In vitro cytokine profiles and their relevance to rejection following renal transplantation.

Graft rejection remains an important cause of renal allograft failure, despite improvements in immunosuppression and HLA typing. Although HLA matching is beneficial, ensuring an exact match it is often impractical. Thus, a reliable in vitro method for quantitating and qualitating alloreactivity is an important goal. In this study, we measured in vitro the cytokine secretion profiles of mononuclear cells from patients prior to renal transplantation by stimulating with anti-CD3 monoclonal antibody and suppressing with cyclosporine. Mononuclear cells from patients who subsequently developed acute cellular rejection secreted higher mean levels of interleukin (IL)-2 and gamma-interferon (IFN-gamma) than those from patients who had no rejection episodes. IFN-gamma secretion was significantly associated with rejection (P = 0.002), whereas IL-2 secretion did not quite reach statistical significance. There was no significant correlation between IL-4 levels and rejection. Although cyclosporine suppressed the secretion of both IL-2 and IFN-gamma, there was no difference in sensitivity to suppression between rejectors and nonrejectors. These results further emphasize the importance of the TH1 lymphocyte subset in renal allograft rejection. The IFN-gamma secretory capacity of alloreactive T cells may influence the outcome of a renal allograft by (1) activating graft infiltrating macrophages and/or (2) up-regulating HLA molecules on the graft.

An automated enzymatic inulin assay, capable of full sinistrin hydrolysis.

Renal inulin clearance remains the standard by which other methods of measuring glomerular filtration rate are judged. A fully automated enzymatic assay capable of use with linear configuration inulin was recently published (Summerfield AL, et al. Clin Chem 1993; 39:2333-7). Sinistrin, a readily soluble preparation of polyfructan with side branching, is more suitable for clinical use and far more widely used in Europe. By modifying the incubation phase of samples with inulinase, incorporating a kinetic modification to the method of fructose analysis, and increasing the buffer strengths, we report a fully automated system, with minimal sample prehandling capable of complete sinistrin hydrolysis, and adapted for use on the Cobas Mira.

Urinary protein excretion and the diagnosis of graft rejection or renal dysfunction in renal transplant patients.

Urinary proteins have been found to be a sensitive marker of renal damage caused by nephrotoxic agents. An electrophoretic method was used to investigate the potential value of the pattern of urinary protein excretion in 14 cyclosporin-treated renal transplant patients, to differentiate between graft rejection episodes and other causes of renal dysfunction. Urinary protein excretion consistent with renal damage was observed in all of the patients studied, with no marked differences between those with signs of graft rejection, those with renal dysfunction, or those with stable renal function.

Cellular versus vascular rejection in transplant kidneys. Correlation of radionuclide and Doppler studies with histology.

The presence of two distinct subtypes of renal allograft rejection are well documented by histological studies. The differentiation between vascular rejection (VR) and cellular rejection (CR) is essential for proper management by avoiding the need for unnecessary and potentially harmful immunosuppressive treatment of VR. A histological pattern with features that are similar and confusable with some cases of rejection may be seen in cyclosporin A toxicity (CyT). To evaluate the efficiency of Guy's perfusion index (GPI) and the Doppler pulsatility index (DPI) in differentiating these two histological subtypes, a prospective study was designed in which a total of 140 radionuclide tests and 133 ultrasounds scans performed on the same day on 58 patients during the first 3 months post-transplant were analysed, and the results correlated with the histological findings of 84 renal biopsies. Results show that the GPI had a sensitivity of 86.5% and a specificity of 94% in differentiating VR and CyT from CR, while the DPI had values of 83% and 69%, respectively. Chi-squared analysis showed a higher significant association between the GPI and histology (P < 0.0001) compared to that of the DPI and histology (P < 0.005), while Youden's index (J) showed a significant difference (P < 0.05) between GPI and DPI. It is concluded that GPI is more sensitive and specific than DPI in differentiating transplants that are well perfused from those with poor perfusion (VR and CyT).

Peritoneal dialysis catheters: a comparison between percutaneous and conventional surgical placement techniques.

Percutaneous peritoneal dialysis catheter (PDC) placement is a well-tolerated, rapidly performed side-room procedure that allows the rapid initiation of dialysis without the delay imposed in co-ordinating a surgeon, theatre time, and theatre staff. We retrospectively reviewed the clinical outcome of 230 PDC inserted over a 30-month period. Fifty were placed percutaneously (group P) and 180 were placed using conventional surgical techniques, 107 in patients commencing CAPD (group A) and 73 in patients commencing CAPD (group A) and 73 in patients previously established on CAPD (group B). Total experience accumulated was 2563 patient months: 270 patient months group P, 1381 patient months group A, 912 patient months group B. Percutaneous PDC insertion was non-elective, and reserved for patients unfit for general anaesthesia or haemodialysis. Group P patients were older (P < 0.001) and had increased early mortality (P < 0.005) due to underlying pathology. Death and early mechanical failure contributed to a shorter mean duration of catheter use in group P (9.0 +/- 2.3 months compared to 15.3 +/- 9.6 months group A and 17.3 +/- 9.7 group B) (P < 0.05). The peritonitis rate was similar in group P (1 per 6.75 patient months) and group B (1 per 7.4 patient months) but significantly lower in group A (1 per 15.7 patient months) (P < 0.01). We conclude that percutaneous PDC placement provides a safe, reliable access for peritoneal dialysis and is especially suitable for ill patients who would not tolerate general anaesthesia.

Partial correction of dialysis-associated anaemia does not reduce erythropoietin dose or the incidence of side effects.

We examined the hypothesis that administering epoetin to maintain a lower target haemoglobin (Hb) results in a reduced side effect profile and a lower maintenance epoetin dose. We report a prospective study of 14 haemodialysis patients assessing epoetin dose efficiency and side effect profile of partially correcting dialysis-associated anaemia. Initial Hb was 6.2 +/- 0.6 g/dl (mean +/- 1 SD). Intravenous epoetin was commenced at 120 IU/kg/week in 3 divided doses and titrated to achieve a target Hb of 8 g/dl. Follow-up was 24 weeks. The final Hb was 8.7 +/- 0.8 g/dl. The peak epoetin dose was 196 +/- 86 IU/kg/week with a maintenance dose of 141 +/- 71 IU/kg/week. Therapy was associated with hypertension--5 patients (32%); seizures--1 patient (6%) (withdrawn from therapy), and temporary iron deficiency--4 patients (35%). Iron deficiency was corrected with oral therapy. There was 1 treatment failure. Comparable conventional regimens use 100-200 IU/kg to maintain the Hb at 10-13 g/dl and have a similar incidence of side effects. We concluded that reducing the target Hb in order to decrease epoetin requirements is not justified as it offers no benefit over conventional Hb targets in terms of dose requirements or side effects.

NMR spectroscopy as a novel approach to the monitoring of renal transplant function.

High field 1H NMR spectroscopy was used for the rapid multicomponent analysis of low molecular wt compounds in urine in order to investigate the patterns of metabolic changes associated with early renal allograft dysfunction. Urine samples were collected daily for 14 days from 33 patients who underwent primary renal allograft transplantation, and analyzed by 500 and/or 600 MHz 1H NMR spectroscopy. All patients received 20 mg prednisolone and 5 mg/kg b.d. oral cyclosporin A (CsA) solution. In this study no patient showed clinical or histopathological evidence of CsA nephrotoxicity. For each patient the NMR-generated metabolite data were correlated with the clinical observations, graft biopsy pathology, and data from conventional laboratory techniques for assessing renal function. The NMR spectra of urine from patients with immediate functioning grafts were similar with respect to their patterns of amino acids, organic acids and organic amines, whereas the patients with delayed or non-functioning grafts showed significantly different metabolite excretion patterns. In longitudinal studies on individual patients there were increased urinary levels of trimethylamine-N-oxide (TMAO), dimethylamine (DMA), lactate, acetate, succinate, glycine and alanine during episodes of graft dysfunction. However, only the urinary concentration of TMAO was statistically significantly higher (P < 0.025) in the urine collected from patients during episodes of graft dysfunction (410 +/- 102 microM TMAO/mM creatinine) than in patients with good graft function (91 +/- 18 microM TMAO/mM creatinine) or healthy control subjects (100 +/- 50 microM TMAO/mM creatinine). These findings suggest that graft dysfunction is associated with damage to the renal medulla which causes the release of TMAO into the urine from the damaged renal medullary cells. This provides a possible novel urinary marker for post-transplant graft dysfunction. This study shows that NMR spectroscopy of biofluids, when used in combination with conventional laboratory techniques, is a valuable aid to renal transplant monitoring.

Carbamylated haemoglobin in normal, diabetic and uraemic patients.

Carbamylated haemoglobin arises from the non-enzymic modification of haemoglobin monomers by isocyanate derived from the spontaneous dissociation of urea. We measured carbamylated haemoglobin by high performance liquid chromatography in healthy subjects, non-uraemic hospital patients, diabetics, and different groups of uraemic patients. Carbamylated haemoglobin levels were found to be raised in uraemic subjects, but were independent of age, sex, glycaemic state and haemodialysis procedure. There was no significant difference in carbamylated haemoglobin levels between two groups of patients having different modes of dialysis treatment, probably indicating a similar degree of uraemic exposure in these patients.

