Dealing with prognostic signature instability: a strategy illustrated for cardiovascular events in patients with end-stage renal disease.

BACKGROUND: Identification of prognostic gene expression markers from clinical cohorts might help to better understand disease etiology. A set of potentially important markers can be automatically selected when linking gene expression covariates to a clinical endpoint by multivariable regression models and regularized parameter estimation. However, this is hampered by instability due to selection from many measurements. Stability can be assessed by resampling techniques, which might guide modeling decisions, such as choice of the model class or the specific endpoint definition. METHODS: We specifically propose a strategy for judging the impact of different endpoint definitions, endpoint updates, different approaches for marker selection, and exclusion of outliers. This strategy is illustrated for a study with end-stage renal disease patients, who experience a yearly mortality of more than 20 %, with almost 50 % sudden cardiac death or myocardial infarction. The underlying etiology is poorly understood, and we specifically point out how our strategy can help to identify novel prognostic markers and targets for therapeutic interventions. RESULTS: For markers such as the potentially prognostic platelet glycoprotein IIb, the endpoint definition, in combination with the signature building approach is seen to have the largest impact. Removal of outliers, as identified by the proposed strategy, is also seen to considerably improve stability. CONCLUSIONS: As the proposed strategy allowed us to precisely quantify the impact of modeling choices on the stability of marker identification, we suggest routine use also in other applications to prevent analysis-specific results, which are unstable, i.e. not reproducible.

Survival and distribution of injected haematopoietic stem cells in acute kidney injury.

BACKGROUND: Endogenous bone marrow-derived cells are known to incorporate into renal epithelium at a low rate. Haematopoietic stem cells (HSCs) rather than mesenchymal stem cells (MSC) are responsible for this phenomenon. MSCs have the potential to ameliorate kidney function after acute kidney injury (AKI) without directly repopulating the tubules. However, little is known about the short-term effect of HSCs. METHODS: In this article, we analysed the survival rate and organ distribution of isolated rat HSCs injected into the renal artery after ischaemic renal injury, using quantitative real-time PCR, as well as their impact on renal function and histomorphology. RESULTS: Intra-arterially injected Lin(-)CD90(+) HSCs were detected in the kidney at significant amounts only within the first 24 h after injection and were virtually absent by Day 2. Compared with control animals, no differences were seen after HSC administration with respect to kidney function or histomorphologic changes of AKI. At Day 7 HSCs were again readily detectable in the kidney suggesting a redistribution of cells at later time points. Of note, HSCs did not seem to have an exclusive tropism for the injured kidney but were detectable in the lungs, liver, spleen, heart and brain at all time points. CONCLUSIONS: Injected HSCs do not appear to significantly contribute to tubular repair or ameliorate renal damage in ischaemic AKI although they may show considerable engraftment in various organs. These data further challenge the concept that injection of HSCs may be used as a therapeutic approach in treating AKI.

Indicators of acute and persistent renal damage in adult thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathies (TMA) in adults such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are life-threatening disorders if untreated. Clinical presentation is highly variable and prognostic factors for clinical course and outcome are not well established. METHODS: We performed a retrospective observational study of 62 patients with TMA, 22 males and 40 females aged 16 to 76 years, treated with plasma exchange at one center to identify clinical risk factors for the development of renal insufficiency. RESULTS: On admission, 39 of 62 patients (63%) had acute renal failure (ARF) with 32 patients (52%) requiring dialysis treatment. High systolic arterial pressure (SAP, p = 0.009) or mean arterial pressure (MAP, p = 0.027) on admission was associated with acute renal failure. Patients with SAP>140 mmHg on admission had a sevenfold increased risk of severe kidney disease (OR 7.464, CI 2.097-26.565). MAP>100 mmHg indicated a fourfold increased risk for acute renal failure (OR 4.261, CI 1.400-12.972). High SAP, diastolic arterial pressure (DAP), and MAP on admission were also independent risk factors for persistent renal insufficiency with the strongest correlation for high MAP. Moreover, a high C-reactive protein (CRP) level on admission correlated with renal failure in the course of the disease (p = 0.003). At discharge, renal function in 11 of 39 patients (28%) had fully recovered, 14 patients (23%) remained on dialysis, and 14 patients (23%) had non-dialysis-dependent chronic kidney disease. Seven patients (11%) died. We identified an older age as risk factor for death. CONCLUSIONS: High blood pressure as well as high CRP serum levels on admission are associated with renal insufficiency in TMA. High blood pressure on admission is also a strong predictor of sustained renal insufficiency. Thus, adult TMA patients with high blood pressure may require special attention to prevent persistent renal failure.

