Harald D. Rupprecht

Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Factors controlling growth and matrix production and matrix production in vascular smooth muscle and glomerular mesangial cell

The vasculature wall is an active, integrated organ composed of endothelial cells and vascular smooth muscle cells, as well as other cell types depending on the specific vascular segment (e.g. fibroblasts in many vascular regions). The vascular wall is not static; the vascular components (cells and extracellular matrix) dynamically increase, decrease or reorganize, or both, in response to physiological and pathological stimuli. The vascular smooth muscle cells are the final common pathway for many of these dynamic changes in vascular wall structure. In the renal glomerulus, however, the glomerular mesangial cells - a cell phenotypically related to the vascular smooth muscle cells - also participate. Although sometimes beneficial, changes in vascular or glomerular structure often lead to cardiovascular (e.g. atherosclerosis, restenosis, intimal hyperplasia) and renovascular (e.g. glomerulosclerosis) diseases. Consequently, much effort has been expended to elucidate the mechanisms that control growth and extracellular matrix production by vascular smooth muscle cells and glomerular mesangial cells. The purpose of this review is to discuss recent developments. Center for Clinical Pharmacology, Department of Medicine, University

Identification and Validation of Urinary Biomarkers for Differential Diagnosis and Evaluation of Therapeutic Intervention in Anti-neutrophil Cytoplasmic Antibody-associated Vasculitis

Renal activity and smoldering disease is difficult to assess in anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) because of renal scarring. Even repeated biopsies suffer from sampling errors in this focal disease especially in patients with chronic renal insufficiency. We applied capillary electrophoresis coupled to mass spectrometry toward urine samples from patients with active renal AAV to identify and validate urinary biomarkers that enable differential diagnosis of disease and assessment of disease activity. The data were compared with healthy individuals, patients with other renal and non-renal diseases, and patients with AAV in remission. 113 potential biomarkers were identified that differed significantly between active renal AAV and healthy individuals and patients with other chronic renal diseases. Of these, 58 could be sequenced. Sensitivity and specificity of models based on 18 sequenced biomarkers were validated using blinded urine samples of 40 patients with different renal diseases. Discrimination of AAV from other renal diseases in blinded samples was possible with 90% sensitivity and 86.7–90% specificity depending on the model. 10 patients with active AAV were followed for 6 months after initiation of treatment. Immunosuppressive therapy led to a change of the proteome toward “remission.” 47 biomarkers could be sequenced that underwent significant changes during therapy together with regression of clinical symptoms, normalization of C-reactive protein, and improvement of renal function. Proteomics analysis with capillary electrophoresis-MS represents a promising tool for fast identification of patients with active AAV, indication of renal relapses, and monitoring for ongoing active renal disease and remission without renal biopsy.

Transcription Factor Egr-1 Regulates Glomerular Mesangial Cell Proliferation

Increase of glomerular mesangial cells (MCs) is a prominent histopathological finding in many types of glomerulonephritis. We have shown previously that expression of the zinc-finger transcription factor, early growth response gene-1 (egr-1), is closely correlated with the proliferation of cultured MCs. To elucidate whether Egr-1 is required for MC proliferation, we inhibited serum-induced Egr-1 expression by phosphothioate-modified antisense oligonucleotides (ODNs). Uptake of antisense ODNs into MCs was demonstrated, and five different egr-1 antisense ODNs were tested for their impact on serum-induced egr-1 mRNA and protein levels and on MC growth. The most potent egr-1 antisense ODN inhibited serum-induced egr-1 mRNA by 68%, protein induction by 58%, and MC replication as measured by [3H]thymidine uptake and cell counts by 78 and 46%, respectively. The effects of antisense ODNs on MC growth correlated closely with their ability to inhibit Egr-1 protein. ODNs acted in a dose-dependent manner, the minimal effective concentration being 1 μM. Control ODNs had no significant effects. In addition, antisense ODNs against egr-1 potently inhibited endothelin-1-induced Egr-1 expression and MC growth. Heparin, a known inhibitor of MC growth, suppressed serum-induced [3H]thymidine uptake by 39% and egr-1 mRNA expression by 44%. We conclude that Egr-1 is an essential part of the mitogenic signal transduction cascade in cultured MCs.

Noninvasive diagnosis of chronic kidney diseases using urinary proteome analysis

Background In spite of its invasive nature and risks, kidney biopsy is currently required for precise diagnosis of many chronic kidney diseases (CKDs). Here, we explored the hypothesis that analysis of the urinary proteome can discriminate different types of CKD irrespective of the underlying mechanism of disease. Methods We used data from the proteome analyses of 1180 urine samples from patients with different types of CKD, generated by capillary electrophoresis coupled to mass spectrometry. A set of 706 samples served as the discovery cohort, and 474 samples were used for independent validation. For each CKD type, peptide biomarkers were defined using statistical analysis adjusted for multiple testing. Potential biomarkers of statistical significance were combined in support vector machine (SVM)-based classifiers. Results For seven different types of CKD, several potential urinary biomarker peptides (ranging from 116 to 619 peptides) were defined and combined into SVM-based classifiers specific for each CKD. These classifiers were validated in an independent cohort and showed good to excellent accuracy for discrimination of one CKD type from the others (area under the receiver operating characteristic curve ranged from 0.77 to 0.95). Sequence analysis of the biomarkers provided further information that may clarify the underlying pathophysiology. Conclusions Our data indicate that urinary proteome analysis has the potential to identify various types of CKD defined by pathological assessment of renal biopsies and current clinical practice in general. Moreover, these approaches may provide information to model molecular changes per CKD.

