bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Excitability of Vasopressin Neurons in the Supraoptic Nucleus Following 48 hr. Water Deprivation
Authors: Farmer George E. , Little Joel T. , Knapp Blayne , Cunningham J. Tom
Published: November 2023

Arginine vasopressin (AVP) is a neurohypophyseal hormone released from the posterior pituitary by magnocellular neurosecretory cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. AVP is involved in the regulation of body fluid homeostasis and influences blood pressure, plasma osmolality, and blood volume. The regulation of AVP release is critical for body fluid homeostasis and is dependent on the activity of MNCs. However the cellular mechanisms leading to changes in AVP release are not fully understood. Water deprivation (WD), a progressive physiological challenge, was used to examine changes in excitatory neurotransmission in MNCs using patch‐clamp electrophysiology. Male Sprague‐Dawley rats weighing 250 – 350 g received bilateral SON infusions of an adeno‐ associated virus (AAV) construct containing AVP gene promoter and GFP reporter using isoflurane anesthesia. Two weeks following AAV infusions, rats were water deprived for 24 hr. or 48 hrs. Controls were always allowed ad libitum access to water. For electrophysiological experiments, rats were anesthetized with isoflurane and coronal brain slices (300 μm) containing the SON were prepared using standard procedures. Whole‐cell patch clamp recordings were obtained from slices superfused with aCSF containing tetrodotoxin (TTX; 0.5 μM) and bicuculine methbromide (Bic; 10 μM). Following baseline mEPSCs recordings, individual NMDAR and AMPA mediated components were pharmacologically isolated with 5 minutes drug application of AMPA antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 10uM) or the NMDAR antagonist 2‐amino‐5‐phosphonopentanoic acid (APV; 50 uM). Parameters measured for mEPSCs were amplitude (pA), rise time (ms), decay time (ms), charge transfer, and mEPSC frequency. In GFP labeled SON neurons (i.e. confirmed vasopressinergic neurons), 24 (n = 9; P < 0.05) and 48 hr (n = 9; P <0.05) water deprivation increased the frequency of mEPSCs when compared to controls (n = 13). In cells from the 48 hr WD group, the increase in frequency was accompanied by a reduction in amplitude. The increase in frequency associated with both 24 and 48 hr WD was mediated by an increase of AMPA mediated EPSCs (p < 0.05) whereas the reduction in amplitude specific to 48 hr WD was mediated by a reduction in the amplitude of NMDA mediated EPSCs (p < 0.05). The results suggest that water deprivation may be associated with presynaptic mechanisms that increase AMPA excitatory events that after 48 hr may also be associated with postsynaptic mechanisms that reduce NMDA excitation. Further studies are necessary to understand the individual AMPA and NMDA receptor mechanisms contributing to increases in SON excitability and subsequent vasopressin release in response to water deprivation.

Support or Funding Information

Supported by R01 HL119458‐04
Maternal High Fat‐High Sucrose Diet is Associated with Elevated Offspring Hypothalamic Oxidative Stress and Blood Pressure Responses to Psychological Stress.
Authors: Jayaratne Sachini , Prestipino Louise , Polson Jaimie W
Published: November 2023

There is mounting evidence that diets high in fat and sucrose (HFS) during pregnancy adversely affect offspring cardiovascular health. The aims of the present study were to assess the level of oxidative stress (OS) in brainstem cardiovascular control sites and compare blood pressure (BP) responses to psychological stress in HFS and control offspring.

Dams were fed a HFS diet (21% fat, 34% sucrose; control, 4.8% fat, 0% sucrose) from 4 weeks prior to mating until weaning. Three‐month old male offspring (n=7) were assessed for levels of the antioxidant glutathione and the OS marker protein carbonyl (PCs) in brainstem cardiovascular nuclei. At 9–12 months, cardiovascular variables were acquired from both males (control n=5; HFS=6) and females (control n=4; HFS=3) at rest and in response to mild (air jet, AJ) and moderate (restraint, RS) psychological stress using telemetry. Male rats were perfused and brains were processed immunohistochemically for Fos protein expression. Data are expressed as mean ± SE, statistical significance was taken as p<0.05.

HFS offspring exhibited increased retroperitoneal adiposity as a percentage of wet weight (2% vs 3.7%). Markers of OS were elevated in the hypothalamus, comprising a two‐fold increase in PCs and 40% reduction in glutathione. No significant differences were observed in the medulla or midbrain of programmed offspring.

Resting mean blood pressure (BP) was higher in both male and female HFS offspring than controls (male; 102.0±3.0 vs 94.2±2.6 mmHg, female; 104.4±3.7 vs 93.1±2.9 mmHg). BP responses to psychological stress were also enhanced. In HFS males, both stressors elicited greater initial increases in systolic BP (AJ; +30±3.3 vs +17±2.1 mmHg, RS; +39±1.9 vs +26±2.8 mmHg), while recovery was slower in AJ only (45 min post‐AJ, +13±1.6 vs +3±2.4 mmHg from baseline). A larger increase in blood pressure variability (low frequency component) was also observed in HFS males during both stressors, while no difference in heart rate or heart rate variability was observed between groups.

In HFS females, the initial systolic BP response to RS was greater (+41.3±1.9 vs 33.3±0.9 mmHg), however, there was no difference in AJ (HFS, 25.0±2.3 vs 24.3±4.3 mmHg). In contrast to males, recovery was similar to controls in HFS females.

HFS offspring also showed reduced Fos expression in the midbrain periaqueductal grey (PAG) in response to stress. Following AJ, the number of Fos‐positive neurons was reduced by ~40% in the lateral and ventrolateral PAG, while post‐RS a decrease in Fos expression was observed in the dorsomedial (70% reduction), dorsolateral (43%) and lateral (47%) PAG. No difference in Fos expression was observed in the hypothalamus.

