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The gut microbiome regulates many important host physiological processes associated with cardiovascular health and disease; however, the impact of the gut microbiome on aldosterone is unclear. Investigating whether gut microbiota regulate aldosterone can offer novel insights into how the microbiome affects blood pressure. In this study, we aimed to determine whether gut microbiota regulate host aldosterone. We used enzyme-linked immunosorbent assays (ELISAs) to assess plasma aldosterone and plasma renin activity (PRA) in female and male mice in which gut microbiota are intact, suppressed, or absent. In addition, we examined urinary aldosterone. Our findings demonstrated that when the gut microbiota is suppressed following antibiotic treatment, there is an increase in plasma and urinary aldosterone in both female and male mice. In contrast, an increase in PRA is seen only in males. We also found that when gut microbiota are absent (germ-free mice), plasma aldosterone is significantly increased compared with conventional animals (in both females and males), but PRA is not. Understanding how gut microbiota influence aldosterone levels could provide valuable insights into the development and treatment of hypertension and/or primary aldosteronism. This knowledge may open new avenues for therapeutic interventions, such as probiotics or dietary modifications to help regulate blood pressure via microbiota-based changes to aldosterone. NEW & NOTEWORTHY: We explore the role of the gut microbiome in regulating aldosterone, a hormone closely linked to blood pressure and cardiovascular disease. Despite the recognized importance of the gut microbiome in host physiology, the relationship with circulating aldosterone remains largely unexplored. We demonstrate that suppression of gut microbiota leads to increased levels of plasma and urinary aldosterone. These findings underscore the potential of the gut microbiota to influence aldosterone regulation, suggesting new possibilities for treating hypertension. [ABSTRACT FROM AUTHOR]

This article explores the potential of leveraging the gut microbiota to combat essential hypertension. It acknowledges the strengths of previous research, including the discovery of gut dysbiosis in hypertension and the role of gut microbiota-derived metabolites in blood pressure regulation. The article also discusses the challenges in studying the gut microbiota and the need for a diverse workforce in this area of research. It suggests the establishment of a consortium or network of researchers to collaborate on gut microbiota research in the context of hypertension. The article highlights the importance of considering dietary components, disparities in study standards, and the need for diversity in microbiota data. It identifies opportunities for advancing research, such as using metagenomics, artificial intelligence, and CRISPR-Cas9. The article explores various areas of research, including the gut-brain axis, gut epithelial permeability, and the role of other biota members in hypertension. It also discusses the interaction between gut microbiota and antihypertensive medications and the potential for personalized interventions for regulating blood pressure. The document emphasizes the reciprocal relationship between diet and the gut microbiota, as well as the impact of exercise on gut microbial diversity and metabolic diseases. It explores emerging therapeutic approaches like fecal microbiota transplantation and genetic engineering of bacteria for hypertension treatment. The article concludes by calling for increased funding, collaboration, and education to advance research on the gut microbiota and its potential for personalized antihypertensive therapies. [Extracted from the article]

Next-generation sequencing (NGS) technology has advanced our understanding of the human microbiome by allowing for the discovery and characterization of unculturable microbes with prediction of their function. Key NGS methods include 16S rRNA gene sequencing, shotgun metagenomic sequencing, and RNA sequencing. The choice of which NGS methodology to pursue for a given purpose is often unclear for clinicians and researchers. In this Review, we describe the fundamentals of NGS, with a focus on 16S rRNA and shotgun metagenomic sequencing. We also discuss pros and cons of each methodology as well as important concepts in data variability, study design, and clinical metadata collection. We further present examples of how NGS studies of the human microbiome have advanced our understanding of human disease pathophysiology across diverse clinical contexts, including the development of diagnostics and therapeutics. Finally, we share insights as to how NGS might further be integrated into and advance microbiome research and clinical care in the coming years., Introduction The number of microbial cells that reside on and in us rivals the number of our own cells (1). In health, we, the host, and microbes live in symbiosis. [...]

