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Background: Although there is evidence that the CYP3A4*22 variant should be considered in tacrolimus dosing in renal transplantation, its impact beyond tacrolimus dose requirements remains controversial. Methods: In a cohort of 121 kidney transplant recipients, we analyzed the CYP3A4*1B, CYP3A4*22, and CYP3A5*3 alleles and the ABCB1 variants 1236C>T, 2677G>T/A, and 3435C>T for their impact on exposure and dose requirement. Relevant clinical outcome measures such as acute rejection within the first year after transplantation, delayed graft function, and renal function at discharge (estimated glomerular filtration rate) were evaluated. Results: Extensive metabolizer (n = 17, CYP3A4*1/*1 carriers with at least one CYP3A5*1 allele) showed significantly higher tacrolimus dose requirement (P = 0.004) compared with both intermediate metabolizer (IM, n = 93, CYP3A5*3/*3 plus CYP3A4*1/*1 or CYP3A4*22 carriers plus one CYP3A5*1 allele), and poor metabolizer (n = 11, CYP3A4*22 allele in combination with CYP3A5*3/*3) after onset of therapy. Significantly higher dose requirement was observed in CYP3A5 expressers (P = 0.046) compared with non-expressers again at onset of therapy. Using the log additive genetic model, the area under the curve for the total observation period up to 16 days was significantly associated with the CYP3A5*3 genotype (P = 3.34 × 10-4) as well as with the IM or extensive metabolizer phenotype (P = 1.54 × 10-4), even after adjustment for multiple testing. Heterozygous carriers for CYP3A4*22 showed significantly higher areas under the curve than the CYP3A4*1/*1 genotype in the second week post-transplantation (adjusted P = 0.016). Regarding clinical outcomes, acute rejection was significantly associated with human leukocyte antigen mismatch (≥3 alleles; OR = 12.14, 95% CI 1.76, 525.21, P = 0.019 after correction for multiple testing). Graft recipients from deceased donors showed higher incidende of delayed graft function (OR 7.15, 95% CI 2.23, 30.46, adjusted P = 0.0008) and a lower estimated glomerular filtration rate at discharge (P = 0.0001). Tested CYP3A4 or CYP3A5 variants did not show any effects on clinical outcome parameters. ABCB1 variants did neither impact on pharmacokinetics nor on clinical endpoints. Conclusion: At our transplantation center, both CYP3A5*3 and, to a lesser extent, CYP3A4*22 affect tacrolimus pharmacokinetics early after onset of therapy with consequences for steady-state treatment in routine clinical practice.

Background and objectives Donor dopamine improves initial graft function after kidney transplantation due to antioxidant properties. We investigated if a 4 µg/kg per minute continuous dopamine infusion administered after brain-death confirmation affects long-term graft survival and examined the exposure-response relationship with treatment duration. Design, setting, participants, & measurements Five-year follow-up of 487 renal transplant patients from 60 European centers who had participated in the randomized, multicenter trial of dopamine donor pretreatment between 2004 and 2007 (ClinicalTrials.gov identifier: NCT00115115). Results Follow-up was complete in 99.2%. Graft survival was 72.6% versus 68.7% (P=0.34), and 83.3% versus 80.4% (P=0.42) after death-censoring in treatment and control arms according to trial assignment. Although infusion times varied substantially in the treatment arm (range 0–32.2 hours), duration of the dopamine infusion and all-cause graft failure exhibited an exposure-response relationship (hazard ratio, 0.96; 95% confidence interval [95% CI], 0.92 to 1.00, per hour). Cumulative frequency curves of graft survival and exposure time of the dopamine infusion indicated a maximum response rate at 7.10 hours (95% CI, 6.99 to 7.21), which almost coincided with the optimum infusion time for improvement of early graft function (7.05 hours; 95% CI, 6.92 to 7.18). Taking infusion time of 7.1 hours as threshold in subsequent graft survival analyses indicated a relevant benefit: Overall, 81.5% versus 68.5%; P=0.03; and 90.3% versus 80.2%; P=0.04 after death-censoring. Conclusions We failed to show a significant graft survival advantage on intention-to-treat. Dopamine infusion time was very short in a considerable number of donors assigned to treatment. Our finding of a significant, nonlinear exposure-response relationship disclosed a threshold value of the dopamine infusion time that may improve long-term kidney graft survival.

Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR).The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR). The assay was compared to conventional flow cytometry and the agreement of results between two laboratories using a comparable qPCR protocol was assessed. In addition, the effect of gender, age, and medications was investigated.Within and between series imprecision was20% (n=6). Whole blood samples are suitable for analysis within 3days when stored at room temperature; both whole blood and DNA samples - within 12months when frozen at -80°C. A significant correlation between the qPCR results and flow cytometry was lacking both with samples from HI and KTR. qPCR results between laboratories showed a bias of 76% but correlated well (r=0.645; p=0.0002, n=29). nTregs determined by qPCR were significantly (p0.05) higher in HI (0.73%±0.23%, n=60) than in KTR (0.45%±0.21%, n=60) and in female HI (1.0%±0.27%, n=30) than in male HI (0.45%±0.23%, n=27). No effect of drugs or age was observed.The qPCR assay for nTregs provides reproducible results and is of sufficient quality for working with patient samples although inter-laboratory differences can be encountered due to a lack of method standardization. It was confirmed that gender-specific reference ranges are required.

Therapeutic drug monitoring is a well-established approach in transplantation medicine to guide immunosuppressive therapy. However, it cannot always predict the effects of immunosuppressive drugs on immune cells, because it does not reflect any aspect of an individual patient's immune system. Pharmacodynamic monitoring is a more recent strategy to provide information about the biologic effect of a specific drug or drug combination on the individual transplant patient. Currently, there is a large number of different biomarkers that either directly (specific markers) or indirectly (global markers) relate to the pharmacodynamic effects of immunosuppressive drugs and are under investigation as potential candidates to be introduced in clinical practice. Such biomarkers may be useful to identify patients at risk of developing acute rejection, infection, or cancer as well as patients who are suitable for minimization of immunosuppressant therapy and may be helpful to manage the timing and rate of immunosuppressant weaning. Serial longitudinal monitoring may allow maintenance of an individualized immunosuppressive regimen. Thus, biomarker monitoring is a potential complementary tool to therapeutic drug monitoring. This review summarizes the current state of knowledge about the use of a number of global or drug-specific pharmacodynamic biomarkers. It is not a comprehensive overview of the literature available, but rather an evidence-based reflection by experts who are intensively involved in scientific work in this field.

