bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc Deficiency Stimulates NADPH Oxidase‐2 Upregulation in Mouse Kidneys
Authors: Li Mirandy S , Gooch Jennifer L , Hoover Robert S , Williams Clintoria R
Published: November 2023

Background

Zn2+ deficiency (ZnD) is common in chronic kidney disease, including diabetic nephropathy. Experimental studies show that ZnD worsens diabetic kidney complications. Oxidative stress caused by increased reactive oxygen species (ROS) plays a role in the harmful effects of ZnD. However, the sources of oxidative stress continue to be identified. In the kidney, NADPH oxidases (Nox) are the predominant enzymes that promote oxidative stress.

Hypothesis

The objective of this study is to determine the role of Nox in ZnD‐induced oxidative stress. We hypothesize that ZnD upregulates Nox, resulting in sustained ROS levels and kidney damage (see proposed schema).

Methods

WT mice were pair‐fed a ZnD or control diet for 6 weeks. After collecting kidneys and urine, kidney damage (urinary albumin:creatinine ratios), ROS levels (Amplex Red and 2′,7′‐dichlorofluorescein diacetate) and Nox expression (qRT‐PCR and Western blot) were examined. To further investigate the effects of Zn2+ bioavailability on Nox, mouse distal convoluted tubular (mDCT) cells were exposed to the Zn2+ chelator N,N,N′,N′‐Tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) or vehicle (DMSO) for 24 hours ± Zn2+ supplementation for an additional 24 hours.

Results

Mice fed a Zn2+‐restricted diet develop microalbuminuria that is accompanied by elevated renal Nox2 expression and ROS levels compared to control‐diet fed mice. In mDCT cells, intracellular Zn2+ depletion with TPEN increases Nox2 expression and ROS levels compared to vehicle‐treated cells. Nox inhibition with diphenyleneiodonium (DPI) prevents TPEN‐induced ROS increases. Furthermore, intracellular Zn2+ repletion reverses Nox2 upregulation induced in TPEN‐exposed mDCT cells.

Conclusions

Taken together, these findings identify Nox2 as a possible mediator of ZnD‐induced oxidative stress and kidney damage.

Significance

Understanding the mechanisms by which ZnD contributes to kidney injury may have important impact on the treatment of diabetic kidney disease.

Support or Funding Information

R25DK101390 (MSL), VA‐MERIT (JLG), R01DK085097 (RSH), T32‐DK00756 (CRW)
Aldosterone Blockade Reduces Low Salt‐induced Renal Oxygen Consumption
Authors: Patinha Daniela , Pihl Liselotte , Fasching Angelica , Palm Fredrik
Published: November 2023

Low salt (LS) diet activates the renin‐angiotensin system, in order to maintain volume homeostasis, which increases active electrolyte transport and thus kidney oxygen consumption (QO2). However, the role of aldosterone in low salt‐induced QO2 and tissue hypoxia is presently unknown.

Male Sprague Dawley rats were fed normal (NS, 0.4%) or low (LS, 0.01%) salt diet during two weeks. Thereafter, catheters were placed in the carotid artery and femoral vein for blood pressure measurement and infusion of 3H‐inulinin thiobutabarbital‐anesthetized animals. The left kidney was immobilized. Measurements were obtained before and after intravenous administration of the aldosterone antagonist canrenoic acid (20 mg/kg). QO2 was estimated from the arterio‐venous difference in oxygen content multiplied by renal blood flow (RBF; Transonic flow probe). Renal cortical and medullary partial pressures of oxygen (PO2) were measured using Clark‐type electrodes.

Mean arterial blood pressure, glomerular filtration rate and RBF were similar in both groups. LS rats had lower fractional excretion of Na+ (FENa) and less efficient Na+ transport (TNa), defined as TNa/QO2 together with increased QO2 and markers of oxidative stress compared to NS. LS intake also reduced cortical PO2 and increased medullary PO2. Acute aldosterone inhibition doubled FENa, normalized QO2 and reduced oxidative stress only in LS.

In conclusion, LS‐induced QO2 and oxidative stress are caused by increased intrarenal aldosterone signaling. However, if the beneficial effects of aldosterone blockade for long‐term kidney function in chronic kidney disease are due to normalized oxygen metabolism remains to be determined.

Support or Funding Information

This work was supported by the Swedish Heart and Lung Foundation and the Swedish Research Council.
Increased Susceptibility to Cisplatin‐Induced Acute Kidney Injury in the Kidneys of Na‐H Exchanger Regulatory Factor Isoform 1 (NHERF1) Deficient Mice
Authors: Sherwood Amanda Renee , Khundmiri Syed Jalal , Conklin Caryl Anne , Siskind Leah Renee , Bushau Adrienne , Dupre Tess , Barati Michelle , Merchant Michael , Lederer Eleanor DeLand
Published: November 2023

