Biallelic loss-of-function variants in KCNJ16 presenting with hypokalemic metabolic acidosis

KCNJ16 encodes Kir5.1 and acts in combination with Kir4.1, encoded by KCNJ10, to form an inwardly rectifying K+ channel expressed at the basolateral membrane of epithelial cells in the distal nephron. This Kir4.1/Kir5.1 channel is critical for controlling basolateral membrane potential and K+ recycling, the latter coupled to Na-K-ATPase activity, which determines renal Na+ handling. Previous work has shown that Kcnj16−/− mice and SSKcnj16−/− rats demonstrate hypokalemic, hyperchloremic metabolic acidosis. Here, we present the first report of a patient identified to have biallelic loss-of-function variants in KCNJ16 by whole exome sequencing who presented with chronic metabolic acidosis with exacerbations triggered by minor infections.

Direct inhibition of basolateral Kir4.1/5.1 and Kir4.1 channels in the cortical collecting duct by dopamine

It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about the molecular targets of dopamine actions on water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine, which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K+ channels in the distal renal tubule are critical for K+ recycling and controlling basolateral membrane potential to establish the driving force for Na+ reabsorption. Here, we demonstrate that Kir4.1 and Kir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCD. Using patch-clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40-pS and scarce 20-pS single channel conductances, most likely representing Kir4.1/5.1 and Kir4.1 channels, respectively. Dopamine reversibly decreased the open probability of both channels, with a relatively greater action on the Kir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. PKC blockade abolished the inhibition of basolateral K+ channels by dopamine. Importantly, dopamine significantly decreased the amplitude of Kir4.1/5.1 and Kir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential, as directly monitored using current-clamp mode in isolated CCDs. Therefore, we demonstrate that dopamine inhibits basolateral Kir4.1/5.1 and Kir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of the basolateral membrane and a decreased driving force for Na+ reabsorption in the distal renal tubule.

Dopamine stimulates salivary duct cells in the cockroach Periplaneta americana

This study examines whether the salivary duct cells of the cockroach Periplaneta americana can be stimulated by the neurotransmitters dopamine and serotonin. We have carried out digital Ca2+-imaging experiments using the Ca2+-sensitive dye fura-2 and conventional intracellular recordings from isolated salivary glands. Dopamine evokes a slow, almost tonic, and reversible dose-dependent elevation in [Ca2+]i in the duct cells. Upon stimulation with 10(−)6 mol l-1 dopamine, [Ca2+]i rises from 48+/−4 nmol l-1 to 311+/−43 nmol l-1 (mean +/− s.e.m., N=18) within 200–300 s. The dopamine-induced elevation in [Ca2+]i is absent in Ca2+-free saline and is blocked by 10(−)4 mol l-1 La3+, indicating that dopamine induces an influx of Ca2+ across the basolateral membrane of the duct cells. Stimulation with 10(−)6 mol l-1 dopamine causes the basolateral membrane to depolarize from −67+/−1 to −41+/−2 mV (N=10). This depolarization is also blocked by La3+ and is abolished when Na+ in the bath solution is reduced to 10 mmol l-1. Serotonin affects neither [Ca2+]i nor the basolateral membrane potential of the duct cells. These data indicate that the neurotransmitter dopamine, which has previously been shown to stimulate fluid secretion from the glands, also stimulates the salivary duct cells, suggesting that dopamine controls their most probable function, the modification of primary saliva.

Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells

We examined the mechanisms of cellular Na+ transport, both Cl− dependent and Cl− independent, in the mammalian esophageal epithelium. Rabbit esophageal epithelium was dissected from its muscular layers and mounted in a modified Ussing chamber for impalement with ion-selective microelectrodes. In bicarbonate Ringer, transepithelial potential difference was −14.9 ± 0.9 mV, the transepithelial resistance ( R TE) was 1,879 ± 142 Ω ⋅ cm2, the basolateral membrane potential difference ( V mBL) was −53 ± 1.5 mV, and the intracellular activity of Na+([Formula: see text]) was 24.6 ± 2.1 mM. Removal of Na+ and Cl− from the serosal and luminal baths decreased [Formula: see text] to 6.6 ± 0.6 mM. Readdition of Na+ to the serosal bath in the absence of Cl− increased[Formula: see text] by 21.8 ± 3.0 mM, whereas V mBL and R TE remained unchanged. When serosal Na+ was readded in the presence of amiloride the increase in[Formula: see text] and the rate of Na+ entry were decreased by ∼50%. 5-( N-ethyl- N-isopropyl)amiloride mimicked the effect of amiloride, whereas phenamil did not. Subsequent readdition of Cl− to the serosal bath further increased [Formula: see text] by 4.4 ± 1.9 mM. When the cells were acid loaded by pretreatment with[Formula: see text] in nominally[Formula: see text]-free Ringer, intracellular pH measurements showed a pHi recovery that is dependent on the presence of Na+ in the serosal bath and that can be blocked by amiloride. These data indicate that esophageal epithelial cells possess a Na+-dependent, amiloride-sensitive electroneutral mechanism for Na+entry consistent with the presence of a basolateral Na+/H+ exchanger. The ability of Cl− to further enhance Na+ entry supports the existence of at least one additional Cl−-dependent component of basolateral Na+entry.

