bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow‐Induced Activation of NO Production is Calcium‐ and HDAC1‐Dependent in Inner Medullary Collecting Duct Cells
Authors: Sedaka Randee S. , Hyndman Kelly A. , Pollock Jennifer S.
Published: November 2023

High salt intake mediates an increase in sodium delivery and flow within renal tubules, especially the inner‐medullary collecting duct (IMCD). We previously reported that isolated IMCD from mice on high salt diet produce significantly more NO compared to mice on normal salt diet without a change in NO synthase (NOS) expression suggesting that high salt and/or flow increases NO production through increased NOS activity. NOS activity is activated by intracellular calcium signaling in other cell types, thus we hypothesized that flow increases NO production through increased intracellular calcium. Mouse IMCD cells were grown to confluency on glass slides, then placed under static conditions or high flow (10 dynes/cm2) for 1 hour at 37°C in the absence or presence of calcium‐free media or BAPTA. NO production was analyzed by HPLC. NO production increased in response to flow >5‐fold (static: 653.8±165.9, flow: 3357.3±1024.7 pmol/mg pr/hr; n=4–9/group, p<0.001). Flow‐induced NO production was abolished in the absence of calcium (BAPTA: 609.6±152.5 pmol/mg pr/hr; calcium‐free media: 1440.0±907.0 pmol/mg pr/hr; n= 4–9/group, p<0.001). Previous reports from the literature show that histone deacetylase (HDAC) 1 is activated with intracellular calcium and is flow‐responsive. Thus, we further hypothesized that flow‐mediated increases in NO production occur via HDAC1. HDAC1‐specific inhibition with MS‐275 (300 nM) significantly decreased NO production in response to flow (static: 215.8±20.6, flow + vehicle: 1619.2±44.1 pmol/mg pr/hr, flow + MS‐275: 381.9±77.6 pmol/mg pr/hr; n=5–10/group, p<0.0001). HDAC1 expression was unchanged with flow (static: 2.86±0.9, flow + vehicle: 2.91±0.3, flow+MS‐275: 3.26±0.6 RDU; n=3/group, p>0.05). Additionally, preliminary HDAC1‐specific immunofluorescence staining of post‐flow IMCD cells suggests the presence of HDAC1 in both the nuclear and cytosolic compartments. Therefore, our data suggest that NO production in response to flow is calcium‐ and HDAC1‐dependent. Future studies will elucidate HDAC1 expression and activity in nuclear and cytosolic cell fractions as well as HDAC1 translocation utilizing immunofluorescence staining.

Support or Funding Information

Supported by NIH T32 HL007918 to RSS and P01 HL95499 to JSP.
Radiofrequency Renal Denervation Decreases Inflammation in Kidney Medulla in Spontaneously Hypertensive Rats (SHR)
Authors: Gao Juan , Xiao Liang , Polhemus David J , Smart Frank , Mernaugh Raymond , Goodchild Traci T , Lefer David J , Harrison David G , Kapusta Daniel R
Published: November 2023

We have shown that radio frequency ablation (RF‐ABL) of the renal arteries decreases blood pressure and sympathetic activity in hypertensive SHR. Recently, it has been shown that in angiotensin II‐induced hypertensive mice, surgical renal denervation reduces renal inflammatory cell infiltration, fibrosis, albuminuria and nephrinuria independent of blood pressure. The purpose of this study was to determine if RF‐ABL affects regional localization of inflammatory markers in kidneys of spontaneously hypertensive rats (SHR). SHR (n=8 /group) received bilateral RF‐ABL or Sham‐ABL (Biosense Webster Stockert 70 generator and RF‐probe). After 4wks, kidneys were collected, cortex and medulla separated, and processed for either flow cytometric determination of infiltrating tissue numbers of B cells and T cells (n=8/group) or for measurement of tissue cytokine levels (Bio‐Plex; n=10/group) including Interleukin 2 (IL‐2), IL‐5, IL‐6, IL‐7, IL‐10, IL‐17, Interferon gamma (INF‐γ), Granulocyte Monocyte Colony Stimulating Factor (GM‐CSF), Macrophage Inflammatory Protein‐3a (MIP‐3a), and Tumor Necrosis Factor alpha (TNF‐α). RF‐ABL significantly decreased B cells (56% of Sham‐ABL), CD4+ T cells (55% of Sham‐ABL) and CD8+ T cells (61% of Sham‐ABL) in the medulla, but had no effect on the numbers of B and T cells in the cortex. At 4 weeks following RF‐ABL, tissue levels of IL‐2, IL‐6, IL‐7, IL‐10, IL‐17, INF‐γ, MIP‐3a, and TNF‐α were decreased in the medulla as compared to Sham‐ABL. These findings demonstrate that RF‐ABL is associated with a preferential reduction of inflammatory markers in the kidney medulla, which may contribute to blood pressure independent effects of RF‐ABL in hypertensive rats.

