Regulation of the Cl−/HCO3 − Exchanger AE2 in Rat Thick Ascending Limb of Henle’s Loop in Response to Changes in Acid-Base and Sodium Balance

Abstract

Abstract. The Cl Ϫ /HCO 3 Ϫ exchanger AE2 is believed to be involved in transcellular bicarbonate reabsorption that occurs in the thick ascending limb of Henle's loop (TAL). The purpose of this study was to test whether chronic changes in acid-base status and sodium intake regulate AE2 polypeptide abundance in the TAL of the rat. Rats were subjected to 6 d of loading with NaCl, NH 4 Cl, NaHCO 3 , KCl, or KHCO 3 . AE2 protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex and the outer medulla of treated and control rats. In the renal cortex, AE2 abundance was markedly increased in response to oral loading with NH 4 Cl or with NaCl. In contrast, AE2 abundance was unchanged in response to loading with KCl or with NaHCO 3 and was decreased by loading with KHCO 3 . The response of AE2 in the outer medulla differed from that in the cortex in that HCO 3 Ϫ loading increased AE2 abundance when administered with Na ϩ but had no effect when administered with K ϩ . Immunohistochemistry revealed that NaCl loading increased AE2 abundance in the basolateral membrane of both the cortical and the medullary TAL. In contrast, NH 4 Cl loading increased AE2 abundance only in the cortical TAL but not in the medullary TAL. These results suggest that regulation of the basolateral Cl Ϫ /HCO 3 Ϫ exchanger AE2 plays an important role in the adaptation of bicarbonate absorption in the TAL during chronic acid-base disturbances and high sodium intake. The present study also emphasizes the contribution of cortical TAL adaptation in the renal regulation of acid-base status.The kidney controls systemic acid-base balance through the coordinated regulation of multiple transport processes along the length of the nephron that together allow adaptation of HCO 3 Ϫ reabsorption [for review, see ref (1)]. Eighty-five percent of filtered bicarbonate is reabsorbed by the proximal tubule (PT), and the thick ascending limbs reabsorb the remaining 15%. In response to metabolic acidosis, HCO 3 Ϫ reabsorption by PT cells is stimulated by an increase in expression and activity of the apical Na ϩ /H ϩ exchanger NHE3, in conjunction with increased activity of the basolateral Na ϩ -(HCO 3 Ϫ ) n co-transporter kNBCe1 (2-4). Conversely, chronic metabolic alkalosis leads to the opposite regulation (5,6). Although bicarbonate absorption occurs along the entire course of the thick ascending limb (TAL), its regulation has been studied most extensively in the medullary part of the TAL (MTAL). As in PT, chronic metabolic acidosis is associated with adaptive increases in MTAL bicarbonate reabsorption and enhanced apical NHE3 activity and expression (2,7,8). The MTAL response to chronic metabolic alkalosis depends on the experimental model. In rats with chronic chloride depletion metabolic alkalosis with unaltered sodium intake, the HCO 3 Ϫ absorptive capacity of the MTAL is reduced (9). However, in rats with chronic metabolic alkalosis induced by chronic ingestion of NaHCO 3 , the HCO 3 Ϫ absorptive capacit...

Amelogenins as Potential Buffers during Secretory-stage Amelogenesis

“…In Xenopus oocytes transfected with Ae2 (Humphreys et al 1994) and in mouse LS2 ameloblast-like cell line cultures, Ae2 expression is sensitive for and responds to intraand extracellular pH changes with a maximum expression at pH 6.8 (Paine et al 2008). In vivo chronic metabolic acidosis enhances Ae2 protein levels 6-fold in the renal cortex of rats, while metabolic alkalosis reduces Ae2 protein levels by 50% (Quentin et al 2004). Alternatively, the upregulation of Ae2 in AmelX -/-mice could be explained by the absence of amelogenins per se, which could change the production of other proteins.…”

Exposure to Maternal Diabetes Induces Salt-Sensitive Hypertension and Impairs Renal Function in Adult Rat Offspring

“…Kidneys of both groups were removed from the anesthetized rats and cut into 5-mm slices. To identify Na transporters, membrane fractions from the cortex and the outer medulla (inner stripe) were prepared, and Western blot analysis was performed as previously described (20,21) (primary antibodies used were anti-NHE3, 1:1,000; anti-NCC, 1:50,000; anti-BSC1, 1:5,000; anti-␣ ENaC, 1:3,000; anti-␤ENaC, 1:20,000; anti-␥ ENaC, 1:2,000; and anti-Na ϩ /K ϩ ATPase, 1:20,000). After incubation with the appropriate peroxidase-conjugated secondary antibodies, blots were washed, and luminol-enhanced chemiluminescence (ECL; Perkin Elmer Life Science Products, Boston, MA) was used to visualize bound antibodies before exposure to Hyperfilm ECL (Amersham, Arlington Heights, IL).…”

Regulation of WNK1 Expression by miR-192 and Aldosterone

“…Immunoblotting procedures was performed as described previously 45 using an antibody directed against the N-terminal domain of L-WNK1 (Novus Biological Ltd., 1:500). Coomassie blue-stained polyacrylamide gels were used to control equality of protein loading for each series.…”