NHERF1 is a PDZ binding protein that plays a critical role in defining both the renal proximal tubule brush border membrane (BBM) composition and regulating ion transport through modulation of trafficking and anchoring functions. In a NHERF1‐deficient opossum kidney (OK) cell line, we have observed slower cell growth, a decrease in total mRNA expression, diminished BBM protein glycosylation, and higher levels of phosphorylated PERK, a marker of ER stress. We hypothesized that kidneys from NHERF1 deficient mice were more susceptible to acute kidney injury as a result of increased ER stress. To test this hypothesis, we treated 2 month old WT and NHERF1 deficient mice with 20 mg/kg IP cisplatin for 72 hours. Hemotoxylin and eosin staining of the kidney cortex of NHERF1 deficient mice showed greater injury manifested by BBM sloughing, tubular cell detachment and vacuolization, and intratubular proteinaceous debris. NHERF1 KO mice treated with cisplatin also demonstrated decreased expression of GRP78, a prosurvival ER chaperone that is upregulated during ER stress. Using ID‐RP (C18) nanoflow ultra high performance liquid chromatography and nanoelectrospray‐mass spectrometry (MS), we examined the composition of the BBM of proximal tubule cells from 4 month old NHERF1 KO mice. Compared to WT mice, NHERF1 KO mice demonstrated decreased abundance of several proteins involved in cell survival including ACADS, CDK18, and PRKAR2A in the BBM, while the abundance of proteins specific to renal cell proliferation and survival such as FGFR2, GRN, and NOS3 were decreased 3 to 25 fold. Interestingly, proteins such as DAPK1, a positive mediator of gamma interferon‐induced programmed cell death, and GAK, a direct transcriptional target of p53 that is itself a stress‐induced protein involved in cell cycle arrest and apoptosis, were increased 2 and 19 fold in the NHERF1 KO BBM. We conclude that NHERF1 deficiency increases susceptibility to cisplatin‐induced AKI due to the presence of underlying ER stress and reduced expression of signaling pathways that promote cell survival.

Support or Funding Information

University of Louisville, NIH‐NIA Grant # 5R21 AG047474‐02
Proximal‐tubule‐select dual knockout of insulin and insulin‐like‐growth factor (type I) receptors in mice alters responses to dietary fructose
Authors: Ecelbarger Carolyn M. , Doh Kwame , Lee Hwal , Tiwari Swasti , Li Lijun
Published: November 2023

The monosaccharide, fructose is a common dietary constituent, but metabolized quite differently from glucose. One site of fructose metabolism is the renal proximal tubule (PT). How and whether insulin or the related hormone, insulin‐like‐growth‐factor, type 1 (IGF1), modulate fructose metabolism at this site is not known. To address this, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) from PT by Cre‐lox recombination using a γ‐glutamyltransferase promoter. Male (M) and female (F) wild‐type (WT) and KO mice were fed control (C) or high fructose (Fr) diet (60%) for 4 weeks (n = 10–12/group). KO mice had marginally lower body and/or kidney weights. Fructose feeding did not affect final body weight. Glucose tolerance was significantly impaired by fructose feeding, but only in the WT mice. KO mice also had a phenotype suggesting renal tubular acidosis, i.e., with significantly elevated blood chloride levels, and decreased blood total CO2 levels. Plasma sodium was also reduced in male KO by fructose, but not in the WT or females. Western blotting of cortex homogenates revealed significantly reduced (2‐way ANOVA) band densities for IR, IGF1R, and NaPi‐2 (sodium phosphate cotransporter) in the KO mice. However, no genotype differences were found for band densities of the fructose transporter (GLUT5) and ketohexokinase (KHK), the first enzyme in fructose metabolism, although the abundances of these two proteins were significantly increased in males and in fructose‐fed animals. The sodium bicarbonate cotransporter (NBC) was reduced in KO, but not WT males by fructose‐feeding. Females, overall, had significantly lower levels of NBC. Overall, genotype differences in the metabolic responses to fructose suggest a role for PT IR/IGF1R expression as a modulator of PT metabolism and/or acid‐base homeostasis.

Support or Funding Information

Marriott Cardiovascular Fellowship and Georgetown Internal funding
Functional Dynamics of Cysteines in the Epithelial Na+ Channel Finger Domains
Authors: Blobner Brandon Michael , Kashlan Ossama B
Published: November 2023

The epithelial Na+ channel (ENaC) is a member of the ENaC/Degenerin family of ion channels. The structure of one member of this protein family, acid sensing ion channel 1 (ASIC1), has been resolved. ASIC1 shares high sequence homology with ENaC. ENaC has a set of cysteines in the extracellular domains of each subunit that form disulphide bonds, as is characteristic of this protein family. ENaC subunits contain an additional pair of cysteines in the poorly conserved extracellular finger domains that are not present in ASIC1. The disposition of these cysteines is unclear. Using bifunctional methanethiosulfonate (MTS) crosslinkers and the oxidizing agent copper phenanthroline, we probed the region surrounding the finger domain cysteines. Our data shows that the finger domain cysteines of both the alpha and gamma subunits are positioned in close proximity to the finger‐thumb domain interface of the respective subunits. Crosslinking to these cysteines affected ENaC currents, suggesting mechanical linkage between this peripheral site and the channel gate. Our data also shows that removal of either gamma finger domain cysteine results in the loss of irreversible ENaC inhibition by MTS reagents, an affect that we previously reported. Our data suggests that ENaC finger domain cysteines may be experimentally modified in a manner that regulates ENaC activity.

Support or Funding Information

This work is supported by NIH grants DK098204 and DK061296‐12.
Ankyrin G Alters ENaC Membrane Delivery to Increase Na+ Transport in the Distal Kidney Nephron
Authors: Klemens Christine Anne , Kightlinger Lindsay , Liu Xiaoning , Edinger Robert , Butterworth Michael Bruce
Published: November 2023

The heterotrimeric epithelial sodium channel (ENaC) comprised of α, β, and γ subunits, is the predominant aldosterone regulated sodium (Na+) channel in the kidney distal nephron, and plays an essential role in fine‐tuning Na+ reabsorption into the blood. Stimulation by aldosterone induces a number of changes to increase urinary Na+ reabsorption, including increasing ENaC subunit translation and modifying key regulators that alter ENaC function and/or membrane density. We identified ankyrin G (AnkG) to be a novel, aldosterone stimulated, ENaC regulator. Knock down of AnkG expression reduces the endogenous amiloride sensitive (ENaC) current by 69%±1.57% (n=15, p<0.0001), while over expression of AnkG increases ENaC current by 67%±11% (n=12, p=0.0001) in mouse cortical collecting duct (mCCD) cells. Membrane permeabilization verified that this alteration of Na+ transport was through ENaC and not a secondary effect of intracellular driving force via altered Na+K+ATPase. The aim of this study was to determine the mechanism behind AnkG's regulation of ENaC activity, whether through increased surface delivery, altered channel open probability, increased membrane dwell time, increased intracellular recycling, or blunted internalization and degradation. We propose that AnkG expression modulates Na+ reabsorption in collecting duct cells by increasing ENaC delivery to the surface, thereby increasing the number of channels present at the apical membrane.

