bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bicarbonate therapy has no effect on renal T‐cell infiltration or blood pressure but markedly reduces tubular casts/fibrosis and is associated with an M1 to M2 polarization in Dahl salt‐sensitive rats
Authors: O'Connor Paul , Chen Jian‐Kang , Taylor Lia , Ray Sarah , Baban Babak , Sullivan Jennifer Case 
Published: November 2023

Immune cell infiltration into the kidney has been suggested to occur in response to renal injury but may also contribute to the development of hypertension in the Dahl salt‐sensitive (SS) rat model, initiating a feed‐forward cascade of progressive hypertension, hypertension induced kidney injury, and immune cell infiltration. Experimentally, it has been difficult to separate these factors, given the normally close relationship between blood pressure and renal injury in this model. We have recently reported that HCO3− supplementation limits the development of tubular casts and fibrosis in the kidneys of high salt fed Dahl SS rats, independent of lowering systemic arterial pressure. Therefore, in the current study we leveraged this phenotype to better delineate the roles of hypertension and renal tubular injury/fibrosis on the renal immune cell profile.11 week old Dahl SS rats were either treated with vehicle (0.1M NaCl; n=5) or NaHCO3−(0.1M; n=5) in drinking water for the length of the study. At the beginning of the study, rats were maintained on a 0.4% NaCl diet (Dyets AIN76A) for 4 days before being switched to an 8% NaCl diet for 14 days. On day 14 of 8% NaCl feeding, rats were anesthetized, arterial blood gas measurements taken and both arterial blood and the left kidney excised for flow cytometry analysis. Data are presented in the table below.Results and ConclusionsBicarbonate treatment markedly reduced tubular injury and fibrosis accompanied with significantly higher levels of M2 macrophages and a significantly lower levels of M1 macrophages, without altering renal T‐cell infiltration or blood pressure. Thus, our data for the first time suggest that the potential therapeutic effect of bicarbonate treatment might be mediated by induction of a phenotypic change of pro‐inflammatory M1 macrophage to tissue reparative M2 macrophage in Dahl salt‐sensitive ratsSupport or Funding InformationThis work was supported by NIH NIDDK grant 5R01DK099548

KIDNEY
CD3+
CD4+(% CD3)
CD44+(% CD3)
CD69+ (% CD3)
FOXP3+ (% CD3)
M1
M2

Vehicle (n=5)
1.44±0.23
55.2±1.6
59±2
0.6±0.1
1.1±0.2
0.30±0.03
0.16±0.04

Bicarbonate (n=5)
1.44±0.39
51.4±3.1
52±3
0.8±0.2
1.8±0.4
0.14±0.02
0.3±0.03

p (t‐test)
0.4
0.3
0.08
0.44
0.17
0.004**
0.03*

BLOOD

Vehicle (n=4)
54±1.2
55±1.9
19±2.2
1.2±0.3
2.5±0.3

Bicarbonate (n=5)
57.4±2
52.6±1.4
14.2±1.9
0.9±0.3
4.4±0.5

p (t‐test)
0.22
0.33
0.14
0.45
0.02*
Role of the Novel Kinase TNIK on NKCC2 Surface Expression, Phosphorylation and Na Reabsorption in the Thick Ascending Limb
Authors: Caceres Paulo S , Ortiz Pablo A 
Published: November 2023

