Charlotte Reichardt

PhD Student

Peter Mertens

Project Leader

Our previous data demonstrate that following acute kidney injury cold shock proteins Y-box binding protein-1 (YB-1) and DNA binding protein-A (DbpA) control cell recruitment to activated tubular cells and direct tubular cell phenotypes and survival. The function of YB-1 is highly context-specific, as mice with heterozygote YB-1 knockdown show diverse responses to injuries: following ischemia/reperfusion (I/R) the tubular damage is enhanced, whereas following tubular obstruction (UUO) damage is markedly reduced. Furthermore, we recently showed that high salt diet (HSD)-induced proximal tubular phenotypic activation and sodium-glucose cotransporter-2 expression are coordinated by YB-1. In addition, we identified Notch-3 as receptor for extracellular YB-1. In the absence of receptor Notch-3 tubular cells are unresponsive to cell stress and lack canonical NF-κB activation. Renal tubular epithelial cells are amongst the most metabolic active cells in humans with susceptibility to injuries (e.g. toxins, hypoxia) and orchestrate immune cell recruitment. If perpetuated, these processes lead to maladaptive responses, cell death and breakdown of the barrier functions in isolated nephrons. Tissue architecture devoid of tubular barrier function results in vascular rarefication and organ fibrosis. Collectively, cold shock proteins determine tubular cell fate decisions in an injury-specific manner. Elucidation of the underlying mechanism(s) during acute kidney injury is the focus of this project.
Within this project, we already showed that mice with a genetic deletion of DbpA revealed no apparent kidney pathology and a normal life span. However, DbpA was detected as a mitochondrial protein. Moreover, we already showed that primary tubular cells (TECs) with a genetic deletion of DbpA revealed enhanced basal respiration, proton leak, maximal respiration, spare respiratory capacity and non-mitochondrial respiration. The most striking findings relate to mitochondrial transfer experiments, which improved mitochondrial respiration of recipient wild type TECs (cooperation Project 6). Furthermore, we initiated extensive analyses to assess mitochondria (size, number, outer and inner membrane structure), and glycolysis (glucose uptake), which will be performed by electron microscopy, staining protocols (MitoTracker) and FACS analyses in cooperation with Project 12.

Tubular barrier system. The nephron is the smallest functional unit of the kidney and consists of a glomerular corpuscle and the tubular system. The tubular barrier is formed by epithelial cells and basement membranes and is supplied by adjunct blood vessels. In tubular epithelial cells DbpA is located at the cell membrane and upon tubular cell dedifferentiation may shuttle from the cytoplasm to the nucleus and coordinate proliferation. Furthermore, DbpA was detected as mitochondrial protein  as it co-localizes with mitochondrial marker proteins.

Sohail Ahmad

PhD Student

Peter Mertens

Project Leader

Renal tubular epithelial cells are amongst the most metabolically active cells in the human body and are therefore highly susceptible to injury (e.g. toxins, hypoxia). If the insults are sustained, they can lead to maladaptive responses, including cell death and a subsequent loss of barrier function, which ultimately result in reduced kidney function.
Previous studies in mouse models investigate the role of the cold shock proteins Y‑box binding protein-1 (YB-1) and DNA binding protein-A (DbpA) following acute kidney injury. Heterozygote YB-1 knockdown mice showed enhanced tubular damage following ischemia reperfusion (I/R); whereas damage was markedly reduced following tubular obstruction (UUO). Indicating that YB-1 can both prevent and promote renal injury. Furthermore, we recently showed that YB-1 coordinates the proximal tubular phenotypic activation and sodium-glucose cotransporter-2 expression following high salt diet (HSD) induction in mice. Our recent work demonstrates that a DbpA knockout protects mice from acute hypoxic kidney injury. Previous results also show the co-localization of DbpA within mitochondria, indicating its relevance with mitochondrial metabolic activity.  Collectively, cold shock proteins determine tubular cell fate decisions in an injury-specific manner. Since the above-mentioned data is based on in vivo mouse models, the underlying molecular mechanisms remain unclear.
Based upon our initial data, we hypothesized that targeting DbpA in patients could be beneficial for the treatment of acute kidney injury. Therefore, the first step is to reproduce our data in a human system. For this, we will take advantage of our recently established organoid cultures, which reproduce many features of the architecture seen in an intact organ (kidney). Utilizing CRISPR/Cas technology, we will induce a knockout of DbpA in human cells in order to test whether this reproduces the protective effect we observe in mice under hypoxia.   Furthermore, this model system will allow us to test the influence of environmental factors (high salt, high glucose, bacteria) on cold shock protein expression as well as their influence on the metabolic activity of the cells and the cellular responses to injury (e.g. cellular toxicity). This system is also suitable to test small molecule inhibitors to see whether we can reproduce the protective effects that are observed upon genetic deletion. Thus, this system brings us a step closer to developing new therapeutic strategies to treat kidney disease.