Biotinylation of surface βENaC determined that AnkG over expression increases the number of channels at the surface 71%±26% (n=4, p‐value=0.02). Using a combination of electrical and biochemical pulse‐chase assays, we found that AnkG expression does not alter ENaC internalization rates or ENaC recycling; however, mCCD cells with reduced AnkG had significantly slower apical delivery rates (k=0.1361 min−1 ±0.0081vs k=0.1867 min−1±0.0045, n=12, p<0.0001), while AnkG over expression in mCCDs accelerated ENaC delivery (k=0.1724 min−1 ±.0020vs k=0.1298 min−1±.0035, n=9, p<0.0001). Neither AnkG knock down or over expression altered the total amount of ENaC protein in the mCCD cells, suggesting AnkG is impacting ENaC delivery rates, rather than the quantity of ENaC delivered to the surface.

These findings highlight the role of AnkG as an aldosterone induced protein and novel regulator of ENaC that could alter Na+ reabsorption in the distal nephron. Ongoing studies aim to fully characterize the molecular mechanisms underlying this dynamic interaction.

Support or Funding Information

15PRE25680068, DK079307, UPMC Cell Biology Teaching Fellowship
The Role Of Several Prominent Claudin Tight Junction Proteins In Teleost Fish Gill Epithelium Paracellular Permeability
Authors: Kolosov Dennis , Donini Andrew , Kelly Scott P.
Published: November 2023

Because the internal fluids of freshwater (FW) teleost fishes are hyperosmotic relative to their surroundings, these organisms experience obligatory ion loss across epithelia that directly interface with water (e.g. the gill epithelium). In FW fishes, the tight junction (TJ) complex is broadly acknowledged to limit paracellular ion loss across the gill epithelium as it occludes the paracellular cleft and therefore acts as a barrier to impede paracellular solute movement. The vertebrate TJ complex is composed of numerous TJ proteins and the major proportion of these belong to the claudin (Cldn) superfamily. Cldn TJ proteins are not only implicated in regulating general permeability but also ion‐specific permselectivity of various vertebrate epithelia. Several Cldns seem to be particularly important for the establishment and maintenance of the barrier properties of the fish gill epithelium. Additionally several important ionoregulatory hormones in teleost fishes (e.g. cortisol), have been implicated in the regulation of paracellular permeability of FW teleost fish gill epithelia by exerting their actions on specific Cldns and other TJ proteins. Nevertheless, a clear picture of how paracellular movement of specific ions is regulated by the complex molecular architecture of TJs in an architecturally complex epithelium is only beginning to emerge. This has been achieved in part, by using primary cultured ‘reconstructed’ gill epithelia composed of different gill cell types. These architecturally simple models permit endpoints of transepithelial and paracellular permeability to be measured across a tissue that otherwise exhibits a restrictive morphology and problematic cellular heterogeneity. This can be done in the presence of ionoregulatory hormones and measurements of permeability can then be coupled with manipulations of TJ architecture via transcriptional knockdown of TJ proteins using dsRNA. This study will present an examination of the contribution of several Cldns to gill epithelium permeability as well as endocrine mechanisms regulating TJ protein abundance in the gill epithelium. Particular attention has been paid to the compensatory response caused by transcriptional knockdown of specific Cldns in the gill epithelium of fish as this area has been entirely overlooked, despite the fact that the teleost genome is well known to possess a particularly large number of Cldn genes due to teleost‐specific genome duplication events.

Support or Funding Information

This work was funded by National Science and Engineering Research Council of Canada
Heterogeneity of Signal Transduction by Na/K‐ATPase α‐Isoforms: Role of Src Interaction
Authors: Yu Hui , Cui Xiaoyu , Xie ZiJian
Published: November 2023

Among the four α isoforms expressed in mammalian cells, we have shown that the ubiquitous α1 Na/K‐ATPase possesses both ion‐pumping and Src‐dependent signaling functions. In contrast, we have reported that α2 Na/K‐ATPase pumps but does not conduct Src‐dependent signal transduction, which represents a major functional difference between α isoforms. We have identified two putative pairs of domain interactions between α1 Na/K‐ATPase and Src, but others have challenged the concept of a direct interaction between Na/K‐ATPase and Src. In support of our model, a sequence comparison revealed major differences between α1 and α2 in these two putative Src‐interacting domains, in contrast to the overall high (~80%) level of homology between the α1 and α2 polypeptides. In this study, we tested whether targeted point mutations in the two putative Src‐interacting domains could confer “α1‐like” Src‐regulating properties to α2.