Support or Funding Information

AHA 14POST20450173 to JG; Biosense Webster IIS‐175 to DRK & FS; P30GM106392 to DRK. 1R01 HL092141, 1R01 HL093579, 1U24 HL 094373, and 1P20 HL113452 to DJL.
Sex‐Specific Cell Death Response Following Renal Ischemia‐Reperfusion in Spontaneously Hypertensive Rats (SHR)
Authors: Crislip G. Ryan , Sullivan Jennifer C.
Published: November 2023

Males develop a greater extent of ischemia‐reperfusion (IR) induced injury than females. Recent studies have shown that renal IR injury is primarily mediated by necrosis in male mice, and pilot studies in our lab indicate a sex difference in renal cell death in SHR with females having more apoptotic cell death than males under control conditions. Based on the potential protective role of apoptosis vs. necrosis, the goal of this study was to test the hypothesis that males exhibit greater necrotic cell death following IR while female SHR favor apoptotic cell death. 13 week old male and female SHR were studied: control and 45 minute warm bilateral renal ischemia followed by 24 hours reperfusion (N=6–9). Renal injury was assessed by measuring plasma creatinine (PCr) and histological analysis. Kidneys were harvested to measure cell death via TUNEL assay, PCR Arrays, and Western blot analysis. IR increased PCr in both sexes (M: 0.34±0.04 vs. 3.07±0.5; F: 0.41±0.06 vs. 2.81±0.6), although kidneys from males exhibited a greater extent of red blood cell clogging than females when stained slides were scored (M: 1.0±0.0 vs. 2.4±0.2; F: 1.1±0.1 vs. 1.8±0.3). IR increased renal apoptotic cell death in both sexes, although females tended to have greater apoptotic cell death than males (M: 1.6±0.6 vs 12.0±3.9; F: 5.0±1.0 vs 18.3±3.9 cells per area; effect of treatment: p=0.01; effect of sex: p=0.1). RIP3 activates the formation of a pro‐necrotic complex with RIP1 during the cell death process. RIP3 mRNA increased 24 hours post‐IR in both sexes, although the increase only reached significance in males (M: p=0.01; F: p=0.09; n=4). RIP1 mRNA decreased only in females (M: p=0.8; F: p=0.03; n=4), however, the protein expression decreased in both sexes (M: 0.27±0.02 vs. 0.15±0.03; F: 0.31±0.01 vs. 0.20±0.05; effect of treatment: p=0.003; effect of sex: p=0.1). These data support a sex‐specific cell death response following renal IR with greater necrotic cell death in males and greater apoptotic cell death in females. A sex difference in cell death may underlie the sex difference observed in renal IR induced injury.

Support or Funding Information

American Heart Association Predoctoral Fellowship; AHA Award Number: 15PRE25850008 (PI: Sullivan)
Exposure to Cigarette Smoke Worsens Chronic Kidney Disease Induced Cardiac Hypertrophy and Fibrosis
Authors: Drummond Christopher A , Hernandez Dawn Alita , Haller Steven T , Liu Jiang , Shapiro Joseph I , Cooper Christopher J , Tian Jiang
Published: November 2023

Introduction

Although smoking accelerates renal and cardiac disease progression, the mechanisms by which it does so are unclear. Previously our lab has found that platelet activation can lead to increased renal injury. As smoking is a well‐known cause of platelet activation we postulated that smoking would accelerate renal disease progression as well as the associated cardiomyopathy in experimental renal failure.

