P. Richard Grimm

Hypertension of Kcnmb1−/− is linked to deficient K secretion and aldosteronism

Mice lacking the β1-subunit (gene, Kcnmb1; protein, BK-β1) of the large Ca-activated K channel (BK) are hypertensive. This phenotype is thought to result from diminished BK currents in vascular smooth muscle where BK-β1 is an ancillary subunit. However, the β1-subunit is also expressed in the renal connecting tubule (CNT), a segment of the aldosterone-sensitive distal nephron, where it associates with BK and facilitates K secretion. Because of the correlation between certain forms of hypertension and renal defects, particularly in the distal nephron, it was determined whether the hypertension of Kcnmb1−/− has a renal origin. We found that Kcnmb1−/− are hypertensive, volume expanded, and have reduced urinary K and Na clearances. These conditions are exacerbated when the animals are fed a high K diet (5% K; HK). Supplementing HK-fed Kcnmb1−/− with eplerenone (mineralocorticoid receptor antagonist) corrected the fluid imbalance and more than 70% of the hypertension. Finally, plasma [aldo] was elevated in Kcnmb1−/− under basal conditions (control diet, 0.6% K) and increased significantly more than wild type when fed the HK diet. We conclude that the majority of the hypertension of Kcnmb1−/− is due to aldosteronism, resulting from renal potassium retention and hyperkalemia.

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

Background: Full-length SPAK is thought to be necessary and sufficient to activate NCC in the distal convoluted tubule (DCT). Results: SPAK knock-out disrupts a signaling network, involving OSR1, in the DCT but not the TAL, preventing NCC activation. Conclusion: SPAK and OSR1 function interdependently in the DCT to positively regulate NCC. Significance: This study provides insights into the mechanisms whereby SPAK/OSR1 regulates renal salt transport. STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK−/− mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.

Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.

Role of BK channels in hypertension and potassium secretion.

Purpose of reviewThis review summarizes recent studies of hypertension associated with a defect in renal K excretion due to genetic deletions of various components of the large, Ca-activated K channel (BK), and describes new evidence and theories regarding K secretory roles of BK in intercalated cells. Recent findingsIsolated perfused tubule methods have revealed the importance of BK in flow-induced K secretion. Subsequently, mice with genetically deleted BK subunits revealed the complexities of BK-mediated K secretion. Deletion of BK&agr; results in extreme aldosteronism, hypertension, and an absence of flow-induced K secretion. Deletion of the BK&bgr;1 ancillary subunit results in decreased handling of a K load, increased plasma K, mild aldosteronism and hypertension that is exacerbated by a high K diet. Deletion of BK&bgr;4 (&bgr;4KO) leads to insufficient K handling, high plasma K, fluid retention, but with milder hypertension. Fluid retention in &bgr;4KO may be the result of insufficient flow-induced secretion of adenosine triphosphate (ATP), which normally inhibits epithelial Na channels (ENaCs). SummaryClassical physiological analysis of electrolyte handling in knockout mice has enlightened our understanding of the mechanism of handling K loads by renal K channels. Studies have focused on the different roles of BK-&agr;/&bgr;1 and BK-&agr;/&bgr;4 in the kidney. BK&bgr;1 hypertension may be a ‘three-hit’ hypertension, involving a K secretory defect, elevated production of aldosterone, and increased vascular tone. The disorders observed in BK knockout mice have shed new insights on the importance of proper renal K handling for maintaining volume balance and blood pressure.

Intercalated Cell BK-α/β4 Channels Modulate Sodium and Potassium Handling During Potassium Adaptation

The large-conductance, calcium-activated potassium (BK) channels help eliminate potassium in mammals consuming potassium-rich diets. In the distal nephron, principal cells contain BK-alpha/beta1 channels and intercalated cells contain BK-alpha/beta4 channels. We studied whether BK-beta4-deficient mice (Kcnmb4(-/-)) have altered renal sodium and potassium clearances compared with wild-type mice when fed a regular or potassium-rich diet for ten days. We did not detect differences in urinary flow or fractional excretions of potassium (FE(K)) or sodium (FE(Na)) between Kcnmb4-deficient and wild-type mice fed a regular diet. However, a potassium-rich diet led to >4-fold increases in urinary flows for both groups of mice, although Kcnmb4-deficient mice exhibited less urinary flow, higher plasma potassium concentration, more fluid retention, and significantly lower FE(K) and FE(Na) than wild-type mice despite similar plasma aldosterone levels. Immunohistochemical analysis revealed increased basolateral Na-K-ATPase in principal cells of all potassium-adapted mice, but expression of Na-K-ATPase in intercalated cells was >10-fold lower. The size of intercalated cells reduced and luminal volume increased among potassium-adapted wild-type but not Kcnmb4-deficient mice. Paradoxically, this led to increased urinary fluid velocity in potassium-adapted Kcnmb4-deficient mice compared with wild-type mice. Taken together, these data suggest that BK-alpha/beta4 channels in intercalated cells reduce cell size, increasing luminal volume to accommodate higher distal flow rates during potassium adaptation. These changes streamline flow across the principal cells, producing gradients more favorable for potassium secretion and less favorable for sodium reabsorption.

