bluuebunny/crossref_metadata_2025 huggingface dataset:
Sympathetic Changes through the Menstrual Cycle are Absent in Women with Diabetes
Authors: Joshi Hitesh , McGaugh Sarah , Edgell Heather
Published: June 2021

Background

Glycemic variability (GV) can contribute to autonomic dysfunction in diabetes (Jaiswal et al. 2014), and autonomic dysfunction can be apparent as early as childhood (Metwalley et al. 2018). In women with diabetes, GV can differ throughout the menstrual cycle where hyperglycemia and decreased insulin sensitivity are often present in the luteal phase (Brown et al. 2015). Therefore, we investigated the role of the menstrual cycle in autonomic control in women with diabetes.

Methods

Women with diabetes (n=7; age 23.6 ± 1.0years; BMI 24.4 ± 1.1kg/m2) were tested in the early follicular (EF; day 2–5) and mid‐luteal (ML; day 18–24) phases of the menstrual cycle. Blood glucose was 8.6 ± 1.1mmol/L in the EF phase and 8.1 ± 0.7mmol/L in the ML phase (P=0.71). Trials included resting heart rate variability (HRV), paced deep breathing, Valsalva, and 10 minutes of 70‐degree upright tilt. Heart rate (HR), stroke volume index (SVi), and mean arterial pressure (MAP) were measured. Frequency domain analysis of HRV was conducted on 5 minutes of resting ECG (low frequency power (LF) and high frequency power (HF)).

Results

The expiration to inspiration HR ratio during paced deep breathing (EF: 1.40 ± 0.06, ML: 1.43 ± 0.05, P=0.39) and the Valsalva HR ratio (EF: 2.29 ± 0.16, ML: 2.16 ± 0.13, P=0.48) were similar between the phases. During the Valsalva maneuver, the increase in MAP from Phase II minimum to the end of Phase II was similar between phases (EF: +18.6 ± 3.8mmHg, ML: +18.4 ± 1.5mmHg, P=0.64). Upright tilt resulted in higher HR (P=0.005) and lower SVi (P=0.011) with no interaction effects between tilt and menstrual phase (P≥0.64). The maximum HR response to tilt was similar between the phases (EF: 101.2 ± 5.7bpm, ML: 99.5 ± 5.9bpm, P=0.69). There were no differences between phases in resting heart rate variability (LF(nu): EF: 37.5 ± 6.0, ML: 40.2 ± 6.8, P=0.68; HF(nu): EF: 45.0 ± 8.3 ML: 58.0 ± 6.3, P=0.66).

Conclusion

Abidi et al. (2017) found a strong tendency for a greater MAP response to Valsalva in the ML phase compared to the EF phase in healthy women. They also found higher heart rate and cardiac output in the ML phase compared to the EF phase throughout 10min of standing supporting greater sympathetic output in the ML phase. Further, in healthy participants, Brar et al. (2015) and Carter et al. (2013) found higher LF control of HR and higher resting muscle sympathetic nerve activity in the ML phase, respectively. Therefore, we suggest that the expected increase of sympathetic activity in the ML phase is absent in women with diabetes.

Support or Funding Information

Faculty of Health, York University Canadian Foundation for Innovation ‐ Infrastructure Operating Fund Natural Sciences and Engineering Research Council of Canada

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Sensory Nerves Increase Blood Pressure and Sympathetic Nerve Activity in 2‐Kidney 1‐Clip Hypertensive Mice
Authors: Ong Jason , Sved Alan F , Kinsman Brian J. , Tan Roderick J , Rush Brittney M , Carrattino Marcelo D , Stocker Sean D
Published: June 2021

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to determine whether renal sensory nerves contribute to the 2‐Kidney‐1‐Clip (2K1C) model of hypertension. 2K1C hypertension was produced in male C57B16 mice (12–14 weeks, Jackson Laboratories) by placement of a 0.5mm length of PTFE tubing (ID: 0.008” x OD: 0.014”) around the left renal artery. 2K1C mice (n=10) displayed an elevated ABP measured via telemetry (Day 0: 96 ± 1mmHg vs Day 14: 120 ± 3mmHg; P<0.01). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 14 vs Day 0 (Day 0: −35 ± 2mmHg vs Day 14: −57 ± 5mmHg; P<0.01). 2K1C‐clipped kidneys vs 2K1C‐unclipped kidneys had lower mass (105 ± 14mg vs 173 ± 8mg, respectively; P<0.05) and higher mRNA levels of several pro‐inflammatory cytokines (IL1B, IL2, IL10, TNFa; P<0.05). Renal afferent discharge was significantly higher in 2K1C‐clipped kidneys vs 2K1C‐unclipped vs sham mice (2K1C‐clipped: 3.4 ± 0.9 Hz vs 2K1C‐unclipped: 0.7 ± 0.3Hz vs control: 0.3 ± 0.1 Hz, n=6/group; P<0.05). Furthermore, electrical stimulation of renal afferent nerves in control mice (n=6) produced a frequency‐dependent increase in ABP (5Hz: 4 ± 1mmHg, 10Hz: 6 ± 1mmHg, 20Hz: 11 ± 2mmHg; P<0.05). These responses were eliminated after ganglionic blockade with 5mg/kg chlorisondamine (5Hz: 1 ± 1mmHg, 10Hz: 1 ± 1mmHg, 20Hz: 1 ± 1mmHg, n=4/group; P<0.05). Stimulus‐triggered averaging of SNA during stimulation of renal afferent nerves (1 Hz, 200uA) revealed significant (P<0.05, n=4/group) increases in splanchnic (179 ± 11%), renal (195 ± 15%), and lumbar (167 ± 9%) SNA. Interestingly, the latency to the peak SNA (142 ± 10ms) suggests supraspinal pathways mediate the sympathoexcitatory response. Finally, both total renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33mM capsaicin) at time of clipping resulted in lower ABP than 2K1C mice at Day 14 (2K1C: 120 ± 3mmHg, phenol: 106 ± 3mmHg, capsaicin: 108 ± 3mmHg, n=6–10/group; P<0.05). These findings suggest 2K1C hypertension depends on renal sensory nerves and elevated SNA via supraspinal pathways.