To this end, we generated stable mammalian cell lines that express almost exclusively a α2 polypeptide mutated to mimic α1 sequence in the putative Src‐interacting domains. Functional analyses of α2 and α2 mutant cell lines reveal the following. First, the mutant α2 was fully functional as an ion pump. Second, unlike α2, the mutated α2 gained α1‐like signaling function, was capable of Src regulation, and restored caveolin‐1 expression. Because these mutated sequences of α2 are highly conserved across different species, the lack of Src‐regulation by α2 appears to be a conserved property of this isoform, most likely to provide α2‐specific and physiologically important functions that Src interaction could alter. Third, as expected, ouabain activated Src/ERK in the α2 mutant, but not in α2‐expressing cells. Finally, the mutant α2 cells exhibited a growth phenotype comparable to that of α1, which was much faster than that of α2 cells. Taken together, these new findings not only reveal the structural requirements for the Na/K‐ATPase to function as a Src‐dependent receptor, but also provide strong evidence of isoform‐specific Src interaction involving the identified key amino acids.
Pig Renal Na/K ATPase and Bcl‐2 Proteins: A Co‐Immuno‐Precipitation Study
Authors: Maharjan Chandra Kumar , Heiny Judith , Adragna Norma C , Lauf Peter K
Published: November 2023

The Na/K ATPase (NKA) has at least two Bcl‐2 like sequences (Cell Physiol. Biochem. Cell Physiol Biochem 31: 257–276, 2013) and, in human lens epithelial cells, co‐immuno‐precipitates with the Bcl‐2 proteins BclXL and Bak (Am J. Physiol. Cell Physiol 2015: 308: C51–C60). Here we searched for immunochemical evidence of the presence of BH3 motif‐containing Bak co‐immuno‐precipitated with the well‐characterized pig renal NKA (PRNKA) (Klodos et al., Kidney Int. 62:2097–2100, 2002).

Results

PRNKA was prepared by the Klodos procedure omitting the final SDS purification step to preserve NKA activity at 0.45 μmol in 5.54 mg/ml. Mouse (ms) anti‐NKA (α6F) antibody (ab) showed the α1 subunit of ~110 kDa in immunoblots (IB) of the PRNKA input, in the protein‐G coated metal bead (PGCMB) ‐ α6F immuno‐pulldown, as well as in PGCMB‐ms and rabbit (rb) IgG pulldowns. A rb polyclonal anti‐human Bak BH3 motif ab (Cell Signaling #3814) together with a secondary (2nd) goat anti‐Rb Fc’ heavy (H) chain ab detected a low level staining of a ~27 kDa peptide in the PRNKA input and a prominent band in the α6F immuno‐pull down suggesting the presence of Bak in the enzyme preparation. IBs with the 2nd anti‐Fc’ H‐chain ab alone, however, not only detected IgG H chains in α6F ab‐, and ms‐ and rb‐IgG immuno‐pull downs, but unexpectedly also in PRNKA input. These findings suggest the presence of 1) Bak, and 2) NKA in PRNKA inputs and in immune and “non‐immune” pulldowns, with an absence of both proteins in ms and rb IgG input only; and 3) an apparent IgG contamination in PRNKA. Because of the latter, our efforts are now directed to discriminate further between Bak and L chains both of very close molecular mass. Whether the triple occurrence of NKA, Bak and IgG in all pull downs is IgG‐molecular specific but not species‐specific or coincidental due to non‐specific adsorption to the protein G beads, or both, is currently under study.

Support or Funding Information

Supported by Wright State University Foundation to the Cell Biophysics Laboratory, by the Pharmacology and Toxicology Program to C.K.M., and by NIH to JH.
Oxygen Channels in Red Blood Cells
Authors: Zhao Pan , Geyer R. Ryan , Sim Edward S. , Salameh Ahlam I. , Meyerson Howard , Wass Amanda B. , Moss Fraser J. , Boron Walter F.
Published: November 2023

It is well known that there are at least three families of membrane proteins—aquaporins (AQPs), rhesus (Rh) proteins, and urea transporters (UTs)—function as channels for CO2 or NH3. In human red blood cells (RBCs), AQP1 and the Rh complex (mainly RhAG) together account for ~90% of membrane CO2 permeability. We hypothesize that most O2 crosses RBC membranes through known polytopic proteins that also act as O2 channels. We quantitated O2 efflux through mouse RBC membranes using stopped‐flow (SF) absorbance spectroscopy to determine the O2 saturation of hemoglobin (Hb). For each mouse, we correct the raw data for modest degrees of hemolysis using our novel SF hemolysis assay, based on the release of carbonic anhydrase (CA) enzymes from RBCs. We find that SF analysis per se increases hemolysis from ~0.5% before a SF experiment to ~5% during a SF experiment on wide type (WT) mouse RBCs. AQP1‐KO, RhAG‐KO and double knockout (dKO) mouse RBCs exhibit similar degrees of hemolysis. After correction for hemolysis, AQP1‐KO decreases the rate constant for O2 efflux (kO2) by ~11%. RhAG‐KO decreases kO2 by ~15%, and dKO decreases kO2 by ~30%. Hematology results shows that mean corpuscular volume (MCV), compared to WT mice, is 8% and 6% higher in RhAG‐KO and dKO mice (p<0.01), respectively, and 2% higher in AQP1‐KO mice (p>0.05). DIC microscopy of living RBCs and microscopic observation of stained blood smears confirms that RBCs from all four groups are normal biconcave disks, and flow cytometry confirms that the major disk diameter is indistinguishable for all four groups. Thus, RBCs from our KO mice probably are a few % thicker than WT RBCs, differences that would not appreciably affect kO2. Mass spectroscopy of RBC ghosts indicates that the KOs do not unexpectedly affect levels of other proteins. Thus, AQP1 and RhAG, the two dominant CO2 channels in human RBCs, together make an important contribution to O2 permeability in mouse RBCs. Our data provide the first evidence that membrane proteins, presumably acting as channels, can enhance O2 permeability, which would be a major paradigm shift.

