bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing the Pleiotropic Role of PravastatinTM on the Expression of AQP1 in Vascular Endothelial Cells Cultured under Static, Venous, and Arterial Flow Conditions in vitro
Authors: Crum Raphael J. , Krane Carissa M.
Published: November 2023

The current surgical procedure to address coronary artery disease (CAD) involves the grafting of the human saphenous vein (HSV) into an arterial environment in the heart in a process called a coronary artery bypass graft (CABG). However, a high percentage of HSV grafts fail within five years due to the development of intimal hyperplasia (IH). The trigger for IH development is currently unknown. It is possible that difference in exposure to venous vs. arterial shear stress plays a role. Statins, a class of cholesterol lowering drugs, have been shown to suppress the early development of IH. Statins have also been shown to differentially regulate the expression of some members of the aquaporin water channel family of transmembrane proteins. Preliminary results suggest that the expression of aquaporin 1 (AQP1), a water channel abundantly expressed in vascular endothelium, is regulated in part by changes in shear stress in human umbilical vein endothelial cells (HUVECs) in vitro. Based on these observations, it is hypothesized that AQP1 may function as an early environmental sensor in HSV grafts to promote IH development, and therefore, may be a novel target for the early intervention and prevention of IH. The aim of this investigation is to determine the effects of changes in shear stress and Pravastatin™ exposure on the gene regulation of AQP1. The time‐dependent effects of venous and arterial shear stress (6 dynes/cm2 and 16 dynes/cm2) on AQP1 protein expression after 0, 24, 48, and 72 hours of static, venous, and arterial flow conditions were assessed using immunocytochemistry (ICC). The expression of AQP1 mRNA isolated from HUVECs cultured under static and flow conditions for 0, 24, and 48 hours with and without Pravastatin™ was analyzed using qPCR. Combined, the results of these experiments will contribute to an understanding of the relationship between the pleiotropic effects of statins and vascular shear stress and the combinatorial role of the two conditions in the early onset and possible prevention of IH in HSV grafts.

Support or Funding Information

Support for this research was funded by the 2015 APS Undergraduate Summer Research Fellowship and the University of Dayton Department of Biology.
Immunological Memory Exacerbates Responses to Repeated Hypertensive Stimuli
Authors: Itani Hana A. , Xiao Liang , Saleh Mohamed A , Wu Jing , Pilkinton Mark , Dale Bethany L. , Barbaro Natalia , Foss Jason , Kirabo Annet , Montaniel Kim , Norlander Allison , Chen Wei , Sato Ryosuke , Navar L. Gabriel , Mallal Simon , Madhur Meena , Bernstein Kenneth , Harrison David
Published: November 2023

Accumulating evidence supports a role of adaptive immunity and particularly T cells in the pathogenesis of hypertension. Formation of antigen‐specific memory T cells is a cardinal feature of adaptive immunity. We hypothesized that memory T cells contribute to long‐term renal damage in response to repeated hypertensive challenges. To impose repeated episodes of hypertension, we utilized two mouse models of repeated hypertensive challenges (L‐NAME/high salt treatment and repeated ang II stimulation) and show that effector memory T (TEM) cells are formed upon these repeated challenges and contribute to both salt‐sensitive and ang II‐induced hypertension. L‐NAME followed by high salt increased SBP to 147 ± 14 mmHg and caused a two‐fold increase in effector memory T (TEM) cells in the kidney and bone marrow (BM), as identified by the surface marker CD44hiCD62Llo. Intracellular staining showed that memory T cells were predominant sources of the inflammatory cytokines IL‐17A and IFN‐g. Development and reactivation of memory T cells require the interaction of CD27 on T cells with CD70 on antigen presenting cells. Flow cytometry revealed that L‐NAME/High salt increased expression of CD70 on splenic macrophages by 5‐fold and dendritic cells by 3‐fold. Because memory T cells are a major source of IFN‐g, we examined the hypertensive response to the L‐NAME/high salt protocol in IFN‐g−/− mice. The hypertension caused by L‐NAME was identical between WT, CD70−/− and IFN‐g−/− mice. In contrast, the hypertension induced by subsequent salt administration was markedly attenuated in CD70−/− mice (123 ± 1.3mmHg, p<0.02) and IFN‐g−/− (127.6 ± 5.5, p<0.04). Interestingly, CD70−/− and IFN‐g −/− mice failed to develop memory T cell formation in the kidney. The L‐NAME/high salt caused striking albuminuria and increased urinary N‐gal in WT mice, and these were absent in CD70−/− and IFN‐g −/− mice. We also defined a novel role of BM residing TEM cells in hypertension, in that these cells exhibited clonality, proliferated and redistributed to the kidney in response to repeated salt feeding. Adoptively transferred TEM cells from hypertensive mice homed to the BM and spleen, and expanded upon salt feeding of recipient mice in both the BM and kidney. Thus, these studies show that formation of long‐lived hypertension‐specific TEM cells that are major sources of inflammatory cytokines. These cells place the host at risk of developing blood pressure elevation and end‐organ damage upon delayed exposure to mild stimuli such as salt feeding or low doses of ang II. These studies provide further insight into how the adaptive immune system promotes hypertension.

