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[Ca.sup.2+]-activated BK channels in renal intercalated cells (ICs) mediate luminal flow-induced [K.sup.+] secretion (FIKS), but how ICs sense increased flow remains uncertain. We examined whether PIEZO1, a mechanosensitive [Ca.sup.2+]-permeable channel expressed in the basolateral membranes of ICs, is required for FIKS. In isolated cortical collecting ducts (CCDs), the mechanosensitive cation-selective channel inhibitor GsMTx4 dampened flow-induced increases in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]), whereas the PIEZO1 activator Yoda1 increased [[Ca.sup.2+].sub.i] and BK channel activity. CCDs from mice fed a high-[K.sup.+] (HK) diet exhibited a greater Yoda1-dependent increase in [[Ca.sup.2+].sub.i] than CCDs from mice fed a control [K.sup.+] diet. ICs in CCDs isolated from mice with a targeted gene deletion of Piezo1 in ICs (IC-Piezo1-KO) exhibited a blunted [[Ca.sup.2+].sub.i] response to Yoda1 or increased flow, with an associated loss of FIKS in CCDs. Male IC-Piezo1-KO mice selectively exhibited an increased blood [[K.sup.+]] in response to an oral [K.sup.+] bolus and blunted urinary [K.sup.+] excretion following a volume challenge. Whole-cell expression of BK[alpha] subunit was reduced in ICs of IC-Piezo1-KO mice fed an HK diet. We conclude that PIEZO1 mediates flow-induced basolateral [Ca.sup.2+] entry into ICs, is upregulated in the CCD in response to an HK diet, and is necessary for FIKS., Introduction The kidney is subject to variations in tubular (urinary) flow rate that regulate various physiologic functions, including [Na.sup.+] absorption and [K.sup.+] secretion in the aldosterone-sensitive distal nephron (ASDN), composed [...]

K+ secretion in the distal nephron has a critical role in K+ homeostasis and is the primary route by which K+ is lost from the body. Renal K+ secretion is enhanced by increases in dietary K+ intake and by increases in tubular flow rate in the distal nephron. This review addresses new and important insights regarding the mechanisms underlying flow‐induced K+ secretion (FIKS). While basal K+ secretion in the distal nephron is mediated by renal outer medullary K+ (ROMK) channels in principal cells (PCs), FIKS is mediated by large conductance, Ca2+/stretch activated K+ (BK) channels in intercalated cells (ICs), a distinct cell type. BK channel activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), and both PCs and ICs exhibit increases in [Ca2+]i in response to increases in tubular fluid flow rate, associated with an increase in tubular diameter. PIEZO1, a mechanosensitive, nonselective cation channel, is expressed in the basolateral membranes of PCs and ICs, where it functions as a mechanosensor. The loss of flow‐induced [Ca2+]i transients in ICs and BK channel‐mediated FIKS in microperfused collecting ducts isolated from mice with IC‐specific deletion of Piezo1 in the CCD underscores the importance of PIEZO1 in the renal regulation of K+ transport. [ABSTRACT FROM AUTHOR]

The pore-forming a-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channelmediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney. NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo. NEW & NOTEWORTHY: The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets. [ABSTRACT FROM AUTHOR]

