Urine and Kidney Proteomics

Urine and Kidney Proteomics

Acronym:

EUROKUP (European Kidney and Urine Proteomics)

Proposer: Antonia Vlahou Ph.D

Biomedical Research Foundation, Academy of Athens

Soranou Efesiou 4, 11527

Athens, Greece

Tel: 30 210 65 97 506

Fax: 30 210 65 97 545

e-mail: vlahoua@bioacademy.gr

COST National Coordinator: Mrs Katerina Galanou

International Cooperation Directorate

European Union Division

General Secretariat for Research and Technology

14-18 Messogion Avenue, 11527

Athens, Greece

Tel. +30 210 7458092

Fax. +30 210 7714153

e-mail: kgal@gsrt.gr

A. Abstract (up to 200 words)

Renal diseases constitute a major health threat in all societies. Proteomics is the large-scale analysis of the proteins of biological samples. Application of proteomics methodologies in the investigation of renal diseases will catalyze the development of optimal diagnostic and prognostic tests. Despite preliminary successful efforts, the interactions between the multidisciplinary teams of scientists working on kidney diseases and proteomics are still limited in Europe. EUROKUP will foster the generation of a strong and growing multi disciplinary network of scientists from at least 20 European countries, focusing on renal and urine proteomics. The objectives of the Action focus on the identification of reference clinical centers for major kidney diseases and establishment of uniform clinical databases, standardization and optimization of procedures, integration of data to systems biology approaches and dissemination of information for application to diagnostic/prognostic procedures. Multiple scientific, technological and societal benefits to the European Union are expected, including but not limited to setting the urgently needed standards for clinical proteomics and translational research and improving the clinical situation in chronic renal diseases.

Key Words (max 5 keywords): Kidney, Urine, Clinical Proteomics, Standardization, Network

Europ
B. Background (up to 2250 words)

B1. General background

Impaired structure/function of the kidneys is a major health problem of modern societies. Chronic renal diseases, usually progress to end stage renal disease (ESRD) which requires renal replacement therapy, currently costing more than 30.000 € per patient annually. The prevalence of chronic renal disease in adult European populations is above 10%, posing a serious health threat. It may also be one of the leading causes of cardiovascular disease (CVD), as failing renal function is one of the best predictors of CVD. In the future it is expected that the number of patients with chronic renal disease will even increase due to the increased number of patients with type II diabetes and aging of the population.

Research aiming at the development of urgently needed new diagnostic/prognostic biomarkers and understanding the molecular mechanisms underlying kidney diseases is being carried on in many European Clinics and Laboratories. In this effort, Urine plays a pivotal role; Specifically, it is considered the ideal tool to monitor non-invasively the kidney’s health status, since even in normal conditions, 70% of its protein content originates from the kidney or the urinary tract. However, this research area has not yet made full use of the developing field of proteomics. Proteomics is the large-scale analysis of the proteins of biological samples and involves all aspects of protein analysis from the determination of amino acid sequence to the identification of functional partners and ultimately assignment to regulatory pathways. “Clinical Proteomics” defined as the application of proteomic analysis to answer clinical questions is considered one of the most important research fields in our days, promising to discover diagnostic and prognostic biomarkers, therapeutic targets and unravel mechanisms of diseases. In this field, the standardization of clinical specimen collection, of quality controls, as well as the establishment of an interdisciplinary approach involving clinicians and basic scientists are of paramount importance in order to progress towards the development of “personalized medicine”. This need is reflected in the establishment of the Human Proteome Organization (HUPO) in 2001, which is “an international consortium of national proteomics research associations”, aiming at promoting among others “the development and awareness of proteomics research”. In 2005, the Human Urine and Kidney proteome initiative was established within HUPO with the objective to map the urine and kidney proteome in health and disease. This initiative highlights the strong interest of the scientific community in urine and kidney proteomics, but it does not provide funding for regular networking activities, needed to achieve its objectives.

Why COST Action?

The proposed Action provides the unique opportunity to expand the HUPO initiative, enhances its networking activities and promotes research in this field in Europe. As shown by a Medline Search using the terms “proteomics, kidney and urine” out of 379 entries, 175 had originating (corresponding) institutions in North America (46.17%), 142 in Europe (37.46%), and 62 in Asia and Australia (16.35%). These numbers indicate that there is a strong basis in Europe. The proposed Action is the only way to: a) establish maximal networking activity and b) boost research and help establishing Europe as a place of Scientific Excellence in the field of renal and urine proteomics. To demonstrate the competitive nature of this field, it should be noted that the American Society of Nephrology recently organized a series of retreats with the objective to define priorities in renal research and to make specific recommendations to the National Institute of Health for the development of core facilities (including proteomics) for kidney research.

Importantly, the Action is highly complementary to running European projects (see below), and falls within the guidelines of the European Science Foundation (ESF) since it tackles Functional Genomics and their integration into Systems Biology in an interdisciplinary approach; via standardization of approaches from the clinical specimen collection all the way up to the data acquisition, handling and analysis, the Action targets the smooth transition from basic to clinical research and the establishment of translational approaches in the area of kidney diseases. It is obvious that at this stage, these ambitious objectives cannot be achieved through research frameworks with limited partner numbers; to bring them into fruition, the first and most crucial step is to accrue the interested vastly multidisciplinary team of scientists, including clinicians, statisticians, bioinformaticians, protein chemists, molecular/cell biologists, to provide their expert opinion and disseminate their “know-how”. This can be only achieved through the open-minded and flexible COST mechanism.

B2 Current state of knowledge

Kidney proteomics

The application of proteomics methodologies in order to understand the molecular mechanisms of renal diseases and to develop improved biomarkers is continuously expanding. Applications on the investigation of metabolic diseases like diabetic or hypokalemic nephropathy, fibrotic processes including glomerulosclerosis, renal cancer, IgA nephropathy and other diseases have been reported. Proteomics methodologies are extensively used in the investigation of cultured cells obtained from human patients and models for kidney diseases. Collectively these experiments strongly support that (a) extensive proteomics investigations of kidney tissue will provide useful diagnostic/prognostic markers and drug/therapeutic targets for renal diseases; and (b) coordination of these efforts is absolutely necessary.

