bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Expression of the (Pro)renin Receptor in the Subfornical Organ of Hypertensive Humans
Authors: Cooper Silvana G , Yamamoto Rieko , Jensen Dane D , Sorensen Jacob T , Trivedi Darshan , Xiong Zhenggang , Feng Yumei
Published: November 2023

We previous reported that (pro)renin receptor (PRR) is functional important in the subfornical organ (SFO) of the brain, a key brain circumventricular organ that is involved in BP regulation and body fluid homeostasis. The SFO PRR mediates Ang II formation and is critical for hypertension development and body fluid homeostasis in hypertension animal models. However, it is not known whether PRR expression level is altered in hypertensive humans and what is the cellular distribution. We have collected human subfornical organ (SFO) tissues post mortem, of which 14 are clinically diagnosed hypertensive and 7 are diagnosed normotensive. Systolic (SBP) and diastolic (DBP) blood pressure, age, body weight, and medications used information was collected. SFO tissues were fixed in 10% formaldehyde and processed for paraffin embedding and sectioning. The age and body weight patterns are similar between the normotensive and hypertensive subjects. Regardless of anti‐hypertension medications, the SBP was significantly higher in clinical diagnosed hypertensive (142.4 ± 4.2 mmHg) compared with the normotensive (118.1 ± 7.7, P=0.007) subjects. There is no difference in DBP between these two groups (74.1±5. VS. 79.29±3.5). SFO sections were used for immunofluorescence double labeling of human PRR with neuronal marker (anti–human neuronal protein HuC/HuD), astrocyte marker (glial fibrillary acidic protein, GFAP), or microglia marker (ionized calcium‐ binding adaptor molecule 1, Iba1). We found that the human PRR is co‐localized only with the HuC/HuD but not GFAP or Iba1 suggests that PRR is majorly expressed in the neurons of human SFO. To examine whether there is alteration of PRR expression in the hypertensive humans, we performed immunohistochemistry labeling of PRR in these tissues. The PRR immuno‐reactivity was significantly higher in the hypertensive subjects (145.3 ± 1.4 Arbitrary Units, AU) compared with the normotensive subjects (135.5 ±1.2 AU, P=0.0002). In addition, there is significant positive correlation between the PRR expression levels and the SBP (Pearson r=0.55, R2=0.308, P=0.018) among all normotensive and hypertensive subjects. The data suggests that PRR expression level is elevated in hypertension subjects and there is positive correlation of PRR expression level with systolic BP. We conclude that PRR in the SFO may play important role in the body fluid homeostasis and hypertension development in humans.

Support or Funding Information

NIH/NHLBI (1R01HL122770) TO YUMEI FENG; NIH/NIGMS (P20GM103440) TO SILVANA G. COOPER
Assay Translation of Preclinical Cardiovascular (CV) Assessments
Authors: Northcott Carrie , Jenkinson Stephen , Bassyouni Assar , Ramirez David , Schroeter Thomas , Warren Laurie , Fensome Andrew , Bhatt Siddhartha , Steidl‐Nichols Jill
Published: November 2023

Combinations of in vitro, ex vivo and in vivo animal testing have historically played a vital role in the research and the development of new therapeutic treatments. However, understanding the translation of these data across assays, as well as how these data translate to humans has proven to be challenging, especially with regards to the complex and integrated mechanism involved in blood pressure (BP) and heart rate (HR) regulation. We have evaluated the translation of several in vitro and ex vivo assays to in vivo CV assessments, as well as the translation of rat to large animal (LA), and LA to human BP and HR data. To investigate the translation of in vitro and ex vivo findings to in vivo hemodynamic results, 41 compounds with direct, indirect or unknown mechanisms of BP and/or HR modulation as well as 13 negative controls were evaluated in ppMLC (phosphorylation of myosin light chain), functional pharmacology (19 receptors/ion channels), ex vivo aortic vascular reactivity and isolated guinea pig Langendorff heart assays. Results from the in vitro and ex vivo assays were compared to in vivo conscious rat telemetry BP and HR findings. Vascular aortic relaxation and the paced guinea pig isolated heart assay detected 40% and 35%, respectively, of the in vivo positive compounds within 10× of the free Cmax that produced a 5 mmHg change in BP. The guinea pig isolated heart assay had a lower false positive rate than the aortic assay. The ppMLC assay detected 30% of compounds targeted at kinases within 10× the 5 mmHg in vivo Cmax concentration, but did not identify compounds with BP/HR risk with non‐kinase primary targets. Additional translation analyses were performed to more fully understand in vivo rat to large animal hemodynamic changes, as well as large animal preclinical to phase 1 clinical BP and HR measures. 83 compounds were assessed in both rat and LA studies. The specificity observed (true positive rate) was 71% and sensitivity (true negative rate) was 84%. Suggesting that rat is an effective model for evaluating hemodynamic risk. In evaluating 79 compounds in both LA and preclinical Phase 1 trials there was also good specificity (79%) and sensitivity (78%). These studies demonstrate that several in vitro/ex vivo assays are useful for identifying in vivo BP and HR changes; however due to the complexity of the CV system, have limited predictivity. Preclinical in vivo BP and HR testing was demonstrated to translate not only across species but also to the clinic. These data confirm the importance of pre‐clinical in vitro, ex vivo and animal models in the examination of the efficacy and safety of drugs, and the usefulness of these models for selection of compounds for advancement to clinical testing and ultimately the treatment of disease.
Meprin β expression enhances multi bacterial sepsis‐induced acute kidney injury
Authors: Yahaya Ismaila , Niyitegeka Jean‐Marie Vianney , Fernander Mizpha Courtney Simone , Sedighi Rashin , Kenley Sharniece R , Ahmed Faihaa , Boston Ava Millicent , Ongeri Elimelda Moige
Published: November 2023

