Furosemide reduces BK-αß4-mediated K+ secretion in mice on an alkaline high-K+ diet.

Special high-K diets have cardioprotective effects and are often warranted in conjunction with diuretics such as furosemide for treating hypertension. However, it is not understood how a high-K diet (HK) influences the actions of diuretics on renal K+ handling. Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. We previously found that an alkaline urine is required for large conductance Ca2+-activated K+ (BK)-αß4-mediated K+ secretion in mice on HK. We therefore hypothesized that furosemide could reduce BK-αß4-mediated K+ secretion by acidifying the urine. Treating with furosemide (drinking water) for 11 days led to decreased urine pH in both wild-type (WT) and BK-ß4-knockout mice (BK-ß4-KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-ß4-KO. Western blotting and immunofluorescence staining showed that furosemide treatment decreased cortical expression of BK-ß4 and reduced apical localization of BK-α in connecting tubules. Addition of the carbonic anhydrase inhibitor, acetazolamide, to furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to control levels. Acetazolamide plus furosemide also restored the cortical expression of BK-ß4 and BK-α in connecting tubules. These results indicate that in mice adapted to HK, furosemide reduces BK-αß4-mediated K+ secretion by acidifying the urine.

Net K+ secretion in the thick ascending limb of mice on a low-Na, high-K diet.

Because of its cardio-protective effects, a low-Na, high-K diet (LNaHK) is often warranted in conjunction with diuretics to treat hypertensive patients. However, it is necessary to understand the renal handling of such diets in order to choose the best diuretic. Wild-type (WT) or Renal Outer Medullary K channel (ROMK) knockout mice (KO) were given a regular (CTRL), LNaHK, or high-K diet (HK) for 4-7 days. On LNaHK, mice treated with either IP furosemide for 12 hrs, or given furosemide in drinking water for 7 days, exhibited decreased K clearance. We used free-flow micropuncture to measure the [K+] in the early distal tubule (EDT [K+]) before and after furosemide treatment. Furosemide increased the EDT [K+] in WT on CTRL but decreased that in WT on LNaHK. Furosemide did not affect the EDT [K+] of KO on LNaHK or WT on HK. Furosemide-sensitive Na+ excretion was significantly greater in mice on LNaHK than those on CTRL or HK. Patch clamp analysis of split-open TALs revealed that 70-pS ROMK exhibited a higher open probability (Po) but similar density in mice on LNaHK, compared with CTRL. No difference was found in the density or Po of the 30 pS K channels between the two groups. These results indicate mice on LNaHK exhibited furosemide-sensitive net K+ secretion in the TAL that is dependent on increased NKCC2 activity and mediated by ROMK. We conclude that furosemide is a K-sparing diuretic by decreasing the TAL net K+ secretion in subjects on LNaHK.

Maintaining K+ balance on the low-Na+, high-K+ diet.

A low-Na+, high-K+ diet (LNaHK) is considered a healthier alternative to the "Western" high-Na+ diet. Because the mechanism for K+ secretion involves Na+ reabsorptive exchange for secreted K+ in the distal nephron, it is not understood how K+ is eliminated with such low Na+ intake. Animals on a LNaHK diet produce an alkaline load, high urinary flows, and markedly elevated plasma ANG II and aldosterone levels to maintain their K+ balance. Recent studies have revealed a potential mechanism involving the actions of alkalosis, urinary flow, elevated ANG II, and aldosterone on two types of K+ channels, renal outer medullary K+ and large-conductance K+ channels, located in principal and intercalated cells. Here, we review these recent advances.

Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with the Centromere through CENP-B and Regulates Chromosome Segregation.

ADA3 (alteration/deficiency in activation 3) is a conserved component of several transcriptional co-activator and histone acetyltransferase (HAT) complexes. Recently, we generated Ada3 knock-out mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. The use of Ada3(FL/FL) mouse embryonic fibroblasts with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with the higher order repeat region of the α-satellite region on human X chromosome centromeres that is consistent with its role in mitosis. Given the role of centromere proteins (CENPs) in mitosis, we next analyzed whether ADA3 associates with the centromere through CENPs. Both an in vivo proximity ligation assay and immunofluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein that binds to the 17-bp DNA sequences on α-satellite DNA. Deletional analysis showed that ADA3 directly associates with CENP-B through its N terminus, and a CENP-B binding-deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting that ADA3 is required for the loading of CENP-B onto the centromeres. Finally, we show that deletion of Ada3 from Ada3(FL/FL) mouse embryonic fibroblasts exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study, together with its known function in maintaining genomic stability and its mislocalization in cancers, suggests an important role of ADA3 in mitosis.

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.

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-α 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.

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-ß4; BK-α/ß4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/ß4-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-ß4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-ß4 knockout (ß4KO) 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 ß4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in ß4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/ß4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease.