtained samples furthermore suggests that transplant-derived sequences are elevated during periods of graft rejection. These results imply that the measurement of graft-derived urinary DNA may serve as a new marker for kidney graft tolerance.

Antigen-dependent and antigen-independent factors have been implicated in the pathophysiology of chronic allograft rejection, but their relative role is not well established. In the Fisher 344-->Lewis rat kidney transplant model, we sought (1) to compare the relative efficacy of the novel immunosuppressant, mycophenolate mofetil (MMF), with that of the AT1 receptor blocker, losartan, in preventing the development of chronic graft rejection when given for 52 wk; (2) to examine whether combining MMF with losartan affords better protection than each of the drugs alone. For comparison, the effect of cyclosporine (CsA) to control chronic graft rejection was also assessed. Administration of MMF alone or losartan alone to the kidney allografted rats resulted in a partial decrease in the amount of proteinuria, preservation of glomerular and tubulo-interstitial graft structure, limitation of intragraft cell infiltration, and improvement of graft survival compared with corresponding parameters in untreated, transplanted control rats. Combined treatment with MMF and losartan completely prevented the development of proteinuria, largely reduced glomerular and tubulointerstitial injury, and suppressed intragraft cell infiltration, and all animals survived at the end of the follow-up. Similarly, CsA treatment largely prevented graft injury but failed to achieve 100% animal survival. We have shown that MMF synergizes with the angiotensin II receptor antagonist, losartan, in simultaneously targeting complementary pathways of chronic allograft rejection. Combining MMF and angiotensin II receptor blocker offers superior long-term renoprotection as compared with CsA. Together, these findings provide the basis to prevent chronic injury and progressive dysfunction after renal transplantation.

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.

Background: The use of urine flow cytometry (UFC) as a noninvasive tool for the diagnosis of acute and chronic rejection of the renal allograft has been previously reported., Methods: We analyzed the expression of various cell surface antigens during a 30-day period after the diagnosis and treatment of 24 acute rejection (AR) episodes. UFC was performed on 59 urine specimens, from 17 patients meeting the diagnostic criteria for AR. UFC analysis was performed blinded to the clinical management utilizing the following fluorescinated monoclonal antibodies: anti-CD3, anti-CD14, anti-HLA-DR, anti-CD54, and anti-interleukin 2 receptor. Results were correlated with the patient's requirement for antilymphocytic drugs and increment in serum creatinine level (mg/dl) on day 30 after AR., Results: HLA-DR was the most prevalent antigen noted during the first 2 days of AR (91.7% of the samples), followed by CD14 (50%) and CD54 (41.7%). After day 4 the degree of expression of HLA-DR-, CD14-, and CD54-positive cells correlated with the need for antilymphocytic drugs. CD54 was the best parameter with a sensitivity=100% and specificity=90.9% (P=0.001). Those patients who had permanent graft injury after treatment of the AR had persistence of CD54- and CD14-positive cells in the urine., Conclusion: Serial monitoring of urine sediments by UFC was predictive of the requirement for antilymphocytic therapy and irreversible graft damage.

Background: The role of nitric oxide (NO) after cadaveric renal graft transplantation has not been yet fully clarified. The aim of our study was to examine NO production into the urine of patients following cadaveric renal graft transplantation with a normal course and complications (acute rejection and cyclosporin toxicity)., Methods and Results: Production of stabile NO metabolites (NO2 and NO3) into urine (U-NOx) was examined in recipients of cadaveric renal transplantation. Only patients with standard triple immunosuppressive therapy (cyclosporin, azathioprine, prednisone) were include into the study. Patients receiving other immunosuppressive agents or drugs affecting NO formation (nitrates, ACE inhibitors) were excluded from the study, as were those with infectious or other serious post-transplant complications. Overall, we examined 33 patients (21 men and 12 women), with acute rejection and cyclosporin-induced toxicity in ten each, and a normal course with no complications in 13. The mean age of the patients was 50.96&#95;11.13 years. U-NOx was examined by biochemistry using Griesse reaction every day after transplantation both in a morning urine sample and in a sample from 24-hour collection over the preceding day and calculated to 1 mmol/l of urinary creatinine (U-Cr). The levels of U-NOx/U-Cr in patients with acute rejection over the past 2 days before its development were lower compared with those in patients with a normal course (p&#95;0.05). No difference was found between the groups of patients with cyclosporin-induced toxicity and a normal course. The levels of U-NOx were inversely correlated (p&#95;0.01) to the levels of serum creatinine (S-Cr), but did not correlate with the blood levels of cyclosporin A., Conclusions: The study demonstrated a decrease in urinary U-NOx production within the past 2 days before renal transplant rejection. The levels of U-NOx in patients with cyclosporin-induced toxicity remain unaltered. U-NOx/U-Cr could possibly become a non-invasive marker of rejection.