The European Tacrolimus versus Ciclosporin-A Microemulsion (CsA-ME) Renal Transplantation Study demonstrated that tacrolimus decreased acute rejection rates at 6 months. Primary endpoints of this investigator-initiated, observational 7-year follow-up study were acute rejection rates, patient and graft survival rates, and a composite endpoint (BPAR, graft loss, and patient death). We analyzed data from the original intent-to-treat population (n = 557; 286 tacrolimus, 271 CsA-ME). A total of 237 tacrolimus and 208 CsA-ME patients provided data. At 7 years, Kaplan-Meier estimated rates of patients free from BPAR were 77.1% in the tacrolimus arm and 59.9% in the CsA-ME arm, graft survival rates amounted to 82.6% and 80.6%, and patient survival rates to 89.9% and 88.1%. Estimated combined endpoint-free survival rates were 60.2% in the tacrolimus arm and 47.0% in the CsA-ME arm (P =

Hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and acute renal failure. Lack of complement inactivating factor H predisposes to the development of atypical HUS. Little is known about mechanisms linking complement activation with loss of erythrocyte integrity during HUS. Recent studies disclosed that increased cytosolic Ca2+ activity and cellular ceramide trigger programmed erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. In the present study, we investigated whether eryptosis occurs during the course of HUS. To this end, erythrocytes from healthy volunteers were exposed to plasma from a patient with severe idiopathic recurrent HUS secondary to factor H depletion. Phosphatidylserine exposure (Annexin binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation [anti-ceramide antibody and enzymatic (diacylgycerol kinase) analysis] were determined. Exposure of erythrocytes to plasma from the patient, but not to plasma from healthy individuals, triggered Annexin binding. The effect of plasma on erythrocyte Annexin binding was abolished by plasmapheresis or filtration at 30 kDa. It was paralleled by formation of ceramide and increase of cytosolic Ca2+ activity. Enhanced Annexin binding of erythrocytes from healthy individuals was observed after exposure to plasma from three other patients with HUS. The proeryptotic effect of patient plasma was mimicked by exposure to the Ca2+ ionophore ionomycin, and eryptosis was potentiated in the presence of cell membrane-permeable C6-ceramide. Furthermore, in vitro complement activation similarly triggered erythrocyte phosphatidylserine exposure, an effect which was blunted by the addition of factor H. In conclusion, our present observations disclose a novel, pathophysiological, factor-H dependent mechanism leading to injury of erythrocytes during the course of hemolytic uremic syndrome.

BACKGROUND: Comparison studies of calcineurin inhibitors as cornerstone immunosuppressants in renal transplantation have demonstrated that tacrolimus consistently reduces acute rejection rates and, in some studies, also improves long-term renal outcome in comparison to cyclosporin A (CsA). The aim of the present 2 year follow-up of the European Tacrolimus vs Cyclosporin A Microemulsion Renal Transplantation Study was to investigate long-term clinical outcome in terms of rate of acute rejection, graft and patient survival and graft function. METHODS: The European Tacrolimus vs Cyclosporin A Microemulsion Renal Transplantation Study was a randomized, comparative 6 month trial of the calcineurin inhibitors tacrolimus and CsA in combination with both azathioprine and steroids. The intent-to-treat population (ITT) consisted of 286 patients in the tacrolimus arm and 271 in the CsA microemulsion (CsA-ME) arm. Whereas whole blood level targets were 10-20 and 5-15 ng/ml for tacrolimus and 100-400 and 100-200 ng/ml for CsA during months 0-3 and 4-6, respectively, during the investigator-driven follow-up after termination of the main study (months 7-24) no specific calcineurin inhibitor target levels were required. Follow-up data were collected at 2 years post-transplantation from 237 (82.9% of the ITT population) patients who received tacrolimus and 222 (81.9% of the ITT population) patients who received CsA-ME. RESULTS: Calculated on ITT populations, mortality (2.0% vs 3.3%; P

Spiral computed tomographic angiography (CTA) is a noninvasive method to diagnose renal artery stenosis (RAS). In digital subtraction angiography (DSA), contrast media (CM) is injected directly into the renal artery; in CTA, a greater amount of CM is injected intravenously, potentially leading to an increased incidence of CM nephropathy.We investigated 80 patients with suspected RAS randomized to either CTA or DSA prospectively. The following parameters were determined: serum creatinine level and single-shot inulin clearance for evaluation of renal function and urine alpha1-microglobulin and beta-N-acetyl-glucoseaminidase (beta-NAG) as markers for tubular toxicity. Data from 16 patients undergoing angioplasty in the same session were excluded.In the CTA and DSA groups, 163 +/- 13 and 104 +/- 56 mL of CM (mean +/- SD; P0.0001) were administered, respectively. Mean serum creatinine levels increased from 1.78 +/- 1.61 to 1.92 +/-1.73 mg/dL (157 +/- 142 to 170 +/- 153 micromol/L; P = 0.00001) in the CTA group and from 1.52 +/- 1.23 to 1.60 +/- 1.28 mg/dL (134 +/- 109 to 141 +/- 113 micromol/L; P = 0.01) in the DSA group. Mean inulin clearance decreased from 63 +/- 28 to 58 +/- 23 mL/min (P = 0.01) and 65 +/- 26 to 62 +/- 26 mL/min (P0.01), median beta-NAG levels increased from 4.6 to 6.0 U/g creatinine (P = not significant) and 2.5 to 8.0 U/g creatinine (P0.001), and median alpha1-microglobulin levels increased from 13 to 17 microg/g creatinine (P0.025) and 11 to 21 microg/g creatinine (P = not significant) in the CTA and DSA groups, respectively. CM nephropathy occurred in 3 of 33 patients in the CTA group and 2 of 31 patients in the DSA group. The increase in creatinine level was reversible in all patients within 7 days.On this study, CTA performed for the detection of RAS is not associated with an increased risk for CM nephropathy compared with intraarterial DSA despite a greater dose of CM.

The aim of this study was to compare renal function between patients with renal angiography and patients with renal angiography and angioplasty (AP) for renal artery stenosis (RAS). Forty-seven patients with suspected RAS were prospectively investigated by digital subtraction angiography (DSA) using non-ionic low osmolar contrast media (CM). In 22 patients RAS was detected and in 16 cases an angioplasty was performed in the same session. The following parameters were determined 1 day prior to and after the DSA, respectively: serum creatinine (S-Crea, micromol/l) and single-shot inulin clearance (In-Cl, ml/min) for the evaluation of renal function; and urine alpha 1-microglobuline (AMG, microg/g Crea) and beta-N-acetyl-glucoseaminidase (beta-NAG, U/g Crea) as markers of tubular toxicity. Serum creatinine was measured additionally 2 days after CM had been injected. In both groups with and without AP 174+/-65 and 104+/-56 ml of CM ( p0.0005) were used, respectively. There were no differences with regard to renal function or risk factors for CM nephrotoxicity between both groups. In the group with AP S-Crea and In-Cl (each: mean+/-SD) did not change significantly (before DSA: 171+/-158 and 61+/-24, after DSA: 189+/-177 and 61+/-25, respectively), beta-NAG (median) rose from 4 to 14 ( p0.05) and AMG from 8 to 55 (n.s., because of high SD). In the group without AP S-Crea increased from 134+/-109 to 141+/-113 ( p0.01), In-Cl dropped from 65+/-26 to 62+/-26 ( p0,01), beta NAG (median) rose from 4 to 8 ( p=0.01), and AMG from 7 to 10 (n.s.). A rise in baseline S-Crea by more than 25% or 44 micromol/l occurred in 4 and 2 patients in the group with and without AP, respectively. Creatinine increase was reversible in all cases within 7 days. In this study using sensitive methods to detect changes of renal function and tubular toxicity no additional renal function impairment in DSA with angioplasty for RAS compared with DSA alone could be demonstrated. Our data suggest that AP performed for RAS has a beneficial effect on renal function.

Hyperlipoproteinemia can aggravate glomerulosclerosis and chronic tubulointerstitial (ti) damage in kidneys without primary immunologic disease. We evaluated whether the effect of hyperlipidemia on progression of renal damage differed between kidneys without preexisting glomerular disease and kidneys with mesangioproliferative glomerulonephritis and whether the renal actions of hyperlipidemia were dependent on oxidant-antioxidant balance. Hyperlipidemia was induced by high-fat and high-cholesterol diet in uninephrectomized rats. In rats without glomerulonephritis, hyperlipidemia led to a rise in glomerular and ti generation of reactive oxygen species (ROS). Oxygen radicals were mainly generated by enhanced xanthine oxidoreductase (XO), which rose with protein concentration and activity during hyperlipidemia; concurrently, glomerulosclerosis and chronic ti injury were noticed during hyperlipidemia [ti damage (% of total tubulointerstitium (TI) after 150 days): normolipidemia 0.1 ± 0% vs. hyperlipidemia 3.4 ± 0.9%; P < 0.05]. In mesangioproliferative Thy-1 nephritis, ti injury was significantly accelerated by hyperlipidemia (ti damage after 150 days: normolipidemic Thy-1 nephritis 2.5 ± 0.6% vs. hyperlipidemic Thy-1 nephritis 12.5 ± 3.1%; P < 0.05). Antioxidant enzyme activities decreased and XO activity rose markedly in the TI (XO activity in TI after 150 days: normolipidemic Thy-1 nephritis 2.2 ± 0.5 vs. hyperlipidemic Thy-1 nephritis 4.5 ± 0.7 cpm/μg protein; P < 0.05). In hyperlipidemic Thy-1 nephritis rats, which had a higher urinary protein excretion than normolipidemic rats, hypochlorite-modified proteins, an indirect measure for enhanced myeloperoxidase activity, were detected in renal tissue and in urine, respectively. During hyperlipidemia, chronic damage increased in renal TI. Enhanced generation of ROS, rise in oxidant enzyme activity, and generation of hypochlorite-modified proteins in renal tissue and urine were noticed. These data suggest that oxidant stress contributed to the deleterious effects of hyperlipidemia on the renal TI.

1. Antioxidant and oxidative enzymes were examined in renal glomeruli and proximal tubules of healthy young rats (10-12 weeks old), and results were related to the superoxide anion generation of these tissues. 2. Activities of superoxide dismutases, catalase, and glutathione peroxidase were 3- to 6-fold higher in proximal tubules than in glomeruli. Similarly, enzyme levels and mRNA levels of superoxide dismutases and catalase were significantly higher in proximal tubules. 3. NADH- and NADPH-dependent oxidase activity and xanthine oxidase activity were not different in glomeruli and proximal tubules. 4. Measurements with lucigenin-enhanced chemiluminescence in vital tissues indicated 10-fold higher rates of superoxide anion in glomeruli than in tubules. 5. Compared with the young rats, tubules of 8-month-old rats had significantly higher superoxide anion rates and lower superoxide dismutase activity, whereas NADH- and NADPH-dependent oxidase activities were unchanged. 6. We conclude that considerable differences in the antioxidant-oxidant balance exist between the glomerulus and proximal tubule. Results from experiments using chemiluminescence in vital tissues suggest that changes in the antioxidant-oxidant balance have an effect on oxygen radical levels. The relevance of the observed differences to glomerular and tubulo-interstitial disease remains to be determined, but a greater susceptibility of the glomerulus to oxidant stress might be anticipated.

Extracorporeal removal of low-density lipoprotein (LDL) cholesterol by dextran sulfate adsorption is indicated in patients with diet and drug resistant hyper-cholesterolemia to prevent or to regress coronary heart disease. Plasma separation is the first step in the process, followed by adsorption of LDL cholesterol and lipoprotein (a) (Lp[a]) to negatively charged dextran sulfate covalently bound to cellulose beads. The reduction per treatment in LDL cholesterol is 65-75% and in Lp(a) 40-60%. In most patients one treatment per week is sufficient to reduce mean LDL to 100-150 mg/dl. Minor side effects occur in 2-6% of treatments. Major side effects are rare. In uncontrolled studies long-term treatment was associated with inhibition of progression and induction of regression of coronary artery disease. LDL apheresis by dextran sulfate may increase blood perfusion of some tissues, and preliminary results indicate a beneficial effect on therapy resistant nephrotic syndrome with hypercholesterolemia.

Objectives Human CD26 is co-stimulatory for lymphocytes, circulates in a soluble form in blood (sCD26), and has intrinsic dipeptidyl peptidase IV (DPPIV) activity. Associations between CD26 expression on the surface of T cells (CD26 +/CD3 +) and acute rejection and between (CD26 +/CD3 +)/DPPIV and clinical immunosuppression have been reported. These results encouraged the investigation of CD26 as a potential biomarker to optimize immunosuppressive therapy. To better understand the significance of CD26, a comparative study of CD26 expression on CD3 + cells, sCD26 concentration, and DPPIV activity in healthy persons (HP) and kidney transplant recipients (KTR) was performed. Design and methods Thirty-one HP and 34 KTR were included in the study. CD26 +/CD3 + was determined by FACS, sCD26 concentration was determined by ELISA, and DPP activity was determined by spectrophotometry. For KTR, these parameters were studied on the day before transplantation (preTx) and 7 ± 1 days after transplantation (postTx). Results There was no significant difference in the CD26 +/CD3 +, sCD26, and DPPIV data regarding gender, donor type (16 living donors), delayed graft function (n = 8), or presence of ≥ 4HLA mismatches (n = 16). Compared to the HP data, preTx CD26 +/CD3 + was 4.5-fold higher, sCD26 was 1.2-fold higher, and DPPIV showed no significant difference. PostTx, CD26 +/CD3 + was 3.8-fold higher, and sCD26 and DPPIV decreased significantly, reaching lower values than that observed in HP. Re-transplanted patients (n = 5) showed significantly lower preTx CD26 +/CD3 + expression than patients receiving their first transplant. Patients with preemptive transplantation (n = 7) showed higher postTx CD26 +/CD3 + expression than patients on dialysis. Conclusions CD26 expression on CD3 + cells was strongly increased in patients with end stage kidney disease compared to HP and remained high early postTx. The differences in sCD26 and DPPIV behavior compared to that of CD26 +/CD3 + postTx may reflect a regulatory response to the new immunological situation and the effects of therapy.

Spiral CT in the form of CT angiography (CTA) provides a new method for the investigation of the large vessels. 52 patients with suspected renovascular hypertension were examined by CTA and arterial DSA. The computer tomographic and angiographic findings in 124 renal arteries were compared. Sensitivity of CTA for demonstrating renal artery stenosis was 95%, and for relevant stenoses it actually reached 100%. Specificity was 92%. Positive/negative prediction value was 85% and 100%. CTA proved outstandingly good for demonstrating or excluding renal artery stenosis and was superior to other imaging methods in several respects. CTA requires high contrast values, optimal timing and careful evaluation. Amongst various forms of image reconstruction, interactive viewing of the primary axial images and multiplanar reconstruction in a cine mode are essential and MIP projections are valuable.

Immunosuppressive therapy is administered to all solid organ transplant recipients to help prevent acute rejection and the loss of allograft function. Adequate immunosuppression is a delicate balance between rejection rates and chronic allograft dysfunction on the one hand and immunological and nonimmunological side effects on the other hand. The general strategy is to minimize the toxicity associated with triple immunosuppressive regimens and possibly enhance long-term allograft survival without compromising short-term allograft survival. There are no criteria that enable the clinician to predict who will do well with the decrease or complete avoidance of immunosuppression. It becomes more and more clear that randomized controlled trials will not be able to solve the problem of defining the optimal immunosuppression for a given individual. The most promising road to achieve this goal and to improve chronic allograft survival may be personalized immunosuppression guided by biomarkers complementary to conventional drug monitoring strategies.

Introduction: Strategies based on monitoring pharmacodynamic effects are increasingly evaluated to individualize immunosuppressive therapy. In the present investigation, both drug-specific and general pharmacodynamic biomarkers were assessed and their association with clinical events early after kidney transplantation was examined. Methods: Thirty-five de novo kidney transplant patients receiving basiliximab, enteric-coated mycophenolate sodium (2 × 720 mg/day), steroids, and tacrolimus (target: 6-8 μg/L) were included. Blood was drawn on days 7(± 1) and 21(± 2) after transplantation. Mononuclear leucocytes were isolated and the following parameters were investigated: inosine monophosphate dehydrogenase activity (high-performance liquid chromatography-diode array detection), cell proliferation (bromodeoxyuridine test), and CD marker cell surface expression (CD25, CD71, CD26) on stimulated (phytohemagglutinin 2.5 μg/10 6 cells) and nonstimulated CD3 + cells. Acute rejection, gastrointestinal adverse effects, leucopenia, and infections were monitored over 3 months. Results: There was no association between clinical events and inosine monophosphate dehydrogenase activity apart from patients with diarrhea showing a significantly higher inosine monophosphate dehydrogenase activity 2 hours after the enteric-coated mycophenolate sodium dose (P < 0.05). Cell proliferation was significantly reduced in patients with leucopenia (P < 0.05). CD71 expression was less inducible in patients with infections (P < 0.05). A lower CD26 expression on non stimulated CD3 + cells predicted freedom from rejection (Day 7; negative predictive value 100%). No associations were found between CD25 expression and events. Conclusions: A potential benefit of pharmacodynamic monitoring to optimize immunosuppressive combination therapy has been demonstrated. In particular, CD26 and CD71 may be promising biomarkers to assess adequate immunosuppression in the early phase after kidney transplantation. The results of this pilot study require verification in further trials with more patients and events as well as with different graft types.