Effect of norepinephrine on intracellular pH in kidney proximal tubule: role of Na+-( HCO 3 − ) n cotransport

We examined the effect of norepinephrine (NE) on intracellular pH (pHi) and activity of Na+([Formula: see text]) in the isolated perfused kidney proximal tubule of Ambystoma, using single-barreled voltage and ion-selective microelectrodes. In control[Formula: see text] Ringer, addition of 10−6 M NE to the bath reversibly depolarized the basolateral membrane potential ( V 1), the luminal membrane potential ( V 2), and the transepithelial potential difference ( V 3) and increased pHi by 0.14 ± 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of CO2/[Formula: see text]. Removal of bath Na+ depolarized V 1 and V 2, hyperpolarized V 3, and decreased pHi. These effects are largely mediated by the electrogenic Na+-([Formula: see text]) n cotransporter. In the presence of NE, the effects of Na+ removal on membrane potential differences and the rate of change of pHi were significantly smaller. Reducing bath [Formula: see text] concentration from 10 to 2 mM at constant CO2 (pH 6.8) depolarized V 1 and V 2, decreased pHi, and lowered[Formula: see text]. These changes are also due to Na+-([Formula: see text]) n . In the presence of NE, reducing bath [[Formula: see text]] caused a smaller depolarizations of V 1 and V 2, and the rate of pHi decrease was significantly reduced. Our results indicate: 1) NE causes an increase in pHi; 2) the NE-induced alkalinization is mediated by a SITS-sensitive and[Formula: see text]-dependent transporter on the basolateral membrane; and 3) in the presence of NE, the reduced effects caused by basolateral[Formula: see text] changes or Na+ removal are indicative of an inhibitory effect of NE on Na+-([Formula: see text]) n cotransport.

Relation of membrane potential to basolateral TEA transport in isolated snake proximal renal tubules

We measured the effects of changes in bath K+ concentration ([K+]) on basolateral membrane potential difference (PD) and [3H]tetraethylammonium (TEA) transport in isolated snake (Thamnophis) proximal renal tubules (25 degrees C; pH 7.4). Increasing bath [K+] from 3 to 65 mM decreased PD from -60 mV (inside of cells negative) to -20 mV and 2-min uptake of [3H]TEA by approximately 25%, indicating that PD influences TEA entry into the cells. Uptake of [3H]TEA was inhibited similarly at both K+ concentrations by unlabeled TEA, indicating that uptake is carrier mediated. Kt (approximately 18 microM) for 2-min uptake of [3H]TEA in 3 mM K+ increased significantly in 65 mM K+, suggesting that the decrease in PD or the increase in [K+] alters the affinity of the transporter for TEA. The steady-state cell-to-bath ratio for [3H]TEA with 3 mM K+ (-60 mV PD) was approximately 16, significantly above the ratio of 10 predicted for passive distribution at electrochemical equilibrium. With 65 mM K+ (-20 mV PD) this ratio decreased to approximately 6, again significantly above the predicted ratio of 2. These data suggest that the PD can account for much, but not all, of the steady-state uptake of TEA. Efflux of [3H]TEA across the basolateral membrane was identical with either 3 or 65 mM K+ in the bath but was almost completely inhibited in either case by tetrapentylammonium, a potent inhibitor of TEA uptake. These data indicate that virtually all TEA transport across the basolateral membrane is carrier mediated and that transport out of the cells is unaffected by PD.

Adaptive responses to Na(+)-coupled solute transport and osmotic cell swelling in salamander proximal tubule

Measurements of basolateral membrane potential and relative K+ conductance were performed in isolated perfused proximal tubules from Ambystoma. To investigate adaptive increases in basolateral membrane K+ conductance (gK) associated with Na(+)-solute cotransport, measurements were made comparing transport of glucose and alanine, with changes caused by hypotonicity- and solute-induced cell swelling. Luminal perfusion with alanine produced results consistent with an adaptive increase in gK; perfusion with glucose failed to show this response. Hypotonic peritubular solutions also produced results consistent with an adaptive increase in gK, but isosmotic increases of peritubular glucose sufficient to swell the cells failed to produce this. No changes in the responses to luminal perfusion with alanine or glucose were induced by hypotonic peritubular solutions. With a high concentration of glucose in isosmotic peritubular solutions, perfusion of the lumen with glucose now produced results consistent with an adaptive increase in gK. Isosmotic peritubular solutions containing urea produced adaptive changes similar to those observed using hypotonic peritubular solutions, but when glucose was subsequently added to the lumen, no further adaptive response occurred. We conclude that cell swelling alone is insufficient to explain the mechanisms involved in the adaptive responses of gK occurring during Na(+)-solute cotransport in the salamander proximal tubule.