bluuebunny/crossref_metadata_2025 huggingface dataset:
Exercise Induced Irisin Alleviates Type 1 Diabetic Nephropathy by Promoting Mitochondria Biogenesis and Function
Authors: Majumder Suravi , Pushpakumar Sathnur , Sen Utpal
Published: June 2021

Mitochondrial dysfunction is known to play an important role in the development and progression of chronic kidney disease. Improving mitochondrial homeostasis therefore has the potential to restore renal function in diabetic kidney disease. Exercise training was shown to be beneficial in improving skeletal muscle and cardiovascular system pathology by improving mitochondrial function, reduction of oxidative stress and metabolic reprogramming. Recent studies have shown that Irisin, a molecule secreted by the skeletal muscles during exercise protects the mitochondria from ischemia reperfusion injury in the lung and heart. In clinical studies, involving type 1 diabetes, high and low levels of Irisin has been reported however, its functional significance remains unclear. Exercise induces the expression of PGC‐1α, leading to the expression of the fibronectin type III domain containing 5 (FNDC5). Irisin derives from the proteolytic cleavage of the peptide FNDC5 by yet unknown enzymes. The purpose of our study was to investigate whether exercise induced Irisin improves mitochondrial biogenesis and function in type 1 diabetic nephropathy. C57BL/6‐Ins2Akita/J (Type 1 diabetes, Akita) and C57BL/6J (Wild type, WT) mice were subjected to 16‐week exercise regimen of 280m run on treadmill at speed of 7m/min for 5 days/week for 2 weeks and increased to 12m/min for the remaining 14 weeks. In vitro experiments were done in mouse glomerular endothelial cells (MGECs) treated with recombinant Irisin. In Akita and WT mice, exercise training improved renal vascularity, cortical blood flow and glomerular filtration rate compared to sedentary groups. Exercise upregulated Irisin expression in Akita and WT mice compared to sedentary mice but to a greater extent in WT mice. The protein and mRNA expression of PGC1α, PPARα and PPARγ were decreased in sedentary Akita mice and increased following exercise training suggesting induction of mitochondrial biogenesis. Sedentary Akita mice showed decreased expression of regulatory markers of mitochondrial DNA transcription, Nrf1, Nrf2, and Tfam and exercise normalized their expression. The expression of MnSOD and catalase were decreased in Akita mice suggesting impaired antioxidant defense and exercise restored their levels to normal. In conclusion, our results suggest that exercise induced Irisin protects the kidney in type 1 diabetes by restoring mitochondrial homeostasis.

Support or Funding Information

This study was supported, in part, by NIH grant HL‐104103 and DK 104653 to US and AHA grant: 15SDG25840013 to SP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective effects of Dianthus superbus on Diabetic renal dysfunction in in vivo and in vitro models
Authors: Yoon Jung Joo , Son Chan Ok , Kim Hye Yoom , Lee Hyeon Kyoung , Lee Yun Jung , Yang Sun Nye , Lee Ho Sub , Kang Dae Gill
Published: June 2021

Glomerular fibrosis is caused by accumulation of extracellular matrix proteins in the mesangial interstitial space resulting in fibrosis manifested by either diffuse or nodular changes. Dianthus superbus belongs to the Caryophyllaceae family and has been used in traditional medicine as a diuretic, a contraceptive, and an anti‐inflammatory agent. The aim of this study was to investigate the effects of Dianthus superbus on glomerular fibrosis and renal dysfunction causing diabetic nephropathy in human renal mesangial cells and db/db mice. In diabetic db/db mice models, Dianthus superbus were administrated to db/db mice (n=8) to 10 or 50 mg/kg/day concentrations. The experiment was measured for 8 weeks following Dianthus superbus treatment respectively. As a result, Dianthus superbus significantly ameliorated blood glucose, insulin, HOMA‐IR and HbA1c of diabetic index. Dianthus superbus decreased albumin excretion, creatinine clearance and plasma creatinine. Dianthus superbus ameliorated kidney injury molecules‐1 of kidney function index and C‐reactive protein of inflammation marker. In addition, Dianthus superbus reduced the PAS positivity stain intensity and basement membrane thickening in glomeruli of diabetic nephropathy model. Dianthus superbus suppressed TGF‐β/Smad signaling pathway. Collagen type IV, glomerular fibrosis biomarker, was significantly decreased by Dianthus superbus. Additionally, Dianthus superbus pretreatment attenuated inflammatory factors expression and translocation of nuclear factor kappa B (NF‐κB) in Angiotensin II (Ang II)‐stimulated mesangial cell. These findings suggest that Dianthus superbus has protective effect against renal inflammation, fibrosis and oxidative stress. Therefore, Dianthus superbus may be potential therapies targeting glomerulonephritis and glomerulosclerosis leading to diabetic nephropathy.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Fat Diet Exacerbates Diabetic Kidney Injury in Mice Lacking eNOS
Authors: George Jasmine , Zhao Xueying
Published: June 2021

Diabetic nephropathy is the leading cause of chronic kidney disease and end stage renal disease. It has been shown that deficiency in endothelial nitric oxide synthase (eNOS), and/or reduced eNOS activity is associated with diabetic nephropathy progression in humans. This study was designed to examine whether high fat diet (HFD) would enhance diabetic kidney injury in eNOS deficient mice. To induce diabetes, six‐week old eNOS deficient mice were placed on normal diet (ND) or HFD (42% from fat). Two weeks later, half of them were rendered diabetic with alloxan. Proteinuria/albuminuria and urinary levels of kidney injury markers were measured in control and diabetic animals 4 months post alloxan injection. There is no difference in urinary total protein among four groups. HFD treatment increased urinary albumin as well as proinflammatory cytokine osteopontin, whereas urinary parameters remained normal in alloxan‐induced diabetic mice fed with normal diet. A combination of HFD and alloxan treatment resulted in a more pronounced albuminuria as well as increased glomerular and tubular damage as evidenced by an elevation of urinary synaptopodin, matrix metalloproteinase‐9 and kidney injury molecule‐1. These results demonstrate that HFD accelerates diabetic kidney injury in eNOS deficient mice. Thus eNOS deficient mice may be suitable for studying the roles of albuminuria and renal lipotoxicity in development and progression of diabetic nephropathy.