Sarmad Sleiman

MD Student

Peter Mertens

Project Leader

DbpA (DNA binding protein A) is a member of the human cold shock protein family. Cold shock proteins are characterized by the presence of one or more highly conserved cold shock domains, which possess nucleic acid binding properties. Thus,  DbpA is a multifunctional RNA/DNA binding protein with pleiotropic functions, such as the regulation of cell proliferation, differentiation and stress responses. Evidence links a skewed cold shock protein expression pattern with inflammatory diseases and cancer. Upregulated DbpA expression has been observed in various types of cancer including hepatocellular carcinoma, gastric cancer, breast cancer, ovarian melanoma, colorectal cancer, and chronic myeloid leukemia. In healthy kidney tissue DbpA expression is found to be restricted to vascular smooth muscle cells but profoundly upregulated within the glomerular mesangial compartment in mesangioproliferative glomerularnephritis. This project aims to investigate the functional roles of DbpA in a murine model of mesangioproliferative glomerularnephritis, denoted nephrotoxic serum nephritis.  Mice with genetic ablation of DbpA will be included into the study and compared to DbpA wildtype and DbpA heterozygous mice after disease induction. This will provide an insight into the disease-modulating effects of DbpA and its contribution to disease development and progression. Recent studies have shown modulating effects of DbpA concerning immune response. In order to determine immune cell infiltration, we will use flow cytometric analysis of kidney tissue.  DbpA is further involved in cell-cell communication. Given its interaction with a major tight junction protein, Zonula occludens-1 (ZO-1), DbpA is denoted as ZO-1-associated nuclear acid binding protein (ZONAB) and is significantly involved in the regulation of podocyte interactions in the filtration barrier of the kidney. Malfunctioning podocyte interactions cause continuous leakage of plasma proteins (proteinuria) and is considered to be an important clinical risk factor in the progression of chronic kidney disease. Therefore we will further focus on alternations of podocyte interactions which depend on changes in the DbpA genotype and expression pattern in disease and health. The Podocyte Exact Morphology Procedure PEMP allows visualization of podocyte interactions along the filtration barrier, based on super-resolution microscopy. This method will be conducted in co-operation with the University of Greifswald. Further prospective cooperation partners are the Z-project SFB 854 for MELC analysis of DbpA and its interaction proteins and Project 9 of RTG 2408 for in vitro analysis of podocyte cell cultures. We hypothesize that DbpA is essential for the development of induced mesangioproliferative glomerular nephritis, while the expression level of DbpA influences the phenotype and the severity of the disease.

Leo Hoffmann

MD Student

Peter Mertens

Project Leader

Organoids are three-dimensional cell cultures that resemble functional and structural traits of human organs. These structures have advantages over two-dimensional cell culture experiments given the establishment of complex polarized three dimensional cell-cell interactions that more closely mimic physiological barriers and functional processes in the human body. In our hands organoid structures develop following reprograming induced Pluripotent Stem Cells (iPSCs). Induced Pluripotent Stem Cells have been manipulated through retro- and lentiviral transduction of transcription factors such as Oct3/4, Sox2, Klf4, Lin28, Nanog and c-Myc causing them to regain their pluripotency when incubated with basic fibroblast growth factor (bFGF) and develop into kidney-like organoids following stimulation with a Wnt-agonist in 12 to 20 days. This project first aims to standardize the generation of kidney organoids. Kidney specific proteins and cell types are visualized and quantified by Western blotting, immunofluorescence staining and fluorescent activate single cell sorting. A comparison of different batches and different stages of organoid growth will be performed to assess variability in generating kidney organoids. High glucose and high salt addition to culture media and their influence on organoid generation are another focus of this project. Establishment of an infection model will be initiated after completion of standardization. For that means organoids of a chosen age will be incubated with bacteria such as Proteus mirabilis, Klebsiella pneumoniae, uropathogenic Escherichia coli tribes and Staphylococcus aureus, as the most common pathogens of acute infectious pyelonephritis. The bacterial colonization and infection will be monitored for bacterial numbers (multiplicity of infection) and spatiotemporal distribution throughout the organoid. Thus, novel insights into cellular responses following bacterial dissemination and cell damage as well as alterations in the barrier integrity will be gained. Furthermore, cell damage will be quantified by propidium iodine and annexin V staining as well as examination of damage and pathogen associated molecular patterns. Cooperation with other members of the RTG include Project 8, Prof. Dudeck’s, Prof. Naumann’s and Prof. Kahlfuß’ workgroup, as well as Prof. Kaasch and Prof. Zautner from the Institute of Microbiology.