Carbamylated haemoglobin, urea kinetic modelling and adequacy of dialysis in haemodialysis patients.

Urea kinetic modelling (UKM) has increasingly been used for assessing adequacy of dialysis and protein nutritional status of dialysis patients. Using a precise HPLC method we developed, we measured carbamylated haemoglobin (CarHb) values in 20 stable twice-weekly dialysed patients and attempted to correlate their CarHb values with their UKM-derived indices. Based on these indices, 11 patients were found to have been adequately dialysed with sufficient protein intake, three patients were adequately dialysed but malnourished and six patients were under-dialysed. Estimated dietary protein intake correlated poorly with calculated daily protein catabolic rate in our patients. CarHb values were found to correlate strongly with the time-averaged urea concentrations, suggesting that CarHb might be a time-integrated urea-derived index. Those adequately dialysed patients have a mean (SD) CarHb value of 142 (29) micrograms CV/gHb against the underdialysed patients, 197 (30) micrograms CV/gHb (t-test, P = 0.002). We suggest that a CarHb value less than 175 micrograms CV/gHb may represent satisfactory uraemic exposure, whereas CarHb value greater than 175 micrograms CV/gHb is undesirable.

Determination of carbamylated hemoglobin by high-performance liquid chromatography.

We have developed an HPLC method for measuring carbamylated hemoglobin (CarHb), based on the quantification of valine hydantoin formed from the released NH2-terminal carbamyl valine residue after acid hydrolysis of hemoglobin. In uremia, CarHb is produced by nonenzymatic post-translational modification of the terminal amino group of hemoglobin monomers by isocyanic acid, derived from the spontaneous dissociation of urea. We measured CarHb in 25 nonuremic control subjects, 24 nonuremic diabetic subjects, and 30 patients with stable chronic renal failure. There was no significant difference between the controls and diabetic patients, their mean (SD) CarHb values being 41 (11.5) and 38 (10.8) micrograms of carbamyl valine per gram of hemoglobin (microgram CV/gHb), respectively. Mean (SD) CarHb values in the uremic patients were much greater, 164 (87.7) microgram CV/gHb. There was significant correlation between the concentrations of CarHb and plasma urea in the uremic subjects. Thus CarHb provides a urea-derived index of chronic uremia.

Acute renal failure following accidental cutaneous absorption of phenol: application of NMR urinalysis to monitor the disease process.

An unusual case of acute renal failure is reported following accidental cutaneous absorption of phenol and exposure to dichloromethane. Renal function during the onset of the nephrotoxic episode and the subsequent recovery period was monitored using a combination of standard clinical biochemical techniques and high resolution 1H-NMR urinalysis. The initial urine biochemical patterns (up to 2 weeks following exposure) showed amino aciduria, glycosuria and lactic aciduria consistent with renal cortical necrosis. There followed a period of polyuria revealing a biochemical pattern (succinic aciduria, dimethylaminuria and N,N-dimethylglycinuria) consistent with renal papillary damage. Haemodialysis was required for a period of 3 weeks and the patient was discharged 42 days after admission to hospital when renal function was normal by standard clinical chemistry criteria (urea, potassium, sodium, creatinine, calcium, phosphate, urine glucose and protein). 1H-NMR spectroscopic urinalysis revealed residual renal biochemical abnormalities consistent with renal papillary damage that were not detected by conventional analytical techniques. One year after the incident the patient is still polyuric, passing up to 3 l of urine a day.

Caecal perforation in a renal transplant patient with disseminated histoplasmosis.

A renal transplant patient developed a fatal caecal perforation after Histoplasma capsulatum infection acquired abroad. Disseminated histoplasmosis is an uncommon fungal infection, usually seen in patients with impaired immunity. The diagnosis should be considered in immunosuppressed patients who develop prolonged fever or whose health deteriorates unexpectedly after travelling overseas. The infection is endemic in parts of the United States of America but occurs all over the world. Rapid diagnosis is often possible by histological examination of infected tissues. Treatment if started early may lead to recovery, but if it is not treated it is usually fatal.

Isolation of organisms in CAPD peritonitis: use of nutrient broth cultures and Bactec blood culture media.

Cultures of the first cloudy dialysates from 51 consecutive episodes of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis were carried out using concentrated nutrient broth and Bactec blood culture media in addition to primary culture on solid media. Thirty-seven episodes (73%) were positive on solid media, and 42 episodes (82%) were positive by both nutrient broth and Bactec methods. The value of Gram-stained smears and WBC counts on dialysates is discussed.