Support or Funding Information

Office of Naval Research Awards N00014‐14‐1‐0716 and N00014‐15‐1‐2060
Bicaudal‐C in Drosophila as a model of polycystic kidney disease (PKD) and intersection of oxalate nephrolithiasis
Authors: Knier Catherine Grace , Landry Greg M. , Romero Michael F.
Published: November 2023

Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of end‐stage renal disease in adults affecting 1 in 800 people in the US. Knocking down bicaudal‐C (BicC) in zebrafish and Xenopus has been reported as a method used to model the cystic changes in ADPKD. Gamberi and colleagues [JASN 25: 538A, 2014] recently presented a similar knockdown in Drosophila melanogaster resulting in cystic Malpighian Tubules (MTs) homologous to the human kidney. Additionally, calcium oxalate (CaOx) kidney stones affect ~1 in 10 people in the US, with the incidence increasing ~2‐fold in PKD patients.

Methods

Our group has investigated the PKD phenotype in Drosophila using a Gal4/UAS‐RNAi system to knockdown the expression of BicC in MTs. We initiated CaOx crystal formation by feeding Na‐oxalate or adding to tubule bathing solution [Hirata, AJP 2012].

Results

Penetrance of Uro, CapaR, and c724 drivers in the MTs were compared using UAS‐YFP, resulting in Uro::BicC expression in ~50% of cells with CapaR::BicC approaching 100% and c724 expression specific to MT stellate cells only. MT morphology was analyzed: controls [WT (0%); parental (18%)] occurrence v BicC knockdowns [Uro::BicC (25%); CapaR::BicC (15%); c724::BicC (6.3%)]. Functional tests using Ramsay secretion assays reveal a slower secretion rate in Uro::BicC, CapaR::BicC, and c724::BicC knockdowns compared to WT MTs. In CapaR::BicC knockdowns, slower secretion rates are observed even after stimulation with Drosophila‐kinin. Since there is an increased risk for ADPKD‐related kidney stones, MT calcium oxalate (CaOx) crystallization experiments were performed to quantify crystal formation in BicC knockdown vs WT MTs. While crystals are present in all oxalate exposed MTs, BicC knockdowns using Uro::BicC and c724::BicC form more CaOx crystals than CapaR::BicC and WT. However, crystals formed by Uro::BicC and c724::BicC knockdowns have a significantly smaller total crystal area and average area/individual crystal compared to both CapaR::BicC and WT.

Conclusions

These results suggest that BicC knockdown parallels kidney stone increases associated with human PKD, but does not yet consistently mimic morphologic ADPKD‐induced cystic phenotypes usually observed in PKD models.

Support or Funding Information

DK90278, DK101405, DK101405
Store Operated Ca2+ Entry Suppressed TGFβ1‐ Smad3 Signaling Pathway in Glomerular Mesangial Cells
Authors: Chaudhari Sarika , Li Weizu , Ma Rong
Published: November 2023

Excessive expression of extracellular matrix (ECM) proteins in glomerulus is one of the consistent pathological changes seen in kidney diseases, such as diabetic nephropathy. Transforming growth factor‐ β1 (TGFβ1)‐Smad3 pathway plays a critical role in ECM protein expression and renal fibrosis. Receptor binding of TGFβ1 leads to phosphorylation and subsequent translocation of Smad3 into the nucleus regulating transcription of target genes directly or indirectly.

The Orai1‐mediated store operated Ca2+ entry (SOCE) is associated with many physiological and pathological processes in a variety of cells, including glomerular mesangial cells (MCs) which are a major source of ECM proteins. We have previously demonstrated that SOCE in MCs suppressed ECM protein expression in vitro and in vivo. However, the downstream mechanism underlying the SOCE effect is not known. The present study was conducted to test the hypothesis that SOCE suppressed ECM protein expression by inhibiting TGF β1‐Smad3 pathway in MCs. In cultured human MCs, TGFβ1‐induced phosphorylation and translocation of Smad3 was examined in presence and absence of thapsigargin (TG) (1 μM), a classical activator of store‐operated Ca2+ channel. We found that treatment with TGFβ1 (5 ng/ml for 15 hours) significantly increased the expression level of Phospho‐Smad3 (p‐Smad3) evaluated by Western blot. However, this response was markedly inhibited by TG treatment. Consistently, immunocytochemistry and Western blot showed that TGF β1 significantly increased the expression of nuclear Smad3. Again, this TGFβ1‐induced nuclear translocation of Smad3 was prevented by pre‐treatment with TG. Importantly, the TG effect was reversed by La3+ (5 μM) and GSK‐7975A (10 μM), both of which are selective blockers of store‐operated Ca2+ channel. Furthermore, knockdown of Orai1 using siRNA approach significantly augmented TGFβ1‐induced p‐Smad3 expression. Taken together, our results indicate that SOCE in MCs negatively regulates the TGFβ1/Smad3 signaling pathway, and thus could be a potential therapeutic target of kidney disease with glomerular fibrosis.

Support or Funding Information

UNTHSC Institutional Seed Grant, UNTHSC Doctoral Student Bridge Grant, Sigma Xi Grants ‐in‐Aid research grant.
Expression of KCNJ1 (ROMK) in the Gastrointestinal Tract
Authors: Han Jaeyong , Lee Seung‐Hun , Ishikawa Yasunobu , Guo Xiaoyun , Xu Shuhua , Wang Tong
Published: November 2023