Urine Proteomics

Urine is excreted by the kidneys, is non-invasively and easily attainable and largely reflects renal function, making an ideal substrate for biomarker research. The protein content of urine is low under normal conditions and derives mainly from human plasma proteins filtered through the renal glomeruli and not absorbed at the proximal tubule level, exfoliated cells, degradation of extracellular matrix as well exosomes secreted from epithelial cells of the urinary track.

Multiple individual research efforts in the investigation of the urine proteome are currently being conducted. These involve the application of a series of proteomics technologies including two-dimensional electrophoresis (2DE), various forms of liquid chromatography in combination to mass spectrometry (LC-MS), MALDI-TOF or SELDI-TOF profiling and capillary electrophoresis coupled to mass spectrometry (CE-MS). Nevertheless, in comparison to other proteomics samples, urinary proteome analysis is still in its early stages. It been shown that variations in the protein content of urine exist depending on the collection time, the age, sex, diet and health of the donor. Despite this variability, protein markers for kidney and other diseases of major clinical importance (cancer, diabetes, atherosclerosis etc) have been detected in urine and in many cases have been approved to be utilized in the clinic for disease diagnosis and prognosis. This justifies and triggers a coordinated in-depth analysis of the urinary proteome, particularly with the advent of contemporary proteomics technologies, with the objective to identify novel disease biomarkers. Even more, to fully exploit the potential of urine as a diagnostic/prognostic tool, a concerted effort to develop standardized protocols for urine collection and processing is extremely urgent.

Proteomics methodologies

Undeniably, advances in the fields of chemistry, physics and computer sciences in the last decades have greatly facilitated proteomic studies. Significant improvements in the protein separation methodologies such as planar two-dimensional and capillary electrophoresis and chromatography, as well as in mass spectrometry systems, in the last decades have greatly increased the resolution, sensitivity and accuracy in protein detection. In addition, novel approaches such as CE-MS, MALDI-TOF-MS profiling and Multiple Reaction Monitoring (MRM) expand for the first time the application of mass spectrometry from a research-biomarker discovery tool to a biomarker quantification tool with potential direct clinical applications.

As expected, these technological/methodological improvements have given rise to new research challenges: the reproducibility, portability and general applicability of each methodology as a clinical diagnostic/prognostic tool has to be carefully evaluated by well designed multi-institutional studies. Each separation methodology and MS technology has distinct strengths and limitations and extensive complementarity between them exist. Given the proteome complexity, a combined approach that entails collaboration of different centers of expertise for each methodology, will be therefore required for the successful characterization of the human proteome in the normal and disease state. The detection of low abundance protein molecules remains a big challenge; In this field, nano-technologies and microfluidics, currently in development provide a highly promising avenue for improvement. Last but not least, the use of innovative bioinformatic tools is mandatory to make the most from all data generated by proteomics.

Innovation of the Action:

Coordination of activities and standardization of processes are clearly needed at all levels of investigation in the Renal/Urine Proteomics field. The Action fully addresses this need by bringing together the experts in the field with the objective to synthesize their input into a set of standardized approaches so as to maximize research efficiency and knowledge extraction. In addition, the Action recruits and brings together innovative proteomics, statistical and bioinformatics techniques with the objective to identify new biomarkers and therapeutic targets and develop non-invasive urine-based tests for kidney diseases.

B3 Reasons for the action

The critical mass of researchers working on a) the investigation of kidney diseases, b) exploring urine in disease diagnostic/prognostics, and c) developing/optimizing proteomics technologies for the investigation of clinically important questions, is clearly emerging in Europe. It is of paramount importance to bring these teams of scientists together with a clear focus on urine and kidney proteomics. Such an Action will have immediate and long-term benefits on multiple scientific/technological as well as economic/societal aspects in Europe:

Immediate Scientific/Technological benefits

1) The establishment of appropriate standards and quality controls in all aspects of urine and renal proteomics research, from clinical specimen collection and databasing to proteomics analysis and data processing will greatly increase the effectiveness and comparability of running studies in individual research centers.

2) Optimized protocols for sample processing, fractionation, protein identification and data analysis will be readily disseminated to the scientific community promoting proteomics research in general.

Future scientific/technological benefits

1) The foundation for future biomarker validation and translational studies will be set up, including working out their special statistical, clinical and methodological needs as well as establishing unified clinical biobanks.

2) The basis for the establishment of core facilities dedicated to proteomic kidney research in Europe will be set up. Such effort has already been initiated in the United States and is urgently needed in order to enhance the visibility and excellence of European research in this field.

3) The new proteomic approaches will provide insights in renal disease, which will subsequently lead to innovative therapeutic targets/approaches

4) Through the dissemination of the optimized protocols/novel proteomics approaches and the need for commercialization of the novel findings, enhancement of the activities of the private sector is anticipated.

5) The promotion of collaborations achieved during the Action will give rise to competitive research proposals to the European Framework programs and other funding organizations.

Future societal/economic benefits

1) The foundation for the exploration of urine in the clinical arena will be established with clear societal and economic benefits: urine diagnostic/prognostic tests for the highly prevalent kidney diseases may replace current invasive and costly approaches; they will also facilitate early detection and disease monitoring, reducing the number of patients and associated cost with end-stage disease.

2) Young researchers will be trained on the use of state-of the art proteomics technologies which are widely used in the pharmaceutical and medical industry.

3) Through the dissemination activities of the Action, public awareness on the potential of novel proteomics technologies in disease diagnosis and prognosis will increase.