Background: Intercellular adhesion molecule-1 (ICAM-1) binds to leukocyte adhesion receptors LFA-1 and MAC-1, and mediates leukocyte adhesion to target structures. During acute rejection there is increased expression of ICAM-1 in vascular and tubulointestial cells, and consequently accumulation of inflammatory leukocytes. Soluble ICAM-1 (sICAM-1) is released from ICAM-1 expressing cells and excreted into the surrounding fluid. Increased serum sICAM-1 levels are found in patients with acute rejections of various allografts, and high urinary levels in steroid resistant acute kidney allograft rejection., Methods: Urinary excretion of sICAM-1 was measured by EIA in 136 kidney allograft recipients during the first 1-6 post transplant weeks: 30 patients developed acute rejection, and 106 patients had stable graft function. The molecular weight, binding to hyaluronan, and the origin of urinary sICAM-1 were studied., Results: We show that urinary sICAM-1 circulates as a monomer with a molecular weight between 50 and 100 kD. It binds to immobilized, but not to circulating hyaluronan. About one week after transplantation the mean sICAM-1/creatinine ratio (306 ng/mmol) in transplanted patients was higher than in the healthy controls (167 ng/mmol, P<0.01), and remained basically unchanged during the follow-up in patients with stable graft function, whereas it increased in patients developing rejection, being about 2.5-fold above the initial level a few days before rejection (P<0.01). Urinary sICAM-1 did not correlate with the urinary albumin, whereas in patients developing rejection it correlated with urinary IL-2R (r=0.5146, P<0.001), a marker of lymphocyte activation. In the urinary sediment of rejecting patients ICAM-1 was demonstrated in the tubular epithelial cells, and in the macrophages., Conclusions: Increased urinary excretion of sICAM-1 was demonstrated in kidney transplanted patients a few days before acute rejection. It seems to originate from activated macrophages and/or from the tubular epithelial cells. The fact that urinary sICAM-1 is not bound to hyaluronan or to leukocytes suggests that it is not able to compete with membrane-bound ICAM-1 for these bindings, but may do so for the binding of activated macrophages.

Background: Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation., Methods: We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status., Results: The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed., Conclusions: Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.

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.

Background: After transplantation, even if the graft starts functioning immediately, there are morphological and functional changes in tubular structures. In addition, acute allograft rejection causes damage in the tubular epithelium, tubular basement membrane, and intertubular connective tissue. It also affects the functional capacity of proximal tubular cells resulting in impaired reabsorption and thus increased urinary excretion of low molecular weight proteins., Methods: We present a double-antibody radioimmunoassay for determination of the concentration of alpha1-microglobulin (alpha1 M) in urine. It was used to measure urinary excretion of alpha1 M approximately once a week during the first 1-6 posttransplant weeks in 136 consecutive patients: 30 patients developing acute rejection (75 24-hr urine samples) and 106 patients with stable graft function (223 24-hr urine samples). The results are expressed as alpha1 M/creatinine ratios., Results: Approximately 8 days after transplantation the mean (+/-SD) urinary alpha1 M/creatinine ratio of all patients was 17.0+/-14.8 mg/mmol, being about the same both in patients with uncomplicated posttransplantation course (16.3+/-14.0 mg/mmol) and in those who later developed rejection (19.3+/-15.1 mg/mmol), but about 60-fold higher than in healthy controls (0.27+/-0.15 mg/mmol). At that time, when all patients were included there was a correlation (r=0.3465, P<0.001) between alpha1 M/creatinine ratio and duration of cold ischemia. Thereafter, during the second week alpha1 M/creatinine ratio decreased in 89% of patients with stable graft function, but only in 14% of patients who later developed rejection (P<0.001). On the contrary, a significant increase (P<0.01) of alpha1 M/creatinine ratio was observed 4 to 1 day before rejection in all 15 patients, who had urines collected at that time. At the end of the follow-up period, alpha1 M/creatinine ratio in patients with rejection was 3-fold compared with the nonrejecting patients, and 100-fold compared with the healthy controls., Conclusion: These results show that cadaveric transplantation results in impaired low molecular weight protein reabsorption, the degree of dysfunction relating to the duration of cold ischemia, and suggest that during the posttransplant weeks decreasing alpha1 M/creatinine ratio in consecutively collected urine samples indicates improved tubular function and in most cases rules out development of acute rejection.

The proteinuria in renal allograft recipients has been regarded as a sign of poor prognosis. The causes of post-transplant proteinuria include chronic rejection, chronic transplant glomerulopathy, glomerulonephritis (GN), acute rejection, and cyclosporine nephrotoxicity. Among them, chronic rejection is known to be most frequent. We analyzed the histopathologic findings of renal allograft biopsies in 197 Korean recipients with proteinuria. Among them, 26 patients developed proteinuria over 500 mg/d. All patients received baseline immunosuppression with cyclosporine. From 26 patients with post-transplant proteinuria, 29 biopsies were performed and their histologic diagnoses were immunoglobulin A nephropathy (IgAN) in 17, IgAN combined with chronic allograft nephropathy in 1, focal segmental glomerulosclerosis in 2, crescentic GN in 1, membranous GN in 1, diabetic nephropathy in 1, acute tubulointerstitial nephritis in 1, and chronic rejection in 3 biopsies. The remaining two biopsies showed nonspecific findings. The most common cause of post-transplant proteinuria was IgAN (62% of biopsies). The incidence of chronic rejection was relatively low and predominant cyclosporine-associated changes were not observed. In conclusion, our data suggest that the main causes of post-transplant proteinuria in Korea are primary glomerulonephritides rather than chronic rejection or cyclosporine nephrotoxicity, and the kidney allograft biopsies from patients with proteinuria should be handled as native kidney.

