Pretransplant virtual PRA and long-term outcomes of kidney transplant recipients.

Virtual panel-reactive antibodies (vPRA) have been implemented to gauge sensitization worldwide. It is unclear how it associates with long-term outcomes, and its correlation with peak (pPRA) or actual (aPRA) has not been studied. We retrospectively reviewed data from 18- to 65-year-old kidney-only transplant patients during 1.1.1996-31.7.2011 in our center. PRAs were calculated based on solid-phase techniques. Of the 726 qualified cases, regardless of the PRA type, sensitized patients (PRA > 5%) had more females and previous transplant. Highly sensitized (HS, PRA > 50%) had longer waiting time, lower transplant rate, less living donor, more delayed graft function, and acute rejection. The conformity between vPRA and pPRA in HS was 75%, 57% between pPRA and aPRA. Forty-three percent (61/142) patients whose pPRA was >5% had no detectable aPRA and maintained similar outcomes as sensitized patients. Multivariate analysis showed consistently lower death-censored graft survival in HS defined by vPRA [HR 2.086 (95% CI 1.078-4.037), P < 0.05] and pPRA [HR 2.139 (95% CI 1.024-4.487), P < 0.05]. Both vPRA and pPRA provided reliable way estimating sensitization and predicting long-term graft survival, while aPRA might underestimate true sensitization. vPRA might be the most objective parameter to gauge sensitization.

Safety and efficacy of regional citrate anticoagulation in continuous venovenous hemodialysis in the presence of liver failure: the Liver Citrate Anticoagulation Threshold (L-CAT) observational study.

INTRODUCTION: Regional citrate anticoagulation (RCA) for continuous renal replacement therapy is widely used in intensive care units (ICUs). However, concern exists about the safety of citrate in patients with liver failure (LF). The aim of this study was to evaluate safety and efficacy of RCA in ICU patients with varying degrees of impaired liver function. METHODS: In a multicenter, prospective, observational study, 133 patients who were treated with RCA and continuous venovenous hemodialysis (RCA-CVVHD) were included. Endpoints for safety were severe acidosis or alkalosis (pH ≤7.2 or ≥7.55, respectively) and severe hypo- or hypercalcemia (ionized calcium ≤0.9 or ≥1.5 mmol/L, respectively) of any cause. The endpoint for efficacy was filter lifetime. For analysis, patients were stratified into three predefined liver function or LF groups according to their baseline serum bilirubin level (normal liver function ≤2 mg/dl, mild LF >2 to ≤7 mg/dl, severe LF >7 mg/dl). RESULTS: We included 48 patients with normal liver function, 43 with mild LF, and 42 with severe LF. LF was predominantly due to ischemia (39 %) or multiple organ dysfunction syndrome (27 %). The frequency of safety endpoints in the three patient strata did not differ: severe alkalosis (normal liver function 2 %, mild LF 0 %, severe LF 5 %; p = 0.41), severe acidosis (normal liver function 13 %, mild LF 16 %, severe LF 14 %; p = 0.95), severe hypocalcemia (normal liver function 8 %, mild LF 14 %, severe LF 12 %; p = 0.70), and severe hypercalcemia (0 % in all strata). Only three patients showed signs of impaired citrate metabolism. Overall filter patency was 49 % at 72 h. After censoring for stop of the treatment due to non-clotting causes, estimated 72-h filter survival was 96 %. CONCLUSIONS: RCA-CVVHD can be safely used in patients with LF. The technique yields excellent filter patency and thus can be recommended as first-line anticoagulation for the majority of ICU patients. TRIAL REGISTRATION: ISRCTN Registry identifier: ISRCTN92716512 . Date assigned: 4 December 2008.

Midterm echocardiographic follow-up of cardiac function after living kidney donation.