Support or Funding Information

NIH grant HL113270, AHA Undergraduate Research Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NHERF1 loss results in metabolic stress and increased susceptibility to cisplatin‐induced acute kidney injury
Authors: Bushau‐Sprinkle Adrienne Marie , Barati Michelle , Conklin Caryl , Dupre Tess , Gagnon Kenneth B. , Siskind Leah , Doll Mark , Rane Madhavi , Clark Barbara , Merchant Michael , Klinge Carolyn , Brier Michael , Coventry Susan , Lederer Eleanor
Published: June 2021

Background

Acute kidney injury (AKI) is associated with a high mortality rate and develops in 30% of patients who receive cisplatin. The sodium hydrogen regulatory factor isoform 1 (NHERF1) is a scaffolding protein that anchors multiple membrane proteins, in renal proximal tubules. We have recently demonstrated that NHERF1 loss results in changes in mitochondrial protein abundance and function.

Hypothesis

We hypothesize that NHERF1 loss increases susceptibility to AKI through an underlying metabolic stress.

Materials and Methods

We treated 2 month old male and female wild type (WT) C57BL/6 and NHERF1 knock out (KO) mice with vehicle (VEH) or cisplatin (CIS) (20 mg/kg dose IP) and euthanized after 72 hours. Blood was collected for blood urea nitrogen (BUN) levels. Urine was collected for NGAL. Kidneys were harvested for histology, TUNEL assay, and Western Blot for cleaved caspase 3. Proteomic analysis was conducted on kidney brush border membrane (BBM) of WT and KO kidneys. Metabolic measurements were conducted via the XF Flux analyzer on non‐treated primary proximal tubule cell cultures. Three‐way ANOVA was used to compare the different treatment groups. Ordinal regression was performed for the semi‐quantitative scoring. All statistical analysis was conducted with SPSS software. P values of <0.05 were considered statistically significant.

Results

CIS caused significantly greater severity of kidney injury in KO compared to WT mice by semi‐quantitative injury score (WT: 1.89, KO: 2.8), BUN levels (WT: 97.8 mg/dL +/− 10.1, KO: 151.8 mg/dL +/− 17.2) and NGAL protein (WT: 2.7 μg/mL +/− 0.53, KO: 55.6 μg/mL +/−21.3). Apoptosis markers were significantly increased in CIS treated KO and WT mice compared to respective controls. Proteomic comparison of BBM protein expression in WT and KO showed marked changes in the expression of mitochondrial proteins in KO mice, suggesting alterations in cell metabolism. KO proximal tubule cells also exhibited decreases of 35% in glycolytic reserve capacity, 40% in baseline mitochondrial respiration rate, 37% in ATP‐linked mitochondrial respiration rate, 40% in mitochondrial reserve capacity, and 40% in maximum mitochondrial capacity.

Conclusions

We conclude that the KO mice have increased susceptibility to CIS‐induced AKI, which may be due to altered metabolism and/or mitochondrial dysfunction.

Support or Funding Information

Funding for this research was provided by VA and UofL School of Medicine (to EDL).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AKI to CKD Transition is driven by Alpha 2 Adrenergic Receptors
Authors: Jang Hee‐Seong , Padanilam Babu
Published: June 2021

Background

Enhanced tissue level and activity of norepinephrine (NE) have been recognized as risk factors of development and progression of chronic kidney disease (CKD). Our reports showed renal denervation (DNx) can prevent renal fibrosis and inflammation in renal fibrogenesis models. However, the underlying mechanism by which renal DNx prevents the long‐term sequelae of acute kidney injury (AKI) remains to be elucidated.

Methods

Based on our published data, we hypothesized that NE mediates its effect through activation of alpha 2 adrenergic receptors (α2‐ARs). In this study, we investigated whether genetic and/or pharmacological inhibition of α2‐ARs prevents ischemia/reperfusion injury (IRI)‐induced inflammation and CKD progression.

Results

Renal bilateral IRI resulted in severe kidney fibrosis in medullary region over 2 weeks with persistent tubular injury and massive macrophage infiltration. Renal DNx suppressed renal inflammation, tubular damage, and fibrosis progression, as well as increase in blood pressure, 2 weeks post‐IRI. Pharmacological inhibition of α2‐AR also inhibited fibrosis progression with reduced downstream targets of NE‐α2‐AR axis, including angiotensinogen and fibrogenic factors. Consistent with results in renal DNx and pharmacological inhibition of α2‐AR, inhibition of α2A‐ and α2C‐AR subtypes by pharmacological or genetic manipulation additively prevented macrophage accumulation with downregulation of M1 and M2 macrophage markers, resulting in suppression of persistent renal tubular injury and fibrosis progression.

Conclusion

Taken together, blockage of α2‐AR subtypes prevents IRI‐induced AKI to CKD transition through inhibition of macrophage accumulation, persistent renal tubular injury and fibrosis progression, suggesting that targeting α2‐ARs may be a potential therapeutic strategy to prevent long‐term sequelae of ischemic AKI.