B4. Complementarity with other research programs

The proposed Action, with a clear focus on Clinical Proteomics and specifically on Urine and Kidney has no direct overlap with any other European project; however, it is considered highly complementary with multiple running projects. A search of the CORDIS FP6 website using the keywords “renal proteomics” returned one entry: EUGINDAT. The project employs various –omics technologies with the objective to study Primary Inherited Aminoacidurias, a rare disease that affects renal reabsorption of amino acids. In addition, when individually utilized, the keywords “urine”, “renal”, “kidney” or “proteomics” brought up various projects aiming at investigating renal disease at the genome level (EUROGENE, Cells into Organs), involving urine analysis for prostate cancer diagnostic purposes (P-MARK), or focusing on the development of bioinformatics (ADDNET, BioInfoGrid, Diamonds, EMBnet, EMBRACE and HealthGrid ) or proteomics platforms (NanoSpad, Interaction Proteome, Loccandia, ProteomeBinders) for biological data. In addition, a COST Action on plant proteomics (EuPP) was recently funded. A clear synergy between the Action and these projects is envisaged: Tools developed and experience accumulated in these projects will be of interest to the Action, and vice versa: clearly the standards, proteomics tools and databases developed in the Action will facilitate the realization of the objectives of these projects, leading to more efficient use of European Resources. Leading scientists of these projects are participating or will be invited to the workshops organized by the Action so as to bring this synergy into fruition. As the 2006-10 Strategic Plan of ESF involves translational research and renal diseases, the timing for launching this Action is considered highly appropriate.

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C. Objectives and Benefits (up to 1500)

C.1 Main/primary objectives

The main objective of the Action is to catalyze translational research in kidney diseases via standardization in all aspects of urine and kidney proteomics analysis from specimen collection and databasing up to data processing and analysis.

C.2 Secondary objectives

1) Identify reference clinical centers in Europe and develop unified biobanks and a network for distribution of kidney disease-related clinical specimens (tissue-urine). Specific research areas will include glomerulopathies and tubulointerstitial diseases all leading to chronic renal disease and renal failure.

2) Evaluate the applicability of selected proteomics technologies (SELDI-TOF-MS, MALDI-TOF-MS, CE-MS, MRM) as urinary biomarker quantification and profiling tools to be utilized in the clinical setting. This will be achieved by dissemination of the expertise from the respective centers and designing the appropriate portability and reproducibility studies.

3) Optimize methodologies for the proteomics analysis of pure kidney cell populations. This is a very important research area since the analysis of selected cell types (rather than whole tissues) greatly facilitates the discovery of novel biomarkers and therapeutic targets.

4) Establish bioinformatics infrastructure specialized for urine and kidney proteomics

5) Train young investigators in state of the art proteomics and bioinformatics methodologies employed for Clinical Proteomics applications.

6) Disseminate the information gathered to the scientific community and increase the public awareness about the potential of proteomics in the development of non-invasive diagnostic/prognostic procedures

C.3 How will the objectives be achieved?

The objectives of the Action will be achieved by bringing together scientists working on relevant fields, in a manner that will allow fruitful interactions, dissemination of expertise and promotion of collaborations. It is characteristic that several scientific meetings in the fields of renal research and proteomics take place in Europe and internationally every year. However, the missing factor is a focus on urine and kidney proteomics and exchange of valuable information in this field. The Action will have a very strong interdisciplinary nature and will foster and promote the interaction of basic scientists (biologists, protein chemists, biochemists, statisticians, bioinformaticians), with clinicians (nephrologists, urologists, pathologists, epidemiologists). Centers of expertise in all aspects of the kidney disease proteomics analysis including clinical/patient enrolment centers, proteomics laboratories as well as biostatistical, data analysis and bioinformatics centers in Europe and internationally have been and will be continuously identified and recruited to the Action. Dissemination of the individual “know-how” and promotion of collaborations will be achieved through organization of workshops, summer schools, conferences, short term scientific missions and exchange visits of junior scientists. To this end, special efforts will be made to employ infrastructure developed in European Framework programs (e.g. available e-learning platforms such as the European Multimedia Bioinformatics Educational Resource-EMBER)

C4. Benefits

Considerable scientific, technological and societal benefits are expected.

At the scientific level:

Standardized approaches for tissue and urine collection will be defined and homogeneous clinical databases will be developed that will catalyze sample exchange between different researchers and consequently biomarker validation studies. Methodologies for urine and kidney tissue proteomics analysis will be optimized and respective standards will be set that will increase the effectiveness and comparability of current research efforts. Proteomic databases generated through this effort will be reference points for researchers and clinical nephrologists and will form the basis towards the development of personalized medicine approaches. Cross-correlation of these databases with genomic, transcriptomic and metabolomic data will form the basis for kidney and urine systems biology. The parallel analysis of urine and kidney proteome will lead to a better understanding of the biological processes in renal cells and more efficient exploitation of urine in disease diagnostics/prognostics.

At the technological level, the basis for the establishment of core facilities for kidney research in Europe including reference clinical settings for selected kidney diseases, facilities for tissue and urine specimen processing, proteomics and bioinformatics analysis will be set. Undeniably, such a major task will require further funding for infrastructure expansion. The roots however, for such collaborative research projects will be implanted and grown during the Action. In addition, the protocols and data to be generated will facilitate the development of new therapeutic approaches as well as of novel, non-invasive (and cost effective), diagnostic and treatment monitoring tests, mainly but not limited to renal diseases, since urine frequently reflects metabolic alterations stemming from other diseases. Therefore, extensive interactions with the private sector will be set-up for the exploitation of the findings. The latter interactions will also focus on the dissemination of the new proteomics approaches developed in the Action, which will enhance the activities of existing industries and/or lead to the establishment of new „start-up“companies.

At the level of the society, the Action will contribute to the reduction of the severity of renal pathologies and of the huge ESRD related costs, by setting up the basis for the development of non invasive early diagnostic tests and new therapeutic approaches.