Background: Acute rejection and urinary tract infection (UTI) both increase nitric oxide synthase (NOS) activity in urine from renal transplant patients. Also, with rejection, a regulatory interplay between nitric oxide (NO) and cytokines has been suggested. Thus, measurement of the temporal changes of NOS products and cytokines in urine will provide a strategy for the diagnosis of acute rejection and for its differentiation from UTI., Methods: Soluble interleukins (ILs) and NOS-related products, cyclic GMP (cGMP), nitrate, and nitrite were measured in 192 urine samples consecutively collected from 13 patients within the first three months of transplantation. Sixty-seven additional urine specimens were collected randomly from 24 patients for follow-up analysis of the nitrate test., Results: Among patients who experienced rejection, the percentage (%) binding of IL-2 increased within the first five days (P = 0.0004) after transplantation and one to five days prior to the clinical diagnosis (dx) of rejection (P = 0.02). Tumor necrosis factor-alpha, IL-6, and IL-8 increased at the time of rejection dx (P < or = 0.01). With UTI, IL-2 (P = 0.01) decreased one to five days prior to dx, and IL-10 (P = 0.003) increased one to five days after dx. Although cGMP and nitrate are dependent variables, cGMP increased (P < or =0.0009) with both rejection and UTI, and nitrate increased (P = 0.0001) with rejection and decreased (P = 0.0001) with UTI. Prior to formal dx (1 to 5 days), urine nitrate clearly differentiated rejection (3004 to 7451 micromol/L) from UTI (90 to 885 micromol/L) and controls (1059 to 3235 micromol/L). The additional 67 urines demonstrated that the sensitivity of the nitrate test for rejection and UTI was 100%., Conclusions: In renal transplant patients, specific temporal changes in urine cytokine levels do occur with acute rejection and UTI, but urine nitrate levels are the most precise at differentiating rejection from UTI.

Acute rejection (AR) is the recipient's inflammatory response to the grafted organ. Within the graft-infiltrating cells, a high ratio of IL-6 producing cells can be found, indicating local IL-6 production. Therefore, in cases of kidney transplantation, urinary (u) IL-6 should be detectable. In order to establish the dynamics and diagnostic relevance, uIL-6 levels were determined daily by Quantikine IL-6 immunoassay (R & D Systems, Minneapolis, Minn.) in 101 kidney graft recipients (n = 1915 urine samples) during their post-transplant hospital stay. Immunosuppression consisted of azathioprine, steroids, cyclosporine and an intraoperative high-dose single antithymocyte globulin (ATG)-Fresenius bolus (9 mg/kg). In all the uncomplicated courses (n = 31) mean uIL-6 level was determined, after a post-transplant peak of 174 pg/ml, to be between 4 and 8 pg/ml. In contrast, delayed graft function (n = 16) was always associated with very high uIL-6 levels (> 200 pg/ml), dropping down only with commencement of graft function. Steroid-sensitive AR (n = 14) was consistently associated with significantly increasing uIL-6 levels prior to antirejection therapy (from 23 to 82 pg/ml). In cases of steroid-resistant AR, following antirejection therapy with methylprednisolone (5 days 5 mg/kg), there was no obvious trend towards normalization, indicating the persistence of inflammation (mean uIL-6 peak prior to OKT3 or ATG therapy: 99 pg/ml). In addition, AR-associated uIL-6 levels were found to be of much greater diagnostic relevance than AR-associated serum IL-6 levels. In bacterial urinary tract infections (n = 20), increased uIL-6 levels (peak 53 pg/ml) coincided with the commencement of antibiotic therapy. In mild cytomegalovirus diseases (n = 8), the development of leukocytopenia was associated with a slight increase of uIL-6 (peak 26 pg/ml), showing graft involvement. All increased uIL-6 values returned towards baseline after successful treatment. Thus, uIL-6 provides information about the intragraft inflammatory situation. Its determination is simple, expressive, non-invasive and can be recommended.

Our studies of protocol biopsy studies have shown that normal allograft histology can not be assumed by crude tests of renal function such as the serum creatinine concentration, and that there is a high prevalence of subclinical rejection in the first 6 months post-transplant (7, 13-17). The apparent ability of urine MR and IR spectra to reliably identify patients with normal allograft histology, if confirmed in a larger database, will preclude the need for a protocol biopsy in approximately 20-50% of patients. Conversely, finding urine MR or IR spectra characteristic of subclinical rejection would provide the opportunity for early treatment. The clear separation between patients with normal histology from those with subclinical rejection can be attributed to the use of the whole urine spectrum to develop the classifiers. Additional advantages of using MR or IR spectra of urine as a diagnostic tool compared to the biopsy include simplicity (i.e. no processing is required), low cost, rapid turnaround (i.e. < 15 minutes/sample), and, particularly, low risk, thus allowing for repetitive sampling. The ability to non-invasively diagnose acute inflammation in the kidney would be of great assistance in the post-transplant monitoring of renal transplant patients. Indeed, by following subclinical inflammation as detected in the MR/IR spectra it will be possible to tailor the intensity of the immunosuppression to the inflammatory status of the graft, thus minimising the risks of both insufficient and excessive immunosuppression. Furthermore, by following subclinical inflammation, as detected in the MR/IR spectra, it will be possible to test the hypothesis that subclinical rejection (i.e. persistence of its MR/IR spectral classifier) is a surrogate marker for the development of chronic rejection.