Methods

We used male Sprague‐Dawley rats which were divided into two surgical groups one receiving sham (control) operations the other receiving the 5/6th partial nephrectomy (PNx) operation, a model of Chronic Kidney Disease (CKD). Each surgical group of animals were further split into two groups, one receiving exposure to side stream smoke from five 3R4F combustible cigarettes, delivered over 4 hours per day, 5 days per week for 4 weeks and non‐exposed controls. Blood pressure via tail cuff, renal and cardiac fibrosis, proteinuria, cardiac ejection fraction and Western blots to evaluate CD40 expression in kidney were analyzed 4 weeks following the start of the procedure.

Results

PNx increases systolic blood pressure compared to sham controls (177±16 vs 128±6 mmHg, p<0.05). Cigarette smoke exposure also increased blood pressure in Sham operated animals (142±3 mmHg, p<0.05) and with PNx (191±10 mmHg, p<0.05). PNx alone increased cardiac and renal fibrosis by 2.7‐ and 8.7 fold, respectively (p<0.01) versus sham‐operated animals. Cigarette smoking worsened cardiac and renal fibrosis (4.4‐ and 9.4‐fold, p<0.01) in Sham operated animals. Additionally, smoking significantly increased cardiac and renal fibrosis in PNx‐operated animals, resulting in 2.5‐ and 2.4‐fold higher fibrosis.

Proteinuria increased significantly with PNx alone vs shams (p<0.05). Smoke exposure doubled proteinuria in PNx (p<0.05), a similar trend was not observed in sham animals exposed to smoke vs those that were not. Ejection fraction as determined by echocardiography and cardiac hypertrophy as measured by heart weight to body weight ratio increased significantly in PNx operated animals versus shams (p<0.01) and smoking induced further significant increases in ejection fraction and cardiac hypertrophy vs non‐exposed animals (p<0.01). Finally, western blot analysis revealed that PNx and smoking both individually increased kidney expression of the pro‐inflammatory molecule CD40 and when combined synergized to increase CD40 protein expression over that observed for either treatment alone.

Conclusions

Combustible cigarette smoking significantly worsens progression of experimental cardiac and renal fibrosis and hypertrophy.

Support or Funding Information

This work was supported by NIH grant R01 HL‐105649 and F32 DK‐104615
Lack of collecting duct renin does not protect against DOCA‐salt hypertension and renal injury
Authors: Song Kai , Stuart Deborah , Abraham Nikita , Wang Fei , Wang Shuping , Yang Tianxin , Sigmund Curt D , Kohan Donald E , Ramkumar Nirupama
Published: November 2023

Collecting duct (CD)‐derived renin is involved in the hypertensive response to chronic angiotensin‐II (Ang‐II) administration. However, whether CD renin is involved in Ang‐II independent hypertension is currently unknown. To begin to examine this, 12 week old male and female CD‐specific renin knock out (KO) mice and their littermate controls were subjected to uninephrectomy followed by 2 weeks of deoxycorticosterone acetate (DOCA) infusion combined with a high salt diet. Radiotelemetric blood pressure (BP) was similar between KO and control mice at baseline; BP increased in both groups to a similar degree throughout the 2 weeks of DOCA‐salt. Urinary albumin excretion and plasma blood urea nitrogen were comparable between the two groups after DOCA‐salt treatment. Further, renal fibrosis as assessed by Masson's Trichrome stain and Sirius Red stain were similar between control and KO mice. Taken together, these data suggest that CD derived renin does not play a role in DOCA‐salt hypertension. Further studies are needed to examine the role of CD renin in other Ang‐II independent hypertensive models.
Transferrin Receptor Levels are increased by Interleukin 1β in a Human Kidney Proximal Tubular Cell line
Authors: Bonnemaison Mathilde L , Boesen Erika I
Published: November 2023

Background

Iron is required for several essential cellular processes such as DNA synthesis and energy metabolism, but when present in excess can cause cellular injury. Although kidney iron levels and urinary excretion of iron and iron transport proteins such as transferrin are increased in patients with kidney disease, it remains unclear how iron is handled by the kidneys during inflammation. We hypothesize that inflammation increases renal tubular iron uptake mechanisms, which in turn may promote cellular injury.

