DCT Transcriptional and Structural Remodeling: Impacts on Magnesium Handling

Loss of activity of the NaCl Cotransporter (NCC) in Gitelman syndrome and with chronic thiazide treatment results in hypomagnesemia but the underlying mechanism remains unresolved. The prevailing hypothesis is that it results indirectly from distal convoluted tubule (DCT) atrophy lowering magnesium (Mg)-reabsorbing capacity by this segment. We tested the hypothesis that NCC inhibition directly disrupts a cellular mechanism related to Mg handling independent of DCT atrophy. We used the NCC-Cre+−/−- INTACT+/- mouse line to allow enrichment of DCT nuclei via Cre-dependent GFP expression in DCT nuclei. Eight- to 10-week-old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days; littermate controls receiving only vehicle. After nuclei isolation, GFP+ events (DCT nuclei) were sorted by fluorescence assisted nuclei sorting. Purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat. Tubule morphometrics using optical tissue clearing was performed on kidneys from animals receiving the same treatment. MTZ-treated mice had lower plasma Mg compared to controls recapitulating thiazide-induced hypomagnesemia. Our snRNAseq dataset containing 45,953 cells from 3 MTZ-treated and 3 control mice showed clear separation into early DCT (DCT1) and late DCT (DCT2) based on canonical segment markers. Gene expression analysis showed the DCT1 contained a magnesium cassette consisting of known magnesiotropic genes Trpm6, Trpm7, Egf, Umod, Prox1, Fxyd2, Cnnm2, and Slc41a3 so we focused subsequent analyses on DCT1. NCC inhibition caused DCT1 degeneration as indicated by lower mean DCT1 score of DCT1 cells and a greater proportion of DCT1 cells with low differentiation state in the MTZ group. Similarly, MTZ-treated samples had lower DCT1/DCT2 cell proportion in our snRNAseq dataset and morphometrics revelead significantly shorter DCT1 tubules implying that the degeneration process eventually leads to DCT1 atrophy. Using the aggregate expression of the magnesium cassette genes, we computationally defined a Mg score as a surrogate marker for Mg reabsorption capacity for each individual cells. The average Mg score was lower in the MTZ-treated group compared to the controls. Our data supports the long-standing hypothesis that hypomagnesemia is a secondary consequence of the loss of effective surface area for Mg reabsorption. Additionally, we uncovered that even at the single cell level, the Mg handling capacity of the cell decreases as a result of NCC inhibition implying that a disruption in sodium reabsorption disrupts expression of the Mg reabsorption machinery. NIH DK51496, NIH DK054983, NIDDK DK132066, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP. 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.