Prenatal programming of rat cortical collecting tubule sodium transport

Prenatal insults have been shown to lead to elevated blood pressure in offspring when they are studied as adults. Prenatal administration of dexamethasone and dietary protein deprivation have demonstrated that there is an increase in transporter abundance for a number of nephron segments but not the subunits of the epithelial sodium channel (ENaC) in the cortical collecting duct. Recent studies have shown that aldosterone is elevated in offspring of protein-deprived mothers when studied as adults, but the physiological importance of the increase in serum aldosterone is unknown. As an indirect measure of ENaC activity, we compared the natriuretic response to benzamil in offspring of mothers who ate a low-protein diet (6%) with those who ate a normal diet (20%) for the last half of pregnancy. The natriuretic response to benzamil was greater in the 6% group (821.1 ± 161.0 μmol/24 h) compared with the 20% group (279.1 ± 137.0 μmol/24 h), consistent with greater ENaC activity in vivo ( P < 0.05). In this study, we also directly studied cortical collecting tubule function from adult rats using in vitro microperfusion. There was no difference in basal or vasopressin-stimulated osmotic water permeability. However, while cortical collecting ducts of adult offspring whose mothers ate a 20% protein diet had no sodium transport (−1.9 ± 3.1 pmol·mm−1·min−1), the offspring of rats that ate a 6% protein diet during the last half of pregnancy had a net sodium flux of 10.7 ± 2.6 pmol·mm−1·min−1 ( P = 0.01) in tubules perfused in vitro. Sodium transport was measured using ion-selective electrodes, a novel technique allowing measurement of sodium in nanoliter quantities of fluid. Thus we directly demonstrate that there is prenatal programming of cortical collecting duct sodium transport.

CFTR-dependent and -independent swelling-activated K+ currents in primary cultures of mouse nephron

The role of CFTR in the control of K+ currents was studied in mouse kidney. Whole cell clamp was used to identify K+ currents on the basis of pharmacological sensitivities in primary cultures of proximal (PCT) and distal convoluted tubule (DCT) and cortical collecting tubule (CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT cells, forskolin activated a 293B-sensitive K+ current in WT, but not in KO, mice. In these cells, a hypotonic shock induced K+ currents blocked by charybdotoxin in WT, but not in KO, mice. In PCT cells from WT and KO mice, the hypotonicity-induced K+ currents were insensitive to these toxins and were activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting that the corresponding channel was TASK2. In conclusion, CFTR is implicated in the control of KCNQ1 and Ca2+-sensitive swelling-activated K+ conductances in DCT and CCT, but not in proximal convoluted tubule, cells. In KO mice, impairment of the regulatory volume decrease process in DCT and CCT could be due to the loss of an autocrine mechanism, implicating ATP and adenosine, which controls swelling-activated Cl− and K+channels.

Novel Schering and ouabain-insensitive potassium-dependent proton secretion in the mouse cortical collecting duct

The intercalated (IC) cells of the cortical collecting duct (CCD) are important to acid-base homeostasis by secreting acid and reabsorbing bicarbonate. Acid secretion is mediated predominantly by apical membrane Schering (SCH-28080)-sensitive H+-K+- ATPase (HKA) and bafilomycin-sensitive H+-ATPase. The SCH-28080-sensitive HKA is believed to be the gastric HKA (HKAg). Here we examined apical membrane potassium-dependent proton secretion in IC cells of wild-type HKAg (+/+) and HKAg knockout (−/−) mice to determine relative contribution of HKAg to luminal proton secretion. The results demonstrated that HKAg (−/−) and wild-type mice had comparable rates of potassium-dependent proton secretion, with HKAg (−/−) mice having 100% of K+-dependent H+ secretion vs. wild-type mice. Potassium-dependent proton secretion was resistant to ouabain and SCH-28080 in HKAg knockout mice but was sensitive to SCH-28080 in wild-type animals. Northern hybridizations did not demonstrate any upregulation of colonic HKA in HKAg knockout mice. These data indicate the presence of a previously unrecognized K+-dependent SCH-28080 and ouabain-insensitive proton secretory mechanism in the cortical collecting tubule that may play an important role in acid-base homeostasis.

Regulation of apical K channels in rat cortical collecting tubule during changes in dietary K intake

Long-term adaptation to a high-K diet is known to increase the density of conducting secretory K (SK) channels in the luminal membrane of the rat cortical collecting tubule (CCT). To examine whether these channels are involved in the short-term, day-to-day regulation of K secretion, we examined the density of K channels in animals fed a high-K diet for 6 or 48 h. CCTs were isolated and split open to provide access to the luminal membrane. Cell-attached patches were formed on principal cells with 140 mM KCl in the patch-clamp pipette. SK channels were recognized from their characteristic single-channel conductance (40–50 pS) and gating patterns. Animals fed a control diet had SK channel densities of 0.40 channels/μm2. When the diet was changed for one containing 10% KCl for 6 h, the channel density increased to 1.51 channels/μm2. Maintaining the animals on a high-K diet for 48 h resulted in a further increase in SK channels to 2.29 channels/μm2. Animals fed a low-K diet for 5 days or longer had SK densities of 0.53 channels/μm2, not significantly different from control values. The presence of conducting Na channels in the luminal membrane will also affect K secretion by the CCT by altering the electrical driving force through the K channels. The density of Na channels, measured with LiCl in the pipette, was 0.08 for controls and 1.00 and 1.08 channels/μm2 after 6 h and 48 h on a high-K diet. Plasma aldosterone increased from 15 ± 4 ng/dl (controls ) to 36 ± 8 and 98 ± 23 ng/dl after 6 and 48 h of K loading, respectively. The increase in K channel density could not be reproduced by infusion of the animals with aldosterone. We conclude that regulation of the density of conducting Na and K channels may contribute to day-to-day variation in the rate of renal K secretion and to the short-term maintenance of K balance.

Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct

Extracellular K+-dependent H+ extrusion after an acute acid load, an index of H/K exchange, was monitored in intercalated cells (ICs) from rat cortical collecting tubule (CCT) using ratiometric fluorescence imaging of the intracellular pH (pHi) indicator, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The hypothesis tested was that 12- to 14-day NaCl deprivation increases H-K-ATPase in rat ICs. The rate of H/K exchange in the low-NaCl ICs was double that of controls. In control ICs, H/K exchange was inhibited by Sch-28080 (10 μM). In the low-NaCl ICs, it was partially blocked by Sch-28080 or ouabain (1 mM). Simultaneous addition of both inhibitors abolished K-dependent pHirecovery. The induced H/K exchange observed with NaCl restriction was not due to elevated plasma aldosterone as evidenced by experiments on ICs from rats implanted with osmotic minipumps administering aldosterone such that plasma levels were comparable to those of NaCl-deficient rats. The results suggest that NaCl deficiency induces two isoforms of H-K-ATPase in ICs and that this effect is not mediated by elevated plasma aldosterone.

P2 purinoceptor localization along rat nephron and evidence suggesting existence of subtypes P2Y1 and P2Y2

Effects of extracellular ATP on intracellular free calcium concentration ([Ca2+]i) were examined in rat single nephron segments using the fura 2-AM. ATP (10 μM) induced a significant transient increase in [Ca2+]iin the glomerulus, the early proximal convoluted tubule (S1), the cortical collecting tubule (CCT), and the outer medullary collecting tubule (OMCT). The magnitude of the response was the greatest in the OMCT among four segments. ATP induced an increase in the [Ca2+]iin a dose-dependent manner in S1 and OMCT. In the OMCT, ATP caused a biphasic increase in [Ca2+]iconsisting of an initial rapid rise and a sustained phase. Removal of calcium from the medium resulted in an attenuation of the sustained phase of [Ca2+]iand an ∼30% reduction in the height of the initial [Ca2+]ipeak in response to 10 μM ATP. Effects of ATP, its analogs, and its metabolites were tested in the S1 and OMCT. ATP, 2-methylthio-ATP (2-MeS-ATP), ADP, and UTP increased [Ca2+]idose dependently. AMP and adenosine did not affect [Ca2+]iin the S1 and OMCT. The ATP- or 2-MeS-ATP-induced [Ca2+]iincrease was inhibited by the pretreatment of the S1 and OMCT with suramin or reactive blue 2. Neomycin, a phospholipase C inhibitor, attenuated the ATP-induced [Ca2+]iincrease. To investigate the hormonelike action of ATP in OMCT, a heterologous cross desensitization was performed. The pretreatment of OMCT with ATP inhibited increases in vasopressin-, ANG II-, endothelin-1-, or bradykinin-induced [Ca2+]iincrease. These findings suggest that ATP might affect the above peptidyl agonist-activated calcium mobilizations.

Dissociation of K channel density and ROMK mRNA in rat cortical collecting tubule during K adaptation

The density of conducting K channels in the apical membrane of the rat cortical collecting tubule (CCT) is increased by a high-K diet. To see whether this involved increased abundance of mRNA coding for K channel protein, we measured the relative amounts of mRNA for ROMK, the clone of the gene thought to encode the secretory K channel in the CCT. Tubules were isolated and fixed for in situ hybridization with a probe based on the ROMK sequence. Radiolabeled probe associated with the tubule was quantified using densitometric analysis of the autoradiographic images of the tubules. The densitometry signal was shown to be proportional to the amount of radioactive probe in the sample and to the time of exposure of the film. The technique was able to detect an approximately twofold increase in the abundance of mRNA coding for the water channel aquaporin 3 (AQP3), in response to a 30-h dehydration period. Tubules from rats fed a normal diet or a high-K (10% KCl) diet had equal amounts of ROMK mRNA. This suggests that an increase in the abundance of mRNA does not underlie the increase in channel density observed under these conditions.

Regulation of Na channels in the rat cortical collecting tubule: effects of cAMP and methyl donors

The whole cell patch-clamp technique was used to investigate the interactions of the amiloride-sensitive Na channel of the rat cortical collecting tubule (CCT) with adenosine 3',5'-cyclic monophosphate (cAMP) and with methyl donors. The amiloride-sensitive whole cell current (INa) was measured in principal cells of dissected, split-open tubules from rats maintained either on a control diet or on a low-Na diet to increase endogenous aldosterone secretion. With Na-depleted animals, INa was highest immediately after rupture of the membrane patch and averaged 325 pA at a membrane potential of -60 mV. INa declined over 15 min to approximately 35% of the initial value. With 8-(4-chlorophenylthio)-cAMP in the pipette, INa increased within 5 min of membrane rupture and was maintained for 15 min at levels three- to fourfold higher than the corresponding control values. With Na-replete animals, INa was undetectable (< 10 pA) without cAMP. With cAMP in the pipette, INa averaged 40 pA. In cell-attached patches on tubules from Na-replete rats exposed to cAMP, single Na channels were observed with conductive and kinetic properties similar to those from Na-depleted rats but at lower density. Inclusion of the methyl donor S-adenosyl methionine to the pipette solution did not increase INa in CCTs from Na-replete rats, either in the presence or absence of cAMP. The methylation inhibitor S-adenosyl homocysteine did not affect INa in CCT from Na-depleted animals.