Support or Funding Information

NIH F32 HL124972‐01
Endothelial‐derived Endothelin‐1 distinctly mediates the development of renal apoptosis in male and female mice
Authors: De Miguel Carmen , Battles Kevin D. , Hamrick William C. , Liu Xiaofen , Pollock David M , Pollock Jennifer S.
Published: November 2023

Renal injury is known to be preceded by apoptosis and loss of nephrons. The vasoactive peptide endothelin‐1 (ET‐1) is upregulated in renal disease; however, its role in renal apoptosis is not completely understood. These studies were designed to determine the role of vascular ET‐1 in renal apoptosis in response to tunicamycin (TM) in male and female mice. TM is a nucleoside antibiotic often used to induce acute kidney injury. Male and female mice lacking ET‐1 in vascular endothelial cells (VEET KO) and the corresponding flox controls (n=5–9/group) received a single i.p. injection of either TM (1.5 mg/kg) or saline. 24 hours post‐injection, kidneys were isolated and renal apoptosis was detected by TUNEL staining and quantified. TM treatment significantly increased apoptosis in renal cortex and outer medulla of female flox mice (% TUNEL positive area, flox vs. VEET KO; cortex: 5.0±0.7% vs. 3.0±0.3%; outer medulla: 6.7±0.9% vs. 4.1±0.2%; p<0.05), while TM‐induced apoptosis was abolished in female VEET KO mice. Interestingly, in male mice, TM did not induce apoptosis in either genotype. Endoplasmic reticulum (ER) stress is known to induce cellular apoptosis, and our group has recently implicated ET‐1 in the activation of ER stress pathways in the kidney. ER stress marker expression was assessed by qRT‐PCR in renal vessels isolated from TM‐treated male and female VEET KO and flox control mice. TM significantly increased ER stress in renal vessels of flox mice of both sexes. However, the absence of ET‐1 from the vasculature blunted the effects of TM on vascular ER stress in female VEET KO mice, but not in male VEET KO mice. These results indicate that vascular ET‐1 plays a critical role in the development of TM‐induced renal apoptosis and ER stress in female mice, but not in male mice. We propose that renal injury is mediated via sex‐specific mechanisms.

Support or Funding Information

Funded by NIH T32 DK007545 to CDM and P01 HL95499 to DMP and JSP
Activated Immune Cells Exacerbate Angiotensin II Induced Hypertension and Renal Damage in the Dahl Salt Sensitive Rat
Authors: Wade Brittany M , Mattson David
Published: November 2023

In recent years, there has been a reenergized interest in the role of immune cells in the pathogenesis of hypertension. We recently demonstrated attenuated Angiotensin II (AngII) induced hypertension and renal damage in Dahl Salt Sensitive (SS) rats lacking T‐ and B‐ lymphocytes due to a null mutation in the Rag1 gene (SS‐Rag1em1Mcwi). The present study was performed to analyze the abundance of different renal infiltrating immune cells and assess the cytokines produced by these cells during AngII induced hypertension. The continuous infusion of AngII (5 ng/kg/min, iv) to SS and SS‐Rag1em1Mcwi rats fed low salt (0.4% NaCl) chow led to a significantly greater increase in mean arterial pressure (MAP) in SS rats (189±3 mmHg) than in Rag1 mutant rats (177±3 mmHg) after 12 days of infusion (n=9 rats/group). Renal damage, as assessed by albumin excretion rate, was significantly increased after 12 days of AnglI infusion in the SS (from 33±4 to 81±9 mg/day) and in the Rag1 mutants (from 12±2 to 50±8 mg/day); albumin excretion rate was significantly different between the groups at all points measured. To examine the immune cells involved in the phenotypic response of the SS rats, cells were isolated from the kidneys and analyzed by flow cytometry. Compared to vehicle infused rats, kidneys of AngII treated SS rats had increased CD45+ total leukocytes (1,807,134±235,766 vs. 4,434,147±1,295,814 cells/kidney), CD11b/c+ macrophages/monocytes (1,439,002±163,119 vs. 3,566,048±1,124,655 cells/kidney), and CD3+ T Cells (299,557±81,501 vs. 740,370±155,065 cells/kidney). Further experiments were performed to assess the abundance of cytokine secreting CD3+ cells in the kidneys of AngII and vehicle infused SS rats. These experiments revealed 57% more CD3+/IL17+ T cells from the rats treated with AngII than the vehicle infused group (n=5–6 rats/group). This difference in IL‐17 production was observed in both CD3+/CD4+ and CD3+/CD8+ T cells. There was a tendency for more CD3+/IFNγ and CD3+/TNFα cells from the AngII infused group as well, though the changes were not significant. The present data suggest that infiltrating T cells in the kidney may secrete IL17 or other cytokines to exacerbate renal damage in AngII induced hypertension.

Support or Funding Information

Supported by DK‐96859 and HL116264
Hydrochlorothiazide Does Not Acutely Acidify Urine
Authors: de Bruijn Pauline , Brekelmans Renee , Praetorius Helle , Leipziger Jens
Published: November 2023

Urine acidification is fine‐tuned in the collecting duct. The α‐intercalated cells in this segment express the apical H+‐ATPase, which activity is assumed to be dependent on the transepithelial voltage (Vte). The lumen‐negative Vte generated by Na+ influx via the epithelial Na+ channel (ENaC) is believed to promote H+‐ATPase‐dependent H+ secretion. An increased Na+ delivery to the collecting duct would result in additional Na+ uptake through ENaC, increased lumen‐negative Vte, and thus, favor acid secretion via the H+‐ATPase. This mechanism has been the main explanation for the marked urine acidification by loop diuretics. However, we recently showed that urine acidification by furosemide occurs independent of ENaC activity. We demonstrated that furosemide directly stimulates H+ secretion in the thick ascending limb via the Na+/H+ exchanger NHE3. Here, we investigate whether hydrochlorothiazide, an inhibitor of Na+, Cl− cotransporter (NCC) in the distal convoluted tubule causes acute alterations of urine pH. NCC inhibition should similar to the loop diuretics lead to increased Na+ delivery to the collecting duct and, thus, cause urine acidification.

