Aufdeckung der molekularen Nuancen von TRPC6 bei der fokalen segmentalen Glomerulosklerose in einem In-vitro-Modell menschlicher Podozyten

Focal segmental glomerulosclerosis (FSGS) is a rare disease-causing glomerular lesion in the nephron and inadvertently end stage renal disease. Pathologically, FSGS is characterized by sclerosis in some (focal) parts of the glomeruli. Podocyte injury is the earliest morphological hallmark of FSGS supporting the notion that FSGS is a podocyte disease. The physiological function of podocytes is critically dependent on the proper intracellular calcium levels; excessive or deficient Calcium influx into these cells may result in foot process effacement, apoptosis, and nephron degeneration. A key protein involved in the regulation of Calcium influx is the transient receptor potential cation 6 (TRPC6), which is expressed in podocytes. Rare mutations in the TRPC6 gene were implicated to cause FSGS with high penetrance, presumably by increasing or decreasing calcium influx. Here I studied the effect of gain- and loss-of-function (GOF, LOF) mutations in TRCP6 on intracellular Calcium levels using novel model systems, including podocytes derived from human induced pluripotent stem cells (hiPSC). To establish an inducible human FSGS model system, I generated and phenotypically characterized three transgenic hiPSC lines with controllable overexpression of TRPC6 wild-type and FSGS-associated GOF and LOF mutations (P112Q and G757D). Furthermore, these cell lines were induced to differentiate into podocytes (iPodocytes). Initial experiments in HeLa and HEK293 reporter cell lines confirmed the impact of TRPC6 GOF and LOF mutants on Calcium influx. Although iPodocytes also showed Calcium responses consistent with GOF and LOF phenotypes, these were lower in comparison to data obtained from the reporter lines, probably due to reduced and aberrant expression of TRPC6 during podocyte differentiation. In addition, I reported a novel heterozygous TRPC6 mutation (V691Kfs*) in a large kindred with no signs of FSGS despite the presence of a largely truncated TRPC6 protein. Functional analysis showed that the V691Kfs* truncation inactivated the TRCP6 channel-specific Calcium influx consistent with a complete LOF phenotype. Our data corroborate that one defective dominant-negative TRPC6 gene copy and reduced Calcium influx through TRPC6 are not sufficient to cause FSGS. To increase our capacity to study the effect of mutations in rare disease models other than the kidney, I have established four new hiPSC lines derived from three different patients diagnosed with FSGS, Polycystic Kidney Disease (PKD), and Neurofibromatosis 13 Type 1 (NF1), as well as a healthy relative, respectively. These hiPSC lines can be differentiated into both 2D and 3D nephrogenic models and relevant tubule cell types (FSGS and PKD), or neural crest derivatives (NF1) using standard differentiation protocols to analyze disease-related phenotype and pathology. The newly established hiPSC – lines are available for research through the European Bank for iPSC (EBiSC).Fokale segmentale Glomerulosklerose (FSGS) ist eine seltene Erkrankung, die zu glomerulären Läsionen im Nephron und unweigerlich zum Funktionsverlust der Niere führt. Pathologisch ist die FSGS durch Sklerose in einigen (fokalen) Bereichen der Glomeruli charakterisiert. Die Podozytenschädigung ist das früheste morphologische Merkmal der FSGS. Die physiologische Funktion der Podozyten hängt entscheidend von genauen intrazellulären Ca2+-Spiegeln ab; ein übermäßiger oder fehlender Ca2+-Einstrom in diese Zellen könnte zu einer Tilgung der Fußprozesse, Apoptose und Abbau des Nephrons führen. Ein an der Regulierung des Ca2+-Einstroms beteiligtes Protein ist das in Podozyten exprimierte Transient Receptor Potential Cation 6 (TRPC6). Seltene Mutationen im TRPC6-Gen werden mit der Entstehung von FSGS mit hoher Penetranz in Verbindung gebracht, vermutlich durch Erhöhung oder Verringerung des Kalziumeinstroms.Ich untersuchte den Einfluss von Gain- und Loss-of-Function (GOF, LOF) - Mutationen in TRCP6 auf den intrazellulären Ca2+-Spiegel mithilfe von neuartigen Modellsystemen, einschließlich aus menschlichen induzierten pluripotenten Stammzellen (hiPSC) differenzierte Podozyten. Für die Etablierung des FSGS-Modellsystems wurden drei transgene hiPSC-Linien mit regulierbarer Überexpression von TRPC6-Wildtyp und FSGS-assoziierten GOF- und LOF-Mutationen (P112Q und G757D) erzeugt, charakterisiert und in Podozyten differenziert (iPodozyten). Anfängliche Experimente in HeLa- und HEK293-Reporterzelllinien bestätigten die Auswirkungen der TRPC6 GOF- und LOF-Mutanten auf den Ca2+-Einstrom. Obwohl auch in iPodozyten die mit den GOF- und LOF-Phänotyp übereinstimmenden Ca2+-Reaktionen gezeigt wurden, waren diese verglichen zu den Daten aus den Reporterlinien geringer, was vermutlich an einer reduzierten und abweichenden Expression von transgenem TRPC6 während der Differenzierung lag. Zusätzlich wurde eine neuartige heterozygote TRPC6-Mutation (V691Kfs*) in einer Kohorte, die trotz eines weitgehend verkürzten TRPC6-Proteins keine Anzeichen von FSGS zeigte, generiert. Die funktionelle Analyse zeigte, dass die V691Kfs*-Trunkierung den TRPC6-spezifischen Ca2+-Einstrom inaktiviert, was einem vollständigen LOF-Phänotyp gleicht. Unsere Daten bestätigen, dass eine defekte dominant-negative TRPC6-Genkopie und ein verminderter Ca2+-Einstrom durch TRPC6 nicht ausreichen um eine FSGS auszulösen. 15 Um die Auswirkungen von Mutationen in Modellen für weitere seltene Krankheiten besser untersuchen zu können, wurden vier neue hiPSC-Linien etabliert, die von drei verschiedenen Patienten mit FSGS, polyzystischer Nierenerkrankung (PKD) und Neurofibromatose Typ 1 (NF1), sowie von einem gesunden Verwandten stammen. Diese Zelllinien können mithilfe von Standardprotokollen in 2D- und 3D-Nephronmodelle und Tubuluszelltypen (FSGS und PKD) oder Neuralleisten-Derivate (NF1) für eine Analyse des Krankheitsbilds und der Pathologie differenziert werden und stehen für Forscher über die Europäische Bank für iPSC (EBiSC) zur Verfügung

