bluuebunny/crossref_metadata_2025_split huggingface dataset:
Renal cortical hydrogen peroxide (H2O2) stimulates renin release from juxtaglomerular (JG) cells and increases blood pressure: Role of JG cell NOX4
Authors: Hypertension Mariela Mendez. , Division Vascular Reasearch 
Published: November 2023

Renin plays an essential role in blood pressure control and the development of hypertension. Enhanced hydrogen peroxide (H2O2) levels in the kidney are associated with hypertension. This is thought to be due to direct stimulation of tubular salt transport and enhanced vascular reactivity. However, we previously found that H2O2 can directly stimulate renin release from renal juxtaglomerular (JG) cells suggesting that this pathway may also be responsible for the hypertension. However, it is not clear whether enhanced H2O2 in the renal cortex stimulates renin release and increases blood pressure. We hypothesized that enhancing renal cortical H2O2 stimulates renin release and increases blood pressure. To enhance renal cortical H2O2 in mice, we placed a subcapsular renal catheter connected to an osmotic minipump to reach a concentration of 1 μM, and measured blood pressure by radio telemetry and plasma renin concentration (PRC). Two days after infusion of H2O2, systolic blood pressure increased by 22±2 mmHg (p<0.05) and PRC doubled (from 110± 7 to 214±39 ngAngI/hr/ml, p<0.05). In control mice, with renal cortical infusion of saline, blood pressure and PRC were not different from baseline, indicating that renal cortical H2O2 increased renin release and blood pressure. In vivo, H2O2 produced by surrounding cells or endogenously produced, could enhance renin release. We found that in freshly isolated mouse JG cells, decreasing endogenous H2O2 with catalase (100U/ml) decreased baseline renin release by 45±9% (n = 6; p < 0.05) indicating that endogenously produced H2O2 tonically stimulates renin release. To start dissecting the enzymatic sources of H2O2 production, we studied the NADPH oxidase isoforms present in JG cells. By Western blot, we found that NOX1, NOX2 and NOX4 are expressed in JG cell lysates (n=3). By immunofluorescence and confocal imaging, we observed that NOX4 isoform is highly expressed in JG cell renin granules (n=3). Treating JG cells with apocynin (120 μM), a general inhibitor of NADPH oxidases, decreased renin release by 28±7% (p<0.04). To identify whether NOX4 mediates the endogenous H2O2 production, we used adenovirus mediated gene silencing of NOX4. Silencing NOX4 in JG cells decreased renin release by 34 ±4% (p < 0.05). We concluded that renal cortical H2O2 is a potent stimuli for renin release in vivo. Endogenously produced H2O2 from NOX4 expressed in mouse JG cells stimulates renin release. Our data suggest that enhanced renal cortical reactive oxygen species may induce hypertension by enhancing renin release. NOX4 inhibition in JG cells might be a candidate for renin release and blood pressure control.Support or Funding InformationSDG12070304; RO3DK105300‐01
Lithium Modulates Cilia Length in Renal Collecting Duct Cells
Authors: Mehran Nikki A. , Mallow John F. , Himmel Nathaniel J. , Blount Mitsi A. 
Published: November 2023

Cilia are important regulators of multiple cell processes throughout the body. Optimal cellular function can depend on cell‐specific regulation of cilia length; however, the mechanisms that control cilia length are not well understood. Defects in ciliary length appear to be related to ciliopathic diseases, which often present with renal cysts. Lithium chloride (LiCl) has been shown to affect cilia in multiple cell types, but its effects have not been explored in inner medullary collecting duct (IMCD) cells. We found that following a 24 hr LiCl (25 mM) treatment of IMCD cells in vitro, cilia are significantly elongated by 1.5 fold. Although LiCl can inhibit GSK3β, addition of the GSK3β‐specific inhibitor TDZD‐8 did not increase IMCD cilia length, indicating that LiCl‐mediated cilia elongation is independent of GSK3β. Instead we found that the SQ‐22536‐inhibtion of adenylate cyclases in IMCD cells mimics LiCl‐mediated cilia elongation, whereas addition of a cAMP analog in the presence of LiCl presence prevents elongation. In addition, we observed a similar increase in cilia length in renal collecting duct cells from rats fed dietary lithium for 2 wk coinciding with a decrease in urinary cAMP. We conclude that lithium increases cilia length in IMCD cells by dampening cellular cAMP in a manner independent of GSK3β. Our data provide new insights into the effects of LiCl on renal cilia and suggest that lithium may have therapeutic benefits in the treatment of some ciliopathies.Support or Funding InformationEmory SOM‐Renal Division
Monocyte Mitochondrial Function as a Marker of Calcium Oxalate Stone Formers
Authors: Mitchell Tanecia , Williams Jennifer , Holmes Ross P , Assimos Dean G 
Published: November 2023

