Binding of [125I] Tyr A14 human insulin ([125I] insulin) was measured at 4 degrees C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10(-18) mol [125I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5 +/- 0.3; proximal convoluted tubule (PCT), 12.6 +/- 0.6; pars recta (PR), 4.0 +/- 2.6; thin descending limb (TDL), 0.6 +/- 0.2; thin ascending limb (TAL), 0.6 +/- 0.2; medullary thick ascending limb (MAL), 0.8 +/- 0.1; cortical ascending limb (CAL), 2.1 +/- 0.1; distal convoluted tubule (DCT), 5.6 +/- 1.1; cortical collecting tubule (CCT), 3.2 +/- 0.3 and outer medullary collecting tubule (MCT), 2.3 +/- 0.1. Specific [125I] insulin binding to glomeruli and tubule segments was time and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [125I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone effects are mediated by the mineralocorticoid receptor (MR), a transcription factor highly expressed in the distal nephron. Given that MR expression level constitutes a key element controlling hormone responsiveness, there is much interest in elucidating the molecular mechanisms governing MR expression. To investigate whether hyper- or hypotonicity could affect MR abundance, we established by targeted oncogenesis a novel immortalized cortical collecting duct (CCD) cell line and examined the impact of osmotic stress on MR expression. KC3AC1 cells form domes, exhibit a high transepithelial resistance, express 11beta-hydroxysteroid dehydrogenase 2 and functional endogenous MR, which mediates aldosterone-stimulated Na(+) reabsorption through the epithelial sodium channel activation. MR expression is tightly regulated by osmotic stress. Hypertonic conditions induce expression of tonicity-responsive enhancer binding protein, an osmoregulatory transcription factor capable of binding tonicity-responsive enhancer response elements located in MR regulatory sequences. Surprisingly, hypertonicity leads to a severe reduction in MR transcript and protein levels. This is accompanied by a concomitant tonicity-induced expression of Tis11b, a mRNA-destabilizing protein that, by binding to the AU-rich sequences of the 3'-untranslated region of MR mRNA, may favor hypertonicity-dependent degradation of labile MR transcripts. In sharp contrast, hypotonicity causes a strong increase in MR transcript and protein levels. Collectively, we demonstrate for the first time that optimal adaptation of CCD cells to changes in extracellular fluid composition is accompanied by drastic modification in MR abundance via transcriptional and posttranscriptional mechanisms. Osmotic stress-regulated MR expression may represent an important molecular determinant for cell-specific MR action, most notably in renal failure, hypertension, or mineralocorticoid resistance.

No abstract