Rituximab treatment for adults with refractory nephrotic syndrome: a single-center experience and review of the literature.

BACKGROUND/AIMS: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are common causes of nephrotic syndrome (NS) in adults. However, induction of remission and sustained control of proteinuria is often difficult. Recently, B cell-directed therapy using the anti-CD20 antibody rituximab has been suggested as induction regimen in pediatric FSGS and MCD patients. Data on rituximab use in adults are still limited. METHODS: We report on rituximab use in five consecutively treated adult patients (mean age 42.2 ± 9.9 years) with FSGS or relapsing MCD (2 FSGS, 3 MCD) who failed to respond to standard immunosuppressive treatment. Median follow-up was 8 months (3-25). RESULTS: Rituximab induced complete remission in 2 MCD patients and partial remission in 3 patients. Proteinuria was reduced by 86.8% (42.9-95.2) 3 months and by 73.0% (60.1-95.5) 6 months after therapy. In 1 patient with severe FSGS, partial remission was not evident before 6 months after rituximab treatment. Relapses occurred in 2 patients. No severe adverse events related to rituximab were observed. CONCLUSION: Our findings suggest that B cell-directed therapies are novel treatment options for adults with refractory NS. Response to rituximab varied, with MCD patients exhibiting a faster and more pronounced response compared to FSGS patients.

ABO-incompatible kidney transplantation using regenerative selective immunoglobulin adsorption.

BACKGROUND: ABO-incompatible (ABOi) kidney transplantation is an established procedure relying on the removal of donor-specific isoagglutinine antibodies as part of the recipient preconditioning. At present, current protocols using immunoadsorption apply a single-use selective carbohydrate isoagglutinine adsorber. A regenerative and selective immunoglobulin immunoadsorption could be an alternative but has not been reported for ABOi transplantation. METHODS: Eight patients were treated with the commonly used isoagglutinine carbohydrate epitope adsorber and seven with a regenerative polyclonal sheep anti-immunoglobulin adsorber as part of the preconditioning for ABOi kidney transplantation. An IgG-isoagglutinine titer of less or equal 1:4 qualified for transplantation. Treatment safety, efficiency, length of desensitization, number of postoperative immunoadsorptions, and allograft outcome were retrospectively compared. RESULTS: With the use of the immunoglobulin adsorber the median initial isoagglutinine IgG titers of 1:64 (range 1:32-1:256) were lowered to the target of 1:4 preoperatively with a mean of 6.2 immunoadsorptions (range 5-11). Mean IgG/IgM titer step reduction per IA was 1.98/1.21 for (range 0-4/0-4) and mean titer step rebound 1.31/0.82 (range 0-4/0-3), respectively. The number of immunoadsorptions and length of desensitization was not different from the use of the specific isoagglutinine adsorbers. After transplantation, no rejection occurred and only one postoperative immunoadsorption was necessary. No adverse events in relation to immunoadsorption were observed. Graft function was comparable to the isoagglutinine adsorber group. CONCLUSION: These data suggest that ABOi kidney transplantation can be performed safely and effectively with a selective regenerative immunoglobulin immunoadsorber.

Immunoadsorption in patients with chronic inflammatory demyelinating polyradiculoneuropathy with unsatisfactory response to first-line treatment.

BACKGROUND/AIMS: First-line treatment options for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are corticosteroids, intravenous immunoglobulin, and plasma exchange. In a significant number of patients, first-line therapy fails, and long-term maintenance treatment still remains a therapeutic challenge. Immunoadsorption (IA) may be an alternative to classical plasma exchange in the therapy of immune-mediated neurologic diseases. The aim of this investigation was to evaluate efficacy and safety of IA in patients with CIDP with unsatisfactory response to first-line treatment options. METHODS: CIDP patients received adjunct IA treatment using tryptophan-immune adsorbers. The inflammatory neuropathy cause and treatment disability (INCAT) score was used to grade disability and monitor treatment effects. RESULTS: In total, 14 CIDP patients were analyzed. Ten patients were treated in hospital. After one IA treatment series, the INCAT score decreased significantly in all 10 patients. Four of these 14 patients were treated in outpatient clinics using long-term maintenance IA with 1-2 treatments per week. In these 4 patients, effects of long-term maintenance IA resulted in an improvement of overall disability. In all patients, IA was safe, well tolerated, and no severe adverse effects occurred. CONCLUSION: IA could be an effective and safe option for CIDP patients with unsatisfactory response to first-line treatment options and for long-term maintenance treatment.

Leflunomide: a drug with a potential beyond rheumatology.