Integrated compensatory network is activated in the absence of NCC phosphorylation

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.

BK channels and a new form of hypertension

Large, Ca-activated K channels (BK) are comprised of an α pore (BKα) and one of four β subunits (BKβ1-4). When the gene for BKβ1 is knocked out (BKβ1-KO), the result is increased myogenic tone of vascular smooth muscle and hypertension. We reexamined whether the hypertension is entirely due to increased vascular tone, because most monogenic forms of hypertension have renal origins and BKβ1 resides in renal connecting tubule (CNT) cells. Moreover, BKβ1 is localized in the adrenal glands, where it may control production of aldosterone. This review will summarize our report that a majority of the hypertension of BKβ1-KO is the result of insufficient handling of dietary K, resulting in increased plasma K and hyperaldosteronism, the latter promoting Na and fluid retention. The fluid retention and hypertension are exacerbated by a high-K diet and reduced by eplerenone, an aldosterone receptor inhibitor. Genetic knockout of BKβ4 (BKβ4-KO), which resides in intercalated cells, also exhibits deficient K excretion, fluid retention, and mild hypertension that is not exacerbated when animals are treated with a high-K diet. These results show that the hypertension associated with BKβ1-KO occurs because of enhanced fluid retention, as well as because of the previously described vascular dysfunction.

BK channels in the kidney.

Purpose of reviewLarge, BK (calcium-activated potassium) channels are now regarded as relevant players in many aspects of renal physiology, including potassium secretion. This review will highlight recent discoveries regarding the function and localization of BK in the kidney. Recent findingsPatch clamp electrophysiology has revealed BK in cultured podocytes, glomerular mesangial cells, and in several tubule segments including principal cells (connecting tubules/principal cells), and intercalated cells of connecting tubules and cortical collecting ducts. Flow-induced potassium secretion is mediated by BK in the distal nephron and may be partly the result of shear stress-induced increases in cell calcium concentrations. ROMK−/− and wild-type mice on a high potassium diet exhibit BK-mediated potassium secretion, and studies of BK-α−/− and BK-β1−/− mice suggest that flow-induced potassium secretion is mediated by BK-α/β1, which is specifically localized in the apical membrane of the connecting tubule of the mouse and connecting tubule plus initial cortical collecting duct of the rabbit. SummaryBK channels, located in glomerular cells and in many nephron segments, especially mediate potassium secretion in the combined condition of potassium adaptation and high flow. Understanding the molecular makeup of BK in specific renal cells and the dietary and physiological conditions for their expression can yield improved potassium-sparing compounds.

SPAK-mediated NCC regulation in response to low-K diet

The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low-K(+) diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated whether the NCC response to low-K(+) diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type and SPAK knockout (SPAK(-/-)) mice fed a low-K(+) or control diet for 4 days, we found that low-K(+) diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low-K(+) diet was blunted but not completely inhibited in SPAK(-/-) mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the distal convoluted tubule in response to a low-K(+) diet, but other low-potassium-activated kinases are likely to be involved.

Role of BKβ1 in Na+ reabsorption by cortical collecting ducts of Na+-deprived mice

On a low-Na(+) diet (LNa(+)), urinary Na(+) loss is prevented by aldosterone-induced Na(+) reabsorption through epithelial Na(+) channels (ENaC) in the connecting tubules (CNT) and cortical collecting ducts (CCD). However, the mechanism whereby K(+) loss is minimized and Na(+) reabsorption is maximized in the face of a reduced lumen-to-bath Na(+) gradient is not fully understood. The large-conductance calcium-activated potassium channel (BK)beta1 subunit (gene: Kcnmb1), which has a role in K(+) secretion in the CNT, is absent in the CCD in mice on a control diet. We hypothesized that BKalpha/beta1 helps to maximize Na(+) reabsorption during Na(+) deficiency. With LNa(+), the Na(+) clearance of Kcnmb1-mutant mice (Kcnmb1(-/-)) was 45% greater and the plasma Na(+) concentration and osmolality were significantly reduced compared with wild-type mouse (WT) controls. On LNa(+), Kcnmb1(-/-) exhibited exacerbated volume depletion (higher Hct and weight loss) compared with WT. LNa(+), which did not affect the mean arterial blood pressure (MAP) of WT, significantly reduced MAP of Kcnmb1(-/-). The plasma aldosterone concentration of Kcnmb1(-/-) on LNa(+) was significantly elevated compared with Kcnmb1(-/-) on a control diet but was not different from WT on LNa(+). Immunohistochemical staining revealed that BKalpha and BKbeta1, which were absent in the principal cells (PCs) of the CCD, were localized on the basolateral membrane (BSM) of PCs of WT on LNa(+). Moreover, BKalpha was absent from the BSM of PCs of Na(+)-deficient Kcnmb1(-/-). We conclude that part of the mechanism to maximize Na(+) reabsorption during Na(+) deficiency is the placement of BKalpha/beta1 channels in the BSM of CCD PCs.