Jürgen E. Scherberich

Elevated levels of soluble CD14 in serum of patients with systemic lupus erythematosus

A soluble form of CD14 (sCD14) was assessed with an ELISA assay in the serum of the following three clinical groups: 35 patients with an inactive phase of systemic lupus erythematosus (SLE). 17 patients with SLE relapses, and 65 normal healthy volunteers, Increased levels of sCD14 were observed in all patients suffering from SLE compared with normal controls. In addition, patients with active SLE revealed higher serum concentrations of sCD14 (median 6–9 mg/l) than patients under remission (4.1 mg/l; P< 0.0001). Serum values of sCD14 correlated neither with the number of peripheral blood monocytes bearing the CD 14 membrane antigen, nor with serum concentrations of IL‐1β. Serum sCD14 was compared with other clinical parameters used to monitor the clinical course of patients with SLE, among them complement C3. anti‐dsDNA antibodies and soluble IL‐2 receptor (sIL‐2R). A good correlation emerged between sCD14 and C3 as well as sIL‐2R concentrations. but sCD14 and anti‐dsDNA titres disclosed no significant correlation in both groups of patients with SLE. Serial studies in patients with severe SLE showed that serum sCD14 closely parallels the clinical course as defined by an activity score. Our data suggest that serum sCD14 represents a promising parameter lo monitor disease activity in patients with SLE.

Biochemical and immunological studies on isolated brush border membranes of human kidney cortex and their membrane surface proteins.

Abstract A subfraction rich in brush border (BB) membranes was obtained by differential centrifugation of plasme membranes (PM) of human kidney cortex. In addition to alkaline phosphatase, the usual BB marker enzyme, an alanine specific aminopeptidase (AAP) and a gamma-glutamyltranspeptidase (g-GTP) were associated with this membrane fraction. The three enzymes are considered specific markers for the brush border region of the proximal tubules. Among the proteins released from the BB membranes by proteolytic treatment these three enzymes were found. The results indicate that AAP and g-GTP are components of the outer membrane surface. Antisera prepared against PM reacted in several immunological techniques with soluble proteins (including the three BB marker enzymes) released from the BB membranes by digestion with papain, as well as with those found in the urine of patients with kidney diseases. AAP from both sources (papain digest and urine) were immunologically indistinguishable. The molecular weights and some other biochemical parameters of the three enzymes were determined and compared with those reported in the literature. It is suggested that the methods and systems described might be of use for further studies on the structure and function of the membranes of kidney proximal tubules and are of potential value for diagnostic purposes.

Haemodialysis monocytopenia: differential sequestration kinetics of CD14+CD16+ and CD14++ blood monocyte subsets.

In peripheral blood the majority of circulating monocytes present a CD14highCD16− (CD14++) phenotype, while a subpopulation shows a CD14lowCD16+ (CD14+CD16+) surface expression. During haemodialysis (HD) using cellulosic membranes transient leukopenia occurs. In contrast, synthetic biocompatible membranes do not induce this effect. We compared the sequestration kinetics for the CD14+CD16+ and CD14++ monocyte subsets during haemodialysis using biocompatible dialysers. Significant monocytopenia, as measured by the leucocyte count, occurred only during the first 30 min. However, remarkable differences were observed between the different monocyte subsets. CD14++ monocyte numbers dropped to 77 ± 13% of the predialysis level after 15 min, increasing to ≥ 93% after 60 min. In contrast, the CD14+CD16+ subset decreased to 33 ± 15% at 30 min and remained suppressed for the course of dialysis (67 ± 11% at 240 min). Approximately 6 h after the end of HD the CD14+CD16+ cells returned to basal levels. Interestingly, the CD14+CD16+ monocytes did not show rebound monocytosis while a slight monocytosis of CD14++ monocytes was occasionally observed during HD. A decline in CD11c surface density paralleled the sequestration of CD14+CD16+ monocytes. Basal surface densities of important adhesion receptors differed significantly between the CD14+CD16+ and CD14++ subsets. In conclusion, during HD the CD14+CD16+ subset revealed different sequestration kinetics, with a more pronounced and longer disappearance from the blood circulation, compared with CD14++ monocytes. This sequestration kinetics may be due to a distinct surface expression of major adhesion receptors which facilitate leucocyte–leucocyte, as well as leucocyte–endothelial, interactions.

