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Abstract: FR-PO543

Mechanisms of Thiazide-Induced Magnesium Wasting and Calcium Retention at Single-Cell Resolution

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

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

Authors

  • Reyes, Jeremiah V., University of the Philippines Manila College of Medicine, Manila, Metro Manila, Philippines
  • Su, Xiao-Tong, Oregon Health & Science University, Portland, Oregon, United States
  • Medina, Paul Mark B., University of the Philippines Manila College of Medicine, Manila, Metro Manila, Philippines
  • Yang, Chao-Ling, Oregon Health & Science University, Portland, Oregon, United States
  • Demirci, Hasan, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
  • Bachmann, Sebastian, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
  • McCormick, James A., Oregon Health & Science University, Portland, Oregon, United States
  • Nelson, Jonathan W., Oregon Health & Science University, Portland, Oregon, United States
  • Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
Background

States of NCC inhibition, as in Gitelman syndrome and chronic thiazide treatment, present with hypokalemia, alkalosis, hypomagnesemia, and hypocalciuria. The mechanism by which loss of NCC function affects Mg and Ca handling remains very unclear. Here we combined single nucleus RNA sequencing with physiologic and morphometric analysis to unravel the causes of hypomagnesemia and hypocalciuria secondary to NCC inhibition.

Methods

8 to 10-week-old male NCC-Cre-INTACT mice received 50 mg/kg/day metolazone (MTZ) orally for 4 days. Fluorescence-activated nuclei sorting was performed to select for DCT nuclei. Sequencing was done using 10X Chromium. Reads were analyzed using a transcriptomic bioinformatics pipeline with Seurat. We also performed tubule morphometrics.

Results

MTZ-treated mice had lower plasma Mg and lower urinary Ca excretion compared to controls, consistent with NCC inhibition. Our snRNA-seq dataset showed 2 clusters, DCT1 and DCT2, based on canonical markers consistent with published transcriptomic atlases. We curated Mg and Ca cassettes from known magnesiotropic and calciotropic genes and defined Mg and Ca scores from the pooled expressions of the genes and used them to indicate Mg and Ca handling capacity. The analyses showed that Mg handling is primarily a DCT1 process and Mg score was lower in the MTZ group. We also found that the DCT1 undergoes dedifferentiation leading to reduction in magnesiotropic gene expression. Additionally, 3D morphometrics showed that DCT1 was shorter in the MTZ group. Ca handling is primarily a DCT2 process and Ca score was lower in the MTZ group consistent with low distal delivery due to enhanced proximal Ca reabsorption. Yet, DCT2 cells maintained their cell state and were less vulnerable to NCC inhibition compared to DCT1 cells. Lastly, MTZ-treatment caused hypertrophy of the Ca-transporting connecting tubule (CNT).

Conclusion

The results indicate that hypomagnesemia secondary to NCC inhibition results both from a disruption of genes involved in Mg handling and from DCT1 dedifferentiation and atrophy. While increased proximal tubule Ca reabsorption is required for hypocalciuria, our results also indicate that aldosterone-induced hypertrophy of the CNT and relative preservation of DCT2 contribute to calcium retention.

Funding

  • NIDDK Support