BACKGROUND: Living kidney donation (LKD) has become increasingly important as more patients reach end-stage renal disease. While safety of the donor is of utmost importance, recent data have suggested an increased risk for cardiovascular mortality after LKD. Therefore, we assessed the changes of cardiac structure and function after LKD by advanced echocardiographic methods. METHODS: 30 living kidney donors were evaluated by medical examination, laboratory testing, and echocardiography before and after LKD (median follow-up 19.5 months). Left ventricular (LV) and right ventricular (RV) function was assessed by echocardiographic standard indices. Longitudinal 2D strain of the LV and left atrium (LA) was determined by 2D speckle tracking. RESULTS: Serum creatinine increased significantly from 0.80 ± 0.12 mg/dL to 1.18 ± 0.21 mg/ dL (p < 0.001) after LKD. There was a trend to higher blood pressure after LKD, accompanied with significantly higher intake of antihypertensive drugs. Echocardiographic parameters of LV, LA, and RV function did not change significantly after LKD. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels remained within normal ranges after LKD. CONCLUSION: The rise in serum creatinine and blood pressure indicates that patients have a potentially higher cardiac risk after LKD. However, our pilot study found no evidence for detrimental effects of LKD on cardiac structure and function within a relatively short-term follow-up.

Evaluation of acid-base control, electrolyte balance, and filter patency of a Prismaflex-based regional citrate anticoagulation protocol for pre-dilution continuous veno-venous hemodiafiltration.

BACKGROUND: Regional citrate anticoagulation (RCA) is increasingly used in patients requiring continuous renal replacement therapy (CRRT). This study evaluated a new RCA protocol based on the Prismaflex® dialysis device and an isotonic citrate solution (prismocitrate) for pre-dilution continous veno-venous hemodiafiltration. METHODS: The Prismaflex®/Prismocitrate-based protocol involved an AN69ST® membrane (Prisma Flex ST100), a blood flow of 120 mL/min, 1.8 L/h Prismocitrate (10 mmol/L citrate/2 mmol/L citric acid) substitution fluid in pre-dilution mode, and 0.8 L/h dialysate flow (PrismOcal) at the start. In parallel, infusions of potassium, calcium, and magnesium were initiated. Blood pH, bicarbonate, base excess, and ionized calcium levels were measured in 6 hours intervals and magnesium levels every 24 hours. Scheduled hemofilter run time was 72 hours. RESULTS: A consecutive series of 25 continuous renal replacement treatments was analyzed in 15 patients. After at least 6h of RRT, 69.9% of bicarbonate concentrations and 84.6% base excess (BE) calculations were below normal range. During CRRT, mean bicarbonate decreased from 22.9 to 20.2 mmol/L and mean BE from -1.5 to -4.2 mmol/L. In addition, 66.3% of ionized systemic calcium concentrations were out of the normal range, while 54.1% of the magnesium readings were above normal range. Five filters reached the scheduled run time of 72 hours, 19 treatments stopped prematurely because of RRT related reasons (5 filter clottings, 2 severe metabolic disarrangements, 12 major Prismaflex® hardware or software handling problems). One patient was switched to intermittent hemodialysis. CONCLUSIONS: The evaluated Prismaflex®/ Prismocitrate-based citrate anticoagulation protocol provides insufficient control of blood acid-base and electrolyte balance.

Volume matters: CT-based renal cortex volume measurement in the evaluation of living kidney donors.

Currently, no international standard for the pre-transplant evaluation of living donor renal function exists. Following a standardized questionnaire on current practice in all Eurotransplant (ET) centers, we compared a new CT-based technique to measure renal cortex volume with our standard of DTPA-clearance combined with MAG3-scintigraphy (DTPA × MAG3) and with creatinine-based methods in 167 consecutive living kidney donors. Most ET centers use creatinine-clearance (64%) to measure total renal function and radioistopic methods (82%) to assess split renal function. Before transplantation, CT-measured total cortex volume (r = 0.67; P < 0.001) and estimated GFR using the Cockcroft-Gault formula [eGFR(CG)] (r = 0.55; P < 0.001) showed the strongest correlation with DTPA-clearance. In contrast, the correlation between DTPA-clearance and creatinine clearance was weak (r = 0.21; P = 0.02). A strong correlation was observed between CT-measured split cortex volume and MAG3-measured split renal function (r = 0.93; P < 0.001). A strong correlation was also found between pre-transplant split renal function assessed by eGFR(CG) together with cortex volume measurement and post-transplant eGFR(CG) of both, the donor (r = 0.83; P < 0.001) and the recipient (r = 0.75; P < 0.001). In conclusion CT-based assessment of renal cortex volume bears the potential to substitute existing methods to assess pre-transplant living donor split renal function.