The prevalence of kidney stones (KS) is 8.8% in the US and contributes significantly to health care costs. The most common type of KS is the calcium oxalate kidney stone (CaOx‐KS). Both inflammation and oxidative stress have been suggested to play an integral role in KS pathogenesis. The purpose of this study was to investigate whether inflammation is elevated and cellular bioenergetics is suppressed in immune cells from calcium oxalate (CaOx) stone formers (SF) compared to healthy subjects (HS). Adult age‐matched HS (n=18) and CaOx SF (n=15) were included in the study. Data collection included demographic and clinical values from electronic medical records. Pro‐inflammatory Interleukin‐6 (IL‐6) plasma values were measured using ELISA. Bioenergetic function was assessed in monocytes, lymphocytes, and platelets using the Seahorse XF96 Analyzer. Fifty‐three percent of the CaOx SF had a family history of KS and 60% had a KS event previously. Plasma IL‐6 levels were significantly elevated (p=0.0024) and monocyte mitochondrial function was decreased in CaOx‐SF compared to HS. Specifically, maximal respiration (p=0.0001) and reserve capacity (p<0.0001) were significantly lower. In contrast, lymphocyte and platelet mitochondrial function was similar to monocytes from HS. The bioenergetic health index (BHI), an integrated value of mitochondrial function, was also determined to be significantly lower in CaOx SF monocytes compared to HS (p=0.0191). These data suggest that impaired monocyte mitochondrial function may play a role in CaOx KS pathogenesis and the BHI could potentially serve as a marker in CaOx SF.Support or Funding InformationSupported by NIH grants DK054468, UL1TR001417, P30 DK079337, and the 2015–2016 UAB Faculty Development Grant Program (TM). TM is also supported by DK054468‐S1.
Low Birth Weight Impairs Murine Kidney Development and Function
Authors: Ratliff Brian , Pawar Rahul , Aldumuahdhi Wasan , Nnoli Oluwadara , Zullo Joseph , Rabadi May 
Published: November 2023

BackgroundThe low birth weight (LBW) neonate is at greater risk for morbidity and mortality after birth and during adulthood.MethodsUsing immunohistochemistry, immunofluorescence, PCR, Laser‐Doppler flowmetry, and ELISA assay, we examined survival, kidney development and function, and circulating levels of pro‐inflammatory cyto‐/chemokines in the LBW offspring of malnourished (caloric and protein restricted) pregnant mice.ResultsMaternal malnourishment during gestation led to LBW newborns (40% reduced body weight) with only 46% of these babies surviving the first week after birth. The LBW neonate had elevated serum creatinine, while kidney blood perfusion was reduced by 26%. During gestation the LBW offspring suffered a severe impairment in nephrogenesis, due to a 76% drop in renal vesicle formation that was characterized by a 2.2‐fold increase in apoptosis, a 75% decrease in six2 positive nephron progenitor cells, and a 27% decrease in expression of the renal mesenchymal‐to‐epithelial signaling factor Wnt9b. At the conclusion of nephrogenesis, the LBW neonate had 36% fewer nephrons than controls. At birth, the LBW neonate had elevated plasma levels of IL‐1, IL‐12(p70) and GM‐CSF, further impairing six2 positive progenitor cell competence and nephrogenesis.ConclusionThe kidneys of LBW neonates are underdeveloped, thereby predisposing the neonate to adverse health problems.Support or Funding InformationStudies were supported by AHA grant 12SDG9080006, ASN grant 010973‐101 and The New York Community Trust‐Renal Clinical Fund (BBR)
The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion
Authors: Czogalla Jan , Vohra Twinkle , Ribas David Penton , Kirschmann Moritz , Carrel Monique , Loffing Johannes 
Published: November 2023