It is well understood that the renal outer medullary potassium channel (ROMK; KCNJ1) is expressed in the apical membrane of the renal tubules along the distal nephron and plays an important role in K+ secretion and Na+ and K+ homeostasis. In the gastrointestinal (GI) tract; however, little is known whether ROMK plays a role in K+ secretion. We previously reported that ROMK is functionally expressed in the parietal cells and is involved in gastric acid secretion. Our current study examines ROMK expression and localization in other parts of the GI tract, and also examines whether the expression of ROMK channel is influenced by K+ intake. ROMK expression was examined in the esophagus, ileum, proximal and distal colon of mouse tissue collected from 20‐week old ROMK wild‐type and KO mice. The ROMK mRNA expression was examined by real‐time PCR and immunofluorescence (IF) staining. ROMK specific antibodies were used to study ROMK expression and localization at the protein level. Experimental data show ROMK mRNA expression in esophagus, Ileum, proximal and distal colon in WT mice, but there was no expression detected in these segments of ROMK KO mice. Q‐PCR data showed a slightly increase in ROMK mRNA expression in Ileum, proximal and distal colon in animals treated with high K+ diet (5% K+ diet for 7days) compared with normal K+ dieted animals. IF staining showed very weak ROMK staining in the esophagus, but clearly stained in Ileum, proximal and distal colon. The localization of ROMK is in the epithelial cells; there was no co‐localization of ROMK with the nuclear marker DAPI. In conclusion, ROMK is expressed in the epithelial cells of the Ileum, proximal and distal colon and HK intake might increase ROMK expression in the GI tract.

Support or Funding Information

NIH RO1 DK099284 and PO1 DK17433
Effects of TNFα challenge on sensory neuron ion channel expression and temperature sensitivity
Authors: Staunton Caroline Amy , Kumagai Kosuke Amy , Thippeswamy Thimmasettapp , Djourhi Laiche , Barrett‐Jolley Richard Amy
Published: November 2023

Chronic neuropathic pain is a major worldwide cause of disability with over 116 million adults affected in the US alone. In many cases, direct neuronal effects of cytokines are thought to contribute to the associated temperature and mechanical hypersensitivity. In a previous report we showed that the resting membrane potential of TNFα treated dorsal root ganglion (DRG) neurons was hyperpolarized compared to controls, when measured at 30°C. In this study we extended this work to measure TNFα (24hr, 10ng/ml) induced changes in underlying ion channel biomarker mRNA abundance (with qPCR), absolute resting action potential (AP) frequency and on its temperature sensitivity (using cell‐attached patch, action current recording). DRG neurons were isolated and prepared as described previously [1]. Analysis of whole DRG, glial cell depleted cultures revealed that several ion channels were differentially transcribed following TNFα treatment. Interestingly, there was a statistically significant switch from TRPV4 (transient receptor potential cation channel, subfamily V, member 4) to TRPM2 (transient receptor potential cation channel, subfamily M, member 2) transcript (TRPM2 up >3 fold, TRPV4 down >2 fold; both p<0.001) and significant shifts from KCNA1/2 to KCND2/3). For patch‐clamp experiments, only small DRG neurons (<35pF) were chosen, since they exhibit predominantly temperature and nociceptive phenotypes. Whilst we found that resting action current frequency was lower in TNFα treated neurons than controls, we found that the increase in action current frequency following a +15°C temperature challenge was significantly greater in TNFα treated neurons (1.6 fold increase in control vs 3.5 fold increase after TNFα challenge). These changes are generally smaller than previously reported. Following mathematical simulations, we conclude that this may be due to our use of cell‐attached AP frequency recording. This technique will allow native cellular control of intracellular Ca2+ and may allow endogenous feedback control that may be lost when using conventional whole‐cell patch‐clamp recording.

Support or Funding Information

The authors wish to acknowledge the D‐Board project, which has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 305815
NMDA Receptors Modulate Brainstem Excitatory Neurotransmission in Spinal Trigeminal Nucleus
Authors: Jennings Ernie A
Published: November 2023

Nociceptive inputs from craniofacial regions relayed via trigeminal primary afferents terminate primarily in the trigeminal subnucleus caudalis (the medullary dorsal horn, MDH). We have previously reported that increased glutamatergic neurotransmission induced by purinergic receptor activation can be attenuated in vivo and in vitro with N‐methyl‐D‐aspartate receptor (NMDAR) antagonists D‐AP5 and MK‐801, in addition to the P2X2/3 receptor antagonists A317491 and TNP‐ATP. To further characterise the role of NMDAR in trigeminal nociceptive processing, we investigated the effects of NMDA (100μM) and D‐AP5 (40μM) on pharmacologically isolated, evoked excitatory postsynaptic currents (eEPSCs) by using whole‐cell voltage‐clamp recordings from MDH deep laminae in brainstem slices. eEPSCs were evoked by electrical stimulation of trigeminal spinal tract axons. Application of NMDA significantly decreased eEPSC amplitude (n = 9, p < 0.05: Control: 509.8 ± 179.4 pA, NMDA: 413.7 ± 169.6 pA), whereas D‐AP5 did not significantly depress the eEPSC amplitude (n = 7, p > 0.05: Control: 285.9 ± 47.0 pA, AP5: 249.7 ± 48.4 pA). Paired‐stimuli (70ms interstimulus interval) are traditionally used to indicate whether an effect is either pre‐or postsynaptic. This MDH synapse exhibited paired‐pulse depression with a paired‐pulse ratio (PPR) less than 1 under control conditions, indicating a high probability of release. Bath application of D‐AP5 did not alter the PPR (n = 7, p > 0.5, Control: 0.68 ± 0.08, AP5: 0.66 ± 0.05), whereas NMDA resulted in a significant PPR increase (n = 16, p < 0.05, Control: 0.57 ± 0.05, NMDA: 0.70 ± 0.05). With the inclusion of the NMDAR open‐channel blocker MK‐801(1mM) in the pipette internal solution, to exclude potential contributions by postsynaptic NMDARs, NMDA caused a significant increase in PPR (n = 16, p<0.005) and marked depression of the first eEPSC (p < 0.005, Control: 500.5 ± 62.4 pA, NMDA: 445.4 ± 57.3 pA), suggesting that NMDA was not acting on the postsynaptic neuron. Furthermore, the eEPSC decay kinetics were not altered by either D‐AP5 or NMDA when compared to control (D‐AP5: p > 0.1; NMDA: p > 0.1), further suggesting that postsynaptic NMDARs are not predominantly involved in this attenuation. Together, these results suggest NMDARs, located at sites other than on the postsynaptic neuron, modulate glutamatergic neurotransmission between trigeminal primary afferent fibres and neurons in the deep laminae of the medullary dorsal horn