Plasma Uromodulin Correlates With Kidney Function and Identifies Early Stages in Chronic Kidney Disease Patients

AbstractUromodulin, released from tubular cells of the ascending limb into the blood, may be associated with kidney function. This work studies the relevance of plasma uromodulin as a biomarker for kidney function in an observational cohort of chronic kidney disease (CKD) patients and subjects without CKD (CKD stage 0). It should be further evaluated if uromodulin allows the identification of early CKD stages.Plasma uromodulin, serum creatinine, cystatin C, blood-urea-nitrogen (BUN) concentrations, and estimated glomerular filtration rate (eGFR CKD-EPIcrea-cystatin) were assessed in 426 individuals of whom 71 were CKD stage 0 and 355 had CKD. Besides descriptive statistics, univariate correlations between uromodulin and biomarkers/eGFR were calculated using Pearson-correlation coefficient. Multiple linear regression modeling was applied to establish the association between uromodulin and eGFR adjusted for demographic parameters and pharmacologic treatment. Receiver-operating-characteristic (ROC) analysis adjusted for demographic parameters was performed to test if uromodulin allows differentiation of subjects with CKD stage 0 and CKD stage I.Mean uromodulin plasma levels were 85.7 ± 60.5 ng/mL for all CKD stages combined. Uromodulin was correlated with all biomarkers/eGFR in univariate analysis (eGFR: r = 0.80, creatinine: r = −0.76, BUN: r = −0.72, and cystatin C: r = −0.79). Multiple linear regression modeling showed significant association between uromodulin and eGFR (coefficient estimate &bgr; = 0.696, 95% confidence interval [CI] 0.603–0.719, P < 0.001). In ROC analysis uromodulin was the only parameter that significantly improved a model containing demographic parameters to differentiate between CKD 0° and I° (area under the curve [AUC] 0.831, 95% CI 0.746–0.915, P = 0.008) compared to creatinine, cystatin C, BUN, and eGFR (AUC for creatinine: 0.722, P = 0.056, cystatin C: 0.668, P = 0.418, BUN: 0.653, P = 0.811, and eGFR: 0.634, P = 0.823).Plasma uromodulin serves as a robust biomarker for kidney function and uniquely allows the identification of early stages of CKD. As a marker of tubular secretion it might represent remaining nephron mass and therefore intrinsic “kidney function” rather than just glomerular filtration, the latter only being of limited value to represent kidney function as a whole. It therefore gives substantial information on the renal situation in addition to glomerular filtration and potentially solves the problem of creatinine-blind range of CKD, in which kidney impairment often remains undetected.

Comparative biochemical and immunological studies on gamma-glutamyltransferases from human kidney and renal cell carcinoma applying monoclonal antibodies

We have purified gamma-glutamyltransferases (GGT) from human kidneys and renal cell carcinomas, and fractionated them according to different lectin-binding properties of the isoenzymes. Native polyacrylamide gel electrophoresis and isoelectric focusing revealed different GGT-bands (even after desialylation) not only among kidney and renal carcinoma, but also among Con A-affine tumor fractions separated by ion-exchange chromatography. Mr of native GGTs were between 106 to 161 kDa, the pI ranged from pH 3 to 4 (pH 5 to 6 after desialylation). Monoclonal antibodies to GGT were produced. One of these, of IgG1 class and designed 138H11, recognizes human kidney GGT and, in addition, GGT from renal cell carcinomas and liver carcinomas. The specificity of mAb 138H11 for GGT was confirmed by Western blotting, by immunohistochemistry and by immunoprecipitation. The potential usefulness of mAb 138H11 in monitoring renal cancer patients and in identification of renal cancer metastases is currently being studied.

