bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Internalization and Trafficking of GC‐A/NPRA via Endo‐lysosomal Compartments with Concurrent Generation of cGMP in Mouse Mesangial Cells: Role of FQQI Motif
Authors: Mani Indra , Garg Renu , Tripathi Satyabha , Pandey Kailash N
Published: November 2023

The objective of this study was to delineate the critical role of endocytic signal in intracellular sorting and signaling of receptor into these compartments. We have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AAAA) using QuickChange II site‐directed mutagenesis kit in enhanced green fluorescence protein (eGFP)‐tagged NPRA (eGFP‐NPRA) cDNA sequence. Mouse mesangial cells (MMCs) were transiently transfected with both mutant and wild‐type constructs and cultured at 370C in an atmosphere of 5% CO2 and 95% O2. We utilized immunofluorescence staining and co‐immunoprecipitation (co‐IP) of plasma membrane, adaptor protein‐1, endosomal, lysosomal, and Rab 11 markers to follow subcellular trafficking and signaling by confocal immunofluorescence microscopy and immunoblotting. The treatment of cells with ANP at different times accelerated the internalization of receptor from cell surface to cell interior. The internalization studies revealed the immunofluorescence colocalization of mutated motif (FQQI/AAAA) receptor with pan‐Cadherin, a plasma membrane marker, and showed a decreased internalization of eGFP‐NPRA by 49%±3.4 (P< 0.001) compared with the wild‐type receptor. Interestingly, we show that μ1B subunit of adaptor protein‐1 (AP‐1, μ1B), binds directly to a phenylalanine based FQQI motif in the cytoplasmic tail of receptor. However, subcellular trafficking studies indicated that colocalization of mutated receptor with μ1B of AP‐1, early endosome antigen‐1 (EEA‐1), lysosome‐associated membrane protein‐1 (LAMP‐1), and Rab 11 marker, was decreased by 50%±3.2 (P< 0.001), into early endosome by 57%±4.7 (P< 0.001), lysosome by 32%±1.4 (P< 0.01), and recycling endosome by 40%±1.8 (P< 0.01) respectively, compared with wild‐type receptor. The receptor containing mutated motif (FQQI/AAAA) also produced a significantly decreased level 64%±3.6 (P< 0.001) intracellular cGMP during subcellular trafficking compared to WT receptor. The co‐immunoprecipitation assay confirmed a decreased level of immunofluorescence colocalization of mutant receptor with subcellular compartments during endocytic processes. The results suggest that Phe790 and Ile793 constitute important element in FQQI motif, which are essential for the internalization and trafficking of NPRA. The findings may provide the basis for new molecular association of single nucleotide polymorphisms in Npr1gene with susceptibility to diseases and /or resistance to drugs in translational studies.

Support or Funding Information

This work was supported by NIH grants R01HL057531 and R01HL062147
Localization and Regulation of the Disease‐Causing H+‐ATPase B1 Subunit in the Early Distal Nephron of Human and Murine Kidney
Authors: Dimke Henrik , Chambrey Régine , Zamani Reza , Marcussen Niels , Svenningsen Per , Frische Sebastian
Published: November 2023

The H+‐ATPase B1 subunit is abundantly expressed in intercalated cells (IC) of the collecting system. Loss of function mutations in the gene results in distal renal tubular acidosis, due to insufficient acid secretion into urine. Curiously, mutations in the disease‐causing H+‐ATPase subunits has been reported to cause more severe forms of distal renal tubular acidosis, as compared to dominant mutations in AE1. Using 2 antibodies directed against the N‐terminal domain of the B1 subunit, we found abundant immunoreactivity in human kidney. As expected IC showed strongest signal, but significant signal was also observed in apical membrane aspects of the early distal nephron. Colocalization using known cellular markers confirmed expression of the B1 subunit in NCC positive distal convoluted tubules and NKCC2 positive cortical thick ascending limbs, but much less abundant in ENaC positive connecting tubule cells. Further immunohistochemical evaluation using an antibody directed against the other disease causing H+‐ATPase subunit A4, also showed localization to segments in the early distal nephron in human kidney. Expression was also detectable in macula densa for both subunits. In mouse and rat, B1 localization was investigated using antibodies directed against the N‐ or C‐terminal domains of the B1 subunit. Here subunit expression could be detected in apical membrane domains, beginning from the medullary thick ascending limb and stretching to the distal convoluted segment 2, in addition to the previously described IC. Using mice lacking the B1 subunit to validate the antibodies, complete loss of antigenicity was observed for two antibodies, while one showed minor staining in connecting tubules and IC, likely representing overlap with the B2 subunit epitope. To address whether the B1 subunit was subjected to regulation in the distal tubules, tissue from rats subjected to NH4Cl‐ and hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis were studied by immunohistochemistry. A mildly increased expression of the B1 subunit was noted in early distal nephron segments in kidneys of NH4Cl‐induced acidotic rats. In contrast, no change in expression could be observed in hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis. In conclusion, the B1 subunit is expressed in the early distal nephron in humans and rodents. The physiological consequences of H+‐ATPase expression in these segments remain to be delineated in detail.