Genetic and Cellular Studies of The Podocyte in Focal Segmental Glomerulosclerosis

The podocyte forms the outer layer of the filtration barrier in the glomerulus to prevent albumin leakage. Podocyte damage leads to focal segmental glomerulosclerosis (FSGS), a leading cause of chronic kidney disease. The cause of the majority of FSGS cases is unknown and referred to as sporadic FSGS. Genetic studies have identified genes as monogenic causes of FSGS in patients with a strong family history, but these cases account for only a small proportion of the FSGS population. Whether genetic susceptibility contributes to sporadic FSGS and which cellular process in the podocyte initiates the pathogenesis of FSGS are important questions that remain to be elucidated. To answer these questions, my research followed two different lines of inquiry. I performed a genetic analysis of both familial and sporadic FSGS patients, and I investigated the role of the actin cytoskeleton in podocytes. Based on expression analysis, we identified a new FSGS susceptibility gene, ARHGAP24, and showed that it was mutated in a family with FSGS. Since ARHGAP24 functions to maintain high Rho and low Rac levels, my work suggested that this balance might be important in FSGS. Using an inducible transgenic mouse model and multi-photon intravital microscopy, we validated that high activity of Rac1, one of the Rho family GTPases, is responsible for podocyte foot process effacement, increased membrane dynamics, and podocyte shedding into the urine, three important processes that lead to proteinuria and FSGS. By sequencing a large cohort of sporadic FSGS patients, I identified 16 potential FSGS susceptibility genes that were novel. Using a novel podocyte-specific indicible RNAi mouse model that I developed, four of these genes were validated. Some of these genes function as regulators of the actin cytoskeleton. Our genetic study further reinforces the role of actin cytoskeletal regulation in the pathogenesis of FSGS

