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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 Ca2+ 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 Ca2+ in the distal nephron. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 NH4Cl 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. [ABSTRACT FROM AUTHOR]

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 (π; 10 dynes/cm²) -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 P² receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited T-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 π-induced K efflux. Similarly, carbenoxolone (25 μM), which blocks connexins, putative AlP pathways, blocked T-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 π-induced ATP release in IC. [ABSTRACT FROM AUTHOR]

The rare disease Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in the genes encoding Cullin 3 (CUL3), Kelch-Like 3 (KLHL3), and two members of the With-No-Lysine [K] (WNK) kinase family, WNK1 and WNK4. In the kidney, these mutations ultimately cause hyperactivation of NCC along the renal distal convoluted tubule. Hypertension results from increased NaCl retention, and hyperkalemia by impaired K + secretion by downstream nephron segments. CUL3 and KLHL3 are now known to form a ubiquitin ligase complex that promotes proteasomal degradation of WNK kinases, which activate downstream kinases that phosphorylate and thus activate NCC. For CUL3, potent effects on the vasculature that contribute to the more severe hypertensive phenotype have also been identified. Here we outline the in vitro and in vivo studies that led to the discovery of the molecular pathways regulating NCC and vascular tone, and how FHHt-causing mutations disrupt these pathways. Potential mechanisms for variability in disease severity related to differential effects of each mutation on the kidney and vasculature are described, and other possible effects of the mutant proteins beyond the kidney and vasculature are explored. © 2024 American Physiological Society. Compr Physiol 14:5839-5874, 2024., (Copyright © 2024 American Physiological Society. All rights reserved.)