Support or Funding Information

This work was funded by grants from the Novo Nordisk Foundation, the Carlsberg Foundation, the A.P. Møller Foundation, and the Lundbeck foundation.
Sexual Dimorphic Expression of Renal Claudins, Water Channels and Transporters accounts for the Downstream Shift in Salt and Volume Reabsorption along the Nephron in Female vs. Male Rats
Authors: Veiras Luciana Cecilia , Pei Lei , Yu Alan S.L. , McDonough Alicia Ann
Published: November 2023

Recently, we reported that female rats, as well as mice, have lower abundance of apical proximal tubule (PT) sodium transporters (NHE3, NaPi2) as well as lower abundance of myosin VI, the motor that drives regulated redistribution of the apical transporters. This reduction in PT transporters in females is complemented by higher abundance and phosphorylation of distal tubule Na‐Cl cotransporter (NCC) and higher abundance of cleaved (activated) forms of epithelial sodium channels’ (ENaC) alpha and gamma subunits. At the physiologic level, the impact of the differences is evident as more rapid excretion of a saline load, higher clearance rate of endogenous lithium (a marker of flow from the proximal tubule), and no difference in thiazide sensitive natriuresis in females vs. males. The aim of the current study was to test the hypothesis that a sexual dimorphism in claudins (Cld‐) and water channels (AQP‐) accompanies the observed differences in transporters. Sprague Dawley rats were fasted overnight with water provided before termination. Abundance of claudins and water channels were determine by quantitative immunoblot of cortical or medullary homogenates. Claudin‐2, expressed from the PT through the thin descending limb, forms high‐conductance cation‐selective paracellular pores that play an important role in the paracellular reabsorption of salt in the PT (>32% of the filtered load). Claudin‐2 expression in cortex was lower in females vs. males: 0.32 ± 0.03 vs. 1.0 ± 0.1 (p< 0.002). Aquaporin 1, which confers “leakiness” to the PT, expressed in both apical and basolateral membranes of the PT, was also lower in females vs. males: 37 kD band: 0.44 ± 0.014 vs. 1.0 ± 0.023; 25 kD band: 0.84 ± 0.22 vs. 1.0 ± 0.26 (both p<0.001). Claudin‐7, expressed in the DCT and CCD, functions as either a Cl− barrier or pore; in vivo knockout provokes lethal salt wasting. Claudin‐7 expression in cortex was higher in females vs. males: 1.4 ± 0.01 vs. 1.0 ± 0.03 (p< 0.001). Claudin‐10, detected all along the nephron, and medullary apical Aquaporin‐2 were expressed at similar levels in females and males.

Conclusions

Lower abundance of both Cld‐2 and AQP‐1 in females corresponds to lower Na+ transporter abundance in the proximal nephron and may contribute to their improved ability to excrete a saline load. These factors may contribute to the lower blood pressures and resistance to experimental hypertension in females vs. males. In the distal nephron, higher abundance of Cld‐7 corresponds to the higher NCC and ENaC activities where it may facilitate salt reabsorption in response to the increased volume flow from the proximal nephron.

Support or Funding Information

AHA 15GRNT23160003 and NIH DK083785
A Novel GPCR in the Renal Proximal Tubules, Gprc5c, Is Involved in Systemic pH Regulation
Authors: Rajkumar Premraj , Cha Boyoung , Donowitz Mark , Hirabayshi Yoshio , Pluznick Jennifer L
Published: November 2023

Recent studies have found that novel G‐protein coupled receptors (GPCRs) are utilized as specialized chemosensors to detect metabolites in a variety of tissues, and are often involved in mediating essential physiological processes and metabolic pathways. Our lab aims to uncover the functional role of novel GPCRs in the kidney, and to that end we recently used a Taqman‐based mouse GPCR array to identify GPCRs with unknown function that are expressed at high levels in murine kidney (Rajkumar, et al PLoS One 2014). In this study, we focused on elucidating the functional role of one such novel GPCR, Gprc5c. Gprc5c is an orphan GPCR with no known ligand which we found to be present in the kidney by real‐time PCR at a comparable level to the well‐studied and highly expressed Angiotensin receptor, AT1a (ΔCtAT1a: 6.25±0.24; ΔCtGprc5c: 7.0±0.16). Using immunohistochemistry, we found that Gprc5c localizes to the renal proximal tubule apical brush border in wild‐type (WT) mice, where it colocalized with both LTA (lotus tetragonolobus agglutinin) and megalin (Gprc5c staining was absent in whole‐animal Gprc5c knockout (KO) mice). Because Gprc5c is an orphan receptor with no known ligand, we performed ligand‐induced GPCR internalization assays in vitro to identify activators of Gprc5c in transiently transfected HEK293T cells. We found that alkaline pH triggered Gprc5c activation, with maximal internalization seen above pH 7.4 (pH 8.0>pH 7.4>pH 6.5). Importantly, other GPCRs tested as controls showed no changes in internalization over the pH range tested. In agreement with a role for Gprc5c in responding to changes in extracellular pH, we found that Gpr5c KO mice have alkaline urine pH compared to wild‐type littermates (WT: 5.5±0.02, n=4; KO: 5.76±0.05, n=5; p<0.05). In addition, preliminary studies indicate that the plasma pH of Gprc5c knockout mice trends mildly acidic (WT: 7.44±0.04, n=4; KO: 7.34±0.02, n=4; p=0.07). To begin to understand the mechanism by which Gprc5c may modulate pH regulation, we loaded HEK293T cells with BCECF‐AM and determined that Gprc5c increases the activity and set point of NHE3 (a sodium‐hydrogen exchanger expressed in the brush border of the renal proximal tubule). Under alkaline conditions (pH8), we find that Gprc5c increases sodium proton exchange by 1.6 fold compared to control cells (p<0.05), and that this increase in activity is Tenapanor‐sensitive, implying that it is mediated via NHE3. Together, these results demonstrate that Gprc5c localizes to the renal proximal tubule where it contributes to pH homeostasis via modulation of NHE3 function.
Nitric Oxide (NO) Regulates Paracellular Resistance in Isolated, Perfused Rat Thick Ascending Limbs through cGMP
Authors: Monzon Casandra M , Garvin Jeffrey L
Published: November 2023