Cannabis and synthetic cannabinoid consumption are increasing worldwide. Cannabis contains numerous phytocannabinoids that act on the G protein-coupled cannabinoid receptor type 1 (CB1R) and cannabinoid receptor type 2 expressed throughout the body, including the kidney. Essentially every organ, including the kidney, produces endocannabinoids, which are endogenous ligands to these receptors. Cannabinoids acutely increase urine output in rodents and humans, thus potentially influencing total body water and electrolyte homeostasis. As the kidney collecting duct (CD) regulates total body water, acid/base, and electrolyte balance through specific functions of principal cells (PCs) and intercalated cells (ICs), we examined the cell-specific immunolocalization of CB1R in the mouse CD. Antibodies against either the C-terminus or N-terminus of CB1R consistently labeled aquaporin 2 (AQP2)-negative cells in the cortical and medullary CD and thus presumably ICs. Given the well-established role of ICs in urinary acidification, we used a clearance approach in mice that were acid loaded with 280 mM NH4Cl for 7 days and nonacid-loaded mice treated with the cannabinoid receptor agonist WIN55,212-2 (WIN) or a vehicle control. Although WIN had no effect on urinary acidification, these WIN-treated mice had less apical + subapical AQP2 expression in PCs compared with controls and developed acute diabetes insipidus associated with the excretion of large volumes of dilute urine. Mice maximally concentrated their urine when WIN and 1-desamino-8- d -arginine vasopressin [desmopressin (DDAVP)] were coadministered, consistent with central rather than nephrogenic diabetes insipidus. Although ICs express CB1R, the physiological role of CB1R in this cell type remains to be determined. NEW & NOTEWORTHY: The CB1R agonist WIN55,212-2 induces central diabetes insipidus in mice. This research integrates existing knowledge regarding the diuretic effects of cannabinoids and the influence of CB1R on vasopressin secretion while adding new mechanistic insights about total body water homeostasis. Our findings provide a deeper understanding about the potential clinical impact of cannabinoids on human physiology and may help identify targets for novel therapeutics to treat water and electrolyte disorders such as hyponatremia and volume overload. [ABSTRACT FROM AUTHOR]

Ca2+-activated BK channels in renal intercalated cells (ICs) mediate luminal flow--induced K+ secretion (FIKS), but how ICs sense increased flow remains uncertain. We examined whether PIEZO1, a mechanosensitive Ca2+-permeable channel expressed in the basolateral membranes of ICs, is required for FIKS. In isolated cortical collecting ducts (CCDs), the mechanosensitive cation-selective channel inhibitor GsMTx4 dampened flow-induced increases in intracellular Ca2+ concentration ([Ca2+]i), whereas the PIEZO1 activator Yoda1 increased [Ca2+]i and BK channel activity. CCDs from mice fed a high-K+ (HK) diet exhibited a greater Yoda1-dependent increase in [Ca2+]i than CCDs from mice fed a control K+ diet. ICs in CCDs isolated from mice with a targeted gene deletion of Piezo1 in ICs (IC-Piezo1-KO) exhibited a blunted [Ca2+]i response to Yoda1 or increased flow, with an associated loss of FIKS in CCDs. Male IC-Piezo1-KO mice selectively exhibited an increased blood [K+] in response to an oral K+ bolus and blunted urinary K+ excretion following a volume challenge. Whole-cell expression of BKα subunit was reduced in ICs of IC-Piezo1-KO mice fed an HK diet. We conclude that PIEZO1 mediates flow-induced basolateral Ca2+ entry into ICs, is upregulated in the CCD in response to an HK diet, and is necessary for FIKS. [ABSTRACT FROM AUTHOR]

K + secretion in the distal nephron has a critical role in K + homeostasis and is the primary route by which K + is lost from the body. Renal K + secretion is enhanced by increases in dietary K + intake and by increases in tubular flow rate in the distal nephron. This review addresses new and important insights regarding the mechanisms underlying flow-induced K + secretion (FIKS). While basal K + secretion in the distal nephron is mediated by renal outer medullary K + (ROMK) channels in principal cells (PCs), FIKS is mediated by large conductance, Ca 2+ /stretch activated K + (BK) channels in intercalated cells (ICs), a distinct cell type. BK channel activation requires an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ), and both PCs and ICs exhibit increases in [Ca 2+ ] i in response to increases in tubular fluid flow rate, associated with an increase in tubular diameter. PIEZO1, a mechanosensitive, nonselective cation channel, is expressed in the basolateral membranes of PCs and ICs, where it functions as a mechanosensor. The loss of flow-induced [Ca 2+ ] i transients in ICs and BK channel-mediated FIKS in microperfused collecting ducts isolated from mice with IC-specific deletion of Piezo1 in the CCD underscores the importance of PIEZO1 in the renal regulation of K + transport., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The pore-forming α-subunit of the large-conductance K + (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K + secretion in the kidney is crucial for overall renal K + homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K + loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K + , are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K + loading in mouse kidney. NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K + .

The epithelial Na + channel (ENaC) γ subunit is essential for homeostasis of Na + , K + , and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 , in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 (γ Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of γ Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γ Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in γ Q4/Q4 mice was not altered. γ Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo. NEW & NOTEWORTHY The epithelial Na + channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na + or high K + diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na + or high K + diets.