Support or Funding Information

Funding provided by Australian NHMRC project grant 454606
Mechanisms of Passive Inward Transport in Hyperaldosteronism Mutants and Normal Na/K Pumps
Authors: Meyer Dylan , Spontarelli Kerri , Allen Morgan , Olivera J. Fernando , Artigas Pablo
Published: November 2023

Hyperaldosteronism (HA)‐inducing mutations in the α1 subunit of adrenal Na/K pumps (NKAs) have been found to induce a passive Na influx through the mutated NKAs under near‐physiological conditions (> 100 mM Nao;4.5 mM Ko). Under such conditions, wild type (WT) NKA catalyzes the uphill transport of 3 Na out of the cell in exchange for 2 K using the energy of one ATP molecule, a process that induces an outward current. However, under non‐physiological conditions, WT NKAs passively import H when the [Na]o and [K]o are not enough to saturate externally facing ion bindingsites. Here we use electrophysiology(two electrode voltage clamp and patch clamp) in oocytes expressing mutant NKAs to test the characteristics of the ion pathways through WT and mutant NKAs and whether the inward currents (IIN) induced by HA mutations are large enough to significantly depolarize the adrenal cells, or whether the observed depolarization (~30 mV) is secondary to the distorted ion homeostasis.

WT H IIN occurs through a mechanism where the ions traverse the ion binding site that exclusively binds Na (site III). To test the contribution to IIN of ion binding sites I and II (which bind Na or K during the normal cycle), we simultaneously introduced 3 mutations(E327D, N776T, E779D) to disrupt ion binding there by increasing the size of the ion‐binding cavity. Resembling the HA mutations, the triple mutant presented a large IIN carried by Na under physiological conditions. Evaluation of the single mutants attributed this IIN to E327D in trans membrane segment (TM) 4. It has been proposed that the Na ions transported through IIN in two HA mutations, L97R (in TM1) and V325G (in TM4), involves a pathway for Na ions distinct from the H pathway in WT NKAs. To test this proposal, we concomitantly introduced the site III mutation D926N, known to abolish H permeation in WT pumps, with L97R, V325G, and E327D templates. Large IIN was observed in both L97R/D926N and V325G/D926N, while the E327D/D926N lacked IIN. This suggests that the pathway for Na current through these two HA mutants is different than the pathway for H through WT NKA. Additional studies will further clarify whether the effect of D926N on E327D is due to lack of expression, lack of a required partial reaction, or due to a pathway distinct from the HA mutants. We are also using giant inside‐out patches to compare the intracellular NKA ligand (ATP, Na, K, etc.) requirements for Na current through WT and mutant NKAs.

To evaluate if the IIN observed in mutant NKAs is enough to significantly depolarize the adrenal cells, we are measuring IIN while counting the total number of NKAs present in the membrane. If the total IIN/NKA molecule is similar to the total IP/NKA molecule in WT pump, it is unlikely that the depolarization is a direct reflection of charge influx through the mutated pumps in the cell (half of the total pumps).

Support or Funding Information

Funded by NSF1515434
The Calcium‐Activated Chloride Channel, Anoctamin‐1, is a Critical Regulator of Cell Proliferation Mediated via p44\42 MAPK Phosphorylation and Calcium Signaling
Authors: Franklin Brandon M , Osborn Jeffrey L
Published: November 2023

Soft tissue repair is a critical function of living organisms which requires restoration of anatomical structure and function following tissue damage. A better understanding of the cellular and molecular mechanisms that control critical cellular processes, such as proliferation, is crucial to the development of clinical therapies in regenerative medicine. We have recently shown that chloride channel inhibition, via 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS), 4,4′‐Dinitro‐stilbene‐2,2′‐disulfonic acid (DNDS), T16a(inh)‐A01 or Cl− free environment effectively inhibits or delays tail regeneration in larval Ambystoma mexicanum salamanders. Proliferation was assessed via EdU incorporation assay, which revealed that this effect was manifested through regulation of mesenchymal cell proliferation, with all 3 pharmacological treatments reducing proliferation rate in the mesenchyme (Control: 33.5±3.2%; DIDS: 18.13±2.25%; DNDS: 19.64±3.79%; T16a(inh)‐A01: 19.65±1.85% EdU+ cells). There is no effect on proliferation in either neural or epidermal tissues. Western blot analysis demonstrated that phosphorylation of p44/42 MAPK (Erk1/2) is reduced when the calcium activated chloride channel, Tmem16a, is inhibited with T16a(inh)‐A01 in 72 hpa regenerates. These results indicate that the calcium activated chloride channel, anoctamin‐1, regulates proliferation via p44\42 MAPK. A transgenic HEK 293 cell line that overexpresses the anoctamin‐1 channel is being used to further elucidate the channel mechanisms involved in Cl‐channel mediated cell proliferation. Fluorescent calcium indicators will provide semi‐quantitative evidence regarding the influence of anoctamin‐1 on intracellular calcium signaling.