NaCl reabsorption by the thick ascending limb (TAL) of the loop of Henle maintains NaCl balance and is important for blood pressure regulation. The apical cotransporter NKCC2 mediates NaCl reabsorption in the TAL. In animal models of hypertension, NKCC2 phosphorylation at amino‐terminal Thr‐96,101 and its trafficking to the plasma membrane are enhanced. The kinases SPAK and OSR1 bind NKCC2 and phosphorylate Thr‐96,101 and are important for blood pressure control. In addition to these, we recently showed that a novel kinase, Traf2 and NCK‐interacting kinase (TNIK), also binds and phosphorylates Thr‐96,101 in NKCC2. However, the role of TNIK on NKCC2 trafficking, activity and blood pressure regulation is unknown. We hypothesized that TNIK enhances NKCC2 phosphorylation, surface NKCC2 expression and NKCC2‐dependent water and Na transport. To test this, we generated TNIK knockout (TNIK−/−) mice and measured phosphorylation of NKCC2 and surface expression in isolated TALs. We observed that NKCC2 phosphorylation at Thr‐96,101 was decreased by 23 ± 6% (p<0.05) in TALs from TNIK−/− mice on a normal Na diet. Feeding mice a low‐Na diet increased NKCC2 phosphorylation by 179 ± 38% in WT mice, but only by 56 ± 5% in TNIK−/− mice (p<0.05). The NKCC2 surface‐to‐intracellular ratio was decreased by 58 ± 4% in TALs from TNIK−/− on a normal diet and by 55 ± 16% on a low‐Na diet. When placed in metabolic cages TNIK−/− mice excreted 99% more urine (WT = 1.03 ± 0.01 ml vs. TNIK−/− = 2.05 ± 0.13 ml; p<0.05). Baseline Na excretion was similar between strains on normal Na diet. However, after 48 hours of switching to low‐Na diet, TNIK−/− mice excreted 75% more Na compared to WT (WT = 15 ± 0.7 μmol/day vs. TNIK−/− = 26 ± 5 μmol/day; p<0.05). To test whether TNIK mediates NKCC2‐dependent Na excretion, we measured bumetanide‐induced diuresis and natriuresis in TNIK−/− mice over a six‐hour period following oral administration of bumetanide (1.66 mg/g food). On a normal diet, we did not observe any difference between strains. However, when placed on a low‐Na diet, TNIK−/− mice showed decreased bumetanide‐induced diuresis (WT = 2.66 ± 0.12 ml vs. TNIK−/− = 2.14 ± 0.11 ml; p<0.05) and natriuresis (WT = 239 ± 9 μmol vs. TNIK−/−= 201 ± 7 μmol; p<0.05). Finally, TNIK−/− mice had lower systolic blood pressure than WT mice when fed a low‐Na diet (WT = 123 ± 3.9 mmHg vs. TNIK−/−= 105 ± 3 mmHg; p<0.05). We conclude that TNIK mediates NKCC2 phosphorylation, surface expression and is involved in NKCC2‐mediated Na reabsorption during adaptation to low salt diet. TNIK‐mediated activation of NKCC2 may also be involved in blood pressure control.Support or Funding InformationAmerican Heart Association
Transcription Factor Elf3 Mediates Vasopressin‐Regulated Aquaporin‐2 Expression in Renal Collecting Duct Cells
Authors: Lin Shu‐Ting , Ma Chia‐Ching , Su Ying‐Fang , Yang Chian‐Huei , Weng Shih‐Che , Sun Chin‐Hung , Lin Shuei‐Liong , Yu Ming‐Jiun 
Published: November 2023

Vasopressin is a pituitary peptide hormone responsible for aquaporin‐2 (AQP2) gene expression in the kidney collecting ducts. Low levels of AQP2 gene expression are associated with a number of water balance disorders including lithium induced‐nephrogenic diabetes insipidus; however, the molecular mechanisms are not fully understood. Here, we studied the roles of the ETS‐related transcription factor Elf3 in vasopressin‐mediated AQP2 gene expression. In the mouse collecting duct cell model mpkCCD, vasopressin increased AQP2 mRNA and protein levels in a time‐dependent manner. Stable Elf3 knockdown significantly reduced basal and vasopressin‐induced increases in AQP2 mRNA and protein levels. Overexpression of Elf3 isoform 1 or 2 enhanced basal and vasopressin‐induced AQP2 promoter activity and AQP2 mRNA levels. Vasopressin did not affect Elf3 mRNA levels but induced nuclear translocation of V5‐tagged Elf3 in the mpkCCD cells. Electrophoretic mobility shift assay showed that the V5‐tagged Elf3 interacted with the AQP2 promoter DNA and that the interaction was inhibited with a competitor AQP2 promoter DNA or an antibody against the V5‐tagged Elf3. Lithium reduced Elf3 mRNA level and reduced AQP2 mRNA and protein levels in the mpkCCD cells in the presence of vasopressin. Similar observations were made in the mice that manifested polyuria and polydipsia on a lithium diet for two weeks. Taken together, our data support a role of Elf3 in vasopressin‐mediated AQP2 gene expression and provide a potential explanation to lithium induced‐nephrogenic diabetes insipidus.Support or Funding InformationThis work was supported by Ministry of Science and Technology, Taiwan to MJY (101‐2320‐B‐002‐038‐MY3) and National Taiwan University Hospital to SLL (NTUH103‐S2405).
Internalization of Angiotensinogen in Renal Proximal Tubules: Evidence for Mitochondrial Trafficking
Authors: Wilson Bryan , Cruz‐Diaz Nildris , Su Yixin , Rose James C. , Chappell Mark C. 
Published: November 2023