Support or Funding Information

This work was supported by NIH DK‐083291 and DK‐090332 and UNMC College of Medicine Bridge fund.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Trehalose attenuates mitochondrial dysfunction in cisplatin‐induced acute kidney injury by activating autophagy
Authors: Zhu Lingling , Yuan Yujia , Li Hongxia , Chen Younan , Lu Yanrong , Cheng Jingqiu
Published: June 2021

Objective

Cisplatin, an anticancer drug, always lead to nephrotoxicity via causing mitochondrial dysfunction. As a major catabolic pathway, autophagy has been proved that could protect against cisplatin‐induced acute kidney injury (AKI). Base on the activation of autophagy induced by trehalose, we aimed to investigated whether trehalose could alleviate mitochondrial dysfunction and kidney injury in mice though autophagy‐lysosome mediated clearance of damaged mitochondria.

Methods

Mice were administered a single intraperitoneal (i.p.) injection of cisplatin 16mg/kg to induce AKI, then mice were treated with PBS or trehalose (i.p.). In vitro, HK2 cells were treated with cisplatin 16mg/kg in the presence or absence of trehalose. To evaluate mitochondrial bioenergetic, mitochondrial morphology, membrane potential and ATP content were detected.

Results

In vivo, comparing with the normal mice, BUN and Crea were significantly increased in AKI mice, while decreased after the treatment with trehalose, indicating that trehalose could alleviate kidney injury in AKI mice. In vitro, along with the activation of autophagy (elevated expression of LC3 II and ATG5), mitochondrial fragmentation, depolarization, and reduced ATP generation induced by cisplatin were markedly inhibited in trehalose‐treated HK2 cells. Moreover, the double immunofluorescence staining results showed that the co‐localization between mitochondria and LC3 was increased, indicating that the mitophagy was induced. In addition, we found that the protective effect of trehalose was largely abolished when the autophagy process was inhibited by HCQ.

Conclusion

These results indicated that trehalose can ameliorate cisplatin‐induced mitochondrial dysfunction by activating autophagy, and thus it might be a promising therapy to acute kidney injury.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Involvement of Tubular Mas Receptor in Lipid‐Induced Impaired Autophagy and ER Stress in the Kidney
Authors: Kong YongLun , Zhao Xiaoduo , Li Suchun , Qiu Miaojuan , Lin Yu , Li Chunling , Wang Weidong
Published: June 2021

The local renin‐angiotensin system in the kidney plays an important role in development of obesity‐related kidney diseases. The purpose of current study was to investigate the role of Mas receptor in lipid‐induced kidney injuries. In human proximal tubular epithelial HK2 cells, saturated fatty acid palmitic acid (PA, 0.4mM) treatment induced increased LC3B and P62 protein expression and decreased LAMP1 expression, indicating an impaired autophagy flux and accumulation of autophagosome. This was associated with upregulated BIP and CHOP protein expression, two markers of endoplasmic reticulum stress (ER stress), and upregulated expression of cleaved caspase‐3, a marker of apoptosis. PA‐induced autophagosome accumulation, ER stress, and apoptosis was enhanced by Mas receptor agonists Ang (1‐7) or AVE0991, and suppressed by A779, a synthetic specific Mas inhibitor. PA‐induced impaired autophagy flux, increased ER stress and apoptosis were markedly attenuated by Mas receptor gene knockout in HK2 cells. Knockout Mas receptor also improved HK2 cell vitality and proliferation in response to PA treatment. Moreover, PA treatment caused elevated intracellular calcium levels, mitochondrial depolarization which were also markedly prevented by Mas knockout. Mas receptor gene knockout in HK2 cells was associated with markedly improved mitophagy after PA treatment. In mice with high‐fat diet for 12 weeks, Ang (1‐7) treatment caused enhanced autophagosome accumulation, ER stress, and apoptosis in the kidney cortex. In conclusion, Mas receptor mediated lipid‐induced impaired autophagy and ER stress in the kidney, likely contributing to tubular injuries seen in obesity‐related kidney diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel role of dual oxidase 2 as a mediator of podocyte injury in the diabetic environment
Authors: Abraham Sachin M , Navarro Megan M , Wauquier Fabien M , Lee Doug‐Yoon , Gorin Yves , Ford Bridget M
Published: June 2021

Glomerular injury is a prominent pathological feature of diabetic nephropathy (DN). In glomerular epithelial cells, or podocytes, hyperglycemia alters slit diaphragm proteins and causes foot process effacement, apoptosis and cell detachment. Oxidative stress has emerged as an important pathogenic mechanism in the development of glomerular injury in DN. However, the mechanisms by which these factors exert their action remain poorly understood.

We provide evidence that the NADPH oxidase of the Nox family, Dual oxidase 2 (Duox2), is present in cultured glomerular cells, including podocytes. In mouse kidney cortex, immunostaining for Duox2 shows that a fraction of glomerular Duox2 colocalized with podocyte markers. Exposure of cultured podocytes to high concentrations of glucose (HG) elicited a rapid upregulation of Duox2 protein expression. Inhibition of Duox2 with specific siRNA prevented the HG‐induced increase in intracellular reactive oxygen species (ROS) generation and hydrogen peroxide (H2O2) production in cultured podocytes.

In additional experiments, we established a functional link between Duox2‐derived ROS generation and podocyte injury in response to HG. Impairment of Duox2 function nearly abolished HG‐mediated DNA fragmentation, apoptosis and decrease in/reorganization of slit diaphragm protein expression in podocytes, indicating that Duox2 is required for the deleterious effects of glucose in podocytes.

Our study demonstrates, for the first time, that Duox2 is responsible for increased ROS generation and subsequent alteration of podocyte function in response to HG. Our work serves as proof of concept to demonstrate the utility of targeting Duox2 as a future therapeutic intervention to reduce diabetes‐mediated glomerular lesions.

