Ryan J. Cornelius

Coupled ATP and potassium efflux from intercalated cells.

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (τ; 10 dynes/cm(2)) -induced, BK-dependent, K efflux. We found that 10 μM ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P(2) receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited τ-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-β4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and τ-induced K efflux. Similarly, carbenoxolone (25 μM), which blocks connexins, putative ATP pathways, blocked τ-induced K efflux and ATP secretion. Compared with BK-β4(-/-) mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for τ-induced ATP release in IC.

Regulation of BK-α expression in the distal nephron by aldosterone and urine pH

In the distal nephron, the large-conductance Ca-activated K (BK) channel, comprised of a pore-forming-α (BK-α) and the BK-β4 subunit, promotes K excretion when mice are maintained on a high-K alkaline diet (HK-alk). We examined whether BK-β4 and the acid-base status regulate apical membrane expression of BK-α in the cortical (CCD) and medullary collecting ducts (MCD) using immunohistochemical analysis (IHC) and Western blot. With the use of IHC, BK-α of mice on acontrol diet localized mostly cytoplasmically in intercalated cells (IC) of the CCD and in the perinuclear region of both principle cells (PC) and IC of the MCD. HK-alk wild-type mice (WT), but not BK-β4 knockout mice (β4KO), exhibited increased apical BK-α in both the CCD and MCD. When given a high-K acidic diet (HK-Cl), BK-α expression increased but remained cytoplasmic in the CCD and perinuclear in the MCD of both WT and β4KO. Western blot confirmed that total BK-α expression was enhanced by either HK-alk or HK-Cl but only increased in the plasma membrane with HK-alk. Compared with controls, mice drinking NaHCO3 water exhibited more apical BK-α and total cellular BK-β4. Spironolactone given to mice on HK-alk significantly reduced K secretion and decreased total cellular BK-α but did not affect cellular BK-β4 and apical BK-α. Experiments with MDCK-C11 cells indicated that BK-β4 stabilizes surface BK-α by inhibiting degradation through a lysosomal pathway. These data suggest that aldosterone mediates a high-K-induced increase in BK-α and urinary alkalinization increases BK-β4 expression, which promotes the apical localization of BK-α.

Bicarbonate promotes BK-α/β4-mediated K excretion in the renal distal nephron

Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high-K conditions to remove excess K. Because BK predominantly reside with BK-β4 in acid/base-transporting intercalated cells (IC), we determined whether BK-β4 knockout mice (β4KO) exhibit deficient K excretion when consuming a high-K alkaline diet (HK-alk) vs. high-K chloride diet (HK-Cl). When wild type (WT) were placed on HK-alk, but not HK-Cl, renal BK-β4 expression increased (Western blot). When WT and β4KO were placed on HK-Cl, plasma K concentration ([K]) was elevated compared with control K diets; however, K excretion was not different between WT and β4KO. When HK-alk was consumed, the plasma [K] was lower and K clearance was greater in WT compared with β4KO. The urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and β4KO. Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in β-IC, comparing WT and β4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in β4KO, compared with WT, on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice with an alkaline, Na-deficient, high-K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K handling of β4KO. When WT on LNaHK were given NH(4)Cl in the drinking water, K excretion was reduced to the magnitude of β4KO on LNaHK. These results show that WT, but not β4KO, efficiently excretes K on HK-alk but not on HK-Cl and suggest that BK-α/β4-mediated K secretion is promoted by bicarbonaturia.

