Clonal landscape of human nephrons

Background: Renal cell carcinoma (RCC) subtypes are believed to arise from distinct nephron segments, suggesting that the accumulation and patterns of genetic alterations in normal nephron may also vary by segment. However, due to the intricate architecture and the technical challenges in isolating each nephron segment separately, the mutational landscape within the nephrons remains poorly understood. Methods: We used laser-capture microdissection (LCM) to isolate five distinct nephron segments, including proximal convoluted and straight tubules (PCT and PST), distal convoluted and straight tubules (DCT and DST), and collecting ducts (CD), which were then analyzed using a highly accurate sequencing platform (NanoSeq) and whole exome sequencing (WES). Results: NanoSeq analysis of the 175 LCM samples from 12 patients revealed that single nucleotide variants (SNVs) linearly increased with aging. However, the accumulation rate markedly varied across segments: PCT and PST showed notably higher rates (123 and 132 SNVs per genome/year, respectively), compared with DST (28), DCT (27), and the lowest in CDs (18). Seven SBS signatures (SBS1,5,8,12,40a-c) were detected. The age-related SBS5 was similar across all segments and accounted for most of the mutations in the distal compartments. In contrast, SBS12, SBS40b and SBS40c were largely confined to the proximal segments, mostly explaining their higher mutational burdens. Furthermore, the SBS12 signature was significantly enriched in PCT, showing a strong transcriptional strand bias. WES analyses on the 683 LCM samples from 6 patients showed frequent monoclonal expansion in the proximal tubules and CDs of aged donors, which were rarely seen in distal tubules. Notably, an extensively expanded clone in the PCT harboring a 3p loss and a mutation affecting PBRM1, a common driver gene in clear cell RCC, was observed in morphologically normal PCTs. In total, 11.7% of all LCM samples harbored ≥1 non-synonymous mutations affecting known RCC driver genes. Most of these mutations were detected in proximal tubules, while rarely found in distal tubules and CDs. Copy number alterations were quite common, found in 35.1% of the samples and strongly correlated with donor age. The most frequent alterations included gains of Chromosomes 10 and 7 and losses of Chromosomes 18 and 22. Of note, chromosome 10 gain was mostly confined to proximal nephrons, while Chromosome 18 loss was almost exclusively found in CD. Conclusion: Aging kidney nephrons undergo pronounced remodeling characterized by segment-specific accumulation of mutations and clonal expansions. Proximal tubules exhibit the highest mutation rates and accumulate RCC driver alterations. The strong enrichment of the SBS12 signature in the PCT confirmed the previous view that PCT is the origin of this RCC subtype. The strong strand bias for SBS12 suggests the influence of exogenous mutagens absorbed from PCTs, and identifying the causative agent could be important for RCC prevention.