Evidence for an intrarenal renin‐angiotensin system (RAS) is well established. The renal proximal tubules, which constitute a key functional component of the kidney, express the RAS precursor angiotensinogen (Aogen); however, it is unclear the extent that tubular Aogen reflects local synthesis or internalization. Therefore, the current study determined whether Aogen is internalized by proximal tubules and the intracellular pattern of distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with 125I‐Aogen for 2 hrs at 37°C in DMEM/F12 media. Approximately 10% of exogenous 125I‐Aogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I‐Aogen associated with the mitochondrial fraction, with additional labeling in the nucleus [60 ± 7%], endoplasmic reticulum [4 ± 0.5%] and cytosol [15 ± 4%; n=4]. Subsequent studies determined whether Mito directly internalize 125I‐Aogen using isolated Mito from renal cortex and human HK‐2 cells. Sheep cortical Mito and the HK‐2 Mito internalized 125I‐Aogen at a comparable rate of [33 ± 9 vs. 21 ± 10 fmol/min/mg protein; n=3]. Lastly, unlabeled Aogen (100 nM) competed for 125I‐Aogen uptake to a greater extent than human albumin in HK‐2 Mito [60 ± 2% vs. 16 ± 13%; n=3]. Collectively, our data demonstrate Aogen internalization and subsequent trafficking to the Mito in proximal tubules. We conclude that this pathway may provide a precursor source for the intracellular expression of angiotensin peptides in the tubules.Support or Funding InformationGrant Funding Source: Supported by AHA grant 15PRE25120007 and NIH grants HL56973, HL52972, HL112237, and HD047584.
Involvement of Bile Acids in Regulation of Water Channel Expression in Colon Epithelial Cells
Authors: Yde Jonathan , Moeller Hanne B. , Dalsgaard Peter , Fenton Robert A. 
Published: November 2023

Bile acids (BAs) affect water transport in the colon, as evident in Bile acid malabsorption (BAM). In BAM patients, an increased production of BAs or a decrease in the capacity for BA reabsorption in the ileum cause BAs to overflow into the colon, where they lead to diarrhoea through mechanisms that are yet to be determined. Although there is evidence for expression of specific water channels (AQPs) in the colon, results from different studies have been contradictory. A connection between BAs and AQPs has not been described. We wanted to confirm the expression of AQPs in the colonic epithelium and we wanted to investigate, whether changes in expression of AQPs occur during BAM syndromes.Here we profiled mRNA and protein levels of AQPs in the colons of humans and rodents. As a model for BAM, we fed sodium cholate to rats for 3 days to examine the long‐term effects of naturally occurring BAs on the expression of AQPs in colonic epithelial cells.In colon biopsies from humans, AQP1, −3, −4, −7, −8 and −9 were detected at the mRNA level by reverse transcription polymerase chain reaction (PCR), with AQP1, −3, −4 and −8 confirmed at the protein level by immunohistochemistry (IHC). In colonic epithelial cells isolated from rats, AQP1, −3, −4, −7 and −8 were detected at the mRNA level by PCR, with AQP1, −3 and −8 expression confirmed at the protein level by IHC. In mouse AQP1, −4 and −8 mRNA and protein were detected by PCR and IHC. Feeding rats sodium cholate for 3 days increased the expression of AQP3, −7 and −8 at the mRNA level, which was seen by real‐time PCR, compared to a control group fed standard rat chow.In conclusion, BAs affect the expression of AQPs in the rodent large intestine. Our data suggest that AQPs may play a role in BAM syndromes.Support or Funding InformationThe study was supported by The Danish Council for Independent Research, MEMBRANES, Gangstedfonden and Grosserer L.F. Foghts Fond.
Impact of Phosphorylation Site Mutations on Human Amino Acid Uniporter LAT4 Expression, Localization and Function
Authors: Oparija Lalita , Guetg Adriano , Verrey François 
Published: November 2023

