#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p < 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.