In this study, we investigated the relationship between excretion of urinary nitrate as a stable end-product of nitric oxide (NO) metabolism and hepatic allograft rejection. In experimental rat models, hepatic allograft rejection was associated with increased nitrate excretion with a peak on postoperative day 5. The severity of the hepatic allograft rejection was dependent on the increased urinary nitrate excretion. No significant increase in urinary nitrate excretion was observed in cases in which effective immunosuppression was achieved. Inducible nitric oxide synthase mRNA expression was upregulated parallel to interferon-gamma gene expression in the graft-infiltrating mononuclear cells and spleen cells from the recipients. In clinical cases, urinary nitrate excretion increased parallel to increased serum cytosolic enzymes that accompanied rejection. These results suggest that urinary nitrate excretion is a useful indicator for the surveillance of graft rejection and the monitoring of therapeutic effects of antirejection treatments.

Background: Previous studies have indicated that microchimerism is present in body tissues, peripheral blood, and plasma of recipients after organ transplantation. We hypothesize that donor-derived DNA may also be present in cell-free urine of renal transplant recipients and that the concentrations of urine DNA may be correlated with graft rejection., Methods: Thirty-one female patients who had renal transplantation were enrolled in the study. In women with male organ donors, the SRY gene on the Y chromosome was used as a marker for donor-derived DNA. Real-time quantitative PCR for the SRY and beta-globin genes was carried out on cell-free urinary DNA from these patients. Serial urine samples from a female renal transplant recipient undergoing an acute rejection episode were also collected and analyzed with the beta-globin quantitative PCR system., Results: SRY sequences were detected in the urine of 14 of 17 female patients with male organ donors. None of the 14 patients with female organ donors had detectable SRY sequences in urinary DNA. The median fractional concentration of donor-derived DNA was 8.7% (interquartile range, 1.9-26.4%). During the acute rejection episode, urinary concentrations of the beta-globin gene were markedly increased, with the concentrations returning rapidly to normal following antirejection treatment., Conclusions: Our results demonstrate that urinary DNA chimerism is present following renal transplantation. The measurement of urinary DNA using quantitative PCR may be useful for the diagnosis and monitoring of graft rejection.

Objective: In renal transplant patients having graft dysfunction, it is usually difficult to obtain the accurate diagnosis, such as acute rejection, acute tubular necrosis, infection, or cyclosporin nephrotoxicity. An accurate diagnosis can provide the proper treatment of these patients, thereby lessening the chance of kidney loss., Methods: A total of 42 patients were enrolled. By using the flow-cytometric technique, the white cell populations of urine in these patients were analyzed and linked to their clinical course. All patients underwent sonography-guided biopsy of the transplanted kidney with a definitive diagnosis., Results: When 10% lymphocytes and 15% granulocytes in urine were set as the cutoff point of a normal ratio threshold, the flow-cytometric analysis presented the highest sensitivity and the highest negative predictive rate for acute tubular necrosis. However, a lower sensitivity and positive predictive rate was found in acute rejection cases., Conclusions: Our results suggest that flow-cytometric analysis of the urinary cell population can be used as an adjunct in patient follow-up after kidney transplantation.

Background: Interleukin-1 (IL-1) is produced by activated monocytes/macrophages; highly increased amounts of IL-1 have been found in renal tissue in acute rejection of renal grafts. The endogenous inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1ra), is produced in many cells in response to the same stimulus as IL-1. There is some evidence that the balance between IL-1 and IL-1ra is important in the regulation of inflammatory responses. In many inflammatory diseases in both humans and animals, a high concentration of endogenous IL-1ra or administration of exogenous IL-1ra has been shown to relate to shorter recovery time or to reduced mortality., Methods: We measured the urinary excretion of IL-1ra and IL-1beta during the first 3-6 posttransplant weeks in 23 patients with acute rejection (69 24-hr urine samples) and in 17 patients with stable graft function (51 24-hr urine samples) and expressed the results as cytokine/creatinine ratios., Results: Within the follow-up time, patients with rejection had higher urinary IL-1beta/creatinine (ng/mmol) ratios (median 5.0 vs. 2.7; P<0.005), lower IL-1ra/creatinine (ng/mmol) ratios (median 18.1 vs. 34.2; P<0.1), and consequently lower IL-1ra/IL-1beta ratios (median 3.6 vs. 20.3, P<0.005), compared with patients without rejection. In rejecting patients, IL-1ra/creatinine was constantly low and decreased even further during acute rejection, whereas IL-1beta/creatinine ratios increased from a median prerejection value of 3.5 (range not measurable to 9.0) to a median value of 8.1 (P<0.0005) (range 1.6 to 18.3) during rejection., Conclusion: These results suggest that patients who produce high amounts of IL-1ra in relation to IL-1beta are less prone to acute allograft rejection than patients with low IL-1ra/IL-1beta ratios.

