High urinary flow rates stimulate K secretion in the fully differentiated but not neonatal or weanling rabbit cortical collecting duct (CCD). Both small-conductance secretory K and high-conductance Ca2+/stretch-activated maxi-K channels have been identified in the apical membrane of the mature CCD by patch-clamp analysis. We reported that flow-stimulated net K secretion in the adult rabbit CCD is 1) blocked by TEA and charybdotoxin, inhibitors of intermediate- and high-conductance (maxi-K) Ca2+-activated K channels, and 2) associated with increases in net Na absorption and intracellular Ca2+ concentration ([Ca2+]i). The present study examined whether the absence of flow-stimulated K secretion early in life is due to a 1) limited flow-induced rise in net Na absorption and/or [Ca2+]i and/or 2) paucity of apical maxi-K channels. An approximately sixfold increase in tubular fluid flow rate in CCDs isolated from 4-wk-old rabbits and microperfused in vitro led to an increase in net Na absorption and [Ca2+]i, similar in magnitude to the response observed in 6-wk-old tubules, but it failed to generate an increase in net K secretion. By 5 wk of age, there was a small, but significant, flow-stimulated rise in net K secretion that increased further by 6 wk of life. Luminal perfusion with iberiotoxin blocked the flow stimulation of net K secretion in the adult CCD, confirming the identity of the maxi-K channel in this response. Maxi-K channel α-subunit message was consistently detected in single CCDs from animals ≥4 wk of age by RT-PCR. Indirect immunofluorescence microscopy using antibodies directed against the α-subunit revealed apical labeling of intercalated cells in cryosections from animals ≥5 wk of age; principal cell labeling was generally intracellular and punctate. We speculate that the postnatal appearance of flow-dependent K secretion is determined by the transcriptional/translational regulation of expression of maxi-K channels. Furthermore, our studies suggest a novel function for intercalated cells in mediating flow-stimulated K secretion.

Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngIIinduced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

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Both membrane-bound carbonic anhydrase (CA) (isozyme type IV) and cytosolic CA (type II) activities enhance urinary acidification. We have previously shown that chronic metabolic acidosis (CMA) accomplished by NH4Cl loading with food restriction induces soluble CA activity in rabbit renal cortical homogenates. The present study was designed to assess the effect of CMA on the activity of CA isozymes in cortical and outer medullary homogenates, as well as in major proton-secreting segments of the kidney. Segments were microdissected from proximal convoluted tubules (PCT) proximal straight tubules, cortical collecting ducts, and outer medullary collecting ducts (OMCD). Total CA activity was measured by a colorimetric endpoint method, and CA IV activity was assessed from the sodium dodecyl sulfate-resistant hydratase activity. In controls, CA IV activity accounted for 3% of total CA activity in tissue homogenates. CMA induced a threefold increase in CA IV activity in cortical homogenates, in the absence of renal or tubular hypertrophy. In the PCT, CMA induced a 78% increase in total CA activity, which comprised a 178% increase in CA IV activity, and a 58% increase in CA II activity. In the OMCD, CMA induced a 53% increase in total CA (probably CA II) activity. We conclude that CMA induces CA activity in the PCT (CA II and CA IV) and the OMCD (most likely CA II) of adult rabbit kidneys. The induction of CA activity accompanies the increase in urinary acidification observed in CMA.

TR, Satlin LM. Role of NKCC in BK channel-mediated net K ϩ secretion in the CCD. Am J Physiol Renal Physiol 301: F1088 -F1097, 2011. First published August 3, 2011 doi:10.1152/ajprenal.00347.2011.-Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (JK) and Na absorption (JNa) at slow (ϳ1) and fast (ϳ5 nl·min Ϫ1 ·mm Ϫ1 ) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal JK, JNa, or the flow-induced [Ca 2ϩ ]i transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce JK. Basolateral Cl removal reversibly inhibited FIKS but not basal JK or JNa. Quantitative PCR performed on single CCD samples using NKCC1-and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH4 ϩ at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH4 ϩ addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH4 ϩ entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD. apical membrane voltage; tubular flow rates; bumetanide; intercalated cell; principal cell