Mice were anesthetizes and infused (0.5 ml/h) via a jugular vein catheter with control or hydrochlorothiazide (HCT) (30 mg/kg/h) and bladder was catheterized. Urine pH was measured directly in the outflow of bladder catheter and urine Na+ and K+ were measured using flame photometry.

HCT increased urine flow rate from 8.1±1.8 to 15.8±2.6 μl/h/g BW (n=11). Na+ excretion was increased from 20.81±4.15 to 51.8±7.73 μmol/h, whereas no change in K+ excretion was observed. These results clearly demonstrate the diuretic properties of HCT. Interestingly, urine pH was unchanged during the entire infusion period of HCT (1h) compared to control (pH 6.13±0.23 vs 6.15±0.16).

Hence, despite the natriuresis induced by HCT, an acute increased H+ secretion was not observed. These results therefore challenge the current understanding of loop‐diuretic‐induced urine acidification, because other means for increasing Na+ delivery to the collecting duct do not acidify urine. Thus, this study supports our previous finding that H+ secretion by furosemide takes place already in the thick ascending limb and will contribute to a better understanding of how the kidneys secrete acid.
A Renal Olfactory Receptor Aids in Kidney Glucose Handling
Authors: Shepard Blythe D , Cheval Lydie , Koepsell Hermann , Doucet Alain , Pluznick Jennifer L
Published: November 2023

Olfactory receptors (ORs) are G protein‐coupled receptors which serve to detect odorants in the nose. Recently, ORs have been shown to play novel roles beyond odorant detection in a number of tissues, and we previously determined that a subset of ORs are expressed in the kidney. One of these ORs is Olfr1393, which we now report plays a novel role in renal glucose handling. In the absence of a reliable Olfr1393 antibody, we localized this receptor using reverse‐transcribed RNA from microdissected renal segments. These studies showed that Olfr1393 is exclusively expressed in the proximal tubule (PT; S1, S2 and S3), the site of renal glucose reabsorption (n=3 mice, PCR products sequenced to confirm). When stably expressed in polarized MDCK cells, Olfr1393 localizes to the apical plasma membrane, but not to cilia, indicating that Olfr1393 likely localizes apically in vivo. We generated whole‐animal Olfr1393 knockout (KO) mice and found that they are similar to wild‐type (WT) littermates with regard to plasma electrolytes, blood pressure and GFR (measured via iStat, tail cuff and elimination kinetics of plasma sinistrin, respectively). However, Olfr1393 KOs exhibit urinary glucose wasting (1.4× increase in glucose/creatinine vs WT; P = 0.01) despite being euglycemic under both fed and fasted conditions, with normal insulin levels. Olfr1393 KOs also have improved glucose tolerance as measured by a glucose tolerance test (area under curve: WT 20.4 ± 1.6 vs KO 16.3 ± 1.1; P = 0.008). Consistent with a role in renal glucose reabsorption, Olfr1393 co‐immunoprecipitates with Sglt1 and Sglt2, the two Na+/glucose co‐transporters responsible for apical glucose reabsorption. When Sglt1 and Sglt2 were localized within the kidney, we detected a 22% decrease in the luminal expression of Sglt1 in Olfr1393 KO mice, but no change in Sglt2. These data suggest that Olfr1393 is a novel regulator of Sglt1. In addition, as Olfr1393 is an orphan receptor with no known ligands, we utilized a large scale screen of over 1400 chemicals to determine the ligand profile of Olfr1393 (see abstract 987). Together, this data offers new insight into potential pathways of physiological regulation for this novel signaling pathway.

Support or Funding Information

NIDDK, ASN
Conditional knock‐down of Kidney‐specific Klotho Gene Impairs Sodium Reabsorption by Downregulating Epithelial Sodium Channel Expression
Authors: Ali Quaisar , Lin Yi , Wang Xiuqing , Sun Zhongjie
Published: November 2023

Background & Purpose

Aging is associated with an increased risk of abnormal sodium metabolism. The levels of Klotho, an anti‐senescence gene recedes with age. The Na‐transporters in the distal tubules finely regulate sodium re‐absorption. Klotho is primarily expressed in the renal distal tubules. However it is not known if klotho directly regulates sodium excretion. The purpose of this experiment is to investigate if conditional knockout of renal klotho affects tubular function.

Methods & Results

Briefly, kidney specific cre and klotho floxed mice were bred to generate kidney‐specific Cre‐loxP mice. Daily food and water intakes and urine output were monitored in Klotho floxed (control) and KspKL−/− mice for 12 days using metabolic cages. Food and water intakes, urine flow (UF), urinary sodium (UNaV), sodium balance (Na‐balance) and potassium excretion (UkV) were not different between KspKL−/− and control mice either during the baseline or five days of tamoxifen injection. Daily Injection of tamoxifen for 5 days efficiently knocked down kidney klotho (KspKL−/−). Interestingly, conditional deletion of renal klotho caused significant increases in UF, UNaV, and UkV within 3 days. Hematocrit, plasma and blood volume also significantly decreased during this period, suggesting volume depletion. Western blotting analysis showed a significant decrease in the expression of epithelial Na channel alpha (ENaCα) in kidney cortex while expression of ENaCβ or ENaCγ remains unchanged.

Conclusions

This study demonstrates that depletion of klotho causes impairment in Na reabsorption, probably via suppressing ENaCα expression.

