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.

Generalized lymphedema in a sirolimus-treated renal transplant patient.

Generalized lymphedema is an extremely rare effect of sirolimus therapy in renal transplant recipients. We describe the development of this complication in a 56-yr-old woman, who was given an experimental protocol with cyclosporine, sirolimus, steroids, and basiliximab. Following the protocol, after one month, the patient was randomized to the "sirolimus only" group, while cyclosporine was completely suspended and the oral steroids were continued. Three months later, the patient was admitted for severe lymphedema of the lower limbs, with significant weight increase, massive ascites and dyspnea, but excellent renal function. A chest radiography and an ultrasound study of the heart showed a moderate pleural and pericardial effusion. An abdominal ultrasound scan showed two small lymphoceles next to the transplanted kidney, confirmed with a CT scan. After sirolimus discontinuation the generalized lymphedema started to improve and three months later all the symptoms had disappeared.

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.

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.

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.

Reduction of hypotensive side effects during online-haemodiafiltration and low temperature haemodialysis.

BACKGROUND: This study compares the effect of online-haemodiafiltration (o-HDF, post-dilution mode) with conventional haemodialysis (HD) and 'temperature-controlled' HD (Temp-HD) on the haemodynamic stability of hypotension-prone patients. METHODS: Seventeen patients with a history of frequent hypotensive episodes during dialysis sessions were studied, each patient serving as his or her own control. The first 25 HD treatments in comparison with 25 o-HDF sessions were evaluated using identical dialysate temperature. In the second part of the study, o-HDF (n = 25) was compared with Temp-HD (n = 25). In the latter method, the temperature of the dialysate was adjusted to result in identical energy transfer rates to those in the corresponding o-HDF. The number of hypotensive episodes, blood temperature and blood volume regulation were assessed. RESULTS: Symptomatic hypotension was much more frequent during HD (40%) than during o-HDF (4%) (P < 0.001). During o-HDF, an enhanced energy loss within the extracorporeal system occurred (o-HDF, 16.6 +/- 4.0 W; HD, 5.4 +/- 5.1 W; P < 0.0001), despite identical temperature settings for dialysate and substitution fluid. As a result, the blood returning to the patient was cooler during o-HDF than during HD (o-HDF 35 +/- 0.2 degrees C vs HD 36.5 +/- 0.3 degrees C; P < 0.0001). In o-HDF, even in the patients' circulation, the mean blood temperature was lower (o-HDF 36.7 +/- 0.2 degrees C vs HD 36.9 +/- 0.3 degrees C; P < 0.0001) and blood volume was significantly more reduced (o-HDF, 91.8 +/- 3.1%; HD, 94.0 +/- 3.2%; P < 0.05). Energy transfer rates and blood temperature did not differ significantly between o-HDF and Temp-HD. The rate of hypotensive episodes was low and not different between o-HDF (4%) and Temp-HD (4%). Neither was there any significant difference in blood volume reduction. CONCLUSIONS: O-HDF showed a significant reduction of hypotensive episodes compared with HD. Surprisingly, o-HDF resulted in cooling of the blood via enhanced thermal energy losses within the extracorporeal system, despite use of replacement fluid prepared from pre-warmed dialysate. The incidence of symptomatic hypotension was reduced to that of o-HDF by using cooler Temp-HD. Thus, unexpected blood cooling appears to be the main blood pressure-stabilizing factor in o-HDF.

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.