Sepsis is a systematic inflammatory response syndrome which accounts for 51% of reported cases of acute kidney injuries (AKI) in intensive care units (ICU) and is associated with high morbidity and mortality. The high mortality rate associated with AKI is partly due to the fact that underlying mechanisms are not fully understood. Meprins are metalloproteinases of the astacin family which are most highly expressed in the brush border membranes of proximal kidney tubules. Meprins are also expressed in leukocytes (monocytes and macrophages). Meprins have been shown to play a role in the pathophysiology of acute kidney injury and in leukocyte infiltration. This study was designed to determine how meprin expression impacts the pathology of AKI induced by sepsis. To this end, cecal ligation and puncture (CLP) was used to induce multi‐bacterial sepsis in 12‐week old wild‐type (WT) and meprin β knockout (βKO) mice on a C57BL/6 background. The mice were sacrificed at 18 h post‐CLP. Blood samples were collected by tail nicking at 0 h and by cardiac puncture at 18 h post‐CLP. For biochemical assessment of kidney injury, enzyme linked immunosorbent assays were used to measure plasma neutrophil gelatinase‐associated lipocalin (NGAL) and cystatin C levels. The 18 h post‐CLP survival rates were comparable for WT and meprin βKO mice. The levels of cystatin C at 18 h post‐CLP were significantly higher in both genotypes for mice subjected to CLP when compared to sham operated controls (p≤0.0001). More importantly, WT mice subjected to CLP had significantly higher levels of plasma cystatin C when compared to the meprin βKO counterparts. Furthermore, there was a significant increase in NGAL levels in WT mice subjected to CLP but not in meprin βKO counterparts (p≤0.05). The results indicate that meprin β plays a detrimental role in sepsis‐induced acute kidney injury. The data further suggests that when compared to NGAL, cystatin C is a better biomarker for kidney injury induced by sepsis.

Support or Funding Information

This study was supported by the National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) under Award # SC3GM102049 to Elimelda Moige Ongeri.
Enteral Resuscitation with WHO Oral Rehydration Salts Ameliorates Burn‐Induced Kidney Injury (AKI) and is Protective of the Glomerular Glycocalyx in a Swine Model
Authors: Gomez Belinda I. , McIntyre Matthew K. , Dubick Michael A. , Burmeister David M.
Published: November 2023

Following severe burn injury oral resuscitation fluid may be sufficient to treat patients, especially when i.v. fluid may be delayed such as in mass casualty scenarios. The World Health Organization (WHO) Oral Rehydration Salts (ORS) formulation is designed for treatment of severe dehydration however; its effectiveness at maintaining kidney function and perfusion after burn has not been studied. The objective of this study was to evaluate the effectiveness of ORS and water on post‐burn Acute Kidney Injury (AKI). Anesthetized Yorkshire pigs sustained 40% TBSA full‐thickness contact burns [with brass probes heated to 100°C placed on the skin for 30 seconds]. Animals recovered in metabolic cages and were randomized to one of four treatment groups: no water access (n=7), ad libitum water access (n=6), 15 mL/kg/d of ORS (n=6), or 70 mL/kg/d ORS (n=6) for a 48 hour period. Urine and blood were collected at baseline (BL), 6, 12, 24, 32, and 48‐hour post‐burn for quantification of biochemical markers. Regardless of treatment, all animals experienced AKI as demonstrated by a 0.50±0.10 mg/dL elevation in plasma creatinine by 6 hours following burn. However, animals receiving 70 mL/kg/d ORS or ad libitum water had reduced plasma creatinine by 24 hours, as levels were 1.30±.21 and 1.30±0.13 respectively. Myoglobin was also elevated by 6 hours post‐burn but differences among treatments were not detected. Additionally, preliminary experiments demonstrated elevated free hemin in the urine and plasma following burn in water restricted animals. Animals receiving 70 mL/kg/d ORS also had similar renal wet to dry ratios as the ad libitum group, but both ratios were greater than the water restricted group (p < 0.05), indicating maintained renal perfusion. Moreover, glycosaminoglycan (GAG) concentrations in the urine were elevated post‐burn in all groups suggesting degradation of the glomerular glyocalyx. However, by 48 hours, 70 mL/kg/d animals had reduced urine GAGs when compared to other treatments. Collectively, these data demonstrate that water restriction in burn‐trauma exacerbates burn induced AKI and 70 mL/kg/d of ORS was as sufficient as ad libitum water in alleviating AKI, with preferential protection of the endothelial glycocalyx in the glomerulus. This study highlights the importance of hydration status in response to burn injury, and supports the use of oral resuscitation efforts in burn casualties in prolonged care and mass casualty scenarios.
SCUBE1‐enhanced Bone Morphogenetic Protein Signaling Protects against Acute Kidney Injury
Authors: Liao Wei‐Ju , Lin Heng , Cheng Ching‐Feng , Yang Sung‐Sen , Chen Ann , Yang Ruey‐Bing
Published: November 2023

Acute renal failure represents a common clinical problem associated with high morbidity and mortality. Renal ischemia‐reperfusion (I/R) is the major cause of acute renal failure in the native and transplanted kidneys. It has been reported that bone morphogenetic protein 7 (BMP7) reduces severity of injury via its proliferative and anti‐apoptotic effects on tubular cells after renal I/R insult. Our previous study showed that a novel secreted and membrane‐tethered protein SCUBE1 (signal peptide‐CUB‐EGF domain‐containing protein 1) was up‐regulated in renal tubular epithelium and may be involved in renal regeneration and repair after I/R injury (Nephrol. Dial. Transplan. 25: 1421, 2010). However, the precise mechanism underlying its protective function after renal I/R insult remains largely unknown. To further elucidate the renal pathophysiological roles of SCUBE1, we generated new Scube1 deletion (Δ2) mouse. As compared with wild‐type littermates, Δ2 mice showed severe renal histopathologies (extensive loss of brush border, tubular necrosis, and tubular dilation) and increased inflammation (neutrophil infiltrate and induction of tumor necrosis factor α) during the resolution of I/R damage. They also showed elevated levels of biomarkers of acute renal injury (neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1), and reduced BMP signaling (phosphorylated Smad1/5/8) along with decreased proliferation and increased apoptosis in renal tubular cells. Moreover, molecular and biochemical analyses revealed that SCUBE1 directly binds BMP7 and its receptors, possibly functioning as a BMP co‐receptor to promote BMP7 signaling. Importantly, lentivirus‐mediated overexpression of SCUBE1 enhanced BMP signaling and conferred a concomitant survival outcome for Δ2 proximal tubular epithelial cells after hypoxia–reoxygenation treatment. The protective BMP7 signaling may be facilitated by stress‐ inducible SCUBE1 after renal I/R, which suggests potential targeted approaches for acute kidney injury.