Tyrosine kinases inhibition by Imatinib slows progression in chronic anti-thy1 glomerulosclerosis of the rat.

BACKGROUND: Chronic progressive mesangioproliferative nephropathy represents a major cause of end-stage renal disease worldwide. Until now, effective approaches to stop or even slow its progression are limited. We tested the effects of an inhibitor of PDGF receptor, abl and c-kit tyrosine kinases, Imatinib, in a chronic progressive model of mesangioproliferative glomerulosclerosis. METHODS: Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, according to the degree of proteinuria, animals were stratified and assigned to chronic glomerulosclerosis (cGS) and cGS plus Imatinib (10 mg/kg body weight/day). In week 20, renoprotective actions of Imatinib were analyzed by a set of functional, histological and molecular biological parameters. RESULTS: Untreated cGS rats showed elevation of systolic blood pressure and marked progression in proteinuria, renal fibrosis, cell infiltration, cell proliferation and function lost. Administration of Imatinib went along significantly with lower systolic blood pressure (-10 mmHg) and proteinuria (-33%). Imatinib administration was paralled by significant reductions in tubulointerstitial accumulation of matrix proteins (-44%), collagen I deposition (-86%), expression of TGF-beta1 (-30%), production of fibronectin (-23%), myofibroblast differentiation (-87%), macrophage infiltration (-36%) and cell proliferation (-45%), respectively. In comparison with untreated cGS animals, Imatinib therapy lowered also blood creatinine (-41%) and blood urea concentrations (-36%) and improved creatinine clearance (+25%). Glomerular fibrotic changes were lowered moderately by Imatinib. CONCLUSIONS: Therapy with Imatinib limits the progressive course of chronic anti-thy1 glomerulosclerosis towards tubulointerstitial fibrosis and renal insufficiency. This was paralleled by direct and indirect sign of TGF-ß1 and PDGF inhibition. The findings suggest that the pharmacological principal of inhibition of tyrosine kinases with drugs such as Imatinib might serve as approach for limiting progression of human mesangioproliferative glomerulosclerosis.

Extended daily on-line high-volume haemodiafiltration in septic multiple organ failure: a well-tolerated and feasible procedure.

BACKGROUND: The outcome of patients with septic multiple organ failure (MOF) remains poor. There are experimental and clinical data indicating a beneficial effect of high-volume haemofiltration. Delivering high-volume therapy is only cost effective using on-line devices because of high costs for additional solution bags in conventional continuous renal replacement therapy (CRRT). We investigated feasibility and effectiveness of extended daily on-line high-volume haemodiafiltration (HDF) with technically maximum convective volume in patients with septic MOF in a pilot study. METHODS: We included 21 consecutive critically ill patients with septic MOF having a mortality risk >50% (SAPS II >50, APACHE II >25). Renal replacement therapy (RRT) was applied with extended daily HDF for 6-23 h using the AK 200 Ultra S dialysis machine in the ultracontrol pre-dilution mode. Dialysate and substitution fluid were prepared on-line. Patients underwent 289 treatments. RESULTS: The mean convective volume was 17.8 ± 3.7 L/h and 208 ± 66 mL/kg/h, respectively, median treatment time was 10:15 h/day. Seventeen of 21 patients survived 28 days (81%). The 90-day survival rate was 52% (11/21) versus 19% compared to the survival rate predicted by APACHE II (33.6 mean) and SAPS II (68.6 mean) scores. Haemodynamics improved significantly during the treatment procedures. Material costs per treatment amounted to 35 €. CONCLUSIONS: Extended daily on-line HDF using maximum convective volume seems to improve the outcome of septic MOF, especially in the early phase. The investigated mode of treatment proved to be feasible, well tolerated and highly cost effective compared to conventional CRRT. At present, this procedure would be applicable at every ICU facility with nephrological support.

Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection.

BACKGROUND: Antibody-mediated rejection (ABMR) following kidney transplantation is associated with poor allograft survival. Conventional treatment based on plasmapheresis (PPH) and the administration of intravenous immunoglobulins (IVIG) is not satisfactory. Two compounds, more specifically targeting B cells and plasma cells, may help to improve the prognosis: rituximab, a B-cell-depleting monoclonal antibody, and bortezomib, a proteasome inhibitor causing apoptosis of plasma cells. METHODS: Starting in February 2009, we treated 10 consecutive patients with ABMR according to current Banff criteria with one cycle of bortezomib [1.3 mg/m(2) intravenously (i.v.), Day 1, 4, 8, 11]. This group was compared to a historical control group of patients (n = 9) treated with a fixed single dose of rituximab (500 mg i.v.). All patients received PPH (6×) and IVIG (30 g). Patients with acute ABMR additionally received methylprednisolone (3 × 500 mg i.v.). RESULTS: Patient survival in both groups was 100%. At 18 months after treatment, graft survival was 6/10 in the bortezomib group as compared to 1/9 functioning grafts in the rituximab group (P = 0.071). Renal function was superior in patients treated with bortezomib as compared to rituximab-treated patients (serum creatinine at 9 months: 2.5 ± 0.6 versus 5.1 ± 2.1 mg/dL, P = 0.008). Proteinuria was not different between both groups (9 months: 1.3 ± 1.0 versus 1.6 ± 1.6 g/day, P = n.s.). CONCLUSIONS: Treatment of ABMR with bortezomib in addition to standard therapy was partially effective, whereas treatment with a fixed dose of rituximab in addition to standard therapy with PPH and IVIG did not result in sufficient long-term graft survival. In the future, new strategies including the combination of both substances and the application of higher doses must be discussed.

Effects of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function and activation.

Humoral rejection processes may lead to allograft injury and subsequent dysfunction. Today, only one B-cell-specific agent is in clinical use and the effects of standard and new immunosuppressant substances on B-cell activation and function are not fully clarified. The impact of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function was assessed by analysing proliferation, apoptosis, CD80/CD86 expression and immunoglobulin and IL-10 production in primary stimulated B cells. In addition, B-cell co-cultures with pre-activated T cells were performed to evaluate the effect of the different immunosuppressive agents on T-cell-dependent immunoglobulin production. Sotrastaurin did not inhibit B-cell proliferation, CD80/CD86 expression, and IgG production and had only minor effects on IgM levels at the highest concentration administered. In contrast, mycophenolic acid and everolimus had strong effects on all B-cell functions in a dose-dependent manner. All immunosuppressive agents caused decreased immunoglobulin levels in T-cell-dependent B-cell cultures. The data provided here suggest that mycophenolic acid and everolimus, but not sotrastaurin, are potent inhibitors of human B-lymphocyte function and activation.

Differential impact of the CYP3A5*1 and CYP3A5*3 alleles on pre-dose concentrations of two tacrolimus formulations.

OBJECTIVES: We investigated the pharmacokinetic and pharmacogenetic implications of conversion from a twice-daily (P-Tac) to a once-daily (A-Tac) tacrolimus (Tac) formulation. METHODS: We analyzed Tac levels in a cohort of 41 renal transplant patients with a stable graft function over a period of 1 year before and after conversion. RESULTS: After conversion, the patients had, on average, significantly lower Tac trough and dose-normalized trough levels (14%, P=0.0004 and 23%, P=0.001, respectively) despite similar doses. CYP3A5*3/*3 patients (n=27) required significantly lower Tac doses with both the formulations to reach Tac target levels (P-Tac 39%, P=0.011; A-Tac 36%, P=0.003) compared with *1/*3 patients (n=13). Interestingly, after the conversion, mean Tac trough levels and dose-normalized trough level remained almost constant in *1/*3 patients, but decreased significantly in *3/*3 patients (16%, P=0.001 and 25%, P=0.006). CONCLUSION: This study provides further evidence that the CYP3A5*1/*3 polymorphism significantly impacts Tac pharmacokinetics. Moreover, we show for the first time a pharmacogenetic effect on two different Tac formulations, as Tac trough levels of *3/*3 patients declined significantly after conversion to identical A-Tac doses.