Dextrans undergo glomerular filtration and are taken up by endocytosis into lysosomes of proximal tubule cells. We measured the activities of the intralysosomal proteolytic enzymes cathepsin B and L and the nonproteolytic acid phosphatase in microdissected segments of rat proximal tubules by means of fluorescent ultramicroassays. Cathepsin activities were increased following dextran injection in a dose-dependent manner. Cathepsin stimulation was not dextran-specific since enhanced proximal albumin uptake also increased cathepsin activities. Acid phosphatase remained almost unchanged. We conclude that increased proximal tubule uptake of macromolecules is accompanied by a rather specific stimulation of proteolytic enzymes in the lysosomes. For proteins this constitutes an adequate response to increased protein uptake to maintain the steady state between uptake and catabolism of proteins in lysosomes. For the nondigestible dextran the cathepsin induction is an inadequate response. Since lysosomal cathepsins are involved in breakdown of intracellular proteins, it is conceivable that the increased activities reflect increased cellular autophagy leading to proximal tubule damage.

Renal hypertrophy in streptozotocin diabetic rats: Role of proteolytic lysosomal enzymes. Renal protein mass increases in diabetic renal hypertrophy. Accretion of protein may he the result of increased protein synthesis and/or decreased protein degradation. The lysosomal proteases, cathepsins B and L, are key enzymes in cellular protein catabolism. To evaluate the role of protein degradation in diabetic renal hypertrophy, the activities of cathepsins B and L were measured in microdissected proximal tubule segments and in kidney cortex homoge-nates. In rats four and ten days following induction of diabetes by streptozotocin, the kidney weight was increased and the cathepsin activities were reduced in proximal tubule segments. Treatment with insulin prevented both changes. The liver weight in diabetic rats was decreased and the activity of cathepsins B and L was increased, while the activity in kidney cortex was reduced. This excluded that diabetes per se may be accompanied by decreased cathepsin activities independent of organ hypertrophy. Renal hypertrophy as a cause rather than as the consequence of reduced cathepsin activities was excluded by the finding of unchanged cathepsin activities in proximal tubule segments from rats with compensatory renal hypertrophy four days and ten days following unilateral nephrectomy. Decreased activities of cathepsins B and L may reflect decreased intracellular protein degradation. Decreased protein breakdown in proximal tubules may contribute to diabetic renal hypertrophy. In agreement with this interpretation are the results from rats six months following induction of diabetes. Renal hypertrophy is complete at that time. No further accretion of protein occurs and the cathepsin activities in the proximal tubule were not different from controls.

Alterations in lysosomal enzymes of the proximal tubule in gentamicin nephrotoxicity. Gentamicin accumulates in proximal tubule lysosomes, increases their number, and changes their structure. An important lysosomal function is degradation of intracellular proteins. To evaluate the effect of gentamicin on this lysosomal function, we measured the activity of the key lysosomal proteinases, cathepsin B and L, in microdissected S1, S2, and S3 segments of rat proximal tubules by means of a fluorometric microassay. The cathepsin activities were decreased in S1 and S2 following one and four gentamicin injections of 100 mg/kg body weight. The lysosomal enzyme, acid phosphatase, was also measured and was not decreased by gentamicin. The urine excretion of cathepsins B and L was decreased after gentamicin. This excludes an increase in urinary loss of cathepsins as the cause of decreased tubule activity. Structural changes of the lysosomes per se were excluded as the factor responsible for the reduced cathepsin activity by demonstrating increased cathepsin B and L activity in proximal tubule segments from rats injected with dextran, since dextran induces an increase in number and size of proximal tubule lysosomes. In vitro incubation of urine and tubule segments with gentamicin demonstrated a concentration-dependent reversible inhibition of cathepsin B and L. We conclude that gentamicin per se decreased cathepsin B and L activities in proximal tubule segments as early as 24 hours following one injection due to either enzyme inhibition or reduced generation of active intralysosomal cathepsin B and L. Gentamicin may, therefore, reduce renal protein catabolism by decreasing the activity of the key proteolytic enzymes, cathepsin B and L. Since cathepsin B and L are proteolytic activators of other lysosomal enzymes, their reduced activity may also decrease the activities of other lysosomal enzymes.

Background. The European tacrolimus versus ciclosporin A microemulsion (CsA-ME) renal transplantation study showed that tacrolimus was significantly more effective in preventing acute rejection and had a superior cardiovascular risk profile at 6 months.Methods. The endpoints of this investigator-initiated, observational, 36-month follow-up were acute rejection incidence rates, rates of patient and graft survival and renal function. An additional analysis was performed using the combined endpoints BPAR, graft loss and patient death. Data available from the original ITT population (557 patients; 286 tacrolimus and 271 CsA-ME) were analysed.Results. A total of 231 tacrolimus and 217 CsA-ME patients participated. At 36 months, Kaplan-Meier-estimated BPAR-free survival rates were 78.8% in the tacrolimus group and 60.6% in the CsA-ME group, graft survival rates were 88.0% and 86.9% and patient survival rates were 96.6% and 96.7%, respectively. The estimated combined endpoint-free survival rate was 71.4% with tacrolimus and 55.4% with CsA-ME (P ≤ 0.001, chi-square test). Significantly more CsA-ME patients crossed over to tacrolimus during the 3-year follow-up: 21.2% versus 2.6%, P ≤ 0.0001, chi-square test. Most patients in the tacrolimus arm discontinued steroids and received monotherapy and fewer tacrolimus patients remained on a triple regimen. Mean serum creatinine concentration was 145.4 ± 90.9 μmol/L with tacrolimus and 149.0 ± 92.1 μmol/L with CsA-ME. Significantly more CsA-ME patients had a classified cholesterol value >6 mmol/L (26.3% versus 12.6%, P ≤ 0.0003, chi-square test).Conclusions. Patients treated with tacrolimus had significantly higher combined endpoint-free survival rates and lower acute rejection rates with less immunosuppressive medication at 36 months.

A patient developed relapsing inflammatory demyelinating polyradiculoneuropathy associated with nephrotic syndrome. Renal biopsy showed focal-segmental glomerulosclerosis. The review of the literature disclosed that glomerulonephritis with and without nephrotic syndrome seems to be not uncommon in inflammatory demyelinating polyradiculoneuropathy, such as Guillain-Barré syndrome. membranous glomerulonephritis is the most frequent histologic diagnosis while minimal change nephropathy, 'acute glomerulonephritis' and postinfectious type glomerulonephritis are not often present. This is the first case of inflammatory demyelinating polyradiculoneuropathy associated with nephrotic syndrome due to focal-segmental glomerulosclerosis. Larger prospective studies are necessary and may contribute to the understanding of the pathogenesis of both, glomerulonephritis and inflammatory demyelinating polyradiculoneuropathy.

