Resolving the thiazide-induced hypocalciuria conundrum using single cell transcriptomics
Authors: Reyes Jeremiah , Su Xiao-Tong , Yang Chao-Ling , Medina Paul Mark , McCormick James , Nelson Jonathan , Ellison David 
Published: May 2023

Thiazide diuretics lower urinary calcium excretion and are used to prevent calcium stone recurrence. Thiazides inhibit the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Hypocalciuria also occurs in Gitelman syndrome, caused by mutations in SLC12A3, encoding NCC. The mechanism by which loss of NCC function affects calcium handling remains very controversial. The two prevailing hypotheses invoked are 1) hypocalciuria results from volume-dependent increases in proximal tubule reabsorption; 2) thiazides activate calcium reabsorption along the DCT. Here we combined single nucleus RNA sequencing with physiologic analysis to unravel the causes of hypocalciuria.Eight to 10-week old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days. Littermates receiving only vehicle were used as controls. As DCT cells comprise a small percentage of the kidney mass, we used the NCC-Cre+/-- INTACT+/- mouse line to enrich for DCT via Cre-dependent GFP expression in DCT nuclei. After nuclei isolation, GFP+ events (DCT nuclei) were enriched by fluorescence assisted nuclei sorting. The purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat.The MTZ-treated mice showed lower urinary calcium excretion compared to controls recapitulating thiazide-induced hypocalciuria. Our snRNAseq integrated dataset contained 51,876 mapped genes across 45,953 samples from 3 MTZ-treated and 3 control mice. Roughly 99% of the nuclei were singlets expressing Slc12a3 showing successful enrichment for the DCT. Dimensional reduction analysis and projection confirmed two clusters, DCT1 and DCT2, based on canonical segment markers consistent with previously published transcriptomic atlases. Gene expression analysis showed the DCT2 contained a calcium cassette consisting of the known calciotropic genes Trpv5, Calb1, S100g, Slc8a1, Ryr2, Vdr, Casr, and Atp2b1. These genes were also expressed in the connecting tubule (CNT), as documented using the Calb1-Cre-INTACT system. From this set of genes, we defined a calcium score computed from the pooled average expression of all these genes in a single cell. Although all calcium cassette genes, and the average calcium score, were lower in the MTZ-treated group, the proportion of DCT2/DCT1 cells increased (0.27 ± 0.03 vs 0.35 ± 0.03; p = 0.027). These data suggest that lower calcium delivery to the DCT reduces calcium cassette expression per cell, but that distal fractional reabsorption is nonetheless increased owing to hypertrophy and elongation of the calcium transporting segments, DCT2 and CNT.We propose a modified mechanism to account for thiazide -induced hypocalciuria: 1) volume-dependent increase in proximal tubule reabsorption; 2) increased fractional (not absolute) DCT2 reabsorption owing to structural remodeling of DCT2 and CNT providing more surface area for calcium reabsorption. Funding: NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP This is the full abstract presented at the American Physiology Summit 2023 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.