Support or Funding Information

This project was funded by the University of the Incarnate Word, Office of Research and Graduate Studies and the following grants: NRSA 5T32HL007446‐32, NIH 1RO1DK07999601A2, and NIH UL1 TR001120/IIMS Pilot Project

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Concept for Origin and Treatment of Diabetic Nephropathy
Authors: Svensson Daniel , Zhang Liang , Panizza Elena , Nilsson Linnéa , Fontana Jacopo Maria , Moruzzi Noah , Scott Lena , Brismar Hjalmar , Aperia Anita
Published: June 2021

Objectives

Diabetic nephropathy (DN) is the most common cause of end‐stage renal failure and affects around 40% of all diabetic patients. This project aims to investigate the loss of proximal tubular cells (PTCs) in DN which are epithelial cells of the nephron leading away from the Bowman's capsule. Hyperglycemia induced damage to PTCs is attenuated by the Na/K‐ATPase ligand ouabain. Our second objective was thus to investigated the underlying mechanisms of ouabain in vitro and the function of Na/K‐ATPase as a signaling transducer.

Methods

PTCs were isolated from mouse and rat kidney cortex and cultured for up to a week. COS‐7 kidney cells were used for some experiments. Protein expression and phosphorylation was studied by Western blotting and proteomics/phosphoproteomics. Gene ontology analysis was performed to find distinct cellular processes regulated by ouabain in the phopshoproteomic analysis. Seahorse was used for metabolic studies. Gene expression was analyzed by qPCR and apoptosis assessed by the TUNEL assay.

Key results

High glucose (HG, 15 mM) triggers apoptosis in PTC due to their expression of sodium dependent glucose transporter 2 (SGLT2), SGLT2 siRNA abolishes HG induced apoptosis. Apoptosis does not appear to coincide with a switch in glucose metabolism. Low concentrations of ouabain (10 nM) completely inhibits the apoptosis. Phosphoproteomic analysis revealed 2580 ouabain‐regulated phosphorylation sites, many of which were associated with cell adhesion and proliferation. Out of these, CaMK2γ were shown to be involved in the anti‐apoptotic effect of ouabain, siRNA silencing of CaMK2γ eliminates the protective effect of ouabain.

Conclusions

Loss of tubular cells in DN might be driven by hyperglycemia due to the PTC expression of SGLT2. Low concentrations of ouabain protects from the detrimental hyperglycemic effects through an anti‐apoptotic mechanism involving CaMK2γ.

Support or Funding Information

The study was funded by the Swedish Research Council and Erling‐Persson Family Foundation. D.S. and N.M. is supported by a Novo Nordisk postdoctoral fellowship run in partnership with Karolinska Institutet.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Absence of Circadian Clock Gene Per1 Causes Increased Heart Rate and Hypertension in 129 Mixed Strain Mouse Model
Authors: Roig Emilio M , Masten Sarah , Miller Amber , Douma Lauren G , Lynch I Jeanette , Cheng Kit‐Yan , Wingo Charles S , Gumz Michelle L
Published: June 2021

According to recently updated blood pressure guidelines, nearly half of Americans are affected by hypertension. A subset of these patients is salt sensitive, indicating that their blood pressure is more prone to acute changes in response to salt intake. Healthy individuals experience 24‐hour cycles in blood pressure, which is regulated by the molecular circadian clock. This clock is comprised of four core transcriptional factors, including period (PER) and BMAL. Our lab has previously demonstrated that male PER1 knockout mice (KO) on a C57B1/6 background become “non‐dippers” (failure to lower blood pressure during the inactive period) following a high salt (HS) diet plus an aldosterone analogue treatment (HS/DOCP, Solocinski et al. 2017). To test the role of PER1 in blood pressure regulation on a different background strain, we obtained global PER1 KO male mice on a 129/sv/S1S4 background. Similar to C57B1/6 KO, male KO on a 129/sv/S1S4 background lose their rhythm in mean arterial pressure (MAP) following the same HS/DOCP treatment. Based on this previous observation, we wanted to test the hypothesis that heart rate, systolic blood pressure, and diastolic blood pressure would also exhibit a loss of circadian rhythm compared to control mice (WT). Telemetry data from KO (n=7) and WT (n=4) following HS/DOCP treatment were collected. Analysis was performed using Cosinor software to fit data to a cosine wave, determining amplitude, mesor, and period. Male KO mice on a 129/sv/S1S4 background strain exhibited approximately twenty percent greater heart rate mesor compared to WT (KO: 488 ± 15; WT: 411 ± 10; P=0.005). The period and the amplitude for heart rate were similar between the groups (P=0.74, P=0.49). The systolic blood pressure mesor for KO was approximately 32 mmHg higher than WT (KO: 135 ± 6; WT: 103 ± 5; P=0.0054). No significant differences were found between period and amplitude for systolic blood pressure (P=0.65, P=0.58). Lastly, KO mice displayed 17 mmHg higher diastolic blood pressure mesor than WT (KO 113 ± 3; WT 96 ± 3; P=0.0025). No significant differences were found between period and amplitude for diastolic blood pressure (P=0.62, P=0.88). While individually the circadian rhythms of systolic and diastolic blood pressure are unaffected by PER1 KO in male 129/sv/S1S4 mice, MAP rhythms are dampened.