Passive Heymann nephritis (PHN) in rats is a model of human membranous nephropathy characterized by formation of subepithelial immune deposits in the glomerular capillary wall and complement activation. Oxygen radicals have been implicated in the subsequent glomerular damage which leads to proteinuria. This study examines the involvement of xanthine oxidase in this process. Xanthine oxidase activity was increased nearly twofold in glomeruli isolated 1 and 12 d after induction of PHN, and this was associated with increased glomerular superoxide anion generation. Analysis of glomerular samples by Northern and Western blotting revealed no quantitative changes in xanthine oxidoreductase expression in PHN, suggesting conversion of xanthine dehydrogenase to the oxidase form as the cause of increased activity. Treatment of rats with tungsten, an inhibitor of xanthine oxidase, before induction of PHN resulted in a marked decrease in glomerular xanthine oxidase activity and superoxide anion generation, and decreased proteinuria by 80% (day 12: 423+/-245 mg/d in PHN versus 78+/-53 mg/d in tungsten-treated PHN animals, P < 0.01). These findings point to a pivotal role of xanthine oxidase in the pathophysiology of PHN and could be of importance in the therapy of human membranous nephropathy.

The inability to separate irreversible lesions of tubular epithelia from reversible tubular atrophy constitutes a major problem in histopathology and in decisions for revascularization of shrunken kidneys with renal artery stenosis. In order to characterize reversible tubular atrophy ('kidney hibernation') we studied the physiological and biochemical parameters and morphology including histochemistry in rat kidneys made atrophic by renal artery stenosis and treatment with the angiotensin-converting enzyme inhibitor, enalapril. Renal artery stenosis was induced by a 0.2-mm clip around the left renal artery. Following 7 weeks of clipping and 2 concomitant weeks of enalapril treatment, the kidney length decreased from 17.8 +/- 0.3 to 13.7 +/- 0.7 mm (mean +/- SEM). Renal blood flow and glomerular filtration rate decreased to 39 +/- 3% and to approximately 3% of control values, respectively. The activities of the intracellular proteolytic enzymes cathepsin B and L and of Na-K-ATPase in microdissected proximal tubular segments decreased to values below 50 and 10%, respectively. All changes were significant (p0.05). Histochemical staining for ATPase activity in the distal tubule segments remained unchanged. Tubular cells were atrophic but not necrotic. Histochemical staining of alkaline phosphatase in the tubular brush border and of acid phosphatase and peroxidase in lysosomes was greatly reduced. All observed changes were reversible within 2-3 weeks following removal of the clip and withdrawal of enalapril either with or without contralateral nephrectomy. Thus, a form of kidney hibernation with readily reversible tubular atrophy has been described. Based on this description it may be possible in consecutive experiments to differentiate between reversible and irreversible tubular atrophy.

Acute and chronic proteinuria were studied in rats, using lysosomal cathepsin B and L as marker enzymes for tubular protein degradation. The activity of cathepsin B and L has been determined in microdissected segments S1, S2 and S3 of the proximal tubule by an ultramicroassay. Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as a substrate. In normoproteinuric Sprague-Dawley rats, induction of acute unselective glomerular proteinuria with Adriamycin (5 mg/kg body weight) revealed a moderate activity increase of cathepsin B and L in the S2 segment, reaching 12.6 ± 5.6 versus 8.6 ± 4.2 pmol·mm-1·min-1 in controls. In contrast, Munich Wistar Frömter (MWF) rats, that are characterized by a genetically determined, chronically elevated glomerular protein excretion, showed a very high activity of cathepsin selectively in S2 of 25.0+12.1 pmol· mm-1 min-1. Acute proteinuria induced by Adriamycin in chronic proteinuric MWF rats could increase cathepsin activity in the S3 segment only, showing 12.0 ± 8.3 versus 6.8 ± 4.0 pmol-mm-1 min-1 in MWF control rats. In conclusion, chronically increased protein filtration changes the functional reserve capacity of the proximal tubule. While acutely induced glomerular proteinuria in normoproteinuric rats stimulates lysosomal proteolytic activity mainly in S2 segment, chronic proteinuric MWF rats may display already a maximally stimulated cathepsin activity in this segment probably due to long-term increased tubular protein load. In case of acute elevation of chronic proteinuria, the consecutive S3 segment shows increased lysosomal function for protein conservation.

Cyclosporin (CsA) induces autophagolysosomes in proximal tubules. A major lysosomal function is degradation of cell proteins. Cathepsin B and L are marker enzymes for the activity of lysosomal protein degradation. Therefore we measured cathepsin activities in microdissected proximal tubule segments and urine from rats treated with CsA, 30 mg/24 h by gavage for 42 days. Cathepsin activity was increased in proximal tubule by 117% and in 24-h urine by 37% compared to controls. Unchanged values of serum cathepsin activity and of proteinuria excluded increased glomerular filtration or decreased tubular absorption as major source of urine cathepsins. Therefore urine cathepsin excretion reflected tubular cathepsin activity. Urine cathepsin excretion was also measured in patients without renal disease treated with CsA for multiple sclerosis (MS). It was increased by 59% in patients with CsA serum values > 120 ng/ml compared to MS patients without CsA or with serum CsA < 120 ng/ml. Urine cathepsin excretion increased linearly with serum CsA (P < 0.001). The data suggest that cathepsin activity is increased in proximal tubules of CsA-treated patients. Hence CsA may stimulate protein degradation in proximal tubules of men and rats.

