Adrenergic Repression of the Epigenetic Reader MeCP2 Facilitates Cardiac Adaptation in Chronic Heart Failure.

RATIONALE: In chronic heart failure, increased adrenergic activation contributes to structural remodeling and altered gene expression. Although adrenergic signaling alters histone modifications, it is unknown, whether it also affects other epigenetic processes, including DNA methylation and its recognition. OBJECTIVE: The aim of this study was to identify the mechanism of regulation of the methyl-CpG-binding protein 2 (MeCP2) and its functional significance during cardiac pressure overload and unloading. METHODS AND RESULTS: MeCP2 was identified as a reversibly repressed gene in mouse hearts after transverse aortic constriction and was normalized after removal of the constriction. Similarly, MeCP2 repression in human failing hearts resolved after unloading by a left ventricular assist device. The cluster miR-212/132 was upregulated after transverse aortic constriction or on activation of α1- and ß1-adrenoceptors and miR-212/132 led to repression of MeCP2. Prevention of MeCP2 repression by a cardiomyocyte-specific, doxycycline-regulatable transgenic mouse model aggravated cardiac hypertrophy, fibrosis, and contractile dysfunction after transverse aortic constriction. Ablation of MeCP2 in cardiomyocytes facilitated recovery of failing hearts after reversible transverse aortic constriction. Genome-wide expression analysis, chromatin immunoprecipitation experiments, and DNA methylation analysis identified mitochondrial genes and their transcriptional regulators as MeCP2 target genes. Coincident with its repression, MeCP2 was removed from its target genes, whereas DNA methylation of MeCP2 target genes remained stable during pressure overload. CONCLUSIONS: These data connect adrenergic activation with a microRNA-MeCP2 epigenetic pathway that is important for cardiac adaptation during the development and recovery from heart failure.

Epidemiology of autosomal-dominant polycystic kidney disease: an in-depth clinical study for south-western Germany.

BACKGROUND: As we emerge into the genomic medicine era, the epidemiology of diseases is taken for granted. Accurate prevalence figures, especially of rare diseases (RDs, ≤50/100,000), will become even more important for purposes of health care and societal planning. We noticed that the numbers of affected individuals in regionally established registries for mainly hereditary RDs do not align with published estimated and expected prevalence figures. We therefore hypothesized that such non-population-based means overestimate RDs and sought to address this by recalculating prevalence for an important 'common' hereditary disease, autosomal-dominant polycystic kidney disease (ADPKD) whereby presumed-prevalence is 100-250/100,000 METHODS: The Else-Kroener-Fresenius-ADPKD-Study in south-west Germany with a population of 2,727,351 inhabitants was established with the cooperation of all nephrology centres. Furthermore, general practitioners, internists, urologists, human geneticists and neurosurgery centres were contacted with questionnaires for demographic, family and kidney function data. Germline-mutation screening of susceptibility genes PKD1 and PKD2 was offered. Official population data for 2010 were used for overall and kidney function-adjusted prevalence estimations. RESULTS: A total of 891 subjects, 658 index-cases and 233 relatives, aged 10-89 (mean 52), were registered, with >90% response rate, 398 by nephrologists and 493 by non-nephrologists. Molecular-genetic analyses contributed to confirmation of the diagnosis in 57%. The overall prevalence of ADPKD was 32.7/100,000 reaching a maximum of 57.3/100,000 in the 6th decade of life. CONCLUSIONS: Prevalence of ADPKD is overestimated by 2- to 5-fold and close to the limit of RDs which may be of broad clinical, logistic and policy implications.

Long-term outcome of ABO-incompatible living donor kidney transplantation based on antigen-specific desensitization. An observational comparative analysis.

BACKGROUND: ABO-incompatible living donor kidney transplantation based on specific conditioning has been successfully adopted by transplant centres worldwide. Excellent short-term results have been reported in small cohorts. However, long-term data and comparative analyses are still sparse. We report on the outcome of 40 consecutive ABO-incompatible living donor kidney transplant recipients and compare their clinical course to a control group of 43 ABO-compatible living donor transplant patients transplanted during the same time period. METHODS: This is an observational single-centre analysis of 40 consecutive patients undergoing ABO-incompatible kidney grafting between April 2004 and April 2009, using a protocol of rituximab, antigen-specific immunoadsorption, intravenous immunoglobulin, basiliximab induction and oral triple immunosuppression with tacrolimus, mycophenolic acid and prednisone. Forty-three ABO-compatible kidney transplant recipients served as controls. The control group had also received basiliximab induction and an identical initial maintenance immunosuppression. The two groups were observed for an average of 39 and 19 months, respectively. RESULTS: There was a significantly higher incidence of lymphoceles requiring surgical revisions in the ABO-incompatible group. However, this surgical complication did not affect patient or graft survival. Mean serum creatinine, estimated glomerular filtration rate and proteinuria did not differ between the two groups. Furthermore, ABO-incompatible and ABO-compatible patients had the same incidence of humoral and cellular rejections. Despite a more aggressive induction therapy, no differences in infectious or malignant complications were observed. CONCLUSIONS: ABO-incompatible living donor kidney transplantation utilizing a combination of rituximab and antigen-specific immunoadsorption yielded results identical to ABO-compatible transplantation despite a significantly higher number of human leukocyte antigen mismatches.