As a result of their training through the workshops, short-term scientific missions, and exchange visits during the Action, young researchers will increase their competitiveness in job hunt in medical and pharmaceutical industries. Public awareness on the exciting new field of proteomics and its potential applications in the clinical setting will increase through the dissemination activities of the Action.

In summary, by bringing together experts from a variety of scientific fields and bridging the existing gap in information exchange between them, the Action will reach the following high quality outputs:

1. Increase efficiency of current and future research activities involving urine and kidney proteomics by optimizing and standardizing processes.

2. Set the pavement for the discovery and validation of novel therapeutic targets and diagnostic and prognostic biomarkers/biomarker profiles for the non-invasive detection of the highly prevalent kidney diseases.

3. Set the foundation for kidney and urine systems biology.

4. Catalyze translational research in this field.

C5. Target groups/ End users

The anticipated end users of the results of the Action include:

1. Basic scientists working in the Clinical Proteomics field,

2. Physicians and nurses involved in biological sample collection for research purposes,

3. Private sector which in collaboration with basic scientists will be involved in the commercialization of the novel diagnostic/prognostic approaches,

4. Patients suffering from kidney diseases and their families

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D. Scientific focus and workplan (up to 3000 words)

D1. Scientific focus (guidelines: describe the most important research tasks; provide a structured (not too detailed) workplan, explain the human and technical means- in lay terms)

To reach its major objective, the scientific focus of the Action includes the following 3 major research tasks:

1) Establishment of standardized procedures for acquiring, databasing and storing urine and kidney samples related to clinically important research questions.

This task tackles one of the main problems of current renal research which is lack of comparability of individual research studies due to differential use or misuse of clinical terminology, different biological specimen processing and storage mechanisms, and improper case-control study design including insufficient statistical power and sub-optimal control selection.

2) Definition of quality controls and optimized protocols for the various proteomics methodologies in use in urine and kidney tissue research.

This task addresses the optimization/development of protocols for the proteomic analysis of pure populations of kidney cells by the use of innovative approaches; it also tackles major difficulties that current researchers encounter in evaluating and reproducing the results from individual proteomics laboratories. The latter problem partially stems from the lack of appropriate quality controls and of knowledge on the technical characteristics (accuracy, resolution, reproducibility, linearity etc) of each proteomics technology. Another factor contributing to this problem is the presence of a plethora of technical protocols for protein extraction, fractionation and separation. The use of different protocols is undeniably needed due to the enormous complexity of the human proteome. At the same time however, optimization of a single protocol for each specific type of experiment and coherence of different laboratories to that is needed to allow direct data comparability.

3) Formation of bioinformatics infrastructure specialized for kidney and urine proteomics data and establishment of procedures for its continuous maintenance and updating.

The vast amount of data acquired by the contemporary proteomics methods require databases and mining tools that will allow comparisons of different samples, generation of reference profiles as well as both statistical and graphical information about the sample under examination. Along the same lines, various types of kidney or urine related 2DE protein maps have been generated and become available through the world wide web. These tools greatly facilitate research by providing protein map resources and efforts are underway towards their expansion. There is a definite need for urine and kidney database integration from multiple sources and development of user interfaces that allow new data entry, visualization and retrieval. Simply stated, an “open-minded” database has to be established to allow continual additions of data and integration of information acquired by different centers of expertise.

Summary of workplan

The Action will achieve its objectives by bringing together scientists with an in-depth knowledge on the various aforementioned aspects of renal disease, proteomics and bioinformatics analysis. As expected, the Action is characterized by a very strong multidisciplinary character since it includes nephrologists, urologists, pathologists, epidemiologists, statisticians, molecular/cell biologists, protein chemists and bioinformaticians. The “know-how” and the necessary infrastructure, including a wide range of state-of the art proteomics technologies (2DE, quantitative 2DE, various quantitative LC-MS approaches, CE-MS, SELDI/MALDI MS platforms) are available by the participating investigators. As described in detail below (section D2), four working groups (WG) will be formed; dissemination of the expertise and individual results, and synthesis of the input from various partners will be carried out through scientific meetings and short term scientific missions always with a clear focus on reaching the objectives outlined above.

D2. Scientific workplan .

The working plan has been discussed with all partners. To reach optimal efficiency in achieving the objectives, four working groups (WG) have been conceived:

WG1: Focusing on standardization in the clinical setting.

WG2: Addressing the special needs and protocol optimization for kidney tissue proteomics analysis.

WG3: Focusing on method optimization and standardization for urine proteomics analysis.

WG4: Addressing the bioinformatics issues pertinent to the urine and kidney proteomics analysis.

These working groups will be strongly linked together as shown on Figure 1. Clearly each of them has distinct tasks which also define the main type of expertise required in each case: clinical and expertise on statistics in WG1, kidney proteomics (WG2), urine proteomics (WG3) and bioinformatics (WG4). It should be emphasized however, that completion of the tasks of each group can only be achieved through continuous information exchange between them. For example, the clinical and statistical information accumulated in WG1 will have to be passed on to the basic researchers of WG 2 and 3 so as to design their studies accordingly, and to the bioinformaticians of WG4 to formulate the specific domains required for databases and ontologies. Similarly, WG2 and 3 will have to disseminate their expertise to WG 1 and 4 so as define the requirements for sample collection and storage (WG1) and the special needs for proteomics data deposit and analysis (WG4). Finally WG4 will have to work closely with the rest of the groups to integrate the accumulated data and increase knowledge extraction from them.

Figure 1: WGs in (acronym). The major objectives and main research tools within each group are shown. The WGs are strongly linked together and extensive information exchange between exists.