The results suggest that maternal HFS elicits increased OS in the hypothalamus of offspring and programs a pro‐hypertensive phenotype characterised by exaggerated cardiovascular responses to psychological stress.
Endogenous Annexin‐A1 is a Protective Determinant in HFD‐induced Insulin Resistance and Diabetic Nephropathy
Authors: Purvis Gareth S.D. , Chiazza Fausto , Collino Massimo , Solito Egle , Thiemermann Chris
Published: November 2023

Introduction

Type‐2 diabetes mellitus is a key driver of cardiovascular disease often leading to renal failure, infarction or stroke. Here we investigate the role of Annexin‐A1 (ANXA1), an endogenous anti‐inflammatory peptide, in a mouse model of high‐fat diet (HFD) induced insulin resistance and diabetic nephropathy.

Methods

10‐week old male C57BL/6 (wild‐type) and ANXA1 null (ANXA1−/−) mice were fed a normal (chow) diet or high‐fat high‐sugar (HFD) diet for 10 weeks. Mice were administrated either human recombinant (hr) ANXA1 (1 μg hrANXA1, Hepes buffer, i.p.) or vehicle (100 μl Hepes buffer, i.p.) from weeks 5–10 of dietary manipulation.

Results

When compared to WT‐mice fed on chow diet, WT‐mice fed on HFD exhibited significantly lower serum levels of ANXA1 (1.259±0.01 vs. 1.154±0.03 ng/ml, P<0.05) and reduced ANXA1 expression in the kidney (0.96±0.02 vs. 0.30±0.04 O.D, P<0.05). When compared to WT‐mice fed on chow diet, WT‐mice fed on HFD and subjected to OGTT demonstrated a significant impairment in their oral glucose tolerance test (OGTT; AUC analysis: 84.42±4.51 vs. 110.70±3.89, P<0.05), which was improved with hrANXA1 treatment (110.70±3.89 vs. 89.40±0.99, P<0.05). The OGTT of ANXA1−/− mice fed on HFD was significantly worse than that of WT‐mice. When compared to WT‐mice fed on chow diet, WT‐mice fed on HFD demonstrated a significant increase in albumin‐to‐creatinine (ACR) ratio (20.75±2.434 vs. 56.10±5.80 mg/mg, P<0.05), suggesting the development of proteinuria, which was improved with hrANXA1 treatment (56.10±5.80 vs. 35.28±6.04 mg/mg, P<0.05). ANXA1−/− mice fed on HFD demonstrated a significant increase in ACR compared to WT‐mice fed on HFD (56.10±5.80 vs. 79.38±4.33 mg/mg, P<0.05) suggesting more severe proteinuria. When compared to WT‐mice fed on chow diet, WT‐mice fed on a HFD had decreased phosphorylation Ser188 of RhoA in the liver (1.00±0.004 vs. 0.575±0.002 O.D, P<0.05) and kidney (0.95±0.02 vs. 0.030±0.04 O.D, P<0.05) which was associated with increased phosphorylation of MYPT1 (liver, 1.15±0.006 vs. 4.15±0.07 O.D, P<0.05; kidney, 1.025±0.09 vs. 2.60±0.09 O.D, P<0.05). Both of these signalling events was prevented by treatment with hrANXA1. Interestingly, ANXA1−/− mice demonstrated constitutively activated RhoA and phosphorylation of MYPT1 suggesting that ANXA1 could be a major regulator of this GTPase activity.

Conclusion

We have shown for the first time that ANXA1 expression is reduced in a mouse model of HFD‐induced insulin resistance and diabetic nephropathy. This down‐regulation removes an important tissue‐protective factor allowing for exacerbation of renal dysfunction, which can be attenuated with treatment with hrANXA1. Most notably, hrANXA1 reduces both the metabolic derangements and the renal dysfunction associated with T2DM.

Support or Funding Information

This work is funded by the Britsish Heart Foundation grant number FS/13/58/30648.
Evidence for Mitochondrial AT2 Receptor‐Mediated Inhibition of Intracellular Angiotensin II‐induced Respiratory and Glycolytic Responses in Mouse Proximal Tubule Cells
Authors: Li Xiao Chun , Zhuo Jia Long
Published: November 2023

It is well recognized that endocrine and paracrine angiotensin II (ANG II) is involved in mitochondrial dysfunction in cardiovascular, renal and hypertensive diseases, but whether intracrine or intracellular ANG II plays an important role in the regulation of mitochondrial function remains virtually unknown. We hypothesize that in addition to activation of its cell surface receptors, ANG II and its receptors are internalized and trafficked to the mitochondria, where it may induce significant mitochondrial respiratory and glycolytic responses. As a proof of concept study, we constructed adenoviral vectors encoding an intracellular ANG II fusion protein, ECFP/ANG II (a gift from Dr. Julia Cook of Ochsner Health System), or a GFP‐tagged AT2 receptor, AT2R/GFP, a mitochondria targeting sequence, and a proximal tubule cell‐specific sglt2 promoter for proximal tubule‐ and mitochondria‐specific overexpression of mito‐ECFP/ANG II or mito‐AT2R/GFP in cultured mouse proximal tubule (mPCT) cells (a gift from Dr. Ulrich Hopfer of Case Western Reserve University). The mitochondrial respiratory and glycolytic responses to overexpression of mito‐ECFP/ANG II or mito‐AT2R/GFP were measured using Seahorse XF Cell Mito and XF Glycolysis Stress Test Kits, respectively. Fluorescent microscopic imaging revealed robust overexpression of mito‐ECFP/ANG II and/or mito‐AT2R/GFP in mPCT cells 48 h after transfection, and confirmed their colocalization with a mitochondrial marker MitoTracker® Red FM. Overexpression of mito‐ECFP/ANG II alone markedly increased oxygen consumption rate (OCR) (Control: 130.4 ± 8.2 vs. mito‐ECFP/ANG II: 232.3 ± 10.6 pmol/min; p<0.01, n=10) and extracellular acidification rate (ECAR) (Control: 8.8 ± 0.3 vs. mito‐ECFP/ANG II: 11.8 ± 1.3 mpH/min; p<0.01, n=10), respectively. The AT1 receptor blocker losartan (10 μM) completely blocked the effects of mito‐ECFP/ANG II on OCR and ECAR, whereas the AT2 receptor blocker PD123319 (10 μM) had no significant effect. However, overexpression of mito‐AT2R/GFP with mito‐ECFP/ANG II in mPCT cells significantly attenuated the effects of mito‐ECFP/ANG II on OCR (119.4 ± 10.5 pmol/min; p<0.01 vs. mito‐ECFP/ANG II, n=10) and ECAR to control (5.4 ± 0.37 mpH/min, p<0.01 vs. mito‐ECFP/ANG II, n=10). Interestingly, PD123319 significantly blocked the effects of mito‐ECFP/ANG II and mito‐AT2R/GFP overexpression on OCR (184.5 ± 4.3 pmol/min; p<0.01 vs. mito‐ECFP/ANG II and mito‐AT2R/GFP, n=5) and ECAR (8.2 ± 0.31 mpH/min, p<0.01 vs. mito‐ECFP/ANG II and mito‐AT2R/GFP, n=5). Our results strongly support the proof of concept that activation of AT1 receptors by ANG II in the mitochondria stimulates, whereas activation of AT2 receptors by ANG II in the mitochondria inhibits, mitochondrial respiratory and glycolytic responses in mouse proximal tubule cells.