Interacting influence of diuretics and diet on BK channel-regulated K homeostasis

Large conductance, Ca-activated K channels (BK) are abundantly located in cells of vasculature, glomerulus, and distal nephron, where they are involved in maintaining blood volume, blood pressure, and K homeostasis. In mesangial cells and smooth muscle cells of vessels, the BK-α pore associates with BK-β1 subunits and regulates contraction in a Ca-mediated feedback manner. The BK-β1 also resides in connecting tubule cells of the nephron. BK-β1 knockout mice (β1KO) exhibit fluid retention, hypertension, and compromised K handling. The BK-α/β4 resides in acid/base transporting intercalated cells (IC) of the distal nephron, where they mediate K secretion in mammals on a high K, alkaline diet. BK-α expression in IC is increased by a high K diet via aldosterone. The BK-β4 subunit and alkaline urine are necessary for the luminal expression and function of BK-α in mouse IC. In distal nephron cells, membrane BK-α expression is inhibited by WNK4 in in vitro expression systems, indicating a role in the hyperkalemic phenotype in patients with familial hyperkalemic hypertension type 2 (FHHt2). β1KO and BK-β4 knockout mice (β4KO) are hypertensive because of exaggerated epithelial Na channels (ENaC) mediated Na retention in an effort to secrete K via only renal outer medullary K channels (ROMK). BK hypertension is resistant to thiazides and furosemide, and would be more amenable to ENaC and aldosterone inhibiting drugs. Activators of BK-α/β1 or BK-α/β4 might be effective blood pressure lowering agents for a subset of hypertensive patients. Inhibitors of renal BK would effectively spare K in patients with Bartter Syndrome, a renal K wasting disease.

Low Na, High K Diet and the Role of Aldosterone in BK-Mediated K Excretion

A low Na, high K diet (LNaHK) is associated with a low rate of cardiovascular (CV) disease in many societies. Part of the benefit of LNaHK relies on its diuretic effects; however, the role of aldosterone (aldo) in the diuresis is not understood. LNaHK mice exhibit an increase in renal K secretion that is dependent on the large, Ca-activated K channel, (BK-α with accessory BK-β4; BK-α/β4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/β4-mediated K secretion in LNaHK mice. We found that the plasma aldo concentration (P[aldo]) was elevated by 10-fold in LNaHK mice compared with control diet (Con) mice. We subjected LNaHK mice to either sham surgery (sham), adrenalectomy (ADX) with low aldo replacement (ADX-LA), or ADX with high aldo replacement (ADX-HA). Compared to sham, the urinary flow, K excretion rate, transtubular K gradient (TTKG), and BK-α and BK-β4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-β4 knockout (β4KO) and WT mice exhibited similar K clearance and TTKG in the ADX-LA groups; however, in sham and ADX-HA, the K clearance and TTKG of β4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in β4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/β4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease.

Increased Epithelial Sodium Channel Activity Contributes to Hypertension Caused by Na+-HCO3− Cotransporter Electrogenic 2 Deficiency