The specific objectives and workplan for each of the WGs are outlined below

WG1: Standardization in the clinical setting

This working group will address and reach a consensus on the following issues:

Definition of major kidney diseases and establishment of universally accepted pertinent terminology (staging system, definition of high risk patients etc); Depending on the specific interest of the members of the Action and tissue availability from several biobanks EUROKUP will focus on (a)congenital diseases (obstructive or cystic); (b) diseases affecting primarily the renal parenchyma, either glomeruli (IgA nephropathy, focal segmental glomerulosclerosis, membranous glomerulonephritis) or tubules (acute necrosis); (c) systemic diseases affecting the kidney like diabetes mellitus and lupus; and (d) other diseases like tumors and rare renal diseases.
Review of state of the art in proteomics research for the selected renal diseases and definition of the special research needs in each case. The latter include: a) definition of the most important clinically research questions (early diagnosis versus prognosis versus drug efficacy endpoint etc) and b) assessment of the stage of current proteomics (biomarker) research (discovery phase, versus phases of confirmation or validation of already discovered biomarkers);
Definition of optimal controls for each disease type so as to increase reliability of current and future biomarker discovery, confirmation or validation studies;
Definition of the sample sizes needed to reach statistically sound results for each of the biomarker discovery, confirmation and validation studies. This is an area of special interest since inappropriate use or even complete negligence of statistics resulted in an array of insignificant or even misleading data in the past;
Development of protocols for patient enrolment as well as for monitoring and evaluating participating centers.
Standardization of biological sample collection (in collaboration with WG2 and 3-described below). This also includes an assessment of the ability of participating centers to follow uniform protocols for sample collection and databasing.
Establishment of criteria for the quality assessment of existing body fluid and tissue banks for kidney disease
Develop a network for the distribution of kidney disease-related biological material

These research questions will be addressed by a series of teaching activities (summer schools, Working Group meetings) focusing on each of these issues. It should be emphasized that courses on the principles of statistical design of biomarker discovery and translational studies are urgently needed in order to increase the efficiency of the large number of discovery and validation studies currently being conducted and anticipated to be conducted in the future.

WG2: Method optimization for kidney tissue proteomics analysis and imaging mass spectrometry

The field of biomarker and therapeutic target discovery for kidney diseases can greatly benefit from the development of innovative approaches for the proteomics analysis of pure kidney cell populations. The Action addresses this need by setting as a very important scientific goal to optimize and standardize methods for the proteomics analysis of pure kidney cell populations either by coupling to laser capture microdissection techniques or by imaging mass spectrometry.

The urgent need to address this issue stems from the multiple distinct structural elements forming the kidney: There is a cortex, containing glomeruli and tubules, the medulla, containing tubules with different characteristics than the ones in the cortex, and finally there is the vascular system that provides blood supply to both the cortex and the medulla. All these structural differentiations reflect functional differences.

Proteomic approaches so far have been hampered by our limited ability to study each of these compartments separately and within its biological context. Most of the studies performed so far, deal with a piece of biopsy tissue or a part of an animal model renal tissue, analyzed by the classical methodologies. Therefore, these analyses cannot discriminate between the different cell types and their specific structural/functional alterations in a pathologic process.

In an effort to overcome this problem, some studies have introduced the purification of either glomeruli (where filtration takes place) or tubules (where re-absorption takes place). This is a clear improvement, however, this approach requires lengthy isolation procedures that lead to stress and therefore alter substantially the proteomic profile of the compartments under study.

The Action will address the above mentioned limitations by coupling the proteomics analysis to the Laser Capture Microdissection (LCM) technology. Alternatively peptide/protein imaging approaches of kidney tissue slices by mass spectrometry will also be developed. With these approaches, the proteomic profiles of specific compartments or even specific cell types can be determined greatly facilitating the study of specific categories of renal diseases (cystic diseases, glomerular diseases either of primary or systemic aetiology, tubular/tubulointerstitial diseases, vascular diseases).

Groups with expertise on the combinatorial use of LCM with proteomics or imaging mass spectrometry are participating in the Action. They will disseminate their “know-how” to other partners by organization of workshops, short-term scientific missions and summer schools. In parallel, in collaboration with the clinicians and the proteomics experts in the Action the improvement of several aspects of the technique will be addressed. Specifically, the optimal storage, fixation and staining of tissue sections need to be determined and standardized. Current protocols employ primarily frozen fixed tissue as starting material. However, in many occasions, renal tissue from humans even dating back to the middle of the 20^th century and classified by expert pathologists as well as tissue from animal models for diseases have been fixed in formalin and embedded in paraffin. The Action will focus on the development of novel techniques for the proteomics analysis of such material. This would allow retrospective prognostic and diagnostic biomarker investigations of a vast archive of pathologically characterized clinical samples that exist in most Pathology departments worldwide.

The LCM technology has a low yield of proteins and their further analysis is consequently quite challenging. It is anticipated that for obtaining the maximal information, the coordinated use of different techniques (e.g. 2DE, SELDI and LC-MS) as well as the recruitment of developing nano-scale proteomics devices (such as lab-on chip miniaturized 2DE systems) is necessary. Regardless of whether the analysis involves kidney pure cell populations or whole tissue, the detection of low abundance molecules such as growth factors, transcription factors, as well as of specific protein classes such as hydrophobic/membrane proteins or post-translationally modified proteins (glycosylated, phosphorylated etc) remains technically difficult. To address these issues, the expertise from groups working on the development of proteomics nano-devices as well as on sample enrichment techniques for specific type of proteins (glycoproteins etc) will be indispensable; It is anticipated that workshops and training activities for young investigators on these issues will be organized throughout the course of the Action.

Collectively, objectives of the activity of WG 2 include:

1. Method optimization and standardization for the preparation of pure populations of all renal compartments from a) frozen and b) paraffin tissue sections following the use of Laser Capture Microdissection and imaging/profiling mass spectrometry;

2. Method optimization for protein extraction, separation, quantification and detection of post-translational modifications of kidney proteins;

3. Identification of existing infrastructure on nano-scale proteomics devices such as 2DE- lab on chip mini-devices and evaluation of their potential application for the analysis of starting material of limited amount, such as the protein extracts from LCM-isolated cells.