Unlabelled: The urinary excretion of 99mTc-mercaptotriacetylglycine (MAG3), like that of 131I-orthoiodohippurate (OIH), can be used to identify acute renal transplant rejection and measure its severity. This parameter is often quantitated as the excretory index (observed excretion/predicted excretion). A new method for predicting the urinary excretion of 99mTc-MAG3 is presented., Methods: The expected excretion was calculated from multisample plasma time-activity curves in 122 subjects, with correction for the first pass of the initial bolus. The resulting formula was tested prospectively against actual urine measurements in an additional 466 subjects., Results: Least-squares fitting led to the following equation: Predicted excretion = 0.79(1-exp(-0.0066CMAG3), with residual s.d. 0.06, where CMAG3 is MAG3 clearance in ml/min and the predicted excretion is expressed as a fraction of the administered dose. Tested prospectively in the additional 466 subjects, the s.d. was 0.09., Conclusion: A new formula to predict the urinary excretion of 99mTc-MAG3 has been developed and prospectively validated. Based on our data, the normal range for the excretory index using MAG3 is the same as that of 131I-OIH, 0.8-1.2.

Early diagnosis of rejection is a pivotal problem in renal transplantation. Recent advances in urinary cell analysis using flow cytometry are still burdened with difficulties concerning urine lymphocyte (UL) isolation. The analysis of lymphocytes washed out with the urine from the kidney transplant offers a tool to monitor noninvasively the intragraft immune response. However, the demand for optimal isolation of UL with high viability and good separation of other cell types has not, as yet, been met. The present study was undertaken to evaluate the optimal conditions for harvesting UL in order to perform adequate UL analysis by flow cytometry. We found that UL viability is mainly dependent on the time of urine harvesting. Low UL viability was caused by high urine osmolality due to high concentrations of urea and glucose. In contrast, high protein concentrations protected UL viability. Hence, the following algorithm of adequate UL isolation for flow cytometric analysis was established: (1) Collection of morning urine directly onto foetal calf serum (FCS: 30% v/v): (2) UL isolation within 2 h; (3) Erythrocyte lysis with subsequent two-step density gradient isolation of UL from residual erythrocytes, granulocytes (Ficoll-Isopaque, 1.077 g/cm3) and from uroepithelial cells (30% methylglucamine 3,5-diacetomido-2,4,6-triiodobenzoicum, 1.085 g/cmn3); (4) Flow cytometric analysis of UL using the 'live gate' setting in the area of blood lymphocyte cluster. Adequate UL isolation and special settings of the flow cytometer may provide a useful tool for early diagnosis and the noninvasive monitoring of renal transplant rejection.

Background: Despite marked improvements in the success of solid organ transplantation, a significant percentage of transplanted organs is lost due to recurrent episodes of acute cellular rejection. The mechanisms that govern allograft rejection likely include a complex regulatory network of multiple cytokines and growth factors., Design and Method: This study investigated the kidney gene (in situ hybridization) and protein (immunohistochemistry) expression and the urinary excretion rate of IL-6 and EGF in 29 renal transplant recipients: 16 with acute cellular rejection (AR) and 13 with acute tubular damage/cyclosporine toxicity (ATD)., Results: AR patients displayed a 4-fold increase of renal IL-6 expression, which localized chiefly to proximal tubular cells and monocytes/macrophages, whereas EGF signal was extremely weak or even absent. In ATD patients, EGF expression was markedly reduced, while IL-6 specific signal was unchanged. In all the patients examined the renal expression of IL-6 and EGF strictly correlated with their urinary excretion rate (r:0.459, P:0.001). Thus, urinary IL-6/EGF ratio was markedly increased in the former group (> 20-fold at day 1), where it paralleled the modifications of plasma creatinine over time (r:0.603, P < 0.0001), and was only slightly increased in the latter group (< 3-fold)., Conclusion: Kidney transplanted patients with acute cellular rejection or acute tubular damage/CyA nephrotoxicity exhibit a distinctly different pattern of intragraft expression of IL-6 and EGF, which is closely reflected by their rate of urinary excretion.

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A2 (TXA2) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA2 metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B2 (i-TXB2) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB2 was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 +/- 372 vs. 526 +/- 57 pg/mL). The urine i-TXB2 (704 +/- 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB2 were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 +/- 11 vs. 39 +/- 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB2 is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB2 may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.

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.