Support or Funding Information

NIH R01 HL118558, DK093403 HL105302 and HL102074
Lack of an Effect of Collecting Duct‐Specific Knockout of Nitric Oxide Synthase 3 on Renal Sodium and Water Excretion or Blood Pressure During Physiological Conditions
Authors: Gao Yang , Stuart Deborah , Takahashi Takamune , Kohan Donald
Published: November 2023

Nitric oxide (NO) can act in an autocrine manner to inhibit collecting duct (CD) sodium and water reabsorption. Mice with CD‐specific knockout (KO) of NO synthase 1 (NOS1) have impaired ability to excrete a salt load and have salt‐sensitive hypertension. The CD also expresses NOS3, however the role of NOS3 in regulating CD salt and water reabsorption is unknown. Consequently, mice with CD‐specific NOS3 KO were generated and renal sodium and water excretion and blood pressure assessed. Mice were developed that were homozygous for the loxP‐flanked exons 9–12 (encodes the calmodulin binding site, removal of which renders the protein non‐functional) of the NOS3 gene and hemizygous for a transgene with the aquaporin‐2 promoter driving Cre recombinase; control mice were homozygous for loxP‐flanked NOS3. An organ panel showed predominant kidney‐specific gene targeting, while acutely isolated inner medullary CD from CD‐specific NOS3 KO mice showed complete target gene recombination. Control and CD‐specific NOS3 KO mice were fed a normal (0.3% sodium), high (3.17% sodium) or low (0.03% sodium) salt diet for 7 days each while in metabolic cages. There were no significant differences between control and CD‐specific NOS3 KO mice in food intake, water intake, urine volume, urinary sodium excretion, or urinary potassium excretion on any of the 7 days of normal, high, or low salt diets. Mice were implanted with telemetry devices and blood pressure determined during 7 days of normal, high or low salt intake. Systolic and diastolic blood pressure and pulse rate were not detectably different between control and CD‐specific NOS3 KO mice on any of the 7 days of normal, high or low salt intake. Plasma renin concentration was determined on day 7 of each salt diet and there were no significant differences between genotypes on the varied salt intakes. Mice underwent 1 day of water deprivation or 7 days of water loading (5% sucrose in drinking water). There was no significant difference between control and CD‐specific NOS3 KO mice in food intake, water intake, urine volume or urine osmolality after 1 day of water deprivation or on any of the days of water loading. In summary, these data suggest that CD NOS3 does not play a critical role in the physiological regulation of renal sodium and water excretion or blood pressure. Whether CD NOS3 is involved in the regulation of renal function under hypertensive conditions remains to be determined.

Support or Funding Information

This research was supported by NIH P01 HL095499
AQP1 in Peritoneal Dialysate as Predictive Biomarker of Integrity of the Peritoneal Barrier and Ultrafiltration Efficiency
Authors: Procino Giuseppe , Nicoletti Maria Celeste , Corciulo Simone , Corciulo Roberto , Russo Roberto , Grandaliano Giuseppe , Carmosino Monica , Svelto Maria , Gesualdo Loreto
Published: November 2023

Background

The water channel Aquaporin 1 (AQP1) plays a pivotal role in the mechanism of free water ultrafiltration during peritoneal dialysis (PD). Wether or not AQP1 is exclusively expressed in peritoneal capillaries or also in the mesothelial cells (MC) is still debated. It has been hypothesized that decreased expression or function of AQP1 may be responsible for some cases of ultrafiltration failure (UFF). Plasma membrane proteins are released in biological fluids through the exosome pathway to an extent proportional to their abundance at the plasma membrane.

Methods

In this work we investigated the localization of AQP1 in human peritoneum and its presence in exosomes isolated from PD effluent.

Results

Proteomic analysis of peritoneal‐derived exosomes showed a significant expression of AQP1. Interestingly, the same samples were devoid of the endothelial marker CD31 but were positive for the mesothelial marker mesothelin, thus suggesting a mesothelial, rather than endothelial origin for these vesicles. In human omental biopsies, AQP1 was localized in peritoneal capillaries and at the plasma membrane of MC, where it co‐localized with mesothelin.

Conclusions

Our results suggest that AQP1 is expressed in MC and released in the peritoneal cavity through the exosome pathway. This evidence opens the debate on the role of MC in regulating the efficiency of PD and suggests that AQP1 released in the PD effluent may represent a potential non‐invasive biomarker of integrity of the peritoneal barrier.

Support or Funding Information

Non‐U.S.
Regulation of Calcium Transport Pathways in Kidney and Intestine During Lactation
Authors: Dimke Henrik , Beggs Megan R , Alexander R. Todd
Published: November 2023

Significant alterations in maternal calcium balance occur postpartum during lactation. Demineralization of the skeleton mobilizes calcium, in order to supply this mineral to the neonate via breast milk. Changes in intestinal and renal calcium transport processes also promote this. However, the specific molecular alterations in transcellular and paracellular calcium transport pathways across renal and intestinal epithelia during lactation have not been fully delineated. To ascertain which changes occur during lactation, female mice were divided into 3 groups, non‐pregnant controls, lactating mice with litters maintained for 12 days after birth, or mice undergoing involution with litters removed after birth and maintained for 12 days. Induction of lactogenesis was confirmed histologically in mammary fat pads. Bodyweight increased in lactating mice as did urinary volume, measured during 9‐hr collections performed on day 11. Both total urinary excretion and fractional excretion of calcium and magnesium were elevated during lactation. Enlargement of the intestinal tract was confirmed visually in lactating animals. Renal 1‐alpha hydroxylase and 24‐OHase mRNA expression increased dramatically. This was accompanied by significant increases in the gene expression of Trpv6 and Calbindin‐D9K in duodenum and proximal large bowel of lactating mice. No significant alterations in the calcium permeable claudins (−2, −12 or −15) were observed in any intestinal segments. A similar gene expression profile was observed in kidney, which showed no change in claudins (−2, −12, −14, −15, −16 or −19), while marked increases in Trpv5 and Calbindin‐D28K were observed. Increased Calbindin‐D28K protein expression was found by immunohistochemical analysis, while no changes in claudin‐2 were noted, in line with observations from mRNA expression analysis. In conclusion, proteins driving transcellular calcium transport pathways increase during lactation, likely due to increased Vitamin D dependent‐stimulation, while proteins mediating paracellular transport appear unchanged. Increased urinary mineral excretion can be explained on the basis of intestinal hyperabsorption and bone demineralization, although selective calcium uptake is maintained in kidney.