Methods and Results

To understand how iron is handled by proximal tubule cells during inflammation, we turned to HK‐2 cells, a human proximal tubular cell line, and investigated the levels and distribution of transferrin receptor and divalent metal transporter 1 (DMT‐1) in response to interleukin 1β, a pro‐inflammatory cytokine. Transferrin receptor allows the uptake of iron after binding and internalization of iron‐loaded transferrin while DMT‐1 carries divalent metals like iron, zinc and copper inside the cells. Our data with HK‐2 cells indicated that transferrin receptor levels doubled upon treatment with 0.1 ng/mL interleukin 1β for 24 hours (P<0.005 by one‐way ANOVA, followed by Dunnett's test). Immunofluorescent staining of HK‐2 cells showed no apparent change in transferrin receptor distribution. In contrast, DMT‐1 levels in HK‐2 cells remained unchanged upon treatment with interleukin 1β for 24 hours at concentrations as high as 50 ng/mL. Increased intracellular levels of iron may lead to mitochondrial oxidative stress, which in turn may impact ATP production. After treating HK‐2 cells with 1 ng/mL interleukin 1β for 24 hours, intracellular ATP levels were measured and indicated no significant difference with untreated cells.

Conclusions

These data suggest that under inflammatory conditions, the uptake mechanism for the key iron transport protein transferrin is increased in the proximal tubule. This could have important implications in the setting of glomerular injury, which permits greater amounts of transferrin to be filtered, resulting in increased exposure of proximal tubular cells to transferrin and thus iron uptake.
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 Conclusions

Bicarbonate 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 rats

Support or Funding Information

This 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*
SH2B3 Mutation Attenuates T Lymphocyte Intracellular Ca2+ Release in Response to Direct Angiotensin‐II Stimulation in Dahl S Hypertensive Rats
Authors: Blass Gregory , Palygin Oleg , Rudemiller Nathan P. , Mattson David , Staruschenko Alexander
Published: November 2023

T lymphocytes were shown to enhance the progression of salt‐sensitive hypertension and the associated kidney damage. These cells express SH2B3 (LNK), an adapter protein that was linked to hypertension by a Gene Wide Association Study, and is involved in regulation of several ligand‐induced transduction pathways. Angiotensin II (AngII) is a peptide hormone of the renin‐angiotensin system and is a major effector of blood pressure. AngII was found to stimulate the production and release of T‐cell inflammatory mediators via the AT1 receptor. Additionally, SH2B3 was shown to be involved in AngII‐induced hypertension in mice, where the knockout of SH2B3 increased T‐cells cytokine production and potentiated the development of hypertension and kidney damage. The lack of direct measurements of T‐cell response to AngII precludes conclusions of how AngII and the SH2B3 adaptor protein signaling cascade operate together to affect T‐cell function and further modulate the production of inflammatory mediators during the development of hypertension. To explore the interaction of AngII and T‐cells, we used SH2B3 mutant rats (SH2B3em1Mcwi) created on the Dahl salt sensitive (SS) rat background. The mutation, produced by zinc‐finger nuclease technology, resulted in a predicted 3 amino acid change in the evolutionarily conserved Src homology 2 (SH2) domain. This mutation in the SH2B3 gene was shown to cause attenuated development of high salt‐induced hypertension and kidney damage that was mediated by T cells. SH2B3 mutant and SS littermate control rats were kept on a 0.4% NaCl diet. At age 12 weeks, splenocytes from each group were isolated by sieving and centrifugation, labeled with anti‐CD5 FITC antibody, and loaded with the intracellular Ca2+ indicator Fura‐2 AM for further epifluorescence imaging experiments. Splenic T‐cells were then treated with Concanavalin A to engage the T Cell Receptor for cell activation. Bath applications of 10 μM of AngII rapidly produced an intracellular Ca2+ transient in both types of T‐cells. However, SH2B3 mutant CD5+ T‐cells had significantly attenuated response to AngII (0.41 ± 0.13 Fura340/380 ratio, n = 81) compared to SS littermates (0.92 ± 0.05 Fura340/380 ratio, n = 62) (P < 0.05). This result indicates that AngII is directly involved in the signal transduction pathway of CD5+ T‐cells and this pathway can be significantly modulated by SH2B3 adapter protein function. We conclude that the SH2 domain of SH2B3 may be a potential target for the control of T‐cell mediated oxidative damage and inflammation, and control salt‐sensitive hypertension. Further studies are required to define the role of SH2B3 in signaling and molecular mechanisms underlying T‐cells function and the development of hypertension.
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 Information

American 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 Information

This 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 Information

Grant 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 Information

The 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 Information

Supported 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

Rationale

Factors 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.