Leflunomide, an inhibitor of the dihydroorotase dehydrogenase and thereby pyrimidine synthesis, was introduced and licensed for the treatment of rheumatoid arthritis in 1998. In the following years, its antiviral properties were discovered and the drug was used in solid organ transplantation for polyomavirus type BK or cytomegalovirus infection. Owing to its long half-life and weak interaction with the cytochrome system, special considerations apply in the use of this drug. This article summarizes the clinical experience with leflunomide in rheumatology and in the evolving field of transplantation.

Combination of immunosuppressive drugs leaves specific "fingerprint" on gene expression in vitro.

Following organ transplantation many patients suffer from drug-related side effects, or receive more immunosuppression than necessary to prevent rejection. Hence, parameters are needed to tailor the immunosuppressive therapy to the individual needs of an organ recipient. The aim of this study was to determine whether drug combinations provoke specific gene expression patterns in a simple assay system in vitro. Stimulated peripheral blood lymphocytes were cultured in the presence of cyclosporine A, tacrolimus, mycophenolic acid, everolimus and sirolimus, or combinations thereof. Using a cDNA microarray, we found that all samples clustered in drug-specific groups. Gene expression profiles were almost identical in PBL treated with either cyclosporine A or tacrolimus, and with either sirolimus or everolimus. More than 50 genes were synergistically affected by combinations of calcineurin-inhibitors and TOR-inhibitors and drug-specific regulated genes could be identified for both substance groups. Our data suggest that in vitro gene profiling can be used to describe synergistic effects of immunosuppressive drugs. Furthermore, our approach may help to identify marker genes urgently needed to optimize and individualize immunosuppressive drug regimens after organ transplantation.

Increased expression of secreted frizzled-related protein 4 in polycystic kidneys.

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease associated with progressive renal failure. Although cyst growth and compression of surrounding tissue may account for some loss of renal tissue, the other factors contributing to the progressive renal failure in patients with ADPKD are incompletely understood. Here, we report that secreted frizzled-related protein 4 (sFRP4) is upregulated in human ADPKD and in four different animal models of PKD, suggesting that sFRP4 expression is triggered by a common mechanism that underlies cyst formation. Cyst fluid from ADPKD kidneys activated the sFRP4 promoter and induced production of sFRP4 protein in renal tubular epithelial cell lines. Antagonism of the vasopressin 2 receptor blocked both promoter activity and tubular sFRP4 expression. In addition, sFRP4 selectively influenced members of the canonical Wnt signaling cascade and promoted cystogenesis of the zebrafish pronephros. sFRP4 was detected in the urine of both patients and animals with PKD, suggesting that sFRP4 may be a potential biomarker for monitoring the progression of ADPKD. Taken together, these observations suggest a potential role for SFRP4 in the pathogenesis of ADPKD.

ABO-incompatible kidney transplantation-proposal of an intensified apheresis strategy for patients with high initial isoagglutinine titers.

Since Tydén's description of ABO-incompatible (ABOi) kidney transplantations based on antigen-specific immunoadsorption (IA) and rituximab (Tydén et al., Am J Transplant 2005;5:145-148), this technique has been successfully adopted by many transplant centers worldwide. The majority of centers strictly adhere to the Swedish protocol and perform IAs with a target volume of 1.5-2 plasma volumes on preoperative days -6, -5, -2, and -1, and postoperative days +3, +6, and +9, respectively. Patients who initially present with an IgG anti-A/B titer higher than 1:128 are not considered suitable candidates for ABOi transplantation by the Swedish protocol. Our center has gone beyond these suggestions and follows a slightly different strategy: We do not exclude patients with initial IgG anti-A/B titers higher than 1:128 and we perform as many preoperative antigen-specific extracorporeal treatments as needed to reach a threshold isoagglutinine titer of 1:4 or less. To intensify isoagglutinine clearance preoperatively, the total target volume per treatment was increased to 2.5-3 plasma volumes. Preconditioning IAs are performed every other day, instead of daily. Postoperatively we perform IAs only, if titers mandate us to do so (Wilpert et al., Nephrol Dial Transplant 2007;22:3048-3051). We report on 11 "high-titer patients" who entered our ABOi kidney transplant program with initial titers of 1:256 or above. Seven of 11 patients (64%) could successfully be transplanted with our modified ABO-apheresis protocol. Four of 11 high-titer patients did not reach target isoagglutinine titers of 1:4 or less and therefore did not undergo transplantation. We conclude that intensified preoperative IA renders a majority of high-titer patients suitable candidates for ABOi kidney transplantation.

On-demand strategy as an alternative to conventionally scheduled post-transplant immunoadsorptions after ABO-incompatible kidney transplantation.