The presence of autophagolysosomes in proximal tubule cells and the increased urine excretion of the lysosomal enzyme N-acetyl-glucosaminidase following administration of cyclosporine suggests involvement of the lysosomes in tubular toxicity of CsA. To evaluate the effect of CsA on lysosomal function, the activity of the lysosomal proteinases cathepsin B and L was measured in microdissected segments of the proximal tubule by means of a fluorometric microassay. Rats received oral doses of 30 mg/kg CsA for eight weeks. Controls received olive oil. CsA reduced renal blood flow, glomerular filtration rate, and kidney weight. Hence, a second control group was included where the left renal artery was clipped to reduce RBF and GFR. CsA administration was accompanied by a 130% increase in cathepsin activities in the S1 segments of the proximal convoluted tubule. The activity remained unchanged in the pars recta. Enzyme activities in convoluted proximal tubules and pars recta from the control groups were not increased irrespective of reduced RBF, decreased GFR, and decreased KW. Hence, cathepsin B and L stimulation was induced by CsA per se. Since lysosomes are involved in cellular protein catabolism, the increased cathepsin activities may reflect an increased rate of protein breakdown. The tubular atrophy induced by CsA may be related to increased intracellular protein degradation.

Effect of low molecular weight proteins and dextran on renal cathepsin B and L activity. Renal extraction of low molecular weight proteins (LMWP) accounts for 30% to 80% of their total metabolic clearance. Extraction includes glomerular filtration, proximal tubular uptake, and intralysosomal proteolysis. To characterize the anatomic sites and enzymes involved in digestion of reabsorbed LMWP, the lysosomal proteases, cathepsin B and L, were measured by ultramicroassay in isolated S1, S2 and S3 segments of the proximal tubule of proteinuric rats. Increased glomerular filtration and tubular uptake of LMWP were induced by i.v. and i.p. injections of myoglobin and cationic and anionic lysozyme. Both cationic lysozyme and myoglobin increased cathepsin B and L activities in the proximal tubule, while anionic lysozyme had no effect. Morphologic examination of kidney tissue suggested that proximal tubular uptake of anionic lysozyme was negligible in comparison with the cationic form. Hence, only LMWP absorbed by the proximal tubule cells stimulated cathepsin B and L activities. Proximal tubular uptake of cationic lysozyme was determined by measurement of lysozyme activities in S1, S2, and S3. S1 segments contained the highest lysozyme activity, while S2 and S3 had much lower activities, and cathepsin B and L activity following cationic lysozyme injection was stimulated only in S1 segments. These results suggest that cathepsin B and L participate in lysosomal digestion of certain LMWP. Furthermore, the activities of cathepsin B and L adapt to increased uptake of LMWP. To gain additional insight into the mechanism of cathepsin adaptation, the cathepsin B and L activities were measured following injection of dextran with a similar low molecular weight. Dextran uptake in proximal tubules was confirmed by morphologic examination of kidney tissue. Dextran increased cathepsin B and L activities in the proximal tubule. Hence, increased endocytic activity of proximal tubule cells or increased lysosomal load of macromolecules or both rather than direct protein-enzyme interaction seem to be involved in cathepsin stimulation.

The in vivo functional characteristics of continuous arteriovenous hemofiltration (CAVH) were studied in 21 intensive-care patients with acute renal failure. FH-66 hemofilters were applied. The relationships between prefilter blood pressure (BP), blood flow (QB) and filtration rate (QF) were evaluated by stepwise clamping of the arterial access and simultaneous measurements of these parameters. The correlations between BP and QB, and between QB and QF, were linear (p less than 0.001). The total pressure drop across the extracorporeal circuit was 90 +/- 12 mmHg with Scribner shunt and 70 +/- 13 mmHg with femoral catheters as vascular access. The relative pressure drops across arterial access, hemofilter and venous access for Scribner shunt and for femoral catheter were 30%, 43% and 27% and 12%, 74% and 14%, respectively. At a given BP, QB was lower and transmembrane filtration pressure (TMP) higher in CAVH with Scribner shunt. QB was 102 +/- 38 ml/min; QF was 20 +/- 7 ml/min. The effects of hemofilter geometry and membrane material on functional parameters of CAVH were evaluated by applying four hemofilters (Amicon D-20 HP, D-30 HP, Gambro FH-66, Fresenius AV-400) consecutively in the same patient. The filters were different with respect to hollow fiber length, its internal diameter, number of fibers and membrane material. BP, hematocrit (Hct) and plasma protein remained constant during measurements. QB increased with decreasing filter resistance. QF did not increase with increasing QB. QF was also not closely related to membrane surface area. The hydraulic permeability (Lp) had a major impact on QF.(ABSTRACT TRUNCATED AT 250 WORDS)

A patient with severe Crohn's disease was treated with 5-aminosalicylic acid (5-ASA). Following initiation of treatment, serum creatinine increased slowly from 105 to 530 mumol/l (creatinine clearance 16 ml/min). The diagnosis of an interstitial nephritis was made based on normal urinary findings and the renal biopsy histology of interstitial mononuclear infiltrates and normal glomeruli, 5-ASA was discontinued and serum creatinine decreased to 245 mumol/l (creatinine clearance 40 ml/min) during the following 3 months. Partial reversibility of renal failure following discontinuation of 5-ASA and the absence of other drugs possibly causing interstitial nephritis suggest a causal relationship between 5-ASA and interstitial nephritis.

The intralysosomal proteinases cathepsins B and L were measured in microdissected segments of rat nephron. Z-Arg-Arg-NMec served as the substrate for cathepsin B and Z-Phe-Arg-NMec for cathepsin B and L together. Individual S1, S2, and S3 segments of proximal tubules, TDL, MTAL, CTAL, DCT, CCD, and OMCD were dissected from young female rats weighing 130 +/- 11 g with low protein excretion (0.68 +/- 0.1 mg/24 h), from older female rats weighing 289 +/- 9 g with protein excretion of 10 +/- 6.3 mg/24 h, from older male rats weighing 404 +/- 11 g with protein excretion of 22 +/- 6 mg/24 h, from female rats weighing 198 +/- 10 g with albumin-induced proteinuria (411 +/- 134 mg/24 h), and from female rats weighing 203 +/- 11 g with low protein excretion (2.7 +/- 0.4 mg/24 h). The distributions of cathepsin activities along the nephron were similar. In all five groups, S1 and S2 segments had enzyme activities three times higher than in all remaining segments. In S2 and S3, enzyme activities were two to three times higher in proteinuric animals. These findings suggest that in proteinuric animals the increase in the protein load delivered to the proximal tubules selectively stimulated cathepsin activities in the S2 and S3 segments, presumably because of an increase in protein uptake, and that cathepsins B and L participate in lysosomal digestion of protein reabsorbed from the glomerular filtrate via endocytosis.