Support or Funding Information

This work was supported by Ministry of Science and Technology Grants of Taiwan [grant number NSC 102‐2320‐B‐001‐015‐MY3 (to R.‐B. Y.)]
Rifaximin Effects on Serum Trimethylamine‐n‐oxide in Chronic Kidney Disease
Authors: Johnson Cassi , Zhang Shiqin , Omede Faith , Stubbs Jason
Published: November 2023

Introduction

Trimethylamine‐n‐oxide (TMAO) is a byproduct of intestinal bacteria metabolism of choline and L‐carnitine. In patients with chronic kidney disease (CKD), TMAO accumulates in the bloodstream and is thought to perpetuate kidney fibrosis and promote cardiovascular disease (CVD). We hypothesized that rifaximin, a broad‐spectrum oral antibiotic with poor bioavailability, would significantly lower serum TMAO levels and slow kidney disease progression in CKD mice.

Design

CKD mice were generated by feeding C57BL/6J mice a diet containing 0.2% adenine; non‐CKD mice received the same diet without adenine supplementation. To test whether acute rifaximin therapy could lower serum TMAO levels, mice were fed the 0.2% adenine or control diet for 7 weeks. Starting at week 6 of the study, mice were randomized to receive 50 mg/kg rifaximin or vehicle once daily for the final week. In a separate study to assess whether long‐term rifaximin therapy could lower serum TMAO levels and attenuate kidney disease, 50 mg/kg rifaximin or vehicle therapy was administered 5 days/week (M‐F) at the onset of CKD induction by 0.2% adenine diet and continued for 13 weeks.

Results

Acute rifaximin therapy lowered post‐treatment serum TMAO levels in both non‐CKD (0.76 ±0.41 μM for rifaximin vs 3.78±1.45 μM for vehicle; p=NS) and CKD mice (8.91±2.20 μM for rifaximin vs 15.92±1.67 μM for vehicle; p<0.01). We observed shifts in microbiome composition with rifaximin therapy reflected by a decrease in microbiome diversity when comparing stool samples collected pre‐ and post‐rifaximin treatment that could account for decreased serum TMAO (Shannon H test: 2.51±0.01 for rifaximin vs 3.42±0.15 for vehicle in non‐CKD mice; 2.37±0.09 for rifaximin vs 3.53±0.25 for vehicle in CKD mice). However, long‐term rifaximin therapy did not lower serum TMAO levels in non‐CKD (6.87±1.13 uM for rifaximin vs 10.21±1.58 for vehicle; p=NS) or CKD mice (41.08±5.78 for rifaximin vs 45.51±3.89 for vehicle; p=NS). Likewise, rifaximin therapy failed to alter CKD progression as assessed by both serum markers of renal function and gene markers of kidney fibrosis.

Conclusion

Our data suggests that rifaximin therapy may be a viable option for short‐term lowering of serum TMAO in CKD; however, long‐term administration of this drug does not sustain this TMAO‐lowering effect or alter CKD progression in mice.
Urine Osmolality as a Diagnostic Tool for High Risk for Kidney Stone Development in Patients with Recurrent Kidney Stones
Authors: Suh Hyun‐Gyu , Kavouras Stavros A , Gharbi Kakam , Mauromoustakos Andy A , Vallet Marion , Tack Ivan
Published: November 2023

Formation of kidney stone affects approximately 1 in 11 people in the U.S., and it can develop into various adverse renal outcomes. Although American Urological Association guideline recommends having at least 2.5 L of urine daily by having a high fluid intake to reduce 50% of recurrence rate in stone formers, few studies have evaluated the association between urine concentration as a marker of hydration status and the risk of kidney stone formation.

PURPOSE

To identify the threshold of optimal hydration in order to classify patients as high risk for kidney stone formation.

METHODS

Total of 302 subjects from Southern France with a history of recurrent kidney stones were retrospectively included in the study (female: 40.4 %, age: 46±14 y, BMI: 25.3±5.0 kg·m−2). Tiselius calcium oxalate crystallization risk index (CRI) was calculated based on urinary calcium (6.42±3.60 mmol·24h−1), oxalate (0.36±0.14 mmol·24h−1), citrate (32.0±17.5 mmol·24h−1), and magnesium (4.31±1.74 mmol·24h−1) from 24 h samples. The ability of 24 h urine osmolality to classify patients as high risk for kidney stone crystallization was examined through the receiver operating characteristic (ROC) curve analysis. High risk for kidney stones crystallization was defined as CRI >1.61 and >1.18, for males and females, respectively. The correlation coefficient was also examined between 24 h urine osmolality (538±198 mOsm·kg−1) and 24 h urine volume (1,948±823 mL·24h−1) and 24 h CRI (1.23±0.71).

RESULTS

The overall accuracy for diagnosing high risk of stone formation (AUC, area under the curve) was 84.9%, with threshold of 577 mmol·kg−1 (sensitivity: 78.4%, specificity: 81.6%) for the entire sample. When data where analyzed by gender, AUC for females was 84.6%, with cut‐off point of 501 mmol·kg−1 (sensitivity: 83.3%, specificity: 76.0%). Males had AUC of 85.8% with thresholf of 577 mmol·kg−1 (sensitivity: 85.5%, specificity: 77.6%). A moderate negative association was found between urine volume and urine osmolality (r=−0.632, P<0.05). Also a moderate but positive association was found between 24 h urine osmolality and Tiselius CRI index (r=0.638, P<0.05).