Support or Funding Information

NIH/NIDDK 1R01DK109570 (MLG) and The Gatorade Trust through the UF Department of Medicine

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping chromatin accessibility in mouse nephron segments
Authors: Chen Lihe , Chou Chung‐Lin , Knepper Mark A
Published: June 2021

Active DNA regulatory elements recruit transcription machinery to drive gene expression and generate nephron heterogeneity, which is crucial for renal physiology and pathophysiology. Such heterogeneity poses a significant challenge in the precise mapping of these DNA regulatory elements in each renal segment. Whole kidney chromatin accessibility analysis averages the signal from multiple cell types, thus it is necessary to carry out such analysis at the single‐tubule level.

Here, we report single‐tubule ATAC‐seq, a method using hyperactive Tn5 transposase to simultaneously fragment and tag the regions of accessible chromatin with adapters for high‐throughput DNA sequencing. We adapted this method to the microdissected mouse proximal tubule S2 segment (PTS2), cortical thick ascending limb (cTAL), and cortical collecting duct (CCD). DNA libraries were sequenced to saturation and around 20M unique high‐quality reads were recovered for downstream analysis. Our data are consistent with the expected nucleosomal pattern and enrichment of reads in the transcription start site (UCSC, TSS, mm10), indicating the high quality and robust ATAC‐seq. Overall, we identified more than 95,000 narrow peaks for each segment (MACS2, ‐q 0.05 ‐‐nomodel). Coupled with single‐tubule RNA‐seq, for those transcripts uniquely expressed in these segments, our data revealed a high correlation of chromatin accessibility with gene expression. Initial analysis revealed unique chromatin openness in PTS2 including Slc22a7, Aqp1, Slc34a1, Kap; in cTAL including Hnf4a, Egf, Kcnj1, Umod, Slc12a1, Cldn16; and in CCD including Aqp2, Aqp3, Slc4a1, Fxyd4, and three ENaC subunits.

Our results provide a comprehensive chromatin accessibility map for the nephron segments. The identification of these regulatory elements along the nephron will provide additional information beyond transcriptome and point to gene regulation in the kidney.

Support or Funding Information

The work was primarily funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute (project ZIA‐HL001285 and ZIA‐HL006129, M.A.K.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Hemodynamic Function is Impaired in Female Mice with SLE
Authors: Dent Elena , Ryan Michael
Published: June 2021

Systemic lupus erythematosus (SLE) is a chronic multi‐system autoimmune disease characterized by the presence of circulating autoantibodies, systemic and localized inflammation, prevalent hypertension, renal injury, and cardiovascular disease. Onset of the disease typically occurs in young women of child‐bearing age, a time when women are typically protected from cardiovascular risk factors like hypertension. Although kidney involvement is common to patients with SLE, and renal hemodynamic dysfunction is evident in patients with active renal disease, surprisingly little is known about early changes in renal hemodynamic function that may contribute to the pathogenesis of hypertension during autoimmune diseases. We hypothesize that the loss of immunological tolerance and subsequent production of autoantibodies in SLE leads to impaired renal hemodynamic function that precedes the development hypertension.

Female NZBWF1 mice, an established experimental model of SLE, and female NZW (control) mice were instrumented with carotid artery and jugular vein catheters to determine mean arterial pressure (MAP) and glomerular filtration rate (GFR) respectively at ages 15, 20, 28, 31, and 34 weeks (n>3 control/age, n>4 SLE/age). MAP was measured in conscious, freely‐moving mice. GFR was measured by the clearance of fluorescein isothiocyanate‐inulin (FITC‐inulin) after achieving steady state through continuous infusion.

From 15 weeks of age to 34 weeks of age, there is a progressive increase in the levels of circulating autoantibodies in female mice with SLE, whereas autoantibodies are unchanged in control mice (Figure 1). Compared with control mice, female mice with SLE have higher MAP at 15, 20, 28, 31, and 34 weeks of age, with an increase in pressure that occurs by 34 weeks of age (Figure 2). GFR remains moderately higher in SLE mice compared with control mice until 28 weeks of age at which time GFR sharply declines in the mice with SLE (Figure 3). GFR is unchanged in control mice across all ages.

These data suggest that changes in renal hemodynamic function occur in female SLE mice prior to changes in MAP suggesting an important mechanistic role for autoimmunity to directly impair renal hemodynamic function and promote the development of hypertension.

Support or Funding Information

VA Merit BX002604, American Heart Association Predoctoral 19PRE34380830, NIH P20GM104357, NIH 5U54GM115428, NIH 5P01HL051971

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Decrease in Tregs Exacerbates DOCA‐Salt Hypertension and Renal Injury in Female Rats
Authors: Belanger Kasey Marie , Gillis Ellen , Musall Jaqueline , Sullivan Jennifer
Published: June 2021

Background

T regulatory cells (Tregs) have previously been shown to protect against increases in blood pressure (BP) in male experimental models of hypertension, however less is known regarding the role of Tregs in BP control in females. Our lab has previously reported that there is a sex difference in the renal T cell profile in DOCA, where DOCA results in a decrease in Tregs in males while females maintain their Tregs. The current study tested the hypothesis that Treg maintenance attenuates the increase in blood pressure (BP) to DOCA‐salt in females.

Methods

Female (n=4–7) 13‐week‐old Sprague Dawley rats underwent an uninephrectomy followed by the implantation of a time release 200mg Deoxycorticosterone Acetate (DOCA) pellet and 0.9% saline to drink ad libitum. Rats were then randomized to receive a vehicle or anti‐CD25 injection (IP 250 μg/week) weekly for 3 weeks and BP was measured by telemetry. 24‐hour urinary protein excretion was measured via Bradford Assay at baseline and weekly thereafter for the duration of the study. At the end of the study, kidneys were isolated for flow cytometric analysis of T cells and a terminal plasma sample was collected to measure the kidney injury marker Tim1/Kim1.