Process of generating kidney organoids. After transforming adult somatic cells into iPSCs, these are held in culture on a basement membrane matrix. To set the conversion into kidney organoids in motion the iPSCs are stimulated for three days. Following that period, the organoid bodies will start to grow in size. On day 7 first tubular structures start appearing. Between day 12 and day 20 the differentiation of tubular segments reaches its peak and characterization as well as infection experiments are conducted in this time. From day 21 and onwards central fibrosis will arise due to deficient nutrient perfusion of the continuously growing organoid body.

Anna Krause

MD Student

Peter Mertens

Project Leader

Excessive sodium chloride ingestion is continuously rising in the western world. High salt diet (HSD) damages the kidneys leading to fibrosis, tubulointerstitial injury and renal hypertrophy. Furthermore HSD aggravates albuminuria and decreases the glomerular filtration rate, which is associated to a worse CKD prognosis. A focus of our group are the roles that cold shock proteins play in inflammatory diseases, with the most prominent being Y-box binding protein-1 (YB-1). Members of this protein family regulate cell proliferation, matrix synthesis and the inflammatory milieu. Within this project we plan to further investigate the role of YB-1 in an HSD-induced damage model. To achieve this we use an tamoxifen-inducible Ybx1 knockout (KO) strain and compare wildtype (WT) and KO mice that receive either a normal salt diet (NSD; standard chow, tap water) or a high salt diet (HSD; 4%NaCl in chow, 1% NaCl in water) for up to 16 months. So far it remains unclear whether a direct link between high salt intake and kidney fibrosis exists autonomously from arterial hypertension. Therefore our mouse model is remarkable as we could show the absence of elevated blood pressure in our animals. Our previous findings demonstrated that HSD-induced proximal tubular phenotypic changes and sodium-glucose cotransporter-2 expression are coordinated by cold shock Y-box binding protein-1 (YB-1). Furthermore we could show that HSD leads to kidney fibrosis and that this fibrosis is aggravated in YB-1 KO animals. To further determine the damage pattern of HSD-induced fibrosis we plan to perform immunohistochemical staining of kidney tissue for different markers of fibrosis. In addition we found that immune cell infiltration is increased under HSD while this immune response is blunted in YB-1 KO animals. To further understand the effect of excessive sodium intake on the immune system fluorescent activated single cell sorting of different infiltrating cells, that are influenced by HSD, will be performed.

Ronnie Morgenroth

Clinician Scientist

Peter Mertens

Project Leader

The most common neurodegenerative disease-causing dementia is Alzheimer's disease. Current understanding of pathophysiology links progressive memory loss with neuronal cell death due to extracellular amyloid plaque formation and intracellular deposition of neurofibrillary tangles. Despite intensive research, the disease remains an enigma, although there is substantial evidence that both proinflammatory cytokines and autoantibodies not only play a minor role but can also be linked to causal factors. YB-1 is the prototypical member of cold shock proteins in humans and fulfills pleiotropic functions in the cell cycle, differentiation, stress response, DNA repair and inflammation response. In an Alzheimer's animal model, nasal immunization with YB-1 resulted in amyloid fibril destruction and cognitive improvement. Also, YB-1 could be linked within the process of senescence. In our own preliminary work, specific YB-1 autoantibody epitopes have already been disclosed in tumor patients. In addition, preliminary examinations in patients with Alzheimer's disease show another specific autoantibody epitope. The research project aims to investigate the hypothesis of whether the specific YB-1 autoantibody epitope (i) is established as a diagnostic marker in a large cohort of Alzheimer's patients or (ii) can be used as an early diagnostic marker (MCI, SCI), whether (iii) Autoantibodies play a role in the YB-1 biology and (iv) if they can influence the neuroinflammatory process. In this context, patients are recruited with the DZNE. Then experiments in protein biochemistry and cell culture are carried out.

Plasma samples from Alzheimer's patients show a specific epitope in the N-terminal section of the cold shock protein YB-1. YB-1 interacts with amyloid-b  fibrils. (A) The N-terminally located autoantibody epitope is mapped to the minimal binding sequence in the plasma and liquor of patients with Alzheimer's disease using an exceptionally fine peptide array. (B) That for the YB-1:b-amyloid interaction responsible YB-1 fragment should be determined using specific YB-1 protein deletion fragments. In addition, the interaction is examined in the presence of YB-1 antibodies and tested for inflammatory changes in the cell model.