Previous investigations have shown that the determination of two acute-phase proteins in the urine, C-reactive protein (CRPu) and alpha2-macroglobulin (alpha2-MGu), allows a noninvasive diagnosis of acute renal graft dysfunction. A reliable differentiation between rejection and urinary tract infection can be made only when considering the C-reactive protein in serum and urine at the same time (CRPs:CRPu ratio). Therefore, a diagnostic procedure independent of parameters other than urinary proteins is needed. As granulocytes play only a minor role in graft rejection but are a common feature in urinary tract infection, we determined a marker of granulocytes (myeloperoxidase) in urine (MPOu). Eighty-nine renal transplant recipients were included in the study. In normal courses, CRPu, alpha2-MGu, and MPOu were within the normal range. In 15 cases of acute interstitial rejection, an increased excretion of CRPu and alpha2-MGu could be confirmed, but MPOu could not be detected. On the occasion of acute vascular rejection (n=6), with the exception of one case, MPOu could not be observed. The pattern of the three urinary proteins differed in urinary tract infections (n=40): MPOu could be detected in all cases, CRPu in 50% of cases, and alpha2-MGu in 73% of cases. In patients with cytomegalovirus infection (n=7), no MPOu, CRPu, or alpha2-MGu was found. In conclusion, the simultaneous measurement of the three proteins allows a complete, noninvasive, differential diagnostic procedure of renal graft dysfunction.

Background: The significance of noninvasive techniques to the early diagnosis of acute rejection in kidney transplants remains elusive. In this study, we examined whether an early posttransplant increase in serum- and urine-soluble interleukin (IL) 2 receptor (sIL-2R) and IL-6 levels predicted acute rejection., Methods: Sequential determinations of serum and urine sIL-2R and IL-6 were performed in the first 30 postoperative days in 40 renal transplant patients. Changes during the posttransplant period observed in 26 patients who had one or more episodes of acute rejection (group A) were compared with those recorded in 14 patients who did not experience acute rejection of their graft (group B)., Results: Serum sIL-2R was higher than normal in patients of groups A and B without statistical differences between the two groups. In the first 3 days after transplantation, urinary sIL-2R was higher than normal in group A but not in group B. Urinary sIL-2R at days 2 and 3 was significantly higher (P<0.05) in group A than in group B. In the first 5 days after transplantation, urinary IL-6 was persistently higher than normal in group A, whereas it progressively decreased to normal value on day 4 in group B. Sudden increases (doubling within 24 hr) in urine IL-6 preceded clinical diagnosis of acute rejection by a mean period of 2 days, with an 87% sensitivity and a 64% specificity, and also predicted recurrent rejection episodes., Conclusions: Sequential monitoring of urinary sIL-2R and IL-6 levels does allow very early diagnosis of rejection without invasive procedures. Specifically, high urinary sIL-2R in the first 5 posttransplant days identifies the subgroup of patients at risk. In the subsequent days, a sudden increase in urinary IL-6 occurs in those of the above patients who will indeed reject their graft.

In children, the early detection and accurate diagnosis of acute renal allograft rejection (AR) may be difficult. A delay in the diagnosis and treatment of AR might engender a poor outcome. Core needle biopsy is diagnostic but invasive. However, only a few less-invasive means to diagnose AR are sufficiently accurate to warrant their routine use. One such method is urine cytology. Investigators have demonstrated in more than 800 patients that urine cytology is simple and reliable for diagnosing graft failure. These observations may be important for children, for whom a non-invasive, accurate, and easily repeated indicator of AR may be particularly useful. While not a substitute for transplant biopsy, urine cytology may reduce diagnostic uncertainty and indicate the need for core needle sampling in a timely manner.

Mononuclear cell infiltration is a common histopathological feature of acute renal transplant rejection, in which it seems to play a key role in the pathogenesis of tubulointerstitial lesions. Monocyte chemotactic peptide-1 (MCP-1) is a specific chemotactic and activating factor for monocytes. Thus, the present study was aimed at evaluating MCP-1 gene and protein expression in renal biopsies of kidney transplant recipients with acute deterioration of graft function, and to correlate it with the extent of monocyte infiltration. We studied 20 kidney transplant recipients with acute graft dysfunction (13 with acute rejection, seven with acute tubular damage). MCP-1 gene and protein expression were analyzed by in situ hybridization and immunohistochemistry, respectively. CD68-positive cells were identified as monocytes. CD68-positive cell number and MCP-1 expression were quantified by a computerized image analysis system. MCP-1 gene expression, undetectable in normal human kidneys, was strikingly increased in patients with acute rejection. The chemokine localized mainly to the proximal tubular cells and to mononuclear-infiltrating cells. In patients with acute tubular damage, the MCP-1 expression, even if higher than in controls, was significantly lower than in acute rejection. The expression of the chemokine strictly correlated with the number of infiltrating monocytes (r=0.87, P<0.05). Moreover, we measured MCP-1 urinary excretion by ELISA, in eight normal subjects (36+/-16 pg/mg urine creatinine), in 13 clinically stable transplant recipients (33+/-9 pg/mg, ns vs. normal patients), in 12 transplant recipients with acute rejection (250+/-46 pg/mg, P<0.01 vs. normal patients), and in five transplant recipients with acute tubular damage (97+/-33 pg/mg, P<0.05 vs. controls and patients with acute rejection). Urinary MCP-1 excretion directly correlated with renal MCP-1 gene expression (r=0.65, P=0.05). Finally, we observed a significant reduction in MCP-1 urine levels in patients with acute rejection, who responded to the antirejection treatment. In conclusion, our data suggest that MCP-1 may play a critical role in modulating monocyte influx and consequent tubulointerstitial damage in acute rejection. Therefore, an increase in urinary MCP-1 excretion may represent an early signal of ongoing acute graft rejection.

