Proximal renal tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for stabilizing bicarbonate transport (original) (raw)
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The Journal of General Physiology, 2003
Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wildtype and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using  -galactosidase staining. TASK2 was only localized in PCT cells. K ϩ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K ϩ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 M 293B, but blocked by 500 M quinidine and 10 M clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K ϩ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K ϩ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.
P. Extracellular pH alkalinization by Cl Ϫ /HCO 3 Ϫ exchanger is crucial for TASK2 activation by hypotonic shock in proximal cell lines from mouse kidney. We have previously shown that K ϩ -selective TASK2 channels and swelling-activated Cl Ϫ currents are involved in a regulatory volume decrease (RVD; Barriere H, Am J Physiol Renal Physiol 284: F812-F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl Ϫ and K ϩ currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K ϩ currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pH i) and increased the external pH (pH e) in wild-type but not in cftr Ϫ/Ϫ cells. The inhibitory effect of DIDS suggests that the acidification of pH i and the alkalinization of pHe induced by hypotonicity in wild-type cells could be due to an exit of HCO 3
Cloning, renal distribution, and regulation of the rat Na+-HCO3- cotransporter
The American journal of physiology, 1998
We recently reported the cloning and expression of a human kidney Na+-HCO3- cotransporter (NBC-1) (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111-19114, 1997). To expedite in vivo experimentation, we now report the cDNA sequence of rat kidney NBC-1. In addition, we describe both the organ and nephron segment distributions and the regulation of NBC-1 mRNA under three models of pH stress: chloride-depletion alkalosis (CDA), metabolic acidosis, and bicarbonate loading. Rat NBC-1 cDNA encodes an open reading frame of 1,035 amino acids, with 96 and 87% identity to human and salamander NBC-1, respectively. Rat NBC-1 mRNA is expressed at high levels in kidney and brain, with lower levels in colon, stomach, and heart. None appears in liver. In the kidney, NBC-1 is expressed mainly in the proximal tubule, with traces found in medullary thick ascending limb and papilla. HCO3- loading decreased NBC-1 mRNA levels, which were unchanged either by metabo...
Proximal Tubule Function and Response to Acidosis
Clinical Journal of the American Society of Nephrology, 2013
The human kidneys produce approximately 160-170 L of ultrafiltrate per day. The proximal tubule contributes to fluid, electrolyte, and nutrient homeostasis by reabsorbing approximately 60%-70% of the water and NaCl, a greater proportion of the NaHCO 3 , and nearly all of the nutrients in the ultrafiltrate. The proximal tubule is also the site of active solute secretion, hormone production, and many of the metabolic functions of the kidney. This review discusses the transport of NaCl, NaHCO 3 , glucose, amino acids, and two clinically important anions, citrate and phosphate. NaCl and the accompanying water are reabsorbed in an isotonic fashion. The energy that drives this process is generated largely by the basolateral Na 1 /K 1-ATPase, which creates an inward negative membrane potential and Na 1-gradient. Various Na 1-dependent countertransporters and cotransporters use the energy of this gradient to promote the uptake of HCO 3 2 and various solutes, respectively. A Na 1-dependent cotransporter mediates the movement of HCO 3 2 across the basolateral membrane, whereas various Na 1independent passive transporters accomplish the export of various other solutes. To illustrate its homeostatic feat, the proximal tubule alters its metabolism and transport properties in response to metabolic acidosis. The uptake and catabolism of glutamine and citrate are increased during acidosis, whereas the recovery of phosphate from the ultrafiltrate is decreased. The increased catabolism of glutamine results in increased ammoniagenesis and gluconeogenesis. Excretion of the resulting ammonium ions facilitates the excretion of acid, whereas the combined pathways accomplish the net production of HCO 3 2 ions that are added to the plasma to partially restore acid-base balance.
Similar chloride channels in the connecting tubule and cortical collecting duct of the mouse kidney
AJP: Renal Physiology, 2006
Using the patch-clamp technique, we investigated chloride channels on the basolateral membrane of the connecting tubule (CNT) and cortical collecting duct (CCD). We found a ~10-pS channel in CNT cell-attached patches. Replacing NaCl by Na-gluconate in the pipette shifted the reversal potential by +25 mV, whereas NMDG-chloride had no effect, indicating anion selectivity. On inside-out patches, we determined a selectivity sequence of Cl -> Br -~ NO 3 -> F, which is compatible with that of ClC -K2, a ClC chloride channel present in the distal nephron. In addition, the NPo measured in cell-attached patches was significantly increased when the calcium concentration or the pH in the pipette was increased, which is another characteristic of the ClC -K. These findings suggest that this channel is underlain by ClC -K2. A similar chloride channel was found in CCD patches. Since CNT and CCD are heterogeneous tissues, we studied the cellular distribution of the chloride channel using recording conditions ( KCl-rich solution in the pipette) that allowed us to detect simultaneously chloride channels and inwardly-rectifying potassium channels. We detected chloride channels alone in 45 and 42%, and potassium channels alone in 51 and 58% of CNT and CCD patches respectively. Chloride and potassium channels were recorded simultaneously from two patches (4% of patches) in the CNT and from no patch in the CCD.
Properties and Regulation of Organic Cation Transport in Freshly Isolated Human Proximal Tubules
Journal of Biological Chemistry, 2001
The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP ؉). Depolarization of the cells by rising extracellular K ؉ concentration to 145 mM reduced ASP ؉ uptake by 20 ؎ 5% (n ؍ 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K i ؍ 63 M) and cimetidine (K i ؍ 11 M) as well as the inhibitor quinine (K i ؍ 2.9 M) reduced ASP ؉ uptake concentration dependently. Maximal inhibition reached with these substances was ϳ60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 M) or ATP (100 M) inhibited ASP ؉ uptake by 30 ؎ 3 (n ؍ 16) and 38 ؎ 13% (n ؍ 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 M, n ؍ 7). Activation of adenylate cyclase by forskolin (1 M) decreased ASP ؉ uptake by 29 ؎ 3% (n ؍ 10). hANP (10 nM) or 8-bromo-cGMP (100 M) also decreased ASP ؉ uptake by 17 ؎ 3 (n ؍ 9) or 32 ؎ 5% (n ؍ 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP-and the cGMP-dependent protein kinases.
Growth, immortalization, and differentiation potential of normal adult human proximal tubule cells
In Vitro Cellular and Developmental Biology--Animal, 2004
Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase-polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K- adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide- and adenosine triphosphate-regulated mechanisms of ion transport. Transcripts for Na(+)-bicarbonate cotransporter, Na(+)-H(+) exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.
Cloning and Expression of a Novel pH-sensitive Two Pore Domain K+ Channel from Human Kidney
Journal of Biological Chemistry, 1998
A complementary DNA encoding a novel K ؉ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K ؉ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18 -22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K ؉ currents with activation potential thresholds that closely follow the K ؉ equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K ؉ concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mM K ؉ . TASK-2 currents are blocked by quinine (IC 50 ؍ 22 M) and quinidine (65% of inhibition at 100 M) but not by the other classical K ؉ channel blockers tetraethylammonium, 4-aminopyridine, and Cs ؉ . They are only slightly sensitive to Ba 2؉ , with less than 17% of inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6.5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K ؉ transport. The abbreviations used are: TMS, transmembrane segments; 2P, two pore-forming; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; pBS, pBluescriptII SKϪ; pS, picosiemens.