Universitätsklinik für Innere Medizin IV

Methodology

In recent projects we have successfully established Laser-capture microdissection (LCM) for isolation of specific cells of interest - such as proximal renal tubule cells - from kidney cryosections. Due to the minute amount of material isolated by LCM we have generated and optimized protocols for T7-based linear RNA amplification, quality control (QC) and microarray hybridization of RNA isolated from tubule cells.

Array based genome wide gene expression profiling is combined with state of the art methodologies such as quantitative real-time PCR, immunohistochemistry, immunofluorescence and western blotting of candidate genes and proteins. Furthermore, we test regulatory mechanisms of genes of interest in vitro in collaboration with the group of Prof. Herbert Schramek at our Department.

Genomics and biomarker research

Transcriptional profiling of tubule cells from patients with proteinuric nephropathies and of tubule cells from control tissues revealed significant differential regulation of pro-fibrotic and tubulo-protective (e.g. BMP-7) mechanisms already at an early stage of kidney disease. We also compared renal gene expression patterns from patients with stable disease and progressive renal failure during follow-up.

Interestingly, an attenuated tubular VEGF-A expression was noted despite a strong tubular hypoxia response and the activation of VEGF signalling pathways. These gene expression profiles serve also as a basis for further research on biomarkers in chronic kidney disease. For example, the expression levels of HIF-1a and VEGF-A were significantly superior in predicting clinical outcome as compared with proteinuria, renal function, and degree of tubular atrophy and interstitial fibrosis.

Further research interests

Another area of interest of our laboratory is the influence of ageing on transcriptomics in the kidney. This area is of special importance in the field of renal transplantation as chronological donor age is one of the major determinants of long term outcome. A currently ongoing project tries to improve the predictive power by assessing biological donor age.

Cooperations

We perform our projects in close collaboration with many institutions, amongst others emergentec biodevelopment GmbH (www.emergentec.com), the Medical University of Vienna (www.meduniwien.ac.at/nephrogene/) and the University of Katowice. Furthermore, we cooperate closely with the Department of Transplant Surgery at the Medical University in Innsbruck and the Department of General, Visceral and Transplant surgery at the University of Tübingen.

Our involvement in the large-scale integrating European research project “SysKid“

Our goal for many years has been the identification of novel biomarkers to assess the risk to develop CKD and to predict the rate of progression. Syskid for us is the unique opportunity to find new collaborators, which will allow us the combine our transcriptomics data with state of the art bioinformatics as well as animal models and cell culture experiments. All –omics techniques are extremely valuable hypothesis generating tools, however the data obtained need confirmation and we are looking forward to the able to combine and share whatever is necessary to improve our understanding of CKD.

Reference Papers

Read more about this group and our cooperating partners at www.microarray.at and www.syskid.eu.

The research group of Gert Mayer (from left to right):
Michael Rudnicki, Hannes Neuwirt, Gert Mayer, Susie-Jane Noppert,
Christian Koppelstätter, Judith Sunzenauer, Susanne Eder, Georg Kern

The respective specific research interests can be summarized as follows:

Regulation of tubular epithelial cell differentiation, survival, and proliferation; mechanisms of tubular epithelial cell protection; regulation of epithelial-mesenchymal-transition (EMT), mesenchymal-epithelial-transition (MET), and tubulointerstitial repair; role of oncostatin M for the differentiation and proliferation of renal tubular epithelial cells when compared with profibrotic mediators such as TGF-β1 and IL-1β the regulation of these processes by intracellular signaling molecules with a special emphasis on mitogen-activated protein kinases (MAPK), Akt/PKB, NFκB, Smad and Stat signaling.

A second large field of research interest of this laboratory represents diagnosis, therapy and prevention of myeloma-associated kidney disease.
In this context, the lab members are predominantly interested in tubular injury mechanisms induced by nephrotoxic monoclonal light chains and on tubular effects of the proteasomal inhibitor bortezomib.

Within WP5.1 of SysKid, the Cellular and Molecular Nephrology group uses advanced in vitro human epithelial renal cell culture models with the objectives of:

1. mechanistic analysis of the effects of candidate biomarkers and mediators known or identified in WP 2, 4, and 5.2 on the progression of CKD;
2. the identification and functional characterization of candidate target genes in in vitro models,
3. the identification of signaling processes, interacting partners and regulation of selected candidate targets.

Real-time PCR and Western blot analyses are utilized to study intrinsic expression of genes of interest in distinct cell culture models in order to identify those cell types, which are most suitable for subsequent over-expression and knock-down experiments.

As powerful research tools for assessing gene function as well as its regulation the group primarily uses gene knockdown and overexpression strategies utilizing RNA interference (RNAi) technology and transient/stable transfections, respectively. In addition, the Cellular and Molecular Nephrology Lab of P3-IMU is able to provide various well established assays in order to study functional endpoints such as cell phenotype, cell differentiation, EMT/MET, cell survival/protection, proliferation and apoptosis.

Selected References

The research group of Herbert Schramek (left to right):
Markus Pirklbauer, Rita Sarközi, Viktoria Haller,
Christine Hauser, Herbert Schramek

Most interestingly, both populations acted in the lymph node rather than locally in the kidney and had the potency to downregulate the pathogenic Th1- and Th17-response (Wolf D, JASN, 2005; Hochegger K, Eur J Immunol, 2005; Eller K, JASN, 2010).

We are now evaluating the interplay of these two populations in NTS. Additionally, we have recently provided data on the effects of the immunosuppressive substance Rapamycin. When started early Rapamycin effectively protects from NTS by limiting the B and T cell response. But when Rapamycin was started when proteinuria was already evident, proteinuria significantly increased (Hochegger K, JASN, 2008). We are now evaluating the exact mechanisms of rapamycin-induced proteinuria by performing laser capture microdissection in cooperation with the research group of Prof. Mayer.

Senescence, apoptosis and inflammation in renal IRI

We have focused on the one hand on senescence/apoptotic and on the other hand on inflammatory pathomechanisms in renal IRI. We found p21 and mouse telomerase reverse transcriptase – both involved in senescence - to be stable markers for long and short ischemic time intervals, respectively (Hochegger K, Am J Physiol – Renal Physiol, 2007).

To gain further insights in the function of p21, we pharmacologically influenced p21 by the proteasome-inhibitor Bortezomib. Bortezomib is known as a potent drug to treat multiple myeloma including its renal complications. In renal IRI Bortezomib turned out to aggravate renal function by increasing apoptosis of tubular cells even though renal inflammation was effectively inhibited (Huber JM, Am J Physiol - Renal Physiol, 2009). Currently, we try to upregulate p21 in vivo and thereby evaluate its function in renal IRI.

Immunoregulatory mechanisms and calcification in DN

We are currently working in db/db transgenic mice and evaluate the impact of regulatory T cells in this model.
Additionally, we treat the diabetic mice with a high phosphate-diet to evaluate vascular and tissue calcifications. This project is part of our FWF grant.

If you need further information please visit the homepage of our research group: www.rosenkranz-lab.com

Reference Papers