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Translational research (training) in Polycystic Kidney Disease

Final Report Summary - TRANCYST (Translational research (training) in Polycystic Kidney Disease)

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited kidney disease characterised by the development of cysts in both kidneys, ultimately leading to kidney failure. The therapeutic strategies available today remain limited, hence translational research using innovative approaches from basic sciences to clinical applications are necessary to unravel the disease mechanism and to develop interventions, reliable monitoring of cystic renal disease growth and to slow down renal cystic disease progression. The broad objectives of our multidisciplinary training network included the characterisation of cellular cystic changes and validation of preclinical disease models, preclinical drug testing, identification of new tools for early prediction and monitoring of cystic renal disease progression, as well as evaluating ongoing clinical trials. Bioinformatics and biosemantic approaches supported, enriched and connected the different themes.

Research activities and Results
Different animal (mouse, zebrafish) and cell models have been generated and used to get insight into Polycystic Kidney Disease (PKD) mechanism and have been validated for preclinical testing. We studied several critical molecular processes involved in establishing and maintaining renal polarity and cellular integrity. We focused on several specific pathways/processes but we also performed and combined more comprehensive profiling experiments. These different approaches combined with drug screening experiments revealed several potential drugs/drug-targets.
We established highly differentiated cell culture systems from several PKD-models and showed that PKD is associated with specific defects of transport mechanisms in the kidney, which are essential to retain various solutes such as sugar, proteins and vitamins that are otherwise lost in the urine. Animal models showed that these defects occur early in the progression of the disease before the development of massive cysts. Additionally we showed that the impairment of the transport mechanisms is associated with alterations of the primary cilium, a structure present at the cell surface which sense extracellular stimuli and affect the cells behaviour accordingly. We performed proof-of-concept experiments which demonstrate that pharmacological and genetic manipulations of primary cilia lead to impaired endocytosis activity.
In parallel, we focused on the role of two specific signaling pathways, i.e. planar cell polarity and Hippo signaling processes involved in epithelial polarity and tissue size. Our data indicated that Planar Cell Polarity (PCP) and Hippo pathway are deregulated in PKD. These pathways are indirectly controlled by upstream regulator Four-jointed (Fjx1). However, altered PCP and Hippo signaling cannot explain the difference in disease progression that we observed in single Pkd1 versus Fjx1/Pkd1 double knock-outs mice. Another cystic disease gene, HNF1B encodes for a transcription factor called 'Hepatocyte Nuclear Factor 1 beta' (HNF1b) and regulates expression of several PKD-genes. Our results indicated that HNF1b bookmarking activity may be impaired by naturally occurring mutations found in patients and suggest a functional link between the nuclear import system and the mitotic localization/translocation of bookmarking factors. Furthermore, our results have demonstrated in HNF1b-deficient embryos structural alterations, which lead to obstructive dilation and formation of glomerular cyst. Interestingly, the same aberration can be observed in fetuses carrying HNF1B mutations.
For a more comprehensive analysis of tissue alterations messenger RNA, extracted from mice at various stages of PKD progression, was analysed using the next generation sequencing technology. A meta-analysis of PKD expression profiles from different models identified a robust PKD expression signature. We found that up to 65% of the molecular pathways and functions involved in PKD, are involved in injury repair related processes. We dissected the expression patterns involved in PKD cyst formation over time into gene clusters and we identified clusters that correlate strongly either positively or negatively with cyst formation and disease severity. Using these data, we managed to identify gene modules that are involved in PKD progression and are responsive to drug treatments. These modules are further analysed and combined with publicly available drug activity profiles from ChEMBL to identify novel drug targets for PKD treatment. In addition, non-coding (mir-RNA) transcripts were compared to mRNA profiles. This way, we have identified changes in a number of novel genes and their regulation by specific mir-RNA. These genes represent novel drug targets or potentially useful biomarkers of disease progression for future investigation.
Complementary to the pathway related approaches to identify potential new treatments, we conducted a comprehensive search for new drug candidates using the zebrafish pkd2 mutant embryos. This resulted in the identification of several candidate compounds, which were also confirmed to have the same predicted effects on human ADPKD cysts in culture. These compounds therefore represent clear drug leads and we the most promising ones will be tested in mouse models. We also showed that pkd2 regulates calcium signals in the embryonic zebrafish kidney, and that pkd2 has a functional interaction with another essential cilia protein in regulating embryonic kidney development.
Both, animal models and patient materials, have been used to search for biomarkers in urine, blood or tissues. The markers may aid decision making since ultimate goal of interdisciplinary care clinics is to provide well-timed prevention from the unnecessary therapeutic intervention and/or keeping the long-term function of the kidneys. Using a targeted approach we investigated multiple urinary markers for tubular injury and their association with disease burden in ADPKD patients at early disease course. Our results indicate that the urinary biomarkers osmolality, UACR and KIM-1 have the property to assess disease state at early ADPKD stage.
For a comprehensive analysis we performed urine proteomic analysis using capillary electrophoresis coupled to mass spectrometry (CE-MS) technology comparing ADPKD patients with relatively stable disease condition to those progressed towards renal failure. We identified 20 significant biomarkers and developed a urinary prognostic test that accurately predicts relevant clinical endpoints in ADPKD. Using the same approach in our experimental animal models, we found 30 significant peptide biomarkers, which showed dose-dependent differences upon preclinical drug treatments.
Total kidney volume (TKV) was earlier identified as a potential imaging biomarker in ADPKD patients. We quantitatively compared all of the state-of-the-art methods for kidney volume computation. We found that whole kidney contouring methods should be preferred for accurately monitoring ADPKD progression and assessing drug treatment efficacy on MRI, while fast, simplified methods can provide only a rough estimate of kidney volume. We computed TKV using whole kidney contouring methods on all CT images from ADPKD patients involved in SIRENA-II and ALADIN 2 clinical trials. These measurements were used along with other measures of kidney function, to measure drug efficacy in these trials, i.e. Sirolimus and a long-acting Somatostatin analogue, respectively. Moreover, we estimated TKV on a set of MR images from ADPKD patients involved in the EUROCYST initiative.
Another cohort, the Suisse ADPKD registry, was set-up and we showed the long-term effect of coffee consumption on ADPKD progression. We also performed a temporal and geographical external validation study and updating of the Mayo Clinic Prediction Model in a Swiss ADPKD Study for selecting patients for the treatment with Tolvaptan.

In the TranCyst project young researchers were trained in multi-disciplinary translational research. They provided a better understanding of the early and advanced disorders occurring in PKD and mechanisms involved. They identified potential compounds and drug targets for PKD treatment, and they contributed to new patient registries and complementary methods to monitor disease progression. Over-all the students acquired knowledge and skills, which are beneficial for the development of new therapeutic strategies for polycystic kidney disease. (