Comparing the tube and gel techniques for ABO antibody titration, as performed in three European centers.

Data from 60 consecutive ABO-incompatible kidney transplantations performed in Stockholm, Sweden; Freiburg, Germany; and Uppsala, Sweden, revealed significant variation in preoperative A/B antibody levels, with median titers of 1:32, 1:128, and 1:8, respectively. We wanted to investigate whether these differences were method-related. The same samples from 21 healthy blood donors were analyzed in the three centers using current local methods. Results confirmed method-related differences, with higher A/B titers in Freiburg and lower titers in Uppsala compared with Stockholm. Results for the same sample differed by a median of three (range 0 to 6) titer steps. When the same number of samples were analyzed in the three centers using the same gel method and the same test erythrocytes, results differed by a median of one titer step (range 0 to 4) for the same sample. In conclusion, gel hemagglutination technique significantly decreases intercenter variation compared with tube technique.

Implementation of a Protocol for ABO-incompatible kidney transplantation--a three-center experience with 60 consecutive transplantations.

BACKGROUND: A new protocol for ABO-incompatible kidney transplantation has recently been introduced. We report here on the joint experience of the implementation in Stockholm and Uppsala, Sweden and Freiburg, Germany. METHODS: The new protocol utilizes antigen-specific immunoadsorption to remove existing ABO-antibodies, rituximab, and intravenous immunoglobulin to prevent the rebound of antibodies, and conventional tacrolimus, mycophenolate-mofetil, and prednisolone immunosuppression. Sixty consecutive ABO-incompatible kidney transplantations were included in the study. The outcome is compared with the results of 274 ABO-compatible live donor transplantations performed during the same period. RESULTS: Two of the ABO-incompatible grafts have been lost (non-compliance and death with functioning graft). All the remaining 58 grafts had good renal function at a follow-up of up to 61 months. We did not observe any late rebound of antibodies and there were no humoral rejections. Graft survival was 97% for the ABO-incompatible compared with 95% for the ABO-compatible. Patient survival was 98% in both groups. There was a significant variation in preoperative A/B-antibody titer between the centers, with a median 1:8 in Uppsala, median 1:32 in Stockholm and median 1:128 in Freiburg. More preoperative antibody adsorptions were therefore needed in Freiburg than in Stockholm and Uppsala. CONCLUSIONS: The new protocol was easily implemented and there were no graft losses that could be related to ABO-incompatibility. A significant inter-institutional variation in the measurement of anti-AB-antibodies was found, having a substantial impact on the number of immunoadsorptions and consequently on the total cost for the procedure. A standardized fluorescence-activated cell sorting technique for antibody quantification is much needed.

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.

cDNA microarray analysis of adaptive changes after renal ablation in a sclerosis-resistant mouse strain.

BACKGROUND: 5/6 nephrectomy (Nx) in susceptible animals causes glomerular sclerosis and interstitial fibrosis in the remnant kidney. Oxidative stress, transforming growth factor-beta (TGF-beta), and the de novo synthesis of collagen seem to contribute to this process. However, these factors might also be required for tissue repair without fibrosis. METHODS: We examined dynamic changes after nephron loss in a mouse strain capable of complete recovery. C57BL/6 mice underwent single-session Nx and were followed for 40 weeks. Gene expression was monitored over 20 days using 22,000 cDNA microarrays. RESULTS: The mice developed transient hypertension and glomerular hypertrophy after Nx but failed to progress to glomerular sclerosis or renal failure. Gene expression profiles revealed three stages of recovery, an early phase of injury response, an intermediate phase of extracellular matrix (ECM) production and a later phase of reconstitution. Surprisingly, oxidative stress responses and collagen production were strongly upregulated soon after Nx. Furthermore, TGF-beta(1) and connective tissue growth factor were rapidly upregulated and remained elevated. CONCLUSION: We suggest that oxidative stress, collagen production, profibrotic growth factors and ECM turnover are part of the comprehensive adaptation to nephron loss and not necessarily associated with progressive loss of renal function.