Results

Anti‐CD25 injections in DOCA hypertensive females significantly decreased renal (4.2 ± 0.6 vs. 6 ± 0.7; p=0.03) and circulating Tregs compared to DOCA alone (3.6 ± 0.5 vs. 5.5 ± .6 p=0.05). 3 weeks of DOCA‐salt treatment significantly increased BP (101 ± 3 to 160 ± 5; p=<.0001), and decreasing Tregs exacerbated the DOCA‐salt induced increase in BP (109 ± 1 to 188 ± 4; p=0.002). We calculated area under the curve for each BP tracing, and the overall BP load in the rats treated with anti‐CD25 was significantly greater than in rats with DOCA‐salt alone (3327 ± 37 vs. 3662 ± 39; p=0.033). DOCA‐salt also increased urinary protein excretion from baseline to week 3 (3 mg/day ± 0.4 to 34 mg/day ± 9; p=0.01) and this increase was exacerbated with anti‐CD25 treatment (2 mg/day ± 0.5 to 86 mg/day ± 17; p=0.003). Tim1/Kim1 was also increased in DOCA hypertensive females compared to uninephrectomized controls, and this increase was greater in rats treated with anti‐CD25 (control: 292 ± 25 pg/ml; DOCA: 415 ± 57pg/ml; DOCA+anti‐CD25: 589 ± 105; p=0.04).

Discussion

Our data suggests that the maintenance of Tregs in DOCA‐salt hypertensive females attenuates increases in BP and renal injury. Future studies will determine if this effect if mediated by the anti‐inflammatory and anti‐hypertensive effects of IL‐10.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Dexamethasone Therapeutic Doses Treatment in Electrocardiographic Abnormalities and Cardiac Remodeling of Rats: Role of Oxidative Stress
Authors: Macedo Fabricio Nunes , Souza Diego Santos , Santos João Eliakim Araújo , Vasconcelos Carla Maria Lins , Silva‐Filha Elizabete Santos , Quintans‐Júnior Lucindo José Santos , Mesquita Thassio Ricardo Ribeiro , Santana‐Filho Valter Joviniano
Published: June 2021

Dexamethasone is the most clinically used glucocorticoid with an established role in the treatment of a wide spectrum of inflammatory‐related diseases. While the therapeutic actions are well known, dexamethasone treatment causes a number of cardiovascular side effects, which are complex, frequent and, in some particular cases, clinically unnoticeable. Thus, we investigated whether a therapeutic regimen of dexamethasone affects cardiac arrhythmogenesis and/or promote cardiac remodeling, focusing on the contribution of reactive oxygen species (ROS). Male Wistar rats were treated with dexamethasone (2 mg/kg, i.p.) for 7 days. Afterward, hemodynamic measurements, autonomic modulation, left ventricular function, cardiac fibrosis, superoxide (O2·−) generation, superoxide dismutase (SOD) activity, lipid peroxidation (TBARS) and arrhythmias were evaluated. Here, we show that dexamethasone increases blood pressure, associated with enhanced cardiac and vascular sympathetic modulation. Moreover, a marked increase in the cardiac O2·− generation was observed, whereas the enhanced SOD activity did not prevent the higher levels of lipid peroxidation in dexamethasone group. Although preserved ventricular contractility and β‐adrenergic responsiveness, we found that dexamethasone‐treated rats display greater interstitial and perivascular fibrosis than control. Besides that, despite the absence of arrhythmias at basal condition, we demonstrated by in vivo and ex vivo approaches that dexamethasone‐treated rats are more susceptible to develop harmful forms of ventricular arrhythmias, when challenged with pharmacological drugs or burst pacing‐induced arrhythmias. Notably, concomitant treatment with apocynin, an inhibitor of NADPH oxidase, prevented these ectopic ventricular events. Taken together, our data revealed that the dexamethasone treated rats were hypertensive and their hearts become arrthymogenic, in addition, our results uncover the pivotal role of ROS‐generating NADPH oxidases in the arrhythmias vulnerability.

Support or Funding Information

Fapitec/SE ; CAPES ; CNPq and Estácio University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcription Factor HNF4A Regulates Urate Transporter ABCG2
Authors: Kuhns Victoria Lea Halperin , Lewis Raychel , Marten Jonathan , Vitart Veronique , Tin Adrienne , Consortium CKDGen , Köttgen Anna , Woodward Owen M
Published: June 2021

Elevated urate levels in the serum can contribute to the development of many diseases, including gout, the most prevalent form of inflammatory arthritis. Our previous work identified ABCG2 as a key urate transporter of the kidney and gut epithelium and its dysfunction significantly increases risk of hyperuricemia and gout. A new collaborative meta analysis of genome wide association studies, integrating the results from 457,690 individuals, identified 183 loci associated with serum urate. A further analysis of associated loci to identify independent and potentially causal variants created a credible set of variants for experimental follow up including variants in ABCG2, HNF1A, and HNF4A. HNF1A and HNF4A are transcription factors that are key regulators of liver, renal, and gut transport proteins. Here we sought to examine if HNF1A or HNF4A regulated the expression of ABCG2, and the effects of the identified HNF1A and HNF4A missense variants. To determine any direct regulation of ABCG2 by HNF1A or HNF4A, we used a luciferase assay reporter system where the ABCG2 promoter was used to drive expression of a luciferase reporter. HEK293T cells were co‐transfected with this ABCG2 construct, and plasmids expressing either HNF1A or HNF4A, as well as a GFP reporter used to determine cell number and transfection efficiency. HNF4A increases expression of ABCG2 driven luciferase in a dose dependent and significantly reproducible manner. No increase is observed with either HNF1A co‐transfection, or in the empty vector negative control. Next, the missense mutations were examined in the same luciferase assay for both HNF1A (A98V) and HNF4A (T139I). HNF4A‐T139I demonstrated an increase in ABCG2 luciferase expression to a significantly greater extent than the wild‐type protein, again in a dose dependent and reproducible manner. The increased ABCG2 promoter activation observed with the HNF4A‐T139I was not attributable to increased abundance of the mutant protein. In addition, the HNF4A‐T139I amino acid resides in an area critical for interacting with target DNA promoter regions. Our findings that HNF4A‐T139I is a gain of function variant resulting in increased abundance of ABCG2, the primary secretory transporter, is consistent with our observation that this variant associates with decreased serum urate levels in humans. Thus, we conclude that HNF4A is likely directly regulating expression of ABCG2, and thereby influencing serum urate levels.