Stimulation of soluble guanylate cyclase improves renal recovery after relief of unilateral ureteral obstruction.

PURPOSE: The antifibrotic effects of soluble guanylate cyclase stimulation and cyclic guanosine monophosphate production have been observed in cases of anti-thy1-induced renal disease. We analyzed the action of the specific soluble guanylate cyclase stimulator BAY 41-8543 on the renal recovery phase in rats with unilateral ureteral obstruction after obstruction was relieved. MATERIALS AND METHODS: Sprague-Dawley® rats underwent reversible unilateral ureteral obstruction for 5 days, after which obstruction was relieved. Rats were randomly assigned to unilateral ureteral obstruction and unilateral ureteral obstruction plus BAY 41-8543 (10 mg/kg body weight daily). Seven days after relief of obstruction we determined treatment effects on renal atrophy, apoptosis, fibrosis and nitric oxide/cyclic guanosine monophosphate signaling. RESULTS: Untreated obstructed rats showed mildly increased systolic blood pressure, marked tubular atrophy and apoptosis, tubulointerstitial macrophage infiltration and fibrosis. Plasma cyclic guanosine monophosphate levels were unaltered in untreated rats with obstruction while renal soluble guanylate cyclase mRNA expression was increased. BAY 41-8543 administration significantly increased plasma cyclic guanosine monophosphate, which was paralleled by significant decreases in systolic blood pressure, renal tubular diameter, apoptosis and renal macrophage infiltration. Also, soluble guanylate cyclase stimulation decreased tubulointerstitial fibrosis, as shown by tubulointerstitial volume, matrix protein accumulation, α-smooth muscle actin expression, collagen IV deposition and transforming growth factor-ß1 mRNA expression. CONCLUSIONS: Soluble guanylate cyclase stimulation by BAY 41-8543 increases cyclic guanosine monophosphate production and subsequently enhances renal recovery after unilateral ureteral obstruction relief through an array of pathways. This finding suggests that soluble guanylate cyclase stimulation may serve as a novel treatment approach to restore or preserve renal structure and function in cases of obstructive kidney disease.

Inosine 5'-Monophosphate Dehydrogenase Activity for the Longitudinal Monitoring of Mycophenolic Acid Treatment in Kidney Allograft Recipients.

BACKGROUND: Mycophenolic acid (MPA) is a standard immunosuppressant in organ transplantation. A simple monitoring biomarker for MPA treatment has not been established so far. Here, we describe inosine 5'-monophosphate dehydrogenase (IMPDH) monitoring in erythrocytes and its application to kidney allograft recipients. METHODS: IMPDH activity measurements were performed using a high-performance liquid chromatography assay. Based on 4203 IMPDH measurements from 1021 patients, we retrospectively explored the dynamics early after treatment start. In addition, we analyzed the influence of clinically relevant variables on IMPDH activity in a multivariate model using data from 711 stable patients. Associations between IMPDH activity and clinical events were evaluated in hospitalized patients. RESULTS: We found that IMPDH activity reflects MPA exposure after 8 weeks of constant dosing. In addition to dosage, body mass index, renal function, and coimmunosuppression affected IMPDH activity. Significantly lower IMPDH activities were found in patients with biopsy-proven acute rejection as compared to patients without rejection (median [interquartile range]: 696 [358-1484] versus 1265 [867-1618] pmol xanthosine-5'-monophosphate/h/mg hemoglobin, P < 0.001). The highest IMPDH activities were observed in hospitalized patients with clinically evident MPA toxicity as compared to patients with hospitalization not related to MPA treatment (1548 [1021-2270] versus 1072 [707-1439] pmol xanthosine-5'-monophosphate/h/mg hemoglobin; P < 0.001). Receiver operating characteristic curve analyses underlined the usefulness of IMPDH to predict rejection episodes (area, 0.662; confidence interval, 0.584-0.740; P < 0.001) and MPA-associated adverse events (area, 0.632; confidence interval, 0.581-0.683; P < 0.001), respectively. CONCLUSIONS: IMPDH measurement in erythrocytes is a novel and useful strategy for the longitudinal monitoring of MPA treatment.

