Differential Cell Types in the Thick Ascending Limb of the Kidney: Insights into Functionally Distinct TAL Claudin 10 and TAL Claudin 16 Cells

Abstract

Study objective, hypothesis: The Thick Ascending Limb (TAL) in the kidney is crucial for reabsorbing Na+ and divalent cations like Ca2+ and Mg2+. This reabsorption depends on the apical cotransporter NKCC2, encoded by the Slc12a1 gene. The TAL extends from the outer medulla to the cortex. While typically considered a single functional cell type, recent evidence suggests the presence of two distinct TAL cell types, distinguished by the expression of Claudin 10 and Claudin 16 (PMID: 28028216). Despite these insights, the lack of correlation with functional and morphological data and the absence of a comprehensive model for the cooperative regulation of NaCl and Ca2+/Mg2+ transport remain significant knowledge gaps. Our objective is to couple immunohistochemistry with single nuclei (sn) RNA sequencing technology to uncover unique cell features that are obscure, when a single approach is employed. Methodology: Immunofluorescence analysis was performed on samples from mice, rats, and humans. Additionally, Slc12a1-positive cells were analyzed using sn-RNA sequencing datasets that contained spatial information about cell localization. These datasets were sourced from published studies on mouse (PMID: 31689386) and human samples (PMID: 37468583). Data Immunofluorescence images from mice, rats, and humans revealed mutually exclusive expression patterns of Kir4.1 and ROMK. Apical ROMK was observed only in cells that express Claudin 10, while Kir4.1 was expressed only in cells that express Claudin 16. phospho NKCC2 and CaSR displayed a mosaic pattern, with expression in the Claudin 16 cells throughout the entire TAL and, in both Claudin 10 and Claudin 16 cells only in the cortex. Unsupervised clustering of mouse sn-RNA seq data identified two distinct clusters, TAL Cldn10 and TAL Cldn16, present in both the cortex and medulla. Key Differentially Expressed Genes (DEG) for TAL Cld10 included Avp2r, Wnk4, Spak, Kcnt1 (encoding KCa4.1 or Slo2.2), and genes related to arachidonic acid signaling such as Ptger3 (encoding Ep3), Pla2g7, Cox7a1. Kcnj1 (encoding ROMK) transcripts were found in both clusters, even though ROMK is located apically only in TAL Claudin 10 cells. TAL Claudin 16 cells showed a higher expression of calcium-related genes, including Casr, Pth1r, Vdr, Kl (encoding Klotho), as well as transcripts for Kcnj10 (encoding Kir4.1), Kcnj16 (encoding Kir5.1), Wnk1, Clcnkb, and Bsnd (encoding Barttin). This transcriptional heterogeneity was also observed in human TAL cells. Summary of results Combining morphological and transcriptomic analyses identified two functionally distinct cell types within the TAL, present in both cortex and medulla. Each cell type appears to be associated with specific transport pathways: TAL Claudin 10 cells, express ROMK apically and are thus responsible for generating the transepithelial voltage and for paracellular Na+ reabsorption. They also express more Na+ transport-regulatory genes, including Avp2r and Wnk4, along with genes related to arachidonic acid signaling. TAL Claudin 16 cells mediate Ca2+ & Mg2+ transport via the paracellular pathway, driven by the voltage generated by claudin 10 cells; they express calcium-regulatory genes, including Casr, Pth1r, and Vdr. Interestingly, they also express genes associated with transcellular Na+, K+, and Cl− transport, including Kcnj10 (Kir4.1), Kcnj16 (Kir5.1), Wnk1, and Clcnkb. Together, these results present a new model of discrete but coupled ion transport pathways in the TAL. NIH DK51496 | NIH DK054983 | VA 1I01BX002228 | Global Research Network of Excellence, Leducq Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.