Thick ascending limbs reabsorb30% of the filtered Na. About 50% traverses the paracellular pathway. We showed that exogenously‐added nitric oxide reduces the Na/Cl permeability ratio(PNa/PCl) of the paracellular pathway via cGMP/PKG, but the effects on absolute per meabilities are unknown. We hypothesized that endogenously‐produced NO decreases paracellular PNa in thick ascending limbs. To test this we measured the effect of L‐arginine (0.5 mM; NO synthase substrate) and cGMP (0.5 mM; NO's second messenger) on PNa and PCl calculated from PNa/PCl and transepithelial resistance. PNa/PCl was 2.0 ± 0.2 in the control period and 1.6± 0.1 after L‐arginine treatment (p<0.05). Transepithelial resistance (Rt) was 7722 ± 1554 ohm‐cm in the control period and 6318 ± 1757 ohm‐cm after L‐arginine (p<0.05). Calculated PNa and PCl were 0.361 ± 0.049 μm/sec and 0.181 ± 0.025μm/sec, respectively, in the control period. After L‐arginine they were 0.621 ± 0.158μm/sec and 0.388± 0.099 μm/sec, respectively. In the presence of the NOS inhibitor L‐NAME (5mM), L‐arginine had no significant effect on paracellular resistance (L‐NAME alone: 7924 ± 1964 ohm‐cm; vs L‐NAME + L‐arginine: 8463 ± 1725ohm‐cm). Next we tested the effect of cGMP. PNa/PCl was 2.6 ± 0.6 in the control period and 1.6 ± 0.2 after cGMP treatment (p<0.06). Rt in the control period was 7592 ± 1470 ohm‐cm and 4796 ± 847 ohm‐cm after cGMP (p<0.035). PNa and PCl were 0.390 ± 0.071 μm/sec and 0.150± 0.028 μm/sec, respectively, for the control period, and 0.550± 0.170 μm/sec and 0.340 ± 0.110 μm/sec, respectively, after db‐cGMP. We conclude that contrary to our hypothesis NO increased both PNa and PCl, and thus decreased transepithelial resistance. NO's effects are likely mediated by cGMP. The increases in PNa and PCl most likely contribute to elevated NaCl flux from bath to lumen along the thick ascending limb due to their electrochemical gradients in vivo and thus reduce net NaCl reabsorption.

Support or Funding Information

HL‐28982
Extreme blood pressure polymorphisms influence the bicarbonate‐independent conductance of NBCn1 but not its Na/HCO3 cotransport activity
Authors: Moss Fraser J , Zeise Brian , Parker Mark D , Yu Bing , Morrison Alanna C , Boron Walter F
Published: November 2023

Genome‐wide association studies identified SLC4A7 as a locus associated with extreme blood pressure levels (Ehret G.B. et al. Nature. 2011 vol. 478 p.103). The product of this gene, the electroneutral Na+‐coupled HCO3− cotransporter NBCn1 is an important mediator of acid–base movement wherever it is expressed, which includes epithelial tissues, neurons and vascular smooth muscle. In addition to Na/HCO3 cotransport, NBCn1 mediates a steady‐state conductance (Gn1) that is independent of HCO3− transport. Exome sequencing of SLC4A7 was conducted among African and European American individuals from the Atherosclerosis Risk in Communities (ARIC) study. Individuals with whole exome sequence data were selected from the extremes (above upper quartile and below lower quartile) of the systolic and diastolic blood pressure distributions. In total, 42 single‐nucleotide polymorphisms (SNPs) associated with extremes of blood pressure were found within SLC4A7. Six SNPs located in the NBCn1 coding sequence (these result in the E252D, L261M, E326K, T563A, N756S, and P896S missense mutations) were selected for insertion into the human NBCn1‐B variant to test their function when expressed in Xenopus oocytes. All six NBCn1‐B mutants exhibited a near‐normal Na/HCO3 cotransport activity as determined by the rate of intracellular pH (pHi) recovery from a CO2‐induced acidification. However, when the Gn1 is normalized to cotransport activity to control for surface presentation, five of the mutants displayed a 12–35% reduction in the measured Gn1. The sixth mutation, P896S, which resides in the fourth extracellular loop (EL4) of NBCn1, caused a reduction in Gn1 of 84% on the wild‐type E326 background and 76% on the extreme blood‐pressure–associated K326 background. We conclude from this work that the polymorphisms we tested have little, if any effect on the Na/HCO3 cotransport rate, but can have substantial effects on the Gn1. Thus, these polymorphisms would not be expected to have a major, direct effect on pHi, but would be expected to affect cell membrane voltage (Vm). In particular, reduced Gn1 would be expected to hyperpolarize cells (i.e., shift Vm toward more negative voltages). Such shifts in Vm would be expected to secondarily affect a wide range of ion channels and electrogenic solute carriers in the SLC superfamily.

Support or Funding Information

R01‐HL090969, R01‐DK30344, R01‐DK81567
Salt‐Losing Nephropathy in Mice with a Null Mutation of Clcnkb
Authors: Grill Alexandra , Schießl Ina Maria , Hammer Anna , Castrop Hayo
Published: November 2023