CONCLUSION

Urine osmolality >501 and >577 mmol·kg−1 was assosicated with greater risk for kidney stone formation in patiens with history of recurrent kidney stone.
Gender Differences in the Renal Toxicity of the Neurotoxin Domoic Acid Griffin Thompson, Hernan Grenett, Lan He, Noor Alsirafi, Amanda Hanninen, and P. Darwin Bell University of Alabama Birmingham, and Birmingham VA, Birmingham, AL
Authors: Thompson Robert Griffin
Published: November 2023

The algae‐derived neurotoxin, domoic acid (DA), is an ionotropic receptor agonist and is currently considered an increasing threat to mammals and other species. Intraperitoneal administration (IP) of 4–5 mg/KgBW in mice has been found to cause neurological symptoms\damage. However, we recently reported (JASN 2104) that the kidney was 100 times more susceptible to injury with DA compared to brain. With IP injection, the kidney has a 4‐fold higher concentration of DA compared to brain and prior probenecid treatment leads to even further accumulations of DA suggesting that DA is secreted by the kidney. The purpose of these studies was to determine if there is a gender difference in the renal injury response to DA. Doses of 0.05 mg/Kg and 0.005 mg/Kg were IP injected into C57BL6 mice and 30 mins later the early injury‐response genes c‐fos, Junb, and EGR‐1were assessed using quantitative RT‐PCR. At this early time point both male and female mice exhibited an increase in gene expression. Surprisingly, female mice expression of early response genes was significantly higher, by approximately 2 fold, compared to male mice. Further studies were performed by implanting osmotic minipumps and infusing DA at a rate of 0.005 mg/KgBW per 24 hrs for 7 days. Urine DA levels were measured during this time by mass spectrometry. Over this period of time female mice excreted 50% less DA compared to male mice. Since the renal elimination of DA is both through both filtration and secretion, this suggests that female mice may have a reduced capacity to secrete DA. Our studies demonstrate that female mice are more susceptible to acute renal injury which maybe due to a reduced capability to excrete DA compared to male mice.
DPP4 Gene Deletion Protects Mice from Larp6‐mediated Fibrosis in Progressive Kidney Injury Models
Authors: Nistala Ravi , An Jianzhong , Kandikattu Hemanth Kumar , Bysani Chandrasekar
Published: November 2023

Obesity and heightened activity of the renin‐angiotensin‐aldosterone system (RAAS) are known risk factors for kidney injury and proteinuria. Progression of kidney injury occurs due to increase in inflammation, tubulointerstitial fibrosis and loss of nephron mass. Dipeptidyl peptidase 4 (DPP4) inhibition has been shown to improve proteinuria in both humans with Type 2 diabetes (SAVOR‐TIMI) and rodent models of obesity and diabetes. We and others have previously reported that DPP4 may be upregulated with increased activity in diabetic humans and rodent models of obesity and diabetes. DPP4 is known to bind collagens and fibronectin. Larp6 is a profibrotic gene that upregulates collagen synthesis via binding to the 5′ stem‐loop (5'SL) region of collagen mRNA. Therefore, we hypothesized that DPP4 gene deletion will prevent Larp6‐mediated fibrosis in a model of progressive kidney disease. To test our hypothesis, we fed a cohort of mice (DPP4+/+ and DPP4−/−) for 6 months on a Western diet (46% Kcal fat, 34% Kcal refined sugars) and a second cohort with angiotensin II (Ang II; 1000ng/kg/min via osmotic pump) plus deoxycorticosterone (DOCA, 50mg pellet) plus 0.9% salt for 2 weeks. We observed a significant increase in proteinuria in DPP4+/+ mice in both treatment groups, while a reduction in creatinine clearance (CrCl) was observed in the Western diet‐fed mice only. Pro‐inflammatory IL‐2 and IFN‐γ secretion was increased in kidney CD4+ T cells, while anti‐inflammatory IL‐10 secretion was decreased in the obese mice. This was accompanied by an increase in kidney fibrosis as assessed by picrosirius red staining and Collagen III/fibronectin expression as assessed by immunoblots. Importantly, an increase in kidney Larp6 protein was observed in both the obese and the RAAS activation models. DPP4 gene deletion abrogated the increases in proteinuria and the decrease in CrCl concurrently with increases in kidney inflammation and fibrosis. Furthermore, Larp6 protein levels were significantly lowered in the DPP4 knockout mice. These findings suggest that in conditions of progressive kidney injury, both DPP4 and Larp6 are activated. Moreover, DPP4 may regulate Larp6 expression and kidney fibrosis, thereby identifying DPP4 inhibition as a viable strategy to mitigate progressive kidney injury leading to end stage kidney disease and dialysis.

Support or Funding Information

Ravi Nistala was supported by research grant from Dialysis Clinics Inc., MU Research Board and Department of Medicine Research Council. Chandrasekar Bysani was supported by a VA Career Research Scientist Award
Aerobic exercise training prevents renal damage in insulin resistance mice
Authors: Muller Cynthia Rodrigues , Americo Anna Laura Viacava , Fiorino Patricia , Evangelista Fabiana Sant'Anna
Published: November 2023