WG3: Standardization of urine proteomics analysis

To promote the use of urine as a biomarker discovery but also as the body fluid highly suitable for diagnostic/prognostic and disease monitoring tool several aspects of the proteomics analysis have to be improved. The scientific focus of this WG is method optimization and standardization of protocols for the study of the different types (soluble, exosomal, proteins included in exfoliated cells) of urinary proteins.

Protocols employed for the study of each of the above protein types will be discussed so as to establish clinically feasible guidelines for urine specimen collection and processing (in collaboration with WG1). It should be emphasized that the analysis of the urinary proteome is considered challenging due to the variations in urine composition (intra-individual and inter-individual variabilities) as well as due to the presence of many proteins in low abundance and in modified forms. Various protein preparation/extraction methodologies are being applied in the investigation of the urinary proteome including, ultra-filtration, precipitation, solid phase extraction, dialysis, ultracentrifugation or combinations thereof. Each of these protein preparations methods may be combined with different protein separation techniques (2DE, LC-MS, SELDI, CE-MS, MALDI profiling etc). Partners in the Action have an in-depth knowledge on various urinary protein preparation techniques; their input will be synthesized into a set of optimized urinary protein extraction protocols to be shared among all partners and to the wider scientific community.

Besides optimization/standardization of sample collection, storage and urinary protein extraction protocols, this WG will also address the employment of urine in multiplex protein profiling studies. Recent reports support the potential of proteomics platforms such as MRM, CE-MS and MALDI/SELDI-MS in biomarker validation and even direct clinical application, as novel diagnostic/prognostic approaches. In several cases, application of these techniques has provided significantly higher accuracy rates in disease detection compared to available diagnostic/prognostic means. In the course of the Action, experts in the field will bring in their experience from the application of these methods with the objective to disseminate their “know-how” through workshops and training activities and set the pavement for future method portability and validation studies. In addition, studies involving the analysis of the same set of urine samples by the different technologies will be conducted, so as to directly compare the generated proteomic profiles.

Summarizing, objectives of WG3 include:

1) Optimization and standardization of protocols for sample collection and storage for the analysis of the urinary soluble, exosomal and cellular proteins. This includes determination of the impact of sample preparation methods as well as of intra-individual and inter-individual variability on protein patterns.

2) Method optimization for the enrichment of low abundance urinary proteins and their separation and identification.

3) Optimization of quantification approaches for urinary proteins

4) Evaluation of the applicability of multiplex profiling proteomics platforms as routine urinary biomarker validation and/or renal disease detection tools.

As described above, WG2 and WG3 have distinct objectives pertinent to the special needs of the biological sample under investigation (kidney tissue in WG2, urine in WG3). Nevertheless, these two working groups share a very important main objective: To get a clear understanding and establish the analytical performance characteristics of each of the employed proteomics methodologies (various types of LC-MS approaches, CE-MS, MALDI profiling, 2DE in combination to different detection methods), as applicable. These characteristics are extremely important for the design of appropriate quality controls and include:

1) Reproducibility of analysis, ie. repeatability and long-term stability

2) Achievable separation efficiency (protein resolution)

3) Limit of detection, limit of quantification, linearity

4) Mass accuracy

5) In collaboration with WG4, data normalization and interpretation approaches.

WG4: Development of a bioinformatics infrastructure for specialized research in kidney and urine proteomics.

This WG will focus on computational issues pertinent to renal and urine proteomics data representation, sharing, retrieval, quality assurance and control, integration and analysis. Specific tasks that will be carried out include:

1) Identification of all available knowledge and data sources concerning kidney and urine proteomics (e.g. UroProt, http://intramural.niddk.nih.gov/research/uroprot/) and other -omics. This will be followed by the development of an integrated system for querying heterogeneous information sources relevant to the topic (structured knowledge bases (e.g. UniProt), document collections (e.g. MedLine), image databases, etc.), in view of retrieving and extracting specific information related to kidney and urine proteomics. To this end, software tools developed under running European projects (e.g., UTOPIA (User-friendly Tools for OPerating Informatics Applications)) will be recruited.

2) Creation, maintenance and update of a specialized “urine and renal proteomics” database that will integrate existing and newly acquired proteomics data. This task includes a review of the state-of the art in available pertinent bioinformatics platforms, definition of the special needs that such a dedicated database entails and establishment of standardized approaches for proteomics data deposit. The database schema will be designed in conformity with the ontology to be developed in Task 3.

3) Development of a specialized ontology on kidney and urine -omics and all related diseases. The ontology will be anchored to top level concepts of reference ontologies such as Gene Ontology (for gene products) or FMA (for anatomy), as well as other available ontologies (myGrid, UTOPIA) and terminological resources used by kidney/urine specialists (e.g., MeSH, ICD) . The initial or core ontology on kidney and urine proteomics will be developed by domain specialists together with computer scientists experienced in the development and deployment of biomedical ontologies. The latter will also develop web-service based mechanisms for incremental and collaborative refinement of this ontology by domain experts.

4) Design, implementation and optimization of a set of tools for pre-processing of, and knowledge discovery from, proteomics experimental data. Issues to be addressed include: the data high dimensionality-small sample size problem, the inherently noisy nature of the data, the stability and reproducibility of the produced models, the incorporation of domain knowledge into the knowledge discovery process.

5) Investigation of strategies and development of a set of procedures to control for the quality of the experimental data and learned models.

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E. ORGANIZATION

E1. Coordination and Organization

The Action will be coordinated by the Management Committee (MC), according to the published rules and procedures and with the support of the Scientific Secretariat in Brussels. To better organize and promote interactions between the multidisciplinary teams of scientists participating in the Action, 4 working groups will be established as described below (E2).