A reliable, noninvasive indicator of pancreatic allograft rejection is urgently needed. In this study, serum (S), plasma (P), and urine (U) levels of pancreas-specific protein (P-PASP, U-PASP), neopterin (S-NEOP, U-NEOP), amylase (U-AMYL), and amyloid A (SAA) were measured daily in ten type I diabetic patients following simultaneous pancreas and kidney transplantation (SPK). Rejection episodes occurred in three isolated pancreas, nine isolated kidney, and five simultaneous pancreas and kidney transplants. In the case of the eight pancreas rejections, SAA was the rejection marker with the highest diagnostic accuracy (94%). Using P-PASP and U-PASP, an accuracy of 81% and 79%, respectively, was achieved. During viral infections, U-NEOP levels increased to a maximum level of 1904 mumol/mol creatinine, whereas during bacterial infections, SAA levels increased to a maximum value of 43 mg/dl. SAA, measured for the first time in SPK, appears to be a valuable rejection parameter. In combination with U-NEOP and U-AMYL, a differential diagnosis between rejection, bacterial infection, and viral infection was possible. Neither U-PASP nor P-PASP monitoring led to a significant improvement in the results.

Acute renal allograft rejection continues to have a negative effect on graft survival despite a better understanding of the molecular basis of renal allograft rejection. Nitric oxide (NO) has important biological functions in cell defense and injury and some evidence exists that it may act as an immunomodulator in allograft transplantation. To determine if NO has any role in acute renal allograft rejection in pediatric patients, acute rejection episodes in pediatric renal transplant recipients were evaluated. Four out of eleven patients who received a renal allograft in 1995 at Children's Kidney Center at The Children's Hospital of Buffalo had eight episodes of acute rejection. One patient received a living-related and three received cadaveric grafts. Stable metabolites of NO (NO-2 + NO-3 = NOx) were measured in the serum and urine samples of the patients daily. Serum levels of NO did not change significantly during acute rejection episodes. Urinary NOx levels decreased by 73+/-9% of the baseline values during episodes of acute rejection: mean +/-SE urinary nitric oxide/creatinine ratio (NOx/Cr) of 0.17+/-0.05 at baseline vs. 0.05+/-0.01 during rejection (P=0.02). Successful treatment of acute rejection by administration of high dose i.v. steroids or OKT-3 induced acute rises in the urinary NO levels to baseline values: NO/Cr = 0.17+/-0.04 (mean +/-SE). We conclude that urinary NO excretion decreases significantly during acute renal allograft rejection and that NOx concentration in the urine increases in response to successful antirejection therapy.

Acute allograft rejection in patients after cardiac transplantation is associated with intense infiltration of inflammatory cells. It has been shown in animal studies that this inflammatory process is accompanied by an induction of nitric oxide syndrome (NOS) enzyme within the grafted organ. We sought to test the hypothesis that acute rejection in cardiac transplanted patients is associated with an increased production of nitric oxide. The radical nitric oxide is unstable in blood and oxidized to nitrite and nitrate; nitrate is secreted by the kidneys. Therefore, nitric oxide production was indirectly assessed by measuring urinary nitrate excretion. During a period of 1 yr, 86 consecutive patients after cardiac transplantation underwent one or more right heart biopsies (total 194) in order to assess for acute graft rejection. At the time of biopsies, urine was collected under sterile conditions. Urinary nitrate excretion was expressed as the ratio of nitrate to creatinine concentration in the urine (mumol/ mmol). Urinary nitrate excretion was 99.7 +/- 9.2 (mean +/- SEM) for patients without evidence of rejection, and tended to increase for patients with moderate/severe rejection (128 +/- 15.1). In a subgroup of patients who underwent several biopsies and showed different degrees of rejection (7 patients, 48 biopsies), urinary nitrate excretion significantly increased by about 99% during episodes of acute rejection. It is concluded that urinary nitrate excretion appears to be elevated in a subgroup of cardiac transplanted patients during episodes of acute rejection. Results are consistent with the hypothesis that acute graft rejection stimulates the production/release of nitric oxide. Because of a large inter- and intraindividual variation in urinary nitrate excretion, this parameter appears not to be suitable for diagnosis of acute rejection in cardiac transplanted patients.