The basolateral chloride channel CLCKb facilitates Cl reabsorption in the distal nephron. Mutations in clcnkb were associated with Bartter's syndrome type 3 in humans. To address the function of clcnkb for renal salt reabsorption, we generated a mouse line with a null mutation of clcnkb by the use of the Zn finger technology. Clcnkb‐deficient mice were viable and were born in Mendelian ratio. At the age of 4 weeks, clcnkb−/− mice showed a slight growth retardation, with a body weight of 14.0±0.5 g compared with 19.7±0.5 g in WT (p<0.0001). Ambient urine osmolarity was markedly reduced in Clcnkb‐deficient mice (590±39 vs. 2216±132 mosmol/L in WT; p<0.0001). During water restriction (12 hrs), urinary osmolarity increased to 1633±153 mosmol/L and 3769±129 mosmol/L in clcnkb−/− and WT mice (n=12; p<0.0001), which was accompanied by a loss of body weight of 12±0.4% and 8±0.2%, respectively (p<0.0001). Fractional urinary K+ excretion averaged 0.21±0.02 mmol/mosmol (clcnkb−/−) and 0.11±0.01 mmol/mosmol (WT; p=0.0008), suggesting increased K secretion in the collecting duct of clcnkb−/− mice. The salt‐losing phenotype of clcnkb−/− mice was associated with a reduced plasma volume (2.5±0.6% of body weight vs. 3.9±0.2% in WT; p=0.0230), a reduced GFR (1040±97 vs. 1395±129 μl/min/100g bw in WT; n=9; p= 0.0411), and massively increased plasma renin concentrations (35720±8941 vs. 56±8 ng Ang I/ml/min in WT, respectively; n=11; p<0.0001). Our data suggest that clcnkb is crucial for renal salt reabsorption and concentrating ability. Clcnkb‐deficient mice appear as a suitable model of Bartter's syndrome type 3.
TRPM4 Is Expressed in and Carries a Nonselective Cation Current Through the Apical Membrane of Mouse Cortical Collecting Duct Principal Cells
Authors: Wu Ming Ming , Thai Tiffany L , Zhai Yujia , Eaton Douglas C , Zhang Zhiren , Ma Heping
Published: November 2023

Maintaining daily water, K+‐ and Na+‐balance is an important function of principal cells of the mouse cortical collecting duct (CCD). Under physiological conditions, these cells express a Ca2+ ‐activated nonselective cation channel (NSCCa), the molecular identity of which has been unknown. This study sought to identify whether the nonselective cation channel, TRPM4, functions as a NSCCa in principal cells of mouse cortical collecting duct.

Using immunohistochemistry and western blotting, we show the presence of TRPM4 channel protein in the mouse cortical collecting duct cell line mpkCCDc14. Single channel recordings showed a current that is carried by monovalent cations and is Ca2+ sensitive and exhibits equal permeability for Na+ and K+. The corresponding current–voltage relationship of this channel was linear with a slope conductance 23.4 pS, which is the same as that observed from heterologously expressed TRPM4 channels. TRPM4 is inhibited by ATP and stimulated by PIP2. Application of 1 mM ATP to the “cytoplasmic” bath solution of an inside‐out patch almost completely inhibited channel activity but washing out ATP fully restored the channel activity. The channel activity significantly increased after adding 1 μM PI(4,5)P2 to the “cytoplasmic” bath solution. The TRPM4 inhibitor 9‐phenanthrol (100 μM) strongly inhibited the endogenous current. These biophysical properties of NSCCa are identical to those described for TRPM4. Acute application of H2O2 (100 μM and 500 μM) was unable to stimulate TRPM4 activity inside‐out patches; however, using a cell surface biotinylation assay, we showed that treating the mpkCCDc14 with 100 μM H2O2 for 24hrs significantly decreased cell surface levels of TRPM4 protein.

Overall, we conclude that TRPM4 is the molecular identity of NSCCa in mpkCCDc14. Further study will be needed to determine the physiological relevance of this channel.

Support or Funding Information

NIH R01‐DK100582 to HM
Elevated Level of Hypoxia Inducible Factor 1α Can Alter Renal Epithelial Function
Authors: Nag Subhra S , Resnick Andrew
Published: November 2023

In the United States, about 600,000 people have Polycystic Kidney Disease (PKD) characterized by fluid filled structures (cysts) formed inside the kidney. Cysts interfere with normal renal function and lead to kidney failure. In the cystic epithelia, hypoxia inducible factor 1α (HIF1α) and HIF2α were found to be elevated. It is hypothesized that kidney cystic epithelia transform from normal absorption to secretion leading to accumulation of fluid within the cyst lumen. The fluid accumulation and insufficient vascular supply result in localized chronic hypoxia i.e., elevated HIF1α levels which may further promote cyst growth. We hypothesize that increased level of HIF1α can promote switching of renal epithelial function from absorptive to a secretory phenotype, possibly due to loss of epithelial tight junction integrity and altered active ion transport.

HIF1α level was pharmacologically elevated by cobalt chloride (CoCl2) in monolayers of mouse cortical collecting duct cell line (mCCD 1296 (d)) grown on suspended permeable membranes in the presence or absence of fluid flow. In our analysis, transepithelial electrical resistance (TEER) and voltage values were recorded using an Endohm chamber. Higher TEER values reflect reduced paracellullar permeability indicating tight junction integrity which was verified by paracellular permeability assay using FITC‐conjugated dextran molecules. Short circuit current (Isc) was calculated using Ohm's law (I= V/R) to compare active ion transport. MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same level of cellular viability. We found that elevated levels of HIF1α by CoCl2 caused loss of tight junction integrity and increased paracellular permeability. Increased HIF1α also resulted in decreased sodium transport which was determined from ameloride‐sensitive Isc. Currently, we are investigating the effect of increased HIFα by CoCl2 or deferoxamine on possible shift of active ion transport across the epithelial monolayer using specific sodium and chloride ion channel and transporter blockers. We are trying to understand the effect of increased level of HIF 1α on the localization and expression of tight junction proteins (e.g., zonula occludin 1 (ZO1) and claudins) and cotransporter proteins (e.g., Na+K+ATPase and NKCC1), leading to altered paracellular permeability and active ion transport. Thus, this study may provide a basis to understand how HIF1α or hypoxia may cause loss of normal epithelial function leading to cyst progression. This work could provide fresh insight into new treatment strategies for PKD.