BACKGROUND: Since 2001, approximately 100 ABO-incompatible kidney transplantations have been performed in Europe. The standard protocol, employed by most transplant centres, uses rituximab and scheduled pre-emptive antigen-specific immunoadsorption on post-operative days 3, 6 and 9. METHODS: Our centre has performed 22 ABO-incompatible kidney transplantations since 2004, using a different approach; like in Sweden, all patients received immunoadsorptions preoperatively, but instead of scheduling pre-emptive post-transplant immunoadsorptions, we submitted patients to immunoadsorptions post-operatively only, if their isoagglutinine titers (IgG-Anti-A or -B) exceeded certain thresholds. These thresholds were greater than 1 : 8 in the first post-operative week and greater than 1 : 16 in the second post-operative week, respectively. RESULTS: A shorter pre-operative length on dialysis, a blood-type constellation of donor A1/recipient 0 and 9a high initial starting-titer were identified as predictors for post-operative immunoadsorptions. CONCLUSION: Using this on-demand strategy, our data reveal that a titer-dependent protocol reduces costs at no additional risk for the patient.

Preemptive postoperative antigen-specific immunoadsorption in ABO-incompatible kidney transplantation: necessary or not?

Several standard protocols for ABO-incompatible kidney transplantation use scheduled preemptive antigen-specific immunoadsorption during the postoperative period. Our center has developed a different approach. Our patients undergo antigen-specific immunoadsorption postoperatively only if their isoagglutinine titers (immunoglobulin G anti-A/B) exceed 1:8 in the first postoperative week and 1:16 in the second postoperative week. Using this strategy, 22 ABO-incompatible kidney transplantations have been performed at our center since 2004. Only 32% of these patients (7 of 22) needed to undergo postoperative immunoadsorption (mean 4.1 immunoadsorption sessions per patient). The renal outcome in patients receiving postoperative immunoadsorption treatment versus the outcome in patients without postoperative immunoadsorption remained equal at a mean follow-up of 17 months. We identified a shorter pretransplant time on dialysis, a blood type constellation of donor A1/recipient O, and high initial starting titers as predictors for the need for postoperative immunoadsorption treatment. A more detailed version of this study, with modified tables and figures, has been accepted for publication in Nephrology Dialysis Transplantation.

Characterization of leakage activity in exudative chorioretinal disease with three-dimensional confocal angiography.

PURPOSE: A novel angiographic technique providing topographic imaging of chorioretinal fluorescence is applied to the characterization of leakage dynamics in exudative chorioretinopathy. The three-dimensional imaging is evaluated with respect to results with conventional two-dimensional fluorescence angiography. DESIGN: Prospective observational case series. PARTICIPANTS: Thirty eyes of 30 patients with different exudative maculopathies (pigment epithelium detachment, branch retinal vein occlusion, central serous chorioretinopathy, each n = 10) and 11 eyes of 10 patients with clinically normal appearance. METHODS: Depth-resolved fluorescence angiography using a confocal scanning laser system was performed after complete ophthalmologic examination. The axial distribution of fluorescein and indocyanine green fluorescence at each x/y position within a tomographic scan of 32 images was analyzed. The chorioretinal fluorescence topography was reconstructed by localizing a defined threshold value of fluorescence and displayed as topographic relief. Qualitative description and quantitative measurements of exudation or structural alterations were performed topographically and conventionally. MAIN OUTCOME MEASURES: Qualitative and quantitative analysis of structural or exudative changes in time course in topographic illustration compared with conventional angiography. RESULTS: Clinically physiologic eyes were presented topographically as a smooth concave surface of fluorescence with defined illustration of retinal vascular structures and the optic disc. Retinal vascular pathologic conditions induce a precisely demarcated pattern of intraretinal edema with a characteristic temporal evolution. In central serous retinopathy the underlying pathologic condition was identified as a perfusion defect, which was subsequently filled with a peak of exudation with differences in the time of maximum in fluorescein/indocyanine green angiography. Pigment epithelium detachment appeared as a high and well defined elevation, with the origin of exudation within the base of the detachment. Differences in the time of maximum prominence were found in indocyanine green angiography within the pigment epithelium detachment group. CONCLUSIONS: Confocal topographic angiography allows for the first time precise three-dimensional functional imaging of fundus alterations caused by leakage or barrier dysfunction. Compared with conventional angiography, depth-resolved angiographic imaging is less impaired by masking phenomena or low fluorescence intensity, which improves the diagnostic yield of angiography. The characterization and quantification of leakage activity is a promising tool in the assessment of exudative maculopathy.