The Management Committee will have as main responsibilities:

---Appointment of chair, vice-chair(s), and WG co-ordinators; This will be carried out during the kick-off meeting

---Appointment of a scientist responsible for generating and frequently updating the Action-specific website (web-site coordinator).

---Decision-making on the distribution of funds to the various activities of the Action

---Planning and coordination of several types of meetings and teaching activities, detailed below

---Evaluation of meetings and other activities necessary to meet the set objectives

---Evaluation and report of the progress of the different WGs and the Action as a whole. Preparation of Annual Reports

---Promotion of collaboration between the different WG members

---Establishment of extensive collaborations between members of the WGs and members of other related Actions and Scientific programs in Europe and world-wide.

– Increase the visibility of the Action and promote interactions with medical and proteomics societies.

--Promote interactions with the private sector and dealing with issues related to exploitation of results

A Steering Group (SG) consisting of the Chair, vice-chair(s), web-site coordinator and the WG coordinators will be established. Members of this group will be in frequent (at least once every two-months) communication via e-mail and/or telephone conferences to discuss on the progress and ensure good coordination of the activities of the different WGs. The Chair will be contacting members of the MC during inter-meeting periods to inform them about SG discussions as needed, and to recruit the necessary elements for achieving the milestones. Besides these checkpoints, the MC/WG meetings will also play a very crucial role in evaluating the progress of the Action (see below)

MC/WG meetings

The Management Committee will convene at least four times, (once or twice every year) to ensure efficient coordination, evaluate the progress and make specific plans for future activities. These meetings with the exception of the first one (kick-off meeting) will coincide with the Working Group meetings. They will take place in different locations reflecting the geographical distribution of the current and future members of the Action. Efforts will be made so that the MC/WG meetings coincide with larger meetings in the field (for example, the Human Proteome Organization (HUPO), Siena Proteomics meetings etc) so as to increase the visibility of the Action and attract more participants. Their duration is expected to be 3-days. To ensure efficiency in meeting the WG-specific needs as well as in promoting the communication and reaching maximum possible exchange of information among different WGs, the meetings will include 1-2 day-long WG-specific sessions and at least 1 day-long plenary sessions involving the participation of representatives of all WGs. These will involve presentations of individual research results, as well as presentations and discussions on later developments in the field. Emphasis will be given on the inclusion of young scientists and women in these activities (oral presentations, chairmanship of sessions etc). Based on these presentations, the MC will be evaluating the progress of the Action towards reaching its objectives and will be deciding on future plans accordingly.

Short Term Scientific Missions and other teaching activities

The STSMs consist a major tool for the dissemination of “know-how” to young investigators and the promotion of collaborations between different research teams. Candidates to participate in these activities will be selected following an application process and assessment by the MC members. In addition the organization of about 5-day long summer schools and workshops for young investigators will be conducted. These will include (but will not be limited to) presentations on biomarker discovery and translational study design, proteomics analysis of LCM cells and urine proteomics. For these training activities special efforts will be made to utilize available e-learning infrastructures, such as EMBER (European Multimedia Bioinformatics Educational Resource), developed under European Framework programs. It should be emphasized that participants in the Action have the infrastructure and “know-how” on a very wide range of state-of the art methodologies and technologies. These teaching activities provide the unique opportunity to disseminate this knowledge to young researchers in Europe, promoting scientific Excellence in the fields of clinical proteomics and translational research.

E2. Working Groups

Four working groups will be formed based on the scientific workplan described in section D2 of the Technical Annex:

WG1: Focusing on standardization in the clinical setting.

WG2: Addressing the special needs and protocol optimization for kidney tissue proteomics analysis.

WG3: Focusing on method optimization and standardization for urine proteomics analysis.

WG4: Addressing the bioinformatics issues pertinent to the urine and kidney proteomics analysis

Each Working Group will have a co-ordinator to be appointed by the Management Committee during the kick-off meeting. The WG-coordinator will play a pivotal role in reaching the objectives of the Action.

Specifically, the main responsibilities of the WG- coordinators include:

--Interact frequently with the other SG members and the members of the respective WG to discuss on WG-related issues

--Coordinate the activities within each WG so as to meet the objectives of the Action

--Suggest to the MC the organization of scientific activities (for example workshops, summer schools, STSM) related to the specific WG.

--Promote interactions and collaborations of different researchers within the WG and with the other WGs.

--Report on the progress of the WG to the Action chair and the MC.

E3. Liaison and interaction with other research programmes

Interactions with scientists participating in Framework Programs and Actions with complementary interests to the Action will be of major interest to achieve maximum efficiency in using the European Resources. As described in section B leading participants of projects focusing on the development of proteomics tools (NanoSpad, Interaction Proteome, Loccandia, ProteomeBinders), or bioionformatics platforms for biological data (ADDNET, BioInfoGrid, Diamonds, EMBnet, EMBRACE and HealthGrid ), that involve urine analysis (P-MARK), or study kidney diseases at different –omics (genomics etc) levels (EUROGENE, Cells into Organs) as well as from the Action on plant proteomics (EuPP) are already participating in the Action or will be invited to the activities organized by the Action. Through these interactions the experience and tools acquired in these projects will be recruited for the investigation of kidney diseases and the development of urinary tests, and vice versa: the standards, optimized protocols, databases, ontologies etc generated in the Action will facilitate the realization of the objectives of these projects.

E4: Gender balance and involvement of early-stage researchers

This COST Action will respect an appropriate gender balance in all its activities and the Management Committee will place this as a standard item on all its MC agendas. This Action will also be committed to considerably involve early-stage researchers. This item will also be placed as a standard item on all MC agendas.

It is worth emphasizing that early stage female scientists have already played a major role in the conception and delineation of the objectives of the Action. It is therefore expected that young female scientists will also play leading roles in the management of the Action. As described above, gender balance will be observed in the Workshops, Teaching Activities and Short Scientific Missions where also early stage researchers are expected to form the overwhelming majority of participants.