Support or Funding Information

The project is supported by National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716 to AR and Cleveland State University Dissertation Research Award to SN
Nox4 regulates the mTOR signaling pathway in the Dahl salt sensitive (SS) model of hypertension
Authors: Kumar Vikash , Staruschenko Alexander , Cowley Allen W
Published: November 2023

Nox4 is the most highly expressed NADPH‐oxidase isoform in the kidney and is involved in a variety of diseases such as idiopathic pulmonary fibrosis, pulmonary arterial hypertension, diabetic nephropathy and metastatic carcinoma. Remarkably little is known about the role of Nox4 in hypertension, renal function, Na+ homeostasis or the pathophysiology of hypertension. In our previous studies, knockout of Nox4 (SSNox4−/−) in the Dahl salt sensitive rat (SS) was developed by our group using a zinc finger nuclease (ZFN) technique. This resulted in an 8 base‐pair frame‐shift deletion of the Nox4 gene. SSNox4−/− exhibited significant attenuation of blood pressure and reduced renal injury in response to a high salt diet compared to SS rats. The signaling pathways underlying these protective effects are unknown so we investigated the role of the mTOR signaling pathway in salt‐sensitive hypertension. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in several phenomena including cell proliferation, cell size and survival. mTOR has emerged as an important pathway in several forms of renal disease but is poorly studied in salt‐sensitive hypertension. The present study investigated the changes in mTOR in the kidney of SS and SSNox4−/− after 21 days of 4.0% NaCl diet. The expression levels of phosphorylated Akt, p70S6 kinase, and S6 in the renal cortex were examined by western blot and immunohistochemistry. Phosphorylation of Akt, p70S6 kinase, and S6 was found to be greatly reduced in SSNox4−/− compared to SS rats. In contrast, phosphorylation of Akt, p70S6 kinase, and S6 in rats fed 0.4% NaCl diet was observed to be similar in both strains. In conclusion, Nox4 appears to regulate the activation of mTOR in the kidney in response to high salt. Further studies are needed to establish the in vivo relevance of mTOR in salt‐sensitive hypertension.
Hyperspectral Imaging Approaches for Measuring Three‐Dimensional FRET
Authors: Annamdevula Naga S , Sweat Rachel , Britain Andrea , Rich Thomas C , Leavesley Silas J
Published: November 2023

Förster Resonance energy transfer (FRET) imaging has become a standard method for assessing protein‐protein interactions as well as changes in the concentration of signaling molecules, such as cAMP. However, many FRET probes or pairs have low signal‐to‐noise ratios and limited dynamic range. Our previous work suggested that hyperspectral imaging and analysis allows quantitative measurement of FRET efficiency in 3D (x, y, and t) within single cell. Here, we present data demonstrating that hyperspectral imaging approaches allow 4D (x, y, z, and t) assessment of FRET efficiency in living cells. In these studies, we have utilized a FRET biosensor consisting of a cAMP binding domain from Epac sandwiched between donor and acceptor fluorophores, Turquoise and Venus. Binding of cAMP induces a conformational change that reduces FRET efficiency. The FRET probe was transfected into HEK293 or pulmonary microvascular endothelial cells. To further demonstrate the potential of 4D hyperspectral approaches to simultaneously assess multiple fluorescence signals in single cells, we co‐transfected the calcium probe RCaMP into cells. 4D hyperspectral images were acquired using a Nikon A1R confocal microscope as follows: Excitation was provided using 405 and 561 nm laser lines. Spectral emission was detected from 424 to 724 nm in increments of 10 nm using a 32‐wavelength spectral detector on a Nikon A1R confocal microscope. A 3D (x, y, z) hyperspectral image was acquired every 15 seconds for 15 minutes. Following one minute measurement of baseline signals, we treated the cells with either 50 μM forskolin (an adenylyl cyclase activator), 0.1 μM or 1 μM isoproterenol (a beta adrenergic agonist), 10 μM rolipram (a PDE4 inhibitor), 1 μM prostaglandin, and/or calcium modulating agents such as 0.1 or 1 μM thapsigargin. Spectral images were processed using custom MATLAB scripts. Briefly, spectral images were unmixed to individual endmembers including Turquoise, Venus, DRAQ5 (nuclear label), WGA‐TRITC (plasma membrane label), and RCaMP. 4D FRET images were calculated from unmixed images. Data were re‐sliced to visualize fluorescence signals in different orientations/projections through each cell. This overall approach allowed assessment of changes in FRET signals in 4D. We observed little or no gradients in the lateral direction with real time 2D (x, y, and t) imaging. However, with 3D imaging (x, y, and z), we found axial gradients (from apical to basolateral) in the cell. With 4D (x, y, z, and t) imaging, we observed that cAMP gradients move from apical to basolateral side over time. Our data suggest that 3D (x, y, and t) imaging approaches may provide only a limited perspective of cAMP gradients and that 4D (x, y, z and t) studies are required to study cAMP gradients and signaling mechanisms in a cell.