Minimally invasive diagnosis of renal artery stenosis by spiral computed tomography angiography. We prospectively compared in a blinded fashion spiral computed tomography angiography (CTA) with arteriography in 62 consecutive patients with suspected renal artery stenosis (RAS). For CTA 150 ml of contrast material were injected intravenously. Arteriography was performed by DSA technique with selective catheterization of renal arteries. Of the 157 visualized renal arteries 155 could be evaluated with DSA and a total of 157 with CTA. Sensitivity of CTA for RAS ≥ 50% was 98% and the specificity was 94%. Comparison of the grade of stenosis as evaluated by DSA versus CTA showed: identical gradation in 59 arteries (DSA ≥ 50%/CTA ≥ 50%), underestimation by CTA in one artery (DSA 50 to 75%/CTA < 50%), and overestimation by CTA in six arteries (DSA < 50%/CTA 50 to 75%). Factors that may contribute to these differences include impaired renal function and possibly “underestimation” of ostial RAS by arteriography. One artery not evaluable by arteriography showed a 70% stenosis by CTA. CTA showed no major side effects. We conclude that CTA has the same accuracy for the diagnosis of RAS ≥ 50% as arteriography. However, CTA is only minimally invasive, safe, and causes less discomfort to patients.

Cathepsin B and L in nephron segments of rats with puromycin aminonucleoside nephrosis. The intralysosomal proteinases, cathepsins B and L, were measured in microdissected segments of rat nephrons following a single injection of puromycin aminonucleoside (PAN). Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as substrate. Enzyme activities, proteinuria, creatinine clearance and renal morphology were determined at specific time intervals following induction of PAN nephrosis. During the first three days following PAN injection, enzyme activities in S 2 and S 3 segments, protein excretion, creatinine clearance and appearance of the renal parenchyma resembled control animals. The enzyme activity in S 1 segments was slightly decreased, but returned to control levels at day six after injection. Days four through eight post-PAN injection were characterized by a dramatic increase in protein excretion and an increase in cathepsin B and L activity in S 2 and S 3 segments of the proximal tubule. During days 9 through 15 enzyme activity decreased significantly in S 2 segments despite continued proteinuria. Overt necrosis and cell injury were seen in the proximal tubule and probably account for the decrease in proteolytic activity. After day 15 following PAN injection, the level of proteinuria decreased, restoration of cathepsin activities occurred and a histopathologic picture of healing was present. The data suggest a positive relationship exists between stimulation of cathepsin B and L activity in S 2 and S 3 segments of the proximal tubule and increased protein filtration in PAN nephrosis. The increased enzyme activity reflects enhancement of the proteolytic capacity of the lysosomal system that is necessary for increased protein catabolism.

Gentamicin is an antibiotic with a broad spectrum of antibacterial activity. It is particularly valuable in severe infections. Nephrotoxicity, however, limits its use in just those critically ill patients in whom it is most needed. Nephrotoxicity has been related to a selective accumulation of gentamicin in proximal tubules. It is generally believed that gentamicin enters proximal tubule cells by endocytosis at the luminal cell membrane. Subsequently, the endocytic vacuoles fuse with primary lysosomes to form heterolysosomes (1) (Fig. 1).

Lysosomal acid phosphatase (AcPase) activity was measured in individual segments of rat and rabbit nephrons employing 4-methylumbelliferyl phosphate as the substrate. Generation of reaction product was linear with incubation time up to 127 min and with tubule length. Activity was much higher in glomeruli and proximal tubules of rat than rabbit kidney. In both rat and rabbit there were higher activities in juxtamedullary than in superficial glomeruli. In rats, AcPase activity decreased from S1 to S3 segments, which parallels the known decrease in the number of lysosomes. Surprisingly, in rabbits of both sexes AcPase activity in the cortical collecting duct (CCD), which contains a limited number of lysosomes, was comparable to levels measured in the S1 and S2 segments of the proximal tubule. Similarly, in the male rat values for AcPase activity in the cortical thick ascending limb, distal convoluted tubule, CCD, and medullary collecting duct paralleled those in the S3 segment. These findings suggest that a considerable amount of AcPase in the distal nephron is either extralysosomal or that the amount of lysosomal AcPase activity per unit volume is greater in distal nephron segments compared with the proximal tubule. Different K'm values for AcPase in S1 segments and CCD were found in the rabbit, suggesting the presence of different isoenzymes.

Results from several radical scavenger studies indirectly suggested an involvement of reactive oxygen species in the pathogenesis of puromycin aminonucleoside glomerulopathy. In this study, generation of reactive oxygen species was examined directly in glomeruli isolated from rats in the acute phase of puromycin aminonucleoside nephrosis and related to the changes in the glomerular antioxidant defense. Five and nine days after puromycin aminonucleoside injection, gross proteinuria, reduced creatinine clearances, and typical changes of glomerular morphology were present. Levels of reactive oxygen species were increased eightfold in glomeruli isolated 15 min after puromycin aminonucleoside injection, returned to baseline levels on days 1 and 5 after injection, and rose again to 14-fold on day 9 after injection, as determined by chemiluminescence with luminol. Further analysis of increased glomerular radical generation, using the chemiluminescence enhancer lucigenin and different radical scavengers, suggested a predominant involvement of hydroxyl radical and hydrogen peroxide in the initial increase in reactive oxygen species 15 min after puromycin aminonucleoside. Nine days after induction of nephrosis, primarily superoxide anion and hydroxyl radical were found to contribute to increased reactive oxygen species. Despite oxidative stress, antioxidant enzymes were not induced in the course of nephrosis. On the contrary, catalase and glutathione peroxidase activities declined 9 d after puromycin aminonucleoside injection. The results indicate that a transient increase in glomerular reactive oxygen species is sufficient to induce the oxidative glomerular injury observed in this model and that the glomerulus may not necessarily respond to oxidative stress with an induction of antioxidant enzymes.