Support or Funding Information

This work was supported in part by 2R01DK067299, 1R01DK102429, and 1R56HL130988.
The Role of CD70 in Vascular Function
Authors: Itani Hana A. , Pandey Arvind , Brown Jonathan D. , Harrison David G.
Published: November 2023

We have recently shown that CD70 is important in formation of effector memory T (TEM) cells in hypertension and TEM cells enhance the hypertensive response and end organ damage caused by repeated hypertensive stimuli. CD70 has traditionally been identified on antigen presenting cells and interacts with CD27 on T cells, promoting proliferation and memory cell formation. The precise cell types that express CD70 and the role of CD70 on non‐immune cells has not previously been investigated. Here we discovered a new role of CD70 in vascular function. Studies of mesenteric vascular reactivity showed that CD70−/− mice have markedly impaired endothelium‐dependent vasodilatation to acetylcholine compared to WT mice (44± 3 vs 25 ± 4%) at baseline. In contrast, there were no differences in relaxation responses to sodium nitroprusside. Pre‐incubating vessels with the endothelial nitric oxide synthase (eNOS) inhibitor L‐NAME blocked endothelium‐dependent vasodilatation to acetylcholine in WT mice by 50%, in contrast, L‐NAME had no effect on endothelial vessel relaxation in CD70−/− mice. This suggests that vessels of CD70−/− mice lack the capacity to produce nitric oxide. Western blots for the endothelial nitric oxide synthase (eNOS) showed a marked reduction of this enzyme in the aortas of CD70−/− as compared to wild type mice. In additional experiments, we showed that human umbillical vein endothelial cells express CD70 mRNA and that this is increased by > 30 fold by laminar (15 dynes/cm2) compared to oscillatory shear. Finally, using immunohistochemistry, we identified CD70 protein localized to resistance vessels of the kidney of ang II‐treated mice. These data define a new role of CD70 in modulating vascular function. Expression of CD70 might contribute to the previously identified ability of endothelial cells to present antigen to T cells and promote T cell activation and TEM cell formation. Moreover, CD70 seems to be linked to eNOS expression by mechanisms that remain to be defined.
Sodium butyrate suppresses Angiotensin II‐induced hypertension by inhibition of renal (pro)renin receptor and intrarenal RAS
Authors: Wang Lei , Zhu Qing , Lu Aihua , Zhang Linlin , Xu Chuangmin , Liu Xiyang , Yang Tianxin
Published: November 2023

Background

Butyrate, one of Short‐chain fatty acids, is end product produced from the fermentation of complex carbohydrates by the gut microbiota. Recently, sodium butyrate (NaBu) is found to play protective roles in a number of chronic diseases. However, it is still unclear whether NaBu processes a therapeutic potential in hypertension. The present study was aimed to investigate the role of NaBu in Ang II‐induced hypertension and further explore the underlying mechanism.

Methods

Ang II (200 ng/min) was infused in uninephrectomized Sprague‐Dawley rats plus intramedullary infusion of NaBu (1μg/kg·min) or not for 14 days. Blood pressure were monitored by the telemetry system. The renal tissues and serum samples as well as the last 24‐hour urine samples were collected to examine renal injury and regulation of (pro)renin receptor (PRR) and renin.

Results

Intramedullary infusion of NaBu in Sprague‐Dawley rats lowered the Ang II‐induced mean arterial pressure from 128.8 ± 6.2 mmHg to 108.0 ± 3.9 mmHg (P<0.01). This was paralleled with improvement of Ang II‐induced renal injury, including urinary albumin (μg/24h: Ctrl 270.7±49.2 vs AngII 1012.0±219.2 vs AngII‐NaBu 449.4±73.3, P<0.05), glomerulosclerosis (GDI: Ctrl 1.48±0.08 vs AngII 2.70±0.06 vs AngII‐NaBu 1.68±0.08, P<0.01), and renal fibrosis (α‐SMA protein: Ctrl 1.00±0.06 vs AngII 1.38±0.05 vs AngII‐NaBu 0.64±0.05, P<0.01), as well as the expression of inflammatory mediators TNF‐α and IL‐6. The renal protein expression of (pro)renin receptor, urinary excretion of soluble (pro)renin receptor and renin were all increased by AngII infusion but decreased by NaBu treatment. In cultured IMCD cells, NaBu treatment attenuated AngII‐induced expression of PRR and renin.

Conclusion

These results demonstrated NaBu exerts antihypertensive action likely via suppressing PRR‐mediated intrarenal RAS.

