Kidney Week

Abstract: TH-OR62

Facilitation of Mg2+ Extrusion by the Murine Distal Convoluted Tubule-Specific Isoform of SLC41A3

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Back to the Basics
  October 24, 2024 | Location: Room 4, Convention Center
  Abstract Time: 05:40 PM - 05:50 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

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

Authors

• Franken, Gijs AC, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Gijs AC Franken, MS, MSc, PhD

• Bosman, Willem, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Willem Bosman, MS

• Jung, Hyun Jun, Johns Hopkins Medicine, Baltimore, Maryland, United States

Hyun Jun Jung, PhD

• Bos, Caro, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Caro Bos

• Latta, Femke, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Femke Latta

• Bindels, René J., Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

René J. Bindels, PhD

• Knepper, Mark A., National Institutes of Health, Bethesda, Maryland, United States

Mark A. Knepper, MD, PhD

• Hoenderop, Joost, Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Joost Hoenderop, PhD

• De Baaij, Jeroen H.F., Radboud Universitair Medisch Centrum, Nijmegen, Netherlands

Jeroen H.F. De Baaij, PhD

Background

The distal convoluted tubule (DCT) plays an indispensable role in magnesium (Mg²⁺) reabsorption in the kidney. Here, transcellular Mg²⁺ transport takes place, yet the extrusion mechanism is currently unknown. The solute carrier 41 A3 (SLC41A3) has been suggested to be involved in Mg²⁺ extrusion. Yet in vitro and in vivo studies could not demonstrate the molecular function of SLC41A3. In this study, we describe two distinct SLC41A3 isoforms with unique expression patterns and functions.

Methods

Using available RNA-sequencing data and RT-qPCR, expression of the two alternative Slc41a3 transcripts, encoding isoform (iso) 1 or -2, were assessed in kidney and isolated DCT tubules. HEK293 or HAP1 cells were transfected with plasmids expressing either of the isoforms, followed by ²⁵Mg²⁺ uptake and extrusion studies. Identification of DCT-specific cis-regulatory elements (CRE) was achieved by combining data from available ATAC sequencing data and luciferase assays in HeLa cells.

Results

Expression studies revealed the presence of a distinct transcript of Slc41a3 in the DCT with an alternative promoter, leading to a shorter protein with a distinct N-terminus (iso2). HEK293 cells overexpressing SLC41A3-iso2 exhibited 2.7-fold higher ²⁵Mg²⁺ uptake compared to mock. In contrast, SLC41A3-iso1-transfected cells increased ²⁵Mg²⁺ uptake by 1.7-fold, while on average protein expression of SLC41A3-iso2 was ~35 fold lower dan SLC41A3-iso1. SLC41A3-iso2 overexpressing cells showed 1.5-fold higher ²⁵Mg²⁺ extrusion compared to mock-transfected cells, whereas iso1-transfected cells did not show this increase. Using TRPM7 KO HAP1 and CNNM3 and -4 KO HEK293 cell models, ²⁵Mg²⁺ extrusion by SLC41A3-iso2 was demonstrated to be independent of these Mg²⁺ transporting proteins. Via available ATAC sequencing data, a CRE accessible in the DCT was identified, ±3.3kb upstream of the TSS of Slc41a3-iso2. Luciferase assays demonstrated that the presence of the CRE increased the Slc41a3-iso2 promoter activity ±4-fold, indicating the CRE contains an enhancer function.

Conclusion

In conclusion, we identified two alternative transcripts of Slc41a3 in mouse. Slc41a3-iso2 is enriched expressed within the DCT with specific gene regulatory elements. We speculate that specifically in the DCT, SLC41A3-iso2 orchestrates Mg²⁺ extrusion.