Support or Funding Information

This work was funded by grants from the Novo Nordisk Foundation (HD), the Carlsberg Foundation (HD), the A.P. Møller Foundation (HD), the Lundbeck foundation (HD), the Kidney Foundation of Canada (RTA), and the Canadian Institute of Health Research (CIHR) (RTA). A Clinician Scientist Award from CIHR and an Alberta Innovates Health Solutions Clinical Investigator Award supports Dr. R.T. Alexander.
Down‐regulation of Kir4.1 eliminates the basolateral K conductance in the distal convoluted tubule (DCT) and inhibits NCC activity
Authors: Su Xiao‐Tong , Wang Ming‐Xiao , Wu Peng , McCormick James A. , Ellison David E. , Wang Wen‐Hui
Published: November 2023

Loss‐of‐function mutations of the inwardly rectifying potassium channel (Kir), KCNJ10 (Kir4.1), caused EAST or SeSAME syndrome. The renal phenotype includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which KCNJ10 mutations cause the tubulopathy is not completely understood. Our previous studies demonstrated that Kir4.1 was a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and the disruption of Kir.4.1 inhibited NCC expression. However, global deletion of Kir.4.1 impaired the animal growth and those mice could not survive more than two weeks after birth. Thus we have developed inducible kidney specific KCNJ10 knockout mice (Ks‐Kcnj10 KO) using a Pax8+/Cre+/Kcnj10f/f system. We fed 8 week old mice with doxycycline or vehicle for 10 days. Immunofluorescence and western analysis confirmed that more than 90% Kir4.1 protein was knocked down by this method. The patch‐clamp study detected a 40 pS K channel (a Kir4.1/5.1 heterotetramer) in the basolateral membrane of the DCT1 of wildtype (WT) mice, but the probability of finding the 40 pS K channel in doxycycline‐treated mice (KO) is lower (10% of the control) than that of WT mice. Moreover, the whole‐cell Ba2+‐sensitive K currents are largely abolished and the membrane potential is less negative in the DCT1 of KS‐Kcnj10 KO mice in comparison to that of WT mice. Thus, we have successfully generated KS‐Kcnj10 KO mice. Finally, we used western blot analysis to examine the expression of NCC in WT and KS‐Kcnj10 KO mice. The down regulation of Kir4.1 significantly inhibited the expression of total NCC protein or phosphor‐NCC. We conclude that Kir4.1 is a major type of K channel in determining the basolateral K conductance and membrane potential of DCT and determines the apical NCC expression.
Effects of PYY on IL‐6 Signaling in Primary Human Myotubes
Authors: Blum Jamie Elaine , Gheller Brandon JF , Roman Heather , Thalacker‐Mercer Anna
Published: November 2023

Rationale

Life expectancy is increasing and as a result, the older adult population in the United States is growing. Diminishing skeletal muscle function can impact the lives of older adults by limiting mobility and independence. Inflammation is thought to contribute to the development skeletal muscle metabolic dysfunction. Thus, identifying and understanding biological molecules that affect inflammatory signaling will inform interventions designed to support preservation of healthy skeletal muscle metabolism. Peptide Tyrosine Tyrosine (PYY), a novel skeletal muscle factor identified by our laboratory, is a potential mediator of inflammation in skeletal muscle. The effects of PYY in skeletal muscle need to be elucidated. Skeletal muscle gene expression of PYY is down‐regulated with aging and in individuals whose skeletal muscles do not hypertrophy in response to resistance exercise training. Exogenous administration of PYY decreases inflammatory signaling in various cell‐lines.

Hypothesis

Based on observations from microarray analysis conducted on RNA from skeletal muscle biopsies as well as preliminary data from our laboratory, we hypothesized that PYY down‐regulates inflammatory signaling in human primary skeletal muscle cells.

Methods

Biopsies were taken from the vastus lateralis muscle of two healthy, young females. Primary human skeletal muscle progenitor cells were isolated. Cells were pooled and cultured in Ham's F12 supplemented with 20% HI FBS and bFGF (GM). Once cells reached 75% confluence (t=0 h) all cultures were switched to differentiation medium [(DM), Ham's F12 + 2% HI Equine Serum] without (CONT) or with 500 pM PYY3‐36 (pPYY). At t=72 h, the medium on CONT and pPYY plates was replaced with DM + 0.5% Fatty Acid Free BSA (FAF‐BSA) without or with 100 ng/ml IL‐6 (CONTinflame and pPYY inflame) for 24 h. A subset of the cells from all treatment groups were exposed to a 20 min 200nM insulin challenge (CONT+I, pPYY+I, CONTinflame+I, and pPYY inflame+I) prior to harvesting the cells for RNA. Gene expression was measured using quantitative PCR.

