Superoxide Anions Are Involved in Mediating the Effect of Low K Intake on c-Src Expression and Renal K Secretion in the Cortical Collecting Duct

Abstract

It is well known that K restriction suppresses renal K excretion (2). This is achieved, at least in part, by decreasing the apical K conductance in the cortical collecting duct (CCD) 1 and by stimulating K absorption in the outer medullary collecting duct (3, 4). However, the mechanism by which low K intake suppresses the apical K channels is not completely understood. We previously demonstrated that low K intake increased the expression of Src family protein tyrosine kinase (PTK) such as c-Src and c-Yes (1) and that inhibition of PTK increased the apical ROMK-like small conductance (SK) channels (1). This suggests that PTK is involved in mediating the effect of low K intake on the apical K channels and that increases in PTK activity and expression are important for suppression of renal K secretion during K depletion.Low K intake has been reported to increase the production of O 2. anion in rabbit carotid arteries (5). Moreover, it has beenshown that H 2 O 2 stimulates the phosphorylation of c-Jun in endothelial cells, an indication of activation of transcription factor (6). Therefore, it is possible that increases in O 2 . or related products induced by low K intake may be an upstream signal responsible for mediating the effect of low K intake on PTK expression and K secretion in the kidney. This hypothesis was tested in the present study by examining whether O 2 . and related products such as H 2 O 2 can mimic the effect of low K intake and stimulate the expression of PTK in the CCD. We also examined whether decreases in O 2 . and related products with tempol could attenuate the effect of low K intake on c-Src expression, ROMK channel activity, and renal K excretion. EXPERIMENTAL PROCEDURESAnimals-Sprague-Dawley rats (6 -8 weeks, either sex) were purchased from Taconic Farms (Germantown, NY). Rats were housed in metabolic cages for 7 days to study urinary K excretion. After 3 days of training in the cage, rats were divided into three groups: 1) control group in which animals were kept on a normal K (1.1%) diet and had a daily intraperitoneal injection of saline for 1 week; 2) the low K group in which rats were maintained on a K-deficient (KD) diet and received a daily intraperitoneal injection of saline for 7 days; and 3) the tempol-treated group in which rats were also fed with KD diet and had a daily intraperitoneal injection of tempol (15 mg/kg) for 1 week. Data regarding the 24-h food intake, body weight, and urine output were recorded. Urinary Na and K concentrations were measured by a flame photometer, and daily Na and K excretion were calculated as mEq/24 h. Animals were anesthetized with pentobarbital (60 mg/kg), and blood samples were drawn from the heart to measure the plasma K and Na concentrations. Rats were then killed, and the abdomens were opened to remove the kidneys.Tissue Preparation-The renal cortex and the outer medulla were separated under a dissecting microscope and suspended in radioimmune precipitation assay buffer solution (1:8 ratio, w/v) containing 1ϫ phosphate-buffered saline, 1% No...

Angiotensin II Inhibits the ROMK-like Small Conductance K Channel in Renal Cortical Collecting Duct during Dietary Potassium Restriction

“…The AngII-induced PTK-independent reduction of ROMK channel activity presumably reflects other PKC-associated effects on the channel, such as that regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (26). Of note was our observation that application of PMA to HA-treated CCDs failed to inhibit the ROMK channel (data not shown), suggesting that PTK is required for activation of PKC (27,28 anions, reduces expression of cSrc in CCDs, which increases ROMK channel activity and potassium excretion in LK rats (30). The immunodetection of components of NADPH oxidase in the CCD suggests that NADPH oxidase may be a source of intracellular ROS in this nephron segment (31).…”

Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

“…The observation that the addition of H 2 O 2 increased the expression of the P110␣-subunit suggests that increases in superoxide anions and related species are partially responsible for the stimulation of PI3K expression induced by low K intake. Also, we previously demonstrated that low K intake increases superoxide anion levels, which stimulate the expression of Src family PTK (2). Thus this suggests that increases in superoxide anion levels induced by low K intake are responsible for stimulation of expression of PI3K and PTK.…”

Inhibitor of growth 4 (ING4) is up-regulated by a low K intake and suppresses renal outer medullary K channels (ROMK) by MAPK stimulation

“…1B). Next, we explored the role of superoxide anions in mediating the effect of low K intake on the expression of ING4 because K restriction increases the level of superoxide in the renal tubules (4). We examined the effect of K restriction on ING4 expression in rats treated with tempol, an agent that decreases superoxide production in the kidney (4) and other tissues (13).…”

“…Low K intake has been shown to activate ERK, p38 MAPK, and Src family PTK, which inhibit ROMK channel activity (4,5). Therefore, we examined whether expression of ING4 stimulates the phosphorylation of p38 and ERK in Xenopus oocytes.…”

“…The inhibitory effect of low K intake on renal K secretion is partially achieved by removing renal outer medullary potassium (ROMK)-like small conductance K channels from the apical membrane of principal cells in the cortical collecting duct (CCD) through a protein-tyrosine kinase (PTK)-dependent mechanism (3). We have demonstrated previously that low K intake stimulates the production of superoxide anions and its related products, which in turn increase PTK expression and activity (4,5). Increased PTK activity stimulates tyrosine phosphorylation of ROMK channels and subsequently enhances the internalization of the ROMK channels (6,7).…”