Support or Funding Information

NIH (SC1DK112151), NIH/NCRR/RCMI (8G12MD007602) and (8U54MD007588)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The protein expression of TNF‐α receptor type 1 (TNFR1) is reduced by nitric oxide (NO) synthase inhibition in cultured renal proximal tubular (HK‐2) cells
Authors: Majid Dewan Syed Abdul , Ekpo Peace Effiong , Talwar Sudha , Abdel‐Mageed Asim B , Castillo Alexander
Published: June 2021

Tumor necrosis factor‐alpha (TNF‐α) production, stimulated by chronic high salt (HS) intake, has been implicated in salt sensitive hypertension (SSH) though the mechanism is not yet clearly defined. TNF‐α exerts its biological responses via interaction with two cell surface receptors, TNF‐α receptor type 1 (TNFR1) and type 2 (TNFR2). To determine the roles of TNF‐α receptors during the development of SSH induced by NO deficiency, experiments in our laboratory demonstrated earlier that chronic HS intake alone in mice increases TNFR1 protein expression in renal cortical tissues. However, HS intake in mice chronically treated with a non‐specific pharmacological inhibitor of NOS or in knockout mice lacking endothelial NOS decreases TNFR1 protein expression in the renal cortical tissues. We have also demonstrated that chronic angiotensin induced intrarenal angiotensinogen (AGT) generation is augmented in TNFR1 knockout mice, indicating that TNFR1 activity suppressed AGT formation in the kidney. The present study examines the hypothesis that NO linearly regulates TNFR1 activity in the renal tubular cells in a way that NO deficiency decreases TNFR1 expression. The cultured HK‐2 cells (human kidney‐2 cells; HK‐2 (ATCC® CRL‐2190™) are used in these in‐vitro experiments. These cells were plated at a 125,000 cell per density and grown RPMI medium containing 10% FBS and 1% Penstrep, Non‐essential amino acids, Glutamine, Amphotericin B, Gentimicin at 37°c 5% CO2, 99% relative humidity for 24–48h until they reach confluency. Cells were then split into petry‐dishes and treated with drugs Nitro‐L‐arginine methyl ester(L‐NAME) at 0.5 and 1.0 mM concentrations as well as vehicle for 24 hours, harvested, and lysed. Protein concentration was calculated (BCA assay), and the predetermine volume was loaded in each well of the gel (mini‐Protean TGX gel 456–1096). Blot was treated with TNFα receptor 1 (19139 Abcam) antibody and GAPDH. It was observed that TNFR1 expression significantly decreases to 60 ± 25% (n=3) at 0.5 mM and 35 ± 15 % (n=5) at 1.0 mM conc. of L‐NAME treatment, compared to vehicle treatment in HK‐2 cells. The results show that L‐NAME reduces TNFR1 protein expression in HK‐2 cells after treatment for 24 hours in dose dependent manner. As proximal tubular cells are the prime site for intrarenal AGT generation, these data suggest that such decreases in TNFR1 activity induces enhanced AGT production which is involved in the exaggerated hypertensive responses to HS intake in NO deficient condition.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bilateral renal denervation does not prevent the kidney damage caused by a high fat diet in rats
Authors: Flores‐Sandoval Omar , Cárdenas‐Romero Skarleth , Salgado‐Delgado Roberto , Saderi Nadia
Published: June 2021

One of the hallmarks of the metabolic syndrome (MS) is the increase of renal sympathetic nerve activity (RSNA), which leads to the upregulation of the renin‐angiotensin system (RAS). Interestingly, the sympathetic hyperactivity in the kidney of obese humans often occurs in absence of overt systemic hypertension. The stimulation of RSNA contributes to the glomerular lesions and the progressive loss of nephron functionality, resulting in acute renal injury (AKI) and, in the long term, chronic kidney disease (CKD), which represent the most common complication of MS. Clinical evidence and animal models of MS have shown that renal sympathetic denervation (RSDN) induces a transient attenuation of the hypertensive symptoms, while the markers of kidney damage continue worsening. Therefore, it is likely that the renal derangement in MS might also depend on the metabolic impairment, such as the upregulation of plasma leptin and insulin, independently from the RSNA. To investigate this issue, we explored the effect of MS over blood pressure and kidney functionality, in intact and renal denervated rats. For that, animals were either fed with a standard diet (SD) or a high‐fat diet for 12 weeks (HFD), with or without bilateral renal denervation performed at the beginning of protocol (n = 6). Results indicate that, as expected, HFD in intact animals promotes fat accumulation, glucose intolerance and hypertension, but also decreases water consumption and induces marked proteinuria from week 7, slight hematuria from week 9, with respect to SD rats. Remarkably, the renal denervation prevents the changes that HFD triggered in blood pressure and renal parameters only in the short and medium term, but it is ineffective in the long term. On the bases of these results, we suggest that the hypertension associated to MS is not only due to hyperactivation of RSNA, but it could involve hormonal/hemodynamic factors, typical of metabolic impairment, which could contribute to renal injury.