Support or Funding Information

This work is supported by P01HL066299, S10RR027535, S10OD020149, R01HL058506, and the Abraham Mitchell Cancer Research Fund.
Contribution of Oligomeric Forms of Tripartite Motif E3 Ubiquitin Ligase Proteins to Plasma Membrane Repair in Striated Muscle
Authors: Gushchina Liubov V. , Alloush Jenna , Choi Jin Hyuk , Bhattacharya Sayak , Beck Eric , Weisleder Noah
Published: November 2023

Defects in the intrinsic plasma membrane repair function underlie the progression of many human diseases associated with membrane repair disruptions, such as ischemia/reperfusion damage and similar injuries to the cardiovascular system. Our previous experimental results showed that mitsugumin 53 (MG53), a novel muscle enriched protein from the tripartite motif (TRIM) family (known as TRIM72), is an essential component of the cell membrane repair machinery that protects injury to skeletal muscle and the myocardium. We recently determined that recombinant human MG53 (rhMG53) protein can be applied directly to cells or animal models to increase membrane repair in muscle cells, showing the potential of rhMG53 as a protein therapeutic agent. At the molecular level, MG53 can form dimers to allow movement of intracellular vesicles to patch membrane injury sites. Dimerization through their central coiled‐coil domain is a common property of TRIM proteins, which open the possibility that other TRIM proteins could form heterodimers with MG53. We screened for novel TRIM proteins that can increase membrane repair capacity and found that one such TRIM, TRIM27, can interact with MG53 to form heterodimers. The role of these homo‐ and heterodimers in the cardiac membrane repair is unclear and has to be elucidated in future studies. To study whether selected TRIM family proteins mediate membrane repair in the heart and to understand the molecular mechanism of this process, we generated several versions of human MG53 and TRIM27 cDNAs, which were optimized for expression in different host organisms for protein production, including E. coli and CHO cells. To create MG53 and TRIM27 and their homo‐ and heterodimers, we performed structural analysis of all TRIM family proteins and their separate domains that are available in the RCSB Protein Data Bank (PDB). Our analysis showed that PRY‐SPRY domains of protein are within several angstroms spatially. This allowed us to propose that the distance between domains can be covered by a linker to generate proteins without losing their activity. Thus, oligomeric forms of TRIM family proteins were created by fusing their human monomeric variants in anti‐parallel orientation (tail‐to‐tail). The therapeutic effects of TRIM proteins and their oligomeric forms on cardiac function and vulnerability to ischemia‐reperfusion injury will be further defined using in vitro and in vivo models.

Support or Funding Information

This work was supported by grant AR063084 from the National Institutes of Health and the Muscular Dystrophy Association (N.W.), Internal Muscle Health and Neuromuscular Disorders Program (L.G.) and the American Heart Association (AHA) program (14POST19990020, L.G.).
Dysregulation of ZnT2‐Mediated Zn Homeostasis Impairs Epithelial Polarity And Breast Differentiation
Authors: Lee Sooyeon , Kelleher Shannon L
Published: November 2023

Epithelial polarity is essential for the architecture and function of the mammary gland. We previously found that the loss of the vesicular zinc (Zn) transporter ZnT2 in ZnT2‐null mice compromises polarity, reduces STAT5 activation and impairs mammary gland differentiation. Here, we aimed to determine how the loss of ZnT2 impairs polarity and mammary gland differentiation. We used ZnT2‐attenuated mammary epithelial cells (MECs) in vitro and hypothesized that the loss of ZnT2‐mediated Zn transport dysregulates (PTEN), an epithelial polarity factor that disrupts spatial orientation of polarity, which subsequently impairs prolactin‐mediated Jak2/STAT5 signaling. First, we found that without the ability to sequester Zn into vesicles via ZnT2, Zn accumulated in the cytoplasm and significantly reduced PTEN expression. Moreover, ZnT2‐attenuated MECs had reduced membrane expression of, Annexin A2, a protein involved in apical membrane transport, and disrupted localization of ZO‐1, a protein critical for tight junction assembly, indicating impaired spatial orientation. Consequently, ZnT2‐attenuated MECs failed to differentiate into 3D mammospheres with expanded alveolar lumen. Importantly, we showed that the loss polarity disrupted cell surface localization of the principal lactogenic hormone receptor, prolactin receptor, and reduced downstream Jak2/STAT5 signaling, collectively indicating that cytoplasmic Zn accumulation dysregulates epithelial polarity and mammary differentiation. Our recent studies in breastfeeding women identified several non‐synonymous genetic variants in ZnT2 that result in “loss‐of‐function”. To investigate effects of these variants (ZnT2K66N and ZnT2Q71H) on MEC polarity, we transfected MECs to express these ZnT2 variants and found that expression increased cytoplasmic Zn and reduced PTEN expression in MECs, implicating defects of ZnT2 function in breast differentiation in women. Taken together, these results establish that ZnT2‐mediated Zn homeostasis is critical for maintaining epithelial polarity and regulating prolactin signaling for proper mammary gland differentiation and this suggests that “loss‐of‐function” variants of ZnT2 may be associated with impaired breast differentiation and lactation insufficiency in women.

Support or Funding Information

Intramural funds to SLK
Glucocorticoid Receptor‐mediated Lipolysis Increases following Chronic Adrenocorticotropin Infusion in Northern Elephant Seals Pups through its Imperative Interaction with Heat Shock 90 Chaperone Protein
Authors: Juarez Pablo , Lee Debby , Crocker Daniel , Vasquez‐Medina Jose Pablo , Ortiz Rudy
Published: November 2023