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.

Expression profiling on chronically rejected transplant kidneys.

BACKGROUND: Chronic transplant nephropathy remains a poorly defined inflammatory process that limits the survival rate of most renal transplants. We analyzed the gene profile of chronically rejected kidney transplants to identify candidate genes that characterize chronic transplant nephropathy. METHODS: To distinguish genes present in normal renal tissue or specific for end-stage renal failure, we compared the gene profiles of 13 chronically rejected kidney transplants with 16 normal kidneys and 12 end-stage polycystic kidneys using a 7K human cDNA microarray. After elimination of genes with signals close to background, 2190 genes were available for statistical analysis. RESULTS: More than 20% of the examined genes were significantly regulated when compared with the expression level of normal renal tissue (P<0.0003). Hierarchic clustering based on 571 genes differentiated normal and transplant tissue, and transplant and polycystic kidney tissue. Most of these genes encoded proteins involved in cellular metabolism, transport, signaling, transcriptional activation, adhesion, and the immune response. Notably, comprehensive gene profiling of chronically rejected kidneys revealed two distinct subsets of chronically rejected transplants. Neither clinical data nor histology could explain this genetic heterogeneity. CONCLUSIONS: Microarray analysis of rejected kidneys may help to define different entities of transplant nephropathy, reflecting the multifactorial cause of chronic rejection.

Adult patients with sporadic polycystic kidney disease: the importance of screening for mutations in the PKD1 and PKD2 genes.

BACKGROUND: ADPKD is one of the most common inherited disorders, with high risk for end-stage renal disease. Numerous patients, however, have no relatives in whom this disorder is known and are unsure whether they may transmit the disease to their offsprings. The aim of this study was to evaluate whether germline mutation analysis adds substantial information to clinical symptoms for diagnosis of ADPKD in these patients. METHODS: Clinical data included renal function and presence of liver or pancreas cysts, heart valve insufficiency, intracranial aneurysms, colonic diverticles, and abdominal hernias. Family history was evaluated regarding ADPKD. Germline mutation screening of the PKD1 and PKD2 genes was performed for intragenic mutations and for large deletions. RESULTS: A total of 324 adult patients with ADPKD including 30 patients without a family history of ADPKD (sporadic cases) were included. PKD1 mutations were found in 24/30 and PKD2 mutations in 6 patients. Liver cysts were present in 14 patients and intracranial aneurysms in 2 patients. Fourteen patients (45%) had no extrarenal involvement. Compared to the 294 patients with familial ADPKD, the clinical characteristics and the age at the start of dialysis were similar in those with sporadic ADPKD. CONCLUSION: The clinical characteristics of patients with sporadic and familial ADPKD are similar, but sporadic ADPKD is often overlooked because of the absence of a family history. Molecular genetic screening for germline mutations in both PKD1 and PKD2 genes is essential for the definitive diagnosis of ADPKD.

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.

ABO-incompatible kidney transplantation using antigen-specific immunoadsorption and rituximab: a single center experience.

BACKGROUND: For years ABO-incompatible kidney transplantations were preferentially performed in Japanese centers. In order to overcome the increased risk of humoral rejections, patients were treated with multiple sessions of plasmapheresis, intensified immunosuppressive therapy and splenectomy before transplantation. Despite good long-term results regarding patient and organ survival rates, increased morbidity during the early post-transplant period prevented a broad application of this method. Recently, a new protocol including the anti-CD20-antibody (Ab) rituximab and blood group-specific immunoadsorption instead of splenectomy and plasmapheresis was published with excellent short-term results. METHODS: From April 2004 to September 2005, 11 patients were prepared for ABO-incompatible transplantation. All patients received 375 mg/m2 rituximab intravenous 3 to 4 weeks before transplantation. Immunosuppressive therapy consisted of tacrolimus, mycophenolate mofetil and prednisone and was started at least 7 days before transplantation. Intravenous immunoglobulins (0.5 g/kg) were administered the day before transplantation. Immunoglobulin G (IgG)-anti-A or -B Ab titers before starting immunoadsorption treatment ranged between 1 : 4 and 1 : 1024. Immunoadsorption treatment was started in parallel with immunosuppressive medication and was continued until the anti-A or anti -B Ab titers (IgG and IgM) were lowered to the aimed pre-transplant threshold of <1 : 8. During the early postoperative period, additional immunoadsorption treatments were performed, if the titers increased again above 1 : 8 (days 0 to 7) or 1 : 16 (days 8 to 14), respectively. RESULTS: Transplantation could be conducted in eight of 11 patients (two females, six males, mean recipient age 52+/-11 yr). The mean follow-up was 7.0 months (range 4 to 17). The blood group constellation was A1 to 0 in four cases, A2 to 0 in two cases, B to A in one case, and A1 to B in another case, respectively. On average, each patient received seven immunoadsorption treatments. All transplants showed primary function and no humoral rejections occurred. Three of our 11 patients showed rapid increases of isoagglutinin titers after each immunoadsorption treatment and thus could not be transplanted. One patient died 4 months after transplantation with a functioning graft due to sepsis secondary to pseudomembranous enterocolitis. The mean creatinine value of the remaining seven patients now is 1.6 mg/dl. SUMMARY: The use of antigen-specific immunoadsorption and an immunosuppressive regimen consisting of a conventional triple immunosuppressive therapy has shown excellent short-term results. The immunoadsorption treatment using antigen-specific columns is highly effective and even patients with high isoagglutinin titers can be transplanted. This protocol is an option for end-stage renal disease patients who have no blood group-compatible donor.

