To reach the project objectives, we studied primary cells derived from human kidney tubules and clonally expanded in vitro. From each clone, we have obtained whole genome sequencing data, together with expression data of selected genes. For some clones we have obtained metabolomic data. To test our hypothesis on hypoxia signaling as underlying cause of alterations (objective 3), we have used a genetic model, i.e. kidney cells from individuals that carry an inherited pathogenic mutation in the VHL (Von Hippel Lindau) gene. The VHL gene is a tumor suppressor and master regulator of hypoxia signalling. Loss of VHL causes ectopic activation of hypoxia signaling and, according to our hypothesis, should enhance mutagenesis. Of note, the genetic loss of VHL is very often the first event in kidney tumorigenesis. The project has been carried out in collaboration with the Urological Research Institute and the Laboratory of Clinical Molecular Genetics of San Raffaele Hospital.
The project has achieved the following results:
1) Establishment of a method for detection of somatic mutations in single genomes from human kidneys avoiding the need for kidney biopsies and using, instead, non-invasive liquid biopsies (urine samples).
2) Establishment of a protocol for concomitant assessment of somatic mutations, gene expression and metabolite quantification in in vitro-expanded, clonal populations from human kidneys.
3) Detailed characterization of proliferating kidney cells found in human urines and able to grow in vitro. This characterization has been obtained by culturing cells from the urine of 6 healthy individuals in the age range (24-63) and 6 VHL patients (age range 39-56) and is focused on describing changes associated with aging and heterozygous loss of VHL.
4) Generation of somatic mutation catalogues (n=19) of normal kidney genomes obtained from the urine of 3 healthy individuals and 1 patient with VHL disease.
5) Analyses of additional somatic mutation catalogues, obtained from 4 cysts and 7 ccRCC,from the kidney of the same patient with VHL disease.
6) Evidence of increased mutagenesis in normal kidney cells of an individual carrying a germline heterozygous pathogenic mutation in VHL.
7) Evidence, based on mutational profiles, that metabolic changes downstream hypoxia signalling are a cell-type specific cause of mutagenesis in kidney tubule epithelia.
Results have been disseminated both within the host institution and at international events. This work has been presented multiple times at internal congresses and retreats within the host institution (San Raffaele Hospital); at the “14th International VHL Medical/Research Symposium”, organized by the “VHL alliance”, which gathers researchers, clinicians and patients with VHL disease; at the Congress of the European Society of Human Genetics (ESHG) 2022; at the FASEB congress on Polycystic Kidney Disease in July 2022.