Support or Funding Information

CONACYT‐CB‐243298

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of Salt‐Sensitive Hypertension in a Mouse Model of ACTH‐Dependent Cushing Syndrome
Authors: Costello Hannah M , Stewart Kevin , Jones Natalie K , Grenier Celine , Ralph Ailsa F , Livingstone Dawn E W , Dhaun Neeraj , Bailey Matthew A
Published: June 2021

Salt‐sensitive blood pressure (BP) reflects underlying renal and vascular (endothelial) dysfunction. People with abnormal glucocorticoid homeostasis, such as in Cushing syndrome and obesity, are often salt‐sensitive but the underlying mechanisms are not clearly defined1,2. Here, we used a mouse model to examine the effect of ACTH excess on blood pressure and salt‐sensitivity. To test this, radio telemetry was used to track BP longitudinally and measure the effect of high salt diet before and after chronic ACTH treatment in C57BL/6J mice (n=6). Data are mean ± SD. After 7 days of baseline measurements, mean BP was 105.8 ± 6.5mmHg before being placed on a high salt diet (3 % Na+ by weight) for a further 7 days. BP increased ~12 mmHg over the first 3 days before declining to 113.1 ± 7.2mmHg. BP returned to 107.7 ± 10.4mmHg, similar to baseline, after a 5‐day washout period. Mice were then treated with ACTH (2.5μg/day). ACTH increased mean BP to 115.5 ± 5.0mmHg with a flattening of the diurnal rhythm following 13 days infusion. When placing these mice on a high salt diet, mean BP increased and stabilised to 136.1 ± 13.4mmHg after 14 days. These data show that chronic ACTH excess contributes to salt‐sensitivity. To investigate potential mechanisms of salt‐sensitivity, the acute pressure natriuresis (PN) response of renal artery function were examined in separate cohorts of mice treated with either vehicle or ACTH (2.5μg/day; n=6 per group) for 14 days. PN response was induced in anaesthesised mice (120mg/kg Inactin) by sequential arterial ligation. BP increased ~40mmHg following ligations in both vehicle and ACTH groups. In vehicle‐treated mice, fractional sodium excretion increased from 0.2 ± 0.2 to 5.0 ± 4.2μmol/min. The natriuretic response was significantly blunted in ACTH‐treated mice, reaching 2.1 ± 1.1μmol/min at its peak. We next investigated vascular function in ACTH‐excess ex vivo by wire myography in isolated mesenteric and renal arteries. Vascular reactivity to phenylephrine (PHE), acetylcholine (ACh) and sodium nitroprusside (SNP) was assessed. ACTH‐excess caused vascular dysfunction in the renal artery but not in the mesenteric artery. ACTH‐excess resulted in a reduction in sensitivity to vasoconstrictor PHE (p=0.0005) and endothelium dependent (ACh) and endothelium independent (SNP) vasodilators (p=0.0128 and p=0.0002, respectively). These data show that chronic glucocorticoid excess contributes to salt‐sensitivity with potential mechanisms behind this being impairment of the acute natriuretic and diuretic response to increased BP and renal vascular dysfunction.

Support or Funding Information

Supported by British Heart Foundation PhD studentships FS/16/54/32730 & FS/15/63/32033.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GYY4137 Modulates Renal Remodeling in Hyperhomocysteinemia
Authors: John AM Sashi Papu , Sen Utpal
Published: June 2021

Introduction

Elevated plasma levels of homocysteine, also known as hyperhomocysteinemia (HHcy), is associated with chronic kidney disease and end‐stage renal failure. HHcy is also a vascular risk factor that causes glomerular remodeling by inducing and accumulating extracellular matrix (ECM) in the glomerular and peri‐glomerular spaces. This remodeling is, in part, due to Hcy‐mediated imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) and disruption of cell‐to‐cell gap junction protein, connexins (Cxs). HHcy also contribute to renal microvascular impairment and vasoconstriction due to decreased endogenous hydrogen sulfide (H2S) production and homocysteinylation of endothelial nitric‐oxide synthase (eNOS). However, the molecular events targeting Caveolin, eNOS, MMPs/TIMPs, Cxs and the role of H2S in HHcy associated ECM turnover and renovascular remodeling remain elusive.

Hypothesis

We investigated whether GYY4137, slow releasing H2S donor ameliorates HHcy effect and renal remodeling.

Methods

Caveolin protein 1 knockout B6.Cg‐Cav1 (Cav1−/ −), C57BL6/J (WT) and hyperhomocysteinemic CBS+/− mice were supplemented without or with Hcy (2% in diet) and treated without or with GYY4137 (0.5 mg/Kg/d, IP) for a period of 8 weeks.

Results

HHcy decreased plasma H2S levels, upregulated Cav1 and decreased eNOS activity in the kidney. HHcy also resulted in the upregulation of mRNA and protein levels of MMPs −1, 2, −9, −8, −13 as well as connexins – 40 and −43 and decreased the activities of (TIMPs) −1, −2, −3, −4. Immunohistochemistry results indicated increased collagen IV deposition and expression of connexins in the glomerular and interstitial areas. GYY4137 treatment mitigated these changes in HHcy. In conclusion, our data suggests that H2S alleviates renovascular remodeling by modulating Cav‐1, eNOS, MMPs/TIMPs and Cxs in HHcy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Dysfunction and Metabolic Reprogramming in Chronic Kidney Disease
Authors: Li Ying , Pham Hai , Singh Prabhleen
Published: June 2021

Chronic kidney disease (CKD) is a leading cause of death in the U.S. With limited treatment options, the disease progresses to end‐stage renal disease (ESRD), which is also associated with significant morbidity and mortality. Better understanding of the mechanisms which underlie the progression of CKD is indispensable for developing novel treatments to slow or halt the progression of CKD. Towards this goal, we examined the role of mitochondrial function and metabolic reprogramming in renal tubules in the development and progression of CKD using the subtotal nephrectomy (STN) model of CKD.