Northern elephant seal (NES) pups experience a prolonged 2–3 month fasting period and rely primarily on the oxidation of fatty acids to meet its energetic needs. Heat shock 90 chaperone protein (HSP90) is a fundamental part of the glucocorticoid‐cortisol (GR) complex, inducing an up regulation in GR‐mediated lipolysis upon chronoic adrenocorticotropin infusion. This fast is characterized by an increase in cortisol, HSP 90 and non‐esterified fatty acids (NEFA's). However, the functional relevance of this increase in cortisol via the GR is not well defined. We hypothesized that ACTH infusion increases lipolysis in NES. The contributions of cortisol and its receptor (GR) were assessed by exogenous infusion of ACTH and/or concurrently blocking the glucocorticoid receptor in the following groups: 1) control (no treatment), 2) ACTH (40 units of ACTH gel), 3) GR‐blocker (400mg mifepristone in time release pellets), and 4) ACTH+GR‐blocker. Plasma and adipose biopsy samples were collected at days 0 (T0; immediately prior to infusion) and 6 (T6). Mean plasma cortisol concentrations decreased 41±5% in the ACTH‐infused group and 21±3% in the combo group, but remained constant in the control and GR‐blocker groups suggestive of adrenal exhaustion. In contrast, mean plasma aldosterone increased 135%± 4 in the ACTH‐infused group suggesting that the adrenal gland in NES pups is differentially responsive (zona fasciculata vs zona glomerulosa). Mean NEFA levels increased 38%±4 in the ACTH‐infused group suggesting that ACTH‐mediated effects contribute to increased lipolysis. Mean heat shock protein 90 levels increased 119±1 in the ACTH infused group, 31±3 in the GR‐blocker and 192± 4 in the ACTH+GR‐Blocker group suggesting that mifepristone has no residual effect on the binding of HSP90 to the glucocorticoid receptor, making this interaction imperative in GR‐mediated lipolysis. Collectively, this data suggests that the fasting‐associated increase in cortisol is an important contributing factor in stimulating lipolysis potentially through the glucocorticoid receptor.
Transcriptional Regulation of S1PR1 Through FAK and KLF2 is Required for Maintaining Endothelial Barrier Integrity
Authors: Yazbeck Pascal , Schmidt Tracy , Tauseef Mohammad , Mehta Dolly
Published: November 2023

Formation of leaky blood vessels remains a persistent pathology in several diseases including acute lung injury. Sphingosine‐1‐phosphate (S1P) is a lipid mediator well known for its ability to induce endothelial barrier function. Formed through metabolism of sphingomyelin, S1P regulates endothelial barrier function by binding to S1P receptor 1 (previously known as endothelial differentiation gene (Edg1). However, mechanisms upregulating S1PR1 expression enabling formation of endothelial barrier function remain unclear. Focal adhesion kinase (FAK), a non‐receptor tyrosine kinase, regulates focal adhesion formation. We recently showed that loss of FAK impairs barrier formation leading to spontaneous vascular leak in mice lung vasculature. Here we found that FAK forms endothelial barrier by maintaining S1PR1 expression. We showed that FAK depletion significantly reduced the expression of S1PR1 both at mRNA and protein level without changing the expression of other S1PRs. Consistently, FAK depleted ECs failed to anneal adherens junctions as determined by VE‐cadherin immunostaining of the barrier. Additionally, loss of FAK impaired basal endothelial barrier function (as determined by measuring transendothelial electrical resistance (TEER)) under basal conditions and these cells could not strengthen endothelial barrier even after S1P application. Rescuing S1PR1 expression restored barrier function in FAK null ECs indicating S1PR1 function downstream of FAK. We identified KLF2 (Krüppel‐like transcription factor 2) as the transcription of S1PR1 which regulate S1PR1 transcription downstream of FAK. Our data provide a new insight of FAK regulation of S1PR1 expression and signaling through KLF2 to restore the formation of stable endothelial barrier.
Unbuckling the Buccal oscillator in the American Bullfrog
Authors: Baghdadwala Mufaddal I , Trask William M , Wilson Richard JA
Published: November 2023

The American Bullfrog is a remarkable model organism to study rhythm generation underlying breathing. It exhibits a breathing pattern that can be divided into four distinct ventilatory phases: lung‐priming, lung‐powerstroke, buccal‐dilation, and buccal‐constriction. We recently established that lung‐priming and lung‐powerstroke are generated by two separate burst generating substrates in the brainstem—the Priming area and Powerstroke area—located in rhombomere 4–5. The neural origin of the two phases of buccal, on the other hand, remains elusive. Our previous experiments suggest that a Buccal area located in rhombomere 7 is responsible, at least in part, for generating the biphasic buccal rhythm; however, whether any additional burst generating substrates are involved is yet unknown.

The objective of this study was to characterize areas in the frog brainstem that generate the biphasic buccal rhythm. We utilized the isolated frog brainstem preparation and recorded fictive respiratory motor output from the trigeminal and facial cranial nerves using extracellular suction electrodes. We progressively transected the brainstem in both directions (caudal‐to‐rostral, and rostral‐to‐caudal) to identify regions where transection eliminated one or both buccal burst phases.

Our preliminary results suggest that both phases of the buccal rhythm are generated by substrates located at the same level as the previously identified Buccal area. Nevertheless, using our current methodology, we are unable to discern whether multiple substrates or a single substrate is generating the buccal rhythm. To address this question, we developed a ‘buccal slice’ preparation at the level of the Buccal area which, when exposed to 100nM AMPA, produces buccal‐like bursts. We continue to develop this preparation as it holds promise for further dissecting the properties of this system and exploring it at the cellular and molecular levels.

Our analysis suggests that the Buccal area is an evolutionary homolog of the mammalian preBötzinger complex. Both are located in rhombomere 7, are resilient oscillators, being staunchly insensitive to CO2, and co‐opted for sniffing. We anticipate that having two comparable preparations, i.e. ‘buccal slice’ and ‘preBötC slice’, will allow for more direct comparisons between these oscillators, further testing their homology and thus take us closer towards understanding the evolution of breathing.