Differential diagnosis of histogenetically distinct human epithelial renal tumours with a monoclonal antibody against γ-glutamyltransferase

SummaryThe localization of membrane-bound γ-glutamyltransferase with monoclonal antibody (mAb) 138H11 proved to be of value for differential diagnosis of renal cancer, since it correlated with the histogenetic profile of human epithelial renal tumours. Immunoreactive γ-glutamyltransferase was located in the proximal tubule in all normal human kidneys (15/15) examined thus far by both ultrastructural and immunohistochemical techniques. From 68 epithelial renal cancers tested, 31/31 clear-cell carcinomas and 15/16 chromophilic carcinomas expressed the target epitope of mAb 138H11. In contrast, 0/11 oncocytomas, 0/9 chromophobic carcinomas, and 0/1 Duct-Bellini carcinoma were immunoreactive. These results support a model of histogenesis and classification of epithelial renal tumours, according to which clear-cell and chromophilic renal carcinomas originate from transformed proximal tubule cells, whereas oncocytomas, chromophilic and Duct-Bellini carcinomas originate from cells of the collecting duct.

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography.

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.

Isolation and characterization of dipeptidyl aminopeptidase IV from human kidney cortex

Intact dipeptidyl aminopeptidase IV (DAP IV) was solubilized by bromelain treatment from human kidney brush border plasma-membranes. Purification of DAP IV was performed by a 3-step method, applying lectin-affinity chromatography on WGA-Sepharose, gel filtration and anion-exchange chromatography. DAP IV from human kidney cortex showed a pH optimum of 8.7 and was totally inhibited by 1 mmol/l Zn2+. Isolated DAP IV revealed a relative molecular mass of 250 kDa as determined by the native-PAGE method and of 220 kDa by the gel filtration method. Analytical isoelectric focussing of DAP IV revealed an isoelectric point of pH 5.3. Ultrastructural analysis of isolated DAP IV fractions, using the negative staining technique, disclosed the presence of numerous globular particles with an average diameter of 5 nm which correspond to the structural substrate of the purified protein.

Development of a radioimmunoassay for a high molecular mass tubular antigen in urine — Its application for early detection of tubular damage

In order to isolate urinary kidney antigens, the gammaglobulins fraction of an antiserum against human kidney cortex plasma membranes was coupled to Sepharose 4B. By immunospecific affinity chromatography an antigen fraction was obtained from the urine of a patient suffering from severe kidney disease. After gel filtration, the main fraction, eluted with the exclusion volume of a Sephadex G-200 column and enriched 16 000-fold, was labelled with 131I and used in a radioimmunoassay system. Soluble kidney antigens, presumably of proximal tubular origin, could be detected and quantified by the assay system in urine samples of patients with various diseases. The samples did not need to be treated, either concentrated or dialyzed, before application. The results of our experiments show a correlation between antigen excretion and kidney damage. Rejection episodes in patients with kidney transplants could be recognized early by enhanced antigen excretion. Potentially nephrotoxic drugs caused antigen excretion as well. In normal, healthy subjects output of the antigen was very low. The assay system might be of value for monitoring renal diseases.

Immunological and ultrastructural analysis of loss of tubular membrane-bound enzymes in patients with renal damage

The pathophysiological background of shedding of membrane-bound enzymes from the proximal tubule was assessed in urine specimens of patients with renal damage applying immunospecific affinity-chromatography, immunotitration, ultracentrifugation, electroimmunodiffusion, immunohistology, as well as negative staining technique. Compared to healthy controls, patients with kidney injury, e.g. after administration of potentially nephrotoxic drugs (cytostatics, contrast media) revealed an increased excretion rate of vacuolar membrane fragments (50-500 nm) into urine. The brush border (BB) of the proximal tubule was identified as a main source of urinary blebs as concluded from immunoelectrophoretic and immunohistochemical analysis. In addition, the marker enzyme profile of urinary vacuolar blebs was similar to that of the BB membrane from human kidney. The results further evidenced that, during the initial phase of tubular injury, 5-10 nm surface glycoproteins of the BB, among them Ala-Leu-aminopeptidase and portions of gamma-glu-transpeptidase, are released into urine; this might be followed by increased blebbing of macromolecular BB fragments, indicating more severe membrane disruption.