Insulin resistance is associated with lipid accumulation in the kidney which leads to renal damage. We investigated the potential of aerobic exercise training (AET) to prevent renal damage in mice with diet‐induced insulin resistance. Adult male C57BL6/J mice were assigned into chow‐fed controls (C, n=7), cafeteria diet (CAF, n=7), chow‐fed trained (T, n=7), and cafeteria diet plus trained (CAFT=7). The standard chow diet contained 4% of kilocalories from fat, 55% from carbohydrate and 22% from proteins (Nuvilab®, Paraná, Brazil). The cafeteria diet contained 18.7% of kilocalories from fat, 56% from carbohydrate and 14.8% from proteins. PT was performed simultaneously with diet and consisted of 8‐wk running session of 60 min at 60% of maximal speed, 5 days/wk. Experimental procedures were approved by Ethics Committee from Faculty of Medicine of University of São Paulo (#18/2014). Cafeteria diet induced insulin resistance in CAF group compared to C group during insulin tolerance test, which was counteracted by AET in CAFT group. The glomerular filtration rate (GFR) increased by 88% in the CAF group compared to other groups. The water intake was lower in the CAF group (3.5 ± 0.3 mL) compared to the C (5.4 ± 0.5 mL), but there was no difference in urinary flow, urinary excretion of Na +, K +, Cl‐, total protein and urinary osmolarity. Histological analysis showed that CAF group increased intra‐renal lipid deposition (4.12 ± 0.48 %/area) compared to C group (1.70 ± 0.55 %/area), and that AET was able to prevent this increase in CAFT group (2.11 ± 0.51 %/area). There was a reduction in the Bowman's space in CAF group (104.4 ± 5.2 μm) compared to C group (154.3 ± 15.0 μm) and the AET prevented this decrease in CAFT (117.3 ± 5.3 μm). No changes were observed in the glomerular tuft area, however the CAF group showed a tendency to reduce Bowman's capsule area compared to C group (405.04 ± 21.23 vs. 470.7± 16.5 μm; p= 0.07). Protein expression of p‐AMPK increased in the CAFT group compared to the T group but there were no changes in t‐AMPK, t‐ACC, p‐ACC, PGC1‐α, SIRT‐1 expressions. In conclusion, our results showed that AET was efficient to prevent lipid deposition, the exacerbation of GFR and morphological impairments associated with insulin resistance. These responses may be associated, at least in part, with the increase in p‐AMPK in the kidney.

Support or Funding Information

FAPESP # 2015/04948‐4.
YAP‐mediated mechanotransduction determines the podocyte's response to injury
Authors: Rinschen Markus , Grahammer Florian , Huber Tobias , Benzing Thomas , Schermer Bernhard
Published: November 2023

Podocytes are terminally differentiated cells at the kidney filtration barrier and exposed to considerable mechanical strain. Podocyte injury causes morphological alterations as a result of cytoskeletal reorganizations. This becomes clinically apparent as proteinuria owing to leakage of the renal filter. The transcriptional co‐activators YAP/TAZ are tightly controlled through Hippo signaling and responsive to mechanical cues. Here, we show that YAP is upregulated upon podocyte injury in vivo to induce YAP‐dependent target genes. This activation preceded the development of proteinuria. In contrast, similar perturbations of podocytes in cell culture revealed decreased YAP/TAZ activity when cells were grown on stiff surfaces. Notably, culture on soft matrix or inhibition of stress fiber formation both allowed recapitulation of the damage‐induced YAP upregulation observed in vivo indicating a mechanotransduction‐dependent mechanism of YAP hyper‐activity. Yap overexpression enhanced the proteome‐wide expression of proteins related to extracellular matrix generation. Interestingly, increased expression of the bona fide YAP target CTGF was confirmed in renal biopsies from patients with glomerular disease. Consistently, genetic activation of YAP could induce mild proteinuria while pharmacological inhibition of YAP/TEAD activity ameliorated glomerular disease and damage‐induced mechanosignaling in vivo. Therefore, our findings suggest that perturbation of the mechanosensitive Hippo signaling pathway could be used as a therapeutic principle in podocyte disease.

Support or Funding Information

DFG
Peri‐implantation Ozone Exposure Alters Uterine Artery Flow and Induces Fetal Growth Restriction in Rats
Authors: Miller Colette N , Ledbetter Allen D , Schladweiler Mette C , Richards Judy E , Wood Charles , Snow Samantha , Johnstone Andrew , Jaskot Richard , Henriquez Andres , Thompson Leslie C , Farraj Aimen , Hazari Mehdi , Dye Janice A , Kodavanti Urmila
Published: November 2023

Epidemiological studies suggest a relationship between air pollutant exposures and various adverse pregnancy outcomes. Exposure to elevated ambient ozone levels during the first and second trimesters has been associated with increased risk for preeclampsia, gestational diabetes, and intrauterine growth restriction. As many of the adverse pregnancy outcomes are hypothesized to initiate from poor implantation, we sought to determine if an acute exposure to ozone during the implantation period would generate such outcomes later in pregnancy. Timed‐pregnant, Long‐Evans female rats were exposed to filtered air, 0.4ppm, or 0.8ppm ozone for 4 hours on each morning of gestational days 5 and 6. Tail cuff blood pressure and Doppler ultrasound of the uterine artery were measured following the phase of arterial remodeling on gestational days 15, 19, and 21. Rats were euthanized on gestational day 21 and offspring growth parameters were measured. While ozone exposure failed to impact maternal blood pressure at the times measured, multiple indices of blood flow in the uterine artery were adversely impacted as a result of early ozone exposure. Rats exposed to 0.8ppm ozone had increased uterine artery resistance index, systolic to diastolic ratio, pulsatile index, and end diastolic notching as pregnancy advanced compared to air controls. There was no evidence of systemic inflammation or kidney markers of preeclampsia in the dams. Ozone exposure decreased male and female offspring total weight, fat mass, and lean mass at the end of gestation. Further, 7 out of 9 dams exposed to 0.8ppm ozone during implantation had an average female offspring weight under the 10th percentile of air control pregnancies, suggestive of severe intrauterine growth restriction. Together these data suggest that peri‐implantation exposure to ozone adversely impacts uterine artery flow, which may have contributed to blunted fetal growth. These findings suggest a plausible biological mechanism for the epidemiological findings linking air pollutant exposure and adverse pregnancy outcomes. This abstract does not reflect US EPA Policy.