Hypothesis

Based on previous reports and observations, we hypothesized that PYY plays a role in myogenic mechanisms and thus, skeletal muscle health.

Methods

Biopsies 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.

Results

In 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.

Conclusions

This 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 Information

Cornell 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 Information

Supported 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 Information

NIH T32 HL007534, R01 GM66817, and HL085763
A PRIME Labeling Approach For Investigating Dopamine Transporter Endocytic Trafficking In Dopaminergic Cell Lines And Neurons
Authors: Wu Sijia , Uttamapinant Chayasith , Ting Alice , Melikian Haley
Published: November 2023

The presynaptic dopamine transporter (DAT) facilitates synaptic DA reuptake that spatially and temporally constrains DA neurotransmission. DAT is inhibited by addictive psychostimulants, and the rewarding properties of those psychostimulants require their direct binding to DAT. More recently, aberrant DAT function has been identified in Attention Deficit/Hyperactivity Disorder, Autism Spectrum Disorder and Infantile Parkinsonism, emphasizing the critical role DAT plays in maintaining normal DA tone. DAT robustly traffics to and from the cell surface via the endocytic pathway. Numerous stimuli, including protein kinase C (PKC) activation and amphetamine (AMPH) exposure acutely decrease DAT surface levels in dopaminergic terminals by promoting DAT endocytosis. However, DAT's post‐endocytic fate is unclear and conflicting studies report that DAT targets to either recycling or degradative endocytic compartments, or that DAT may not traffic in bona fide DAergic neurons. These discrepancies may stem from the various approaches used examine DAT trafficking, which rely primarily on either antibody or inhibitory ligand‐based methods that could potentially mistarget internalized DAT. In the current study, we take advantage of a novel PRIME (Probe Incorporation Mediated by Enzyme) labeling strategy to track the DAT endocytic itinerary under basal and stimulated conditions. This approach covalently couples fluorophore to surface proteins that encode a ligase acceptor peptide (LAP), thereby facilitating direct protein visualization without the need for inhibitory ligands or bulky antibodies. We engineered the LAP peptide into the DAT 2nd extracellular loop (LAP‐DAT) and observed highly specific fluorophore labeling at the cell surface in cells expressing LAP‐DAT, but not in cells expressing wildtype DAT. LAP‐DAT expresses, functions, and exhibits PKC‐mediated downregulation comparable to wildtype DAT. Moreover, LAP‐DAT robustly internalized in the immortalized mesencephalic cell line, AN27, allowing us to generate a dynamic flow profile that tracks DAT's movement through the endocytic pathway. Ongoing studies using AAV2‐expressed LAP‐DAT in the striatum of TH‐Cre mice, will permit a rigorous examination of DAT endocytic trafficking in bona fide native dopaminergic neurons in ex vivo tissue samples.

Support or Funding Information

NIH R01DA035224 to H.E.M.
Hydrogen Sulfide Inhibits Store‐Operated Calcium Entry by Targeting Orai3
Authors: White Carl , Kipp Jingjing , Fresquez Adriana
Published: November 2023

Store‐operated Ca2+ entry (SOCE) is activated in response to depletion of endoplasmic reticulum (ER) Ca2+ stores. The molecular machinery consists of the ER‐localized stromal interaction molecule (STIM), which upon store depletion, binds to and opens the plasma membrane channel Orai. Activation of SOCE is subject to redox modifications of both STIM and Orai. The gaseous signaling molecule hydrogen sulfide (H2S) is a known redox modifier of ion channels, however, its potential to affect SOCE has not been explored. To study this we generated HEK293 cells stably overexpressing human STIM1 and then transiently expressed either human Orai1, 2 or 3. The STIM1 and Orai proteins were tagged with YFP and CFP respectively to identify co‐transfected cells and SOCE quantified by Ca2+ imaging. Treatment with the H2S donor GYY4137 (500 μM) had no effect on SOCE mediated by Orai1 or 2 but inhibited the SOCE mediated by Orai3. It is established that H2S functionally affects proteins by modifying the thiol groups of reactive cysteine residues. Sequence analysis of human Orai1, 2 and 3 revealed four cysteine residues in the human and mouse Orai3 protein. Two of these (C101 and C118) are conserved in the Orai1 and Orai2 sequence; however, C226 and C232 are unique to Orai3. To assess there role in mediating H2S‐dependent inhibition these residues were mutated to serine. The SOCE generated by Orai3 C226S and C232S mutants was not significantly different from Orai3 wild type; however, the inhibitory effect of H2S was lost. These data are consistent with a model in which H2S inhibits SOCE mediated only by STIM1/Orai3 and not STIM1/Orai1 or STIM1/Orai2 by targeting specific cysteine residues in Orai3.