Support or Funding Information

This work was supported by National Natural Science Foundation of China Grant No. 91439205, No. 31330037 and No. 81600322; Fundamental Research Funds of Sun Yat‐sen University 16ykpy47; Veterans Affairs Merit Review, and National Institutes of Health Grant DK094956. T. Yang is Research Career Scientist in the Department of Veterans Affairs.
Renal‐Specific Dendritic Cell Depletion Reduces Urinary Interleukin 6 Levels Following Angiotensin II‐Mediated Hypertension and Dietary High Salt
Authors: Wynne Brandi Michele , Hecht Gillian G , Kronk Trinity Augusta , van Elst Henrieke , Tansey Malu , Eaton Douglas C , Hoover Robert S.
Published: November 2023

During hypertension, dendritic cells (DCs) have been shown to activate other immune cells and secrete cytokines, such as interleukin 6 (IL‐6). Recently, DCs were found to form an intricate network within the renal cortex and especially along the distal nephron. Studies reveal that renal‐specific DCs (rDCs) play an important role in the progression of hypertension. Previously, our data suggest that IL‐6 can increase sodium chloride cotransporter (NCC) expression and activity. We hypothesize that rDCs modulate intra‐renal levels of IL‐6 during hypertension or high salt (HS), which may affect distal nephron sodium reabsorption.

To test this hypothesis, mice lacking a functional CX3CR1 chemokine receptor (CX3CR1‐EGFP+/+ or rDC mice) were used. CX3CR1 is required for DC localization to the kidney; thus these mice have a renal‐specific DC‐depletion. Mice were either given: vehicle or angiotensin II (AngII) (60ng/min, 28 days + 4% high salt) via osmotic minipump or fed a high salt (4%, 7 days) or normal diet. Systolic BP measurements were obtained with tail‐cuff plethysmography. Metabolic cage studies were performed and urine collected. Urine and serum were used to perform a cytokine array via Meso‐Scale Discovery system. Whole kidney cortex lysates were used for mRNA isolation and RT‐PCR.

Mice with renal‐specific loss of DCs had a decreased systolic blood pressure at baseline. Following AngII hypertension, similar BP increases were observed (Δ38mmHg rDC vs. Δ36mmHg Wt). Baseline serum IL‐6 levels were not different; however, AngII‐infusion resulted in ≈3 fold decrease in IL‐6 mRNA in rDC mice when compared to Wt mice. At baseline, urinary IL‐6 levels were not different between rDC and Wt (4.41 ± 0.85 pmol rDC vs. 4.14 ± 1.20 pmol Wt, n=7–10) mice. However, HS feeding increased urinary IL‐6 levels in Wt mice, which was prevented in rDC mice (2.03 ± 0.20 pmol rDC vs. 7.34 ± 2.51 pmol Wt, n=6, p<0.05).

These data suggest that baseline BP is modulated by resident innate immune cells specific to the kidney and rDC loss reduces systolic BP; this reduction is maintained during AngII‐hypertension. Notably, our data demonstrate that while systemic IL‐6 levels are unchanged with rDC‐depletion, renal mRNA and urinary IL‐6 levels are decreased during hypertension or HS feeding. Thus together, these data propose a model for rDC‐mediated increases in IL‐6 levels during hypertension or increased salt consumption, altering the pro‐inflammatory milieu and increasing distal sodium reabsorption.

Support or Funding Information

NIDDK085097 and Department of Veteran Affairs MERIT Award I01BX002322‐01 to RSH; 2T32DK7656‐21 Emory Renal Division to BMW; R37 DK037963 to DCE;
Intracellular Cl− and the Scaffold Protein Mo25/Cab39 Regulate WNK‐SPAK/OSR1 Signaling in Transepithelial Ion Transport
Authors: Rodan Aylin , Wu Yipin , Sun Qifei , Humphreys John , Schellinger Jeffrey , Goldsmith Elizabeth
Published: November 2023

Objective

The WNK (with‐no‐lysine kinase)‐SPAK (Ste20/SPS1‐related proline/alanine‐rich kinase)/OSR1 (oxidative stress responsive‐1) kinase cascade regulates SLC12 family cation chloride cotransporters. Here, we investigate mechanisms of pathway activation in the regulation of transepithelial ion transport in the Drosophila melanogaster renal tubule.

Methods

Transepithelial K+ flux in the Drosophila tubule main segment was measured using the Ramsay assay and ion‐specific electrodes in control and mutant tubules. Intracellular Cl− concentration was measured using the transgenic sensor ChlopHensor. Phosphorylation of transgenically expressed kinase‐dead rat SPAK was used as a readout of tubule WNK activity. Drosophila WNK autophosphorylation was measured using mass spectrometry. The effect of Mo25 on Fray activity was measured using in vitro kinase assays.

Results

We have previously demonstrated that transepithelial K+ flux is increased in Drosophila renal tubules bathed in hypotonic medium in a WNK‐SPAK/OSR1 (fray in flies)‐NKCC dependent manner. We hypothesized that in hypotonic conditions, intracellular Cl− is decreased, leading to WNK activation. Intracellular Cl− was 30±4 mM in isotonic conditions; after switching to hypotonic medium, Cl− decreased to 20±3 mM at 10 minutes, and 16±2 and 15±2 mM at 30 and 60 min. Cl− inhibits Drosophila WNK kinase domain autophosphorylation in vitro: 13% of Drosophila WNK was autophosphorylated at Ser434 after 4 hours in 150 mM NaCl, compared to 52% in 50 mM NaCl. Paralleling the fall in Cl− in the tubule in hypotonic conditions, tubule WNK activity increased at 30 and 60 minutes after the tubules were transferred to hypotonic medium. However, overexpression of a predicted Cl−‐insensitive WNK, WNKL421F, during adulthood was not sufficient to increase transepithelial K+ flux in isotonic conditions. We hypothesized that the scaffold protein, Mo25/Cab39, may additionally be required. Using purified Drosophila proteins, we showed that Mo25 stimulates Fray phosphorylation of the fly NKCC in vitro. Mo25 knockdown in the tubule did not alter transepithelial K+ flux in isotonic conditions, but abolished hypotonic stimulation of K+ flux, indicating that Mo25 is required for maximal activation of the pathway. Furthermore, while overexpression of Mo25 in adult tubules did not increase K+ flux in isotonic conditions, simultaneous overexpression of Mo25 and WNKL421F increased K+ flux.

Conclusion

Intracellular Cl− and Mo25 act together to stimulate WNK‐SPAK/OSR1‐regulated transepithelial ion transport.