The origin, turnover and removal and normal glomerular basement membrane

A comprehensive account of the natural history of normal glomerular basement membrane is prerequisite to elucidating the pathogenesis of numerous renal diseases. The experimental argyric technique was investigated, adapted and applied in a long term sequential, electron microscopic study of normal glomerular basement membrane in the rat. The results demonstrate that a major component of glomerular basement membrane is secreted by the visceral epithelial cells. This component is laid down on the epithelial side and slowly moves towards the endothelial side of the basement membrane as new basement membrane material continues to be secreted. The old basement membrane material is removed from the endothelial aspect of the membrane and passes by way of the lamina rara interna to the mesangial matrix for subsequent ingestion by the mesangial cells. This process is continuous and slow; the time for complete renewal of the glomerular basement membrane in the rat is of the order of twelve months. Secretion of this component, by the epithelial dells, is effected by a vascular-coated pit mechanism and removal, by the mesangial cells, is effected by a phagocytic mechanism. The results further indicate the presence of a second component in glomerular basement membrane. This second component is probably of endothelial origin and has a much faster turnover rate than the main, or epithelial derived, component. Study was also made of glomeruli from two cases of human argyria and though the observations perforce are limited, the results show that human glomerular basement membrane has a natural history essentially similar to rat glomerular basement membrane. On the basis of these experimental observations, correlated with the results of previous investigations, a model of the functional morphology of glomerular basement membrane is proposed. The potential applications of this model are briefly indicated

Investigating the mechanisms of renal fibrosis following ischaemia and reperfusion injury

PhD ThesisIschaemia-reperfusion injury (IRI) is the major cause of acute kidney injury (AKI) and predisposes to the development of chronic kidney disease (CKD). The role of TGF-β in extracellular matrix deposition and renal fibrosis has been well established. This study was designed to establish an in vitro model of renal tubular IRI, evaluate the role of TGF-β in IRI in human proximal tubular epithelial cells (HKC8 and HK2 cells) and further determine the role of αvβ6 integrin in IRI. Initially an in vitro model of hypoxia and free radical stress by treating HKC8, HK2 and fibroblasts (MRC-5 cells) with CoCl2 and H2O2 respectively was established. These treatments led to pro-fibrotic changes characterised by increased expression of fibrotic marker α-SMA and reduced expression of epithelial cell marker E-Cadherin at mRNA and protein level. Binding of TGF-β to its receptor leads to activation of the kinase ALK5. ALK5 inhibition prevented the changes induced by H2O2 or CoCl2 suggesting the involvement of TGF-β to the cellular response to IRI. To confirm that TGF-β is released after treatment of cells to mimic IRI, media transfer and co-culture studies were performed. These experiments confirmed that bioactive TGF-β was being released. Lastly, the role of αvβ6 integrin was studied post H2O2 or CoCl2 treatment. Expression of αvβ6 integrin was elevated in conditions mimicking IRI in vitro and in biopsy samples acquired from patients with acute tubular necrosis and in mouse kidney following IRI. Knockdown of αvβ6 integrin in HKC8 cells decreased the bioavailability of active TGF-β following CoCl2 or H2O2 treatment and therefore the pro-fibrotic changes that were seen. This study confirms that bioactive TGF-β is produced following IRI and αvβ6 plays an important role in its release