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F. TIMETABLE

The proposed duration for this Action is 4 years. The Kick-off meeting will mark the start-point of the Action where the chair, co-chair(s), WG co-ordinators and web-site coordinator will be selected. The Action-specific website will be generated the first trimester and will be updated every trimester. Reports will be generated at the end of every year. Details about the frequency and the timing of MC meetings, WG meetings, Workshops, Short-Term Scientific Missions and Teaching activities are indicated in the table below. Please note that WG/MC meetings are planned to take place once a year; nevertheless depending on the available budget and the specific needs of the Action their frequency may increase to twice a year.

YEAR 1

YEAR 2

YEAR 3

YEAR 4

ACTIVITY

Coordination

■

Kick-off meeting

■

Website generation

■

Website update

■

Reports

■

Management Committee meetings

■

WG1 meetings

■

WG2 meetings

■

WG3 meetings

■

WG4 meetings

■

Workshops/summer schools

■

Short-term scientific missions

■

Final conference

■

Based on the description in Section D and the above timetable, the following Milestones will be reached

Type of Milestone	Description	Month(s)
Evaluation	Progress Report	12, 24, 36, 48
Dissemination	Website Generation	3
Dissemination	Generation of brochure describing the Action	4
Didactic	Working Group Meetings, Workshops, Summer Schools, Final conference	12, 24, 36, 48
Dissemination	Publication of WG meeting and final conference proceedings on special issues of peer-reviewed scientific journals	12, 24, 36, 48
Dissemination	Publication of material of summer schools to the website	18, 28, 40, 48
Research	Standardizing clinical terminology for major kidney diseases	12, 24
Research	Definition of principles for study design	12
Research	Identification/Establishment of Biobanks for kidney diseases	12, 24, 36, 48
Research	Definition of optimized protocols/standards for analysis of pure kidney cell populations	12, 24, 36, 48
Research	Definition of optimized protocols/standards for the analysis of urinary proteins	12, 24, 36, 48
Research	Definition of optimized bioinformatics tools for proteomics data pre-processing and analysis	12, 24, 36, 48
Research	Establishment and updating of kidney and urine specialized proteomics database and ontology	12, 24, 36, 48

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G. Economic dimension

The following 20 COST countries have actively participated in the preparation of the Action or otherwise indicated their interest: AT, BE, BG, CY, CZ, DK, FI, FR, DE, GR, HU, IE, IT, MK, NL, PT, ES, SE, CH, UK.

It is expected that more participants will join this Action.

On the basis of national estimates provided by representatives of these countries, the economic dimension of the activities to be carried out under the Action has been estimated at roughly EUR 32 Millions for the total duration of the Action.

This estimate is valid under the assumption that all the countries mentioned above, but no other countries, will participate in the action. Any departure from this will change the total cost accordingly.

H. DISSEMINATION PLAN

H.1 Who?

The activities of the Action will be disseminated as widely as possibly to diverse groups of people including basic scientists from academic- research institutions and industrial settings, clinicians and general public. Target groups specifically include:

---Fellow researchers working in the fields of Clinical Proteomics and Proteomics, in general.

---Fellow researchers working on renal diseases and urine diagnostics, not using proteomic approaches

---Other European research groups participating in projects with complementary interests to the Action (described in B4)

---Hospitals and Clinics specializing in renal and urological diseases as well as medical societies (European Renal Association, European Association of Urology (EAU), Board of the European Society for Urological Research, Medical Research Council etc)

---Small and Medium Enterprises focusing on the development of novel diagnostic kits

---European, National and Regional policy makers

---The general public

H.2 What?

---An Action-specific website will be constructed in order to provide information to the international scientific community, to industries (proteomics technologies providers, pharmaceutical, biotechnology settings) and general public. The Management Committee will assign this task to a partner (web-site coordinator). Part of the website will be accessible to the general public, whereas a section will be password-protected for the exchange of specific information and unpublished data between partners. The website will also contain information on the Action activities (meetings, workshops, etc), proceedings of meetings, links to publications of participants, job/STSMs announcements as well as material and presentations from the didactic activities.

---A brochure will be generated at the beginning of the Action describing its objectives and planned activities. This will be distributed to scientists, representatives from the industry and society in major international conferences on proteomics (for example HUPO) and kidney diseases (for example European Renal Association- European Dialysis and Transplant Association meetings etc)

---Scientific publications in peer-reviewed scientific journals generated as a result of collaborative research during the Action either in the form of original, review, or technical articles. To increase the visibility of the Action, the publication of the proceedings of WG-meetings and final conference to highly cited peer-reviewed journals in the field will be pursued.

---Combined Management Committee and Working Groups meetings and other scientific conferences; The MG/WG meetings are planned to take place on a regular basis, ideally every six months, in various geographic regions, in order to encourage participation of all interested members. To increase the visibility of the Action, they will be preferably organized as satellites to major scientific conferences in the field, such as the HUPO, the Siena Proteomics, and the European Renal Association-European Dialysis and Transplant Association (ERA-EDTA) meetings.

---Short-Term Scientific Missions, targeting young scientists especially originating from developing regions, in order to foster exchange of ideas and technology transfer.

---Teaching activities (Workshops and summer schools), in order to disseminate the latest developments in the proteomics field and also combine hands-on practical training with theoretical information. These will be offered mainly to young investigators.

H.3 How?

The Management Committee (MC) will be responsible for implementing all of the above activities. The representative members of each country will be responsible for disseminating the activities of the Action to research groups within their countries, industrial partners, medical societies and representatives of the society. Each MC member is therefore, expected to generate, regularly update and circulate to other MC members a list of target groups with contact information. For regional meetings and other activities, the MC will delegate responsibilities to WG-coordinators and members of the WGs depending on their specialty. In addition, the MC will be responsible for providing all necessary information regarding the above mentioned activities and their outcome as well as revise the dissemination plan according to the Domain Committee (DC) recommendations.

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