The mineralocorticoid receptor (MR) and its ligand aldosterone increase renal sodium reabsorption via regulation of the epithelial Na+ channel (ENaC) and the Na‐K‐ATPase in the renal collecting system. Previous studies suggested that aldosterone also regulates the thiazide‐sensitive cotransporter in the renal distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations such as changed plasma potassium, remained controversial. We have now generated a mouse model carrying a targeted deletion of MR in ~16% of cells randomly scattered along the renal tubule (MRko mice). In this model, MR‐positive and MR‐negative cells can be studied next to each other in the same physiological context via immunofluorescence. To evaluate microscope images objectively, we further developed an ImageJ script allowing radial quantification of optical density, thus allowing comparison of apical staining intensities. Although newborn MRko mice need NaCl supplementation to thrive, adult MRko do not show any differences to wildtype littermates in terms of general physiology, mRNA and protein expression of renal ion transport proteins and urine concentrating capacity, under both normal and low salt diet. MR‐negative cells in the renal collecting system of MRko mice do not show any detectable alphaENaC expression, while gammaENaC is diffusely distributed over the cytoplasm. In MR‐positive cells, these ENaC subunits are targeted to the apical cell surface. Although dietary Na+ restriction increases NCC abundance and phosphorylation to a similar extent in control and MRko mice, no differences in NCC regulation were observed between MR‐negative and MR‐positive DCT cells. In conclusion, MR is crucial for ENaC regulation but dispensable for the maintenance of NCC expression and phosphorylation under control conditions and for NCC upregulation in response to dietary Na+ restriction.Support or Funding InformationThis work was supported by a collaborative project grant from the Zurich Center for Integrative Human Physiology (ZIHP), research funds from the Swiss National Center for Competence in Research “Kidney.CH” and by a project grant (310030_143929/1) from the Swiss National Science Foundation.
γ subunit proteolytic fragment binds the epithelial Na+ channel at the finger‐thumb domain interface of the γ subunit
Authors: Kashlan Ossama B , Thompson Rebecca N 
Published: November 2023

Epithelial Na+ channel (ENaC) maturation and activation entail proteolysis of both the α and γ subunits. Cleavage at multiple sites in the finger domain of each subunit liberates the subunit's autoinhibitory peptide. Synthetic peptides derived from the proteolytically released fragments inhibit the channel, likely by reconstituting key interactions removed by proteolysis. We previously showed that a peptide derived from the α‐subunit's autoinhibitory sequence binds at the α subunit's finger‐thumb domain interface. Despite low sequence similarity between the α and γ subunit finger domains, we hypothesized that a peptide derived from γ subunit's autoinhibitory sequence inhibits the channel through an analogous mechanism. We tested our hypothesis using a crosslinking approach. We used bifunctional methane thiosulfonate (MTS) reagents to cross‐link experimentally‐introduced cysteines in the γ inhibitory peptide, and at select sites within the ENaC γ subunit. Our data show that the γ inhibitory peptide can be linked to both the finger and thumb domains of the γ subunit, consistent with our hypothesis. Our data also suggest that direct manipulation of the finger‐thumb interface using MTS reagents influences channel gating.Support or Funding InformationThis work is supported by NIH grant DK098204
Role of the NEDD4‐2 adaptor protein NDFIP2 in salt transport regulation
Authors: Vacle Sarah , Cheval Lydie , Doucet Alain , Fenton Robert A. , Staub Olivier 
Published: November 2023

Proper Na+ and K+ homeostasis by the kidney is a key process in the prevention of hypertension, the most prevalent disease in Western societies, and a primary risk factor for cardiovascular disease. The distal part of the nephron, referred to as the Aldosterone Sensitive Distal Nephron (ASDN), plays an important role in this process. There are at least two main regulatory pathways that are involved in the regulation of ions transporters (NCC, ENaC and ROMK or BK channels). One involves the WNK/SPAK/OSR1 kinase cascade, and the other the concerted action of SGK1 kinase and the ubiquitin‐protein ligase NEDD4‐2. Both pathways have been extensively studied, but there remain numerous open questions with respect to their regulation and possible crosstalks. In this project, we investigate the role of a putative adaptor protein (NDFIP2), which may provide the link between NEDD4‐2 and NCC. We first assessed the expression of Ndfip2 mRNA in microdissected tubules, and found that it is predominantly expressed in the proximal tubule and the ASDN. In MDCK‐hNCC cells, expressing human NCC under the control of a tetracycline inducible promotor, as well as endogenous NDFIP2 and NEDD4‐2, we demonstrated by co‐immunoprecipitation that NCC, NEDD4‐2 and NDFIP2 form a complex together. In parallel, we generated inducible, nephron‐specific KO mice (Ndfip2Pax8/LC1). This model allows the doxycycline‐sensitive expression of Cre‐recombinase along the nephron and consequently the deletion of exon 3 of Ndfip2. As the deletion is induced at the age of 3 weeks, compensatory mechanisms that may occur during nephrogenesis are avoided. In whole kidney lysates, only weak Ndfip2 mRNA expression and nearly complete suppression of the NDFIP2 protein is observed, which persist at least up to 2 months after deletion. Our data suggest decreased circulating levels of aldosterone, but no observable changes in NCC expression. When animal are kept under a high Na+ diet (HSD), they demonstrate difficulties to handle Na+ excess and adapt their water consumption.Support or Funding InformationThis work was supported by the Swiss National Science Foundation Grant # 310030_159735 (to OS), the Swiss Kidney.ch NCCR.
Vasopressin‐dependent regulation of potassium channel distribution in thick ascending limb of Henle's loop
Authors: Smorodchenko Alina , Sharkovska Yuliya , Böhlick Alexandra , Dittmayer Carsten , Cornejo‐Müller Alejandro , Mutig Kerim , Bachmann Sebastian 
Published: November 2023

