Developmental expression of ROMK in rat kidney

The apical secretory K+ (SK) channel in the principal cell represents the r ate-limiting step for Kt secretion across the cortical collecting duct (CCD ). Patch clamp analysis of maturing rabbit principal cells identifies an in crease in number of conducting SK channels after the 2nd week of life [L. M . Satlin and L. G. Palmer. Am. J. Physiol. 272 (Renal Physiol. 41): F397-F4 04, 1997], similar to 1 wk after an increase in activity of the amiloride-s ensitive epithelial Na+ channel (ENaC) is detected. To correlate the postna tal increase in channel activity with developmental expression of ROMK, the molecular correlate of the SK channel, we used gene-specific probes to sho w a developmental increase in abundance of renal ROMK mRNA and a ROMK-speci fic antibody to examine the nephron distribution, localization, and abundan ce of this protein in developing rat kidney. Using antibodies directed agai nst aquaporin-3 (AQP-3) and Tamm-Horsfall protein (THP), we confirmed that ROMK was expressed along the apical membranes of principal cells and thick ascending limbs of Henle (TALH) in adult kidney. Within the midcortex of th e neonatal kidney, ROMK-positive segments revealed weak coincident staining with the TALH-specific antibody but did not colabel with an antibody direc ted against distal and connecting tubule (CNT)-specific kallikrein or the l ectin Dolichos biflorus agglutinin (DBA), which labels proximal tubules and collecting duets. In inner cortex and outer medulla of kidneys from 1-wk-o ld animals, ROMK protein was identified in medullary TALH (MTALH) and DBA-p ositive collecting ducts. By 3 wk of age, coincident ROMK and DBA expressio n was detected in midcortical and outer cortical CNTs and CCDs. Immunoblot analysis of plasma membrane-enriched fractions of maturing rat kidney revea led a developmental increase in a similar to 40-kDa band, the expected size for ROMK. Immunolocalization of alpha-ENaC showed apical staining of a maj ority of cells in distal nephron segments after the Ist week of postnatal l ife. The beta-and gamma-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abu ndance; gamma-ENaC showed some apical polarization. These results suggest t hat the postnatal increases in a principal cell apical SK and Nat channel a ctivity are mediated, at least in part, by increases in abundance of ROMK m essage and protein and ENaC subunit proteins.