ER-Stress und der UPR-Signal Kaskaden in der Pathogenese von Nierenfibrose
Die Ziele dieses Projektes sind:
Eine genauere molekulare und funktionelle Charakterisierung der Rolle der ER-Stress Schlüssel Proteine und der UPR Kaskaden in der Expressionsregulierung von ECM Proteine und deren extrazellulären Akkumulation,
der Einfluss der Regulierung der ER-Stress Schlüsselproteine auf der Fibrose Progression und die ECM Synthese /Degradation zu studieren,
des Weiteren wird der Einfluss der auf der ER-Stress Inhibition basierten Therapie auf die ECM Synthese und Akkumulation wie auch auf die Progression der Nerenfibrose in Tiermodellen untersucht.
Etablierung eines Diagnosetestes für Diabetische Nephropathie (DM-NP) basierende auf die Massenspektrometrie
Validierte Urin-Biomarker, die das Auftreten einer krankhaften Veränderung der Niere bei Diabetiker frühzeitig (vorm Auftreten der Mikroalbuminuria) und sicher anzeigen, stellen ohne Zweifel eine Bereichung der klinischen Diagnostik der DM-NP dar.
Im Rahmen einer Studie zu Identifizierung von Biomarker für DM-NP könnten wir mittels "Clinical Proteomics" drei unterschiedliche Proteine (Ubiquitin m/z 6188, Beta2-microglubulin, UbA52) identifizieren, die die Diabetiker mit Nephropathie von den Diabetikern ohne Nephropahtie, von den Nephropathie Patienten ohne Diabetes und von den Kontrollgruppe unterschieden. Die identifizierten Proteine besonders Ubiquitin m/z 6188 und UbA52 haben Potentiale als Diagnose Marker für DM-NP zu dienen.
In den weiteren Studien wird die Möglichkeit der Entwicklung eines Testes basierend auf die Massenspektrometrie getestet.
Die Rolle der ER-Calcium bindenden Proteine in der Etablierung des Calcium
Gleichgewichtes in Nierenepithel Zellen unter osmotischen Stress
Die Rolle der Calcium Kaskaden in der Progression der Nierenfibrose.
ER-stress pathways in the pathogenesis of renal interstitial fibrosis: Inhibition of protein disulfide isomerases as regulative molecules and therapeutic targets
Renal diseases are very common and rapidly increasing in western populations. Numerous risk factors (e.g. inflammation, arterial hypertension, diabetes mellitus, proteinuria, toxins, drugs, hypoxia, hyperfiltration etc.) are known risk factors to induce renal diseases, which often lead to endstage renal failure (ERF) via a common pathway of renal interstitial fibrosis (RIF). Up to now diagnostic tools for early recognition of RIF, as a major cause of ERF, in the clinical setting are insufficiently developed and do not allow early discrimination of irreversibly progressive renal diseases from reversible damages. There is still incomplete understanding of the RIF process in addition to a lack of effective therapy to cure RIF.
Among the mechanisms involved in renal fibrosis, stress pathways were reported to inhibit ECM degradation through up-regulation of the plasminogen activator inhibitor-1 system (PAI-1), which plays an important role in modulating the degradation of ECM (1, 2, 9). Despite the progress reported in this field (8, 10, 11), the mechanisms leading to renal fibrosis still remain poorly understood. Functional proteomics provides a new perspective to highlight the changes in protein expression associated with the generation of fibrosis and thereby will lead to a better understanding of the pathophysiology of RIF.
In this research project, scientific and clinical expertise will be combined to evaluate the relevance of ER-stress regulatory pathways in the progression of RIF in human cells as well as in specific animal models presenting with renal interstitial fibrosis. The major research goals are
Characterization of the role of ER-stress proteins especially the protein disulfide isomerase (PDI, ERP57, ERP72) in ECM synthesis and accumulation,
to study the impact of protein disulfide isomerase activity regulation on ECM synthesis and accumulation and on RIF progression,
use of protein disulfide isomerase inhibitors based therapy to reduce or stop ECM synthesis, accumulation and to impair RIF progression in animal models.
Characterization and validation of two ubiquitin forms as urine protein markers for diabetic nephropathy: establishment of new diagnosis test based on mass spectrometry and antibodies
Diabetic nephropathy (DM-NP) is a major complication of diabetes and remains a common health problem worldwide. Currently, microalbuminuria is the only reliable non-invasive marker available for diagnosis of diabetic nephropathy. It is considered a marker for prediction of later avert diabetic kidney disease. However, the use of microalbuminuria measurement is problematic as it also gives high false positive rates (up to 10-12% of general healthy population). Therefore, novel biomarkers for earlier diagnosis of DM-NP are crucially needed.
In an attempt to overcome the described limitations, we performed a pilot study in order to identify patients with diabetes mellitus at the highest risk for developing diabetic nephropathy. We identified three different proteins as discriminating markers between urine from patients with diabetic nephropathy and three other analyzed groups (Dihazi et al. Clin. Chem. 2007). A processed form of ubiquitin (m/z 6188) was missed in the urine of diabetic nephropathy patients. In contrast the level of UbA52, an ubiquitin ribosomal fusion protein was higher in urine of diabetic nephropathy patients. Beta2-microglobulin was the third protein, which level was found to be altered in the urine of diabetic nephropathy patients. The aims of this project are
Purification and identification of the protease that process ubiquitin,
Establishment of new diagnosis test based on mass spectrometry and antibodies and quantification of the processed form of ubiquitin m/z 6188 and its protease during a progressive course of the diabetic nephropathy,
Validation of the usefulness of the established test in human urine samples,
Creation of UbA52 and the identified protease knock out mice to investigate the role of ubiquitin/proteasome system in progression of diabetic nephropathy.
Role of ER-calcium binding proteins in establishment of calcium homeostasis in renal epithelial cells under osmotic stress: Impact of calcium signalling regulation on renal fibrosis progression
Renal diseases lead often to endstage renal failure via a common pathway of renal fibrosis (RF). Among the mechanisms involved in RF, stress was reported to play an important role in modulating the degradation of extra cellular matrix (ECM) and RF progression (4-6). Our preliminary results highlight the important role of the down regulation of ER proteins in establishment of calcium homeostasis by epithelial cells under hypersomotic stress. In parallel we could measure and increase in expression of fibrosis marker. Never then less the molecular mechanisms connecting the osmotic stress ER- calcium binding protein and the calcium signalling to the progression of RF are still unclear.
This project aims to understand the molecular mechanisms underlying the establishment of calcium homeostasis under osmotic stress in cell line and in fibrotic kidney, and to characterize the role of ER calcium binding proteins in this regulation. Furthermore the effect of disturbance of calcium homeostasis through osmotic stress on the progression Renal Fibrosis (RF) will be investigated on cell and animal models.
Development of new mass spectrometry and citrullin specific antibody based tests to diagnose rheumatoid arthritis
Rheumatoid arthritis (RA) is a systemic autoimmune disease. New therapeutic strategies using so called "biologicals" (e.g. TNF-alpha antagonists, receptor blockers) have dramatically improved the outcome of RA. The earlier the diagnosis is made, the better the outcome of the patients. Thus it is necessary to get the diagnosis as early as possible before radiological features (e.g. erosions) are detectable. For this reason we try to establish new diagnostic tools based on mass spectrometric analysis of citrullinic peptides/proteins which are probably targets of diagnostic relevant autoantibodies in RA.
Prof. Dr. rer. nat. H. Dihazi