Support or Funding Information

National Science and Engineering Council of Canada (NSERC) and Alberta Innovates Health Solutions (AIHS) provided funding for this study.
Disturbances in central respiratory rhythm generation may contribute to breathing disturbances in prematurely born mice
Authors: Ramirez Sanja Carina , Koschnitzky Jenna E. , Baertsch Nathan Andrew , Anderson Tatiana M , Smith Charles V , Ramirez Jan‐Marino
Published: November 2023

Nearly one million premature infants die annually, due to respiratory distress. While immaturity of the lungs is a major contributor to morbidity and mortality, much less is known about the potential contribution of disturbances in central neuronal networks. Here we tested the hypothesis that central neuronal mechanisms contribute to neonatal apneas and breathing disturbances occurring within the first few hours of life. We used an animal model of prematurity, in which we administered lipopolysaccharide (LPS) into pregnant dams eliciting a maternal inflammatory response that resulted in mouse pups being born prematurely. This experimental model parallels the human condition because a large fraction of human premature births are associated with Chorioamnionitis and other forms of maternal infections. Administration of 5 micrograms of LPS to C57BL/6 mice at G17.5 resulted in delivery of pups at G18.5, about half of which were viable for at least 2 h post delivery. In a flow, whole‐body plethysmograph, premature mice from LPS‐treated dams showed abnormally large breathing patterns occurring at low frequency, which in some pups transitioned to more mature eupneic respiratory activity. Transverse, rhythmically active slices containing the pre‐Bötzinger complex, the putative respiratory rhythm generator, obtained within 30 minutes after birth from LPS pups, showed large amplitude bursts occurring at a slow and irregular frequency. Conversely term and C‐sectioned pups exhibited respiratory rhythmic activity with a smaller and more regular frequency and amplitude. Bath applied caffeine effectively stimulated and regularized burst activity. The in vitro preparations will enable us to compare the cellular mechanisms that underlie the abnormally large breathing patterns seen primarily in premature mouse pups, with the respiratory patterns generated in term and c‐sectioned pups. Understanding the central mechanisms that underlie disordered breathing in prematurity may lead to novel treatment strategies for neonatal apneas and breathing failures in human infants.

Support or Funding Information

Investigators Inter‐center seed funds
Hypercapnic acidosis acts on two opposing mechanisms to modulate the efficacy of opioid neuromodulation of inspiratory drive
Authors: Garcia Alfredo J , Andreson Tatiana M , Ramirez Jan‐Marino
Published: November 2023

Exogenous neuromodulation of central respiratory networks by opioids disrupts the normal orchestration of central respiratory control and thereby depresses the drive to breathe. Hypercapnic acidosis (HA) is a well‐recognized avenue that enhances central respiratory drive. Using the in vitro rhythmic slice preparation, this ongoing study examines how HA influences opioid neuromodulation of inspiratory drive from the preBötzinger complex (preBötC). In the majority of experiments, rhythms appeared to be HA‐insensitive, yet in a minority of cases, HA stimulated the frequency of the preBötC rhythm. When HA stimulated rhythmogenesis, the ability for 25nM DAMGO to suppress the preBötC was attenuated. However, in HA‐insensitive rhythms, HA augmented the suppressive action of 25nM DAMGO on rhythmogenesis. Enhancing inspiratory drive with 2μM norepinephrine stimulated the rhythm and caused a right‐shift in the dose response of rhythmogenesis to DAMGO, but did not appear to affect the ability for HA to augment the suppressive action of the opioid. These findings suggest that HA acts on two opposing arms of a push‐pull mechanism important to orchestrating opioid neuromodulation in the respiratory network. Thus, when intact, such a mechanism may be important to dynamically control respiratory drive and if imbalanced, this mechanism may perpetuate respiratory instability during opioid overdose and/or in other conditions such as neonatal abstinence syndrome.
Mapping projections of excitatory and inhibitory preBötzinger Complex neurons
Authors: Yang Cindy F , Feldman Jack L
Published: November 2023

Understanding the neural control of breathing requires a detailed neuroanatomical and functional map of the breathing central pattern generator (CPG). The logical entry point in constructing such a map is the “engine” of respiratory rhythm, the preBötzinger Complex (preBötC), a bilateral microcircuit of ~3000 interconnected neurons in the ventrolateral medulla. The preBötC generates respiratory rhythm in rodents (and presumably all mammals, including humans); rhythmic activity in the preBötC is required to produce inspiratory movements. To delineate the breathing CPG circuit in mice, we used a viral strategy to identify projections from molecularly‐defined populations of preBötC neurons. Using stereotaxic delivery of Cre‐dependent AAV expressing EGFP (AAV‐flex‐EGFP) into the preBötC of somatostatin‐Cre or GlyT2‐Cre mice, we located efferent projections emanating from two broad, functionally distinct classes of neurons: excitatory neurons that express somatostatin (SST) and inhibitory neurons that express the glycine transporter GlyT2. Critically, we optimized the viral delivery method to restrict viral transfections within the preBötC, while sufficiently labeling neurons to visualize long distance projections, enabling reliable identification of preBötC projections. Glutamatergic SST+ preBötC neurons project to brainstem nuclei implicated in the control of breathing including the contralateral preBötC, parahypo‐glossal/nucleus of the solitary tract, ventral respiratory group, and parabrachial/Kölliker‐Fuse nuclei, similar to efferent projections in rats; these neurons also had suprapontine projections to multiple hypothalamic nuclei, periaquaductal grey, as well as sparse projections to amygdala, suggesting that the preBötC relays respiratory information to higher brain regions that may modulate physiological or behavioral processes. Strikingly, glycinergic preBötC neurons send projections in parallel with glutamatergic SST+ neurons to the same brainstem and suprapontine targets.

Support or Funding Information

A.P. Giannini Foundation, NIH T32 NS007101, NIH F32 HL126522‐01, NIH RO1 HL070029‐13