Support or Funding Information

Supported by Biomedical Sciences Research Group, LLC.
Cercariae of Proterometra macrostoma may use catecholamines as endogenous neurotransmitters
Authors: Bumpus Erin Nicole , Runyan Keith R , Weitekamp Samuel L , Bartlow Kathleen Marie
Published: November 2023

Furcocystocercous cercariae of the digenetic trematode parasite Proterametra macrostoma exhibit a characteristic vertical swimming behavior to attract their centrarchid hosts. Previous studies showed that swimming behavior is entirely controlled by the cercarial tail, and may be modulated by factors including light wavelength and mechanical stimulation. We conducted studies using a vertical swim‐up assay to examine the behavioral effects on P. macrostoma cercariae of exogenous application of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Catecholamine neurotransmitters increased the speed of swimming. Norepinephrine also increased the number of swims per cercaria in a five‐minute period and the time each cercaria spent off the bottom of the water column, implying that behavioral effects of noradrenergic signaling in this species may be ecologically relevant. Studies conducted in whole‐mounted cercariae, using immunofluorescent staining to detect the neuronal marker beta‐III‐tubulin, reveal dense superficial innervation in the posterior cercarial tail in close apposition to striated muscle fibers. Cryosections immunostained for tyrosine hydroxylase (TH) display TH immunoreactivity throughout the distome body and tail of cercariae, particularly in the papillae of the tail, which are thought to be sensory organs. Together, these results suggest that catecholamines are produced and used by P. macrostoma as a neurotransmitter modulating cercarial swimming.

Support or Funding Information

These studies were funded by the Indiana Academy of Science, and by the following funds associated with Earlham College: Gerald Bakker Collaborative Research Endowment Fund, Matthews Student/Faculty Research in Physics/Biological Science, Harry Todd Costello Fund, Stevenson Fund, and Collaborative Research Fund.
The costs of aggressivity and the benefits of cooperation
Authors: Fox Trevor , Klok Jaco , Harrison Jon , Fewell Jennifer
Published: November 2023

Queens of the harvester ant Pogonomrymex californicus have been documented as having two distinct behavioral phenotypes controlling both aggression and the tendency to form foundress associations. Pleometrotic queens found colonies cooperatively with nest‐mates while haplometrotic queens found solitary nests. Haplometrotic queens are larger and more aggressive to other queens, and produce eggs at a higher rate during colony founding. We tested for possible metabolic differences between these types, predicting that the non‐aggressive pleometrotic queens would have lower metabolic rates, and that pairs of pleometrotic queens would have even lower rates (per ant) as prior studies have shown reduced work and per capita egg production in pairs relative to solitary founders. CO2 emission and O2 consumption was measured using a stop‐flow respirometry system. We tested individual haplo‐ and pleometrotic individuals and pairs of pleometrotic queens. Respiratory quotients averaged. 68 and did not differ among treatments, suggesting that these queens were primarily metabolizing lipids. Indeed, our results showed that the paired pleometrotic queen had a significantly lower metabolic rate (.0066 mlCO2/hour·animal) than the individual pleometrotic queens (.0077 mlCO2/hour·animal), while haplometrotic queens had the highest metabolic rate of the three groups (.009 mlCO2/hour×animal), suggesting that the aggressivity and high egg‐laying rates of the haplometrotic queens has a measurable and significant cost, and that cooperative founding reduces per capita energy use. These differences in energy use may partially explain differences in survival of queens, as queens do not forage during the prolonged period of colony founding.

Support or Funding Information

This research was partially supported by NSF IOS 1122157 and NSF DMS 1313312.
Hatchling Phenotype Of Lake Whitefish Incubated At Increased Temperature During Critical Windows Of Development
Authors: Mueller Casey , Eme John , Manzon Richard , Somers Christopher , Boreham Douglas , Wilson Joanna
Published: November 2023

Phenotypic plasticity is the ability of an animal to modify its phenotype in response to the environment. Embryos may have critical windows during development when they are particularly plastic or susceptible to the environment. We examined whether there are critical windows during Lake whitefish (Coregonus clupeaformis) embryonic development when temperature has an especially strong effect on phenotype. Lake whitefish are a cold‐water fish species that inhabits the North American Great Lakes. This species lays eggs that develop over winter when lake temperatures are near freezing. With 2°C as the overall control temperature, embryos were incubated at either 5°C, 8°C or 11°C for all of embryonic development or only during one of four distinct windows of development: gastrulation, organogenesis, fin development, and late growth. Survival was monitored throughout development. Hatching characteristics, including time to hatch, mass at hatch, and hatchling oxygen consumption rate, were measured to examine if warmer temperatures during the four windows of development altered hatchling phenotype. No embryos survived to hatch when incubated constantly at 11°C, and survival was reduced at constant 8°C. Survival was also reduced when embryos were incubated at 11°C during the windows of gastrulation, organogenesis and fin development, but not during the late growth window. Time to 50% hatch decreased with an increase in constant incubation temperature, and only during the late growth window did an increase in temperature decrease time to hatch. Increased incubation temperature during the late growth window resulted in smaller hatchlings and reduced yolk conversion efficiency compared to embryos incubated constantly at 2°C or embryos incubated in warmer temperatures during earlier windows of development. Incubation at 11°C during organogenesis, fin development and the late growth window also reduced yolk conversion efficiency. Mass‐specific oxygen consumption rate increased with an increase in constant incubation temperature, but incubation at either 5°C, 8°C or 11°C during gastrulation, organogenesis, fin development or the growth window did not have substantial effects on metabolism at hatch. Overall, critical windows for survival in response to increased temperature were present during gastrulation, organogenesis and fin development. However, hatchling size was most influenced by incubation at warm temperatures during the late growth window prior to hatch, due to increased temperature inducing early hatching.

Support or Funding Information

C.A.M. and J.E. were supported by a MITACS Accelerate grant to D.R.B. and J.Y.W. Funding was provided by Bruce Power® and a Collaborative Research and Development Grant from the Natural Sciences and Engineering Research Council of Canada to J.Y.W., R.G.M., and C.M.S.