Z. Marchewka

Enzymuria and low molecular weight protein excretion as the differentiating marker of complications in the early post kidney transplantation period

AbstractRenal function in the early post-transplantation period depends largely on factors affecting the kidney prior to implantation. Function of the graft may be also disturbed by the most common complications of the early post-operative period such as acute graft rejection (AGR), acute tubular necrosis (ATN) and may be modified by nephrotoxic action of cyclosporine A (CsA). Evaluation of excretion of enzymes and low molecular weight proteins (LMWP) may help in the differentiation of these complications.n Aim Comparison of the urinary excretion of markers of tubular injury in patients with AGR, ATN, or patients with stable graft function (SGF) was made and differences between groups and correlations between markers and cold ischemia time (CIT), warm ischemia time (WIT) and blood trough level of cyclosporine A (CsA0) were determined.n Material and methods In 60 cadaveric renal allograft recipients in the early post-transplantation period urinary excretion of N-acetyl-β-d-glucosaminidase (NAG) and B isoenzyme (NAG-B), alanylaminopeptidase (AAP), γ-glutamyltransferase (GGT), α and π isoenzymes of glutathione S-transferase (α-GST, π-GST), retinol binding protein (RBP) and β2- microglobulin (β2M), were analyzed.n Results NAG and NAB-B activities were higher in ATN (P<0.05, P<0.01) and in AGR (P<0.005, P<0.02) than in SGF. Excretion of π-GST was higher in AGR than in SGF (P<0.0002) or ATN (P<0.007). CIT and WIT in patients with ATN were higher (P<0.05) than in SGF group. In ATN patients, correlations of CIT with RBP (P<0.05) and π-GST (P<0.05), and WIT with RBP (P<0.05), and π-GST (P<0.001) were found.n Conclusions High urinary NAG and NAG B excretion characterizes ATN and AGR patients. Evaluating urinary excretion of π-GST may be helpful in differentiating AGR from ATN. However, taking into account ischemia time is necessary in interpreting the π-GST value in early post transplant period.

Etiology of Increased Enzymuria in Different Morphological Forms of Glomerulonephritis

Background: High urinary excretion of lysosomal enzymes is thought to reflect tubulointerstitial damage and is observed both in the acute and chronic phases of various morphological forms of glomerulonephritis (GN). It is related to the degree of proteinuria and secondary interstitial inflammatory process. N-acetyl-β-D-glucosaminidase (NAG) and β-glucuronidase (β-GR) are the most commonly used markers of tubulointerstitial injury. NAG and β-GR are also contained in azurophilic granulations of polymorphonuclear leukocytes (PMNs) and may be released during the activation of PMNs. Aims: The aim of this study was to elucidate the role of PMN degranulation in causing the increase of urinary excretion of lysosomal enzymes that is observed in glomerulonephritis. Material and Methods: We analyzed the urinary excretion of NAG, its B isoenzyme NAG-B, β-GR and leukocyte elastase (EL), in 91 patients with morphologically different primary and secondary glomerulopathies, and in 12 healthy controls. Results: Excretion of NAG, NAG-B and β-GR were statistically significantly higher in all GN patients in comparison to healthy controls. In the whole analyzed GN population significant correlations between amount of proteinuria and excretion of NAG, NAG-B and β-GR were ascertained. In subgroup analysis NAG excretion was significantly correlated with proteinuria in patients with diffuse proliferative GN (PGN), mesangiocapillary GN (MCGN), and minimal change disease (MCD). There was a significant correlation between NAG-B and proteinuria in MCD and PGN patients. There was a significant relationship of β-GR and EL with proteinuria and EL with NAG in the PGN group. Significant relationships between serum creatinine and excretion of EL but not NAG, NAG-B, or β-GR were observed in the whole examined group. Conclusions: Increased urinary excretion of elastase, with concomitant high proteinuria and NAG excretion in patients with proliferative GN may indicate that leukocyte degranulation is an additional source of enzymuria in the primary i.e. glomerular inflammatory process. Significant relationship between EL excretion and serum creatinine may indicate that EL released from PMN may also participate in the secondary i.e. interstitial injury that is decisive in the progression of GN.