Assessment of renal vasoconstriction in vivo after intra-arterial administration of the isosmotic contrast medium iodixanol compared to the low-osmotic contrast medium iopamidol

BACKGROUND Low-osmotic contrast media (LOCM) such as iopamidol are known to increase the renal resistance index (RRI). The aim of our study was to evaluate in vivo the different effects of intra-arterial administration of LOCM in comparison to isosmotic contrast medium (IOCM) such as iodixanol on the human RRI. METHODS Twenty patients (16 males, 4 females; 66 years on average) with normal renal function (mean creatinine 1.0 mg/dl) had digital subtraction angiography (DSA) of the abdominal and lower-limb arteries. Ten patients received LOCM, and 10 patients IOCM (150 ml on average, 20 ml/s). The RRI was assessed by an experienced nephrologist with duplex ultrasound from 15 min before until 30 min after the first injection with delays of 1-5 min. The basic value of the RRI and differential RRI were calculated. RESULTS The basic value of the RRI was 0.69 in the LOCM group and 0.71 in the IOCM group. After LOCM a significant increase of the RRI to 0.73 on average (P < or = 0.001) 2 min after the first injection was found, whereas IOCM did not result in a significant change of the RRI (RRI remained 0.71 on average, P > or = 0.1). In the LOCM group, the RRI returned to the basic value after 30 min (+/-2.3 min). CONCLUSIONS Intra-arterial administration of IOCM had no influence on renal vascular resistance as expressed by the RRI, unlike LOCM, which induced a highly significant increase of the RRI for up to 30 min.

Regulation of osteopontin expression in rat mesangial cells

Hypercellularity and accumulation of extracellular matrix are common responses of renal glomeruli to inflammatory stimuli. Using the differential display approach, we compared the gene expression patterns of proliferating and differentiating rat mesangial cells in two‐ and three‐dimensional cultures. Osteopontin, an extracellular matrix protein, was found to be transcribed, synthesized, and secreted by rat mesangial cells. Osteopontin transcription was not associated with cell proliferation and was found to be FCS‐inducible in proliferating cells. Osteopontin expression was independent of exogenously supplied FCS in differentiating cells. The presented data indicate that osteopontin is differentially regulated in proliferating and differentiating mesangial cells.

Der Transkriptionsfaktor Egr-1 reguliert das Wachstum glomerulärer Mesangiumzellen

ZusammenfassungHintergrundDer Transkriptionsfaktor Early-growth-response-gene-1 (Egr-1) wird in kultivierten glomerulären Mesangiumzellen (MZ) der Niere durch verschiedene Mitogene schnell und transient induziert.Methode und ErgebnisseHier zeigen wir, daß es auch in vivo, im Modell einer mesangioproliferativen Glomerulonephritis (GN), zu einer Hochregulierung von Egr-1 kommt. Im Northern Blot fand sich ein 14,9facher Anstieg der Egr-1-mRNA am Tag 6 der Glomerulonephritis. Mittels Immunzytochemie wurde gleichzeitig ein Anstieg an Egr-1-Protein gemessen. Egr-1 war dabei vorwiegend nukleär und in mesangialer Lokalisation nach-weisbar. Um zu testen, ob Egr-1 direkt die Mesangiumzellproliferation reguliert, präinkubierten wir kultivierte Mesangiumzellen mit gegen Egr-1 gerichteten Antisense-Oligonucleotiden. Es zeigte sich eine deutliche Hemmung des Platelet-Derived-Growth-Factor-(PDGF-)induzierten Anstiegs von Egr-1-mRNA und-Protein um 75%, bzw. 74%. Gleichzeitig hemmten Egr-1 antisense Oligonucleotide dosisabhängig die PDGF-induzierte Mesangiumzellproliferation, gemessen mittels Thymidinaufnahme, um maximal 75%. Entsprechende Kontrolloligonucleotide hatten keinen Einfluß auf Egr-1-mRNA,-Protein oder Mesangiumzellenwachstum.SchlußfolgerungEgr-1 ist demzufolge ein notwendiger Bestandteil der mitogenen Signalübermittlung in glomerulären Mesangiumzellen der Niere.SummaryBackgroundThe transcriptional regulator Early growth response gene-1 (Egr-1) is rapidly and transiently induced by various mitogens in cultured rat mesangial cells (MCs).Method and ResultsHere we show Egr-1 induction in an in vivo model of mesangioproliferative glomerulonephritis (GN). A 14.9-fold increase in Egr-1 mRNA was observed 6 days after disease induction. A concomitant increase in Egr-1 protein was demonstrated by immunocytochemistry. Egr-1 was mainly localized to the nuclei of cells in mesangial localization. To test whether Egr-1 directly regulated MC proliferation, we preincubated cultured MCs with antisense oligonucleotides directed against Egr-1. The platelet-derived growth factor (PDGF)-induced increase in Egr-1 mRNA and protein levels was inhibited by 75% and 74%, respectively. At the same time Egr-1 antisense oligonucleotides dose-dependently inhibited MC-proliferation as determined by thymidine-uptake by up to 75%. Control oligonucleotides were without effects on Egr-1 mRNA, protein or MC growth.ConclusionWe conclude that Egr-1 induction is a necessary step in the mitogenic signaling cascade in glomerular MCs.