System L amino acid transporter LAT4 (SLC43A2) is a sodium‐independent uniporter that transports branched chain‐ and few other essential amino acids. It localizes to the basolateral membrane of kidney epithelial cells in proximal tubule, thick ascending limb of Henle's loop, distal tubule and collecting duct, and also to small intestine epithelial cells and several other tissues. Proteomic studies indicate several possible phosphorylation sites on LAT4, therefore we hypothesized that LAT4 surface expression and/or function might be regulated by phosphorylation. To test this hypothesis, we replaced the serine residues S274, S274+S278, S278 and S297 of human LAT4 with alanine (A) or glutamic acid (E) to mimic non‐phosphorylated and phosphorylated states, respectively, and expressed these mutants in X. laevis oocytes. Measurements of the overall expression of the mutant proteins by Western blot indicated reduced levels for all alanine mutants, in particular for their non‐glycosylated form, with the exception of S278A. Immunofluorescence experiments showed changes also in subcellular localization. Whereas the alanine mutants S274A and S274A+S278A localized mostly near or at the oocyte surface, corresponding glutamate mutants appeared to localize more intracellularly. Compared with the S274 and S274+S278 mutants, S278 mutants displayed opposite localization pattern. Surprisingly, the S297 mutants exhibited different localization on the animal pole (similar to the S274 and S274+S278 mutants) compared to the vegetal pole. Functional measurements of phenylalanine uptake suggested that all alanine mutants, with the exception of S297A, display a slightly increased apparent affinity; whereas the apparent affinity of most of the glutamic acid mutants was unchanged and their maximal transport velocity (Vmax) reduced. The only mutant exhibiting an increase in transport function was S274A; the double mutant S274A+S278A displayed the most similar kinetics to the wild type hLAT4, whereas the S297A mutant had severely reduced transport function. These results suggest that the abundance, surface localization and transport kinetics of hLAT4 may be regulated by phosphorylation.Support or Funding InformationSupported by Swiss National Science Foundation.
PYY, a novel factor in human myogenesis
Authors: Gheller Brandon John‐Forde , Roman Heather , Blum Jamie , Thalacker‐Mercer Anna 
Published: November 2023

RationaleFactors intrinsic and extrinsic to skeletal muscle cells account for a significant portion of the regenerative dysfunction in aging skeletal muscle. Molecules synthesized by skeletal muscle are key factors in regenerative programming and subsequent formation of the repaired tissue. Identification and mechanistic understanding of skeletal muscle factors involved in regenerative processes will aid in developing therapies to improve skeletal muscle‐related disorders. Our lab recently identified Peptide Tyrosine Tyrosine (PYY) as a novel skeletal muscle peptide with a likely role in skeletal muscle regeneration. Skeletal muscle gene expression of PYY is down‐regulated with aging and in individuals who do not hypertrophy after resistance exercise training. PYY increases in circulation after exercise and energy intake; however its role in skeletal muscle is unclear.HypothesisBased on previous reports and observations, we hypothesized that PYY plays a role in myogenic mechanisms and thus, skeletal muscle health.MethodsBiopsies from the vastus lateralis muscle of young females (YF) were taken and skeletal muscle progenitor cells were harvested and grown in culture. Study 1: Cells from YF (n=3) proliferated in growth media (GM) until ~80% confluent. GM was then removed and replaced by fresh differentiation media (DM) every 24h. Cells were harvested for RNA at −6h (6h before switching from GM to DM), 0h (immediately before switching to DM), 6h, 24h, 48h, 72h, and 96h. Study 2: Cells from YF (n=1) were sorted to ensure myoblast heterogeneity. Sorted cells were cultured in GM as described above. The PYY gene was silenced with siRNA (200 pmol, siPYY) transfection 24h prior to switching to DM. siPYY resulted in a 60% knockdown of PYY mRNA levels. Cells were harvested at 48h for RNA. For Studies 1 and 2, expression of the PYY gene and myogenic regulatory factor genes, myogenin and MyoD, were measured using quantitative PCR.ResultsIn study 1 PYY mRNA levels were transiently detected in human myoblasts during differentiation. In study 2 the PYY knockdown resulted in 7.5‐ and 2.3‐fold greater myogenin and MyoD mRNA levels, respectively, at 48h DM compared to controls. Myogenin gene expression from the siPYY cells was elevated at 48h to levels comparable to the 96h time‐point in untreated, differentiating cells.ConclusionsThis is the first evidence that PYY is expressed is human skeletal muscle progenitor cells. Additionally, silencing PYY in primary human progenitor cells may result in premature differentiation suggesting a potential key role of PYY in maintaining skeletal muscle regenerative mechanisms.Support or Funding InformationCornell Start‐up
GPCRomic Analysis of Human Lung Fibroblasts
Authors: Michkov Alexander Vladimirovich , Sriram Krishna , Aroonsakool Nakon , Insel Paul A 
Published: November 2023