Plasma and urine leukocyte elastase–α1protease inhibitor complex as a marker of early and long-term kidney graft function

BACKGROUNDnNeutrophils are mediators of ischaemia/reperfusion (I/R) injury following kidney transplantation (kTx). Leukocyte elastase (LE) complex with alpha(1)protease inhibitor (LE-alpha(1)PI) is a marker of neutrophil degranulation. The aim of this study was to evaluate LE-alpha(1)PI as a marker of I/R kidney damage and to search for correlations between leukocyte activation and post-transplant complications.nnnMETHODSnPlasma and urine LE-alpha(1)PI were estimated in 55 deceased-donor kidney graft recipients on postoperative days (POD) 1, 3 and 7, as well as in the late post-transplant period.nnnRESULTSnThe plasma LE-alpha(1)PI level peaked on POD 1 after kTx, and the urine LE-alpha(1)PI peaked on POD 3. On POD 1 and POD 3, the urine LE-alpha(1)PI levels were higher in delayed graft function (DGF) patients than in patients with immediate graft function (IGF: P < 0.001 and P < 0.003, respectively). Urine LE-alpha(1)PI excretion on POD 1 was significantly higher in patients with longer cold ischaemia time (CIT) than in patients with shorter CIT, P < 0.002. Multivariate regression model revealed two factors influencing the occurrence of early acute rejection-urine LE-alpha(1)PI complex on POD 3 and human leukocyte antigen (HLA) mismatches. There was a significant association between the plasma LE-alpha(1)PI on POD 3 and serum creatinine level 6 and 12 months after kTx (r(2) 0.24; P < 0.005 and 0.19; P < 0.005, respectively).nnnCONCLUSIONSnThis study is the first presentation of a simple, non-invasive measurement of neutrophil activation after kTx. It also demonstrates a strong correlation between the early post-transplant LE-alpha(1)PI complex level and kidney graft function.

Enzymatic Markers of Cyclosporine Nephrotoxicity in Patients after Renal Transplantation

Toxicity of cyclosporine (CsA), an immunosuppressive drug widely used in transplantation, to the transplanted kidney creates a serious side effect. Therefore, searching for sensitive indicators of nephrotoxic action is well worth the effort. In this work we decribe the results of estimation of urine concentration of lysosomal enzymes widely present in the kidney: N-acetyl-β-D-glucosaminidase (NAG), its isoenzyme NAG-B and β-glucuronidase (β-Gr). The studies were conducted in various periods after transplantation of kidneys, on patients under various treatments and receiving different doses of CsA. The results indicate a substantial dependence of the activity of NAG and NAG-B on CsA doses and the period after transplantation. The enzyme proved to be also a sensitive indicator of graft rejection. No such dependence was observed in the case of β-Gr.

N-acetyl-B-D-glucosaminidase isoenzymes in the diagnosis of poisoning and kidney diseases

Damaged lysosomes from renal tubular cells are, to the greatest degree, the source of activity of NAG in urine. Besides this, the enzyme can appear as a result of degranulation of granulocytes (PMN), active infection of the urinary system as also from serum as a result of glomerular filtration during damage to the glomerular basement membrane.For the purpose of explaining the source of origin of the enzyme in urine, NAG was separated into isoenzymes from the kidneys, granulocytes and serum for comparison with isoenzymes in physiological and pathological urines after ethylene glycol poisoning, and glomerulonephritis, respectively.The separation was made by column ion-exchange chromatography on DEAE-52 cellulose and by electrophoresis in 7% polyacrylamide gel. In addition, the thermostability of isolated isoenzymes was compared.

Kidney Graft Function in Long-term Cyclosporine and Tacrolimus Treatment: Comparative Study With Nephrotoxicity Markers

Calcineurin inhibitors improve kidney allograft survival in the posttransplantation period; however, they may cause nephrotoxicity. The objective of this study was to compare the effect of cyclosporine (CsA) and tacrolimus (Tac) treatment on the transplanted kidney. The study included 219 patients aged 21 to 65 years. Of these, 120 (39 women and 81 men) were treated with CsA and 99 (38 women and 61 men) were treated with Tac. Patients were divided into 4 groups depending on the time since kidney transplantation. We evaluated urine markers of nephrotoxicity: proximal tubular cells lysosomal enzymes (N-acetyl-beta-d-glucosaminidase [NAG] and its isoform NAG-B, beta-d-galactosidase, and beta-glucouronidase) and brush border enzymes (alanyl aminopeptidase and gamma-glutamyl transpeptidase). Urine activities of nephrotoxicity markers were compared in CsA- and Tac-treated patients groups depending on the duration of treatment and allograft function as measured by serum creatinine concentration. Correlation studies between CsA and Tac levels and enzyme activities were performed in both groups and in the entire patient cohort. NAG and its isoform NAG-B seemed to be the most reliable markers of nephrotoxicity. Despite the significant correlation between NAG urine activity and serum creatinine concentration in the CsA group, there were no significant differences in NAG or NAG-B activities between CsA- and Tac-treated graft recipients.