Effects of the new immunosuppressive agent AEB071 on human immune cells.

UNLABELLED: BACKGROUND. The novel immunosuppressive agent AEB071 is currently being evaluated for its capability to prevent rejection after kidney transplantation as a potential adjunct to calcineurin inhibitor-based regimen. AEB071 is a selective protein kinase C inhibitor and has been shown to be well tolerated in humans. We here present extensive in vitro studies that contribute to the understanding of AEB071 effects on human lymphocyte, natural killer (NK) cell and dendritic cell (DC) action. METHODS: The impact of AEB071 on several T-cell activation and costimulatory markers was assessed. Furthermore, assays were performed to study the effect on T-cell proliferation and intracellular cytokine production. Additionally, the effect of AEB071 on DC maturation and their capacity to stimulate allogeneic T-cells was examined. Also, an evaluation of AEB071 effects on the lytic activity of human NK cells was performed. RESULTS: We were able to show that T-cell proliferation and cytokine production rates are significantly reduced after AEB071 administration. Also, mitogen-induced T-cell activation characterized by expression levels of surface markers could be significantly inhibited. In contrast, the T-cell stimulatory capacity of AEB071-treated mature monocyte-derived DC (Mo-DC) is not reduced, and AEB071 administration does not prevent lipopolysaccharide (LPS)-induced Mo-DC maturation. It could be demonstrated that AEB071 significantly inhibited the cytotoxic activity of NK cells. CONCLUSIONS: The promising immunosuppressive agent AEB071 has a strong impact on T-cell activation, proliferation and cytokine production as well as NK cell activity, but not DC maturation in vitro, and therefore, seems to function T-cell and NK cell specific via protein kinase C (PKC) inhibition.

Impact of daclizumab, low-dose cyclosporine, mycophenolate mofetil and steroids on renal function after kidney transplantation.

BACKGROUND: Early and long-term use of cyclosporine A (CsA) leads to increased risks of renal toxicity. We hypothesized that administration of daclizumab in combination with mycophenolate mofetil (MMF) allows a relevant reduction in the dose of CsA. METHODS: We carried out a 3-year, prospective, randomized, controlled clinical multi-centre trial in 156 patients. The patients were randomized to standard treatment (CsA, MMF, steroids) or to high-dose daclizumab (first dose: 2 mg/kg), in combination with low-dose CsA, MMF and steroids. We maintained the mean CsA levels of daclizumab patients at 57% of standard patients (132 versus 216 ng/ml) on Day 7 post-transplant, and 84% by 6 months. RESULTS: Primary outcome, creatinine clearance (with imputation of informative dropouts) at 12 months, was significantly better in daclizumab-treated (34 +/- 17) than standard patients (29 +/- 17; P = 0.028, two sided). Only 5 cases of BPAR were recorded in the daclizumab compared to 22 in the standard group (P = 0.0016). Daclizumab patients had 91% event-free survival after 1 year compared to 66% in standard patients (P = 0.00017). CONCLUSION: We demonstrate here that high-dose daclizumab in combination with lower CsA levels in adult renal transplant recipients is as or more effective than standard regimen (CsA, MMF, steroids) and may result in better outcomes at 12 months post-transplant with no increase in adverse reactions.

The lymphocyte migration inhibitor FTY720 attenuates experimental hypertensive nephropathy.

