Periodic Reporting for period 1 - IN-THE-KIDNEY (Functional Measurements IN-THE-KIDNEY to Diagnose, Understand and Treat Renal Tubulopathies)
Reporting period: 2022-09-01 to 2025-02-28
Background | Renal tubulopathies are a large group of genetic disorders that disturb the function of the kidney tubule. A major challenge for diagnostics and treatment of magnesium (Mg2+) wasting tubulopathies, is absence of functional cell models and tools to study the pathophysiology. Diagnostics of hereditary Mg2+ wasting tubulopathies based on genetic screening, rather than biological measurements of kidney processes. Therapy largely depends on oral supplements because therapeutics that target the cause of disease are not available.
Vision | In my vision, better insights in the cellular functional processes in the kidney are the next essential step forward towards better diagnostics, pathophysiological understanding and development of therapy. Development of functional Mg2+ transport measurements and innovative use of patient-specific models is required to increase diagnostic yield and to develop therapy.
Aims and approach | In this project, I aim to develop a urine-based diagnostic approach using scRNA sequencing and extracellular vesicle proteomics to provide biological diagnosis of renal tubulopathies. Using urine-derived adult stem cells, I will perform the first ever functional studies of ion reabsorption in kidney organoids. This approach allows personalized analysis of the pathophysiology as well as patient-specific drug screenings. Lastly, I will be the first to apply intravital multiphoton imaging in mice to measure Mg2+ reabsorption in the kidney tubule. The combination of patient-derived organoids and mice models will be used to test novel therapeutics.
Impact and innovation | IN-THE-KIDNEY establishes the novel concept of biological diagnostics for renal tubulopathies, develops for the first-time patient-derived tissue models for functional analysis and introduces intravital ion transport measurements in mice. The methodology and approaches in this project will result in the identification of novel therapeutics for Mg2+-wasting tubulopathies.
Vision | In my vision, better insights in the cellular functional processes in the kidney are the next essential step forward towards better diagnostics, pathophysiological understanding and development of therapy. Development of functional Mg2+ transport measurements and innovative use of patient-specific models is required to increase diagnostic yield and to develop therapy.
Aims and approach | In this project, I aim to develop a urine-based diagnostic approach using scRNA sequencing and extracellular vesicle proteomics to provide biological diagnosis of renal tubulopathies. Using urine-derived adult stem cells, I will perform the first ever functional studies of ion reabsorption in kidney organoids. This approach allows personalized analysis of the pathophysiology as well as patient-specific drug screenings. Lastly, I will be the first to apply intravital multiphoton imaging in mice to measure Mg2+ reabsorption in the kidney tubule. The combination of patient-derived organoids and mice models will be used to test novel therapeutics.
Impact and innovation | IN-THE-KIDNEY establishes the novel concept of biological diagnostics for renal tubulopathies, develops for the first-time patient-derived tissue models for functional analysis and introduces intravital ion transport measurements in mice. The methodology and approaches in this project will result in the identification of novel therapeutics for Mg2+-wasting tubulopathies.
1. Identification of novel causes of Mg2+-wasting tubulopathies
During the project, we have been able to identify two novel genetic causes of hypomagnesemia (gene X/TRPM7). Additionally, we have extended the phenotypic spectrum of known Mg2+ wasting tubulopathies, by describing novel patient cohorts with mutations in CNNM2/FAM111A. Interestingly, we have demonstrated that intronic mutations in SLC12A3 explain part of the missing hereditability observed in Gitelman syndrome. The identified mutations were demonstrated to cause splicing defects. These findings have been translated into clinical practice, by inclusion of these genes in diagnostic gene panels.
2. Deciphering the mechanisms of cardiomyopathy and tubulopathy caused by RRAGD
We obtained iPSCs carrying the S76L mutations in RRAGD. By differentiating these cells and isogenic control cells into cardiomyocytes and kidney organoids, we have been able to show that cardiac and renal development are retained. However, the mutations cause significant changes in cellular signaling pathways, as demonstrated by RNA-sequencing. Moreover, we demonstrated cardiac dysfunction in a zebrafish model of RRAGD disease, which could be rescued by rapamycin. These findings may provide a therapeutic avenue for RRAGD patients.
3. Intravital kidney imaging
We have established intravital imaging of the kidney in our research institute. Our preliminary data demonstrate that Mg2+ can be measured using the MagZet1 Mg probe in the lumen of kidney tubules. We currently in the process of measuring genetic models of Mg2+-wasting tubulopathies. Our findings have been presented at the American Society of Nephrology Kidney week 2024, where they have resulted in novel collaborations. A first publication will be submitted in 2025.
During the project, we have been able to identify two novel genetic causes of hypomagnesemia (gene X/TRPM7). Additionally, we have extended the phenotypic spectrum of known Mg2+ wasting tubulopathies, by describing novel patient cohorts with mutations in CNNM2/FAM111A. Interestingly, we have demonstrated that intronic mutations in SLC12A3 explain part of the missing hereditability observed in Gitelman syndrome. The identified mutations were demonstrated to cause splicing defects. These findings have been translated into clinical practice, by inclusion of these genes in diagnostic gene panels.