Support or Funding Information

National Institutes of Health
Modularity in the ENaC/Degenerin Family Facilitates Transfer of Functional Properties Between Family Members
Authors: DeMarco Michael A , Wang Xue‐Ping , Balchak Deidra M , Powell Koehler , Mukherjee Anindit , Blobner Brandon M , Kashlan Ossama B
Published: November 2023

Members of the epithelial Na+ channel (ENaC)/Degenerin family are cation selective channels that respond to the extracellular environment. ENaCs and acid sensing ion channels (ASICs) are found in all tetrapods and are involved in diverse processes ranging from renal Na+ reabsorption and regulation of airway surface liquid height (ENaCs) to pain sensation and fear conditioning (ASICs). ASIC and ENaC subunits share significant sequence identity both in the core of the extracellular fold, characterized by β‐sheets, and in the transmembrane helices. The three peripheral helical domains of the extracellular structure, most notably the so‐called finger domain, are poorly conserved. We hypothesized that diversity of function in this protein family rose from an inherently modular architecture. To test our hypothesis, we swapped the finger domain of the mouse ENaC α subunit into mouse ASIC1, and examined its functional and biochemical properties. When we expressed chimeric channels in Xenopus oocytes and assessed function by two‐electrode voltage clamp, we found that the channel had functional attributes of both channels. Like ENaC, channels were constitutively active, and like ASIC1, channels were activated by acidification. Channels exhibited a basal current at pH 8. Upon rapid acidification to pH 4.5, currents substantially increased, and did not desensitize as is characteristic for ASIC1 currents. Consistent with retention of an ASIC1 pore, these channels were permeable to most cations tested, including Na+, K+, and Ca2+, and were blocked by benzamil, an amiloride analog. ENaC α subunits are cleaved at R205 and R231 by furin in the finger domain during biosynthesis, effectively activating quiescent channels. To examine whether the chimeric channel was similarly processed, we added an HA epitope tag to the C‐terminus of the channel and performed an immunoblot. We observed two bands at sizes expected for full length and furin‐cleaved channels. Furthermore, mutating the putative furin‐cleavage sites within the swapped‐in ENaC α subunit finger domain eliminated the lower molecular weight band, but had no apparent effect on channel activity. We also tested whether swapping the ENaC α subunit finger domain into ASIC1 transferred other functional properties, but found none. Our data suggest that the ASIC1 finger domain is critical to channel desensitization, and like the ENaC finger domain, functions as an autoinhibitory domain. Our data suggests a modular architecture for ENaC/Degenerin proteins, and that specific channel functions can be attributed to specific domains.

Support or Funding Information

This work is supported by NIH grant DK098204.
Chronic Loop Diuretic Treatment Mediates Segment‐Specific Hypertrophy in the Nephron
Authors: Krauson Aram , Walczak Elisabeth , Iyer Sonali , Velarde Nona , Nizar Jonathan , Bhalla Vivek
Published: November 2023

Furosemide, a commonly prescribed loop diuretic to treat hypertension and edema, increases salt excretion by inhibiting reabsorption via the sodium‐potassium 2 chloride cotransporter (NKCC2) in the thick ascending limb. Chronic use of loop diuretics can lead to resistance and the need for higher doses and/or additional classes of diuretics with further long term adverse effects. We previously have shown mice chronically fed furosemide develop hyperplasia in the nephron after 7 days and hypertrophy after 21 days. Furthermore, our data suggested the IGF‐1/IGF‐1R mediated pathway to be involved in tubular hypertrophy. From chronic furosemide treatment, cell size increased in the proximal tubule, distal tubule, and connecting tubule/cortical collecting duct segments. In contrast, long term furosemide treatment had no effect on cell size in the furosemide‐inhibitable thick ascending limb. Using novel, inducible kidney tubule‐specific IGF‐1R knockout mice (iT‐IGF‐1R KO), we demonstrate diminished kidney growth in furosemide‐treated knockout mice only in the proximal tubule. In contrast, knocking out IGF‐1R did not affect cell size increase in the distal tubule or the connecting tubule/cortical collecting duct. These results suggest long term furosemide use induces IGF‐1R‐dependent hypertrophy of the proximal tubule but IGF‐1R‐independent hypertrophy of the distal tubule. Interestingly, using immunofluorescence, IGF‐1R is not detected in the proximal tubule but in more distal segments implicating paracrine activation in hypertrophy. Overall, we demonstrate furosemide‐treated mice undergo segment‐specific hypertrophic responses and mechanisms. To uncover the mechanisms of these segment‐specific responses we are using single cell RNA seq analysis and data will be forthcoming.

Support or Funding Information

NIH/NIDDK: 5T32DK007357‐32 (T32), DK09156501A1 (R01)
Calcium‐Mediated Activation of the BK K+ Secretory Channel in Collecting Duct Cells is Dynamically Regulated by the Ca2+‐Activated SK3 and IK1 K+ Channels
Authors: Li Yue , Hu Hongxiang , Tian Jin Bin , Zhu Michael , O'Neil Roger
Published: November 2023