Support or Funding Information

This work was funded by the US EPA.
RhoBTB1 is a Novel Gene Protecting Against Hypertension
Authors: Mukohda Masashi , Ibeawuchi Stella‐Rita C , Hu Chunyan , Lu Ko‐Ting , Nair Anand R , Agbor Larry N , Wu Jing , Quelle Frederick W , Sigmund Curt D
Published: November 2023

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand activated transcription factor regulating metabolic and vascular function. We previously reported that mice (S‐DN) expressing dominant‐negative PPARγ in smooth muscle cells (SMC) are hypertensive, exhibit impaired vasodilation, augmented vasoconstriction and reduced expression of a novel PPARγ target gene, RhoBTB1. RhoBTB1 belongs to class of proteins which act as substrate adaptors for the Cullin‐3 E3 ubiquitin ligase. Delivery of substrates to the Cullin‐3 CRL3 complex leads to ubiquitination of the substrate protein targeting it for degradation by the proteasome. We hypothesized that loss of RhoBTB1 expression in S‐DN mice may be responsible for impaired vasodilation. In this study, we tested the hypothesis that RhoBTB1 may play a protective role by preventing hypertension in response to stressors which cause hypertension, such as Angiotensin‐II (Ang‐II). To test this, we generated double transgenic mice with tamoxifen‐inducible, Cre‐dependent overexpression of RhoBTB1 specifically in SMC (S‐RhoBTB1). S‐RhoBTB1 and non‐transgenic (NT) mice were treated with tamoxifen (Tx; 75 mg/kg, ip, 5 days) or vehicle (corn oil) and then Ang‐II was infused (490 ng/kg/min, 2 weeks). Overexpression of RhoBTB1 did not alter baseline blood pressure (BP) in the absence of Ang‐II. However, increased BP caused by Ang‐II infusion was significantly attenuated by RhoBTB1 overexpression in S‐RhoBTB1 with Tx compared to Ang‐II‐infused control mice (either NT with Tx, NT with corn oil, or S‐RhoBTB1 with corn oil) in which RhoBTB1 was not overexpressed (p<0.05, n=4–5). We also observed increased heart weight in Ang II‐infused control mice, which was reversed in S‐RhoBTB1 with Tx (n=4). Thoracic aorta, carotid and basilar artery from Ang‐II‐infused control mice showed impaired acetylcholine (ACh)‐induced endothelial‐dependent relaxation (p<0.05, n=4–5), which was reversed by overexpression of RhoBTB1 in SMC (p<0.05, n=4–5). Aorta and carotid artery from Ang‐II‐infused control mice also displayed decreased sodium nitroprusside (SNP)‐induced endothelial‐independent relaxation with a right‐shifted dose‐response (p<0.05, n=4–5), which was reversed in Tx‐treated S‐RhoBTB1 mice (p<0.05, n=5). Despite the marked improvement in vasodilation, augmented vasoconstriction to serotonin (5‐HT) was preserved in aorta and carotid artery from Ang‐II‐infused mice and was not reversed in Ang‐II‐infused S‐RhoBTB1 with Tx (n=4–5). We conclude that the novel PPARγ target gene, RhoBTB1, functions in SMC to specifically facilitate vasodilation and through this mediates a protective anti‐hypertensive effect. We are currently assessing which proteins may be substrates for Cullin‐3 through RhoBTB1.

Support or Funding Information

Grants from the NIH and AHA; Support from the Roy J. Carver Trust
Treatment with Lisinopril Delays the Early Progression of Proteinuria in a Novel Model of Prepubertal Obesity
Authors: McPherson Kasi C , Pennington Alyssa , Williams Jan M
Published: November 2023

Prepubertal childhood obesity (PPO) has emerged as an epidemic and major health problem in the United States. Recent studies suggest that PPO is associated with increased risk of renal injury in children. Approximately 37% of obese children develop microalbuminuria. However, an understanding of the mechanisms involved in PPO‐mediated renal disease remains unclear. Recently, we reported that the Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant – PPO model) strain develops proteinuria and podocyte injury independent of hyperglycemia and elevations in arterial pressure prior to puberty. Since renal hyperfiltration is a major characteristic of the early development of proteinuria associated with obesity, the current study determined the time course changes of glomerular filtration rate (GFR) in our model of PPO, the SSLepRmutant rat. GFR was significantly elevated in the PPO model compared to values measured to lean Control rats at 6 weeks of age indicating renal hyperfiltration (2.87±0.29 vs. 2.03±0.25 mL/min/gkwt, respectively). By 18 weeks of age, GFR decreased by 74% in the PPO model and by 46% in Control rats. The standard care of treatment for obese children with hypertension and increased risk of renal disease is lisinopril, an angiotensin converting enzyme inhibitor (ACEi). Therefore, the next set of experiments examined whether treatment with lisinopril prevents the early development of proteinuria and podocyte injury in the PPO model. Experiments were performed on four week‐old Control and PPO rats that were either treated with vehicle or lisinopril (20 mg/kg/day, drinking water) for four weeks. Protein excretion was significantly higher in the PPO model compared to Control rats at baseline (130±29 vs. 14±3 mg/day, respectively) and remained elevated during the course of the study (661±107 vs. 54±10 mg/day, respectively). While treatment with lisinopril had no effect on the progression of proteinuria in Control rats, it reduced protein excretion by 50% in the PPO model by the end of the study. In conclusion, these data indicate that our novel model of PPO, the SSLepRmutant strain, develops proteinuria associated with hyperfiltration at an early age that ultimately progresses to chronic kidney disease later in life. Moreover, treatment with lisinopril slowed the progression of proteinuria in the PPO model indicating that the renin‐angiotensin system plays an important role in the progression of proteinuria during PPO.