Results

Differentiating primary human myoblasts that were pretreated with PYY3‐36 then incubated in IL‐6 and insulin challenged had a −1.87 and −1.79 fold change in AMPK and STAT3 mRNA expression, respectively (pPYYinflame+I vs. CONTinflame+I, p<0.05). This difference however, was not observed in cells pretreated with PYY3‐36 and incubated in IL‐6 without an insulin challenge (CONTinflame vs. pPYYinflame, p>0.05) or in cells incubated without IL‐6 (CONT vs. pPYY, p>0.05 and CONT+I vs. pPYY+I, p>0.05).

Conclusions

PYY pretreatment appears to have a protective effect against IL‐6 inflammatory signaling when an anabolic stimulus is given; PYY down‐regulates IL‐6 signaling through both AMPK and STAT3 pathways in the presence of insulin. Ongoing research in our laboratory is clarifying the role of the insulin stimulus in facilitating this effect of PYY.

Support or Funding Information

Cornell Start‐up
High Glucose Promotes the Pro‐Migratory Phenotype of Retinal Pigment Epithelial Cells through Increased Oxidative Stress
Authors: Farnoodian Mitra , Sorenson Christine M , Sheibani Nader
Published: November 2023

Defects in the outer blood‐retinal barrier have significant impact on pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin for such studies. The mouse models provide the ability to test the impact of various genes with important functions in ocular homeostasis in diabetes and its complications. We recently reported a method for culture of mouse retinal pigment epithelial (RPE) cells from wild type and transgenic mice. The RPE cells are responsible for maintaining the integrity of outer blood‐retinal barrier whose dysfunction during diabetes has significant impact on vision. Here we determined the impact of high glucose conditions on the function of RPE cells. We showed that high glucose conditions minimally impacted the proliferation and apoptosis of both human and mouse RPE cells, but these cells were more migratory and exhibited increased levels of oxidative stress. High glucose minimally affected the cell‐matrix and cell‐cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including tenascin C, thrombospondin‐1 and pigmented epithelium derived factor were increased under high glucose conditions. These cells also exhibited increased nuclear localization of anti‐oxidant transcription factor Nrf2 and reduced phagocytic activity under high glucose conditions. Furthermore, high glucose minimally affected the level vascular endothelial growth factor and activity of AKT, MAPK, and Src kinase signaling pathways. However, incubation of RPE cells under high glucose conditions with anti‐oxidant N‐acetylcysteine restored their normal migratory activity. Thus, high glucose promotes a pro‐inflammatory and oxidative state of RPE cells without a significant effect on their proliferation and apoptosis. The increased migration of RPE cells under high glucose condition is consistent with increased susceptibility to choroidal neovascularization with diabetes.

Support or Funding Information

NIH R24 EY022883; NIH P30 EY016665; NIH R21 EY023024
Nuclear Lemur Tyrosine Kinase‐2 Regulates RNA Polymerase II Dependent Transcription in Prostate Cancer
Authors: Shah Kalpit , Bradbury Neil
Published: November 2023

The Androgen Receptor (AR), a DNA‐binding transcription factor plays a key role in the development and maintenance of prostate epithelia by modulating expression of growth‐promoting genes. AR has been proposed to regulate gene expression by enhancing the efficiency of RNA polymerase II (RNAPII) dependent transcription elongation. Dysregulation of this AR‐dependent transcriptional activity has been implicated, not only in prostate cancer but also androgen‐independent prostate cancer. Recently, we showed that protein expression of Lemur Tyrosine Kianse‐2 (LMTK2), a serine‐threonine kinase is downregulated in prostate cancer. Importantly, our study ascribed a novel role for LMTK2, as a negative regulator of AR‐dependent transcriptional activity in prostate epithelial cells. However, the mechanism through which LMTK2 is able to regulate AR remains to be determined. Here, we show that LMTK2 is not only present in cytoplasmic fraction but also, within the nucleus of mammalian cells where it co‐localizes with AR, newly transcribed mRNA and with RNA polymerase II (RNAPII). Interestingly, our data reveals that LMTK2 colocalizes with elongating RNAPII, phosphorylated on serine 2 of the carboxyl‐terminal domain. This presents an interesting possibility that LMTK2 might be able to regulate transcription of AR‐dependent genes by interacting with the elongating RNAPII. As such, our study identifies LMTK2 as not only a novel regulator of AR but also, possibly an important player regulating the transcription machinery in mammalian cells.

Support or Funding Information

National Institute of Health (1R01HL102208‐01A1) and Cystic Fibrosis Foundation Grants (BRADBU12XXO)
From Protein Folding to Cognition: The Serendipitous Path of Discovery
Authors: Walter Peter
Published: November 2023

A screen for small molecule modulators of the unfolded protein response yielded ISRIB, a drug‐like compound that with high efficacy renders cells insensitive to translational inhibition by phosphorylation of eIF2. ISRIB proved to be a cognitive enhancer in rodents, significantly improving long‐term memory of wild‐type animals in behavioral assays.
TRPC3 serves as a link between osmosensitivity and water transport in the mouse collecting duct
Authors: Tomilin Viktor , Mamenko Mykola , Zaika Oleg , Pochynyuk Oleh M
Published: November 2023