Weight and age‐matched male 129X1/SvJ mice underwent either sham or STN surgeries. Tubular mitochondrial respiration at 2 and 4 weeks post‐surgery were evaluated using a high‐throughput Seahorse XF Analyzer. Renal expression of genes regulating mitochondrial biogenesis, dynamics, and metabolism at 4 weeks post‐surgery were measured by real‐time PCR. The mitochondrial respiration were similar between sham and STN groups at 2 weeks post‐surgery. However, at 4 weeks post‐surgery, the basal, the ATP‐linked, and the maximum respiration were significantly reduced in the STN tubules compared with sham (Figure 1A). Reduction of renal oxidative respiration indicates an impairment of mitochondrial function in STN renal tubules. Interestingly, the expression level of glycolytic mRNA, including phosphofructokinase (PFK, a rate‐limiting enzyme of glycolysis), glycolytic pyruvate kinase (PKM), and Hexokinase‐1 (HK1) were all significantly increased (Figure 1B–D), while expressions of carnitine palmitoyltransferase I (CPT1, a rate limiting enzyme of lipid oxidation), and peroxisomal acyl‐coenzyme A oxidase 1 (acox 1) remained unchanged.

In conclusion, we observed compromised mitochondrial function and metabolic switch from oxidative respiration to glycolysis at 4 weeks post‐STN. Additional studies to further delineate mitochondrial functional changes and switch in tubular metabolism are ongoing. Mitochondrial dysfunction and metabolic reprogramming may contribute to the development and/or progression of CKD. Therapeutic strategies which improve mitochondrial function and inhibit metabolic reprogramming pathway may serve as novel therapeutic targets in CKD treatment.

Support or Funding Information

VA Merit Award BX002175 and NIH R01 DKDK107852

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 TH17 cells are Elevated in Humanized Sickle Cell Mice
Authors: Molina Patrick Axel , Fox Brandon M. , De Miguel Carmen , McMonigle Ryan J. , Botta Davide , Huang Fengyuan , Kasztan Malgorzata , Pollock David M. , Pollock Jennifer S.
Published: June 2021

Sickle cell nephropathy (SCN) is a major complication of sickle cell disease (SCD). However, it is not fully understood what role immune cells play in SCN. Recent studies have implicated IL‐17A+CD4+(TH17) cells in mediating kidney disease outside of SCD. Furthermore, levels of circulating TH17 cells are elevated in SCD patients. We have previously reported that humanized sickle cell (HbSS) mice show progressive age‐dependent nephropathy with proteinuria, glomerulosclerosis, loss of GFR, and elevated CD3+cells compared to control (HbAA) mice. However, a role for renal TH17 cells in SCN has not yet been defined. We hypothesized that HbSS mice have elevated renal TH17 cells compared to HbAA mice. Thus, we phenotyped renal and splenic lymphocytes from 16–24 week‐old male HbAA and HbSS mice by flow cytometry. HbSS mice had increased renal TH17 cells compared to HbAA mice (18.3 ± 2.0% vs. 0.2 ± 0.1%, respectively; p<0.001) but no difference in renal CD4+cells. No differences were observed in splenic TH17 cells between HbSS and HbAA mice, which correlated with similar frequencies of splenic CD4+cells. Interestingly, HbSS mice had a significant increase in renal CXCR3+CD4+(TH1) cells compared to HbAA mice (48.8 ± 2.3% vs. 10.9 ± 3.3%, respectively; p<0.001) but no difference in splenic TH1 cells. Next, we measured changes in circulating proinflammatory mediators by Luminex multiplex technology. HbSS mice had elevated levels of soluble intercellular adhesion molecule 1 (sICAM‐1: 2937 ± 240 ng/mL vs. 2396 ± 57 ng/mL, respectively; p=0.04 ) and plasminogen activator inhibitor 1 (PAI‐1: 546.9 ± 120 ng/mL vs. 69.6 ± 4.5 ng/mL; p=0.001 ) compared to HbAA mice. We conducted a preliminary flow cytometric analysis of activated renal and splenic CD4+cells from aged (8–13 month‐old) mice and observed decreased splenic CD4+cells in aged HbSS mice compared to aged HbAA mice (5.1% vs. 32.5%, respectively; n = 2) but no differences in renal CD4+cells. Activated CD44hiCD4+cells from aged HbSS mice were increased in both the spleen (68.8% vs. 30.8%; n = 2) and the kidney (22.2% vs. 14.0%, respectively; n = 2) compared to aged HbAA mice. We propose that these findings and initial observations suggest an important role for renal T cells, especially TH17 and TH1 cells, in promoting and exacerbating kidney injury and dysfunction in SCD.