Support or Funding Information

This work was supported by funding from PhRMA predoctoral fellowship and the NIH/NIDDK Award Number 1 F31 DK109571‐01 awarded to K.C.M and AHA SDG 12SDG9440034 and by the NIGMS of the National Institutes of Health under Award Number P20GM104357 awarded to J.M.W.
Dairy Cheese Ingestion Does Not Improve Acute Sodium‐Induced Cutaneous Microvascular Dysfunction through Arginase or Inducible Nitric Oxide Synthase Mechanisms
Authors: Alba Billie K. , Stanhewicz Anna E. , Kenney W. Larry , Alexander Lacy M.
Published: November 2023

Epidemiological studies suggest that chronic dairy consumption is associated with improved cardiovascular health; alternately high dietary sodium (Na) ingestion is associated with increased cardiovascular disease risk. Natural cheeses are often high in sodium, thus increasing dairy cheese intake may increase dietary Na intake. We recently demonstrated that single‐meal cheese consumption ameliorates Na‐induced impairments in nitric oxide (NO)‐dependent vasodilation through ascorbate sensitive mechanisms. However, the role of additional inflammatory mediators sensitive to the non‐specific antioxidant ascorbate, including arginase and inducible NO synthase (iNOS), is unknown. We hypothesized that local inhibition of arginase or iNOS would improve NO‐dependent vasodilation following Na ingestion from non‐dairy sources but not Na ingestion from dairy‐based cheese. On 5 separate visits, 14 subjects (61±2 yrs) consumed 85 g natural cheddar cheese (560 mg Na), 85 g soy cheese (560 mg Na), 65 g pretzels (560 mg Na), 170 g natural cheddar cheese (1120 mg Na), or 130 g pretzels (1120 mg Na). Three intradermal microdialysis fibers were inserted in the forearm skin for local delivery of 1) lactated Ringer's solution (control site) 2) 5 mM (S)‐(2‐boronoethyl)‐L‐cysteine (arginase‐inhibited site) and 3) 0.1 mM 1400w (iNOS‐inhibited site). Red cell flux was measured using laser‐Doppler flowmetry during standardized local skin heating (42°C) to stimulate endothelial NOS‐dependent vasodilation. Once a plateau in cutaneous vascular conductance (CVC = flux/mean arterial pressure) was reached, 15 mM L‐NAME (nonselective NOS inhibitor) was perfused at all sites to directly quantify NO‐dependent vasodilation. Data were expressed as a percentage of maximum vasodilation (%CVCmax; 28 mM sodium nitroprusside). NO‐dependent vasodilation was greater following dairy cheese ingestion compared to the same amount of Na in pretzels (560 mg Na: 57±6 vs. 43±4%; p=0.004, 1120 mg Na: 55±5 vs. 46±5%; p=0.04) or soy cheese (57±6 vs. 44±6%; p=0.002). NO‐dependent vasodilation was not different among the control site, the arginase‐inhibited site, or the iNOS‐inhibited site for any of the five dietary treatments (all p>0.05). These results suggest that Na ingestion in dairy cheese is associated with greater NO bioavailability compared to ingestion of an equal amount of Na from non‐dairy sources but that this difference is not mediated by arginase‐ or iNOS‐dependent mechanisms. Rather, macronutrients in dairy cheese likely prevent acute Na‐induced microvascular dysfunction through previously demonstrated antioxidant properties.

Support or Funding Information

Supported by Dairy Management Inc. and T32GM108563
Effect of phytoecdysteroids on protein synthesis and Akt/mTOR signaling after downhill running in skeletal muscle of mice
Authors: Goslen Kevin H. , Perrin Ella C. , Garvick G. Alan , Rice Tyler T. , Gaillard Charleston E. , Rao Shriya D. , Shanely R. Andrew , Zwetsloot Kevin A.
Published: November 2023

Phytoecdysteroids are natural plant steroids synthesized by a variety of hardy plants. Phytoecdysteroids, such as 20‐hydroxyecdysone (20E), possess anabolic properties and previous research indicates that 20E stimulates protein synthesis in cultured muscle cells and increases grip strength in young rats after 28 days of supplementation. The purpose of this study was to investigate the extent to which 20E stimulates protein synthesis and Akt/mTOR signaling in skeletal muscle after an acute bout of downhill running (DHR). Male C57BL6 mice (3–6 mo old) were assigned to four groups. Mice in the DHR groups performed an acute bout of DHR to exhaustion on a rodent treadmill at 17 m•min−1 and −20° decline. After completion of the DHR bout and recovery, mice received an oral gavage treatment of either 50 mg•kg−1 body mass (BM) of 20E (DHR + 20E; n=5) or vehicle (DHR + vehicle; n=6), and then treated daily for the next four consecutive days. Two groups that did not perform DHR, but were treated for five consecutive days with either 50 mg•kg−1 BM of 20E (No DHR + 20E; n=8) or vehicle (No DHR + vehicle; n=6), were used as controls. On the fifth day post‐DHR, mice were not treated with 20E or vehicle; however, an IP injection of puromycin (0.040 μmol•g−1 BM) was administered 30 min prior to sacrifice to assess protein synthesis. Skeletal muscles were harvested and activation of protein synthesis was assessed using the SUnSET method and Western blot and Akt/mTOR signaling activation was measured via Western blot. Mice in the DHR + vehicle group ran for 58.5 min, while mice in the DHR + 20E group ran for 57.0 min. Compared to No DHR + vehicle, DHR + vehicle increased skeletal muscle puromycin incorporation 14%, but DHR + 20E increased puromycin incorporation 32%, five days post‐DHR. Puromycin incorporation was not different between No DHR + vehicle and No DHR + 20E groups. DHR resulted in ~25% increase in phosphorylation of both Akt and p70S6K, compared to No DHR + vehicle; but there was no difference in Akt or p70S6K phosphorylation between DHR + 20E and DHR + vehicle groups, five days post‐DHR. Compared to No DHR + vehicle, DHR + vehicle increased 4EBP1 phosphorylation 35%, but DHR + 20E increased 4EBP1 phosphorylation 70%, and DHR + vehicle increased ribosomal protein S6 (rpS6) phosphorylation 40%, but DHR + 20E increased rpS6 phosphorylation 119%, five days post‐DHR. In conclusion, these findings suggest that DHR stimulates protein synthesis and activates Akt/mTOR signaling five days after DHR, but 20E treatment enhances the pattern of response to an acute bout of DHR in skeletal muscle of young mice.
The Ketogenic Diet Alters Endocrine Regulation of Energy Metabolism in Ultra‐Endurance Athletes
Authors: Miller Vincent J , Hyde Parker N , Dickerson Ryan , LaFountain Richard A , Maresh Carl M , Kraemer William J , Volek Jeff S
Published: November 2023

Purpose

The ketogenic diet induces a profound shift in energy metabolism from carbohydrate oxidation towards fat oxidation, both at rest and during exercise. We compared endocrine regulators of energy metabolism in keto‐adapted and non‐keto‐adapted ultra‐endurance athletes before, during, and after aerobic exercise to better understand the role of endocrine function in adaptation to a ketogenic diet (keto‐adaptation).