Collecting duct serves as the primary site of controlled water reabsorption driven by osmotic gradient in the kidney. Cells of the collecting duct sense the changes of the extracellular osmolarity and react by elevating intracellular calcium via a yet unidentified mechanism. Moreover, the relation between osmosensitivity and water transport is poorly understood. Ca2+ permeable TRPC3 channel has been proposed to participate in mechanosensitive responses in different cell types. Previous evidence demonstrates prominent expression of this channel in the collecting duct cells. Using ratiometric Fura 2‐based [Ca2+]i imaging in freshly isolated split‐opened mouse collecting ducts, we found that pharmacological inhibition of TRPC3 with Pyr3 significantly reduces cellular responses to hypotonicity. Consistently, genetic TRPC3 deletion markedly impairs sensitivity of the collecting duct cells to decreases in extracellular osmolarity. Furthermore, collecting duct cells of TRPC3 −/− mice fail to properly regulate their volume in response to acute hypotonic challenge. Stimulation of water reabsorption with water deprivation exacerbates differences in collecting duct osmosensitivity between wild type and TRPC3 −/− mice and also transiently reduces urinary concentration ability in the mutant animals. We conclude that TRPC3‐mediated Ca2+ influx during hypotonicity is important for proper stimulation of water reabsorption in the collecting duct.

Support or Funding Information

Supported by AHA and NIH grants
Exploring an autoinhibitory domain in the electrogenic Na/HCO3 transporter NBCe1‐B
Authors: Lee Seong‐Ki , Boron Walter F.
Published: November 2023

The SoLute Carrier (SLC) superfamily of membrane transport proteins comprises over 300 genes organized into 52 families. One of the SLC groups is the SLC4 family, the members of which mainly transport HCO3−, or a species related to HCO3− (e.g., CO3=), together with another “coupled” ion across the plasma membrane. Here we study a member of the SLC4 family, the electrogenic Na/HCO3 cotransporter NBCe1 (SLC4A4). This transporter is widely expressed in cells throughout the body. NBCe1 has 5 known splice variants (A—E). A—C variants have been extensively studied. Whereas NBCe1‐A (predominantly expressed in the kidney) is very fast, NBCe1‐B (expressed throughout the body) and NBCe1‐C (mainly expressed in the brain) are very slow. The critical determinant of speed is the extreme NH2‐terminus (eNt), which in NBCe1‐A comprises the first 41 amino acids (aa). In NBCe1‐B and NBCe1‐C, this cassette is replaced by a cassette of 85 aa. Truncation of the first 85 aa of NBCe1‐B/C enhances activity. Thus, it has been suggested that this 85aa cassette in NBCe1‐B/C includes an autoinhibitory domain (AID).

Recently, Shcheynikov et al. (PNAS, 2015) showed that removing 23 aa between residues 40 and 62 stimulates NBCe1‐B. The deleted region contains a unique cluster (residues 40–48) of 9 consecutive positively charged residues. We hypothesize that this positively charged cluster normally inhibits NBCe1‐B. We assess NBCe1 activity by two‐electrode voltage‐clamping of Xenopus oocytes expressing NBCe1‐B constructs in which we systematically mutated or deleted elements of the cluster. We also deleted 3 to 9 aa just before the cluster. All constructs traffic well to the plasma membrane. We show that [1] removing or mutating the cluster of positive charges robustly stimulates NBCe1‐B activity (i.e., relieve autoinhibition), whereas [2] deleting aa before the cluster does not.

Our findings suggest that positively charged residues between residues 40 and 48 of NBCe1‐B are a necessary component of the AID, whereas the preceding 9 residues are not a necessary part of the AID.
The Role of a Common HFE Gene Variant in Brain Iron Accumulation
Authors: Duck Kari Ann , Neely Elizabeth B , Simpson Ian A , Connor James R
Published: November 2023

HFE is an essential protein for regulating iron transport into cells. Two common mutations of HFE, C282Y and H63D, result in loss of this regulation and cause accumulation of iron within the cell. The cellular iron accumulation can reach toxic levels. While much has been studied regarding the role of HFE in iron uptake, it has remained unclear what role the protein plays in the transport of iron into the brain. In fact, it has long been believed that the brain is protected against iron overload by the blood‐brain barrier (BBB). Recently, however, our laboratory and others have correlated HFE gene variants with neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS) and Alzheimer's Disease (AD). A critical review of historic literature, recent MRI studies and a novel more representative mouse model generated in our laboratory all suggest there is elevated iron in the brain associated with HFE gene variants. We generated a mouse model in which the wild‐type (WT) HFE gene is replaced by the H67D gene variant (mouse homologue of the human H63D gene variant). There are elevated levels of brain iron in 3 month old mice with the H67D mutation when compared to their controls, but this difference in levels is decreased at 6 months and no longer present by 12 months of age. Here, we performed an in vivo study to identify brain iron uptake and distribution patterns in 3 month old WT and H67D mice. After injection with 59Fe‐transferrin, mice were sacrificed at 24 hours and 5 days post‐injection. 59Fe levels measured in brain homogenate and isolated microvessels showed no significant differences between genotypes. There was, however, increased 59Fe accumulation in the livers of H67D mice. This study makes two novel discoveries. First, there is iron accumulation in the microvessels in the brain supporting the concept put forth recently by our group that the BBB is not a conduit but a regulatory site for brain iron uptake. Secondly, the similar rate of uptake for iron in the HFE mutant mice, coupled with the increased amount of iron suggests that the activity of the HFE protein is limited at the BBB. The increased iron in the brain in the H67D mice may be the result of altered handling of the iron including decreased export. We cannot rule out the lack of difference in uptake is a compensatory process already in place because of the higher iron levels and this notion is under investigation. The apparent ability of the BBB, unlike the liver for example, to adapt to the HFE gene variant reinforces the importance of understanding the mechanisms and regulation of brain iron uptake and how the pathway is modulated in the diseased brain.

