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Hintergrund und Ziele: Der Hypoxie-Induzierbare Transkriptionsfaktor HIF-1α ist ein wichtiger Regulator im Rahmen zellulärer Hypoxie, der zur Aktivierung einer Vielzahl von Zielgenen führt, die eine Adaptation der Zelle an Sauerstoffmangel bewirkt. Einige dieser Zielgene sind proliferationsfördernd (PDGF, VEGF) oder beeinflussen den Zell-metabolismus (GLUT-1, PDK-1). Die autosomal dominante polyzystische Nierenerkrankung (ADPKD) ist eine Erbkrankheit, die insbesondere zur Bildung zahlreicher größenprogredienter Nierenzysten führt. Interessanterweise ist HIF-1α in hohem Maße im Zystenepithel von ADPKD Patienten nachweisbar. Wir postulieren daher, dass HIF-1α die Proliferation des Zystenepithels begünstigen könnte. Methoden: Zur Untersuchung des Zystenwachstums etablierten wir ein in vitro-Zystenmodell mit MDCK-Zellen und untersuchten, ob HIF-1α in diesem Modell auf Proteinebene nachweisbar ist. Zudem überprüften wir, ob Hypoxie oder pharmakologische „Hypoxie-Mimetika“ wie Diethylfumarat (DEF), Oxalylglycin (OG) und Dimethyloxalylglycin (DMOG) zu einer vermehrten Expression von HIF-1α sowie seines Zielgens Glukosetransporter-1 (GLUT-1) im Zystenmodell führen. Des Weiteren untersuchten wir, ob die vermehrte Expression von HIF-1α Auswirkungen auf das Zystenwachstum und die Zystzellproliferation hat. Außerdem wurden die Effekte des Heat-Shock-Protein-90-Inhibitors 17-Allyl-Amino-Demethoxygeldanamycin (17AAG) und des Topoisomerase-I-Inhibitors Topotecan auf die Expression von HIF-1α, sowie in Korrelation hierzu auf die Zystengröße untersucht. Ergebnisse: HIF-1α ist im in vitro-Zystenmodell unter Kontrollbedingungen auf Proteinebene nachweisbar. Alle 3 Hypoxie-Mimetika sowie Hypoxie selbst führten zu einer vermehrten Expression von HIF-1α sowie des Zielgens GLUT-1. Eine verstärkte Stabilisierung von HIF-1α durch Hypoxie-Mimetika führte zu einer signifikanten Größenzunahme der Zysten. Ursächlich zeigte sich eine gesteigerte Proliferation des Zystenepithels durch Hypoxie-Mimetika. Darüber hinaus war die Glukose-Aufnahme gesteigert, was auf die erhöhte Expression von GLUT-1 zurückzuführen sein könnte. Topotecan sowie 17AAG führten, wie bereits in anderen Modellen beschrieben, zu einer Inhibition von HIF-1α. Topotecan und 17AAG führten darüber hinaus zu einer signifikanten Reduktion des Zystenwachstums. Schlussfolgerungen: Wir konnten zeigen, dass HIF-1α durch Aktivierung seiner Zielgene an der Proliferation des Zystenepithels im MDCK-Zystenmodell beteiligt ist. HIF-1α stellt somit ein potentielles, bislang unbekanntes Ziel pharmakologischer Interventionen zur Verminderung der Zystennierenprogression dar. Introduction: HIF-1α is an important transcription factor and regulator in the context of cellular hypoxia, activating a variety of target genes. These target genes may on the one hand be pro-proliferative (PDGF, VEGF) or on the other hand may affect cell metabolism (GLUT-1, PDK-1). Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disease comprising bilateral renal cyst growth due to augmented cyst cell proliferation. Interestingly, HIF-1α is highly expressed in the cyst-lining epithelium. Therefore, we postulate that HIF-1α may promote cyst cell proliferation in ADPKD. Methods: To examine cyst growth we established an in vitro cyst model with Madin-Darby Canine Kidney (MDCK) cells that form cysts spontaneously in collagen 1. We examined whether HIF-1α and its target gene glucose transporter 1 (GLUT-1) were detectable at the protein level in this model. We further investigated if stabilization of HIF-1α by hypoxia or hypoxia-mimetics like diethylfumarate (DEF), oxalylglycine (OG), or dimethyloxalylglycine (DMOG) had an impact on cyst growth. In addition, we tested if inhibition of the heat shock protein 90 (HSP-90) with 17-allyl-amino-demethoxygeldanamycine (17-AAG), or the use of Topotecan, an inhibitor of topoisomerase-I, could affect HIF-1α protein levels and therefore may influence in vitro cyst growth. Results: HIF-1α can be detected in MDCK cysts under control condition. The three hypoxia-mimetics as well as hypoxia led to increased expression of HIF-1α and its target gene GLUT-1. Increased levels of HIF-1α were accompanied by increased in vitro cyst growth, which was mainly driven by increased cyst cell proliferation. Furthermore, MDCK cysts showed increased glucose uptake in the presence of hypoxia-mimetics, which could have originated from an increased expression of GLUT-1. Topotecan as well as 17AAG inhibited HIF-1α, which resulted in reduced MDCK cyst growth. Summary: We could show that HIF-1α via activation of its target genes may promote in vitro cyst growth. Therefore, HIF-1α may represent a new target for pharmacological intervention studies in ADPKD.