The lymphocyte migration inhibitor FTY720 attenuates experimental hypertensive nephropathy. Infiltration with lymphocytes is found in both immune and nonimmune chronic kidney diseases. In a rat model of immune-initiated progressive glomerulosclerosis, selective inhibition of lymphocyte infiltration by FTY720 showed significant beneficial effects on renal fibrosis. To test whether this translates into hypertensive nephropathy (HN), the lymphocyte migration inhibitor was administered to rats following nephrectomy. Two days after surgery, male Wistar rats were allocated to the following groups: Sham surgery, nephrectomy (HN), and HN + FTY720 (0.3 mg/kg body wt). Therapy was continued for 6 wk. Treatment with FTY720 was found to selectively reduce blood lymphocyte counts by 85% (P < 0.001 vs. HN) and renal lymphocyte infiltration (CD-3 positive cells) by 63% (P < 0.01 vs. HN) as was anticipated. Lymphocyte depletion went along with a significant reduction in proteinuria (-28%), whereas hypertensive systemic blood pressure remained unchanged (160 +/- 5 vs. 161 +/- 5 mmHg, P = not significant). The markedly increased histological tubulointerstitial and glomerular matrix protein accumulation, collagen, laminin, and fibronectin deposition were all significantly impeded in the FTY720-treated animals. The anti-fibrotic effects of FTY720 were paralleled by significant reductions in renal transforming growth factor (TGF)-beta overexpression, macrophage infiltration, and cell proliferation. In conclusion, the lymphocyte migration inhibitor FTY720 significantly limits histological and molecular fibrosis in a model of hypertensive nephropathy without affecting increased systemic blood pressure. Prevention of renal lymphocytes' infiltration by FTY720 was followed by significant reductions in TGF-beta overexpression, macrophage infiltration, and renal cell proliferation. These results suggest that infiltrating lymphocytes play an active, profibrotic role in the progression of hypertensive renal tissue injury.

Chronic allograft nephropathy--a clinical syndrome: early detection and the potential role of proliferation signal inhibitors.

Chronic allograft nephropathy (CAN) leads to the majority of late graft loss following renal transplantation. Detection of CAN is often too late to permit early intervention and successful management. Most current strategies for managing CAN rely on minimizing or eliminating calcineurin inhibitors (CNIs) once CAN has become established. The proliferation signal inhibitors everolimus and sirolimus have potent immunosuppressive and antiproliferative actions, with the potential to alter the natural history of CAN by reducing CNI exposure whilst avoiding acute rejection. Whilst data will be forthcoming from a number of clinical trials investigating this potential, we discuss early detection of CAN and the rationale for a role for this class of agent.

Improved assay for the nonradioactive determination of inosine 5'-monophosphate dehydrogenase activity in peripheral blood mononuclear cells.

Mycophenolic acid (MPA) inhibits the enzyme inosine 5'-monophosphate dehydrogenase (IMPDH). Thus, the measurement of IMPDH activity could serve as a specific pharmacodynamic (PD) tool for monitoring MPA therapy. At present, however, monitoring of pharmacokinetic parameters is preferred over that of PD parameters because, in general, PD assays are labor-intensive and poorly reproducible. Currently, cell count or protein concentration is widely accepted as methods to normalize enzyme activity. In the present study, we have attempted to further improve a method for the determination of IMPDH activity to increase the robustness and reproducibility of the IMPDH activity assay itself, without making the assay more labor-intensive. Therefore, several aspects of the IMPDH method were investigated regarding their influence on the reproducibility and also modified to increase the feasibility and consistency of the assay. The isolation of peripheral blood mononuclear cells (PBMCs) of whole blood samples was found to be the most variable step. Normalization on cell count is labor-intensive and at the same time has a poor reproducibility. Determination of the protein content in cell extracts is impaired by contamination with extracellular proteins and non-PBMCs. Alternatively, the intracellular substance adenosine monophosphate (AMP) was investigated to normalize the newly generated xanthosine monophosphate. Among various subject groups, no significant differences in mean AMP concentration were found. To simplify the procedure, PBMCs were diluted to a fixed volume after isolation from sample of whole blood, and the IMPDH activity was normalized to the AMP concentration quantified in the same high-performance liquid chromatography run as xanthosine monophosphate was quantified. The within-run and total imprecision (coefficient of variation) ranged from 4.2% to 10.6% and from 6.6% to 11.9%, respectively. In conclusion, the modified method described here for the measurement of IMPDH activity can be used reliably in multicenter trials and in longitudinal studies to evaluate the additional value of any PD monitoring among a diversity of patients treated with MPA.