Support or Funding Information

NIDDK R01DK114091

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adaptation to K+ deficiency in mouse: coordinate regulation of muscle and kidney electrolyte transporters
Authors: McFarlin Brandon E , Chen Yuhan , Madhur Meenakshi S , Ralph Donna L , McDonough Alicia A
Published: June 2021

Our previous work in rats subjected to K+ deficient diets demonstrated that K+ shifts from muscle intracellular fluid (ICF) stores to extracellular fluid (ECF) compartment in order to buffer the fall in ECF K+ due to finite losses in urine and feces. The aim of this study was to determine the coordinated renal and muscle adaptations to preserve K+ and Na+ homeostasis during a K+ deficient diet in mice.

Methods

Male C57BL/6 mice were fed control (1K) or K+ deficient (0K) diets for 10 days. Urine was collected overnight at 6 days in metabolic cages. Kidney and muscle were collected at 10 days. [Na+] and [K+] were determined by flame photometry in plasma, urine and muscle (mixed gastrocnemius + soleus) extracts. Transporter abundance was assessed by quantitative immunoblot.

Results

Plasma [K+] fell from 4.8 ± 0.3 (1K) to 2.3 ± 0.3 mM (0K) and plasma [Na+] was maintained at 148.6 ±1.5 mM (0K). K+ deficient diet reduced muscle [K+] from 125 ± 2 (1K) to 92 ± 5 mM (0K) and doubled muscle [Na+] from 30 ± 1 (1K) to 61 ± 1 mM (0K), expressed as tissue wet weight. 0K diet also reduced muscle Na, K‐ATPase α2 and β2 subunit abundance by 35 and 60%, respectively, consistent with the measured K+ loss and Na+ gain. Urinary K+ excretion fell from 0.19 ± 0.1 (1K) to 0.003 ± 0.001 mmol (0K) while urinary Na+ was not significantly altered by 0K indicative of adaptations to conserve K+ and maintain Na+. Renal sodium transporters changed as follows during K+ deficient diet: proximal tubule Na/H exchanger (NHE3) decreased 35% while NHE3pS552 increased 1.5 fold consistent with lower NHE3 activity; distal tubule Na‐Cl co‐transporter (NCC) phosphorylated at T53 and S71 both increased >1.5 fold, consistent with higher distal tubule Na+ reabsorption; epithelial Na+ channel gamma subunit (ENaCγ) increased >1.5 fold (no cleavage), consistent with accumulation of inactive ENaC.

During restricted potassium intake in mice, muscle donates K+ to ECF facilitated by reducing Na, K‐ATPase abundance, and NCC covalent modification coupled with reduced ENaC activity reduces the driving force for K+ secretion. These responses help blunt the fall in plasma [K+], a key determinant of membrane potential. Reduced proximal Na+ reabsorption via NHE3 may drive a tubuloglomerular feedback mediated reduction in GFR to reduce urinary K+ losses. Despite the increase in muscle [Na+] and decrease in proximal Na+ transport there was no evidence for significant changes in sodium excretion.

Support or Funding Information

DK083785

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reduction of circulating renin‐angiotensin following unilateral nephrectomy signals kidney hypertrophy via increase in renal blood flow
Authors: Shimada Satoshi , Cowley Allen W.
Published: June 2021

Rationale

The compensatory hypertrophy of a remaining kidney after nephrectomy has long been recognized and the molecular and functional responses to these events have been well characterized. However, the signal that triggers these events is unknown. Recent studies have found that nephrectomy of the right kidney in rats results in a rapid increase of renal blood flow of the remaining kidney within the first 30 minutes and activation of the mTOR pathway with consequent hypertrophy. In the present study, we hypothesized that a reduction of circulating renin would immediately occur with removal of the right kidney which could produce an immediate peripheral vasodilation and increase of renal blood flow within the remaining left kidney.

Methods

Sprague Dawley rats (male; 7 wk age) were anesthetized, the left kidney was denervated and then the right kidney was removed while monitoring immediate changes of systemic mean arterial blood pressure (MAP), left renal arterial blood flow (RBF), and left urine volume (V). Three groups were studied: 1) No drug treatment (n=5); 2) Pretreated with the AT1 receptor blocker Losartan (n=4); 3) Pretreated with Losartan and Angiotensin II administered to return aortic pressure and RBF to normal before removing the right kidney (n=2).

Results

Left RBF gradually increased from 3.8 to 4.7 ml/min and became stable 30 minutes following removal of the right kidney. A slight decrease of MAP occurred during that time (~4 mmHg) and urine volume nearly doubled from 2.7 to 5.3 μl/min. Rats treated with Losartan exhibited a reduced baseline level of MAP and increased level of RBF but the changes in MAP and RBF following right nephrectomy were abolished. Rats treated with Losartan with Angiotensin II infused to normalize baseline levels of RBF and MAP, also exhibited no increases of RBF following removal of the right kidney.