Olfactory receptors (ORs) are chemosensors that are responsible for one's sense of smell. In addition to this specialized role in the nose, recent evidence suggests that ORs are also found in a variety of additional tissues including the kidney. As this list of renal ORs continues to expand, it is becoming clear that they play important roles in renal and whole-body physiology, including a novel role in blood pressure regulation. In this review, we highlight important considerations that are crucial when studying ORs and present the current literature on renal ORs and their emerging relevance in maintaining renal function., Author(s): Blythe D. Shepard[sup.1] , Jennifer L. Pluznick[sup.1] Author Affiliations: (1) Johns Hopkins University School of Medicine, Baltimore, MD, USA Introduction Olfactory receptors (ORs) are seven transmembrane-domain G-protein-coupled chemosensors that [...]

The gut microbiome impacts host gene expression not only in the colon but also at distal sites including the liver, white adipose tissue, and spleen. The gut microbiome also influences the kidney and is associated with renal diseases and pathologies; however, a role for the gut microbiome to modulate renal gene expression has not been examined. To determine if microbes modulate renal gene expression, we used whole organ RNA sequencing to compare gene expression in C57Bl/6 mice that were germ free (lacking gut microbiota) versus conventionalized (gut microbiota reintroduced using an oral gavage of a fecal slurry composed of mixed stool). 16S sequencing showed that male and female mice were similarly conventionalized, although Verrucomicrobia was higher in male mice. We found that renal gene expression was differentially regulated in the presence vs. absence of microbiota and that these changes were largely sex specific. Although microbes also influenced gene expression in the liver and large intestine, most differentially expressed genes (DEGs) in the kidney were not similarly regulated in the liver or large intestine. This demonstrates that the influence of the gut microbiota on gene expression is tissue specific. However, a minority of genes (n = 4 in males and n = 6 in females) were similarly regulated in all three tissues examined, including genes associated with circadian rhythm (period 1 in males and period 2 in females) and metal binding (metallothionein 1 and metallothionein 2 in both males and females). Finally, using a previously published single-cell RNA-sequencing dataset, we assigned a subset of DEGs to specific kidney cell types, revealing clustering of DEGs by cell type and/or sex. [ABSTRACT FROM AUTHOR]

The diverse functions of each nephron segment rely on the coordinated action of specialized cell populations that are uniquely defined by their transcriptional profile. In the collecting duct, there are two critical and distinct cell populations: principal cells and intercalated cells. Principal cells play key roles in the regulation of water, Na+, and K+, whereas intercalated cells are best known for their role in acid-base homeostasis. Currently, there are no in vitro systems that recapitulate the heterogeneity of the collecting ducts, which limits high-throughput and replicate investigations of genetic and physiological phenomena. Here, we demonstrated that the transcription factor Foxi1 is sufficient to alter the transcriptional identity of M-1 cells, a murine cortical collecting duct cell line. Specifically, overexpression of Foxi1 induces the expression of intercalated cell transcripts including Gpr116, Atp6v1b1, Atp6v1g3, Atp6v0d2, Slc4a9, and Slc26a4. These data indicate that overexpression of Foxi1 differentiates M-1 cells toward a non-A, non-B type intercalated cell phenotype and may provide a novel in vitro tool to study transcriptional regulation and physiological function of the renal collecting duct. NEW & NOTEWORTHY Transfection of M-1 cells with the transcription factor Foxi1 generates cells that express V-ATPase and Gpr116 as well as other genes associated with renal intercalated cells. This straightforward and novel in vitro system could be used to study processes including transcriptional regulation and cell specification and differentiation in renal intercalated cells. [ABSTRACT FROM AUTHOR]

We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular [Ca.sup.2+] concentration [[[Ca.sup.2+]].sub.i] to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 [micro]M SNP, an NO donor, or 100 [micro]M 8-Br-cGMP. Addition of DT-3 (2.5 [micro]M), a specific PKG-1[alpha] inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 [micro]M cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1[alpha] transcript and protein in HMC. Immunocytochemical analysis localized PKG-1[alpha] to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular [Ca.sup.2+] entry, resulting in altered growth and proliferation. Therefore, we used Western blotting and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 min. Coimmunoprecipitation and coimmunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG1[alpha]-mediated inhibition of the TRPC4-SOC response in HMC. cGMP; SOC; nitric oxide; SNP