Background: Acute rejection is characterized histologically by infiltration of the interstitium by mononuclear cells. Monocyte chemoattractant protein 1 (MCP-1) has recently been identified as a monocyte chemotactic factor. This study examined the possible role of MCP-1 in renal transplantation., Methods: The concentration of MCP-1 in urine and serum of 19 renal transplant patients was investigated using an inhibition radioimmunoassay. The patients were divided into a non-rejection (NRj) and a rejection (Rj) group. Normal healthy volunteers were included as controls. Immunoperoxidase staining for MCP-1 and CD14, as a marker for macrophages, was performed in renal biopsies of transplant patients with rejection and six biopsies from histologically normal kidneys, as controls. The size of urinary MCP-1 was determined by gel filtration chromatography and in a number of fractions assessed for monocyte chemotactic activity using a modified Boyden chamber assay., Results: Urinary excretion of MCP-1 in the Rj group ranged between 250 ng/mmol Cr and 3148 ng/mmol Cr with a median of 612 ng/mmol Cr. This is significantly higher than the results in the NRj group, ranging between 47 ng/mmol Cr and 288 ng/mmol Cr with a median of 229 ng/mmol Cr. In the normal control group, urinary MCP-1 levels ranged between 38 ng/mmol Cr and 74 ng/mmol Cr with a median of 50 ng/mmol Cr. The fractional excretion of MCP-1, calculated on the basis of MCP-1 and creatinine clearances, was found also to be significantly higher in the Rj group as compared to the NRj group. However, there was no significant difference in the serum levels of MCP-1 between the Rj, NRj, and normal control group. The intensity of MCP-1 staining in tubular epithelial cells and the degree of CD14+ cells in the interstitium was significantly higher in renal allograft biopsies than in the normal kidneys. In addition, MCP-1 isolated from urine of renal transplant patients with rejection was filtered with apparent molecular weight of 13 kDa and 11 kDa. Both sizes are chemotactically active for monocytes., Conclusions: These data suggest that urinary excretion of MCP-1 can be used as a marker for the episodes of acute rejection. The increase of urinary excretion of MCP-1 most likely is the result of local production by tubular epithelia cells. MCP-1 produced locally may, at least in part, be responsible for the influx of macrophages into the interstitium during rejection.

In recent studies, it has been demonstrated that strict dietary protein restriction has a beneficial effect on renal transplant patients who show chronic rejection, or transplant fibrosis respectively; however, the protein intake in those investigations usually has been below 0.6 g/kg day, and such a strong restriction may be associated with both a negative nitrogen balance, and low patient compliance. Our study was therefore undertaken to investigate whether the same beneficial effect could be attained with a more moderate dietary protein restriction in renal transplant recipients. In a randomized cross-over study, 14 patients with biopsy-proven transplant fibrosis received a mildly protein restricted diet (0.7 g/kg/day), and a normal protein diet (1.2 g/kg/day) respectively during two 3-week periods. In the patients undergoing moderate protein restriction, a significant reduction in urinary albumin, and total protein excretion, as well as a decrease in albumin/creatinine ratio was observed at the end of the 3-week period when compared to the patients on normal protein diet (p < 0.05). The 51Cr-EDTA-clearance did not differ at the end of each of these dietary periods. In contrast to earlier studies with lower protein intake, the moderate protein restriction in our investigation was not associated with a decrease in serum proteins. In conclusion, a mildly restricted protein intake has also proved effective in significantly reducing the urinary protein excretion in patients with renal transplant fibrosis, yet, without causing decreasing serumprotein-concentrations, which are a sign for a negative nitrogen balance.

Thanks to the use of highly sensitive assays for specific proteins in the urine, it is now possible to obtain, non-invasively and reproducibly, relevant differential diagnostic information when rejection is suspected following renal transplantation. The determination of C-reactive protein and alpha-2 macroglobulin, for example, makes it possible to decide whether a bacterial infection is present or not. Certain constellations of other proteins in the urine permit conclusions to be drawn, for example, as to the presence of CMV infection, the nature of the rejection, transplant glomerulopathy or cyclosporine toxicity. Follow-up studies with the aim of checking the success of treatment, are possible.

In 13 supine kidney graft recipients in the late post-transplant period the circadian rhythms of urinary cortisol and aldosterone excretion were studied. An inversion of the circadian rhythm of urinary cortisol excretion was found, with acrophase occuring in the night time. Plasma cortisol level, measured in 6 h intervals, attained the maximal mean value at 24 h. Urinary aldosterone excretion in most of the patients demonstrated also an inversed circadian rhythm with acrophase occuring from 21 to 03 h. In 6 patients the 24 h urinary aldosterone excretion was above the upper limit for healthy persons. The peak plasma aldosterone level was found at 24 h; it greatly exceeded the upper limit of aldosterone level in healthy supine persons. In 5 out of 13 patients the plasma renin activity (PRA) was above the upper limit of normal for supine subjects. The maximal mean PRA level was found at 24 h. The study in kidney graft recipients has established an inversed circadian rhythm of urinary cortisol and aldosterone excretion, probably related to the immunosuppressive treatment and reduced kidney function.

The proximal portion of the human kidney tubular system contains the alpha form, while the distal portion contains the pi form of glutathione transferase. These cytoplasmic proteins are released into the urine under pathological conditions, and an ELISA procedure has been developed for their quantitation. Optimal conditions with respect to concentrations of antibody and antigen and incubation times were determined. The procedure developed can detect as little as 0.5 ng enzyme per ml urine, even in the presence of high concentrations of other proteins. No cross reaction between these two isoenzymes or with a number of other proteins in the urine was observed. Antibodies interacted with these antigens in urine samples in the same manner as they interacted with the purified proteins. Storage of samples without loss of antigen required the presence of low concentrations of detergent, such as Tween 20, which both stabilized the enzymes and prevented their adsorption to the walls of the plastic tubes. The results indicate that increased urinary levels of these two enzyme proteins, as determined by the ELISA procedure, are useful markers for tubular damage.