Support or Funding Information

DePaul‐RFUMS Collaborative Research Grant.
Regulation of Slack KNa Channel Trafficking by p38 MAP Kinase
Authors: Gururaj Sushmitha , Bhattacharjee Arin , Fleites John
Published: November 2023

Nociceptive sensitization is a central component of neuropathic pain, involving an alteration in the membrane properties of the sensory neurons of the Dorsal Root Ganglion (DRG) such that they have a lowered threshold for activation and hence, increased spontaneous activity. This DRG hyperexcitability manifests as the painful physiological symptoms of hyperalgesia and allodynia in patients of neuropathic pain, in whom there is as yet a large unmet need for medical treatment due to a lack of proper understanding of the precise molecular mechanisms involved. The key role of sodium activated potassium (KNa ) channels in the maintenance of firing accommodation in DRG neurons makes them important targets to study in the context of DRG excitability, which then deems mechanisms of KNa channel regulation as potential ways to regulate DRG hyperexcitability. In this context, we examined the KNa channel Slack's extensive intracellular C‐terminus for possible sites of regulatory protein interactions and found that it contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. While there is no direct link between p38 MAPK and KNa channels, there is evidence for p38 MAPK's role in facilitating neuronal regeneration after nerve crush of the sciatic nerve, indicating that the kinase could be an important player in neuropathy and potentially, in nociceptive sensitization. To answer the question of whether p38 MAPK regulates Slack channels via phosphorylation of the C‐terminus, we have performed electrophysiology experiments to demonstrate that Slack current is subject to modulation by p38 MAPK, and biochemical experiments to show that Slack is basally phosphorylated by p38 MAPK. Site‐directed mutagenesis of the p38 MAPK phosphorylation sites indicates that modulation occurs via phosphorylation at both the sites. Finally, biotinylation assays suggest that the mechanism of modulation of Slack current by p38 MAPK is likely at the membrane via a trafficking mechanism. Together, these results are the first direct demonstration of Slack as a p38 MAPK substrate, and provide evidence for p38 phosphorylation playing a role in Slack channel trafficking and hence, DRG membrane excitability.

Support or Funding Information

This work is supported by Grant NS078184 from the National Institute of Health.
The Integration of Gastrointestinal and Renal Function in Nectar‐Feeding Birds
Authors: McWhorter Todd Jason
Published: November 2023

Nectar‐feeding birds face a constant challenge in terms of water balance, experiencing water loading when feeding and potential dehydration when fasting. A growing collection of studies is revealing how species across different orders deal with this osmoregulatory challenge. To meet their high mass‐specific energy demands these birds must often deal with exceptionally high proportionate water fluxes. Birds that ingest large volumes of water may either eliminate excess water in the kidney or regulate the volume of water absorbed in the gastrointestinal tract. Across species, renal filtration during feeding periods generally does not exceed allometric predictions and is not highly responsive to water loading, but renal fractional water reabsorption varies with water load as expected. Hummingbirds (Apodiformes) absorb a constant, high proportion of ingested water across the gastrointestinal tract, while sunbirds and honeyeaters (Passeriformes) modulate intestinal water absorption, potentially avoiding significant water loads when feeding on dilute diets. Data suggest that these passerine nectarivores regulate transepithelial water flux independently of sugar absorption, opening the door to intriguing questions about how water transport is regulated in the vertebrate gastrointestinal tract. Despite reducing their intestinal water absorption, these birds show linear increases in water flux and fractional body water turnover as water intake increases, suggesting that the modulation of fractional water absorption is not sufficient to completely offset dietary water loads. Species in both orders exhibit significant diel variation in renal filtration, essentially shutting down renal filtration during overnight fasts and thus avoiding dehydration. Convergence in diet has led to the evolution of many similar traits in hummingbirds and sunbirds, but the physiological traits of these two groups that allow the processing of a watery and sugary diet may be very different.