Many interstitial lung diseases are associated with increased numbers of fibroblast/myofibroblasts and increased deposition of extracellular matrix (ECM) proteins. Such fibroproliferative responses can lead to pulmonary fibrosis, a disease setting with very limited treatment options. Previous studies have shown that the conversion of fibroblasts into myofibroblasts and excessive deposition of ECM proteins can be regulated by the cellular level of cyclic AMP (cAMP), the synthesis of which is regulated by G‐protein signaling and G protein‐coupled receptors (GPCRs). We thus tested the hypothesis that expression of GPCRs (in particular, previously unrecognized GPCRs) by lung fibroblasts may identify novel regulators of the function of lung fibroblasts and potentially new therapeutic targets for lung fibrosis. We used real‐time PCR GPCR arrays to identify and quantify non‐chemosensory GPCRs expressed in lung fibroblasts and detected on average ~ 100 such commonly expressed receptors in low passage cultures of multiple primary isolates of human lung fibroblasts. To ascertain if the GPCR expression profile changes during the fibroblast to myofibroblast conversion, we treated fibroblasts in vitro with TGFβ1 (10 ng/ml, 48 hr). TGFβ1 treatment prominently increased the expression of pro‐fibrotic markers (e.g., collagens Iα1, Iα2, III; α‐smooth muscle actin; plasminogen activator inhibitor‐1), thus mimicking what occurs in interstitial lung diseases. GPCRomic analysis identified multiple GPCRs that were uniquely expressed in response to TGFβ1. Lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) also revealed the selective expression of a sub‐set of GPCRs. However, TGFβ1 treatment of control fibroblasts does not fully mimic the changes in GPCR expression of fibroblasts from patients with IPF. We conclude that GPCRomic analysis identifies GPCRs associated with (and that may contribute to) the fibrotic state in the lung and thus, may be novel therapeutic targets for lung fibrosis. In addition, our results imply that TGFβ1 is likely not the sole determinant of changes in GPCR expression that occur in the fibrotic state.Support or Funding InformationSupported by Bristol Myers Squibb and NIH T32 HL 098062‐04
Airway epithelial monolayer integrity is a critical target in COPD
Authors: Sidhaye Venkataramana , Brune Kieran , Hansel Nadia , Robinson Douglas 
Published: November 2023

Under healthy conditions, the actin‐myosin II cytoskeleton and apical intercellular junction proteins, such as E‐cadherin, maintain epithelial polarity to separate the apical and basolateral compartments of the epithelium. Even subtle breaches in the monolayer allow for very small proteins to traverse the monolayer and activate epithelial signaling. Such signals include the 5kD, apically restricted protein EGF, which can activate the basolateral EGFR and can release EGFR receptor from the basolateral membrane, allowing it to move to the apical membrane. This leads to a lack of spatial restriction, allowing for promiscuous activation. While alterations in E‐cadherin levels and EGFR activation occur in response to CS exposure and in COPD, the initial mechanisms that lead to disruption in epithelial polarity are largely unknown.We have demonstrated that disrupting the epithelial monolayer by cytoskeletal rearrangements or decreased E‐cadherin, increases EGFR activation. The actin‐myosin cytoskeleton is a principle player in establishing epithelial cell shape and polarity. We observed that CS exposure leads to changes in the subcellular distribution of the myosin II paralogs and a subsequent reduction in E‐cadherin expression in primary human airway epithelium. These changes in the actin‐myosin cytokeletal cause a biphasic effect on the cortical tension of the cells, with acute exposures causing a decrease in cortical tension and repetitive exposures leading to increases. The increased cortical tension corresponds to a reduction in cell‐cell adhesion proteins, potentially due to carboxy‐terminal destabilization of E‐cadherin to the actin cytoskeleton. Histologic sections of airways show decreased E‐cadherin, altered myosin paralog expression, and EGFR activation in COPD patients. In clincial cohorts, E‐cadherin levels inversely correlate with worse emphysema in patients. Thus, low E‐cadherin expression and altered myosin II are strongly associated with chronic bronchitis, although causation remains to be established. By altering these proteins, and breaching epithelial polarization and activating EGFR. EGFR activation, in turn, promotes critical changes in the epithelium and induces mucus production and cell proliferation, helping to drive chronic bronchitis.We have developed an lung restricted, inducible E‐cadherin knockdown using airway adminstration of Adeno‐Cre recombinase in E‐cadherin floxed mice. After seven weeks of E‐cadherin knockdown, histology suggest epithelial remodeling in the mouse indicating loss of cell‐cell adhesion proteins are sufficient to induce epithelial changes.Support or Funding InformationNIH T32 HL007534, R01 GM66817, and HL085763