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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 [...]

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]

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]

Paraoxonase 3 (PON3) is expressed in the aldosterone‐sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+‐coupled co‐transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride‐induced natriuresis and lower plasma [K+] at baseline. Single channel recordings in split‐open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA‐mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+], sodium chloride co‐transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3 has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. Key points: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems.We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model.At baseline, Pon3 KO mice had lower blood [K+], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride‐induced natriuresis, and enhanced sodium chloride co‐transporter (NCC) phosphorylation.Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+] and ‐NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls.Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non‐canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope‐tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti‐ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well‐characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non‐epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti‐α subunit antibody recognizes an intense non‐specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone‐induced full length α‐subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the β‐ and γ‐subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti‐ENaC antibodies. [ABSTRACT FROM AUTHOR]

Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-κB pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are a disintegrin and metalloprotease 17 (ADAM17) substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 knockout (KO) mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1, and ADAM17 levels (and signaling pathways) were assessed by immunoblot analysis. PT localization was assessed by confocal microscopy and an in situ proximity ligation assay. Findings were extended using human kidneys and urine as well as KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and coexpression of KIM-1 and MUC1 in the PT. Compared with WT mice, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in the PT of WT kidneys but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures compared with WT cultures, whereas inflammation was increased in Muc1 KO kidneys compared with WT kidneys. MUC1 was cleaved by ADAM17 in PT cultures and blocked KIM-1 shedding in Madin-Darby canine kidney cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding. [ABSTRACT FROM AUTHOR]

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion. [ABSTRACT FROM AUTHOR]

NADH cytochrome b5 reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism and methemoglobin reduction. While the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil (PTU), a weak inhibitor of CYB5R3 (IC50= ~275 µM), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of PTU, ZINC05626394 (IC50 = 10.81 µM) and ZINC39395747 (IC50 = 9.14 µM), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development. [ABSTRACT FROM AUTHOR]

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 + .

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na + is reabsorbed mainly via the epithelial Na + channel (ENaC) and Na + -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na + and K + homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K + ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K + ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K + diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K + restriction, potentially providing a mechanism to dampen ENaC activity and associated K + secretion. Taken together, our results show that PON3 has a role in renal Na + and K + homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na + channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na + and K + handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K + ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K + diet, Pon3 KO mice had normalized blood [K + ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K + loading or K + restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na + and K + homeostasis in the distal nephron., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)