Support or Funding Information

Support provided by NIH T32 DK116672 to PAM, F30 DK107194 to BMF, NIH P01 HL136267 to CDM, JSP, DMP, NIH T32 AR069516 to RJM, NIH U01 HL117684 to JSP, DMP, and ASN Ben J. Lipps Research Fellowship to MK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acetazolamide Administration Restores the Blood Pressure Lowering Effect of Tempol in Female SHR
Authors: Zuchowski Yvonne , Fernandez Edgar David Torres , Subauste1 Alessandro , Zhang Huimin , Vinson Ruth Michelle , Cardozo Licy Yanes , Reckelhoff Jane F
Published: June 2021

The mechanisms responsible for the sex differences in blood pressure (BP) in humans are not clear. Over the past several years, we have studied the spontaneously hypertensive rat (SHR) as a model of sex differences in BP control. Oxidative stress (OS) plays a major role in BP regulation in preclinical studies. There is a sex difference in the BP responses to pro‐oxidants and antioxidants in the SHR. In contrast to males, BP in female SHR does not decrease in response to Tempol or Apocynin. Acetazolamide (Aceta) inhibits proximal tubular sodium reabsorption, increasing sodium delivery to the distal tubule which increases distal tubule OS. We tested the hypothesis that an increase in sodium delivery in the distal tubule will restore the BP lowering effects of Tempol in female SHR.

Materials and Methods

Female SHR, aged twenty weeks, were implanted with radiotelemetry transmitters and following a two week recovery period, baseline mean arterial pressure (MAP) was measured 24 hrs per day. All rats were then given Aceta (100 mg/kg/day sc) for 14 days (n=14). After this period, a group of rats were randomized to receive Tempol (1mmol/L) in drinking water for an additional two weeks (Aceta + Tempol) (n=7/grp) and BP was compared to controls who received Aceta only (n=7/grp). MAP was measured continuously throughout the treatment periods.

Results

At baseline, MAP was similar in both groups of female SHR (144 ± 1 vs. 145 ± 1 mmHg). Aceta administration for two weeks caused a 10 mmHg reduction in MAP, which was similar in both group of rats (132 ± 1 vs. 131 ± 1 mmHg, p=0.43). Tempol administration significantly lowered MAP in presence of Aceta (138 ± 1vs. 122 ± 1 mmHg, p<0.0001). Body weight was similar between Aceta and Aceta + Tempol groups (200.3 ± 2.0g vs. 192.2 ± 4.0 g).

In conclusion, our data suggest that there are sex differences in proximal sodium reabsorption that may impact OS in SHR. By increasing sodium delivery to the distal nephron with acetazolamide, the BP lowering effect of Tempol is restored in female SHR. These data suggest that different mechanisms may control oxidative stress and blood pressure in male and female SHR.

Support or Funding Information

This project was funded by NIH R01 HL135089, P20 GM12334, & PO1HL51971

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Basolateral Calcium Sensing Receptor Mediated Regulation of TRPV6‐dependent Transcellular Calcium Absorption
Authors: Lee Justin , Liu Xiong , O'Neill Debbie , Beggs Megan R , Weissgerber Petra , Flockerzi Veit , Chen Xing‐Zhen , Dimke Henrik , Alexander Todd
Published: June 2021

The plasma concentrations of ionized calcium (Ca2+) is maintained within a narrow physiological range by amending the release of parathyroid hormone and through direct effects of the calcium sensing receptor (CaSR) on the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption via 1,25‐dihydroxy vitamin D3, bone resorption, and renal Ca2+ excretion. The CaSR is also expressed in intestinal epithelium, but its function with respect to intestinal Ca2+ absorption are unknown. In the present study, we describe a mechanism by which the intestine detects and regulates Ca2+ absorption. Chronic CaSR activation decreased the expression of genes driving transcellular Ca2+ absorption, with the greatest effect observed in the proximal colon. To directly assess Ca2+ fluxes across the proximal colon, radioactive tracer experiments were performed ex vivo in Ussing chambers. Increased extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption acutely. Conversely, Ca2+ absorption was increased when the extracellular buffer was switched to one containing a lower Ca2+ concentration. These responses were absent in mice expressing a non‐functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co‐expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet‐mediated inhibition of Ca2+ flux in both Xenopus ooctyes and murine proximal colon. Together, our results reveal a novel regulatory pathway whereby activation of the CaSR in the basolateral membrane of the proximal large bowel attenuates Ca2+ absorption via genomic and non‐genomic regulation of TRPV6 to prevent hypercalcemia.

Support or Funding Information

This work was funded by the Canadian Institute of Health Research, the Natural Sciences and Engineering Research Council, and the Women and Children's Health Research Institute.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kidney Specific BMAL1 Knockout Exhibit Sex‐dependent Differences in the Expression and Activity of Renal H, K‐ATPases
Authors: Crislip Gene Ryan , Lynch I. Jeanette , Douma Lauren G , Masten Sarah H. , Cheng Kit‐Yan , Esser Karyn A , Wingo Charles S. , Gumz Michelle L.
Published: June 2021

Two isoforms of the Hydrogen, Potassium‐ATPases, HKa1 and HKa2, located in the cortical collecting duct (CCD) are important in acid‐base regulation and potassium handling and are encoded by ATP4a and ATP12a, respectively. Elabida et al. has reported that progesterone mediates antikaliuretic effects via stimulation of HKa2 expression. The circadian clock influences kaliuresis as demonstrated by its 24‐hour cycles. Therefore, the objective of this study was to determine whether expression of the clock gene BMAL1 affected HK activity and if there was a difference in females compared to males.

Kidney‐specific BMAL1 knockout mice (KO) were generated using floxed exon 8 BMAL1 mice crossed with kidney‐specific cadherin Cre+ mice. Floxed Cre− littermates were used as control mice (CNTL). Kidneys were collected for qPCR or CCD isolation. HK‐mediated H secretion in isolated perfused CCD was measured as EIPA‐ and bafilomycin A1‐insensitive intracellular pH (pHi) recovery from an acute H loading (NH4) using BCECF. pHi recovery was measured in A‐ and B‐type intercalated cells (IC) from female and male mice. IC type were differentiated based on the pHi response to peritubular Cl removal.