The gene SLC4A5 encodes the Na+-HCO3− cotransporter electrogenic 2, which is located in the distal nephron. Genetically deleting Na+-HCO3− cotransporter electrogenic 2 (knockout) causes Na+-retention and hypertension, a phenotype that is diminished with alkali loading. We performed experiments with acid-loaded mice and determined whether overactive epithelial Na+ channels (ENaC) or the Na+-Cl− cotransporter causes the Na+ retention and hypertension in knockout. In untreated mice, the mean arterial pressure was higher in knockout, compared with wild-type (WT); however, treatment with amiloride, a blocker of ENaC, abolished this difference. In contrast, hydrochlorothiazide, an inhibitor of Na+-Cl− cotransporter, decreased mean arterial pressure in WT, but not knockout. Western blots showed that quantity of plasmalemmal full-length ENaC-&agr; was significantly higher in knockout than in WT. Amiloride treatment caused a 2-fold greater increase in Na+ excretion in knockout, compared with WT. In knockout, but not WT, amiloride treatment decreased plasma [Na+] and urinary K+ excretion, but increased hematocrit and plasma [K+] significantly. Micropuncture with microelectrodes showed that the [K+] was significantly higher and the transepithelial potential (Vte) was significantly lower in the late distal tubule of the knockout compared with WT. The reduced Vte in knockout was amiloride sensitive and therefore revealed an upregulation of electrogenic ENaC-mediated Na+ reabsorption in this segment. These results show that, in the absence of Na+-HCO3− cotransporter electrogenic 2 in the late distal tubule, acid-loaded mice exhibit disinhibition of ENaC-mediated Na+ reabsorption, which results in Na+ retention, K+ wasting, and hypertension.The gene SLC4A5 encodes the Na+-HCO3− co-transporter electrogenic 2 (NBCe2), which is located in the distal nephron. Genetically deleting NBCe2 (KO) causes Na+-retention and hypertension, a phenotype that is diminished with alkali loading. We performed experiments with acid-loaded mice and determined whether over-active epithelial Na+ channels (ENaC) or the Na+-Cl− co-transporter (NCC) causes the Na+ retention and hypertension in KO. In untreated mice, the mean arterial pressure (MAP) was higher in KO, compared with wild type (WT); however, treatment with amiloride, a blocker of ENaC, abolished this difference. In contrast, hydrochlorothiazide (HCTZ), an inhibitor of NCC, decreased MAP in WT, but not KO. Western blots showed that quantity of plasmalemmal full-length ENaC-α was significant higher in KO than in WT. Amiloride treatment caused a 2-fold greater increase in Na+ excretion in KO, compared with WT. In KO, but not WT, amiloride treatment decreased plasma [Na+] and urinary K+ excretion, but increased hematocrit and plasma [K+] significantly. Micropuncture with microelectrodes showed that the [K+] was significantly higher and the transepithelial potential (Vte) was significantly lower in the late distal tubule (LDT) of the KO compared with WT. The reduced Vte in KO was amiloride-sensitive and therefore revealed an upregulation of electrogenic ENaC-mediated Na+ reabsorption in this segment. These results show that, in the absence of NBCe2 in the LDT, acid-loaded mice exhibit disinhibition of ENaC-mediated Na+ reabsorption, which results in Na+ retention, K+ wasting, and hypertension.

Deficient acid handling with distal RTA in the NBCe2 knockout mouse

In many circumstances, the pathogenesis of distal renal tubular acidosis (dRTA) is not understood. In the present study, we report that a mouse model lacking the electrogenic Na(+)-HCO3 (-) cotransporter [NBCe2/Slc4a5; NBCe2 knockout (KO) mice] developed dRTA after an oral acid challenge. NBCe2 expression was identified in the connecting tubule (CNT) of wild-type mice, and its expression was significantly increased after acid loading. NBCe2 KO mice did not have dRTA when on a standard mouse diet. However, after acid loading, NBCe2 KO mice exhibited complete features of dRTA, characterized by insufficient urinary acidification, hyperchloremic hypokalemic metabolic acidosis, and hypercalciuria. Additional experiments showed that NBCe2 KO mice had decreased luminal transepithelial potential in the CNT, as revealed by micropuncture. Further immunofluorescence and Western blot experiments found that NBCe2 KO mice had increased expression of H(+)-ATPase B1 in the plasma membrane. These results showed that NBCe2 KO mice with acid loading developed increased urinary K(+) and Ca(2+) wasting due to decreased luminal transepithelial potential in the CNT. NBCe2 KO mice compensated to maintain systemic pH by increasing H(+)-ATPase in the plasma membrane. Therefore, defects in NBCe2 can cause dRTA, and NBCe2 has an important role to regulate urinary acidification and the transport of K(+) and Ca(2+) in the distal nephron.

With no lysine kinase 4 modulates sodium potassium 2 chloride cotransporter activity in vivo

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4-/- mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4-/- mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4-/- mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4-/- mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.

Dual gain and loss of cullin 3 function mediates familial hyperkalemic hypertension

Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403-459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403-459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/β1 domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403-459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403-459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.

BK Channels in Epithelia

Large, Ca2+-activated K+ channels (BK) are comprised of the pore-forming α subunit (BK-α) and one of four β subunits (β1–4), which bestows functional diversity. BK channels are localized in the distal nephron of kidneys and a variety of extrarenal, fluid-secreting epithelia. In the kidneys, flow activates BK, which are located in two cell types of the distal nephron. The BK-α/β1 resides in the connecting tubule cells, and the BK-α/β4 is localized in intercalated cells, where they mediate K+ secretion in mice on a high K+, alkaline diet.