Support or Funding Information

This work was supported by NIH P01 AG021190 and P01 HD39386.
Functional analysis of N‐glycosylation in the intestinal peptide transporter PEPT1
Authors: Daniel Hannelore , Stelzl Tamara
Published: November 2023

The intestinal peptide transporter PEPT1 (SLC15A1) mediates electrogenic proton‐coupled influx of dietary di‐ and tripeptides as well as of peptidomimetics into epithelial cells. Western blot analyses performed with protein isolates of mouse intestine revealed the existence of diverse PEPT1 glycoforms (Wuensch, T., et al. Am J Physiol Gastrointest Liver Physiol 305: G66‐73, 2013). In small intestine PEPT1 exhibits a mass of ~95 kDa, while in colon the transporter appears with a mass ~105 kDa. Enzymatic deglycosylation with PNGaseF uniformly reduced the PEPT1 mass in both gut segments to ~65 kDa. These mass differences addressed the question of whether glycosylation has an effect on function. We thus assessed whether different glycosylation states alter PEPT1 transport activity. By targeted mutagenesis, putative N‐glycosylation sites in murine PEPT1 were disrupted and mutant transporters heterologously expressed in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515 and N532 by glutamine (Q) resulted in a decrease of the PEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation‐deficient transporters to be functional with comparable expression levels in oocyte membranes. Remarkably, in the mutant transporter N50Q affinity for glycyl‐sarcosine (Gly‐Sar) decreased from 0.66 ±0.12 mM to 1.33 ± 0.23 mM while maximal inward currents increased 2.5‐fold from 0.85 ± 0.21 nA to 2.11 ± 0.21 nA. Tracer flux studies with [14C]‐Gly‐Sar confirmed the reduction in substrate affinity of the N50Q mutant while maximal influx rate increased from 2.32 ± 0.30 to 4.47 ± 0.32 pmol/min/oocyte. In the human protein (hPEPT1) the N50Q mutant as well presented a lower Gly‐Sar affinity (0.8 ± 0.1 mM in WT versus 1.2 ± 0.2 mM in mutant) and a maximal inward current increased from 1.6 ± 0.2 to 4.1 ± 1.4 nA. Since neither the surface density of the mutant transporter N50Q was changed, nor its conductance, we assume that N‐glcyosylation at N50 constraints the movement of the N‐terminal domain during the transport cycle. Attachment of N‐linked oligosaccharides at sequon N50, with an identified overall mass of 3–5 kDa, seems to attenuate PEPT1 mobility while the absence of the glycans could accelerate the

Support or Funding Information

This work was funded by GRK 1482 by Deutsche Forschungsgemeinschaft
Preliminary examination of a role for insulin in the regulation of glucose excretion in mourning doves (Zenaida macroura)
Authors: Sweazea Karen L , Braun Eldon J
Published: November 2023

Birds have naturally high plasma glucose (PGlu) concentrations and are resistant to the effects of insulin such that high doses must be given to achieve a significant reduction of glucose. In mammals insulin lowers PGlu by increasing uptake into skeletal and cardiac muscle as well as adipose tissues. However, prior studies have shown that high doses of insulin were ineffective at increasing glucose uptake into these tissues or into the kidneys of mourning doves (Zenaida macroura) despite significant decreases in PGlu. In addition, several case studies have shown a correlation between PGlu and urine glucose concentrations in birds. Therefore, the hypothesis of the present study was that insulin lowers PGlu in birds by augmenting renal glucose excretion. Adult male mourning doves were anesthetized using sodium pentobarbital. The brachial vein was cannulated for administration of [C14]‐inulin and insulin and the brachial artery was cannulated for blood collections. Ureteral urine was collected via a catheter inserted into the cloaca. Insulin (400 μg/kg body mass, i.v.) significantly decreased PGlu (p=0.0199) and increased glomerular filtration rate (p=0.0137), urine flow rate (p=0.0074), glucose excretion (p=0.0084) as well as fractional excretion of glucose (p=0.0038) by the kidneys. The findings from the present study suggest for the first time that the mechanism of insulin‐mediated glucose homeostasis in birds is more complex than previously thought and may include glucose excretion in urine. To our knowledge this mechanism of insulin‐mediated glucose excretion has not been demonstrated in other vertebrates.
Feeding status and the rate of crop emptying in the hissing cockroach, Gromphadorhina portentosa.
Authors: Medina Rubie , Contreras Heidy
Published: November 2023

Crop emptying has been studied in only a few species of insects. Generally, it is known that there are a variety of factors that can affect the retention time of a meal in the crop: 1) the availability of food; 2) the nutritional status of the organism, and 3) regulatory mechanisms aimed at meeting the varying nutrient demands of the animal. In this experiment we wanted to investigate whether or not feeding status of fed vs. unfed cockroaches affected the rate at which food moved through the gut. Roaches in the unfed group were starved for 7 days. They were then fed a control diet which had been dyed blue. All roaches were allowed consume 2% of their initial body weight. After roaches had consumed this volume of food, they were placed in a chamber without food for different time periods (0, 30, 60, 120, and 240 minutes). A total of 8 roaches were placed under each time treatment. Cockroaches were then sacrificed using liquid nitrogen. Their digestive system was dissected and measurements were taken to determine how far food had moved through the gut. A similar protocol was used to determine the rate of food movement through the gut of fed roaches. Previous studies suggest that unfed insects retain food in the crop for longer periods of time compared to fed animals. We therefore hypothesize that G. portentosa will show a decreased rate of food movement out of the crop and into the mid gut compared to fed roaches.

Support or Funding Information

ULV Faculty Research Grant