Female mice had 2.3 fold greater ATP12a mRNA in the cortex than males (PSEX=0.008; N=4–6) and greater cortical ATP4a mRNA than males (1.5 fold; PSEX<0.0001), however, there was no genotype difference. Furthermore, the expression of BMAL1 affected ATP4a expression in a sex‐dependent manner (PSEX*KO=0.02). Overall, ICs from female mice exhibited faster rates of HK‐mediated H flux than males (4.3 ± 0.5 vs. 2.9 ± 0.3 H/min, PSEX<0.02, NIC/CCD=108/11 & 103/10). We observed a heterogeneous IC cell population with the number of A‐type ICs increasing (and B‐cells decreasing) from the outer to the inner CCD (P<0.001). We systematically analyzed HK activity in A‐ & B‐type ICs from the outer to the inner CCD. Female KO mice had 2.5 fold greater HK activity than female CNTL in A‐type ICs from mid and inner CCD (2.8 ± 0.6 vs. 6.9 ± 1.0, N=23/4 & 23/4, PKO<0.001).

In conclusion, these data demonstrated a sex‐dependent difference in mRNA expression and HK activity. HKa1 and HKa2 mRNA expression and HK activity in females are greater than in males. HK activity is upregulated in the acid secreting ICs in female BMAL1 KO mice compared with controls. The clock gene BMAL1 has a sex‐specific role in regulating cortical mRNA expression of HKa1 and HK‐activity.

Support or Funding Information

5T32HL083810‐10

NIH/NIDDK 1R01DK109570

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.
Evaluation of Renin Angiotensin System (RAS) in Renal Transplant Patients
Authors: Bochio Filipo , Felipe Claudia Rosso , Aragão Danielle Sanches , Brandão Juliana Dinéia Perez , Pestana José Osmar Medina , Junior Helio Tedesco Silva , Casarini Dulce Elena
Published: June 2021

Kidney transplantation is a surgical process that involves swapping the diseased kidney for a healthy kidney. In Brazil, about 5500 transplants per year are performed. After the transplantation process, the patients started to use immunosuppression therapy for acute rejection prophylaxis. There are two immunosuppressants that are most commonly used, Everolimus, Tacrolimus and Sodium Mycophenolate. Current immunosuppressive regimens are effective in preventing acute rejection but have several adverse events. Among them, hypertension and diabetes probably occurred due to an imbalance in RAS. In view of the above observations, the objective of this study was to verify the expression of the proteins and peptides of the RAS, in order to understand if there were changes in the modulation of the same. Two groups of patients, diabetics (n=10) and non‐diabetics (n=10), all with the underlying disease, hypertension, were analyzed. For this purpose, patients were collected at different times after transplantation: seven days (D7), fourteen days (D14), one month (M1), two months (M2), three months (M3), four months M4), five months (M5) and six months (M6). Patient plasma was prepared separately for albumin removal, and subjected to HiScreen Blue FF (GE Healthcare) (1 × 4.7 ml) affinity column chromatography coupled to Akta FPLC system and equilibrated with 50 mM Monopotassium Phosphate buffer, pH 7.0, under flow rate of 1.0 mL/min. The purification of the samples analyzed for one patient is in the table above.

Purified plasma in different times was submitted to western blotting in order to demonstrate the expression of angiotensin converting enzyme (ACE). In the table above, we can see that the ACE expression increased gradually in the times of D7 to M6, coinciding with the detection of more pronounced hypertension in this patient (M6). This pilot study suggests that the healthy kidney is likely to be physiologically modified as a result of medications received by the patient, immunological modulations, leading to hypertension and that RAS is involved in this profile.

Support or Funding Information

CAPES and CNPq

Purification

Sample (days)
D7
D14
M1
M2
M3
M4
M5
M6

Times (x)
15,65
12,40
15,40
12,87
14,32
16,55
10,72
17,30

Protein Expression (Arbitrary Units)

Expression ACE 190
0,933
1,119
1,390
2,483
2,630
2,517
2,545
4,755

90
0,846
1,200
1,183
1,587
1,839
2,167
2,310
3,158

65
1,625
1,376
1,246
1,420
1,702
1,775
1,687
2,047

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proof of Principle: Transfer of Small Numbers of Bacteria from Mother to Fetus
Authors: Yu Kevin , Zarate Miguel , Keller‐Wood Maureen , Wood Charles E
Published: June 2021

The fetus is thought to develop in a sterile environment in utero. Long standing dogma that “the uterus and the feto‐placental unit is “sterile” is based primarily on microbiological culture‐based techniques that were unsuccessful in growing “culture resistant” bacteria or intracellular bacteria. We have reported the presence of low numbers of bacteria in tissues from normal sheep fetuses in pregnancies not complicated by infection. The exposure of the fetus to bacteria might aid neonatal survival by informing fetal immune development. We propose that the fetus is not sterile and that bacteria or fragments of bacteria can be transferred from mother to fetus. We hypothesize that inoculation in the maternal mouth results in the appearance of bacteria in the fetus. We used S. aureus containing green (GFP), red (RFP), or far‐red (FRFP) fluorescent protein‐expressing plasmids to inoculate late‐gestation pregnant sheep (gestational age= 130–135 days, n=7) intravenously (GFP, 104 cfu), into maternal mouth (RFP, 104 cfu) and vagina (FRFP, 104 cfu). These were small doses of bacteria which did not cause physiological (no fever) or psychological (no anorexia) signs of infection. Five to seven days post maternal inoculation, animals were humanely sacrificed, and fetal tissues were collected, and DNA was extracted from placenta and fetal liver, spleen, and cerebral cortex. We probed for GFP plasmid using several primer pairs for endpoint PCR. While detection of whole‐length plasmids was not successful, PCR reactions probing for smaller fragments of plasmid were successful. We found GFP plasmid DNA in all tissues in 10/10 fetal livers and RFP in 10/10 livers. The appearance of GFP and RFP‐labelled bacteria in fetal liver (p=7.497e‐06) was statistically significant as tested by Chi‐Square analysis. We did not detect significant FRFP plasmid DNA in liver. Analysis of tissues by immunohistochemistry revealed GFP and RFP‐expressing bacteria in fetal tissues, although most appeared to be in aggregates. We conclude that S. aureus, introduced in small numbers in maternal mouth and bloodstream, appear in the fetus and placenta. We were unable to determine whether these bacteria were alive in the fetus.