The Drosophila Nephrocyte - Modeling Podocyte Function and Disease

In modern industrialized nations, chronic non-communicable diseases have become the main reason of mortality. During the last years, the global health burden of chronic kidney diseases (CKD) has grown considerably and in many of these cases, no targeted or personalized therapies are available. To keep up with the rising challenges, the understanding of the causes and mechanisms underlying CKD needs to be improved and novel approaches must be found and refined to support and enrich conventional research methods. This dissertation encompasses four manuscripts concerned with various aspects of utilizing Drosophila nephrocytes for modeling mammalian kidney function and disease and contributing valuable insights, in addition to established kidney models, in a quick and cost-effective way. In general, the morphological characterization of a biological sample via electron microscopy strongly depends on the degree of sample preservation. In the first project, the ultrastructural preservation of nephrocytes after preparation for transmission electron microscopy could be greatly improved by using both an optimized dissection saline and cryofixation techniques. Also, the initially challenging immunogold detection of endogenous Crumbs protein could be realized by an indirect approach detecting GFP-tagged Crumbs using an antibody directed against the tag. The 3D ultrastructure of the complex subcellular structures in nephrocytes was further characterized by creating a 3D model of the outlying network of cell membrane invaginations based upon STEM tomography data. In conventional two-dimensional TEM, only a limited assessment of the spatial arrangement of cellular structures in possible. The question whether foot processes of mammalian podocytes interact with other foot processes of the same cell therefore remained unanswered. The second manuscript arrives at the conclusion, that the interaction exclusively happens between foot processes of different podocytes, by thorough investigation of a 3D model, which was based on a whole podocyte, sectioned and imaged using FIB-SEM technology. The cell-cell contacts between foot processes were further characterized via dual-axis tomography in human and mouse podocytes, as well as Drosophila nephrocytes, leading to the realization that there is both one quasiplanar slit diaphragm spanning the whole slit diameter between two processes and additionally several filamentous contacts, which are locally limited. Additional studies are necessary to identify the proteins these two types of cell-cell contacts are composed of. Podocytes are highly complex polarized epithelial cells. For a better understanding of the molecular fundamentals of development and maintenance of normal podocyte function, the role of Pals1, which acts as scaffolding protein and key factor necessary for apicobasal polarization, was more closely investigated in podocytes in the third manuscript. Therein, Pals1 was found to be an essential, specific and dose-dependent regulator of nephrogenesis, being a negative regulator of Hippo and TGF-β signaling in mouse, which was then also confirmed in the Drosophila model. These findings indicate a connection between renal diseases, especially renal cyst diseases, and apical polarity proteins. The pathogenesis of these diseases needs to be better understood by studying the networks regulated by Pals1. In the course of the last project, the role of Drosophila Crumbs (DmCrb) in nephrocytes was closer examined. DmCrb is an interaction partner of Stardust, the Drosophila homolog of Pals1, and a key determinant of apicobasal polarity. It was found to be involved in the regulation of early endocytosis via interaction with Moesin through its FERM-binding domain, located on its C-terminus. It further contributes to nephrocyte diaphragm assembly and maintenance via its extracellular domain. The functions of its different protein domains and the evolutionary conservation within the Crumbs family was examined by conducting a series of rescue experiments with various deletion constructs of DmCrb as well as its human orthologs. The herein describes mechanisms of nephrocyte development and function are likely also conserved in mammalian podocytes

Role of the Transient Receptor Potential Canonical 6 ion channel in genetic and acquired forms of proteinuric kidney disease