IntroductionNaCl reabsorption via the bumetanide‐sensitive Na‐K‐2Cl‐cotransporter (NKCC2) in the thick ascending limb (TAL) of Henle's loop is crucial for kidney salt and water regulation. The inward‐rectifying potassium channels, luminal renal outer medullary potassium channel (ROMK) and basolateral KCNJ10 (Kir4.1) provide outward movement of potassium; both are essential for the action of NKCC2. A discontinuous pattern of ROMK‐positive and ‐negative cells along TAL has been shown earlier, whereas less is known about TAL Kir4.1 expression. Vasopressin (AVP) may increase TAL function significantly. In this study we investigated how ROMK and Kir4.1 cellular abundances adapt to the activation of TAL via AVP.MethodsControl Long Evans (LE; n=5) and AVP‐deficient Brattleboro rat kidneys (BB) were analyzed. BB rats were treated with V2 vasopressin receptor agonist (dDAVP) (n=10) or saline (n=10) for 72h. Kidneys were perfusion‐fixed or prepared for biochemical analysis. Immunohistochemistry, in situ hybridization, and EM analysis were used for localization and quantification of NKCC2, ROMK, and Kir4.1. Expression was further studied by Western blot.ResultsIn LE, NKCC2 was expressed in all TAL cells throughout the medullary and cortical portions, whereas ROMK and Kir4.1 showed selective expression patterns. Between 60% (medulla) and 40% (cortex) of TAL cells were devoid of ROMK, and respective 35% and 50% were Kir4.1‐negative at steady state as revealed by IHC. Expression of ROMK and Kir4.1 was often mutually exclusive. Administration of dDAVP to BB for 72h led to significant upregulation of both channels and induced an overlapping expression in most cells along the TAL. Changes were confirmed by Western blot, with increases in protein abundance by 220% for ROMK and 25% for Kir4.1, which were restricted to the medulla (p<0.01). ISH revealed no changes at all in mRNA abundances.ConclusionOur results demonstrate expressional cell heterogeneity with respect to ROMK and Kir4.1 signals in TAL. Transepithelial transport activation by AVP coincides with an augmentation of positive cell numbers, presumably via altered protein stability. Changes likely reflect an adaptive mechanism in potassium handling by the TAL to maintain solute homeostasis during changes in transport.
Comparative characterization of the ɛ‐ and α‐subunit of the epithelial sodium channel in Xenopus laevis
Authors: Wichmann Lukas , Perniss Alexander , Althaus Mike 
Published: November 2023

The vectorial transport of sodium ions across most vertebrate epithelia is tightly linked to the activity of the epithelial sodium channel (ENaC) which allows for entry of sodium ions across the apical membrane of epithelial cells. The canonical ENaC is composed of the pore‐forming α‐subunit which assembles with the accessory β‐ and γ‐subunits to form a heterotrimeric ion channel. While α‐ENaC is crucial for proper channel functionality it may be substituted by α‐like subunits such as the delta‐subunit (δ) in humans or the epsilon‐subunit (ɛ) in Xenopus laevis. Although the inherent role of α‐containing ENaC in epithelial sodium absorption is well established, the physiological function of the ɛ‐ENaC subunit remains elusive. This study aimed to examine the expression of ɛ‐ and α‐ENaC subunits in various Xenopus laevis tissues and to comparatively characterize heterologously expressed ɛβγ‐ and αβγ‐ENaCs.Screening of ɛ‐ and α‐ENaC expression in tissue homogenates was performed by reverse transcription PCR. Transcripts for ɛ‐ENaC were predominantly detected in tissues of the urogenital tract (kidneys, urinary bladder, testis, ovary, cloaca). With the exception of the ovary these tissues also expressed α‐ENaC. Functional characterization of heterologously expressed ɛβγ‐and αβγ‐ENaC in Xenopus laevis oocytes was carried out using the two‐electrode voltage‐clamp technique. In comparison with αβγ‐ENaCs, ɛ‐containing channels produced an increased transmembrane current which was more sensitive to inhibition by extra‐ and intracellular sodium ions. Furthermore, activity of ɛβγ‐ENaC was enhanced by acidic extracellular pH but decreased under alkaline conditions, whereas α‐ENaC containing channels were activated in both cases. Additionally, ɛβγ‐ENaC was less sensitive to the ENaC blocker amiloride. A decreased amiloride‐sensitivity of transepithelial sodium transport was also detected in Ussing‐chamber recordings of urinary bladder preparations compared to lung epithelia which did not express ɛ‐ENaC. Since ɛ‐ENaC was the only difference in the ENaC‐subunit transcriptome of both tissues, its presence likely accounts for the distinct amiloride‐sensitivities.These results indicate a functional role for the ɛ‐ENaC subunit in urogenital tissues of Xenopus laevis. The presence of ɛ‐ENaC might facilitate a distinct responsiveness of these tissues to sodium and extracellular pH.
Glycosylated Asparagines Are Important Constituents For The Shear Force Dependent Activation Of The Epithelial Sodium Channel (ENaC)
Authors: Barth Daniel , Knoepp Fenja , Fronius Martin 
Published: November 2023