Methods

Twenty elite ultra‐endurance athletes (male, age 33.5 ± 6.4 yr, BMI 22.1 ± 1.5 kg/m2, VO2max 64.5 ± 4.9 mL/kg/min) who habitually consumed a high‐carbohydrate (HC, 59:14:25 % carbohydrate:protein:fat) or very‐low carbohydrate (LC, 10:19:70) diet for at least 6 months ran on a treadmill for 180 min at 64% of VO2max. Glucagon, cortisol, and leptin were measured from serum collected before exercise (BL); 60 and 120 min after initiation of exercise (RUN60, RUN120); and 0, 60, and 120 min after completion of exercise (IP, P60, P120). After BL and IP, participants consumed a shake with macronutrient ratios similar to their habitual diet (5 kcal/kg body mass, HC: 50:36:14, LC: 5:81:14 % carbohydrate:protein:fat). Glucagon, cortisol, and leptin were further analyzed for correlations with non‐esterified fatty acids (NEFA), glycerol, β‐hydroxybutyrate, total ketones, insulin, glucose, and muscle glycogen.

Results

Glucagon increased with exercise (p<0.0001) and was significantly higher in keto‐adapted athletes at all time points except IP and P120 (p<0.05). Cortisol also increased with exercise (p<0.0001) and was significantly higher in keto‐adapted athletes at RUN60 (p<0.05). Leptin decreased with exercise (p<0.0001), but without any significant effect of diet. Glucagon and, to a lesser extent, cortisol positively correlated with markers of increased fat oxidation (β‐hydroxybutyrate, total ketones, NEFA, and glycerol, p<0.05) and negatively correlated with markers of increased carbohydrate metabolism (glucose, insulin, and lactate, p<0.05). Glucagon's correlations with β‐hydroxybutyrate and total ketones were persistent, occurring at all time points except BL (p<0.01). Glucagon and cortisol also positively correlated with each other at P120 (p<0.005). Muscle glycogen content negatively correlated with glucagon at IP (p<0.05) and positively correlated with leptin at P120 (p<0.05).

Conclusion

In elite ultra‐endurance athletes, a long‐term ketogenic diet is associated with higher glucagon levels before, during, and after aerobic exercise. This is in conjunction with a more than two‐fold higher rate of peak fat oxidation, indicating glucagon may have an important role in keto‐adaptation. The similar but less persistent patterns of cortisol suggest these two hormones may contribute to keto‐adaptation in a coordinated manner.

Support or Funding Information

Support for this work was provided by Quest Nutrition and The Robert C. and Veronica Atkins Foundation.
Dietary Vitamin A Intake Affects the Body Mass Gain and Hepatic Gene Expression in Rats fed a High‐Fat Diet
Authors: Kuang Heqian , Li Yang , Eastep Jennifer , Chen Guoxun
Published: November 2023

Introduction

Vitamin A (VA) plays a role in variety of physiological functions from development to metabolic homeostasis. Feeding of a high‐fat (HF) diet has been shown to cause the development of metabolic diseases such as obesity and fatty liver. The purpose of our study is to determine whether in a HF diet setting, VA status affects and the protein expression levels of hepatic genes involved in glucose, fatty acid metabolism, and insulin signaling pathways.

Methods

Sprague Dawley (SD) rats after weaning were fed an isocaloric HF‐VA sufficient (HF‐VAS) or HF‐VA deficient (HF‐VAD) diet for 8 weeks. Their body mass and food intake were recorded weekly. At the end of feeding, tissue samples were weighted and collected for analysis. The protein levels in liver samples were determined via immunoblotting.

Results

From week 6, body mass of HF‐VAD rats was significantly lower than that of HF‐VAS ones. On the other hand, the total food intake was similar between HF‐VAD and HF‐VAS groups. The liver mass and epididymal fat mass of HF‐VAD group were significant lower than that of HF‐VAS group. The hepatic expression level of cytochrome P450 26A1 protein in HF‐VAS group is higher than that in HF‐VAD group, suggesting reduced retinoic acid production. For glucose metabolism, the expression levels of glucokinase and phosphoenoylpyruvate carboxykinase in HF‐VAS group were higher than the level in HF‐VAD group. For fatty acid biosynthesis, the expression levels of acetyl‐CoA carboxylase (ACC), phosphor‐ACC, fatty acid synthase, and ATP citrate lyase were similar in two groups. The levels of insulin receptor β‐subunit and insulin receptor substrate (IRS) 1 were increased in HF‐VAD rats, while the expression level of IRS 2 were decreased in HF‐VAD rats. The expression level of mitogen‐activated protein kinase of HF‐VAD group was higher than that of HF‐VAS group. The expression levels of protein kinase C ζ, ι, δ, mammalian target of rapamycin, AMP‐activated protein kinase α and β, and protein kinase B were similar between HF‐VAD and HF‐VAS groups. The HF‐VAD rats had higher levels of glycogen synthase kinase 3α (GSK‐3α) and lower GSK‐3β expression levels, but not glycogen synthase, than the HF‐VAS rats did.

Conclusions

VA status reduced the body mass gain in rats fed a HF diet. In a HF setting, VA status still can alter the expression levels of genes for glucose metabolism and insulin signaling pathways. These and our previous results indicate that VA status and its metabolism affect the hepatic expression levels of insulin‐regulated genes in both normal and HF dietary conditions. More studies are needed to determine the underlying mechanisms for these changes.