Podocyte foot processes and the interposed glomerular slit diaphragm are critical components of the permeability barrier in the kidney. Mutations in several podocyte genes have been identified as the cause for progressive kidney failure and focal segmental glomerulosclerosis (FSGS). Podocyte injury is a hallmark of glomerular disease and usually involves the rearrangement of the podocyte actin cytoskeleton. Cell-specific therapies targeting podocyte injury are currently not available. In 2004, a mutation in the TRPC6 ion channel was found to cosegregate with hereditary FSGS. Based on this finding it was hypothesized that TRPC6 is expressed in podocytes, and that TRPC6-mediated Ca2+ signaling contributes to the regulation of the podocyte actin cytoskeleton. According to this model, dysfunction of TRPC6 leads to a disruption of normal cytoskeletal organization, podocyte injury, and proteinuric disease. To test this hypothesis, four specific aims were outlined. First, to explore TRPC6 mutations in genetic FSGS. Second, to investigate its association with the glomerular filtration barrier. Third, to study TRPC6 expression in acquired forms of proteinuric kidney disease. Fourth, to investigate the molecular basis of TRPC6 contribution to the pathophysiology of proteinuric kidney disease In genetic forms of FSGS, additional TRPC6 mutations were identified in five families with a history of FSGS. TRPC6-related FSGS presented as a late-onset disorder in individuals aged 17-57, and was not restricted to certain ethnic groups. All mutations occured in evolutionary conserved sites, and encoded amino acid substitutions at the amino- and carboxy-terminal ends of TRPC6. Two mutants, R895C and E897K, displayed increased current amplitudes, suggesting a pathogenic role of increased channel activity in TRPC6-related FSGS. In an effort to understand the molecular basis for TRPC6 in the kidney, the association of TRPC6 with the glomerular filter was studied. TRPC6 was found to be expressed in podocytes near the glomerular slit diaphragm. TRPC6 colocalized and associated with the slit diaphragm proteins nephrin and podocin. The presence of TRPC6 in podocyte foot processes and its association with slit diaphragm proteins supports a role of TRPC6 in the regulation of glomerular filtration. Since most proteinuric kidney diseases appear not as genetic but acquired disorders, TRPC6 was studied in humans with acquired glomerular diseases and in experimental models thereof. TRPC6 expression was induced in patients with minimal change disease and membranous glomerulopathy, as well as in passive Heymann nephritis (PHN) rats and puromycin aminonucleoside (PAN) rats. PAN-mediated podocyte injury correlated with increased receptor-operated calcium entry in vitro. TRPC6 gene delivery in mice was sufficient to induce proteinuria, and studies in cultured podocytes suggest that TRPC6 overexpression disrupts the actin cytoskeleton. The present data suggest that in both genetic and acquired forms of proteinuric kidney disease, misregulation of TRPC6 – either by presence of mutated hyperactive channels, or by precence of too many wildtype channels – plays a pathogenic role. Together, the results of this work may have broad implications for the pathophysiology of TRPC6-related human kidney diseases, and promote the development of anti-proteinuric drugs interfering with TRPC6 channel function

Improvements in MALDI-Imaging Mass Spectrometry to analyze the lipidome in different tissues. A step forward to clinical application.

305 p.A pesar de los grandes avances de la imagen por espectrometría de masas (IMS), esta técnica aún requiere superar algunas limitaciones antes de despegar como una tecnología potente para su uso en clínica. El objetivo de esta tesis es mejorar algunos aspectos involucrados en el flujo de trabajo MALDI-IMS y enfocar su implementación para resolver cuestiones biológicas relacionadas con la lipidómica. Así, en el Capítulo 4 implantaremos ciertas mejoras en la preparativa de la muestra, así como en el set up óptico del espectrómetro de masas utilizado. El Capítulo 5 presenta la caracterización de las diferencias en el perfil lipídico entre el riñón normal de ratón, un modelo de lesión renal aguda (AKI) y el riñón AKI tratado con ferrostatina (Fer-1). En el Capítulo 6 se muestra la primera caracterización de los diferentes segmentos de la nefrona humana mediante MALDI-IMS de lípidos combinado con protocolos de inmunofluorescencia. Una vez caracterizado el riñón normal murino y humano, en el Capítulo 7 se realizó una comparación de los perfiles lipídicos obtenidos de estas dos especies con el fin de evaluar la calidad del ratón como un buen modelo animal para enfermedades humanas. El Capítulo 8 se centra en el estudio del lipidoma del cáncer renal de células claras (ccRCC) mediante MALDI-IMS y uHPLC y, por último, en el Capítulo 9, se examinará el lipidoma del Glioblastoma además del efecto del agente quimioterapéutico temozolomide (TMZ) sobre tejido cerebral humano

Amyloidosis

Amyloidoses are a heterogeneous group of diverse etiology diseases. They are characterized by an endogenous production of abnormal proteins called amyloid proteins, which are not hydrosoluble, form depots in various organs and tissue of animals and humans and cause dysfunctions. Despite many decades of research, the origin of the pathogenesis and the molecular determinants involved in amyloid diseases has remained elusive. At present, there is not an effective treatment to prevent protein misfolding in these amyloid diseases. The aim of this book is to present an overview of different aspects of amyloidoses from basic mechanisms and diagnosis to latest advancements in treatment