Elastase deposits in the kidney and urinary elastase excretion in patients with glomerulonephritis – evidence for neutrophil involvement in renal injury

Objective. Elastase is a key proteolytic enzyme released during polymorphonuclear leukocyte degranulation. There are abundant data of elastase involvement in the development of injury in experimental models of glomerulonephritis (GN), but scant direct evidence of its involvement in human primary GN. The aims of this study were to determine the immunolocalization of elastase deposits in kidney biopsy specimens from patients with primary idiopathic GN, to attempt to correlate the distribution and intensity of deposits with urinary elastase excretion, and to determine clinical markers of renal injury in several types of primary idiopathic GN. Material and methods. The immunohistochemical localization and intensity of elastase deposits in kidney biopsies, the urinary excretion of leukocyte elastase, and proteinuria and serum creatinine levels were evaluated in 23 patients with primary GN and the associations between these factors were sought. Results. Patients with crescentic proliferative GN had the highest intensity of elastase deposits. In this group of patients, elastase was present in the glomerular endothelium, as well as in the tubular epithelium and interstitium. Patients with a high intensity of elastase deposits within the glomerular endothelium and Bowmans capsule had significantly higher urinary excretion of elastase. Patients with interstitial, mesangial and perivascular elastase deposits had significantly higher serum creatinine than those without. Patients with elastase deposits in the glomerular endothelium and in the interstitium had insignificantly higher proteinuria than those without. Conclusion. Our data provide morphological evidence of leukocyte elastase involvement in renal injury occurring in the course of primary idiopathic GN, in particular in the proliferative types.

Increased Granulocyte Heparanase Activity in Neutrophils from Patients with Lupus Nephritis and Idiopathic Membranous Nephropathy

Heparanase is a β-glucuronidase that cleaves sugar chains of heparan sulfate proteoglycans. It is believed that heparanase may be involved in the pathogenesis of proteinuria. The aim of this study was to assess the significance of heparanase in the pathogenesis of particular glomerulonephritis types. The evaluation of heparanase activity in serum, urine, and granulocytes and superoxide dismutase (SOD) activity in granulocytes of patients with lupus nephritis (nxa0=xa017), membranous nephropathy (nxa0=xa011), IgA nephropathy (nxa0=xa012), focal and segmental glomerulosclerosis (nxa0=xa018), mesangiocapillary glomerulonephritis (nxa0=xa012) and in 19 healthy volunteers were performed. The heparanase activity in granulocytes of patients with lupus nephritis and membranous nephropathy was higher than heparanase activity in granulocytes in the control group (pxa0=xa00.02 in both cases). This is the first observation of this phenomenon. There was no difference between SOD activity in granulocytes of patients with all assessed types of glomerulonephritis and the control group. A positive correlation between heparanase activity in urine and double-strain DNA antibodies (rxa0=xa00.51; pxa0=xa00.04), and reverse correlations between heparanase in urine and hemolytic activity of the complement (rxa0=xa0−0.57; pxa0=xa00.03) in the lupus nephritis group, and between heparanase activity in granulocytes and serum total protein level (rxa0=xa0−0.69; pxa0=xa00.02) in membranous nephropathy were observed. Increase in heparanase activity without changes in superoxide dismutase activity in the granulocytes from patients with lupus nephritis and membranous nephropathy was observed. It may be used as one of the markers of these disease activities.

Diagnostic application of AAP isoenzyme separation.

We describe the separation of alanylaminopeptidase (AAP) from urine into two isoenzymes: a particulate and a soluble form. The separation was accomplished using ion-exchange column chromatography on DEAE-52 cellulose. The purity of the isolated forms was confirmed electrophoretically. We attempted to create a method allowing the quantitative assessment of AAP isoenzymes in urine based on electrophoretic separation in 7.5% polyacrylamide gel with subsequent densitometric analysis. The content of AAP isoenzymes in examined urine was estimated using ultracentrifugation. The differences in the content of cytosolic and microsomal forms were observed suggesting the possibilities of using AAP isoenzymes in diagnostics. Furthermore, the effect of temperature on the activity of AAP separated on DEAE-52 cellulose was studied.