Kidney Week

Abstract: FR-PO557

Heterogeneity in the Renal Thick Ascending Limb Cell Composition: Insights from Ultrastructure, Immunohistochemistry, and Single-Cell Transcriptomics

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Demirci, Hasan, Charité-Universitätsmedizin Berlin, Department of Anatomy, Berlin, Germany

Hasan Demirci, MS, MSc

• Bahena-López, Jessica Paola, Oregon Health & Science University, School of Medicine, Division of Hypertension and Nephrology, Portland, Oregon, United States

Jessica Paola Bahena-López, MD

• Nelson, Jonathan W., University of Southern California, Department of Medicine, Los Angeles, California, United States

Jonathan W. Nelson, PhD

• Su, Xiao-Tong, Oregon Health & Science University, School of Medicine, Division of Hypertension and Nephrology, Portland, Oregon, United States

Xiao-Tong Su, PhD

• Ellison, David H., Oregon Health & Science University, School of Medicine, Division of Hypertension and Nephrology, Portland, Oregon, United States

David H. Ellison, MD, FASN

• Bachmann, Sebastian, Charité-Universitätsmedizin Berlin, Department of Anatomy, Berlin, Germany

Sebastian Bachmann, PhD

Background

The thick ascending limb (TAL) provides the driving force for the renal concentrating mechanism through its capacity to dilute the urine via the “single effect” of reabsorbing NaCl. It also transports divalent cations like Ca2⁺ and Mg2⁺. A single, terminally differentiated cell type with zone-related specificities has so far been defined to line TAL, but recent transcriptomic findings report distinct TAL cell clusters with mosaic expression pattern, illustrated by differential expression of ion transporters and junctional components. Here we couple structure and immunohistochemistry with single-cell (sc) RNA sequencing technology to define unique cell features across kidney zones and local mosaicism.

Methods

Rat, mouse, and human kidney samples were used to examine the morphology of medullary (mTAL) and cortical (cTAL) TAL epithelia. Immunohistochemistry was used to assess the expression of ROMK, NKCC2, pNKCC2, Kir4.1, CaSR, Cldn10 and Cldn16 in TAL. Additionally, scRNA-seq datasets (PMID: 31689386) from mouse kidney were leveraged to define transcriptomic signatures of TAL.

Results

NKCC2 was expressed throughout TAL and macula densa. Mosaic expression of ROMK in TAL showed 46 ± 7% ROMK-negative cells in mTAL, decreasing with cTAL. ROMK-negative cells inversely displayed p-NKCC2 signal chiefly in mTAL, suggesting mosaic NaCl transport activity. Cldn10 and ROMK were colocalized in mTAL and cTAL. CaSR and Kir4.1 were mutually exclusive to ROMK, and a subset of CaSR/Kir4.1 cells expressed Cldn 16 in outer stripe and cTAL cells; here, Cldn 16 dominated over Cldn 10 in mosaic junctional belt conformation. Macula densa expressed exclusively Cldn 10. ScRNA-seq analysis defined Cldn 10 and Cldn 16 as distinct cell clusters, with Cldn 10 coexpressing Stk39, Ptger3, and Cryab, and Cldn16 cells coexpressing Kir4.1, CaSR, Wnk1, and Pth1r.

Conclusion

TAL cells exhibit marked heterogeneity in transporter and tight junction protein expression, correlated with distinct cell clusters from scRNA seq. Modeling TAL function with two distinct cell types, one providing transepithelial voltage generation, the other paracellular divalent cation transport suggests separate functions within TAL cells serving renal concentrating ability and ion transport homeostasis.