Gene profiling of polycystic kidneys.

BACKGROUND: While the genetic basis of autosomal dominant polycystic kidney disease (ADPKD) has been clearly established, the pathogenesis of renal failure in ADPKD remains elusive. Cyst formation originates from proliferating renal tubular epithelial cells that de-differentiate. Fluid secretion with cyst expansion and reactive changes in the extracellular matrix composition combined with increased apoptosis and proliferation rates have been implicated in cystogenesis. METHODS: To identify genes that characterize pathogenical changes in ADPKD, we compared the expression profiles of 12 ADPKD kidneys, 13 kidneys with chronic transplant nephropathy and 16 normal kidneys using a 7 k cDNA microarray. RT-PCR and immunohistochemical techniques were used to confirm the microarray data. RESULTS: Hierarchical clustering revealed that the gene expression profiles of normal, ADPKD and rejected kidneys were clearly distinct. A total of 87 genes were specifically regulated in ADPKD; 26 of these 87 genes were typical for smooth muscle, suggesting epithelial-to-myofibroblast transition (EMT) as a pathogenetic factor in ADPKD. Immunohistology revealed that smooth muscle actin, a typical marker for myofibroblast transition, and caldesmon were mainly expressed in the interstitium of ADPKD kidneys. In contrast, up-regulated keratin 19 and fibulin-1 were confined to cystic epithelia. CONCLUSION: Our results show that the end stage of ADPKD is associated with increased markers of EMT, suggesting that EMT contributes to the progressive loss of renal function in ADPKD.

Host cell responses induced by hepatitis C virus binding.

Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.

Gene expression profiling in polycythaemia vera: overexpression of transcription factor NF-E2.

Summary The molecular aetiology of polycythaemia vera (PV) remains unknown and the differential diagnosis between PV and secondary erythrocytosis (SE) can be challenging. Gene expression profiling can identify candidates involved in the pathophysiology of PV and generate a molecular signature to aid in diagnosis. We thus performed cDNA microarray analysis on 40 PV and 12 SE patients. Two independent data sets were obtained: using a two-step training/validation design, a set of 64 genes (class predictors) was determined, which correctly discriminated PV from SE patients. Separately 253 genes were identified to be upregulated and 391 downregulated more than 1.5-fold in PV compared with healthy controls (P < 0.01). Of the genes overexpressed in PV, 27 contained Sp1 sites: we therefore propose that altered activity of Sp1-like transcription factors may contribute to the molecular aetiology of PV. One Sp1 target, the transcription factor NF-E2 [nuclear factor (erythroid-derived 2)], is overexpressed 2- to 40-fold in PV patients. In PV bone marrow, NF-E2 is overexpressed in megakaryocytes, erythroid and granulocytic precursors. It has been shown that overexpression of NF-E2 leads to the development of erythropoietin-independent erythroid colonies and that ectopic NF-E2 expression can reprogram monocytic cells towards erythroid and megakaryocytic differentiation. Transcription factor concentration may thus control lineage commitment. We therefore propose that elevated concentrations of NF-E2 in PV patients lead to an overproduction of erythroid and, in some patients, megakaryocytic cells/platelets. In this model, the level of NF-E2 overexpression determines both the severity of erythrocytosis and the concurrent presence or absence of thrombocytosis.