Polycystic kidney diseases are the most common genetic disorders; the underlying pathomechanisms are incompletely understood so far. One of the involved in the formation of cystic kidneys genes is DZIP1L. DZIP1L has previously been identified in our group as a new gene for polycystic kidney disease by homozygosity mapping of a family with autosomal recessive polycystic kidney disease. It encodes a homolog of the iguana protein in zebrafish, which is involved in the development of the pronephros and plays a role in the hedgehog signaling pathway. Term of this doctoral thesis was the identification of interaction partners of DZIP1L by a bacterial Two Hybrid system to further characterize the function and role of the protein in the cell. Coimmunoprecipitation experiments and immunfluorescence studies in transiently transfected COS7-, HEK293- and mIMCD-3- cells have been performed for validation of the interaction of DZIP1L with EEF1G, NAGK and PSAP. All three identified binding partners are involved in SMAD-/TGF-ß- signaling. NAGK and PSAP are known to directly interact with R-Smads, whereas for EEF1G an interaction with SnoN has been described. SnoN on his part interacts with an R-Smad too. Consequently, the transcription of TGF-ß target genes will be repressed. Overall, it can be postulated that DZIP1L does not only play a role in hedgehog-signaling but also influences cyst formation through modification of TGF-ß-signaling.

Autosomal recessive polycystic kidney diesease is one of the most common diseases affecting the kidney in childhood. The clinical spectrum is highly variable with ranging from peri- or neonatal demise and survival to adulthood respectively. The ARPKD-gene, PKHD1, is located on chromosome 6p12 and the majority of the ARPKD-cases could be ascribed to mutations in this gene. The longest open reading frame comprises 66 exons and encodes a single-transmembrane-protein, called polyductin/ fibrocystin. A genotype-phenotype-correlation exists, that implicates that no patient carrying two truncating mutations survives the peri-/neonatal period. So at least one missense mutation is indispensable for survival of newborns. Within this doctoral thesis four recently identified truncating mutations in five families leading to a surprising mild phenotype where analysed. It was shown that three of them caused a quantitative or qualitative alteration on transcript level. Additionally a possible effect of nonsense mediated decay (NMD) in ARPKD has been shown for the first time and it seems as if NMD may has been circumvendet in at least one of the analysed clinically milder affected cases. Therefore mechanisms above DNA-level modifying the phenotype of ARPKD were revealed. The other main task of this doctoral thesis was a further characterisation of the protein encoded by PKHD1, polyductin, and possible interaction partners. Antibodies against the intracellular C-terminus were generated and established. In this process it has been shown that polyductin localised to the centrosome in HEK293 cells and that the protein participates in the so called midbody during telophase in mitosis. So an involvement of polyductin in cell division concerning mitotic orientation and/or cytokinesis could be hypothesised. The potential binding partner of polyductin analysed during this work, BBS2, is one of the proteins related to the Bardet-Biedl-Syndrom (BBS). BBS is a multisystemic disorder usually concerning pathological alterations of the kidney among others. Immunofluorescence showed a colocalisation of polyductin and BBS2 during all phases of mitosis as well as the interphase. Additional koimmunoprecipitation experiments using different constructs for both, polyductin and BBS2, affirmed the expected interaction. Thereupon a possible connection between polyductin and the BBSome, a complex of seven BBS-proteins including BBS2, was analyzed. BBS7, another member of the BBSome, was shown to interact with BBS2 and revealed a large homology to this protein. So it was tested wether there was link between Polyductin and BBS7. In immunofluorescence experiments a partial colocalisation within the centrosome during the interphase was observed. ARPKD as well as BBS concern to the group of ciliopathies and the localisation and function of the BBSome is described only for polarised ciliated cells. So the ciliar localisation was checked in polarised mIMCD-3 cells. It turned out that polyductin as well as BBS7 could be detected predominantly at the basis and within the cilium. Continuative CoIP experiments indicated the interaction between polyductin and BBS7. Therefore a direct link between the proteins associated with ARPKD and the Bardet-Biedl-Syndrome could be made for the first time. This allows a deeper insight in the complicated network of ciliary signalling and could help understanding and analysing the pathological changes accompanying these and other ciliopathies.

Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of renal- and liver related morbidity and mortality in neonates and infants, occurring 1 in 20000-40000 live births. Principal histological manifestations involve the fusiform dilatation of renal collecting ducts and hepatobiliary ductal plate malformations. The clinical spectrum is widely variable. About 30 to 50% of affected individuals die in the neonatal period, while others survive into adulthood. ARPKD is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene on chromosome 6p12, which is among the largest human genes known until today. This is the first study analysing mutations and the mutation detection rate in the PKHD1 gene of pediatric ARPKD-patients. The study population consisted of 186 patients from 127 unrelated families. The DNA of each patient was screened for mutations in 66 encoding exons of the longest open reading frame of the PKHD1 gene by DHPLC and sequencing. The significance of each new sequence variation was evaluated by screening a control group of healthy individuals, performing a segregation analysis, and by using bioinformatical methods as well. The mutation detection rate was about 77%. Two mutations were found in 58% families, as could be expected in an autosomal-recessive disorder. One mutation was discovered in 39% families. Conclusively, 96% of patients had at least one mutation, confirming the diagnosis of ARPKD and establishing DHPLC screening as a powerful diagnostic tool in pediatric patients suspected with ARPKD. 78% of the pathogenic variations were missense mutations, only 22% were characterised as truncating ones. No patient carried two truncating mutations corroborating that one missense mutation is indispensable for survival of newborns. More than three quarters of the mutations were “private mutations”, i.e. were detected in one family only. Due to these private mutations and a great number of compound heterozygotes a correlation between genotype and phenotype is hard to establish. This study attempted for the first time to link all known recurrent mutations to date to four different clinical courses.