Although it is generally accepted that ROMK is the [K.sup.+] secretory channel in the mammalian distal nephron, recent in vitro and in vivo studies have provided evidence that large-conductance [Ca.sup.2+]-activated [K.sup.+] channels (BK, or maxi K) also secrete [K.sup.+] in renal tubules. This review assesses the current evidence relating BK channels with [K.sup.+] secretion. We shall consider the component proteins of the BK channel, their localization with respect to segment and cell type, and the electrophysiological forces involved in [K.sup.+] secretion. Although the majority of studies have focused on a role for BK channels in flow-mediated [K.sup.+] secretion, this review also considers a potential role for BK channels in high-K diet-induced [K.sup.+] secretion. The division of workload between ROMK and BK is discussed as a mechanism for ensuring a constant plasma [K.sup.+] concentration. maxi K; distal nephron; potassium secretion; connecting tubule; BK-[beta]1 doi:10.1152/ajprenal.00118.2006

Large, [Ca.sup.2+]-activated [K.sup.+] channels (BK), comprised of [alpha]- and [beta]-subunits, mediate [K.sup.+] secretion during high flow rates in distal nephron segments. Because the BK-[beta]1 subunit enhances [Ca.sup.2+] sensitivity of BK in a variety of cells, we determined its role in flow-induced [K.sup.+] secretion and its localization in the mammalian nephron. To determine the role of BK-[beta]1 in the kaliuretic response to volume expansion, the rate of [K.sup.+] excretion ([U.sub.[kappa]]V) vs. varied urinary flow rates were determined in wild-type and BK-[beta]1 knockout mice (BK-[[beta]1.sup.-/-]). When flow rate was varied by volume expansion (2 ml x [h.sup.-1] x 25 g body [wt.sup.1]) for 30 to 60 min in wild-type mice, we found that the UKV increased significantly with increasing urine flow rates ([r.sup.2] = 0.50, P < 0.00001, n = 31), as demonstrated previously in distal nephron of rats and rabbits. However, in BK-[[beta]1.sup.-/-] mice, [U.sub.[kappa]]V did not vary with changing flow rates ([r.sup.2] = 0.15, P = 0.08, n = 20). Using immunohistochemical techniques, we found that BK-[beta]1 was strongly expressed in the apical membrane of the murine distal nephron and that 98% of BK-[beta]1 protein detected by histochemistry colocalized with NCX, a marker of connecting tubules (CNT). Both BK-[beta]1 and NCX colocalized with BK-[alpha] in separate experiments. Furthermore, we confirmed BK-[beta]1 protein expression in the apical membrane of connecting tubules in rabbits. BK-[beta]1 RNA from rabbit CNT was sequenced and was identical to previously published rabbit muscle sequences. These data show that the BK-[beta]1 accessory subunit is present in the CNT segment of the mammalian distal nephron and has a significant role in the kaliuretic response to increased urinary flow induced by volume expansion. Maxi K; distal nephron; flow-mediated [K.sup.+] secretion

Studies were performed to identify the molecular component responsible for store-operated [Ca.sup.2+] entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise [Ca.sup.2+]-selective and -nonselective cation channels in a variety of cells, we screened TRPC1-TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated [Ca.sup.2+] (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). SOC was measured as the increase in [[[Ca.sup.2+]].sub.i] after extracellular [Ca.sup.2+] was increased from canonical transient receptor potential; TRPC4-[alpha]; TRPC4-[beta]; TRPC1; fura 2; glomerulus