Conclusion

We conclude that rapid reductions of circulating renin‐angiotensin following right kidney nephrectomy produce increased RBF in the remaining kidney which in turn may trigger the compensatory hypertrophy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Primacy of Chymase over Angiotensin Converting Enzyme in the Production of Angiotensin II in Rat Bone Marrow Tissue
Authors: Yamashita Tomohisa , Ahmad Sarfaraz , Wright Kendra N , VonCannon Jessica L , Varagic Jasmina , Sun Xuming , Wang Hao , Groban Leanne , Ferrario Carlos M
Published: June 2021

Background

The bone marrow is attracting attention as not only the nidus of circulating blood cells but the source of stem and progenitor cell‐based therapy. Renin‐angiotensin system (RAS) and angiotensin II (Ang II) receptor type 1 in bone marrow participate in hematopoiesis, a target in the evolution of inflammatory processes involved in cardiovascular disease pathogenesis. Extending the original demonstration of RAS components in the rat bone marrow (Strawn et al. Br J Haematol. 2004;126:120–6), this study investigated the existence of an Ang II‐forming pathway in which chymase rather than angiotensin‐converting enzyme (ACE) cleaves the peptide from the newly described substrate angiotensin‐(1‐12) [Ang‐(1‐12)] which functions as an endogenous tissue substrate for the direct formation of Ang II by chymase.

Methods

Bone marrow tissue was obtained from the tibia of twelve weeks‐old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni's fixative. Bone marrow tissues, homogenized with 0.05 M Tris‐HCl and centrifuged at 28,000 g, were subjected to immunoblot and chymase enzymatic activity using high‐performance liquid chromatography (HPLC). Additional samples were prepared for quantitative RT‐PCR analysis normalized to GAPDH. All data were analyzed using GraphPad PRISM 7.0 with P<0.05 considered statistically significant.

Results

Fluorescent immunohistochemistry showed the existence of immunoreactive (ir‐) Ang‐(1‐12) and ir‐chymase‐positive cells in rat's bone marrow. Immunoblots showed chymase protein expression in cell pellets but not in supernatants. Assessment of 125I‐Ang‐(1‐12) or 125I‐Ang I hydrolysis in cell pellets in the presence and absence of specific chymase or ACE inhibitors, revealed that production of Ang II by chymase was two‐thousand fold higher compared to ACE (4,531 ± 137 fmol/mg/min and 2.3 ± 0.3 fmol/mg/min, respectively, P<0.0001, n = 6). ANOVA and post‐hoc Tukey's test showed that gene expression of rat mast cell protease‐5 (rMCP‐5) was at least twice higher than rMCP‐1, rMCP‐2 and rMCP‐4.

Conclusion

These studies reveal a major role of Ang‐(1‐12) and chymase as the critical pathway for Ang II‐formation in rat's bone marrow. The pleotropic actions of tissue chymase in the degradation of matricellular proteins, activation of matrix metalloproteinases, transforming growth factor‐β, and production of inflammatory cytokines posits this alternate pathway for Ang II‐production as most relevant in understanding and treating cardiovascular disease.

Support or Funding Information

This study was supported by the National Heart, Blood, Lung Institute of the NIH through HL‐051952 grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exercise enhances placental angiogenesis of obese maternal mice
Authors: Son Jun Seok , Liu Xiangdong , Chen Yanting , Tian Qiyu , Zhao Liang , Hu Yun , Chae Song Ah , Deavilla Jeanene , Zhu Mei‐Jun , Du Min
Published: June 2021

Placenta is highly vascularized, which is critically for its function in nutrient transportation. Maternal obesity reduces placental vascularization, thus, negatively affecting fetal development and predisposing offspring to adult diseases. Physical activities have been reported to induce angiogenesis in muscle, and we hypothesized that exercise during pregnancy enhances placental vascularization, which prevents negative changes in placental blood vessel density due to maternal obesity.

PURPOSE

To examine the effects of maternal exercise on placental angiogenesis in high fat diet (HFD)‐induced obese mothers.

METHODS

Female C57BL/6J mice were randomly assigned into four groups: control (CON), exercise (EX), HFD, and HFD with exercise (HFDEX). Mice were fed either a control diet containing 10 kcal% from fat, or a high fat diet containing 60 kcal% from fat for 8 weeks before mating. After mating, exercise intervention was performed during gestation every morning for 60 min at moderate intensity including warming up and cooling down. Maternal mice were euthanized, and placenta and fetuses were collected at embryonic day 18.5 (E18.5). Statistical difference was analyzed using student's t‐test and/or one‐way analysis of variance (ANOVA).

RESULTS

Exercise intervention during gestation reduced body weight (P < 0.05) for both CON and HFD maternal mice, but fasting blood glucose level was only reduced (P < 0.001) in HFDEX group. There was no difference in food intake among groups. The average weight of fetuses was heavier (P < 0.001) in HFD than CON and HFDEX, while no difference among CON, CONEX, and HFDEX. The placental weight was higher in both exercise groups compared to no‐exercise groups. The mRNA expression of vasculogenic markers, Vegfa, Vegfr1, Fgf2, Fgfr2, Hif1a, Ccn1, and Gcm1, was reduced in HFD placenta compared to CON, which was recovered due to exercise. In addition, compared with CON, the blood vessel density was lower in HFD group (P < 0.01) but increased (P < 0.01) in HFDEX group.

CONCLUSIONS

Maternal obesity impaired placental angiogenesis, which was recovered due to exercise, suggesting that exercise during pregnancy is particularly important for obese mothers in order to improve placental vascular development and function, thus offspring health.

Support or Funding Information

Supported by NIH Grant R01HD067449.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.