Support or Funding Information

This work was supported by HD033053, AI120195, and HL083810

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Genes that Comprise the Core Molecular Clock are Altered in the Atrophied Skeletal Muscle by Androgen Deprivation
Authors: Rossetti Michael L , Casero Robert A , Esser Karyn A , Gordon Bradley Scott
Published: June 2021

Skeletal muscle atrophy increases the risk of morbidity and mortality during various pathological conditions. In males, a decrease in the production and/or bioavailability of androgens (termed hypogonadism) directly contributes to muscle atrophy during various pathological conditions. While it is known that androgens prevent muscle atrophy, the mechanism(s) by which androgens mediate this effect are largely undefined. Our laboratory previously showed that mitochondrial turnover is enhanced in the tibialis anterior (TA) muscle of mice by androgen deprivation induced by castration surgery, and the magnitude of turnover was related to the degree of muscle atrophy. These data suggest that potentially dysfunctional mitochondria contribute to the muscle atrophy observed following androgen deprivation. To gain a better understanding of that factors that might contribute to changes in mitochondrial quality control during androgen deprived conditions, we subjected total RNA from the TA of sham and castrated mice to microarray analysis. This unbiased approach identified significant changes in expression of genes that comprise the core molecular clock. qRT‐PCR confirmed that expression of Brain and Muscle Arntl 1 (Bmal1) was decreased, while expression of Period 1, Period 2, and Period 3 (Per1, 2, & 3) were increased in the TA of castrated mice. When measured across a diurnal cycle, the change in expression of Bmal1, Per1, and Per2 exhibited reduced amplitude under androgen‐deprived conditions. Interestingly, strong relationships were observed between the castration‐mediated changes of core clock components and the measures of mitochondrial turnover. Specifically, Bmal1 expression was directly related to BCL2/adenovirus E1B 19 kDA protein‐interacting protein 3 (BNIP3) protein content (R2 = 0.88), and the expression of core clock components were also directly related to the content of various mitochondrial proteins. Expression of core clock components were also related to the autophagy marker, p62, and the mass of the TA. Ex post facto analysis of the microarray also identified changes in genes regulating polyamine biosynthesis. As polyamines are known to alter core clock function, we determined whether androgen deprivation altered polyamine content. While castration did not alter Spermine content, there was a significant reduction in the content of Spermidine in the TA of castrated mice. Overall, these data suggest that reduced Spermidine concentrations may contribute to alterations in the core molecular clock in the skeletal muscle under androgen‐deprived conditions, which may in turn contribute to reduced mitochondrial quality control and subsequent muscle atrophy

Support or Funding Information

NIH‐CA204345 (RAC) and Institute for Successful Longevity (BSG)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of short‐term 17B‐estradiol on low‐flow‐mediated constriction in young women
Authors: Kuczmarski Andrew , Sebzda Kelly , Wenner Megan
Published: June 2021

Low‐flow mediated constriction (L‐FMC) examines vascular responses during a reduction in blood flow and provides a measure of resting shear‐stress dependent vascular tone. L‐FMC is a complementary measure to flow‐mediated dilation (FMD), and is blunted in patients with cardiovascular disease. 17β‐estradiol (E2) exerts positive effects on vascular endothelial health and can improve FMD. However, it is unknown whether E2 impacts L‐FMC.

PURPOSE

The purpose of this study was to test the hypothesis that E2 administration would augment L‐FMC in young women.

METHODS

Eleven young women (24±1 years, 24±1 kg/m2) have completed the study. To isolate the impact of E2, endogenous ovarian hormone production was suppressed with daily subcutaneous injections of a gonadotropin releasing hormone antagonist (GnRHant, 0.25 mg/day) for 10 days. E2 (0.1mg/day patch) was administered during the last 7 days of GnRHant. We measured changes in brachial artery diameter via ultrasound on day 3 of GnRHant and day 7 of E2 add‐back. A blood pressure cuff was placed just distal to the olecranon process and inflated to 200mmHg for five minutes. Images were recorded continuously at baseline (1‐minute), during occlusion (5‐minutes), and after cuff deflation (2‐minutes). L‐FMC was determined from the last 30 seconds of cuff inflation using the lowest average value and was reported as percent change from baseline diameter. FMD was calculated as a percent change from baseline to peak diameter after cuff deflation. Total vessel reactivity was calculated as the absolute value of L‐FMC and FMD. Results are reported as mean ± standard error.