The Ca2+‐activated BK K+ channel is the dominant pathway for flow‐ and Ca2+‐induced K+ secretion/K+ wasting in collecting duct system (CDS) of the kidney. The goal of the current study was to determine the role of two newly identified Ca2+‐activated K+ channels in CDS, SK3 and IK1 (expressed at both luminal and anti‐luminal cell borders), on BK channel activation (expressed at the luminal border). Like BK, these KCa channels are activated by TRPV4‐induced activation of Ca2+ influx (Berrout et al., PLoS ONE 9:e0156368; Li et al., PLoS ONE 11:e0155006, 2016), but have a much higher Ca2+‐binding affinity (300–600 nM) compared to BK (1–11 uM), properties consistent with a “Ca2+ sensing” function of SK3/IK1. In mCCDcl1 cells, a K+ secreting mouse collecting duct cell line, activation of each of these KCa channels following TRPV4 activation was observed to hyperpolarizes Vm, where selective inhibition of SK3, IK1 and BK, by addition of selective blockers apamin (300 nM), TRAM‐34 (300 nM), and IbTX (100 nM), respectively, each depolarized Vm and markedly reduce TRPV4‐mediated Ca2+i levels. Further, activation of BK was observed to be directly proportional to the TRPV4‐mediated increase in Ca2+i levels. Little or no activation of BK were observed when stimulated Ca2+i levels were low and strong activation of BK when Ca2+i levels were high. Inhibition of SK3 and IK1 each reduced the stimulated Ca2+i levels as expected. Addition of both apamin and TRAM‐34 during TRPV4 activation markedly depolarized Vm, reduced the increase in TRPV4‐mediated Ca2+i levels from 766 +/− 25 nM to 348 +/− 40 nM (n = 30), and reduced the initial rate of rise of TRPV4‐mediated Ca2+i from 215 +/− 14 to 30 +/− 6 nM/min (n = 30). Further, TRPV4‐mediated activation of BK was markedly reduced or abolished in the presence of apamin and TRAM‐34. It is concluded that SK3 and IK1 are functionally expressed in collecting duct cells and that both are activated by low levels of TRPV4‐mediated Ca2+ influx that leads to an initial hyperpolarization of Vm and further stimulation of Ca2+ influx. This SK3/IK1‐mediated enhance Ca2+ influx, in turn, is critical for activation of the BK channel where we show that strong dynamic coupling between TRPV4 and SK3/IK1 plays a central role in modulating BK channel activity in the CDS.

Support or Funding Information

This work was supported by the American Heart Association, Grant # 14GRNT20380916 and the National Institutes of Health, Grant #: 1R01DK098401
Role of Sodium Hydrogen Exchange Regulatory Factor‐1 (NHERF1) in Aging: Tale of Two Rat Models
Authors: Ahmad Asrar , Pushpakumar Sathnur , Ketchem Corey J , Sen Utpal , Lederer Eleanor DeLand , Khundmiri Syed Jalal
Published: November 2023

Aging animals develop salt‐sensitive hypertension due, in part, to desensitization of dopamine receptors and sensitization of AT1R in renal proximal tubules. We demonstrated old FBN rats develop salt‐dependent hypertension. Preliminary data showed loss of NHERF1 expression in 22mo old F344 rats. We hypothesized that loss of NHERF1 may contribute to salt reabsorption in aging. To address this hypothesis, Fischer Brown Norway (FBN) rats (1, 4, 12, and 24mo old) were fed 1% or 8% NaCl diet for one week and, urinary volume, sodium, potassium, and chloride and expression of Na‐K ATPase a1 subunit (NKA), NHERF1, NKCC2, NCC, and D1R were measured. Feeding 8% NaCl increased 24 h urine volume compared to rats fed 1% NaCl diet in 1mo (13.6±0.98 vs 29.2±3.8 mL) and 4mo (15.3±1.5 vs 39.2±2.4) but not in 24 mo rats (20.7±4.7 vs 15.9±2.4). UNaV and UClV increased in young rats fed with 8% NaCl but not in old rats. UKV did not change in young rats but significantly decreased in older rats. To our surprise, in contrast to F344 rats, NHERF1 expression increased with age in FBN rats (0.21±.03 (4mo) vs 0.71±0.07 (24mo) AU ratio NHERF1:actin). Increased NHERF1 expression with age in FBN rats was associated with decreased D1R expression (0.8±0.16 vs 0.4±0.04) while in F344 rats D1R expression increased with age (0.6±0.1 vs 0.9±0.1). To determine the role of NHERF1 in the two models of rats, we determined TNFa in kidney homogenates from FBN and F344 rats. TNFa increased with age in both F344 (4.04±0.15 (4 mo) vs 6.9±0.42 (24 mo)) and FBN rats (2.1±0.3 vs 4.05±0.31 ng/mg protein). TNFa levels were significantly higher in young F344 rats as compared to young FBN rats. To determine if lack of NHERF1 is responsible for salt wasting, we measured urine volume and Na in 18 mo old WT and NHERF1 KO mice. The urine volume in WT mice (2.7±0.8 (1% NaCl) vs 5.6±1.2 mL/24 h (8% NaCl)) were significantly lower than KO mice (3.6±0.57(1% NaCl) vs 7.5±0.88 mL/24 h (8% NaCl)). However, there was no significant difference in UNaV between WT and KO mice fed an 8% NaCl diet. D1R expression increased in NHERF1 KO mice as compared to WT mice. TNFa increased significantly in NHERF1 KO mice kidneys as compared to WT mice. We conclude that increased NHERF1 expression as seen in FBN rats result in salt retention while lack of NHERF1 as seen F344 rats and NHERF1 KO mice increases renal inflammation. Further studies are required to delineate the two roles of NHERF1.

Support or Funding Information

NIA grants R21AG047474 and R25AG047843 and AHA grant 16GRNT31030019
Assessing the Role of OCRL1 in Proximal Tubule Function in a Lowe Syndrome Cell Model
Authors: Eshbach Megan L. , Shipman Katherine E. , Raghavan Venkatesan , Rbaibi Youssef , Long Kimberly R. , Labilloy Anatália , Baty Catherine J. , Weisz Ora A.
Published: November 2023

Lowe syndrome is an X‐linked recessive disorder caused by mutations in OCRL, which encodes the phosphatidylinositol 5′‐phosphatase OCRL1. Hallmark symptoms of Lowe syndrome include proximal tubule (PT) dysfunction, typically characterized by low molecular weight proteinuria (LMWP), renal tubular acidosis, aminoaciduria, and hypercalciuria. OCRL1 has been hypothesized to play a role in myriad cellular processes including membrane trafficking, actin dynamics, cell polarity, ciliogenesis, and cell division. However, consensus is lacking for how loss of OCRL1 leads to impaired PT function. To this end, we created a PT cell culture model of the disease. Using the CRISPR/Cas9 genome editing system, we knocked out OCRL1 expression in a human PT cell line. In parallel, we transiently knocked down OCRL1 using siRNA to confirm that chronic depletion did not result in cell adaptation. Neither acute nor chronic depletion of OCRL1 had any effect on endocytic uptake of the megalin/cubilin ligand albumin. However, OCRL1 depleted cells consistently proliferated more slowly and exhibited a higher percentage of multinucleated cells than matched controls. We hypothesize that defects in cell division during kidney development and/or repair after kidney injury may compromise PT length and/or diameter in patients and have developed a mathematical model to assess the effect of such changes on PT albumin uptake. Our data suggest a new explanation for how loss of OCRL1 function results in LMWP as well as the other commonly observed renal manifestations of Lowe syndrome.