2. Deciphering the mechanisms of cardiomyopathy and tubulopathy caused by RRAGD
We obtained iPSCs carrying the S76L mutations in RRAGD. By differentiating these cells and isogenic control cells into cardiomyocytes and kidney organoids, we have been able to show that cardiac and renal development are retained. However, the mutations cause significant changes in cellular signaling pathways, as demonstrated by RNA-sequencing. Moreover, we demonstrated cardiac dysfunction in a zebrafish model of RRAGD disease, which could be rescued by rapamycin. These findings may provide a therapeutic avenue for RRAGD patients.
3. Intravital kidney imaging
We have established intravital imaging of the kidney in our research institute. Our preliminary data demonstrate that Mg2+ can be measured using the MagZet1 Mg probe in the lumen of kidney tubules. We currently in the process of measuring genetic models of Mg2+-wasting tubulopathies. Our findings have been presented at the American Society of Nephrology Kidney week 2024, where they have resulted in novel collaborations. A first publication will be submitted in 2025.
European Collaboration
In the ERC Starting Grant, I had planned to create the largest cohort of Mg2+ wasting tubulopathies worldwide using the European Reference Network for Rare Kidney Diseases (ERKnet). By great effort of several people within my team and collaborators, we have now been able to generate several sub-cohorts of patients with Mg2+-wasting tubulopathies. Unexpectedly, we also identified genes in patients that are often initially diagnosed with endocrine or metabolic disorders. Therefore, we have extended our approach and are also including the European Reference Network for Endocrine Disorders (Endo-ERN) in our studies. This allowed us to identify more patients than initially planned.
Systematic Reviews
Unexpectedly, we identified genetic mutations in patients with isolated hypomagnesemia in genes that had been previously linked to more complex syndromes (FAM111A/mtDNA). This has urged us to perform systematic literature reviews to examine how often hypomagnesemia is present in this group of patients. Although this work was initially not planned within this ERC Starting project, it has resulted in 2 publications. We have now demonstrated that electrolyte abnormalities are quite common in patients in mitochondrial cytopathies. Therefore, we are now discussing with policy makers within ERKNet to see of mtDNA should be included in genetic testing panels for tubulopathies.
Intravital imaging – non labelling structural insights
Multi-photon microscopy can visualise structures in a label-free manner via sample second harmonic generation (SHG), third harmonic generation (THG), and autofluorescence. Although it was not described in the project, we are able to detect collagen (SHG) and vascular structures (THG) in a label-free manner. Moreover, we are able to detect the autofluorescent molecules Nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD), which are important co-enzymes in the central metabolism for each cell. This additional label-free structural information does not only help us judge the anatomic structure and the health of the tissue, but could also be incorporated to study metabolic changes on sub-tubular level. Therefore, these signals will proof beneficial in answering both the proposed and novel scientific questions. Moreover, it is possible that during the progression of the incorporation of long-wavelength microscopy (1650nm), novel signals will be discovered that we currently are still unaware of.
In the ERC Starting Grant, I had planned to create the largest cohort of Mg2+ wasting tubulopathies worldwide using the European Reference Network for Rare Kidney Diseases (ERKnet). By great effort of several people within my team and collaborators, we have now been able to generate several sub-cohorts of patients with Mg2+-wasting tubulopathies. Unexpectedly, we also identified genes in patients that are often initially diagnosed with endocrine or metabolic disorders. Therefore, we have extended our approach and are also including the European Reference Network for Endocrine Disorders (Endo-ERN) in our studies. This allowed us to identify more patients than initially planned.
Systematic Reviews
Unexpectedly, we identified genetic mutations in patients with isolated hypomagnesemia in genes that had been previously linked to more complex syndromes (FAM111A/mtDNA). This has urged us to perform systematic literature reviews to examine how often hypomagnesemia is present in this group of patients. Although this work was initially not planned within this ERC Starting project, it has resulted in 2 publications. We have now demonstrated that electrolyte abnormalities are quite common in patients in mitochondrial cytopathies. Therefore, we are now discussing with policy makers within ERKNet to see of mtDNA should be included in genetic testing panels for tubulopathies.
Intravital imaging – non labelling structural insights
Multi-photon microscopy can visualise structures in a label-free manner via sample second harmonic generation (SHG), third harmonic generation (THG), and autofluorescence. Although it was not described in the project, we are able to detect collagen (SHG) and vascular structures (THG) in a label-free manner. Moreover, we are able to detect the autofluorescent molecules Nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD), which are important co-enzymes in the central metabolism for each cell. This additional label-free structural information does not only help us judge the anatomic structure and the health of the tissue, but could also be incorporated to study metabolic changes on sub-tubular level. Therefore, these signals will proof beneficial in answering both the proposed and novel scientific questions. Moreover, it is possible that during the progression of the incorporation of long-wavelength microscopy (1650nm), novel signals will be discovered that we currently are still unaware of.