NO signaling through cGMP in renal tissue fibrosis and beyond: key pathway and novel therapeutic target.

Nitric oxide (NO) produced by endothelial NO synthase (NOS) in low concentrations is a unique messenger molecule with key homeostatic functions concerning the prevention of pathological vascular and tissue changes such as increases in blood pressure, platelet degranulation, mononuclear cell infiltration, cell proliferation and extracellular matrix protein accumulation. This is in contrast to high levels of NO derived from inducible NOS which act as detrimental effector molecules and free radicals in immune response. Deficiency in NO's protective signaling actions is a major characteristic in numerous experimental and human disease situations. The main function of the NO signaling pathway is activation of the soluble guanylate cyclase (sGC) enzyme with subsequent generation of cyclic guanosine monophosphate (cGMP) as a second messenger and downstream mediator. In the past, attempts to overcome deficiency in endothelial NO effects were focused primarily on increasing the supply with the NO precursor L-arginine or on the use of directly NO-releasing compounds. The clinical impact of these strategies, however, was rather limited. Recent state-of-the-art studies have revealed that NO signaling is highly regulated at the transcriptional level and that deficiency in NO signaling correlates closely with pathological changes. In parallel efforts, novel pharmacological compounds which specifically enhance NO/cGMP signaling have been developed and have demonstrated remarkable efficacy in experimental disease settings. In this review, we summarize the current state of knowledge on the impairment of NO/cGMP signaling and about its pharmacological stimulation. In the first part, experimental renal fibrosis, i.e. the tandem rat model of acute anti-thy1 glomerulonephritis and progressive anti-thy1 renal fibrosis will serve as a paradigm for introducing this new and exciting field. In the second part, we will address the most recent findings on NO signaling in non-renal diseases. Together, these results point out that deficiency in NO/cGMP is a common key pathway as well as a novel therapeutic target in a number of diseases.

Long-term outcomes after acute kidney injury.

Acute kidney dysfunction is a common problem in intensive care units. It is not only associated with increased morbidity and mortality but also with increased healthcare costs. Limited healthcare budgets have now raised the issue of how much therapy should be dedicated to these critically ill patients. A precondition for any further discussion on this topic is the question on the long-term outcome and quality of life of these patients. However, only limited data are available in this field. In this review, we will focus on the existing literature, considering not only acute renal failure patients requiring renal replacement therapy but also those patients with mild or moderate impaired renal function. The intention of this review is to show that acute kidney injury is an important but often underestimated disease and a disease that deserves major attention because it is associated with impaired short- and long-term outcome. We will demonstrate that acute kidney injury patients requiring dialysis have a reasonable long-term survival rate and good quality of life. There is no doubt that aggressive intensive care unit treatment is justified in these patients, irrespective of the health costs.

Pharmacodynamic effects of everolimus on anti-CD3 antibody-stimulated T-lymphocyte proliferation and interleukin-10 synthesis in stable kidney-transplant patients.

Everolimus (rapamycin derivative, RAD) is a new immunosuppressive drug that prevents allograft rejection. Herein, the pharmacodynamics of everolimus in human renal-allograft recipients is evaluated. Single doses of everolimus (0.75-10mg), combined with a maintenance immunosuppressive therapy based on CyA, decreased lymphocyte proliferation. In addition, the effect of multiple doses of everolimus (0.75-10mg) given daily for 21 days, to stable renal-allograft patients (n=11), was investigated. Everolimus treatment resulted in immediate inhibition (25-55%) of lymphocyte proliferation in renal-allograft recipients; values returning to baseline by 14 days after cessation of everolimus treatment. Placebo-treated patients showed no decrease in lymphocyte proliferation. Interestingly, everolimus reduced IL-10 synthesis by 20-60% in renal-allograft recipients. Phagocytosis rates were not changed by everolimus. In vitro, everolimus inhibited lymphocyte proliferation and IL-10 synthesis dose dependently in anti-CD3 mAb and LPS stimulated peripheral blood mononuclear cell cultures derived from human volunteers.