Support or Funding Information

National Institutes of Health R01‐DK101484, R01‐DK100357, P30‐DK079307, T32‐DK061296; Lowe Syndrome Association
Corticosterone activates the thiazide‐sensitive NaCl transporter through the glucocorticoid receptor
Authors: Bailey Matthew A , Ivy Jessica R
Published: November 2023

The Na‐Cl co‐transporter (NCC) is the major route for apical Na entry into cells of the distal convoluted tubule (DCT). Thiazide diuretics inhibit NCC and are a mainstay hypertension treatment. We have previously shown that glucocorticoid excess activates NCC and induces hypertension. The DCT expresses both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) but does not express the glucocorticoid‐metabolizing enzyme 11bHSD2 at high levels. This suggests that glucocorticoids may directly activate the transporter. Here we investigated whether glucocorticoids activate NCC in vivo and whether GR or MR is involved in this process.

Male C57BL6 mice were anaesthetized under isofluorane and implanted with silastic pellets containing spironolactone (50 mg; chronic MR blockade) or RU486 (60 mg; chronic GR blockade). Control mice received no implant. After 5 days, corticosterone (6 mg/kg, s.c.) or vehicle (veh, 2% DMSO, s.c.) was injected at 0800, coincident with the nadir of the endogenous corticosterone circadian rhythm. Mice were culled at 1200 and the kidneys snap frozen. NCC threonine phosphorylation (on T53; pT53‐NCC) was measured by western analysis as an index of transporter activation.

Data are mean±SD. In control mice, acute corticosterone doubled pT53‐NCC abundance (p=0.004, Student's t test, n=6 in each group). In mice receiving chronic MR blockade (n=5), total NCC protein was significantly (P<0.001) reduced compared to controls (n=6); corticosterone still significantly increased pT53‐NCC abundance (1.0±0.46 AU (veh; n=5) vs 3.7±1.4 AU (cort; n=7), p=0.0018, Student's t test). Chronic GR blockade did not affect total NCC abundance (n=6 in each group) but prevented pT53‐NCC phosphorylation induced by acute cort injection (n=6 in each group).

Our main conclusion are: 1) MR activation is required for maintenance of tonic levels of NCC protein; 2) glucocorticoids acutely increase NCC phosphorylation through GR activation. Direct regulation of NCC activity by glucocorticoids has implications for blood pressure control, particularly in individuals with inappropriately elevated glucocorticoids, such as in diabetes, obesity or during chronic stress.

Support or Funding Information

We thank The British Heart Foundation Centre of Research Excellence and Kidney Research UK for funding
Deletion of Xenotropic and Polytropic Retrovirus Receptor 1 in mouse nephron causes renal Fanconi syndrome and hypophosphatemic rickets
Authors: Ansermet Camille , Moor Matthias , Centeno Gabriel , Auberson Muriel , Hu Dorothy , Barron Roland , Nikolaeva Svetlana , Haenzi Barbara , Katanaeva Natalya , Gautschi Ivan , Katanaev Vladimir , Rotman Samuel , Koesters Robert , Schild Laurent , Pradervand Sylvain , Bonny Olivier , Firsov Dmitri
Published: November 2023

Phosphate (Pi) is one of the most abundant minerals in the body and tight control of extra‐ and intracellular Pi concentrations is crucial for many biological processes. The kidney is the main organ involved in the regulation of minute‐to‐minute phosphate homeostasis by controlling Pi reabsorption from the primary urine. This reabsorption primarily occurs in the proximal tubule by the action of the apical sodium‐dependent phosphate transporters NaPi‐IIa, NaPi‐IIc and PiT2. The protein(s) involved in the basolateral phosphate efflux remain unknown. As recent in vitro and genetic studies have linked the xenotropic and polytropic receptor 1 (Xpr1) to Pi efflux, we aimed to assess if it could also be involved in renal Pi reabsorption. To address this question, we used the Pax8‐rtTA‐driven Cre expression system, allowing a conditional inactivation of Xpr1 in mouse renal tubule.

Functional analysis showed that conditional Xpr1 knockout mice (cKO) have hypophosphatemia with transient hyperphosphaturia, an increase in fractional excretion of phosphate (FEPi) along with a decreased TmPi/GFR. These phosphate disturbances leaded us to study the bone phenotype. Micro‐CT analysis of vertebrae revealed significant decrease in bone mineral density, percent bone volume and trabecular thickness and number in male cKO mice, characteristic of osteoporosis and osteomalacia. Histomorphometric analysis of vertebrae also showed an increase in unmineralized osteoid and osteoclastogenesis in cKO mice. Moreover, ex‐vivo experiments using primary cultures of proximal tubule of Control and cKO mice showed that Xpr1 deficiency strongly affects the [33P]phosphate efflux whereas the [33P]phosphate uptake remains similar between Control and cKO cells. Additional phenotypic observations in cKO mice include aminoaciduria, glycosuria, hypercalcuria, hypercalcemia and proteinuria, indicating that these mice are developing a complete Fanconi syndrome, a generalized proximal tubular dysfunction.

Taken together, these results support a significant role for Xpr1 in renal phosphate homeostasis.

Support or Funding Information

This work was supported by the Swiss National Science Foundation Research grants 31003A‐149440 and 310030‐163340
Extreme cousins of ours who are no more: From massive Gigantopithecus to diminutive Flores “hobbits.”
Authors: Laitman Jeffrey T
Published: November 2023

Our closest relatives are other primates, both living and ancestral. While all species have evolved features that separate them from other groups, there are some who, when viewed through the lens of time, stand apart as being “extreme.” Some have effectuated this by evolving to gargantuan or diminutive size, or by accruing unique responses to biological systems such as breathing. Among these, two stand apart for size: the largest primate, Gigantopithecus, and the smallest hominin taxon, Homo floresiensis. Gigantopithecus was a 10‐foot tall orangutan look‐alike first described in 1935 known largely from jaws and teeth. They inhabited SE Asia 9 mya to 100 kya when they succumbed to climate change affecting the plants and fruits these vegetarians ate. Why so big? We don't know. Clearly, however, their size gave them protection from predators and thus freedom to roam and feed as often as needed (which was often, given their size.) On the other extreme is “Flores Man,” discovered in 2003 on the Indonesian Island of Flores. Nicknamed “hobbit” due to their standing only 3.5 feet (cranial and postcranial remains recovered) Flores is argued to exhibit sufficient uniquely derived characters to warrant species‐level distinction separating them from preceding H. erectus or contemporaneous H. sapiens and are placed in the taxon H. floresiensis. Their average brain size is in about 400 cm3, in the range of Plio‐Pleistocene australopiths and chimpanzees, leading some to claim the specimens are microcephalic H. sapiens. Adherents of H. floresiensis attribute their diminutive size to “insular dwarfism” which also affected other island species. Flores died out circa 50 kya, arguably from contact with arriving H. sapiens. While these groups represent size extremes, perhaps the best example of an extinct relative is our closest cousin, the Neanderthals. While Neanderthals are us in many ways, studies have shown that they exhibited extreme specializations in their upper respiratory tracts (e.g., large noses with unique features; large paranasal sinuses; larynges likely higher in the throat than in living humans). This anatomy may have made them ideally equipped to process cold and dry air and be among features that separate them on a species level – H. neanderthalensis ‐ from H. sapiens. Neanderthals may thus have evolved to be “Respiratory Extremists” to conquer their world. Unfortunately, our ancestors lived in that world too, and perhaps what led to their demise was transmission of our respiratory pathogens to them. Whether it was the largest ape, the smallest Homo or our closest relative, our primate extremists seem to all have met the same end: extinction.
Modeling osmotic transients during exercise
Authors: Christmas Kevin M , Jensen Austin , Bassingthwaighte James
Published: November 2023

The body maintains tight osmotic regulation despite deviations from homeostasis at rest or during intense exercise. Maintaining osmotic equilibrium can result in exercising muscle reducing plasma volume by 10–15% depending on intensity, the mass of tissue stimulated, and mode of exercise. Research often references this phenomenon with reduced plasma volume or increased plasma osmolarity during exercise, but more correctly whole body osmolarity goes up with volume shifts occurring in all tissues. Elevated muscle osmolarity from exercising metabolism results in water flux into active muscle from the extracellular compartment, causing muscle cells to swell, decreasing plasma volume, and hemoconcentrating the blood. Assuming all solutes are fixed, impermeable to membranes, we can calculate the increase in extracellular osmolarity due to volume loss from a normal value of 280 mOsM to ~329 mOsM. Assuming de novo formation of intramuscular solutes, exercising metabolism would have to increase the muscle osmolarity by ~94 mM to produce this volume shift, assuming none leaves the cell. This rise in muscle osmolarity results in water influx, diluting the cells concentration to match the bodies osmolarity of 329 mOsM. These simple algebraic calculations fail to incorporate tissue mass, diffusion of metabolic substrates out of the muscle, or anatomical representation of a working human. During intense exercise a 70‐kilogram man generates an osmotic gradient shifting over a liter of fluid towards muscle within 30–60 seconds. Metabolically, glycolytic intermediates are prime candidates with muscle lactate concentration rising ten fold during intense exercise, but cannot be the only factor of importance. This model quantifies osmotic water flux due to metabolic byproducts of glycolysis and ATP breakdown with whole body osmotic balance and recirculation. Additional products of muscle metabolism need to be identified to explain the water fluxes during short term exercise.

Support or Funding Information

Research supported by NIH grants NHLBI T15 088516 and U01‐HL122199, and NIBIB EB08407.
Effect of Hydrogen Peroxide and Glutamate on Muscle Spindle Afferent Receptor Ending Sensitivity to Muscle Stretch in Adult Mice
Authors: Than Kimberly , Kwon Anthony , Wilkinson Katherine A.
Published: November 2023

Proprioception, or the sense of body position and movement, is required for the completion of complex motor tasks. Exercise impairs proprioception, although the mechanism for this is not well understood and likely involves changes to both peripheral signaling and central proprioceptive circuits. The Group Ia and II muscle spindle afferents (MSAs), which report muscle length and movement information to the central nervous system, are thought to be the primary proprioceptors. Substances released during exercise and activity, including reactive oxygen species and glutamate, have been shown to alter MSA activity in rats. Whether these substances alter MSA sensitivity via peripheral and/or central mechanisms is unclear. We tested the hypothesis that reactive oxygen species (hydrogen peroxide) and glutamate can act locally on MSA receptor endings and alter responsiveness to stretch. We used an in‐vitro mouse muscle‐nerve preparation to record MSA firing activity. Briefly, the extensor digitorum longus (EDL) muscle and innervating deep peroneal branch of the sciatic nerve were dissected and perfused in an oxygenated synthetic interstitial fluid tissue bath. An extracellular recording electrode was placed on the nerve and individual MSAs identified by waveform. A series of 4s ramp and hold stretches were applied to the EDL and instantaneous firing frequency before and during stretch was measured. Upon exposure to hydrogen peroxide (1 mM), there was an increase in static stretch response in 10 of 18 MSAs tested (n=9; 23 ± 13% increase). The remaining 8 MSAs showed no change in activity (n=9; 1.5 ± 2.8%). There was also an increase in individual afferent firing rates upon the addition of glutamate in a preliminary sample of MSAs (1 mM; n=4; 30 +/− 18% increase). These results support the hypothesis that hydrogen peroxide and glutamate can act locally in the absence of central nervous system circuits on MSA nerve endings to alter stretch sensitivity. However, not all MSAs respond to locally applied hydrogen peroxide, suggesting different sensitivities in subpopulations of MSAs.

Support or Funding Information

This work was supported by an SJSU RSCA award to KAW.