Jakob Nielsen

Long-term regulation of ENaC expression in kidney by angiotensin II.

Abstract—We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the &agr; subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased &agr;ENaC protein abundance in rat kidney cortex. The decrease in &agr;ENaC protein abundance in response to AT1 receptor blockade was associated with a fall in &agr;ENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT1 receptor blockade on &agr;ENaC expression was not blocked by spironolactone, suggesting a direct role of the AT1 receptor in regulation of &agr;ENaC gene expression. Candesartan administration was also found to increase the abundances of the &bgr; and &ggr; subunits. The increase in &bgr; and &ggr;ENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in &bgr; and &ggr;ENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function.

The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon

The kidneys escape from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism.