The DCT2/CNT is the Major Site of Regulated ROMK-Mediated K⁺Secretion

The Renal Outer Medullary potassium channels (ROMK) are major potassium exit channels in the distal nephron. The Textbook view presents the Collecting Duct (CD) as the chief site of regulated potassium (K + ) secretion mediated by ROMK. This view is challenged by recent studies showing the Late Distal Convoluted Tubules (DCT2) and the Connecting Tubules (CNT) are the main sites of ROMK action. To settle the debate, we developed the first nephron segment-specific DCT2/CNT ROMK knockout (KO) mouse using the novel DCT/CNT Cre-driver, Emx1. ROMK flox/flox, Emx1 Cre-positive (ROMK DCT2/CNT KO, n=6) and Wild-type ROMK wt/wt, Emx1 Cre-positive (ROMK Control, n=6) were randomized to standard (1%K + ) or high K + (HKD: 5%K + ) diets for 2 days. Urinary K + excretion was examined using metabolic cages. Blood electrolytes and protein levels of key components of the K + secretion machinery were analyzed under both standard and HKD. Using immunofluorescence imaging of ROMK and segment-specific markers, we confirmed specific ROMK deletion in the DCT2/CNT while ROMK in the Thick Ascending Limb (TAL) and the CD remained unaffected. In contrast to total ROMK KO mice, ROMK DCT2/CNT KO mice did not exhibit a Bartter syndrome phenotype and hydronephrosis. Instead, ROMK DCT2/CNT KO mice fed the standard K + diet, exhibited higher plasma K + than control mice (4.5 versus 3.8 mmol/L in KOs and controls, respectively; p-value: 0.02) and diminished urinary K + handling despite a compensatory reduction in NCC. On HKD, ROMK DCT2/CNT KO mice became hyperkalemic (6.3 versus 4.6 mmol/L in KOs and controls, respectively; p-value: 0.009). Despite the hyperkalemia, K + excretion in the KO was significantly reduced compared to the control mice. Western blot analysis revealed that total NCC was severely reduced in the hyperkalemic KO compared to the normokalemic control mice. Additionally, aENaC full-length and cleaved forms were higher in the KO compared to control mice, likely reflecting a secondary response to the hyperkalemia and the expected increase of Aldosterone. In conclusion, ROMK in the DCT2/CNT plays a key role in regulated K + secretion. On standard K + intake, mice lacking ROMK in this segment can partially compensate for ROMK deletion by reaching a new balance with higher plasma K + and lower NCC levels. On high K + intake, ROMK DCT2/CNT KO became hyperkalemic due to blunted K + secretion despite the presence of ROMK in the CD, indicating DCT2/CNT is the chief site of ROMK-mediated K + secretion. This work was supported by National Institutes of Health (NIH) Grant R01 90109058 to P.A.W. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.