In vascular smooth muscle and glomerular mesangial cells, relaxing agents such as nitric oxide and atrial natriuretic peptide activate large-conductance [Ca.sup.2+]-activated [K.sup.+] channels (BK) via the cGMP kinase pathway. BK are composed of pore-forming [alpha]-subunits, encoded by the slopoke gene (Slo), and one of four cell-specific accessory [beta]-subunits (h[[beta].sub.1-4]). We used patch-clamp analysis to determine the influence of h[[beta].sup.1], h[[beta].sup.2], and h[[beta].sup.4] on activation of human mesangial BK by cGMP kinase. We found that HEK 293 cells, coexpressing human (h) Slo[alpha] with either h[[beta].sub.1] or h[[beta].sub.2], contained single BK currents activated by db-cGMP in cell-attached patches. However, recombinant BK were not activated by db-cGMP when hSl[alpha] was expressed alone or with h[[beta].sup.4]. DNA-RNA hybridization revealed that mesangial cells contained mRNA for h[[beta].sub.1] but not h[[beta].sub.2] or h[[beta].sub.4]. The BK response to db-cGMP was decreased when h[[beta].sub.1] antisense but not scrambled oligonucleotides were incorporated into mesangial cells. Western blot analysis showed that h[[beta].sub.1] antisense oligo, nucleotide inhibited the amount of h[[beta].sub.1]-V5 fusion protein expressed in HEK 293 cells by ~50%. These results show that mesangial cells contain h[[beta].sub.1], a BK accessory protein, which confers activation of BK by cGMP kinase. large-conductance calcium-activated potassium channels; maxi-potassium; human [beta]-subunit; antisense; patch clamp; guanosine 3',5'-cyclic monophosphate

Large-conductance [Ca.sup.2+]-activated [K.sup.+] channels ([BK.sub.Ca]) are composed of pore-forming [alpha]-subunits and one of four accessory [beta]-subunits. The [beta]1-subunit, found predominantly in smooth muscle, modulates the [Ca.sup.2+] sensitivity and pharmacological properties of [BK.sub. Ca]. [BK.sub.Ca]-[beta]1 null mice (M[[beta]1.sup.-/-]) are moderately hypertensive, consistent with the role of [BK.sub.Ca] in modulating intrinsic vascular tone. Because [BK.sub.Ca] are present in various renal cells including the mesangium and cortical collecting ducts, we determined whether fluid or electrolyte excretion was impaired in M[[beta]1.sup.-/-] under euvolemic, volume-expanded, or high-salt diet conditions. Under euvolemic conditions, no differences in renal function were found between M[[beta]1.sup.-/-] and M[[beta]1.sup.+/+]. However, glomerular filtration rate (GFR) and fractional [K.sup.+] excretion were significantly impaired in M[[beta]1 .sup.-/-] in response to acute volume expansion. In contrast, M[[beta]1 .sup.-/-] exhibited enhanced [Na.sup.+] excretion and fractional [Na. sup.+] excretion responses to acute volume expansion. Differences in renal function between M[[beta]1.sup.+/+] and M[[beta].sup.-/-] were not observed when chronically treated with a high-salt diet. These observations indicate that the [beta]1-subunit of [BK.sub.Ca] contributes to the increased GFR that accompanies an acute salt and volume load and raises the possibility that it is also involved in regulating [K.sup.+] excretion under these conditions. large-conductance, calcium-activated potassium channels; maxi K channel; glomerular filtration rate; volume expansion; potassium excretion

In this study, we elucidate factors that regulate the trafficking and activity of a well-conserved olfactory receptor (OR), olfactory receptor 558 (Olfr558), and its human ortholog olfactory receptor 51E1 (OR51E1). Results indicate that butyrate activates Olfr558/OR51E1 leading to the production of cAMP, and evokes Ca2+ influx. We also find olfactory G protein (Golf) increases cAMP production induced by Olfr558/OR51E1 activation but does not affect trafficking. Given the 93% sequence identity between OR51E1 and Olfr558, it is surprising to note that OR51E1 has significantly more surface expression yet similar total protein expression. We find that replacing the Olfr558 N-terminus with that of OR51E1 significantly increases trafficking; in contrast, there is no change in surface expression conferred by the OR51E1 TM2, TM3, or TM4 domains. A previous analysis of human OR51E1 single nucleotide polymorphisms (SNPs) identified an A156T mutant primarily found in South Asia as the most abundant (albeit still rare). We find that the OR51E1 A156T mutant has reduced surface expression and cAMP production without a change in total protein expression. In sum, this study of a well-conserved olfactory receptor identifies both protein regions and specific amino acid residues that play key roles in protein trafficking and also elucidates common effects of Golf on the regulation of both the human and murine OR. [ABSTRACT FROM AUTHOR]