Spatial Mapping of the Kidney’s Thick Ascending Limb Cellular Diversity

Background: The thick ascending limb (TAL), once considered a uniform epithelium, is now recognized to comprise molecularly and functionally distinct cell subtypes arranged in a conserved mosaic pattern: TAL-α, TAL-β, and TAL-γ (PMID: 40471686; 40358700). The mutually exclusive expression of ROMK and Kir4.1 indicates that only TAL-α cells express apical ROMK to recycle K + , whereas TAL-β and TAL-γ cells express basolateral Kir4.1 and are therefore positioned to mediate K + reabsorption. TAL-α cells also express Claudin-10, enabling them to generate transepithelial voltage and facilitate NaCl transport. In contrast, TAL-β cells, lacking apical ROMK but expressing Claudin-16, basolateral Kir4.1, and CaSR, support Ca 2 + /Mg 2 + homeostasis and K + reabsorption. TAL-γ cells, exhibit α/β features coexpressing Claudin-10 & Kir4.1, and likely contribute to K + reabsorption. Our previous TAL-enriched transcriptomic datasets revealed clear transcriptional heterogeneity within the TAL; however, the precise spatial organization of these cell subtypes across kidney regions remains incomplete. To address this, we tested whether spatial transcriptomics platforms could accurately identify TAL α/β/γ subtypes and define their anatomical distribution within the kidney. Methods: Healthy adult mouse kidneys were processed using the Xenium In Situ platform (10x Genomics). FFPE sections were mounted with consistent cortical–medullary orientation and profiled using the Xenium 5K Prime Gene Panel together with a custom kidney-specific panel. Following high-resolution imaging on the Xenium Analyzer, transcript and segmentation files were imported into Seurat v5, quality-filtered, and normalized using SCTransform. Unbiased clustering and differential gene expression analyses were performed. TAL cells were identified by positive Slc12a1 expression. Subtype-specific spatial distributions were visualized using Xenium Explorer v4.1. Results: Transcriptomic profiling using the Xenium platform recapitulated previously described TAL heterogeneity, identifying TAL-α, -β, -γ subtypes, as well as macula densa (MD) cells. DEG revealed distinct transcriptional programs consistent with specialized functions for each subtype, in agreement with our enriched-TAL dataset. TAL-α & -γ cells showed high expression of genes associated with monovalent cation–transport, like Avpr2, whereas TAL-β cells exhibited higher expression of genes associated with divalent cation–handling, such as Pth1r, Kl, and Vdr. Spatial transcriptomics further demonstrated a region-specific distribution of TAL subtypes. TAL-α cells were present throughout the TAL, spanning both cortical and outer medullary (OM) regions. TAL-β cells were most abundant in the cortex and less represented in the outer stripe of the OM. TAL-γ cells were restricted to the OM, with highest abundance in the inner stripe. Spatial mapping also revealed an intermingled arrangement of TAL subtypes within zones. This gradient-like organization, with different cell types intermingled within zones, supports functional segregation of TAL cells across kidney zones. Conclusion: Spatial transcriptomics, informed by our prior TAL-enriched snRNA-Seq, reliably identified TAL-α/β/γ subtypes and showed an intermingled mosaic distribution of cell types across kidney zones. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.