RESULTS

Baseline diameter was similar between GnRHant (3.14 ± 0.12 mm) and E2 (3.08 ± 0.11 mm; p=0.3). L‐FMC tended to be greater during E2 administration (GnRHant −2.5 ± 0.5% vs. E2 −3.8 ± 0.5%; p=0.11). However, FMD (GnRHant 7.5 ± 0.7% vs. E2 7.6 ± 1.1%; p=0.92) and total vessel reactivity (GnRHant 10.0 ± 0.7% vs. E2 11.5 ± 1.2%; p=0.26) were not altered by E2.

CONCLUSION

These preliminary findings suggest that short term E2 administration may augment L‐FMC in young women. Additional data are needed to understand the impact of ovarian hormones on L‐FMC and its role in regulating vascular health.

Support or Funding Information

NIH Grant P20 GM 103446, P20 GM 113125, U54 GM 104941, The University of Delaware Research Foundation, and AHA Award 16SDG30700015

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Maternal Overconsumption of High Fructose Corn Syrup Causes Hypoxia in Placenta and Fetal Livers of Wistar Rats
Authors: Ahmed Erum , Carrillo‐Sepulveda Maria Alicia
Published: June 2021

Background

American maternal overnutrition negatively impacts fetal development. Several epidemiological and experimental studies have demonstrated that overconsumption of diets rich in fats and sugars can have effects spanning three generations. While the negative impact of high fructose corn syrup (HFCS), a component heavily present in many processed American foods, is well‐recognized, its effects during fetal development remain largely underexplored. Thus, this study aims to examine the placental and fetal hepatic effects of HFCS maternal overconsumption.

Methods

Adult female Wistar rats were randomized in 2 experimental groups: HFCS Group received a 20% HFCS solution in drinking water for 30 days prior to and during pregnancy. Control Group received regular water. Prior to pregnancy, blood pressure was measured in both experimental groups by using tail‐cuff plethysmography. Fasting glucose readings were measured weekly. Terminal experiments were completed on day 21 of gestation. Placentas and fetal livers were isolated and further processed for analysis of Hypoxia‐Inducible Factor‐α (HIF‐α) expression, a classical transcription factor of hypoxia, using Western Blot. Blood samples were collected for biochemical analysis.

Results

Prior to pregnancy, HFCS group exhibited increased serum triglycerides (441.48 vs. 109.47 mg/dL), systolic (159.03 ± 12.04 vs. 136.29 ± 2.57 mmHg, p<0.05) and diastolic blood pressures (114.66 ± 9.47 vs. 99.29 ± 2.49 mmHg, p<0.05) compared to controls. No differences were found in glucose levels (98.17 ± 1.65 vs. 92.5 ± .24 mg/dL controls). At the terminal experimental protocol, HFCS group displayed increased HIF‐α expression in placenta (2.3 fold‐increase, p<0.05) and fetal liver (3.1 fold increase, p<0.001) compared to controls. Therefore, our results show hypoxic changes in both placental and fetal hepatic tissues. Moreover, oil red staining confirmed increased fat deposition in the maternal livers from HFCS group.

Conclusion

Our findings show that overconsumption of HFCS prior to and during pregnancy has a negative impact on offspring, as demonstrated by upregulation of hypoxic markers such as HIF‐α. We will further this study to determine whether other tissues undergo hypoxia and the mechanisms by which HFCS leads to hypoxia in maternal and fetal tissues.

Support or Funding Information

In‐house Grant NYIT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
10‐Week High‐Fat Diet Promotes Endotoxemia and Alters Microbial Taxonomy in Male Adolescent Rats
Authors: Crawford Meli'sa Shaunte , Sweazea Karen
Published: June 2021

Diet‐induced changes in gut microbiota are associated with increased risk of obesity and diabetes. Prior research shows that a proprietary soil‐derived compound decreases weight gain and blood glucose in diabetic mice. The aim of this study was to examine the effects of a similar organometallic complex (OMC) on gastrointestinal inflammation in rats fed high‐fat diet (HFD). Six‐week‐old male Sprague‐Dawley rats (n=36) were divided into two dietary groups: chow (18.9% protein, 57.33% carbohydrates, 5% fat) or HFD (20% protein, 20% carbohydrates, 60% fat) for 10 weeks. Animals were further divided (n=6/group) and administered 0, 0.6 or 3.0 mg/mL OMC in their drinking water. Microbial taxonomy of cecal contents were evaluated by LEfSe analysis and showed a significant abundance of Rikenellaceae, Alistipes, Erysipelotrichaceae and Oceanospirillales in samples from chow rats (LDA <2.0) after 10 weeks. In contrast, HFD cecum contents were more enriched in bacteria classified as “other” and “mitochondria” (LDA <2.0). HFD rats also showed elevated lipopolysaccharide concentrations (p<0.05), which was prevented by OMC. Additionally, HFD rats showed an increase in fat accumulation in the liver as indicated by elevated triglyceride concentrations (p<0.05). Furthermore, plasma ALT activity was increased in HFD rats (p<0.05). Western blot analyses of the cecum showed no changes in protein expression of the inflammatory cytokines NF‐kB, IL‐1B, or IL‐6 (Two‐Way ANOVA, p=0.362, p=0.702, and p=0.746, respectively) in rats fed HFD. Likewise, protein expression of NF‐kB, IL‐1B, and IL‐6 were not altered (Two‐Way ANOVA, p=0.402, p=0.062, or p=0.953 respectively) by OMC‐treatment. Thus, while a 10‐week HFD induces gut dysbiosis and endotoxemia, gut inflammatory biomarkers were not significantly altered by diet or OMC. Further studies are needed to determine whether OMC alters intestinal permeability.

Support or Funding Information

This study was partially funded by a grant from Isagenix International, LLC.

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