The epithelial sodium channel (ENaC) contributes to electrolyte/fluid‐homeostasis and blood pressure regulation and its activity is regulated by shear force (SF). Previous studies have shown that SF sensation of α‐ENaC depends on an interaction with the extracellular matrix (ECM) via N‐linked glycans of two glycosylated asparagines (N312 and N511). To determine whether the asparagines themselves or the attached N‐glycans are required for SF sensation, site‐directed mutagenesis combined with two‐electrode voltage clamp (TEVC) experiments were performed. α‐ENaC subunits were stripped of their N‐glycans by individually disrupting the glycosylation consensus sequence (NXS/T) N312NS to N312NA and N511YT to N511YA. When co‐expressed with β and γ ENaC, a reduced SF‐response was observed compared with wildtype α ENaC. Furthermore, the role of glycosylated asparagines for SF sensation was confirmed in experiments with the δ‐subunit. While functionally related to the α‐subunit (35% of sequence identity), δ ENaC (when co‐expressed with β and γ) is less SF sensitive and lacking the glycosylated asparagines responsible for proper SF sensation in α‐ENaC. Insertion of asparagines corresponding to α N312 and N511, including their glycosylation consensus sequence, at the corresponding positions in the δ‐subunit, generated two δ‐subunit mutants that were more sensitive to SF.These experiments further confirm that glycosylated asparagines and the attached N‐glycans in particular are crucial for SF sensation of ENaC.Support or Funding InformationThe work was supported by University of Otago and Justus‐Liebig‐University Giessen PhD Scholarships and research grants.
COMMD10: A Novel Regulator Of ENaC Trafficking?
Authors: Ware Adam , Burstein Ezra , McDonald Fiona 
Published: November 2023

The Epithelial Sodium channel (ENaC) is a heterotrimeric (consisting of one α, one β and one γ subunit) Na+ channel and is located at the apical membrane of polarised epithelia, such as the distal kidney, lung and colon, where it has an important role in Na+ reabsorption for maintenance of total body Na+, blood pressure and blood volume. ENaC's trafficking to and from the plasma membrane is a highly regulated but not completely understood process. One protein that has been identified as playing a role in ENaC trafficking is COMMD1, a member of the COMMD (Copper Metabolism Murr1 domain containing) family of proteins. The other family members however have not been as extensively studied, including COMMD10, the focus of our study. Preliminary data has suggested an interaction between COMMD10 and Arf1 (a small GTP binding protein with various functions throughout the trafficking pathway) which suggests a role for COMMD10 in protein trafficking. Our work aims to investigate whether COMMD10 is a novel regulator of the protein trafficking pathway, and thus, whether it is involved in ENaC trafficking to and/or from the plasma membrane. A stable COMMD10 knockdown reduces amiloride‐sensitive short circuit current in αβγ‐ENaC transfected FRT epithelia. A COMMD10 knockdown reduces protein expression of some proteins (Vps35 and COMMD1) involved in the retromer complex (a complex implicated in the recycling of ENaC back to the plasma membrane). These results suggest that when COMMD10 is knocked down ENaC trafficking is disrupted. We have also identified that COMMD10 and Arf1, both in its wild‐type form as well as constitutively active and constitutively inactive forms, co‐localise. However, no co‐localisation was observed between COMMD10 and other Arf family members (Arf4 and Arf5) suggesting an Arf1 specific function for COMMD10. Using a cell‐line in which COMMD10 is stabily knocked down we have identified a change in localisation of the active form of Arf1, that when taken with the other results suggests a role for COMMD10 in the protein trafficking pathway.Support or Funding InformationUniversity of Otago PhD Scholarship