Effect of acid/base balance on H-ATPase 31 kD subunit mRNA levels in collecting duct cells

Effect of acid/base balance on H-ATPase 31 kD subunit mRNA levels in collecting duct cells. The cortical collecting duct (CCD) adapts to disturbances of acid/base balance by adjusting the direction and magnitude of its HCO3 transport. The molecular events involved in this adaptation are incompletely understood, but it seems that adaptation is accompanied by changes in the activity and intracellular distribution of the vacuolar H-ATPase. The goal of this study was to examine the effects of metabolic acidosis and alkali load on the expression of the mRNA encoding the 31 kD subunit of the vacuolar H-ATPase in rabbit CCD cells. Pairs of rabbits received either a NH4Cl load or a NaHCO3 load for 16 hours, resulting in a urinary pH of 5.53 ± 0.38 and 8.42 ± 0.10, respectively. CCD cells were isolated by immunodissection and mRNA levels of the H-ATPase 31 kD subunit and of β-actin were determined from the same cDNA samples by quantitative RT-PCR. H-ATPase mRNA levels were significantly higher in CCD cells from acidotic than alkali-loaded rabbits (2.51 ± 1.3 vs. 0.65 ± 0.2; P < 0.05). Similar differences in the H-ATPase 31 kD subunit mRNA levels were observed by Northern blotting. β-actin mRNA levels were comparable in CCD cells of the two groups. The distribution of the H-ATPase 31 kD subunit mRNA was determined among the three cell types of the CCD, that is in α- and β-intercalated cells (α-ICC and β-ICC) and principal cells (PC) isolated by fluorescence-activated cell sorting. The level of expression was comparable in α-ICCs and β-ICCs, whereas PCs contained very low levels of H-ATPase mRNA. In both α-ICC and β-ICC the levels of the 31 kD H-ATPase mRNA were significantly higher in acidotic than in alkaliloaded rabbits. These results indicate that in the rabbit CCD changes in acid/base balance not only regulate the subcellular distribution of the vacuolar H-ATPase but also alter its expression, at least at the mRNA level

Characterization of a mouse cortical collecting duct cell line

Characterization of a mouse cortical collecting duct cell line. A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepi-thelial resistance (885.7 ± 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 ± 3.5mV. The associated short-circuit current (SCC) averaged 71.8 ± 10.3 µA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 ± 0.9 and B = 127.4 ± 0.4; [K+], L = 8.6 ± 0.6 and B = 2.1 ± 0.3; [Cl], L = 68.6 ± 5.8 and B = 101.8 ± 6.6; [HCO3], L = 15.5 ± 1.5 and B = 8.6 ± 1.2; while pH was 7.16 ± 0.03 at the luminal and 6.94 ± 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+. Lactate accumulated predominantly at the basolateral side (L = 7.1 ± 0.44 and B = 17.5 ± 0.52 mM); osmotic concentration was 272 ± 1.4 at the luminal and 290 ±3.0 mOsm/kg at the basolateral side. These data demonstrate that the M-1 cell line retains many phenotypic properties of the CCD epithelium and thus should prove useful as a model in studying mechanisms of ion transport in this segment

Subcellular localization of mineralocorticoid receptors in living cells: Effects of receptor agonists and antagonists

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green fluorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclear-to-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were retained in nuclear matrix preparations after in vivo crosslinking. These data strongly suggest that hormone-activated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids

Regulation of epithelial sodium transport by promyelocytic leukemia zinc finger protein

Aldosterone is the principal regulator of Na homeostasis, and thereby blood pressure. One of the main targets of aldosterone is the epithelial Na channel (ENaC) located in the apical membrane of target cells. Previous studies identified several genes involved in the regulation of ENaC such as SGK1; however, SGK1 knockout mice have only a mild salt-losing phenotype, indicating that further genes must be involved in the action of aldosterone. In our search for further aldosterone-regulated genes, we discovered that aldosterone, at physiological concentrations, induces the expression of the promyelocytic leukemia zinc finger protein (PLZF) in renal cortical collecting duct (CCD) cell lines that stably express mineralocorticoid receptors (MRs). This effect is rapid and does not require de novo protein synthesis, suggesting a direct action. Surprisingly, stable overexpression of human or mouse PLZF isoforms significantly decreased transepithelial Na transport in CCD cells while having no effect on the integrity of the monolayers. In parallel with the decline in Na transport, PLZF suppressed the mRNA levels of β- and γ-ENaC subunits. These observations suggest that PLZF is a negative regulator of ENaC in renal epithelial cells and might be part of a negative feedback loop that limits aldosterone's stimulatory effects on sodium reabsorption